Antigen-specific immune effector cells

Abstract

Provided herein are methods for producing antigen-specific effector T cells and NK cells expressing chimeric antigen receptors (CAR) from pluripotent stem cells. Also provided herein are methods for adoptive cell therapy by administering effector T cells and / or NK cells provided herein.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 62 / 486,875, filed on April 18, 2017, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to methods and compositions related to antigen-specific immune effector cells (eg, T cells and NK cells). Background Art

[0003] Description of related prior art

[0004] Despite technological advances in diagnostic and therapeutic options available to patients diagnosed with cancer, the prognosis generally remains poor, and many patients do not recover. Immunotherapy holds the promise of providing patients diagnosed with a variety of tumors with a potent yet targeted treatment that has the potential to eradicate malignant cells without damaging normal tissue. In theory, T cells of the immune system are able to recognize tumor cell-specific protein patterns and mediate their destruction through a variety of effector mechanisms. Adoptive T cell therapy attempts to harness and enhance the ability of a patient’s own T cells to eradicate tumors and then return these effectors to the patient in a state that is effective in eliminating residual tumors but spares healthy tissue. Although this approach is not new to the field of tumor immunology, a number of shortcomings of adoptive T cell therapy in clinical use have prevented its full utilization in cancer treatment.

[0005] Current adoptive T cell therapies are limited by the scarcity of patient- and tumor-specific T cells, including their rarity in vivo, their inability to overcome multiple tumor immune system evasion mechanisms, and their short lifespan. The often small number of T cells reactive to the desired antigen are difficult to isolate and expand. Therefore, there is an unmet need for therapeutically sufficient and functional antigen-specific immune cells for effective use of immunotherapy. Summary of the invention

[0006] In a first embodiment, the present disclosure provides a method for generating antigen-specific effector T cells and / or NK cells, comprising modifying pluripotent stem cells (PSCs) to express a chimeric antigen receptor (CAR), thereby generating CAR-PSCs; differentiating or forward reprogramming the CAR-PSCs into CD34 + Hematopoietic progenitor cells (HPC); CD34 + HPCs are further differentiated into T cells and / or NK cells; and T cells and / or NK cells are expanded. In some aspects, the expansion includes co-culturing with antigen-specific target cells to produce antigen-specific effector T cells and / or NK cells.

[0007] In certain aspects, the PSC expressed CAR through modification is an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC). In a specific aspect, iPSC is reprogrammed from a somatic cell (e.g., a T cell).

[0008] In some aspects, CAR-PSCs are differentiated into CD34 + The step of HPC includes directed differentiation. In some aspects, directed differentiation includes forming embryoid bodies (EBs) in the presence of blebbistatin, GSK-3 inhibitors, FGF2 and VEGF; contacting EBs with BMP4, VEGF and FGF2 to induce mesoderm induction; differentiating EBs in the presence of Flt-3 ligand, IL-3, SCF and TPO to produce HPCs. In some aspects, differentiation is carried out in a culture medium that is substantially free of or free of BMP4. In other aspects, differentiation is carried out in the presence of BMP4. In particular aspects, the GSK-3 inhibitor is CHIR99021. In some aspects, differentiation also includes the presence of IL-11, cAMP and / or VEGF.

[0009] In some aspects, directed differentiation comprises culturing individualized PSCs on an amine-coated surface in the presence of blebbistatin, BMP4, VEGF, and bFGF. Differentiation is initiated by contacting the PSCs with BMP4, VEGF, and FGF2; and further differentiating the PSCs in the presence of Flt-3 ligand, IL-3, IL-6, SCF, TPO, and heparin to generate HPCs, wherein the method does not include EB formation.

[0010] In certain aspects, the methods include culturing the cells under defined feeder-free conditions, e.g., for the duration of the entire method. In some aspects, the PSCs are substantially transgenic-free or transgenic-free. In certain aspects, the 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, naive T cells Memory T cells and / or γδ T cells.

[0011] In some aspects, the PSC engineered to express CAR is further engineered to express ERG / ETV2, GATA2, and HOXA9, for example, under the control of a single inducible promoter. In some aspects, the method further comprises engineering the PSC to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, and / or HOXA4. Thus, in some aspects, the CAR-PSC is differentiated into a CD34 +The step of producing HPC includes inducing the expression of ERG / ETV2, GATA2 and HOXA9 for a period of time sufficient to generate HPC, and terminating the induction of expression before further differentiating the HPC into T cells and / or NK cells.

[0012] In a specific aspect, CAR-PSCs are differentiated into CD34 + The step of HPC also includes selecting cells expressing CD34 and / or CD43 before differentiating into antigen-specific T cells and / or NK cells. In particular aspects, selection includes performing magnetic activated cell sorting (MACS). In some aspects, cells expressing CD34 and / or CD43 account for at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or higher percentages of the total cell population. In some aspects, the step of selecting CD34 and / or CD43 positive cells with MACS is not performed.

[0013] In certain aspects, at least 2%, 3%, 4%, 5%, 10%, 15%, 20% or more 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% or more HPCs express CAR.

[0014] In some aspects, HPC is differentiated into T cells and / or NK cells and includes: under hypoxic conditions, in the presence of ascorbic acid and nicotinamide, HPC is cultivated on the surface coated with retronectin and Notch DLL-4. In some aspects, the culture also includes SCF, FLT-3 ligand, TPO and IL-7, and optionally includes GSK inhibitor (such as CHIR99021), IL-2 and / or IL-12. In some aspects, the expansion step also includes cultivating antigen-specific T cells in the presence of anti-CD3 antibodies and IL-2. In other aspects, the expansion step also includes cultivating antigen-specific T cells in the presence of anti-CD3 antibodies, IL-2, IL-15 and IL-21. In some aspects, the expansion step includes cultivating antigen-specific T cells in the presence of anti-CD3 antibodies, FLT3- ligand, IL-7, IL-2 and / or IL-15, and optionally also includes SCF, TPO and / or IL-21. In certain 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 of the differentiated CD34 + HPC is CD3 + CAR +In some aspects, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or more of the differentiated CD34 + HPC is CD3 - CAR + NK cells. In some aspects, at least 2% of the expanded CD34 + HPC is CD3 + CAR + In certain aspects, at least 10% of the expanded CD34 + HPC is CD3 - CAR + NK cells.

[0015] In some aspects, CAR and antigen-specific target cells are directed to the same antigen. In a specific aspect, the antigen is CD19. In a particular aspect, the antigen-specific target cell is a tumor cell. In some aspects, the antigen-specific target cell is human. In a particular aspect, the antigen-specific target cell is HLA class I negative. In some aspects, at least 5% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% or higher percentages) of antigen-specific effector T cells show cytotoxic activity to target cells. In some aspects, at least 10% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or higher percentages) of antigen-specific effector NK cells show cytotoxic activity to target cells.

[0016] In some aspects, CAR is encoded by DNA integrated into the PSC genome. In some aspects, CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In specific aspects, the intracellular signaling domain comprises CD3ζ and CD28. In some aspects, the antigen binding region is F(ab')2, Fab', Fab, Fv or scFv.

[0017] In some aspects, the PSC is in the form of HLA homozygosity. In some aspects, the PSC in the form of HLA homozygosity is homozygous for one or more of the locus alleles HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ. In some aspects, the PSC in the form of HLA homozygosity is homozygous for two of the locus alleles HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ. In a specific aspect, the PSC in the form of HLA homozygosity is homozygous for HLA-A and HLA-B. In some aspects, the PSC in the form of HLA homozygosity is homozygous for HLA-A, HLA-B and HLA-C.

[0018] Another embodiment provides a method for producing antigen-specific effector T cells and / or NK cells, which includes modifying PSC to express CAR, thereby producing CAR-PSC. Cultivating CAR-PSC in the presence of blebbistatin, GSK-3 inhibitor, FGF2 and VEGF to form EBs; contacting EBs with BMP4, VEGF and FGF2 to induce mesoderm induction; differentiating EBs in the presence of Flt-3 ligand, BMP4, IL-3, SCF and TPO to produce CD34+HPCs; differentiating CD34 + HPCs are further differentiated into T cells and / or NK cells; and T cells and / or NK cells are expanded, wherein the expansion includes co-culturing with antigen-specific target cells, thereby generating antigen-specific effector T cells and / or NK cells. In a specific aspect, the GSK-3 inhibitor is CHIR99021.

[0019] In yet another embodiment, a method for generating antigen-specific effector T cells and / or NK cells is provided, comprising modifying PSC to express CAR, thereby generating CAR-PSC; culturing personalized CAR-PSC on an amine-coated surface in the presence of blebbistatin, BMP4, VEGF, and initiating differentiation by contacting the CAR-PSC with BMP4, VEGF, and FGF2; further differentiating the CAR-PSC in the presence of Flt-3 ligand, IL-3, IL-6, SCF, TPO, and heparin, thereby generating CD34 + HPC;CD34 + HPCs are differentiated into T cells and / or NK cells; and the T cells and / or NK cells are expanded, wherein the expansion comprises co-culturing with antigen-specific target cells, thereby generating antigen-specific effector T cells and / or NK cells, wherein the method does not comprise EB formation.

[0020] Still another embodiment provides a method for generating antigen-specific effector T cells and / or NK cells, comprising modifying PSC to express CAR, thereby generating CAR-PSC; differentiating CAR-PSC into CD34 + HPC; select CD34 + CD43 + HPC;CD34 + CD43 + The HPCs are further differentiated into T cells and / or NK cells; and the T cells and / or NK cells are expanded, wherein the expansion includes co-culturing with antigen-specific target cells, thereby generating antigen-specific effector T cells and / or NK cells.

[0021] In another embodiment, a population of antigen-specific effector T cells and / or NK cells produced according to the above embodiments is provided. A pharmaceutical composition is also provided herein, comprising antigen-specific effector T cells and / or NK cells produced according to the embodiments described herein. A composition comprising antigen-specific effector T cells and / or NK cells produced according to the embodiments is also provided herein for treating cancer in a subject. A use of antigen-specific effector T cells and / or NK cells produced according to the embodiments is also provided herein for the treatment of cancer.

[0022] In yet another embodiment, a method for 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 produced according to the embodiments described herein. In some aspects, the cancer expresses a tumor antigen, and the antigen-specific effector T cells and / or NK cells are directed against the tumor antigen. In specific aspects, the subject is a human.

[0023] According to the following detailed description, other objects, features and advantages of the present invention will become clear. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, they are only given by way of example, because those skilled in the art will be clear about various changes and modifications within the spirit and scope of the present invention based on the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings constitute part of this specification, and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments described herein.

[0025] Figures 1A-1G :( Figure 1A ) schematically describes methods for generating T cells and NK cells from PSCs, including forward programming methods and directed differentiation methods. ( Figure 1B) represents a diagram of feeder-free and serum-free T and NK cell differentiation of hPCS. Abbreviations: CHIR-CHIR99021 GSK-3 inhibitor; HDM, serum-free hematopoietic differentiation medium; TCDM, T cell differentiation medium; TCEM, T cell expansion medium; HPC, hematopoietic progenitor cells; those marked with an asterisk (*) are optional enhancement components. ( Figure 1C ) through directed differentiation or forward programming from PSC to CD34 + CD43 + HPC efficiency. Figure 1D ) Flow cytometric analysis of T / NK differentiation cultures. Figure 1E )The yield of different cell populations during the entire differentiation process. Figure 1F ) Phenotype of PSC-derived T cells. Figure 1G ) Expansion of PSC-derived T cells. Immobilized anti-CD3 antibody (iCD3) was used to expand PSC-derived T cells (bar graph). T cells proliferating in expansion culture acquired the characteristic morphology of irregularly shaped lymphoblasts (photograph). 2 weeks of T cell expansion obtained CD56 and CD8 expression (flow cytometry dot plot).

[0026] Figure 2 : CAR expression in PSC-derived T / NK cells. CAR expression throughout differentiation stages assessed by flow cytometry using protein L staining. + T cells were co-stained with lambda chain mouse anti-human CD3 mAb (clone SP34-2). CAR expression by E11 PSCs as well as E11-derived HPCs and T cells is shown.

[0027] Figure 3A -B: In vitro anti-CD19 CAR-mediated cytotoxicity in PSC-derived T / NK cells. ( Figure 3A ) Using CD19 expressing luciferase + In vitro cytotoxicity assays with Daudi and Raji target cells. Figure 3B ) The cytolytic potential of CAR-T cells measured by real-time target cell counting using the IncucyteS3 live cell analysis system (Essen Bioscience).

[0028] Figure 4 : In vitro anti-CD19 CAR-induced cytokine production in PSC-derived T / NK cells.

[0029] Figure 5A-5B :In vivo oncolytic potential of PSC-derived CAR-T / NK cells. Figure 5A )Tumor progression in mice monitored by in vivo bioluminescence imaging. Figure 5B) Survival curves of different groups of mice treated with PSC-derived T (1C) or NK (A16) cells. DETAILED DESCRIPTION

[0030] Description of Illustrative Embodiments

[0031] In certain embodiments, the present disclosure provides an efficient method for producing antigen-specific immune effector cells from human pluripotent stem cells (PSC), wherein the PSC has been modified to express an antigen receptor (e.g., a chimeric antigen receptor (CAR)), referred to herein as CAR-PSC. The immune effector cells produced by the method may include, but are not limited to, T cells, NK cells, and iNKT cells.

[0032] PSC can be obtained by reprogramming (e.g., by retroviral or episomal methods) the starting population of T cells to produce T cell-derived iPSCs (TiPSCs). T cells can be isolated from various sources, such as blood samples. The starting population of T cells can retain its characteristic T cell receptor (TCR) gene rearrangement, and can be cells in the form of HLA homozygous forms (i.e., homozygous forms for MHC class I and class II genes). Therefore, iPSC can be produced from cells isolated from subjects in the form of HLA homozygous forms (herein referred to as HLA super donors).

[0033] CAR-PSCs can then be differentiated or programmed to produce CD34 + Hematopoietic progenitor cells (HPCs). This can be achieved by directed differentiation using a combination of cytokines (e.g., SCF, TPO, FLT-3, IL-6, IL-3, and heparin) (e.g., as described in PCT / US2016 / 057899, which is incorporated herein by reference in its entirety). In an alternative approach, forward programming can be used to differentiate CAR-PSCs into CD34+ cells using an expression construct encoding ETV2 / ERG, GATA2, and HOXA9 (i.e., EGH). + HPCs (e.g., as described in PCT / US2016 / 057893, which is incorporated herein by reference in its entirety). These EGH-PSCs can be further engineered to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, and / or HOXA4 to achieve long-term engraftability.

[0034] Finally, CD34 + CAR-HPCs can be differentiated into CD3 + T cells or CD56 + CD3 -NK cells. Determined by the expression of CD34 and CD43, the optimal time range for lymphoid potential may be 7-11 days during HPC differentiation (e.g., 7-11 days). For example, HPCs with enhanced lymphoid potential can be isolated by sorting cell fractions that are positive for two or more of the markers CD 144, CD34, CD45, and CD7.

[0035] An exemplary method of T cell differentiation includes the use of RetroNectin and DLL-4 as a feeder-free matrix. T cell differentiation can be further enhanced by using ascorbic acid to increase efficiency and maturation, and by culturing under hypoxic conditions.

[0036] In addition, T cells and / or NK cells can be expanded by co-culturing with antigen-specific target cells (e.g., tumor cells) during differentiation. This method was found to increase the cytotoxic activity of T cells and NK cells against target cells, specifically observed by reduced tumor growth and increased survival of mice injected with tumor cells.

[0037] Thus, the methods of the present disclosure can provide unlimited numbers of antigen-specific immune effector cells (eg, T cells and NK cells) for a wide range of applications, such as stable transplantation in vivo, compound screening in vitro, and elucidation of the mechanisms of hematological diseases and injuries.

[0038] I. Definitions

[0039] With respect to a specific component, "substantially free" as used herein means herein that the specific component is not purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specific component resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions from which the amount of the specific component cannot be detected using standard analytical methods.

[0040] As used in the specification herein, "a" or "an" may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one.

[0041] Unless explicitly stated to involve only alternatives or the alternatives are mutually exclusive, the term "or" used in the claims is used to mean "and / or," although the present disclosure supports a definition involving only alternatives and "and / or." As used herein, "another" may mean at least a second or more.

[0042] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value or the variation that exists between study subjects.

[0043] The term "exogenous" when used in connection with 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 a cell or organism by artificial or natural means; or when used in connection with a cell, the term refers to a cell that has been isolated and subsequently introduced into other cells or organisms by artificial or natural means. The exogenous nucleic acid can be from a different organism or cell, or can be one or more additional copies of a nucleic acid that occurs naturally in the organism or cell. The exogenous cells can be from different organisms or from the same organism. As non-limiting examples, an exogenous nucleic acid is a nucleic acid that is in a chromosomal location different from that in a natural cell, or that is flanked by a nucleic acid sequence that is different from that occurring in nature.

[0044] "Expression construct" or "expression cassette" refers to a nucleic acid molecule capable of directing transcription. An expression construct includes at least one or more transcriptional control elements (e.g., promoters, enhancers, or functionally equivalent structures thereof) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements may also be included, such as transcription termination signals.

[0045] A "vector" or "construct" (sometimes referred to as a gene delivery system or gene transfer "vector") refers to a macromolecule or complex of molecules that contains a polynucleotide to be delivered to a host cell in vitro or in vivo.

[0046] "Plasmid" is a common type of vector, which is an extrachromosomal DNA molecule separate from the chromosomal DNA, which can replicate independently of the chromosomal DNA. In some cases, it is circular and double-stranded.

[0047] "Origin of replication" ("ori") or "origin of replication" is a DNA sequence, for example, in lymphotrophicherpes virus, which, when present in a plasmid and in a cell, is capable of maintaining a junction sequence in the plasmid and / or at or near the start site of DNA synthesis. As an example, the ori of EBV (Epstein-Barr virus) includes a FR sequence (20 imperfect copies of 30bp repeats), preferably a DS sequence; however, other sites in EBV bind to EBNA-1, for example, a Rep* sequence can replace DS as a replication origin (Kirshmaier and Sugden, 1998). Therefore, the replication origin of EBV includes a FR, DS or Rep* sequence, or any functionally equivalent sequence modified by nucleic acid or a synthetic combination derived therefrom. For example, the disclosure may also use a genetically engineered replication origin of EBV, such as genetically engineered by insertion or mutation of a single element, as specifically described in Lindner et al., 2008.

[0048] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" that "encodes" a particular protein is a nucleic acid molecule that is transcribed in vitro or in vivo and optionally also translated into a gene product (e.g., a polypeptide) when under the control of appropriate regulatory sequences. The coding region may be in the form of cDNA, genomic DNA, or RNA. When in the form of DNA, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A 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. A transcription termination sequence will typically be located at the 3' end of the gene sequence.

[0049] The term "control element" refers generally to a promoter region, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, a replication origin, an internal ribosome entry site (IRES), an enhancer, a splice junction, etc., which together provide for replication, transcription, post-transcriptional processing and translation of a coding sequence in a recipient cell. These control elements do not need to be present, as long as the selected coding sequence can be replicated, transcribed and translated in a suitable host cell.

[0050] The term "promoter" is used herein with its usual meaning to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that can bind to RNA polymerase and initiate transcription of a downstream (3' direction) coding sequence. It may include genetic elements (e.g., RNA polymerase and other transcription factors) to which regulatory proteins and molecules can bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to a promoter that is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence.

[0051] An "enhancer" refers to a nucleic acid sequence that, when located near a promoter, confers increased transcriptional activity relative to the transcriptional activity produced by the promoter in the absence of the enhancer domain.

[0052] "Operably linked" or "co-expressed" with respect to nucleic acid molecules means that two or more nucleic acid molecules (e.g., nucleic acid molecules to be transcribed, promoters, and enhancer elements) are linked in a manner that allows transcription of the nucleic acid molecules. "Operably 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 a manner that produces a single polypeptide chain (i.e., a fusion polypeptide) having at least one property of each of the peptide and / or polypeptide components of the fusion. The fusion polypeptide is preferably chimeric, i.e., consists of heterologous molecules.

[0053] "Homology" refers to the percent identity between two polynucleotides or two polypeptides. The correspondence between one sequence and another sequence can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that promote the formation of stable duplexes between homologous regions, then digesting with single-stranded specific nucleases, and determining the size of the digested fragments. As determined using the above method, two DNA 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, match over a determined length of the molecule.

[0054] The term "cell" is used herein in its broadest sense in the art to refer to such a living body: it is a structural unit of a multicellular organism's organization, surrounded by a membrane structure that isolates it from the outside world, and has the ability to replicate itself, genetic information, and a mechanism for expressing genetic information. The cell used herein may be a naturally occurring cell or an artificially modified cell (e.g., a fusion cell, a genetically modified cell, etc.).

[0055] The term "stem cell" refers herein to a cell that can differentiate into a variety of specialized cell types under suitable conditions, while being able to self-renew under other suitable conditions and maintain a substantially undifferentiated pluripotent state. The term "stem cell" also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells may be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal genital tissue. Exemplary pluripotent stem cells may also be produced from somatic cells by expressing certain transcription factors associated with pluripotency and reprogramming somatic cells to a pluripotent state; these cells are referred to as "induced pluripotent stem cells" or "iPSC or iPS cells".

[0056] "Embryonic stem (ES) cells" are undifferentiated pluripotent cells that are obtained from an early embryo (e.g., the inner cell mass at the blastocyst stage) or generated by artificial means (e.g., nuclear transfer) and are capable of giving rise to any differentiated cell type in the embryo or adult, including germ cells (e.g., sperm and eggs).

[0057] "Induced pluripotent stem cells (iPSC or iPS cells)" are cells produced by reprogramming somatic cells by expressing or inducing a combination of multiple factors (referred to herein as reprogramming factors). Fetal, postnatal, neonatal, adolescent or adult somatic cells can be used to generate iPS cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog and Lin28. In some embodiments, somatic cells are reprogrammed to reprogram somatic cells into pluripotent stem cells 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.

[0058] "Hematopoietic progenitor cells" or "hematopoietic precursor cells" refer to cells that belong to the hematopoietic lineage but are capable of further hematopoietic differentiation, including hematopoietic stem cells, multipotent hematopoietic stem cells, common myeloid progenitor cells, megakaryocyte progenitor cells, pro-erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells (HSCs) are multipotent stem cells that produce all blood cell types, including myeloid (monocytes and macrophages, granulocytes (neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells) (see, e.g., Doulatov et al., 2012; Notta et al., 2015). "Multilymphoid progenitors" (MLPs) are defined to describe any progenitor cells that produce all lymphoid lineages (B, T, and NK cells) but may or may not have other (myeloid) potentials (Doulatov et al., 2010), and they are CD45RA + / CD10 + / CD7 - Any B, T, and NK progenitor cell can be referred to as MLP. Common myeloid progenitor cells (CMP) are cells that express CD45+ / CD31 + / CD43 + / CD34 - Common myeloid progenitor cells defined by the expression of CD34 are capable of producing granulocytes, monocytes, megakaryocytes, and erythrocytes. Hematopoietic progenitor cells may express CD34. Hematopoietic progenitor cells may co-express CD133 and are negative for CD38 expression. Hematopoietic precursor cells include CD34 + / CD45 + Hematopoietic progenitor cells and CD34 + / CD45 + / CD43 + Hematopoietic precursor cells. CD34 + / CD43 + / CD45 + Hematopoietic precursor cells are highly enriched for myeloid progenitor cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells, including: CD34 - / CD133 + / CD38 -(primitive hematopoietic precursor cells), CD43(+)CD235a(+)CD41a(+ / -)(erythro-megakaryopoiesis), lin(-)CD34(+)CD43(+)CD45(-)(specialized) and lin(-)CD34(+)CD43(+)CD45(+)(myeloid tendency) cells, CD133+ / ALDH+(aldehyde dehydrogenase). It is contemplated that any of these primitive hematopoietic cell types or hematopoietic precursor cells can be converted into iPS cells as described herein. In some aspects, the cells may include mast cells, Langerhan's cells, osteoclasts, NK cells, T cells, CIK T cells, or other subtypes of T cells, NK cells, and B cells.

[0059] As used herein, the term "immune cell" 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, erythrocytes, monocytes, basophils, neutrophils, mast cells, eosinophils, and any combination thereof.

[0060] T cell "activators" or conditions that will activate T cells refer to stimuli that activate T cells, including antigens that can be presented on antigen presenting cells or other surfaces; polyclonal activators, regardless of specificity, which can bind to many T cell receptor (TCR) complexes, including lectins (such as concanavalin A (Con-A) and phytohemagglutinin (PHA)) and agents (such as antibodies) that specifically bind to constant framework epitopes of TCR or CD3 proteins; and superantigens that stimulate large numbers of T cells, including, for example, enterotoxins (such as staphylococcal enterotoxin).

[0061] The terms "T lymphocyte" and "T cell" are used interchangeably and refer to a cell that expresses a TCR that is capable of recognizing an antigen when displayed on the surface of an antigen presenting cell or matrix together with one or more MHC molecules or one or more non-classical MHC molecules.

[0062] The term "T cell" refers to a T lymphocyte as defined in the prior art, and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be CD4 + T cells, CD8 + T cells, CD4 + CD8 + T cells or CD4 - CD8 -Cells. T cells can also be T helper cells (e.g., T helper cells 1 (TH1) or T helper cells 2 (TH2) or TH17 cells), as well as cytotoxic T cells, regulatory T cells, natural killer T cells, naive T cells, memory T cells or γδ T cells (Wilson et al., 2009; Wynn, 2005; Ladi et al., 2006). T cells that differ from each other by at least one marker (e.g., CD4) are referred to herein as "subpopulations" of T cells.

[0063] "CD4 + "T cells" refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by a secretory profile upon stimulation that may include the secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10. "CD4" is a 55-kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but is also present on other cells including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and participates as a cognate recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. They define a helper / inducer subset on T lymphocytes.

[0064] "CD8 + "T cells" refers to a subset of T cells that express CD8 on their surface, are MHC-I class restricted and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found on thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a cognate recognition element in the major histocompatibility complex class I restricted interaction.

[0065] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into all the cells that make up one or more tissues or organs, or preferably have the potential to differentiate into any of the following three germ layers: endoderm (inner stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital) or ectoderm (epidermal tissue and nervous system).

[0066] As used herein, the term "somatic cell" refers to any cell other than a reproductive cell (e.g., an ovum, a sperm, etc.) that does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. Somatic cells used herein may be naturally occurring or genetically modified.

[0067] "Programming" is the process of changing the type of offspring a cell can produce. For example, when a cell is altered, it has been programmed to form offspring of at least one new cell type in culture or in vivo, compared to the cell that would be formed without programming under the same conditions. This means that after sufficient proliferation, a significant proportion of offspring with phenotypic characteristics of the new cell type will be observed, while essentially no such offspring could be formed before programming; alternatively, the proportion with characteristics of the new cell type is significantly higher than before programming. This process includes differentiation, dedifferentiation, and transdifferentiation.

[0068] "Differentiation" is the process of changing a less specialized cell into a more specialized cell type. "Dedifferentiation" is a cellular process that reverts a partially differentiated or terminally differentiated cell to an earlier developmental stage (e.g., pluripotency or multipotency). "Transdifferentiation" is the process of converting one differentiated cell type into another differentiated cell type. Typically, transdifferentiation is performed by programming so that the cell does not go through an intermediate pluripotent stage - that is, the cell is programmed directly from one differentiated cell type to another differentiated cell type. Under certain conditions, the proportion of offspring with characteristics of the new cell type can be at least about 1%, 5%, 25%, or more, in order of increasing preference.

[0069] "Reprogramming" is a process that gives cells a significantly improved ability to form offspring of at least one new cell type in culture or in vivo compared to without reprogramming under the same conditions. More specifically, reprogramming is a process that gives somatic cells pluripotency. This means that after sufficient proliferation, there is a significant proportion of offspring with phenotypic characteristics of new cell types, and before reprogramming, there is essentially no such offspring that may form; otherwise, the proportion with new cell type characteristics is significantly higher than before reprogramming. Under certain conditions, the proportion of offspring with new cell type characteristics can be at least about 1%, 5%, 25% or more in order of increasing preference.

[0070] The term "forward programming" refers to programming of multipotent or pluripotent cells by providing one or more specific lineage determining genes or gene products to multipotent or pluripotent cells, rather than programming differentiated somatic cells without pluripotency. For example, forward programming can describe the process of programming ESC or iPSC to hematopoietic precursor cells or other precursor cells or to hematopoietic cells or other differentiated somatic cells.

[0071] As used herein, the term "subject" or "subject in need thereof" refers to a male or female mammal, preferably a human, of any age in need of a cell or tissue transplant. Typically, a subject is in need of a cell or tissue transplant (also referred to herein as a recipient) due to a disorder or pathological or undesirable condition, state or syndrome, or physical, morphological or physiological abnormality amenable to treatment by cell or tissue transplantation.

[0072] As used herein, gene "destruction" refers to the elimination or reduction of one or more gene products encoded by the subject gene in the cell compared to the expression level of the gene product without destruction. Exemplary gene products include mRNA and protein products encoded by the gene. In some cases, the destruction is transient or reversible, and in other cases it is permanent. Although truncated or non-functional products may be produced, functional or full-length proteins or mRNAs may be destroyed in some cases. In some embodiments herein, gene activity or function is destroyed, rather than expression being destroyed. Gene destruction is usually induced at the DNA level by artificial methods (i.e., by adding or introducing compounds, molecules, complexes or compositions and / or by destroying nucleic acids associated with genes). Exemplary methods for gene destruction include gene silencing, knockdown, knockout and / or gene destruction techniques (e.g., gene editing). Examples include antisense techniques (e.g., RNAi, siRNA, shRNA and / or ribozymes, which usually result in a transient reduction in expression) and gene editing techniques (causing target gene inactivation or destruction, such as by inducing fracture and / or homologous recombination). Examples include insertion, mutation and deletion. Destruction usually causes the expression of normal or " wild type " product encoded by gene to be suppressed and / or not expressed at all.The example of this type of gene disruption is the insertion, frameshift and missense mutation, disappearance, knock-in and knock-out of gene or gene part, including the disappearance of whole gene.This type of destruction can occur in coding region (for example, in one or more exons), causing to be unable to produce full-length product, functional product or any product, for example, by inserting termination codon and this type of destruction occurs.This type of destruction also may occur by promoter or enhancer or the destruction of other regions that affect transcriptional activation, so as to prevent the transcription of gene.Gene disruption includes gene targeting, which includes the targeted gene inactivation carried out by homologous recombination.

[0073] "Notch ligands" are proteins that can bind to Notch receptor polypeptides present in the membranes of many 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) that contains 20 to 22 amino acids at the amino terminus and 3 to 8 EGF-like repeats on the extracellular surface (Furie and Furie, 1988; Knust et al., 1987; Suzuki et al., 1987).

[0074] "Super donors" herein refer to individuals who are homozygous for certain MHC class I and class II genes. These homozygous individuals can be used as super donors, and their cells (including tissues and other materials containing their cells) can be transplanted into individuals who are homozygous or heterozygous for the haplotype. Super donors can be homozygous for each of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ loci / locus alleles.

[0075] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and includes a receptor that is artificially specific and transplanted to a specific immune effector cell. CAR can be used to confer monoclonal antibody specificity to T cells, thereby allowing the production of a large number of specific T cells, such as for adoptive cell therapy. In a specific embodiment, for example, CAR instructs cells to be specific to tumor-associated antigens. In some embodiments, CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In a particular aspect, CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, and its transmembrane domain and endodomain are fused to CD3-ζ. The specificity of other CAR designs can be derived from a ligand of a receptor (e.g., a peptide) or from a pattern recognition receptor (e.g., Dectins). In some cases, the interval of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, CAR comprises a domain for additional co-stimulatory signals, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some cases, molecules can be co-expressed with CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products of conditional ablation of T cells when prodrugs are added, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0076] The term "antigen presenting cell (APC)" refers to a type of cell that can present one or more antigens in the form of a peptide-MHC complex that can be recognized by specific effector cells of the immune system, thereby inducing an effective cellular immune response to the presented antigen. APCs can be intact whole cells, such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells; or other naturally occurring or synthetic molecules, such as purified MHC class I molecules complexed with β2-microglobulin. Although many types of cells may be able to present antigens on their cell surfaces for T cell recognition, only dendritic cells have the ability to present effective amounts of antigens to activate naive T cells to produce a cytotoxic T lymphocyte (CTL) response.

[0077] II. Pluripotent Stem Cells

[0078] In certain embodiments, pluripotent stem cells are modified to express antigen receptors, such as CAR.Pluripotent stem cells can be stem cells, including but not limited to induced pluripotent stem cells and embryonic stem cells. In particular aspects, the pluripotent stem cells used herein are human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), which can proliferate in vitro for a long time while retaining the potential to differentiate into all cell types of the body (including hematopoietic precursor cells disclosed herein).

[0079] A. Embryonic stem cells

[0080] In some aspects, pluripotent stem cells are ESCs. ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiation ability. ES cells can be isolated by removing the outer trophoblast of the developing embryo and then culturing the inner cell mass cells on the feeder layer of non-growing cells. The re-plated cells can continue to proliferate and produce new ES cell colonies, which can be removed, separated, re-plated again and grown. This process of "passing down" undifferentiated ES cells can be repeated many times to produce cell lines containing undifferentiated ES cells (U.S. Patent 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 studying cells and controlling genes for cell differentiation. The pluripotency of ES cells can be combined with genetic manipulation and selection through the production of transgenic, chimeric and knockout mice for in vivo gene analysis research.

[0081] The method for producing mouse ES cells is well-known. In one method, the preimplantation blastocyst from 129 strain mice is treated with mouse antiserum to remove trophectoderm, and the inner cell mass is cultured on the mouse embryonic fibroblast feeder cell layer chemically inactivated in the culture medium containing fetal bovine serum. In the presence of fetal bovine serum, the undifferentiated ES cell colony in development is subcultured on the mouse embryonic fibroblast feeder layer to produce an ES cell group. In some methods, mouse ES cells can be grown in the absence of feeder layers by adding cytokine leukemia inhibitory factor (LIF) to the culture medium containing serum. In other methods, mouse ES cells can grow in serum-free medium in the presence of bone morphogenetic protein and LIF.

[0082] Human ES cells can be produced by or derived from zygotes or blastocyst mammalian embryos, which are produced by fusion of sperm and egg cells, nuclear transplantation, pathogenesis, or by previously described methods (Thomson and Marshall, 1998; Reubinoff et al., 2000) by chromatin reprogramming, then incorporating the reprogrammed chromatin into the plasma membrane to produce embryonic cells. In one method, human blastocysts are exposed to anti-human serum, trophectoderm cells are lysed and separated from the inner cell mass cultured on the feeder layer of mouse embryonic fibroblasts. In addition, the cell mass derived from the inner cell mass is chemically or mechanically dissociated, re-plated, and the colony of undifferentiated morphology is selected by a micropipette, dissociated and re-plated. In some methods, human ES cells can be cultured in the absence of serum by culturing ES cells on a fibroblast feeder layer in the presence of basic fibroblast growth factor. In other methods, the cells can be grown in a protein matrix (e.g., MATRIGEL TM Human ES cells were cultured in the absence of a feeder cell layer by culturing cells on culture medium (Xu et al., 2001).

[0083] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, and from established mouse and human cell lines by previously described methods (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000; U.S. Pat. No. 5,843,780). For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As further examples, established mouse ES cell lines include the CGR8 cell line established from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0084] ES stem cells can be detected by protein markers, including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2 or REX1.

[0085] B. Induced pluripotent stem cells

[0086] In other respects, the pluripotent stem cell used herein is an induced pluripotent stem (iPS) cell, usually abbreviated as iPS cell or iPSC. The induction of pluripotency was initially achieved by introducing the transcription factor associated with pluripotency to reprogram somatic cells using mouse cells (Yamanaka et al., 2006) and human cells (Yu et al., 2007; Takahashi et al., 2007) in 2006. The use of iPSC avoids most of the ethical and practical issues related to the large-scale clinical use of ES cells, and the patient with the autologous transplantation derived from iPSC may not need lifelong immunosuppressive therapy to prevent graft rejection.

[0087] In addition to germ cells, any cell can be used as the starting point of iPSC. For example, the cell type may be a keratinocyte, a fibroblast, a hematopoietic cell, a mesenchymal cell, a hepatocyte, or a gastric cell. T cells can also be used as a somatic cell source for reprogramming (U.S. Patent No. 8,741,648). There is no restriction on the degree of cell differentiation or the age of the animal from which the cells are collected; in the methods disclosed herein, even undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as somatic cell sources.

[0088] Somatic cells can be reprogrammed to produce iPSCs using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells, see, for example, published U.S. Patent Application No. 20090246875, published U.S. Patent Application No. 2010 / 0210014; published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,268,620; PCT Publication No. WO 2007 / 069666 A1 and U.S. Patent No. 8,268,620, which are incorporated herein by reference. Nuclear reprogramming factors are generally used to produce pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0089] Mouse and human cDNA sequences of these nuclear reprogramming materials can be obtained with reference to the NCBI accession numbers mentioned in WO 2007 / 069666 and U.S. Patent No. 8,183,038, which are incorporated herein by reference. Methods for introducing one or more reprogramming materials or nucleic acids encoding these reprogramming materials are all known in the art and are disclosed in, for example, U.S. Patent Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​disclosed U.S. Patent No. 8,900,871 and U.S. Patent No. 8,071,369, which are all incorporated herein by reference.

[0090] After producing iPSC, iPSC can be cultured in a medium sufficient to maintain pluripotency. iPSC can be used together with various culture media and techniques developed to cultivate pluripotent stem cells (more specifically embryonic stem cells), as described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603. In the case of mouse cells, leukemia inhibitory factor (LIF) is added in a common culture medium to be cultivated as a differentiation inhibitor. For human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. As known to those skilled in the art, other methods of cultivating and maintaining iPSC can be used together with the method of the present invention.

[0091] In certain embodiments, non-restricted conditions can be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or on culture medium that has been exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, mouse embryonic fibroblasts treated with radiation or antibiotics are used as feeder cells and cultured with the cells to terminate cell division. Alternatively, a restricted culture system that does not require feeder cells (e.g., TESR TM culture medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8 TM / Essential 8 TM Culture medium (Chen et al., 2011)) to culture and maintain pluripotent cells in a largely undifferentiated state.

[0092] There are many goals to consider when designing plasmids, such as achieving regulated high copy numbers and avoiding potential causes of plasmid instability in bacteria, and providing plasmid selection means that are compatible with use in mammalian cells (including human cells). Particular attention should be 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 amounts of DNA can be produced and purified. Second, they are safe and suitable for use in human patients and animals. The first requirement requires high copy number plasmids that can be relatively easily selected and stably maintained during bacterial fermentation. The second requirement requires attention to elements such as selective markers and other coding sequences. In some embodiments, the plasmid encoding the marker consists of the following parts: (1) a high copy number replication origin, (2) a selective marker, such as but not limited to the neo gene for antibiotic selection with kanamycin, (3) a transcription termination sequence, including a tyrosinase enhancer; and (4) a multiple cloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to a tyrosinase promoter. There are many plasmid vectors known in the art for inducing nucleic acids encoding proteins. These vectors include, but are not limited to, those disclosed in: US Patent No. 6,103,470; US Patent No. 7,598,364; US Patent No. 7,989,425; and US Patent No. 6,416,998, which are incorporated herein by reference.

[0093] The episomal gene delivery system can be a plasmid, an episomal vector based on Epstein-Barr virus (EBV) (U.S. Patent 8,546,140), a yeast-based vector, an adenovirus-based vector, an episomal vector based on simian virus 40 (SV40), a vector based on bovine papilloma virus (BPV), or a lentiviral vector. The viral gene delivery system can be an RNA-based or DNA-based viral vector.

[0094] 1. Cells used to generate iPSCs

[0095] Certain embodiments of the present disclosure relate to the starting population of somatic cells (e.g., blood cells or skin cells) reprogrammed into iPSC. A blood cell population may include peripheral blood mononuclear cells (PBMC), whole blood or a portion thereof comprising a mixed population, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukocyte separation, biopsy tissues, and lymph nodes (e.g., lymph nodes drained from a tumor). Suitable donors include immunized donors, non-immunized (primary immunized) donors, treated donors, or untreated donors. A "treated" donor is a donor that has been exposed to one or more biological modifiers. An "untreated" donor has not been exposed to one or more biological modifiers.

[0096] In some aspects, the blood cell population comprises T cells. The T cells can be a purified T cell population, or alternatively, the T cells can be a population of cells of different types (e.g., B cells and / or other peripheral blood cells). The T cells can be a T cell subset (e.g., CD4 + In some embodiments, the T cells are purified populations of T cells, or they can be T cell populations comprising different T cell subsets. In another embodiment, the T cells are T cell clones that have been maintained in culture for a long time. T cell clones can be transformed to varying degrees. In a specific embodiment, the T cells are T cell clones that have been infinitely propagated in culture.

[0097] In some aspects, T cells are primary T cells. The term "primary T cells" is intended to include T cells obtained from an individual, in contrast to T cells that have been maintained in culture for a long time. Therefore, primary T cells are specific peripheral blood T cells obtained from a subject. A group of primary T cells can be mainly composed of a T cell subset. Alternatively, a primary T cell group can be composed of different T cell subsets.

[0098] T cells can be from previously stored blood samples, from healthy individuals, or alternatively from individuals with a certain condition. The condition can be an infectious disease, such as a condition caused by a viral infection, a bacterial infection, or any other microbial infection, or a hyperproliferative disease, such as cancer (such as melanoma). In a specific embodiment, T cells are from individuals infected with human immunodeficiency virus (HIV). In another embodiment, T cells are from subjects suffering from or susceptible to autoimmune diseases or T cell pathology. T cells can be human, mouse, or any other mammalian species.

[0099] Methods for obtaining a cell population comprising T cells are well known in the art. For example, peripheral blood mononuclear cells (PBMCs) may be obtained as described by methods known in the art. Examples of such methods are described in the Examples and discussed in Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

[0100] In some aspects, the starting population of blood cells includes hematopoietic stem cells (HSCs). HSCs are usually located in the bone marrow, but they can be made to enter the blood, a process known as mobilization, which is clinically used to collect a large number of HSCs in peripheral blood. One selected mobilizing agent is granulocyte colony stimulating factor (G-CSF). CD34 circulating in peripheral blood can be collected by apheresis in a quiet state or after mobilization following external administration of hematopoietic growth factors (such as G-CSF). +Hematopoietic stem or progenitor cells. The number of stem or progenitor cells collected after mobilization is greater than the number of stem or progenitor cells obtained after apheresis in a quiet state. In some aspects, the source of the cell population is a subject whose cells have not been mobilized by exogenously applied factors, because enrichment for hematopoietic stem or progenitor cells is not required.

[0101] Methods for obtaining hematopoietic precursor cells from a cell population are also well known in the art. Hematopoietic precursor cells can be expanded using various cytokines (e.g., hSCF, hFLT3, and / or IL-3) (Akkina et al., 1996), or CD34 can be enriched using MACS or FACS. + As described above, negative selection techniques can also be used to enrich for CD34 + cell.

[0102] The cell population used in the methods described herein can be a mammalian cell, such as a human cell, a non-human primate cell, a rodent cell (such as a mouse or rat), a bovine cell, a sheep cell, a pig cell, a horse cell, a sheep cell, a dog cell, and a cat cell, or a mixture thereof. Non-human primate cells include rhesus monkey cells. The cells can be obtained from an animal, such as a human patient, or can be obtained from a cell line. If the cells are obtained from an animal, they can be used as is, for example, as unseparated cells (i.e., a mixed group); they may have been first cultured and established by, for example, transformation; or they may have been treated with a preliminary purification method. For example, a cell population can be manipulated by positive or negative selection based on the expression of cell surface markers; stimulated with one or more antigens in vitro or in vivo; treated with one or more biological modifiers in vitro or in vivo; or any combination or all of them. In an illustrative embodiment, a cell population is negatively selected to remove non-T cells and / or specific T cell subsets. Negative selection can be performed based on cell surface expression of various molecules, including B cell markers (e.g., CD19 and CD20); monocyte marker CD14; NK cell marker CD56. Alternatively, cell populations can be negatively selected to remove non-CD34 + Hematopoietic cells and / or specific hematopoietic cell subsets. Negative selection can be performed based on cell surface expression of various molecules, such as antibody cocktails (e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, CD235a, and CD41 (e.g., for cells of the megakaryocyte lineage)), which can be used for the separation of other types of cells, such as by MACS or column separation.

[0103] Cell samples can also be obtained from a subject and then enriched for the desired cell type. For example, PBMCs and / or CD34 can be isolated from blood as described herein. +Hematopoietic cells. Countercurrent centrifugation (elutriation) can be used to enrich T cells from PBMC. Various techniques can also be used to separate cells from other cells, such as separation and / or activation with antibodies that bind to epitopes on the cell surface of the desired cell type, such as certain T cell separation kits that use beads conjugated to antibodies to activate cells, and then use the same beads for column separation. Another method that can be used includes negative selection using antibodies against cell surface markers to selectively enrich specific cell types without activating cells by receptor binding.

[0104] Bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, rib or other medullary cavity. Bone marrow can be removed from a patient and separated by various separation and rinsing procedures. Known methods for separating bone marrow cells include the following steps: a) centrifuging a bone marrow suspension into three fractions and collecting the middle fraction or buffy coat; b) centrifuging the buffy coat fraction of step (a) once more in a separation fluid, usually Ficoll (trademark of Pharmacia Fine Chemicals AB), and collecting the middle fraction containing the bone marrow cells; c) rinsing the fraction collected in step (b) to recover the bone marrow cells that can be transfused.

[0105] If a T cell-enriched cell population is desired, such a cell population can be obtained from a mixed cell population by leukocyte separation and mechanical apheresis using a continuous flow cell separator. For example, T cells can be separated from the buffy coat by any known method, including separation by Ficoll-Hypaque TM Gradient separation, Percoll gradient separation or elutriation.

[0106] In some aspects, T cells are activated by agents that bind to T cell receptors to trigger the signal transduction cascade of T cell activation. For example, CD3 antibodies can be used. In order to amplify T cells in large quantities and be in a reprogrammed proliferation state, cytokines, such as IL-2, can also be used. In some aspects, in the case of costimulation, both anti-CD3 and anti-CD28 can be used for T cell activation. In alternative aspects, cross-linking of anti-CD3 can be applied, such as anti-CD3 bound to a plate. If soluble anti-CD3 is used to activate T cells in PBMC, soluble anti-CD3 antibodies may bind to APCs in PBMCs, and then present antibodies to T cells. If soluble anti-CD3 antibodies are used alone for purified T cell populations, no response (anergy) will result due to the above reasons. Certain embodiments include culturing T cells in the presence of anti-CD3 (OKT3) and IL2, which is advantageous and convenient because there is no need to use expensive and cumbersome beads or antibodies bound to a plate; after adding OKT3 and IL2, the cell environment of PBMC will help activate T cells. T cells then crowd out other cell types in the PBMC culture due to their preferential expansion.

[0107] In certain aspects, the starting population of blood cells comprises lymphoblasts, for example from a lymphoblastoid cell line (LCL). Generation of LCL is known in the art, for example, by generating LCL by infecting B cells with Epstein-Barr virus (EBV) (Frisan et al., 2001).

[0108] 2. Somatic Cell Reprogramming

[0109] In some embodiments, the starting cell (e.g., T cell) population is reprogrammed into iPSC, for example, by the method described in U.S. Patent Publication No. 2014 / 0315304; it is incorporated herein by reference in its entirety. In certain aspects of the present disclosure, the reprogramming factor is expressed by an expression cassette contained in one or more vectors (e.g., an integrative vector or an additional vector). On the other hand, the reprogramming protein can be directly introduced into a somatic cell by protein transduction.

[0110] By standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference), one skilled in the art will be skilled in constructing vectors. Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses and plant viruses) and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including replicable, replication-defective and empty shell forms thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors.

[0111] a. Viral vector

[0112] Viral vectors may be provided in certain aspects of the present disclosure. In the production of recombinant viral vectors, non-essential genes are usually replaced with genes or coding sequences of heterologous (or non-native) proteins. Viral vectors are a class of expression constructs that utilize viral sequences to introduce nucleic acids and possible proteins into cells. The ability of certain viruses to infect cells or enter cells through receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for transferring exogenous nucleic acids to cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain aspects of the present disclosure are described below.

[0113] Retroviruses hold promise as gene delivery vectors because they are able to integrate genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines ( Miller, 1992 ).

[0114] To construct a retroviral vector, nucleic acid is inserted into the viral genome to replace certain viral sequences to produce a replication-defective virus. To produce virions, a packaging cell line containing gag, pol and env genes but without LTR and packaging components was constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA and a retroviral LTR and packaging sequence are introduced into a special cell line (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0115] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol and env, also contain other genes with regulatory or structural functions. Lentivirus vectors are well known in the art (see, e.g., Naldini et al., 1996; Zuferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136).

[0116] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells are described in U.S. Pat. No. 5,994,136, in which suitable host cells are transfected with two or more vectors carrying packaging functions (i.e., gag, pol and env, and rev and tat); U.S. Pat. No. 5,994,136 is incorporated herein by reference.

[0117] b. Episomal vectors

[0118] In certain aspects of the present disclosure, the use of extrachromosomal (i.e., episomal) vectors based on plasmids or liposomes may also be provided. Such episomal vectors may include, for example, oriP-based vectors and / or vectors encoding EBNA-1 derivatives. Such vectors may allow large DNA fragments to be introduced into cells and maintained extrachromosomally, replicated once per cell cycle, efficiently distributed to daughter cells, and substantially without causing an immune response.

[0119] In particular, EBNA-1 is the only viral protein required for the replication of oriP-based expression vectors, and it does not elicit a cellular immune response because it has developed efficient mechanisms to bypass the processes required for presentation of its antigens on MHC class I molecules (Levitskay et al., 1997). In addition, EBNA-1 can enhance the expression of cloned genes in trans, inducing expression of cloned genes up to 100-fold in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the production of such oriP-based expression vectors is inexpensive.

[0120] Other extrachromosomal vectors include other vectors based on lymphotrophic herpesvirus. Lymphotrophic herpesvirus is a herpesvirus that replicates in lymphoblasts (e.g., human B lymphoblasts) and becomes a plasmid of its natural life cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpesvirus. Exemplary lymphotrophic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); squirrel monkey herpesvirus (HS) and Marek's disease virus (MDV). Other sources of episomal-based vectors are also included, such as yeast ARS, adenovirus, SV40 or BPV.

[0121] Those skilled in the art will readily construct vectors using standard recombinant techniques (see, eg, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0122] The vector may also contain other components or functions that further modulate gene delivery and / or gene expression, or otherwise provide beneficial properties to the targeted cells. Such other components include, for example: components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect cellular uptake of vector nucleic acids; components that affect intracellular localization of polynucleotides after uptake (e.g., agents that mediate nuclear localization); and components that affect polynucleotide expression.

[0123] Such components may also include markers, such as detectable and / or selective markers that can be used to detect or select cells that have ingested and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (e.g., using certain viral vectors with components or functions that mediate binding and ingestion), or the vector may be modified to provide such functions. A large number of such vectors are known in the art and are generally available. When the vector is maintained in a host cell, the vector can be stably replicated by the cell as an autonomous structure during mitosis, incorporated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

[0124] c. Transposon-based systems

[0125] In some aspects, the delivery of programming factors can use a transposon-transposase system. For example, the transposon-transposase system can be the well-known Sleeping Beauty, Frog Prince transposon-transposase system (described in, for example, EP1507865) or the TTAA-specific transposon PiggyBac system.

[0126] Transposons are DNA sequences that are able to move around to different locations within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons, also once called jumping genes, are examples of mobile genetic elements.

[0127] There are many types of mobile genetic elements that can be grouped according to their transposition mechanism. Class I mobile genetic elements, or retrotransposons, replicate themselves by first being transcribed into RNA, then reverse transcribed back into DNA by the enzyme reverse transcriptase, and then inserted into another location in the genome. Class II mobile genetic elements move directly from one location to another using transposases to "cut and paste" them within the genome.

[0128] In certain embodiments, constructs provided in the present disclosure (e.g., multi-lineage constructs) use the PiggyBac expression system. PiggyBac (PB) DNA transposons are mobilized by a "cut and paste" mechanism whereby a transposase (PB transposase) encoded by the transposon itself cleaves and reintegrates the transposon at other sites within the genome. The PB transposase specifically recognizes the PB inverted terminal repeats (ITRs) that flank the transposon; it binds to these sequences and catalyzes the cleavage of the transposon. The PB then integrates into TTAA sites throughout the genome in a relatively random manner. To create gene trap mutations (or for the generation of transgenic animals), the transposase is provided in trans on a plasmid and co-transfected with a plasmid containing a donor transposon (a recombinant transposon comprising a gene trap flanked by transposase binding sites (ITRs)). The transposase will catalyze the cleavage of the transposon from the plasmid and subsequent integration into the genome. Integration within the coding region will capture the elements necessary for gene trap expression. PB has several desirable properties: (1) it preferentially inserts intragenically (50% to 67% insert intragenically), (2) it does not exhibit localized jumping (broad genomic coverage), (3) it is insensitive to hyperproduction suppression (in which elevated transposase levels lead to reduced transposition), and (4) it excises cleanly from the donor site, leaving no “footprints,” unlike Sleeping Beauty.

[0129] d. Regulatory elements

[0130] Expression cassettes included in reprogramming vectors useful in the present disclosure preferably contain (in the 5' to 3' direction) a eukaryotic transcriptional promoter operably linked to a protein coding sequence, splice signals including intervening sequences, and transcription termination / polyadenylation sequences.

[0131] (i) Promoter / enhancer

[0132] The expression construct provided herein comprises a promoter to drive the expression of a programmed gene. The promoter generally comprises a sequence that acts to locate the start site of RNA synthesis. The most famous example of this sequence is the TATA box, but in some promoters lacking the TATA box (e.g., the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene), the discrete elements covering the start site themselves help determine the start position. Other promoter elements regulate the frequency of transcription initiation. Typically, they are located in the region 30-110bp upstream of the start site, although it has been shown that many promoters also comprise functional elements downstream of the start site. In order to place the coding sequence "under the control of the promoter", the 5' end of the transcription start site of the transcription reading frame will be positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0133] The spacing between promoter elements is often flexible so that promoter function is retained when elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can act either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the activation of transcription of a nucleic acid sequence.

[0134] The promoter may be a promoter naturally associated with a nucleic acid sequence, and may be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or an exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may 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 usually associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually 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, prokaryotic or eukaryotic cells, and promoters or enhancers that are not "naturally present", i.e., different elements comprising different transcriptional regulatory regions, and / or mutations that change expression. For example, the most commonly used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthesizing promoter and enhancer nucleic acid sequences, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can also be used to generate promoter and enhancer nucleic acid sequences. TM ) in combination with the compositions disclosed herein to generate sequences (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). In addition, it is contemplated that control sequences directing transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may also be employed.

[0135] Naturally, it is important to employ promoters and / or enhancers that are effective in directing expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. The use of promoters, enhancers, and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology (see, e.g., Sambrook et al., 1989, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions for directing high-level expression of the introduced DNA segment, which is advantageous, for example, in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0136] In addition, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB) can also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription of certain bacterial promoters if an appropriate bacterial polymerase is provided (either as part of the delivery complex or as an additional gene expression construct).

[0137] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the β-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 multiple response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA) and the response element promoter near the minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (eg, the human growth hormone minimal promoter described in Genbank Accession No. X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from ATCC Product No. ATCC 45007).

[0138] Tissue-specific transgenic expression, particularly reporter gene expression in programmed hematopoietic cells and hematopoietic cell precursors, may be an ideal method for identifying derived hematopoietic cells and precursors. In order to increase specificity and activity, the use of cis-acting regulatory elements has been considered. For example, hematopoietic cell-specific promoters can be used. Many such hematopoietic cell-specific promoters are known in the art, such as the promoters of the hematopoietic genes provided in Table 1.

[0139] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the potential to act in cis, regardless of their orientation, even at considerable distances (up to several thousand bases away from the promoter of interest). However, enhancer function need not be limited to such long distances, as they can also function in close proximity to a given promoter.

[0140] Many hematopoietic cell promoter and enhancer sequences have been identified and may be suitable for use in the methods of the present invention. See, e.g., U.S. Patent No. 5,556,954; U.S. Patent Application No. 20020055144; U.S. Patent Application No. 20090148425.

[0141] (ii) Initiation signal and junction expression

[0142] Specific start signals can also be used in the expression constructs provided by the present disclosure to effectively translate the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art will be able to easily determine this and provide the required signals. It is well known that the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be improved by including suitable transcription enhancer elements.

[0143] In certain embodiments, polygenic or polycistronic information is produced using internal ribosome entry site (IRES) elements. IRES elements can bypass the ribosome scanning pattern of 5' methylated cap-dependent translation and start 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 elements from mammalian information (Macejak and Sarnow, 1991). IRES originals can be connected to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, thereby producing polycistronic information. With the aid of IRES elements, each open reading frame can be approached by ribosomes for effective translation. Using a single promoter / enhancer to transcribe a single message can effectively express multiple genes (see U.S. Patent Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

[0144] In addition, certain 2A sequence elements can be used to generate linked expression or co-expression of programmed genes in constructs provided by the present disclosure. For example, by connecting open reading frames to form a single cistron, a cleavage sequence can be used to co-express genes. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A). In a specific embodiment, an F2A cleavage peptide is used to link the expression of genes in a multi-lineage construct.

[0145] e. Initiation of replication

[0146] In order to propagate the vector in the host cell, it may contain one or more replication site origins (commonly referred to as "ori"), such as a nucleic acid sequence corresponding to the above-mentioned EBV oriP or a genetically modified oriP having similar or enhanced functions in programming, which is a specific nucleic acid sequence at the origin of replication. Alternatively, the replication origins of other extrachromosomal replication viruses as described above or autonomous replication sequences (ARS) may be used.

[0147] f. Selection and screening of markers

[0148] In certain embodiments, cells containing nucleic acid constructs can be identified in vitro or in vivo by including a marker in an expression vector. Such markers confer recognizable changes to cells, thereby allowing cells containing expression vectors to be easily identified. Generally, a selective marker is a marker that confer characteristics that allow selection. A positive selection marker is a marker in which the presence of a marker allows selection, while a negative selection marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0149] Typically, drug selection markers are included to aid in cloning and identifying transformants, for example genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to conferring phenotypes to allow condition-based implementation to distinguish transformants, other types of markers are also contemplated, including screening markers (e.g., GFP), based on colorimetric analysis. Alternatively, screening enzymes can be used as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT). Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. As long as it is possible to express simultaneously with the nucleic acid encoding the gene product, it is believed that the marker used is unimportant. Other more examples of selection and screening markers are well known to those skilled in the art.

[0150] Through the present disclosure, nucleic acid (eg, DNA or RNA) is introduced into pluripotent stem cells to be programmed into hematopoietic precursor cells, and any suitable method for delivering nucleic acid for cell transformation as described herein or known to those of ordinary skill in the art can be used. Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (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 microprojectile bombardment (PCT Application No. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by shaking with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Pat. 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 these methods. By applying techniques such as these, organelles, cells, tissues or organisms can be transformed stably or transiently.

[0151] C.MHC haplotype matching

[0152] The major histocompatibility complex (MHC) is the main cause of immune rejection of allogeneic organ transplantation. There are three major MHC class I haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA locus is highly polymorphic and is distributed over more than 4Mb 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 HLA haplotypes between donor and recipient can affect the clinical outcome of transplantation. HLA corresponding to MHC class I present peptides from inside the cell, and HLA corresponding to MHC class II present antigens to T lymphocytes from outside the cell. Incompatibility of MHC haplotypes between the graft and the host can trigger an immune response against the graft and lead to its rejection. Therefore, patients can be treated with immunosuppressants to prevent rejection. HLA-matched stem cell lines can overcome the risk of immune rejection.

[0153] Due to the importance of HLA in transplantation, HLA loci are usually typed by serology and PCR to identify favorable donor-recipient pairs. Serological detection of HLA class I and class II antigens can be completed using purified T or B lymphocytes to carry out complement-mediated lymphocyte toxicity testing. The method is mainly used for matching HLA-A and -B loci. Molecular-based tissue typing may be more accurate than serological tests usually. Low-resolution molecular methods (such as SSOP (sequence-specific oligonucleotide probe) method) can be used to identify HLA antigens, wherein PCR products are tested for a series of oligonucleotide probes, and these methods are currently the most commonly used methods for class II HLA typing. High-resolution techniques (such as SSP (sequence-specific primer) method) utilizing allele-specific primers to carry out PCR amplification can identify specific MHC alleles.

[0154] If the donor cells are HLA homozygous, i.e., each antigen presenting protein contains the same allele, the MHC compatibility between the donor and the recipient will be significantly improved. Most individuals are heterozygous for MHC class I and class II genes, but these genes are homozygous for some individuals. These homozygous individuals can serve as super donors, and the grafts produced from their cells can be transplanted into all individuals that are homozygous or heterozygous for the haplotype. In addition, if the homozygous donor cells have a haplotype that exists at a high frequency in the population, these cells can be used in transplantation treatments for many individuals.

[0155] Therefore, in some embodiments, the PSC of the present method can be generated by somatic cells of the subject to be treated or another subject having the same or substantially the same HLA type as the patient. In one case, the major HLA of the donor (e.g., the three major loci of HLA-A, HLA-B, and HLA-DR) is the same as the major HLA of the recipient. In some cases, the somatic cell donor can be a super donor; therefore, PSCs derived from super donors in homozygous form of MHC can be used to generate HPCs, and then immune cells (e.g., T cells) can be generated. Therefore, immune effector cells derived from super donors can be transplanted into subjects that are homozygous or heterozygous for the haplotype. For example, immune cells can be homozygous for two HLA alleles (e.g., HLA-A and HLA-B). In this way, immune cells produced by super donors can be used in the methods disclosed herein to produce immune cells that can potentially "match" a large number of potential recipients.

[0156] D. Genetically engineered antigen receptors

[0157] PSCs can be genetically engineered to express an antigen receptor, such as an engineered TCR or CAR. For example, PSCs (e.g., autologous or allogeneic) are modified to express a TCR or CAR that is antigen-specific for a cancer antigen.

[0158] Suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al., Hum Gene Ther. 19:496-510 (2008) and Johnson et al., Blood 114:535-46 (2009).

[0159] Electroporation of RNA encoding full-length TCR α and β (or γ and δ) chains can be used as an alternative method to overcome the long-term autoreactivity problem caused by pairing of retrovirally transduced and endogenous TCR chains. Even if this alternative pairing occurs in a transient transfection strategy, the autoreactive T cells that may be generated will lose this autoreactivity after a period of time because the introduced TCR α and β chains are only transiently expressed. When the expression of the introduced TCR α and β chains decreases, only normal autologous T cells will remain. When the full-length TCR chains are introduced by stable retroviral transduction, this is not the case, and the introduced TCR chains will never be lost, resulting in continuous autoreactivity in the patient.

[0160] In some embodiments, the cell comprises one or more nucleic acids encoding one or more antigen receptors introduced by genetic modification, and genetic modification products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not usually present in the cell or in a sample obtained from the cell, such as a sample obtained from another organism or cell, such as it is not usually present in the organism from which the modified cell and / or such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not present in nature (e.g., chimeric).

[0161] In some embodiments, CAR contains an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the cell surface. In some embodiments, CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule, just like TCR.

[0162] Exemplary antigen receptors including CARs and recombinant TCRs and methods for modifying and introducing receptors into cells, including, for example, those disclosed in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130 149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and those described in European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 April; 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, the genetically engineered antigen receptors include those described in CARs as described in U.S. Pat. No. 7,446,190 and International Patent Application Publication No. WO / 2014055668A1.

[0163] 1. Chimeric Antigen Receptor

[0164] In some embodiments, the CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.

[0165] In some embodiments, the modified antigen receptor includes a CAR, which includes an activation or stimulatory CAR, a co-stimulatory CAR (see WO2014 / 055668) and / or an inhibitory CAR (iCAR, see Fedorov et al., 2013). CAR generally comprises an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components, which is connected by a linker and / or a transmembrane domain in some aspects. Such molecules generally simulate or approximate the signal through a natural antigen receptor, the signal through this receptor combined with a co-stimulatory receptor and / or the signal through the co-stimulatory receptor alone.

[0166] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides, including CAR (hCAR) that has been humanized to reduce immunogenicity, which comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signal motifs. In certain embodiments, CAR can recognize an epitope comprising a shared space between one or more antigens. In certain embodiments, the binding region may include a complementary determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen binding fragment thereof. In another embodiment, specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0167] It is contemplated that human CAR nucleic acid can be a human gene for enhancing cellular immunotherapy of human patients. In a specific embodiment, the present invention includes full-length CAR cDNA or coding region. Antigen binding region or domain may include fragments of VH and VL chains of single-chain variable fragments (scFv) derived from specific human monoclonal antibodies, such as those described in U.S. Patent No. 7,109,304, which are incorporated herein by reference. Fragments may also be any number of different antigen-binding domains of human antigen-specific antibodies. In a more specific embodiment, a fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage, to be expressed in human cells.

[0168] The arrangement can be multimeric, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing the variable parts of the light chain and the heavy chain into a diabody. The hinge portion of the construct can have a variety of options, from complete deletion to retaining the first cysteine ​​to proline instead of serine substitution, until it is truncated to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerized protein can achieve this goal. Only one of the Fc domains can be used, such as the CH2 or CH3 domains of human immunoglobulins. The hinge, CH2 and CH3 regions of modified human immunoglobulins can also be used to improve dimerization. Only the hinge portion of the immunoglobulin can also be used. The CD8α portion can also be used.

[0169] In some embodiments, the CAR nucleic acid comprises sequences encoding other co-stimulatory receptors, such as transmembrane domains and modified CD28 intracellular signaling domains. Other co-stimulatory 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 CD3ζ, the additional signals provided by the human co-stimulatory receptors inserted into the human CAR are also important for the complete activation of NK cells, which can help improve the in vivo persistence and treatment success of adoptive immunotherapy.

[0170] In some embodiments, CAR is constructed to be specific to a specific antigen (or marker or ligand), such as an antigen (e.g., cancer marker) expressed in a specific cell type to be targeted by adoptive therapy and / or an antigen (e.g., an antigen expressed on a normal or non-disease cell type) intended to induce a weakened response. Therefore, CAR generally includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or parts, or one or more antibody variable domains and / or antibody molecules in its extracellular portion. In some embodiments, CAR includes an antigen binding portion of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from a variable heavy chain (VH) and a variable light chain (VL) of a monoclonal antibody (mAb).

[0171] In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain comprising the antigen binding region) comprises a tumor-associated antigen or a pathogen-specific antigen binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors (e.g., Dectin-1). Tumor-associated antigens may be of any kind as long as they are expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigens, melanoma-associated antigens, mutated p53, mutated ras, and the like.

[0172] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR) or a combination thereof. Depending on the size of the genomic DNA and the number of introns, the use of cDNA or a combination thereof may be desirable, as introns have been found to stabilize mRNA. Moreover, the use of endogenous or exogenous non-coding regions to stabilize mRNA may be further advantageous.

[0173] It is contemplated that the chimeric construct can be introduced into immune cells in the form of naked DNA or a suitable vector. Methods for stably transfecting cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to a DNA encoding a chimeric receptor contained in a plasmid expression vector in the correct expression orientation.

[0174] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the methods of the present disclosure are non-replicative in immune cells. Many viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain cell survival, such as vectors based on HIV, SV40, EBV, HSV, or BPV.

[0175] In some aspects, antigen-specific binding or recognition components are connected to one or more transmembrane and intracellular signaling domains. In some embodiments, CAR includes a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of these domains to the transmembrane domains of the same or different surface membrane proteins, minimizing the interaction with other members of the receptor complex.

[0176] In some embodiments, the membrane-spanning domain is derived from a natural or synthetic source. If the source is natural, the domain is derived from any membrane-bound protein or membrane-spanning protein in some aspects. The membrane-spanning region includes those derived from (i.e., at least including its membrane-spanning region) α, β or ζ chains of T cell receptors, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules. Alternatively, in some embodiments, the membrane-spanning domain is synthetic. In some aspects, the synthetic membrane-spanning domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, phenylalanine, tryptophan and valine triplet will be found at each end of the synthetic membrane-spanning domain.

[0177] In certain embodiments, the platform technology for genetically modifying immune cells (e.g., NK cells) disclosed herein includes: (i) non-viral gene transfer using an electroporation device (e.g., a nuclear transfection instrument), (ii) CARs that signal through the internal domain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with variable lengths that link the antigen recognition domain to the cell surface, and in some cases (iv) artificial antigen presenting cells (aAPCs) derived from K562 to enable stable and large-scale expansion of CARs. + Immune cells (Singh et al., 2008; Singh et al., 2011).

[0178] 2. T cell receptor (TCR)

[0179] In some embodiments, genetically engineered antigen receptors include recombinant TCRs and / or TCRs cloned from naturally occurring T cells."T cell receptors" or "TCRs" refer to molecules containing variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRγ and TCRδ, respectively), capable of specifically binding to antigenic peptides bound to MHC receptors. In some embodiments, TCRs are in the αβ form.

[0180] Typically, TCRs in the form of αβ and γδ are usually similar in structure, but the T cells expressing them may have completely different anatomical locations or functions. TCRs can exist on the cell surface or in a soluble form. Typically, TCRs exist on the surface of T cells (or T lymphocytes) and are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also include constant domains, transmembrane domains, and / or short cytoplasmic tails (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p.433, 1997). For example, in some aspects, each chain of a TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex that is involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses complete or full-length TCRs, including TCRs in αβ or γδ form.

[0181] Therefore, for the purposes of this article, the TCR referred to includes any TCR or functional fragment, such as an antigen binding portion of a TCR that binds to a specific antigen peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. An "antigen binding portion" or "antigen binding fragment" of a TCR that can be used interchangeably refers to a molecule that contains a portion of a domain of a TCR but binds to an antigen (e.g., an MHC-peptide complex) bound by a complete TCR. In some cases, the antigen binding portion includes a variable domain of a TCR (e.g., a variable α chain and a variable β chain of a TCR) sufficient to form a binding site to bind to a specific MHC-peptide complex, such as typically each chain comprising three complementary determining regions.

[0182] In some embodiments, the variable domains of the TCR chains associate to form loops or complementary determining regions (CDRs) similar to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. 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 main CDR responsible for recognizing processed antigens, although it has also been shown that CDR1 of the α chain interacts with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the β chain may also include a hypervariable (HV4) region.

[0183] In some embodiments, the TCR chain comprises a constant domain. For example, like an immunoglobulin, the extracellular portion of a TCR chain (e.g., an α chain, a β chain) may comprise two immunoglobulin domains, an N-terminal variable domain (e.g., a V a or Vp; usually amino acids 1 to 116 according to Kabat numbering: Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.) and a constant domain adjacent to the cell membrane (e.g., an α-chain constant domain or Ca, typically amino acids 117 to 259 according to Kabat numbering, and a β-chain constant domain or Cp, typically amino acids 117 to 295 according to Kabat numbering). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby forming a connection between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each α and β chain, such that the TCR contains two disulfide bonds in the constant domain.

[0184] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules (such as CD3). For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunit of the CD3 signaling device or complex.

[0185] Typically, CD3 is a multiprotein complex that can have three different chains (γ, δ and ε) and a ζ chain in mammals. For example, in mammals, the complex can contain a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, a feature that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3γ chain, CD3δ chain, and CD3ε chain each contain a single conserved motif, called an immunoreceptor tyrosine-based activation motif or ITAM, while each CD3ζ 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 signals from the TCR to the cell. The CD3- and ζ chains, together with the TCR, form the so-called T cell receptor complex.

[0186] In some embodiments, TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) connected together, for example, connected together by one or more disulfide bonds. In some embodiments, TCR for target antigens (such as cancer antigens) is identified and introduced into cells. In some embodiments, nucleic acids encoding TCR can be obtained from various sources, such as polymerase chain reaction (PCR) amplification by publicly available TCR DNA sequences. In some embodiments, TCR is obtained from biological sources, such as obtained from cells, such as obtained from T cells (such as cytotoxic T cells), T cell hybridomas or other publicly available sources. In some embodiments, T cells can be obtained from cells isolated in vivo. In some embodiments, high-affinity T cell clones can be isolated from patients, and TCR is isolated. In some embodiments, T cells can be cultured T cell hybridomas or clones. In some embodiments, TCR clones for target antigens have been produced in transgenic mice transformed with human immune system genes (e.g., human leukocyte antigen system or HLA). In some embodiments, phage display is used to isolate TCRs for target antigens. In some embodiments, TCRs or their antigen binding portions can be synthetically produced based on knowledge of TCR sequences.

[0187] 3. Antigen Presenting Cells

[0188] APCs, including macrophages, B lymphocytes, and dendritic cells, are distinguished by the specific MHC molecules they express. APCs internalize antigens and re-express a portion of that antigen on their outer cell membrane along with MHC molecules. MHC is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, known as MHC class I and class II. T helper lymphocytes typically recognize antigens associated with MHC class II molecules, while T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. MHC is known as the HLA complex in humans and the H-2 complex in mice.

[0189] In some cases, aAPCs can be used to prepare the therapeutic compositions and cell therapy products of the embodiments.For general guidance on the preparation and use of antigen presentation systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662.

[0190] The aAPC system may include at least one exogenous auxiliary molecule. Any suitable number and combination of auxiliary molecules can be used. Auxiliary molecules can be selected from auxiliary molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (FcγRI), 41BB ligands and IL-21. Adhesion molecules can include carbohydrate-binding glycoproteins (e.g., selectins), transmembrane-bound glycoproteins (e.g., integrins), calcium-dependent proteins (e.g., cadherins), and single-passing immunoglobulin (Ig) superfamily proteins (e.g., intercellular adhesion molecules (ICAM)), which promote, for example, cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs (e.g., ICAM-1). The selection, cloning, preparation and expression of exemplary auxiliary molecules including costimulatory molecules and adhesion molecules, techniques, methods and reagents are illustrated in, for example, U.S. Patent Nos. 6,225,042, 6,355,479 and 6,362,001.

[0191] 4. Antigen

[0192] The antigen of the antigen receptor targeting of genetic modification is the antigen expressed in the disease, the patient's condition or the cell type environment targeted by adoptive cell therapy.Disease and the patient's condition are hyperplastic, tumorous and malignant diseases and disorders, including cancer and tumor, including blood cancer, immune system cancer, such as lymphoma, leukemia and / or myeloma, such as B, T and myeloid leukemia, lymphoma and multiple myeloma.In some embodiments, compared with normal or untargeted cells or tissues, antigen is selectively expressed or overexpressed on disease or patient's condition cells (such as tumor or pathogenic cells).In other embodiments, antigen is expressed on normal cells and / or expressed on transformed cells.

[0193] Any suitable antigen can be used in the present method. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, self-antigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015).

[0194] Tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens 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 variety of tumor types (e.g., melanoma, lung cancer, sarcoma, and bladder cancer). See, e.g., U.S. Patent No. 6,544,518. Tumor-associated antigens for prostate cancer include, e.g., prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphatase, NKX3.1, and prostate six transmembrane epithelial antigen (STEAP).

[0195] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids, which can be used to treat many cancers.

[0196] Tumor antigens include tumor antigens derived from cancer, and the cancer is characterized by tumor-associated antigen expression, such as HER-2 / neu expression. Interested tumor-associated antigens include lineage-specific tumor antigens, such as melanocyte-melanoma lineage antigens MART-1 / Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-associated proteins. Exemplary tumor-associated antigens include but are not limited to being derived from or comprising any one or more of the following antigens: p53, Ras, c-Myc, cytoplasmic serine / threonine kinases (such as 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, phosphatidylinositol-3 kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, nephroblastoma 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-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling protein white 1 (TACSTD1), TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-kappa B (NF-B), Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA,PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, carbonic anhydrase I (CAI) IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucose GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotype. ,

[0197] Antigens may include epitope regions or epitope peptides derived from genes that are mutated in tumor cells or that are transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences, such as N-acetylglucosamine transferase-V; clonal rearrangements of immunoglobulin genes that produce unique idiotypes in myeloma and B-cell lymphoma; tumor antigens, including epitope regions or epitope peptides derived from oncogenic viral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0198] In other embodiments, antigens are obtained or derived from pathogenic microorganisms or opportunistic pathogenic microorganisms (also referred to herein as infectious microorganisms), such as viruses, fungi, parasites and bacteria. In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0199] III. Immune Effector Cells

[0200] A. Hematopoietic precursor cells

[0201] The PSCs of the present disclosure engineered to express antigen receptors (e.g., CARs) can be differentiated into HPCs by methods known in the art. In one method, CAR-PSCs are differentiated into CD34 + HPC. In another approach, CAR-PSCs were differentiated into CD34 + HPC.

[0202] 1. Directed Differentiation

[0203] Certain embodiments of the present disclosure relate to differentiating CAR-PSC into HPC. CAR-PSC is differentiated into HPC by methods known in the art, such as the method described in U.S. Patent No. 8,372,642, which is incorporated herein by reference. In one method, a combination of BMP4, VEGF, Flt3 ligand, IL-3 and GM-CSF can be used to promote hematopoietic differentiation. In certain embodiments, the cell culture is sequentially exposed to a first culture medium to prepare a PSC for differentiation, including a second culture medium of BMP4, VEGF and FGF, and then cultured in a third culture medium including Flt3 ligand, SCF, TPO, IL-3 and IL-6, so that pluripotent cells can be differentiated into HPC and hematopoietic cells. The second defined culture medium may also include heparin. In addition, including FGF-2 (50ng / ml) in a culture medium containing BMP4 and VEGF can enhance the efficiency of generating hematopoietic precursor cells from pluripotent cells. In addition, inclusion of glycogen synthase kinase 3 (GSK3) inhibitors (eg, CHIR99021, BIO, and SB-216763) in the first defined medium can further enhance HPC production.

[0204] Typically, pluripotent cells can be differentiated into hematopoietic precursor cells using defined or undefined conditions. It is to be understood that in embodiments where the resulting cells are expected to be administered to human subjects, defined conditions are generally preferred. Hematopoietic stem cells can be derived from pluripotent stem cells under defined conditions (e.g., using TeSR culture medium), and hematopoietic cells can be produced from embryoid bodies derived from pluripotent cells. In other embodiments, pluripotent cells can be co-cultured on OP9 cells or mouse embryonic fibroblasts and subsequently differentiated.

[0205] As part of the differentiation process, pluripotent cells can be made to form embryoid bodies or aggregates. For inducing differentiation, the formation of "embryoid bodies" (EBs) or growing cell clusters generally involves human pluripotent stem cells being aggregated into EBs in vitro, and enables human pluripotent stem cells to spontaneously and randomly differentiate into various tissue types representing endoderm, ectoderm and mesoderm origin. Therefore, three-dimensional EBs can be used to produce a portion of hematopoietic cells and endothelial cells.

[0206] EBs can be formed using the following protocol. When confluent, EBs can be harvested using 0.5M EDTA for approximately 8-10 minutes at room temperature. TM Undifferentiated iPSCs are grown without feeder layers on coated plates. After incubation, EDTA is aspirated and EBs can be formed by collecting cells in SFD medium containing rock inhibitor or blebbistatin. The next day, the medium can be changed to EB1 differentiation medium containing different cytokine preparations. Cells are plated at a density of 250,000-500,000 cells per ml to promote aggregate formation.

[0207] To promote aggregate formation, cells were transferred to low-adhesion culture plates and incubated overnight in serum-free differentiation (SFD) medium consisting of 75% IMDM (Gibco), 25% Ham's modified F12 (Cellgro), supplemented with 0.05% N2 and B-27 without RA supplement, 200 mM l-glutamine, 0.05 mg / ml ascorbic acid-2-phosphate magnesium salt (Asc2-P) (WAKO), and 4.5 x 10 -4 MTG. The next day, cells can be collected from each well and centrifuged. Then, in the first four days of differentiation, cells can be resuspended in "EB differentiation medium", which is composed of SFD basal medium supplemented with about 50ng / ml bone morphogenetic factor (BMP4), about 50ng / ml vascular endothelial growth factor (VEGF) and 50ng / ml zb FGF. Half of the cell culture fluid is replaced every 48 hours. On the fifth day of differentiation, the culture medium is replaced with a second culture medium, which comprises SFD culture medium, supplemented with 50ng / ml stem cell factor (SCF), about 50ng / ml Flt-3 ligand (Flt-3L), 50ng / ml interleukin 6 (IL-6), 50ng / ml interleukin 3 (IL-3), 50ng / ml thrombopoietin (TPO). Half of the cell culture fluid is replaced with fresh differentiation medium every 48 hours. The culture medium is replaced by centrifuging the differentiation culture at 300g for 5 minutes and drawing half the volume from the differentiation culture and supplementing with fresh culture medium. In certain embodiments, the EB differentiation medium may include about BMP4 (e.g., about 50 ng / ml), VEGF (e.g., about 50 ng / ml), and optionally FGF-2 (e.g., about 25-75 ng / ml or about 50 ng / ml). The supernatant may be aspirated and replaced with fresh differentiation medium. Alternatively, half of the cell culture medium may be replaced with fresh medium every two days. Cells may be harvested at different time points during the differentiation process.

[0208] HPCs can be cultured from pluripotent stem cells using defined media.+ Stem cell methods are described, for example, in U.S. Application 12 / 715,136, which is incorporated by reference in its entirety. It is contemplated that these methods can be used with the present disclosure.

[0209] For example, defined culture medium can be used to induce hematopoietic CD34 + Differentiation. The defined medium may include growth factors BMP4, VEGF, Flt3 ligand, IL-3 and / or GMCSF. Pluripotent cells may be cultured in a first defined medium comprising BMP4, VEGF and optionally FGF-2, and then cultured in a second medium comprising (Flt3 ligand, IL-3 and GMCSF) or (Flt3 ligand, IL-3, IL-6 and TPO). The first and second culture media may also include one or more of SCF, IL-6, G-CSF, EPO, FGF-2 and / or TPO. Substantially hypoxic conditions (e.g., less than 20% O2) may further promote hematopoietic or endothelial differentiation.

[0210] The enzymes can be cleaved mechanically or enzymatically (e.g., using trypsin or TrypLE TM ) to substantially individualize the cells. A ROCK inhibitor (e.g., H1152 or Y-27632) may also be included in the culture medium. It is contemplated that these methods may be automated using, for example, robotic automation.

[0211] In certain embodiments, substantially hypoxic conditions can be used to promote pluripotent cells to differentiate into hematopoietic progenitor cells. As will be appreciated by those skilled in the art, an atmospheric oxygen content lower than about 20.8% will be considered as hypoxia. Compared with ambient air, the human cells in the culture medium can grow under the atmospheric conditions where oxygen content is reduced. This relative hypoxia can be achieved by reducing the atmospheric oxygen exposed to the culture medium. Embryonic cells are usually grown in vivo under the conditions where oxygen is reduced, typically about 1% to about 6% atmospheric oxygen, and carbon dioxide is at ambient levels. It is not desirable to be bound by theory, it is contemplated that hypoxic conditions can simulate aspects of some embryonic development conditions. As shown in the following examples, hypoxic conditions can be used in certain embodiments to promote pluripotent cells (such as iPSC or hESC) to further differentiate into a more highly differentiated cell type (such as HPC).

[0212] The following hypoxic conditions can be used to promote the differentiation of pluripotent cells into hematopoietic progenitor cells. In certain embodiments, 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 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% of the atmospheric oxygen content can be used to promote differentiation into hematopoietic precursor cells. In certain embodiments, the hypoxic atmosphere comprises about 5% oxygen.

[0213] Regardless of which specific culture medium is used in any given hematopoietic progenitor cell expansion, the culture medium used is preferably supplemented with at least one cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more typically 10 ng / mL to 100 ng / mL. Suitable cytokines include, but are not limited to, c-kit ligand (KL) (also known as steel factor (StI), mast cell growth factor (MGF), and stem cell factor (SCF), IL-6, G-CSF, IL-3, GM-CSF, IL-1α, IL-11, MIP-1α, LIF, c-mpl ligand / TPO, and flk2 / flk3 ligand (Flt2L or Flt3L). In particular, the culture medium will include at least one of SCF, Flt3L, and TPO. More particularly, the culture medium will include SCF, Flt3L, and TPO.

[0214] In one embodiment, the cytokines are included in the culture medium and supplemented by culture medium perfusion. Alternatively, when a bioreactor system is used, the cytokines can be added separately as concentrated solutions through a separate inlet without culture medium perfusion. When cytokines are added without perfusion, they will generally be added as a 10× to 100× solution equal to one-tenth to 1 / 100 of the volume in the bioreactor, and fresh cytokines are added approximately every 2 to 4 days. In addition, fresh concentrated cytokines can also be added separately to the cytokines in the perfusion culture medium.

[0215] In some embodiments, the HPCs exhibit disruption of methyl-CpG binding protein 2 (MeCP2) and are cultured under conditions that promote myeloid differentiation or lymphoid differentiation. In some aspects, the HPCs express non-functional MeCP2 that does not substantially bind to methylated DNA. In certain aspects, the HPCs do not express MeCP2 at levels sufficient to affect MeCP2 DNA binding activity. In specific aspects, MeCP2 is non-functional due to truncations or mutations in the MeCP2 gene. In some aspects, obtaining HPCs that exhibit disruption of MeCP2 comprises contacting the HPCs with siRNA, shRNA, or a small molecule inhibitor of MeCP2.

[0216] (i) Exemplary 3D differentiation methods

[0217] An exemplary method for differentiating PSCs into HPCs comprises maintaining the cells under feeder-free conditions, such as MATRIGEL® in Essential 8 (E8) medium. TM or vitronectin-coated culture plates. Aggregates are prepared from PSCs (particularly 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 / F12 basal medium, ascorbic acid (e.g., 100-500 μM), magnesium 2-phosphate, and sodium selenite), supplemented with 50 ng / ml FGF2, 50 ng / ml VEGF, 2 μM CHIR99021 (GSK-3 inhibitor), and blebbistatin (myosin-II inhibitor) (e.g., 2-10 μM, e.g., 10 μM). Aggregate formation and subsequent steps are performed during 24-hour culture in ultra-low adhesion (ULA) flasks under continuous stirring.

[0218] The formed cell aggregates (i.e., EBs) are further transferred to serum-free differentiation medium (e.g., 50% IMDM, 50% Hams F12 medium, 100 μg / ml polyvinyl alcohol, 100 μg / ml recombinant human serum albumin, 1× non-essential amino acid supplement (Invitrogen), 0.1× chemically defined lipid supplement (Invitrogen), 125 μM ascorbic acid 2-phosphate magnesium, 0.25 μM linoleic acid, trace element supplements A (0.3×), B (0.2×), and C (0.1×) (Corning), 5 mM sodium chloride, 100 μM monothioglycerol, 20 μM ethanolamine, 100 ng / ml heparin, and 10 ng / ml IGF1), supplemented with hematopoietic mesoderm inducing cytokines—25 ng / ml BMP4, 50 mg / ml VEGF, and 50 ng / ml FGF2. Continue culturing (e.g., for 4 days), and replace the complete medium the next day.

[0219] To support hematopoiesis CD34 + For differentiation and expansion of progenitor cells, the cell aggregates are further transferred to a serum-free differentiation medium (as described above) supplemented with hematopoietic supportive 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). The culture is continued (e.g., for 4 days), and the complete medium is replaced the next day.

[0220] The culture is harvested after the differentiation process (e.g., 9 days). A single cell suspension is obtained by digesting the differentiated cell aggregates (e.g., in Accutase). The isolated CD34 + Cells (eg, isolated by MACS) are plated into T / NK differentiation medium or stored frozen for subsequent use within 1 hour of isolation.

[0221] (ii) Exemplary 2D Differentiation Methods

[0222] In an alternative exemplary method, PSCs are subjected to a 2D differentiation protocol to generate HPCs. First, MATRIGEL® is used in feeder-free conditions, such as in Essential 8 (E8) medium. TM PSCs are adapted to hypoxic conditions, e.g., for 5-10 passages, in the presence of vitronectin or vitronectin coating. PSCs are individualized and cultured at 25,000 / cm in the presence of serum-free defined (SFD) medium supplemented with 5 uM blebbistatin (e.g., 2-10 μM, such as 10 μM). 2 The cells were plated at a density of 1:1 on PureCoat amine-coated 6-well plates (Corning Inc.). SFD basal medium may contain 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P / S), 25% Hams F12 (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 was supplemented with 50 ng / ml BMP-4, VEGF and bFGF.

[0223] On day 1, cells were cultured in a 4% culture medium containing, for example, 75% IMDM (Invitrogen 12200-069) with glutamine and 25 mM HEPES + P / S, 25% Hams F12 (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 -4The cells were cultured in SFD basal medium supplemented with 50 ng / ml BMP4, VEGF and bFGF with monothioglycerol to initiate hematopoietic differentiation induction. On day 2, the medium was aspirated and the cells were placed in fresh EB1 medium (e.g., containing 75% IMDM (Invitrogen 12200-069) (containing glutamine and 25 mM HEPES+P / S), 25% Hams F12 (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 The cells were then cultured in SFD basal medium supplemented with 50 ng / ml BMP4, VEGF, and bFGF for an additional 48 hours.

[0224] On days 5-10, the medium was aspirated and the cells were placed in EB2 medium for an additional 48 hours. EB2 medium can contain fresh SFD basal medium containing 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P / S), 25% Hams F12 (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 was supplemented with 50 ng / ml Flt-3 ligand, IL3, IL6, SCF and TPO (50 ng / ml each) and 5000 U / ml heparin. Cells were harvested using TrypLE on days 7, 8, 9, and 10 of differentiation and stained for the presence of HPC markers and lymphoid progenitors.

[0225] 2. Forward Programming

[0226] Certain embodiments of the present disclosure provide HPC by forward programming CAR-PSC, which is performed by expressing a combination of programming genes important for hematopoietic cell differentiation / function. In one method, PSC is modified to express at least three hematopoietic precursor programming genes, such as ETS genes (such as ETC2 or ERG), hematopoietic development genes (such as GATA2) and homeobox genes (such as HOXA9), such as described in PCT / US2016 / 057893, which are incorporated herein by reference in their entirety. In a particular aspect, a bidirectional Tight promoter transfected into CAR-PSC is used to co-express ETV2 / ERG, GATA2 and HOXA9 genes by a vector (such as an inducible PiggyBac vector).

[0227] In addition, EGH-CAR-PSC can be further modified to express additional genes for long-term implantation potential. Exemplary genes include HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350 and / or RBAK. For example, PSC can be transfected with one or more vectors to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1 and HOXA4.

[0228] Preferably, only the ETV2 / GAT2 / HOXA9 gene expression is sufficient to positively program the PSC into hematopoietic precursor cells for a period of time. Therefore, the hematopoietic precursor programming gene can be under the control of an inducible promoter. In this way, the hematopoietic precursor programming gene expression can be induced in the PSC for a period of time sufficient to positively program into multi-lineage hematopoietic precursor cells. This period of time can be about 1 day to about 20 days, for example, about 3, 4, 5, 6, 7, 8, 9 or 10 days. Alternatively, the hematopoietic precursor programming gene can be introduced into the PSC via an episomal vector. In this way, the hematopoietic precursor programming gene can be transiently expressed in the PSC.

[0229] B. Lymphoid cell differentiation

[0230] HPCs can then further differentiate into lymphoid lineage cells, including T cells, NK cells, and T / NK cells. In some aspects, HPCs during differentiation are separated on days 7-12 (e.g., days 8-11) to differentiate into lymphoid cells. HPCs 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 and decrease on day 11, which means that a certain threshold expression level of these markers is required to make the cells tend to lymphoid differentiation in the presence of DLL4.

[0231] In some aspects, HPC separated by differentiation process 7-11 days (such as the 7th day, the 8th day, the 9th day, the 10th day or the 11th day) can be differentiated into lymphoid cells, such as T cells and NK cells. In some aspects, the origin time of lymphocyte progenitor cells coincides with the reduction of hematopoietic endothelial progenitor cells and the appearance of progenitor erythrocytes in the HPC differentiation process. In particular aspects, the HPC at the 9th day can have the efficiency of increased production of T cells. HPC capable of lymphoid differentiation can be separated and / or identified by the expression of some markers. For example, cells with CD34 and / or CD43 surface expression (particularly expressing CD34 and CD43 simultaneously) can be selected for lymphoid differentiation, such as selected by MACS sorting.

[0232] Other markers used to detect lymphoid progenitor cells include DLL4, CD144, CD31, CD34, CD43 lo 、CD45 lo / - , CD235, CD7, Flk-1, APNLR. In a specific aspect, CD34 / CD7, CD235 / CD7, DLL4 / CD34, DLL4 / CD31, DLL4 / CD144 or CD34 / CD43 are used. lo Lymphoid progenitor cells are identified by the presence of a double positive population. CD144 expression on HPCs is co-stained with CD31, CD34 and DLL4. CD7 expression on HPCs is co-stained with CD235, CD34 and CD43. Therefore, HPC co-expression of CD144 and CD7 confirms lymphoid potential-captured precursors that express membrane-bound notch ligand (DLL4) and hemogenic endothelial cell markers and produces phenotypic characteristics of emerging lymphoid progenitors capable of generating definitive hematopoietic cell lineages in vitro. In a specific aspect, HPCs can be further sorted into cells with enhanced lymphoid potential by sorting for surface markers including CD31, CD34, CD144, CD43, CD45, CD6, CD335, Flk-1 and DLL4. In some aspects, the CD114 / CD34, CD144 / CD45, CD144 / CD7 and CD144 / CD34 / CD45 / CD7 positive fractions of HPCs are differentiated into lymphoid cells. In specific aspects, the CD144 / CD7 positive fractions of HPCs are differentiated into lymphoid cells.

[0233] HPCs can be cultured under limited conditions without a feeder layer for lymphoid differentiation. The culture medium may contain one or more matrix components, such as RetroNectin, fibronectin or RGD peptide. Without wishing to be bound by any theory, the matrix components can provide solid support for the growth of embryonic stem cells. In certain embodiments, before the cells are inoculated into the culture medium, the matrix components can be applied to the culture surface and contacted with the culture medium. For example, before the cells are mechanically separated into clumps or the cells are individualized and induced to differentiate into hematopoietic precursor cells, the matrix components can be applied to the culture surface and contacted with the culture medium. TM The cells are cultured in a defined medium (eg, TeSR medium) on the coated plates.

[0234] A variety of matrix components can be used to culture pluripotent cells, including collagen (e.g., collagen IV), laminin, vitronectin, MATRIGEL TM , gelatin, polylysine, thrombospondin (e.g., TSP-1, -2, -3, -4 and / or -5) and / or PRONECTIN-F TM In certain embodiments, it is avoided to treat MATRIGEL alone due to possible adverse effects on cell viability. TM , collagen IV or laminin with cells previously cultured using TeSR; nevertheless, these compositions can be advantageously used in combination with other matrix components. Combinations of these matrix components can provide additional benefits for promoting cell growth and cell viability. In certain embodiments, for example, prior to hematopoietic differentiation, 1, 2, 3, 4, 5, 6 or more of the above matrix components can be used to culture cells.

[0235] In some embodiments, ascorbic acid can be used to enhance lymphoid differentiation. Defined medium can be supplemented with about 10 μM to about 1 mM of ascorbic acid, such as 100 to 500 μM, such as about 50 μM to about 100 μM, such as about 95 μM. Ascorbic acid can be selected from various ascorbates, such as magnesium ascorbyl phosphate. In some embodiments, nicotinamide (e.g., nicotinic acid) can be used to enhance lymphoid differentiation, such as using nicotinamide at a concentration of about 0.1 mM to about 5 mM.

[0236] In some aspects, the endogenous activity of Notch ligands is changed by administering a substance that increases the production of Notch ligands in the subject, and HPCs are differentiated into lymphoid cells (such as T cells). The method also includes culturing cells in a culture medium, wherein the culture medium includes an effective amount of notch ligands and one or more cytokines selected from IL-7, IL-15, SCF, Flt-3 and IL-3. In some specific embodiments, the culture medium may also include IL-6. In some embodiments, the notch ligand is a δ4notch ligand (DLL4), such as a DLL4:Fc chimera.

[0237] Notch ligands that promote and maintain differentiation and proliferation of T cell lineage cells are selected. Notch ligands can be human or can be derived from other species, including mammalian species such as rodents, dogs, cats, pigs, sheep, cows, goats and primates. Specific examples of Notch ligands include the Delta family. The Delta family includes Delta-1 (Genbank accession number AF003522, Homo sapiens), Delta-3 (Genbank accession number AF084576, Rattus norvegicus), Delta-like-1 (Genbank accession numbers NM-005618 and NP-005609, Homo sapiens; Genbank accession numbers X80903, I48324, Mus musculus), Delta-like-3 (Genbank accession number NM-05366 6, N-446118, Rattus norvegicus), Delta-4 (Genbank accession number AF273454, BAB18580, Mus musculus; Genbank accession number AF279305, AAF81912, Homo sapiens) and Delta-like-4 (Genbank accession number Q9NR61, AAF76427, AF253468, NM-019074, Homo sapiens; Genbank accession number NM-019454, Mus musculus). Notch ligands are commercially available or can be produced by recombinant DNA technology and purified to varying degrees.

[0238] The method also includes the step of maintaining the HPC cells in the above-mentioned culture medium for a period of time sufficient to produce differentiated NK cells. In some embodiments, differentiated NK cells appear in culture with T cells, but the NK cells may stop proliferating after the 6th week. Typically, conventional methods known to those of ordinary skill in the art are used to assess the increase in the number of NK cells and / or the confirmation of their differentiation state. For example, the NK cell development of the cultured cells can be monitored by flow cytometry by staining the cells with anti-CD56 and anti-CD3 antibodies. Anti-CD3 antibodies (e.g., OKT3) can be present at a concentration of 0.5 to 2 μg. CD56 + / CD3 -The cells will be indicative of differentiated NK cells.

[0239] In a specific aspect, the expression of CD3 T cells was performed by 2D hypoxic culture on retronectin and DLL4 coated plates. + T cells or CD56 + CD3 - Lymphoid differentiation of NK cells. In certain aspects, non-tissue culture treated culture plates can be coated with DLL4:Fc chimeric protein and RetroNectin (fibronectin fragment CH-296; Takara Shuzo, Japan) to perform lymphoid differentiation of HPCs. Differentiation can include a first stage of T / NK cell differentiation, followed by a second stage of T or NK cell expansion. The first stage of differentiation may last from about 1 week to about 2 weeks, and the second stage of expansion may also last from about 1 week to about 2 weeks. Thus, the entire cycle of lymphoid differentiation and expansion may be about 2-4 weeks.

[0240] In some embodiments, the lymphoid differentiation process may include co-culturing with antigen-specific target cells (e.g., antigen-specific tumor cells) to increase the cytotoxic activity of T cells and NK cells. In certain aspects, the expansion phase of lymphoid differentiation may include co-culturing with antigen-specific target cells. For example, CD19-CAR-T cells or NK cells may be co-cultured with CD19 during expansion. + Tumor cells (e.g. CD19 + Daudi cells) for one to three weeks, in particular two weeks. In some aspects, the expansion period may also include the addition of cytokines (e.g., IL2, IL15 and / or IL21, in particular IL2) to improve expansion. The concentration of the cytokine may be optimized, for example, 10 to 50 μM.

[0241] C. Cell Culture

[0242] In certain embodiments, substantially hypoxic conditions can be used to promote HPC differentiation into myeloid or lymphoid lineages. In certain embodiments, 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 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% of the atmosphere oxygen content can be used to promote differentiation into hematopoietic precursor cells. In certain embodiments, hypoxic atmosphere comprises about 5% oxygen.

[0243] As described herein, one or more defined culture media can be advantageously used to promote the differentiation of HPCs into myeloid and lymphoid lineages. In particular, the exclusion of animal products (such as serum and mouse feeder layers) can reduce the risks associated with cell exposure to animal products, making it possible to generate cells that can be more safely administered to human subjects. Since the development of traditional stem cell culture has always relied on serum products and mouse feeder layers to differentiate stem cells into various cell types, these traditional methods have limited the scale of differentiation that can be carried out, increased biological variability and potential contamination, and severely hindered the use of ES cells in conversion therapy, which might otherwise prove useful.

[0244] In general, the cells of the present disclosure are cultured in a nutrient-rich buffered solution culture medium that can maintain cell growth. According to the methods described herein, the culture medium suitable for separating, amplifying and differentiating pluripotent stem cells into hematopoietic precursor cells and hematopoietic cells includes but is not limited to high glucose Dulbecco modified Eagle culture medium (DMEM), DMEM / F-15, RPMI 1640, Iscove modified Dubelcco culture medium (IMDM) and Opti-MEM SFM (Invitrogen Inc.). The culture medium with chemical composition determination includes the minimum essential culture medium (such as Iscove modified Dulbecco culture medium (IMDM) (Gibco)), supplemented with human serum albumin, human ExCyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and mitogens are also suitable. As used herein, mitogens refer to reagents that stimulate cell division. Reagents can be chemical substances, usually certain protein forms that stimulate cells to start cell division and thus cause mitosis. In one embodiment, serum-free medium (e.g., as described in U.S. Serial Nos. 08 / 464,599 and WO 96 / 39487) and "complete medium" as described in U.S. Pat. No. 5,486,359 can be expected to be suitable for use in the methods described herein. In some embodiments, the culture medium is supplemented with 10% fetal bovine serum (FBS), human autologous serum, human AB serum, or platelet-rich plasma supplemented with heparin (2 U / ml).

[0245] Immune cells can be produced by culturing pluripotent stem cells or hematopoietic precursor cells in culture medium under conditions of increasing the intracellular level of factors sufficient to promote cell differentiation into myeloid or lymphoid lineages. Culture medium may also include one or more hematopoietic cell differentiation and maturation reagents, such as various growth factors. These reagents can help induce cells to form a more mature phenotype-or preferably promote the survival of mature cells-or have both effects. Differentiation and maturation reagents may include soluble growth factors (peptide hormones, cytokines, ligand-receptor complexes and other compounds) that can promote the growth of hematopoietic cell lineage cells. Non-limiting examples of such reagents 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 its isotype or variant.

[0246] IV. Use of Antigen-Specific Immune Cells

[0247] The antigen-specific immune effector cells provided by certain aspects of the methods and compositions can be used in a variety of applications. These applications include, but are not limited to, in vivo transplantation or implantation of cells; in vitro screening of cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, drug compounds, etc.; elucidation of the mechanisms of blood diseases and injuries; study of the mechanisms of action of drugs and / or growth factors; diagnosis and monitoring of cancer in patients; gene therapy; and production of biologically active products, to name just a few.

[0248] A. Test Compound Screening

[0249] The immune cells of the present disclosure can be used to screen factors (such as solvents, small molecule drugs, peptides and polynucleotides) or environmental conditions (such as culture conditions or operations) that affect the characteristics of the lymphoid cells provided herein.

[0250] The specific screening application of the present disclosure relates to the testing of drug compounds in drug research. The reader generally refers to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997 and U.S. Patent No. 5,030,015. In some aspects, as previously performed on hematopoietic cells and precursors in short-term culture, myeloid and lymphoid cells play a role in the standard drug screening and toxicity assays of test cells. The assessment of the activity of candidate drug compounds generally involves mixing the hematopoietic cells or precursors provided in some aspects with the candidate compound, determining any changes in cell morphology, marker phenotype or metabolic activity attributable to the compound (compared with untreated cells or cells treated with inert compounds), and then associating the effects of the compound with the observed changes. Screening can be performed because the compound is designed to have a pharmacological effect on hematopoietic cells or precursors, or because the compound designed to have an effect elsewhere may have an unexpected effect on hematopoietic cells or precursors. Two or more drugs can be tested in combination (by mixing with cells simultaneously or sequentially) to detect possible drug-drug interactions.

[0251] B. Adoptive Cell Therapy

[0252] In some embodiments, the disclosure provides a method for immunotherapy, comprising administering an effective amount of an immune cell of the disclosure. In one embodiment, a medical disease or condition is treated by transferring a population of immune cells that elicits an immune response. In certain embodiments of the disclosure, cancer or infection is treated by transferring a population of immune cells that elicits an immune response. Provided herein is a method for treating or delaying the progression of cancer in an individual, comprising administering an effective amount of an antigen-specific cell therapy to the individual. This method can be used to treat immune diseases, solid cancers, blood cancers, and viral infections.

[0253] The tumors to which this treatment method is applicable include any malignant cell type, such as those found in solid tumors or hematologic tumors. Exemplary solid tumors may include, but are not limited to, tumors selected from organs such as pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Examples of exemplary hematologic tumors include bone marrow tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. More examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0254] The cancer may specifically be of the following histological types, although not limited thereto: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyps; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma carcinoma); oncocytic carcinoma; oncocytic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; cervical gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant testicular blastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo maligna malignant melanoma; acral lentiginous melanoma; nodular melanoma; malignant melanoma in giant pigmented hemorrhoids; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone;Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma ; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; small lymphocytic malignant lymphoma; large cell diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease)NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloid leukemia. ;

[0255] To determine the suitability of the cells provided herein for therapeutic applications, the cells can be first tested in a suitable animal model. The ability of the cells to survive and maintain their phenotype in vivo is first assessed. The cells provided herein are administered to immunodeficient animals (such as NOG mice, or animals that have been immunodeficient by chemical or irradiation) at a site suitable for further observation (e.g., under the kidney capsule, in the spleen, in the liver lobule, or in the bone marrow). After a few days to a few weeks or longer, the tissue is collected and assessed whether the initial cell type (such as red blood cells) is still present. This can be done by providing detectable markers (such as green fluorescent protein or beta-galactosidase) to the administered cells; or by measuring constitutive markers that are specific to the administered human cells. When the cells provided herein are tested in a rodent model, the presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA (using human-specific antibodies) or by RT-PCR analysis (using amplification primers and hybridization conditions that produce specificity for human polynucleotide sequences). Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in this disclosure.

[0256] The ability of immune cells provided by the methods of the present disclosure to treat blood diseases and injuries can be tested in various animal models. For example, sickle cell anemia mouse models or T / B cell-deficient Rag-2 knockout mice may be particularly useful animal models for testing myeloid and lymphoid cells disclosed herein.

[0257] Immune cells that demonstrate desired functional characteristics or efficacy in animal models provided in certain aspects of the present disclosure may also be suitable for direct administration to human subjects in need thereof. For the purpose of hemostasis, cells may be administered at any site sufficient to enter the circulation. Hematopoietic cells or their precursors may also be delivered at the site of injury or disease.

[0258] The cells provided in certain aspects of the present disclosure can be used to treat any subject in need thereof. Human conditions that may be amenable to such treatment include various anemias and hemoglobinopathies, as well as diseases characterized by a decrease in the number of hematopoietic cells (e.g., myelodysplastic syndrome, myelofibrosis, neutropenia, agranulocytosis, Glanzmann's thrombasthenia, thrombocytopenia, and acquired immunodeficiency syndrome). For human treatment, dosages are generally in the range of about 10 9 Up to 10 12 cells, usually about 5×10 9 Up to 5×10 10The final responsibility for determining the treatment modality and appropriate dosage lies with the attending clinician.

[0259] A therapeutically effective amount of immune cells can be administered by a variety of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection or infusion.

[0260] The therapeutically effective amount of immune cells used in adoptive cell therapy is the amount that achieves the desired effect in the treated subject. For example, this can be the number of immune cells necessary to inhibit progression, or cause regression of autoimmune or alloimmune diseases, or to alleviate symptoms (such as pain and inflammation) caused by autoimmune diseases. It can be the amount necessary to alleviate symptoms associated with inflammation (such as pain, edema, and elevated body temperature). It can also be the amount necessary to reduce or prevent transplant organ rejection.

[0261] The immune cell population can be administered according to a treatment regimen consistent with the disease, such as a single or several administrations within one to several days to improve the disease state, or periodic administration over an extended period of time to inhibit disease progression and prevent disease recurrence. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the judgment of the practitioner and the circumstances of each patient. The therapeutically effective amount of immune cells will depend on the subject being treated, the severity and type of the disease, and the mode of administration. In some embodiments, the therapeutic dose for human subjects may be at least 3.8×10 4 , at least 3.8×10 5 , at least 3.8×10 6 , at least 3.8×10 7 , at least 3.8×10 8 , at least 3.8×10 9 or at least 3.8×10 10 Immune cells / m 2 In certain embodiments, the therapeutic dose for human subjects is about 3.8×10 9 About 3.8×10 10 Immune cells / m 2 In other embodiments, the therapeutically effective amount of immune cells may be about 5×10 6 cells to about 7.5×10 per kilogram of body weight 8 For example, about 2×10 per kg of body weight 7 cells to about 5×10 8 cells, or about 5×10 per kilogram of body weight. 7 cells to about 2×10 8The exact number of immune cells can be easily determined by a person skilled in the art based on the age, weight, sex and physiological condition of the subject. The effective dose can be inferred from a dose-response curve obtained from an in vitro or animal model test system.

[0262] Immune cells can be co-administered with one or more other therapeutic agents to treat immune-mediated disorders. Combination therapy can include, but is not limited to, one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunoscavengers (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids (e.g., dexamethasone or prednisone)), anti-inflammatory agents (e.g., glucocorticoids (e.g., hydrocortisone, dexamethasone, or prednisone)) or nonsteroidal anti-inflammatory agents (e.g., acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin 10 or transforming growth factor-β), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or tolerogenic agents may be administered, including but not limited to calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, Treosulfan, busulfan); irradiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Depending on the desired effect, such additional agents may be administered before, during, or after the administration of the immune cells. Such administration of cells and agents may be performed by the same route or by different routes, and may be administered at the same site or at different sites.

[0263] C. Pharmaceutical Compositions

[0264] Also provided herein are pharmaceutical compositions and preparations comprising immune cells (e.g., T cells or NK cells) and pharmaceutically acceptable carriers. Administration can be autologous or non-autologous. For example, T cells and / or NK cells and compositions comprising them can be obtained from a subject and administered to the same subject or different compatible subjects. Immune cells of the present disclosure can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the therapeutic composition of the present disclosure, it is usually formulated into a unit dose injectable form (e.g., solution, suspension, emulsion).

[0265] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredient (eg, antibody or polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 nd edition, 2012), in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the receptor at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (such as zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968 describe certain exemplary sHASEGPs and methods of use, including rHuPH20. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

[0266] V. Examples

[0267] The following examples are included to illustrate preferred embodiments of the present invention. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and therefore can be considered to constitute preferred modes of its implementation. However, in light of this disclosure, it should be appreciated by those skilled in the art that many changes can be made in the specific embodiments disclosed and similar or similar results can still be obtained without departing from the spirit and scope of the present invention.

[0268] Example 1 - Derivation of T / NK cells derived from PSC expressing anti-CD19 CAR

[0269] To generate pluripotent stem cells expressing chimeric antigen receptors (CARs), two separate methods were used. In one method, 1C cells were produced using a retroviral vector (US20160257939; incorporated herein by reference in its entirety) or E11 cells were produced using an episomal vector, and transgenic-free PSCs were derived from T cells. In the second method, PSCs were transfected with a PiggyBac expression vector encoding the hematopoietic programming genes ETV2 / ERG, GATA2, and HOXA9 (modified H1 ESCs with introduced DOX-inducible ETV2-GATA2-HOXA9 (EGH) hematopoietic programming genes) to produce A16 cells. PSCs from both methods were then genetically modified to constitutively express a second-generation anti-human CD19 chimeric antigen receptor (CAR), which consists of a scFv domain derived from the FMC63mAb that binds to human CD19, a CD28 costimulatory domain, and a CD3ζ signaling domain.

[0270] Unmodified and CAR-modified PSCs were differentiated into CD34 + T / NK progenitor cells ( Figure 1A -B). Using 4 weeks of hypoxic culture on DLL4 / retronectin-coated plates, isolated CD34 + Progenitor cells further differentiate into CD3 + T(1C, E11) and CD3 - CD56 + NK (A16) cells. In parallel T / NK cultures, mitomycin C-treated Daudi cells (β2-microglobulin-deficient HLA class I-negative CD19+ B lymphoblastoid cell line) were added as a source of antigen (CD19)-specific CAR activation during the last 2 weeks of differentiation. The resulting T / NK cells were named as follows: 1C or E11-derived T cells—T, CAR-T, CAR-T / Ag (Daudi co-cultures); A16-derived NK cells—NK, CAR-NK, CAR-NK / Ag (Daudi co-cultures).

[0271] CAR expression was evaluated throughout the differentiation phase by flow cytometry using protein L staining.+ T cells were co-stained with lambda chain mouse anti-human CD3 mAb (clone SP34-2). After differentiation into T / NK cells, CAR expression was significantly silenced, but upon treatment with CAR-specific antigen (Ag; CD19 + After activation and culture with Daudi cells, CAR-positive T and NK cells can be selectively expanded.

[0272] By using CD19 expressing luciferase + In vitro cytotoxicity assays with Daudi and Raji target cells were used to assess the cytotoxic function of PSC-derived T (1C) and NK (A16) cells. Targets were incubated with T / NK effectors at a 1:1 ratio for 24 h, and luciferase activity in culture lysates was quantified by the Steady-Glo luciferase assay system (Promega). The percentage of cytotoxicity was calculated by the following formula: (1-(ET / T)) × 100, where ET – effector + target culture, T – target only.

[0273] Although unmodified T / NK cells lacked any detectable activity, both CAR-expressing T(1C) and NK(A16) cells showed potent responses against CD19. + Targeted cytotoxicity. + After 2 weeks of T / NK co-culture with target (Daudi) cells (CAR / Ag variants), CAR-dependent cytotoxic activity was significantly enhanced ( Figure 3A ).

[0274] The cytolytic potential of CAR-T cells was confirmed by real-time target cell counting using the Incucyte S3 live cell analysis system (Essen Bioscience). + Raji target cells were incubated with unmodified and CAR-transfected PSC (E11 TiPSC)-derived T cells at a 1:1 ratio. GFP expression was performed within 9 h. + Time-lapse imaging and counting of Raji cells. Viable (GFP) cells were detected only in cultures with CAR-T cells. + )A significant decrease in Raji cells ( Figure 3B ).

[0275] CD34 derived from PSCs (E11TiPSCs) by 2 weeks of T / NK differentiation followed by 1 week of anti-CD3-induced T cell expansion culture + HPC-derived CAR-T cells were fused with mouse P815 mastocytoma cells transfected with human CD19 antigen (CD19 +P815) and untransfected P815 were incubated for 24 h to assess CAR-inducible and constitutive cytokine production, respectively. Cytokines ( Figure 4 ). CAR-T cells demonstrated specific CAR-inducible production of IFNγ, TNFα, IL2, IL13, and GM-CSF. Constitutive secretion of granzyme B also demonstrated cytotoxicity of PSC-derived CAR-T cells.

[0276] To test the in vivo efficacy of CAR-T cells, 5×10 4 8-week-old NSG mice were injected intraperitoneally (ip) with 10 Raji cells expressing luciferase. 7 T (1C-derived) and NK (A16-derived) cells were injected (ip) at a dose of 100 ng / mouse. T / NK injections were repeated on days 3 and 5. During the T / NK injection period (days 1-5), all mice were additionally injected (ip) with a combination of IL2 and IL15 cytokines (both 500 ng / mouse). Tumor progression of mice was monitored every 2 weeks by in vivo bioluminescence imaging ( Figure 5A Anesthetized mice injected (ip) with 150 mg / kg InVivo-Glo luciferin (Promega) were analyzed within 15 min after luciferin injection using a Pearl Trilogy in vivo imager (LiCor). Figure 5B Shown are the survival curves of different groups of mice treated with PSC-derived T (1C) or NK (A16) cells.

[0277] Significant tumor suppression was detected at 2 weeks, and the average survival time of mice was extended by about 2 times by one course of treatment (3 injections 2 days apart) with CAR-expressing PSC-derived T or NK cells. The in vivo oncolytic potential can be significantly improved by pre-coculturing CAR-expressing PSC-derived T or NK cells with antigen-positive tumor cells in vitro.

[0278] Example 2 - Directed Differentiation Method

[0279] PCS differentiation into CD34 + Lymphoid hematopoietic progenitor cells: The T cell-derived PSCs of Example 1 (1C and E11 TiPSCs derived from peripheral blood T cells by retroviral and episomal reprogramming, respectively) were differentiated into CD34 + Hematopoietic progenitor cells. Maintaining PSCs on Matrigel in feeder-free conditions TMor Essential 8 (E8) medium on vitronectin-coated 6-well plates. Aggregates were prepared from subconfluent PSCs (<80% confluence) at a density of 500,000 cells per ml in E8 medium supplemented with 2 μM CHIR99021 (GSK-3 inhibitor) and 5 μM blebbistatin (myosin-II inhibitor). Aggregates were formed by culturing in ultra-low adhesion (ULA) flasks for 6 hours with continuous stirring at 15-20 rpm on a rocker platform (including all subsequent culture steps).

[0280] Cultures with preformed cell aggregates were gradually transferred to serum-free hematopoietic differentiation medium (HDM: 50% IMDM, 50% Hams F12 medium, 100 μg / ml polyvinyl alcohol, 100 μg / ml recombinant human serum albumin, 1× non-essential amino acid supplement (Invitrogen), 0.1× chemically defined lipid supplement (Invitrogen), 125 μM magnesium ascorbate 2-phosphate, 0.25 μM linoleic acid, trace element supplements A (0.3×), B (0.2×) and C (0.1×) (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 of medium at 6 and 24 hours. On day 2, cell aggregates were pelleted by sedimentation for 15 minutes, the medium was aspirated, and the culture was transferred to HDM supplemented with hematopoietic mesoderm-inducing cytokines: 25 ng / ml BMP4, 50 mg / ml VEGF, and 50 ng / ml FGF2. The culture was continued for 3 days, with complete medium replacement every day.

[0281] To support hematopoiesis CD34 + For differentiation and expansion of progenitor cells, the cell aggregates are further transferred to HDM supplemented with hematopoietic supportive cytokines: 50 ng / ml SCF, 50 ng / ml TPO, 20 ng / ml FLT3L and 20 ng / ml IL-3. The culture is continued for 3-5 days, and the complete medium is replaced every day. To improve the hematopoietic transition process, the following enhancement components can be added: IL11 (5-20 ng / ml), 8Br-cAMP (100-300 μM) and / or VEGF (20-50 ng / ml).

[0282] After 2+3+3-5 days of differentiation (8-10 days in total), HPCs identified as floating single cells or small clusters in the suspension were harvested. HPCs were isolated by filtering the entire differentiation culture through 70 μm and 30 μm cell strainers (Corning). HPCs collected from the filtrate by centrifugation were washed once in HDM and resuspended in TCDM (T cell differentiation medium). To enrich for HPCs with T / NK potential, CD34 can be separated by magnetic activated cell sorting (MACS) using direct CD34 microbeads (Myltenyi Biotec) according to the manufacturer's recommendations. + Isolation of HPC fractions. HPCs were plated into T / NK differentiation medium or stored frozen for subsequent use within 1 hour of isolation.

[0283] T / NK differentiation cultures: For T / NK differentiation, non-tissue culture treated plastic plates were coated with Notch ligand hDLL4-Fc chimeric protein and retronectin diluted in PBS (0.5 μg / cm 2 Before plating cells, aspirate the coating solution, wash the culture plate once with PBS, and fill it with 0.25 ml / cm 2 PSC-derived HPCs were cultured at 5000 cells / cm 2 Plate at a density of 10000 cells / cm and culture in a hypoxic (5% O2) CO2 incubator for 2 weeks, add an equal volume of TCDM culture on the 3rd day, and replace half of the volume of culture medium on each following second day. Harvest total differentiated cells by gently resuspending and collecting non-adherent cells, and then collect loosely attached cells by treating with PBS-EDTA (0.5mM) for 5-10 minutes. To continue the differentiation process and increase the yield of T / NK cells, the cells can be cultured at 10,000 cells / cm 2 The harvested cells were re-plated onto freshly prepared DLL4 / retronectin culture plates at a density of 100 μM to repeat the T cell differentiation culture until the desired T / NK cell yield was achieved. The efficiency of T / NK differentiation can also be improved by adding the following enhancement components to TCDM: CHIR99021 (1-3 μM), IL2 (2-10 ng / ml), IL12 (10-50 ng / ml).

[0284] T cell expansion cultureFor T cell expansion, non-tissue culture treated plates were coated with anti-CD3 mAb (clone OKT3), hDLL4-Fc, and retronectin (0.5 μg / cm each) diluted in PBS. 2 Before plating cells, aspirate the coating solution, wash the culture plate once with PBS, and fill it with 0.25 ml / cm 2 T cell expansion medium (TCEM) consists of StemSpan SFEM (Stem Cell Technologies), GlutaMax (1 / 100), magnesium ascorbyl phosphate (250 μM), nicotinamide (2 mM), and cytokines SCF, TPO, FLT3L, and IL7 (50 ng / ml each) and IL2, IL15 (10 ng / ml each). IL21 (5-20 ng / ml) can also be added to improve expansion. Cells harvested from T / NK differentiation cultures were plated at 10,000 cells / cm 2 The cells were plated at a density of 100 μg / mL and cultured in a hypoxic (5% O2) CO2 incubator for 2 weeks, with an equal volume of TCEM added on day 3 and half of the volume of medium replaced on each subsequent second day. The expanded T cells were harvested by gently resuspending and collecting non-adherent cells.

[0285] Example 3 - Characterization of directed differentiation methods

[0286] PSCs were first differentiated into CD34 in suspension aggregate cultures through sequential steps of WNT-induced differentiation priming, mesoderm induction, and HPC differentiation over 8-10 days. + HPC Figure 1B ). The HPC fraction was separated by filtration. Although CD34 can be optionally performed using direct CD34 paramagnetic beads (Myltenyi Biotec) + HPCs were then transferred to human DLL4-Fc + T / NK differentiation was performed on retronectin-coated plates for 2-4 weeks. T cells were expanded in culture on anti-CD3 mAb (OKT3 clone)-coated plates for an additional 1-2 weeks.

[0287] After 2 weeks of T / NK differentiation, PSC (1C TiPSC)-derived CD34 + The cells formed a typical lymphoid cell population as determined by the low FSC / SSC parameters ( Figure 1D , left dot plot). This lymphoid population mainly contains CD3 + T and CD56 + CD3 - NK cells ( Figure 1D , middle dot plot). T cell populations include CD4 + and CD8 + Single-positive and double-positive cells and a significant proportion of double-negative cells ( Figure 1D , click the picture on the right).

[0288] The yield of each corresponding cell type was calculated as the ratio of the absolute cell number of output to input at each stage of cell derivation. + HPC yields indicate that on average 1.5 (output) CD34 + HPC. Therefore, a T cell yield of 102 indicates that one (input) CD34 + HPC yields 102 (output) T cells ( Figure 1E ).

[0289] PSC (1C TiPSC) derived T cells (CD3 + ) were differentiated and expanded over a 4-week period expressing the α / β TCR (not γ / δ nor the invariant Vα24 NKT TCR) and the typical T cell markers CD5, CD27, and CD7 ( Figure 1F ). They also expressed cytotoxic T / NK-related (CD161, CD94) and activation (CD69) markers.

[0290] The minimum required is immobilized anti-CD3 antibody (iCD3) and sufficient for expansion of PSC-derived T cells ( Figure 1G , bar graph). Soluble stimulation with CD3 and CD28 mAbs (sCD3, sCD28) alone or in combination (sCD3+sCD28) or when added to iCD3 (iCD3 + T cells proliferating in expansion culture acquired the characteristic morphology of irregularly shaped lymphoblasts ( Figure 1G , photo). In contrast to the relatively heterogeneous input cell population, cells harvested from the 2-week T cell expansion were essentially pure CD3 + T cells, which also express CD56 and have acquired CD8 expression ( Figure 1G , flow cytometry dot plots). Thus, the directed differentiation approach efficiently generated T cells.

[0291] ***

[0292] All methods disclosed and claimed herein can be performed and executed without undue experimentation in accordance with the present disclosure. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that the methods and the steps or sequence of steps of the methods can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain reagents related to chemistry and physiology can be substituted for the reagents described herein while obtaining the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to fall within the spirit, scope and concept of the present invention as defined by the appended claims.

[0293] References

[0294] The following references are expressly incorporated herein by reference, to the extent they provide exemplary procedural or other details supplementary to those set forth herein.

[0295] Akkina et al., J. Virol., 70:2581-2585, 1996.

[0296] Alexander et al., Proc. Nat. Acad. Sci. USA, 85:5092-5096, 1988.

[0297] Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., MA, 1996.

[0298] Biswas et al., Annals NY Acad. Sci., 590:582-583, 1990.

[0299] Biswas, et al., J. Clin. Microbiol., 29: 2228-2233, 1991.

[0300] Blomer et al., J. Virol., 71(9):6641-6649, 1997.

[0301] Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752,1987.

[0302] Chen et al., Nature Methods 8:424-429, 2011.

[0303] Chothia et al., EMBO J. 7:3745, 1988.

[0304] Doulatov et al., Cell Stem Cell. 10:120-36, 2012.

[0305] Ercolani et al., J. Biol. Chem., 263:15335-15341, 1988.

[0306] Evans, et al., In: Cancer Principles and Practice of Oncology, Devita et al. (Eds.), Lippincot-Raven, NY, 1054-1087, 1997.

[0307] Fechheimer et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987.

[0308] Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979.

[0309] Frisan et al., Epstein-Barr Virus Protocols, Part III, 125-127, 2001

[0310] Furi and Furie, Cell 53:505-518,1988.

[0311] Graham and Van Der Eb, Virology, 52:456-467, 1973.

[0312] International Publication No. WO 2007 / 069666

[0313] International Publication No. PCT / US2016 / 057893

[0314] International Publication No. PCT / US2016 / 057899

[0315] International Publication No. WO 94 / 09699

[0316] International Publication No. WO 95 / 06128

[0317] International Publication No. WO 96 / 39487

[0318] Jores et al., PNAS USA 87:9138, 1990.

[0319] Kaeppler et al., Plant Cell Reports 9:415-418, 1990.

[0320] Kaneda et al.,Science,243:375-378,1989.

[0321] Karin et al., Cell, 36:371-379, 1989.

[0322] Kato et al., J. Biol. Chem., 266:3361-3364, 1991.

[0323] Kim et al., J. Virol., 66:3879-3882, 1992.

[0324] Knust et al., EMBO J. 761-766, 1987.

[0325] Ladi et al., Nature Immunology, 7:338–343, 2006.

[0326] Langle-Rouault et al., J. Virol., 72(7):6181–6185, 1998.

[0327] Lefranc et al., Dev. Comp. Immunol. 27:55, 2003.

[0328] Levitskaya et al., Proc. Natl. Acad. Sci. USA, 94(23):12616–12621, 1997.

[0329] Linnemann, C. et al. Nat Med 21, 81–85, 2015.

[0330] Ludwig et al., Nat. Biotechnol., 24(2):185-187, 2006b.

[0331] Ludwig et al., Nat. Methods, 3(8):637-46, 2006a.

[0332] Macejak and Sarnow, Nature, 353:90-94, 1991.

[0333] Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988.

[0334] Mann et al., Cell, 33:153-159, 1983.

[0335] Nabel et al., Science, 244(4910):1342-1344, 1989.

[0336] Naldini et al., Science, 272(5259):263-267, 1996.

[0337] Zufferey et al., Nat. Biotechnol., 15(9):871-875

[0338] Ng,Nuc.Acid Res.,17:601-615,1989.

[0339] Nicola et al., Blood, 54:614-627, 1979.

[0340] Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and their uses, Lab. Press, 2001.

[0341] Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988.

[0342] Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.

[0343] Nicolau et al., Methods Enzymol., 149:157-176, 1987.

[0344] Notta et al., Science, 218-221, 2011.

[0345] Paskind et al., Virology, 67:242-248, 1975.

[0346] Patent publication number EP1507865

[0347] Pelletier and Sonenberg, Nature, 334(6180):320-325, 1988.

[0348] Potrykus et al., Mol. Gen. Genet., 199(2):169-177, 1985.

[0349] Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.

[0350] Quitsche et al., J. Biol. Chem., 264:9539-9545, 1989.

[0351] Remington's Pharmaceutical Sciences 22nd edition, 2012.

[0352] Reubinoff et al., Nat. Biotechnol., 18:399-404, 2000.

[0353] Richards et al., Cell, 37:263-272, 1984.

[0354] Rippe, et al., Mol. Cell Biol., 10: 689-695, 1990.

[0355] Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor 1997.

[0356] Suzuki et al., EMBO J. 6:1891-1897, 1987.

[0357] Takahashi et al., Cell, 131:861-872, 2007.

[0358] Temin, In: Gene Transfer, Kucherlapati (Ed.), NY, Plenum Press, 149-188, 1986.

[0359] Thomson and Marshall, Curr.Top.Dev.Biol.,38:133-165,1998.

[0360] Thomson and Odorico, J. Trends. Biotechnol., 18:53-57, 2000.

[0361] Thomson et al. Proc. Natl. Acad. Sci. USA, 92:7844-7848, 1995.

[0362] Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986.

[0363] U.S. Patent Application No. 08 / 464,599

[0364] U.S. Patent Application No. 12 / 715,136

[0365] U.S. Patent Application No. 61 / 088,054

[0366] U.S. Patent No. 4,683,202

[0367] U.S. Patent No. 5,030,015

[0368] U.S. Patent No. 5,302,523

[0369] U.S. Patent No. 5,322,783

[0370] U.S. Patent No. 5,384,253

[0371] U.S. Patent No. 5,464,765

[0372] U.S. Patent No. 5,486,359

[0373] U.S. Patent No. 5,538,877

[0374] U.S. Patent No. 5,538,880

[0375] U.S. Patent No. 5,550,318

[0376] U.S. Patent No. 5,556,954

[0377] U.S. Patent No. 5,563,055

[0378] U.S. Patent No. 5,580,859

[0379] U.S. Patent No. 5,589,466

[0380] U.S. Patent No. 5,591,616

[0381] U.S. Patent No. 5,610,042

[0382] U.S. Patent No. 5,656,610

[0383] U.S. Patent No. 5,702,932

[0384] U.S. Patent No. 5,736,524

[0385] U.S. Patent No. 5,780,448

[0386] U.S. Patent No. 5,789,215

[0387] U.S. Patent No. 5,843,780

[0388] U.S. Patent No. 5,843,780

[0389] U.S. Patent No. 5,928,906

[0390] U.S. Patent No. 5,945,100

[0391] U.S. Patent No. 5,981,274

[0392] U.S. Patent No. 5,994,136

[0393] U.S. Patent No. 5,994,624

[0394] U.S. Patent No. 6,013,516

[0395] U.S. Patent No. 6,200,806

[0396] U.S. Patent No. 6,225,042

[0397] U.S. Patent No. 6,355,479

[0398] U.S. Patent No. 6,362,001

[0399] U.S. Patent No. 6,544,518

[0400] U.S. Patent No. 6,790,662

[0401] U.S. Patent No. 7,029,913

[0402] U.S. Patent No. 8,058,065

[0403] U.S. Patent No. 8,129,187

[0404] U.S. Patent No. 8,268,620

[0405] U.S. Patent No. 8,268,620

[0406] U.S. Patent No. 8,372,642

[0407] U.S. Patent No. 8,741,648

[0408] U.S. Patent Publication No. 20020055144

[0409] U.S. Patent Publication No. 20050260186

[0410] U.S. Patent Publication No. 20060104968

[0411] U.S. Patent Publication No. 20090148425

[0412] U.S. Patent Publication No. 20090246875

[0413] U.S. Patent Publication No. 2010 / 0210014

[0414] U.S. Patent Publication No. 20120276636

[0415] U.S. Patent Publication No. 2014 / 0315304

[0416] U.S. Patent Publication No. 20160257939

[0417] Wilson et al., Nature Reviews Immunology, 9:91-105, 2009.

[0418] Wilson et al.,Science,244:1344-1346,1989.

[0419] Wong et al., Gene, 10:87-94, 1980.

[0420] Wynn, Nature Immunology, 6:1069–1070, 2005.

[0421] Xu et al., Nat. Biotechnol., 19:971-974, 2001.

[0422] Yamanaka et al., Cell, 131(5):861-72, 2007.

Claims

1. A method for producing antigen-specific effector T cells and / or NK cells, comprising: (a) Engineering induced pluripotent stem cells (iPSCs) to express chimeric antigen receptors (CARs) to generate CAR-iPSCs; (b) Directed differentiation of CAR-iPSCs into CD34 cells by forming embryoid bodies (EBs) in the presence of GSK-3 inhibitor, FGF2, and VEGF + Hematopoietic progenitor cells (HPCs), which trigger mesoderm induction of CAR-iPSCs; (c) contacting the EBs with BMP4, VEGF and FGF2 to induce mesoderm induction; (d) differentiating the EBs in the presence of Flt-3 ligand, IL-3, SCF and TPO to produce CD34 + HPC; (e) further allowing the CD34 + HPC differentiation into T cells and / or NK cells involves culturing CD34 cells on surfaces coated with fibronectin and the Notch ligand DLL-4 in the presence of magnesium ascorbyl phosphate and nicotinamide and the cytokines SCF, FLT-3 ligand, TPO, and IL7 under hypoxic conditions below 20% O2. + HPC; and (f) expanding the T cells and / or NK cells to generate antigen-specific effector T cells and / or NK cells.

2. The method of claim 1, wherein the iPSCs are reprogrammed from T cells.

3. The method of claim 1, wherein the differentiation in step (b) is free of BMP4, and the differentiation in step (d) is free of BMP4, VEGF, and FGF2.

4. The method of claim 1, wherein the GSK-3 inhibitor is CHIR99021.

5. The method of claim 1, wherein the T cell is a regulatory T cell, a naive T cell, a memory T cell, or a γδ T cell.

6. The method of claim 1, wherein the method comprises culturing cells under defined feeder-free conditions.

7. The method of claim 1, wherein step (b) further comprises selecting cells expressing CD34 and CD43 before differentiating into antigen-specific T cells and / or NK cells.

8. The method of claim 7, wherein cells expressing both CD34 and CD43 comprise at least 65% of the total cell population.

9. The method of claim 1, wherein at least 5% of the HPCs express the CAR.

10. The method of claim 1, wherein the culturing in step (e) further comprises a GSK-3 inhibitor, IL-2 and / or IL-12.

11. The method of claim 1, wherein the expanding the T cells in step (f) further comprises culturing the antigen-specific T cells in the presence of anti-CD3 antibodies, IL-2, and IL-15.

12. The method of claim 11, wherein the T cells are expanded to produce CD3 + T cell populations.

13. The method of claim 1, wherein the expanding of step (f) further comprises culturing the antigen-specific T cells in the presence of anti-CD3 antibody, FLT3-ligand, IL-7, IL-2, IL-15 and / or IL-21.

14. The method of claim 1, wherein at least 1.5% of the differentiated CD34 + HPC is CD3 + CAR + T cells.

15. The method of claim 1, wherein at least 5% of the antigen-specific effector T cells exhibit cytotoxic activity against target cells.

16. The method of claim 1, wherein the iPSCs are homozygous for one or more of the locus alleles HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ.

17. The method of claim 1, wherein in step (e), CD34 + HPC, in which RetroNectin is a fragment of fibronectin.

18. The method of claim 1, wherein the T cell is a cytotoxic T cell or a natural killer T cell.

19. The method of claim 1, wherein the T cells are CD4 + T cells or CD8 + T cells.

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