Cancer immunotherapy using engineered cells

Abstract

The present disclosure provides hematopoietic cells in which one or more selected second signal molecules are knocked out such that stimulation of the hematopoietic cells to heterologous CD8 + T cells is significantly reduced.

Description

Technical Field

[0001] The present disclosure relates to immunotherapy for treating cancer using engineered hematopoietic cells (eg, natural killer (NK) cells or T cells). Background Art

[0002] Currently, the vast majority of cell therapy products are autologous CAR-T cells, the preparation of which is time-consuming, labor-intensive, and costly.

[0003] Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system, whose natural function is to kill infected microorganisms and / or cancer cells. NK cell-mediated immunotherapy has been attempted in patients with leukemia and other cancers. Initially, autologous NK cells were used by isolating hematopoietic cells from the patient, amplifying NK cells, and reintroducing NK cells into the patient. There have been descriptions of NK cells containing transgenes to enhance activity and / or reduce immunogenicity, for example, by providing enhanced IL-15 expression, or using CD64 / CD16A fusion protein for CD16 signaling, or expressing low immunomodulatory polypeptides (e.g., comprising one or more members selected from PD-L2, TGF-β, CD46, CD55, and CD59), or engineering to include heterologous transcription factors (e.g., STAT) and reduce the activity of endogenous cytokine receptors (e.g., endogenous IL receptors such as IL-17R), for example, as described in WO2022095902A1 (the contents of which are incorporated herein by reference to the maximum extent permitted by law). Using autologous NK cells rather than allogeneic NK cells avoids the need for immunosuppressive therapy to prevent rejection of the engineered cells. However, perhaps due to their compatibility with the patient's immune system, autologous NK cells are often ineffective against cancer, for example, due to inhibitory interactions between autologous NK cells and self-MHC I molecules.

[0004] For many years, “off-the-shelf” allogeneic NK cell products that can be used for patients have been the goal, but engineering allogeneic NK cells to reduce immunogenicity (hypoimmunity) has proven challenging. Currently, gene editing for hypoimmunity typically involves knocking out MHC-I and MHC-II to escape the targeting and killing of allogeneic NK cells by host T cells. The problem is that knocking out MHC-I may lead to the killing of other NK cells induced by “loss of self”, a phenomenon sometimes called fratricide. In order to overcome this fratricide, NK inhibitory molecules such as HLA-E / G are introduced to inhibit the killing of other allogeneic NK cells. However, due to the heterogeneity of NK cells, it is difficult to inhibit all allogeneic NK cells by expressing these NK cell inhibitory molecules. Therefore, although allogeneic NK cells are protected from the attack of the host immune system due to the lack of MHC-I and MHC-II, they will still decrease rapidly due to the inability to effectively inhibit the killing of fraternal NK cells.

[0005] Allogeneic T cells, such as allogeneic CAR T cells, may also encounter rejection by CD8 T cells. Current approaches to address this issue include blocking or reducing the expression of HLA class I molecules on T cells. For example, the conserved gene β2-microglobulin is deleted to completely eliminate the surface expression of HLA class I. However, although this approach reduces immunogenic recognition by CD8 T cells, the complete absence of HLA class I molecules increases the risk of allogeneic CAR T cells being recognized by NK cells due to a “loss of self” mechanism. CD4 T cells can also promote the rejection of allogeneic T cells by recognizing HLA class II molecules.

[0006] Better methods are needed to engineer hypoimmunogenic allogeneic hematopoietic cells. Summary of the Invention

[0007] We found that by knocking out selected secondary signaling molecules (such as CD58, CD86, or ICAM1) in hematopoietic cells that retain MHC-I molecules (such as NK cells or T cells), these cells can avoid stimulating CD8+ T cells. Therefore, by simply knocking out these secondary signaling molecules, we can make cells hypoimmunogenic.

[0008] Induced pluripotent stem cells (iPSC) can provide cell products with low immunogenicity (including NK cells or T cells), which can be prepared in large quantities, have high uniformity and low cost. Low immunogenic cell products derived from iPSC differentiation can overcome the limitations of autologous CAR-T and can be prepared in large quantities in advance, have high uniformity and low cost. Low immunogenic cell products can resist the killing of immune cells in the patient's body, which can increase the survival rate of the product in the body, thereby making the product more effective. Since low immunogenic cell products do not trigger immune rejection, they can be administered multiple times on demand. Current cell therapy products need to be infused after lymphocyte clearance. Lymphocyte clearance itself has huge toxic side effects on patients and makes patients susceptible to infection. Low immunogenic cell products can reduce or even eliminate these complex steps, making the administration of cell therapy products simpler and more convenient. In short, the low immunogenic cell products described in this article can become off-the-shelf products.

[0009] Therefore, the present disclosure provides induced pluripotent stem cells (iPSCs) and hematopoietic cells derived therefrom (e.g., NK cells or T cells), in which the second signaling molecule is knocked out so that the cells have low immunogenicity, methods for producing such cells, and methods for treating cancer using such cells.

[0010] More specific embodiments are described below in the detailed description and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Flow cytometric analysis of CD86, ICAM1, and CD58 knockout is presented, showing that the CRISPR-Cas9 construct efficiently knocks out protein expression of these genes.

[0012] Figure 2 The killing capacity of NK cells knocked out for CD86, ICAM1 or CD58 is shown, which indicates that the knockout does not affect the ability of NK cells to kill cancer cells (in this case, Raji cells).

[0013] Figure 3 It was shown that knockout of CD58, CD86, and ICAM1 did not affect MHC-I expression in eNK cells.

[0014] Figure 4 It was shown that knockout of CD58, CD86, and ICAM1 exhibited significantly reduced stimulation of CD8+ cells.

[0015] Figure 5 The effects of knocking out various genes and gene combinations on CD8+ stimulation are shown.

[0016] Figure 6The effects of QN-019eNK cells containing CD19-CAR, CD16, and IL15 transgenes with and without knockout of ICAM1, CD86, and CD58 were demonstrated, showing that these knockouts also reduced QN-019eNK stimulation of CD8+ T cells. DETAILED DESCRIPTION

[0017] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Various changes, modifications, and substitutions may be made by those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0018] The terms "reprogramming", "dedifferentiation", "increasing cell potential" or "increasing developmental potential" used interchangeably herein generally refer to methods of increasing cell potential or dedifferentiating cells to a lower differentiated state. For example, cells with increased cell potential have stronger developmental plasticity (i.e., can differentiate into more cell types) compared to the same cells in a non-reprogrammed state. In other words, reprogrammed cells are cells in a lower differentiated state compared to the same cells in a non-reprogrammed state.

[0019] The term "differentiation" generally refers to the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell (e.g., an immune cell). A differentiated or differentiation-induced cell is a cell that has taken on a more specialized ("committed") position within a cell lineage. The term "committed" generally refers to a cell that has progressed to a point in a differentiation pathway where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into a different cell type or revert to a less differentiated cell type.

[0020] The term "pluripotent" generally refers to the ability of a cell to form all lineages of a body or somatic cell (i.e., embryoid body). For example, an embryonic stem cell is a pluripotent stem cell that can form cells from each of the three germinal layers: ectoderm, mesoderm, and endoderm. Pluripotency can be a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (e.g., epiblast stem cells) that cannot produce a complete organism to more primitive, more capable cells (e.g., embryonic stem cells) that can produce a complete organism.

[0021] The term "induced pluripotent stem cell" (iPSC) generally refers to a stem cell derived from a differentiated cell (e.g., a differentiated adult cell, a neonatal cell, or a fetal cell), which has been induced or changed (i.e., reprogrammed) to differentiate into tissues of all three germinal layers, mesoderm, endoderm, and ectoderm, or the dermis. The iPSC produced does not refer to cells found in nature. In some cases, iPSC can be engineered to directly differentiate into definitive cells (e.g., natural killer (NK) cells). In some cases, iPSC can be engineered to first differentiate into tissue-specific stem cells (e.g., hematopoietic stem cells (HSC)), which can further induce differentiation into definitive cells (e.g., NK cells).

[0022] The term "embryonic stem cell" (ESC) generally refers to the naturally occurring pluripotent stem cell of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and produce all derivatives of the three primitive germ layers of ectoderm, endoderm and mesoderm during development. In some cases, ESC can be engineered to directly differentiate into definitive cells (e.g., NK cells). In some cases, ESC can be engineered to first differentiate into tissue-specific stem cells (e.g., HSC), which can further induce differentiation into definitive cells (e.g., NK cells).

[0023] The term "isolated stem cell" generally refers to any type of stem cell disclosed herein (e.g., ESC, HSC, mesenchymal stem cell (MSC), etc.) isolated from a multicellular organism. For example, HSC can be isolated from a mammal (e.g., a human). In another example, embryonic stem cells can be isolated from an embryo.

[0024] The term "isolated" generally refers to a cell or cell population that has been separated from its original environment. For example, the new environment of the isolated cell is substantially free of at least one component found in the environment in which the "unisolated" reference cell is present. An isolated cell can be a cell from which some or all of its components have been removed relative to its state of existence in its natural environment, for example, a cell isolated from a tissue or biopsy sample. The term also includes cells from which at least one, some or all of its components have been removed relative to the state of existence of the cell in its non-naturally occurring environment, for example, a cell isolated from a cell culture or cell suspension. Thus, an isolated cell has been partially or completely separated from at least one component (including other substances, cells, or cell populations) relative to its state of existence in nature or relative to its state of growth, storage, or maintenance in a non-naturally occurring environment.

[0025] The terms "hematopoietic stem / progenitor cell (hematopoietic stem and progenitor cell)", "hematopoietic stem cell", "hematopoietic progenitor cell" or "hematopoietic precursor cell" used interchangeably herein generally refer to cells that are characterized by hematopoietic lineage but can further hematopoietic differentiation (e.g., differentiate into NK cells or T cells), and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells and lymphoid progenitor cells. Hematopoietic stem / cells (HSC) are multipotent stem cells that can produce all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). In some cases, HSC can be a CD34+ hematopoietic cell that can produce mature myeloid and lymphoid cell types (including T cells, NK cells and B cells).

[0026] The term "immune cell" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells may include NK cells, T cells, monocytes, innate lymphocytes, tumor infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0027] The term "NK cell" or "natural killer cell" generally refers to a subset of peripheral blood lymphocytes, which is defined as expressing CD56 or CD16 and not having T cell receptor (CD3). In some cases, NK cells are phenotypically CD3- and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57 or both expressed to the same or varying degrees) and optionally present CD16, but lack the expression of one or more of the following: PLZF, SYK, FceRγ and EAT-2. In some cases, the isolated CD56+ NK cell subset can show expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A and / or DNAM-1.

[0028] The term "gene" generally refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) encoding an RNA transcript and its corresponding nucleotide sequence. For genomic DNA, the term used herein includes a non-coding region and a regulatory region at intervals, and may include 5' and 3' ends. In some applications, the term encompasses transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region may contain an "open reading frame" encoding a polypeptide. In some applications of the term, a "gene" only includes the coding sequence (e.g., "open reading frame" or "coding region") necessary for encoding a polypeptide. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only a transcribed sequence, but also a non-transcribed region, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a natural gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not in their natural location in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences (e.g., non-natural sequences) that contain mutations, insertions, and / or deletions.

[0029] The term "expression" generally refers to one or more processes in which a polynucleotide is transcribed from a DNA template (e.g., transcribed into mRNA or other RNA transcripts) and / or the process in which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If a polynucleotide is derived from genomic DNA, expression can include the splicing of mRNA in a eukaryotic cell. For expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or a polypeptide sequence relative to its expression level under a wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or a polypeptide sequence relative to its expression under a wild-type state. The expression of a transfected gene can occur transiently or stably in a cell. In the "transient expression" process, the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to the transfected cells, the expression of the gene can be lost over time. In contrast, the stable expression of a transfected gene can occur when a gene is co-transfected with another gene that gives a selective advantage to the transfected cell. Such a selective advantage may be resistance to a certain toxin presented to the cell.

[0030] The terms "peptide", "polypeptide" or "protein" used interchangeably herein generally refer to a polymer of at least two amino acid residues connected by a peptide bond. The term does not imply a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis or is naturally occurring. These terms are applicable to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. These terms include amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). These terms also encompass amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operation (e.g., conjugation with a labeling component). As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural and non-natural amino acids that are chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.

[0031] As used herein with respect to polypeptides, the terms "derivative," "variant," or "fragment" generally refer to a polypeptide that is related to a wild-type polypeptide, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide may comprise one or more amino acid changes (e.g., mutations, insertions, and deletions), truncations, modifications, or a combination thereof, compared to the wild-type polypeptide.

[0032] With respect to polypeptide molecules (e.g., proteins), the terms “engineered,” “chimeric,” or “recombinant” as used herein generally refer to polypeptide molecules having heterologous amino acid sequences or altered amino acid sequences due to the application of genetic engineering techniques to nucleic acids encoding polypeptide molecules, and cells or organisms expressing the polypeptide molecules. With respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms “engineered” or “recombinant” as used herein generally refer to polynucleotide molecules having heterologous nucleic acid sequences or altered nucleic acid sequences due to the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be partially modified or altered by gene editing.

[0033] The term "gene editing portion" generally refers to a portion that can edit a nucleic acid sequence (whether exogenous or endogenous to the cell containing the nucleic acid sequence). In some embodiments, the gene editing portion regulates the expression of a gene by editing a nucleic acid sequence. In some cases, the gene editing portion can regulate the expression of a gene by editing a genomic DNA sequence. In some cases, the gene editing portion can regulate the expression of a gene by editing an mRNA template. In some cases, editing a nucleic acid sequence can change the basic template for gene expression.

[0034] Alternatively or additionally, the gene editing portion may be able to regulate the expression or activity of the gene by specifically binding to a target sequence (or a target sequence within a gene) that is operably coupled to the gene and regulating the production of mRNA from DNA (such as chromosomal DNA or cDNA). In some cases, the gene editing portion may recruit or include at least one transcription factor that binds to a specific DNA sequence, thereby controlling the transcription rate of genetic information from DNA to mRNA. The gene editing portion itself can bind to DNA and regulate transcription through physical barriers, such as preventing proteins (such as RNA polymerase and other related proteins) from assembling on the DNA template. The gene editing portion can regulate the expression of the gene at the translation level, for example, by regulating the production of protein from the mRNA template. In some cases, the gene editing portion can regulate gene expression by affecting the stability of the mRNA transcript.

[0035] The term "antibody" generally refers to a protein binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, different classes of immunoglobulins (Ig) (i.e., IgA, IgG, IgM, IgD, and IgE) and different subclasses of immunoglobulins (Ig) (e.g., IgG1, IgG2, etc.). Its derivatives, variants, or fragments may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), miniantibodies, diabodies, and single-domain antibodies ("sdAbs" or "nanoantibodies" or "camelids"). The term antibody includes optimized, engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity-matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in the fragment crystallizable region) and multispecific antibodies (eg, bispecific antibodies).

[0036] The term "chimeric polypeptide receptor" generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each of which is capable of binding to a specific antigen. A chimeric polypeptide receptor can be monospecific (i.e., capable of binding to one type of specific antigen). Alternatively, a chimeric polypeptide receptor can be multispecific (i.e., capable of binding to two or more different types of specific antigens). A chimeric polypeptide receptor can be monovalent (i.e., comprising a single antigen binding moiety). Alternatively, a chimeric polypeptide receptor can be multivalent (i.e., comprising multiple antigen binding moieties). In some cases, a chimeric polypeptide receptor can comprise a T cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR).

[0037] The term "antigen binding domain" generally refers to a construct that exhibits preferential binding to a specific target antigen. An antigen binding domain can be a polypeptide construct, such as an antibody, a functional variant thereof (e.g., a designed ankyrin repeat protein (DARPin)), a modification thereof, a fragment thereof, or a combination thereof. An antigen binding domain can be any antibody disclosed herein or a functional variant thereof. Non-limiting examples of antigen binding domains can include mouse antibodies, human antibodies, humanized antibodies, camel Ig, shark heavy chain antibodies (VNARs), Ig NARs, chimeric antibodies, recombinant antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), miniantibodies, double antibodies, three antibodies, four antibodies, single-domain antigen-binding fragments (sdAb, nanoantibodies), recombinant heavy chain antibodies (VHHs) and other antibody fragments that maintain the binding specificity of intact antibodies.

[0038] The term "safety switch" generally refers to an engineered polypeptide construct designed to prevent the potential toxicity or other adverse effects of cell therapy. When expressed in a cell, the safety switch can induce host cell death, thereby inactivating the activity of cells in the host (e.g., in the subject). Therefore, the safety switch can be a suicide part. In some cases, the cell can be programmed to express the suicide part (e.g., time programming) at a certain stage of its life cycle. In some cases, the expression of the suicide part in the cell can be conditional or inducible. In some instances, the conditional regulation (e.g., expression) of the suicide part can include post-translational activation mediated by small molecules and control of tissue-specific and / or temporal transcriptional regulation. Therefore, the safety switch can be an inducible suicide part. The safety switch can mediate the induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcription and post-transcriptional gene regulation and / or antibody-mediated depletion. In some cases, the safety switch can be activated by an exogenous molecule (e.g., a drug or prodrug), which triggers apoptosis and / or cell death of the cell (e.g., an engineered NK cell as disclosed herein) when activated.

[0039] The term "immunomodulatory polypeptide" generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secreted polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc.) that can regulate or control one or more properties of an immune cell (e.g., NK cell). One or more properties of an immune cell can include the differentiation of the immune cell, the morphology of the immune cell, the expression of a polynucleotide or polypeptide construct within the immune cell, or the activity of the immune cell (e.g., the cytotoxic activity of engineered NK cells against diseased cells (e.g., cancer cells). The immunomodulatory polypeptide can be endogenous to the host cell. Alternatively or additionally, the immunomodulatory polypeptide can be heterologous to the host cell. In some cases, one or more properties of the control immune cell can be mediated by down-regulating the expression of the immunomodulatory polypeptide (e.g., inhibiting, knocking down, or knocking out). Alternatively or additionally, one or more properties of the control immune cell can be mediated by up-regulating the expression of the immunomodulatory polypeptide (e.g., up-regulating an endogenous gene or knocking in a heterologous gene encoding an immunomodulatory polypeptide). Alternatively or additionally, one or more properties of controlling immune cells can be mediated by maintaining expression of an immunomodulatory polypeptide for a period longer than the natural or normal expression profile of the immunomodulatory polypeptide in the host cell. In some cases, the immunomodulatory polypeptide may comprise a low immunomodulatory agent. In some cases, the immunomodulatory polypeptide may comprise an immune checkpoint inhibitor.

[0040] The term "low immunity regulator" generally refers to a polypeptide construct in a cell, wherein the enhanced expression of the low immunity regulator in the cell (for example, by knocking in a heterologous gene) or reduced expression (for example, by knocking out or knocking low an endogenous gene) can help the cell to reduce or avoid the immune response (for example, immune attack, such as adaptive immune rejection) in the host body when it is applied to the host body. In some cases, cells (for example, engineered NK cells as disclosed herein) can be modified to show enhanced expression or reduced expression of low immunity regulators so that the cells can escape host immune attack when the cells are infused into the host (i.e., recipient) for the second time or further. Therefore, these cells (i) will not be rejected by the host's immune system (for example, antibody-mediated complement cytotoxicity or antibody-dependent cellular cytotoxicity (ADCC)) and / or (ii) compared with the control cells without low immunity regulators enhanced expression or reduced expression, the speed of being rejected by the host's immune system is slower. The cells showing low immunity regulators enhanced expression or reduced expression can be referred to as showing "low immunity" or "immune pardon". For example, the enhanced hypoimmunity (e.g., enhanced resistance to ADCC) of the engineered immune cell populations disclosed herein (e.g., engineered NK cell populations) can be determined in vitro (e.g., in the presence of human serum or human complement and antibodies (e.g., SSEA-4 antibodies)) or in vivo (e.g., upon administration to the bloodstream of a subject).

[0041] In some cases, engineering of immune cells (e.g., NK cells) disclosed herein can enhance the resistance of immune cells to immune rejection (e.g., ADCC) by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%.

[0042] In some cases, enhanced resistance to immune rejection (e.g., ADCC) can be determined in vitro in a culture medium comprising at least or at most about 5%, at least or at most about 10%, at least or at most about 15%, at least or at most about 20%, at least or at most about 25%, at least or at most about 30%, at least or at most about 40%, at least or at most about 50%, at least or at most about 60%, at least or at most about 70%, or at least or at most about 80%.

[0043] The term "immune checkpoint inhibitor" generally refers to a group of molecules presented on the cell surface of immune cells (e.g., T cells, myeloid cells, NK cells, B cells, etc.), which can regulate the immune response of cells by lowering or suppressing the immune response (i.e., anticancer or antitumor immune response) of immune cells to target cells (such as cancer cells). Target cells can express receptors or ligands of immune checkpoint inhibitors presented on the surface of immune cells to engage with immune checkpoint inhibitors and lower or suppress the immune response of immune cells to target cells. Therefore, in some cases, lowering or suppressing the expression of immune checkpoint inhibitors in immune cells can enhance or prolong the immune response of immune cells to target cells.

[0044] The term "immune response" generally refers to a T cell-mediated and / or B cell-mediated immune response produced by the host's immune system to a certain object (e.g., a foreign body). Examples of immune responses include T cell responses, such as cytokine production and cytotoxicity. In some cases, the immune response can be indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0045] The terms "enhanced expression," "increased expression," or "up-regulated expression" generally refer to the production level of a portion of interest (e.g., a polynucleotide or polypeptide) that is higher than the normal expression level of the portion of interest in a host strain (e.g., a host cell). The normal expression level can be substantially zero (or absent) or higher than zero. The portion of interest can comprise an endogenous gene or polypeptide construct of the host strain. The portion of interest can comprise a heterologous gene or polypeptide construct introduced into or imported into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of the host strain to enhance expression of the polypeptide of interest in the host strain.

[0046] The terms "enhanced activity," "increased activity," or "up-regulated activity" generally refer to a modification of the activity of a portion of interest (e.g., a polynucleotide or polypeptide) to a level higher than the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level may be substantially zero (or absent) or higher than zero. The portion of interest may comprise a polypeptide construct of the host strain. The portion of interest may comprise a heterologous polypeptide construct introduced into or introduced into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of the host strain to enhance the activity of the polypeptide of interest in the host strain.

[0047] The terms "reduced expression," "lowered expression," or "down-regulated expression" generally refer to a portion of interest (e.g., a polynucleotide or polypeptide) produced at a level below the normal expression level of the portion of interest in a host strain (e.g., a host cell). Normal expression levels are above zero. The portion of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest can be knocked out or knocked down in the host strain. In some instances, reduced expression of the portion of interest can include completely inhibiting such expression in the host strain.

[0048] The terms "reduced activity," "lowered activity," or "down-regulated activity" generally refer to a modification of the activity of a portion of interest (e.g., a polynucleotide or polypeptide) to a level below the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level is greater than zero. The portion of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest can be knocked out or knocked down in the host strain. In some instances, the reduced activity of the portion of interest can include completely inhibiting the activity in the host strain.

[0049] The terms "subject," "individual," or "patient," used interchangeably herein, generally refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, rodents, simians, humans, farm animals, sports animals, and pets. Also encompassed are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0050] The terms "treatment" or "treating" generally refer to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, treatment can comprise administering the systems or cell populations disclosed herein. A therapeutic benefit refers to any treatment-related improvement or effect on the disease or diseases, conditions, or symptoms being treated. For a prophylactic benefit, a composition can be administered to a subject at risk for developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

[0051] The term "effective amount" or "therapeutically effective amount" generally refers to an amount of a composition, for example comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), comprising a system of the present disclosure, sufficient to produce the desired activity when administered to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" generally refers to an amount of a composition sufficient to delay the manifestation of, prevent the progression of, alleviate or reduce at least one symptom of a condition being treated by the methods of the present disclosure.

[0052] The present disclosure describes systems and methods for immunotherapy. Immune cells can be engineered to show an enhanced half-life compared to control cells (e.g., non-engineered immune cells). Immune cells can be engineered to show enhanced proliferation compared to control cells. Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that control cells are insufficient or unable to target. The engineered immune cells disclosed herein can be engineered in vitro, in vitro, and in some cases in vivo. Engineered immune cells prepared in vitro or in vitro can be administered to a subject in need to treat a disease (e.g., myeloma or solid tumor). Engineered immune cells can be autologous to the subject. Alternatively, engineered immune cells can be allogeneic to the subject.

[0053] In some cases, the engineered immune cells disclosed herein (e.g., engineered NK cells) can be derived from isolated stem cells (e.g., isolated ESC). In some cases, the engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSC). In some cases, the engineered immune cells disclosed herein are hematopoietic stem cells or hematopoietic cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, monocytes, such as NK cells or T cells). In some cases, the engineered immune cells are the progeny of any of the above-mentioned cells.

[0054] In some cases, the stem cells disclosed herein (e.g., isolated stem cells, induced stem cells) can be autologous cells or cells derived from autologous cells. Autologous cells can be obtained from subjects suffering from a disease or suspected of having a disease. Alternatively, autologous cells can be obtained from a subject before the subject is found to have a disease. In some cases, autologous cells can be allogeneic cells, such as universal stem cells with reduced immunogenicity and requiring a reduced amount or not requiring immunosuppressive drugs. Autologous cells can be obtained from healthy donors.

[0055] In some cases, engineered immune cells (for example, engineered NK cells) can be autologous cells. Engineered immune cells can be obtained from a subject suffering from a disease or suspected disease. Alternatively, engineered immune cells can be obtained from a subject before it is found that the subject suffers from a disease. In some cases, engineered immune cells can be allogeneic cells, for example, for having reduced immunogenicity and needing to reduce or not needing a general allogeneic immunotherapy of immunosuppressive drugs. Engineered immune cells can be obtained from a healthy donor.

[0056] In some aspects, NK cells can be engineered to show an enhanced half-life compared to control cells (e.g., isogenic non-engineered NK cells). NK cells can be engineered to show enhanced proliferation compared to control cells. NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that control cells are insufficient or cannot target. The engineered NK cells disclosed herein can be engineered in vitro, in vitro, and in some cases in vivo. Engineered NK cells prepared in vitro or in vitro can be administered to a subject in need to treat a disease (e.g., myeloma or solid tumor). Engineered NK cells can be autologous to the subject. Alternatively, engineered NK cells can be allogeneic to the subject.

[0057] In a first embodiment, the present disclosure provides a hematopoietic cell population (cell 1), wherein one or more selected second signaling molecules are knocked out, such that the stimulation of allogeneic CD8+ T cells by the hematopoietic cells is significantly reduced.

[0058] For example, the present disclosure provides:

[0059] 1.1. Cell 1, wherein the cell is derived from an induced pluripotent stem cell (iPSC) in which one or more selected second signaling molecules are knocked out.

[0060] 1.2. Cell 1 or 1.1, wherein the cell is a hematopoietic stem cell.

[0061] 1.3. Cell 1-1.2, wherein the cell is a natural killer (NK) cell.

[0062] 1.4. Cell 1-1.2, wherein the cell is a T cell.

[0063] 1.5. Any of the preceding cell populations, wherein the cells or their progeny persist in the circulation for at least 30 days, such as at least 60 days, after implantation into a recipient.

[0064] 1.6. Any of the aforementioned cell populations, wherein stimulation of allogeneic CD8+ T cells by the hematopoietic cells is significantly reduced by at least 20%, such as at least 30%, such as at least 50%, relative to syngeneic cells in which one or more selected second signaling molecules are not knocked out.

[0065] 1.7. Any of the foregoing cell populations, wherein the cells or their progeny do not exhibit significant levels of cannibalism, such as killing due to "loss of self"-induced killing.

[0066] 1.8. Any of the aforementioned cell populations, which express normal levels of MHC-I.

[0067] 1.9. Any of the aforementioned cell populations, wherein the one or more second signaling molecules are selected from one or more of CD48, CD80, CD86, LAF-1, ICAM1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.

[0068] 1.10. Any of the aforementioned cell populations, wherein the one or more second signaling molecules are selected from at least two of CD48, CD80, CD86, LAF-1, ICAM1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.

[0069] 1.11. Any of the aforementioned cell populations, wherein the one or more second signaling molecules are selected from one or more of CD86, ICAM1, and CD58.

[0070] 1.12. Any of the preceding cell populations, wherein the one or more second signaling molecules comprise two or more of CD86, CD58, and ICAM1.

[0071] 1.13. Any of the preceding cell populations, wherein the one or more second signaling molecules comprise CD86 and CD58.

[0072] 1.14. Any of the preceding cell populations, wherein the one or more second signaling molecules comprise CD58 and ICAM1.

[0073] 1.15. Any of the preceding cell populations, wherein the one or more second signaling molecules comprise CD86 and ICAM1.

[0074] 1.16. Any of the preceding cell populations, wherein the one or more second signaling molecules comprise CD58, CD86, and ICAM1.

[0075] 1.17. Any of the aforementioned cell populations, wherein the one or more second signaling molecules are knocked out by disrupting one or more genes encoding the one or more second signaling molecules.

[0076] 1.18. Any of the aforementioned cell populations, wherein the one or more second signaling molecules are knocked out by targeted disruption of one or more genes encoding the one or more second signaling molecules by CRISPR / Cas 9.

[0077] 1.19. Any of the aforementioned cell populations, wherein the cells further comprise one or more transgenes, such as a transgene expressing a CAR construct and / or a low inflammatory antigen or cytokine, such as a transgene selected from one or more of the CD19-CAR, CD16, and IL15 transgenes.

[0078] 1.20. Any of the aforementioned cell populations, wherein the cells are CAR-T cells or CAR-NK cells.

[0079] 1.21. Any of the aforementioned cell populations, wherein the cells exhibit at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50% reduced stimulation of allogeneic CD8+ T cells relative to a control, for example in an in vitro assay as described in the Examples below.

[0080] 1.22. Any of the aforementioned cell populations for use in a method of treating cancer, the method comprising administering a composition comprising any of the aforementioned cells to a patient in need thereof.

[0081] 1.23. Any of the aforementioned cell populations for use in a method of treating cancer (e.g., blood cancer, lung cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, or breast cancer, such as acute myeloid leukemia, acute lymphoblastic leukemia, Burkitt lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia).

[0082] 1.24. Any of the aforementioned cell populations for use in a method of treating an autoimmune disease or disorder, eg, a method comprising administering a composition comprising any of the aforementioned cells to a patient in need thereof.

[0083] 1.25. Any of the foregoing cell populations for use in a method of treating an autoimmune disease or disorder (e.g., lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn's disease, myasthenia gravis, stiff-person syndrome, Sjögren's syndrome, rheumatoid arthritis, and / or a pulmonary disorder).

[0084] 1.26. Any of the foregoing cell populations for use in a method of treating a disease or condition with multiple on-demand administration.

[0085] 1.27. Any of the aforementioned cell populations for use in a method for treating a blood cancer or solid tumor (e.g., a myeloma or lymphoma), e.g., the method comprising administering a composition comprising iPSCs, differentiated immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes), or differentiated immune cells derived from the iPSCs.

[0086] 1.28. Any of the aforementioned cell populations, for use in a method for treating an autoimmune disease, e.g., the method comprising administering a composition comprising iPSCs, differentiated immune cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, or monocytes), or differentiated immune cells derived from the iPSCs.

[0087] 1.29. Any of the aforementioned cell populations, for use in a method for treating diabetes, eg, the method comprising administering a composition comprising iPSCs, pancreatic islet cells, or pancreatic islet cells derived from the iPSCs.

[0088] 1.30. Any of the aforementioned cell populations for use in regenerative medicine therapy, such as cardiomyocyte transplantation for cardiac injury or failure, pancreatic islet cell transplantation for diabetes, neural progenitor cell transplantation for stroke or central nervous system disorders / injuries, and endothelial cells for treating ischemia or ischemic injury, for example, the method comprising administering a composition comprising iPSCs, NK cells, T cells, B cells, macrophages, monocytes, cardiomyocytes, pancreatic islet cells, neurons, neural progenitor cells, endothelial cells, mesenchymal cells, or the cells derived from the iPSCs.

[0089] 1.31. Any of the aforementioned cell populations for use in the preparation of a medicament for treating cancer or an autoimmune disease or disorder, eg, wherein the treatment comprises administering a composition comprising any of the aforementioned cells to a patient in need thereof.

[0090] 1.32. Progeny of any of the foregoing cell populations.

[0091] 1.33. A pharmaceutical composition comprising an engineered cell according to any of the foregoing cell populations in a pharmaceutically acceptable carrier suitable for injection (e.g., suitable for intravenous infusion), e.g., for use in a method of treating a disease or condition in a human patient, e.g., for use in a method of treating cancer or an autoimmune disease or condition.

[0092] In another embodiment, the present disclosure provides induced pluripotent stem cells (iPSCs) in which one or more selected second signaling molecules are knocked out, for example, such that stimulation of allogeneic CD8+ T cells by the iPSCs or their progeny is significantly reduced, for example, wherein the progeny of the iPSCs comprise a cell population according to any one of Cell 1 and subsequent items.

[0093] In another embodiment, the present disclosure provides a pharmaceutical composition comprising any one of cells 1 and subsequent items in a pharmaceutically acceptable carrier suitable for intravenous infusion (e.g., selected from saline solution, such as 0.9% w / v saline solution and lactated Ringer's solution); for example, wherein the cell population is allogeneic to the patient, and wherein the cells or their progeny persist in the circulation for at least 30 days, such as at least 60 days, after being implanted into the recipient; for example, wherein the cell population comprises NK cells or T cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1 and CD58.

[0094] In another embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a patient in need thereof any one of cell 1 et seq., or a pharmaceutical composition comprising any one of cell 1 et seq., for example, wherein the cells are allogeneic to the patient, and wherein the patient's CD8+ T cells are not significantly stimulated by the administration; for example, wherein the cells or their progeny persist in the circulation for at least 30 days, for example, at least 60 days, after being implanted into the recipient; for example, wherein the cell population comprises NK cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58, for example, CD86 and CD58, or CD86 and ICAM1, or ICAM1 and CD58; or wherein the cell population comprises T cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58, for example, CD86 and CD58, or CD86 and ICAM1, or ICAM1 and CD58.

[0095] In another embodiment, the present disclosure provides a method for preparing hematopoietic cells, wherein one or more selected second signal molecules are knocked out, so that the stimulation of the cells to allogeneic CD8+T cells is significantly reduced, and the method includes culturing a population of induced pluripotent stem cells (iPSCs) under conditions that induce iPSCs to differentiate into hematopoietic cells, wherein the one or more selected second signal molecules are knocked out, for example, wherein the hematopoietic cells are a cell population according to any one of cell 1 and subsequent items; for example, wherein the one or more second signal molecules that are knocked out include two or more of CD86, ICAM1 and CD58; for example, wherein the hematopoietic cells are NK cells, and the conditions for inducing iPSCs to differentiate into hematopoietic cells are conditions that further induce differentiation into NK cells; or wherein the hematopoietic cells are T cells, and the conditions for inducing iPSCs to differentiate into hematopoietic cells are conditions that further induce differentiation into T cells.

[0096] Example

[0097] Example 1: Second signaling molecule knockout cells

[0098] eNK cells were derived from iPSCs and cultured in NK medium, which includes 95% lymphocyte serum-free medium KBM 581 (Corning, catalog #88581CM), 5% human AB serum (Access Bio, catalog #515), 1x non-essential amino acid solution (Gibco, catalog #11140050), 2mM L-glutamine (Gibco, catalog #25030081), and 200IU / ml IL-2 (R&D, catalog #202-GMP). The CD86, ICAM1, and CD58 genes in eNK cells were knocked out using the CRISPR / Cas9 system. CRISPR / Cas9 consists of two components, Cas9 and sgRNA. Cas9 protein was purchased from Thermo Fisher Scientific (catalog #A36499), and sgRNA was synthesized by Genescript Biotech Corp. Cas9 protein and sgRNA were mixed together to form a ribonucleoprotein complex (RNP) and electroporated into eNK cells using P3 Primary Cell 4D-Nucleofection Solution (Lonza Biosciences, Catalog #: V4XP-3032) and 4D-Nucleofection Program CM137 according to the manufacturer's instructions. sgRNA was selected from the following sequences:

[0099]

[0100] Flow cytometric analysis performed 7 days after electroporation showed that these genes were effectively knocked down at the protein level. hCD86-sg3, ICAM1-sg1, and hCD58-sg3 had the highest efficiencies of 85.95%, 88.83%, and 90.32%, respectively. Figure 1 , which showed that CD86, ICAM1, and CD58 could be efficiently knocked out using CRISPR-Cas9 constructs.

[0101] eNK cells with the indicated gene knockdown were mixed with a GFP-labeled cancer cell line (Raji) at a ratio of 0.3:1 and expressed by using The GFP signal was continuously monitored by the live cell analysis system. We observed that the killing ability of these edited eNK cells against Raji cells was comparable to that of wild-type (WT) eNK cells, indicating that gene knockout did not affect the killing ability of eNK cells against tumor cells. Figure 2 , which showed that knocking out CD86, ICAM1, or CD58 did not affect the ability of eNK to kill cancer cells.

[0102] After knocking out the ICAM1 / CD86 / CD58 genes, we tested MHC-I in these eNKs and found that MHC-I expression was unaffected. MHC-I is a key inhibitory ligand for NK cells, and the presence of MHC-I protects eNKs from attack by the patient's allogeneic NK cells. Figure 3 , which showed that CD58 / CD86 / ICAM1 knockout did not affect MHC-I expression in eNK cells.

[0103] T cell proliferation was used to evaluate the stimulation of edited eNK cells on T cells. More specifically, frozen human PBMCs purchased from SAILYBio (Shanghai, China) were thawed, labeled with the cell tracking dye carboxyfluorescein succinimidyl ester (CFSE, catalog #C34554, from Thermo Fisher Scientific), and co-cultured with edited eNKs in NK culture medium containing 20 IU / ml IL2 (instead of 200 IU / ml) for 6 days, with the culture medium replaced every other day. On day 6, the percentage of proliferating CD3+CD8+T cells that became negative for CFSE labeling was measured by flow cytometry. The stimulation of CD8+T cells by NK cells after knockout of the ICAM1 / CD86 / CD58 genes was significantly lower than that by WT and SH sg1 (sgRNA targeting a safe harbor that does not knock out any functional protein) on CD8+T cells, where CD58 knockout was comparable to B2M knockout (MHC-I stimulates CD8+T cells by binding to the TCR on CD8+T cells). B2M is a component of MHC-I. B2M knockout will lead to the loss of MHC-I, thereby preventing the stimulation of CD8+ T cells. Figure 4 , which showed that CD58, CD86, and ICAM1 knockout exhibited significantly reduced stimulation of CD8+ cells.

[0104] Different combinations of ICAM1, CD86, and CD58 knockout were tested. Figure 5 As shown, CD58 / CD86 / ICAM1 triple knockout exhibited significantly lower stimulation of CD8+ T cells, and CD58+CD86 knockout had the lowest stimulation of CD8+ T cells.

[0105] Example 2 - Second Signal Knockout in Cells Expressing a Transgene

[0106] In QN-019 eNK cells (containing CD19-CAR, CD16 and IL15 transgenes), we tested and found that ICAM1 / CD86 / CD58 knockout reduced eNK stimulation of CD8+ T cells, indicating that knockout of these second signaling molecules can indeed reduce the immunogenicity of another eNK cell line. Figure 6 .

Claims

1. A hematopoietic cell population, wherein one or more selected second signaling molecules are knocked out, so that the stimulation of allogeneic CD8+ T cells by the hematopoietic cells is significantly reduced.

2. The cell population of claim 1, wherein the cells are derived from induced pluripotent stem cells (iPSCs). The cell population according to claim 1 , wherein the cells are natural killer (NK) cells. The cell population according to claim 1 , wherein the cells are T cells.

5. A cell population according to any preceding claim, wherein the cells or their progeny persist in the circulation for at least 30 days, such as at least 60 days, after implantation into a recipient.

6. A cell population according to any preceding claim, wherein the cells express normal levels of MHC-I.

7. The cell population according to any one of the preceding claims, wherein the one or more second signaling molecules are selected from one or more of CD48, CD80, CD86, LAF-1, ICAM 1, VLA4, VCAM 1, CD2, CD58, B7, CD155 and CD122.

8. The cell population according to any one of the preceding claims, wherein the one or more second signaling molecules are selected from at least two of CD48, CD80, CD86, LAF-1, ICAM 1, VLA4, VCAM 1, CD2, CD58, B7, CD155 and CD122.

9. The cell population according to any preceding claim, wherein the one or more second signaling molecules are selected from one or more of CD86, ICAM1 and CD58.

10. The cell population according to any preceding claim, wherein the one or more second signaling molecules comprise two or more of CD86, ICAM1 and CD58.

11. The cell population according to any preceding claim, wherein the one or more second signaling molecules comprise CD86 and CD58.

12. The cell population according to any preceding claim, wherein the one or more second signaling molecules comprise CD86 and ICAM1.

13. The cell population according to any preceding claim, wherein the one or more second signaling molecules comprise CD58 and ICAM1.

14. The cell population according to any preceding claim, wherein the one or more second signaling molecules comprise CD86, CD58 and ICAM1.

15. The cell population according to any preceding claim, wherein the one or more second signaling molecules are knocked out by any means including disrupting one or more genes encoding the one or more second signaling molecules.

16. The cell population according to any preceding claim, wherein the one or more second signaling molecules are knocked out by targeted disruption of one or more genes encoding the one or more second signaling molecules by CRISPR / Cas 9.

17. A cell population according to any of the preceding claims, wherein the cells further comprise one or more transgenes, such as a transgene expressing a CAR construct and / or a low inflammatory antigen or cytokine, such as a transgene selected from one or more of CD19-CAR, CD16 and IL15 transgenes.

18. The cell population according to any preceding claim, wherein the cells are induced pluripotent stem cells (iPSCs) or stem cells.

19. The cell population according to any preceding claim, wherein the cells are immune cells, cardiomyocytes, pancreatic islet cells, neural cells, hematopoietic cells, such as hematopoietic stem cells, natural killer (NK) cells, T cells, macrophages or monocytes; for example, wherein the cells are derived from the iPSC or stem cells of claim 18.

20. A cell population according to any preceding claim for use in a method of treating cancer, an autoimmune disease or for use in a regenerative medicine treatment comprising administering a composition comprising any of the preceding cells to a patient in need thereof.

21. A pharmaceutical composition comprising a cell population according to any preceding claim in a pharmaceutically acceptable carrier suitable for intravenous infusion.

22. The pharmaceutical composition according to claim 21, wherein the pharmaceutically acceptable carrier suitable for intravenous infusion is selected from saline solution, such as 0.9% saline solution and lactated Ringer's solution.

23. The pharmaceutical composition of claim 21 or 22, wherein the cell population is allogeneic to the patient, and wherein the cells or their progeny persist in the circulation for at least 30 days, such as at least 60 days, after being implanted into the recipient.

24. The pharmaceutical composition according to any one of claims 21-23, wherein the cell population comprises NK cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58.

25. The pharmaceutical composition according to any one of claims 21-24, wherein the cell population comprises NK cells, wherein the one or more second signaling molecules that are knocked out comprise CD86 and CD58, or CD86, ICAM1, and CD58.

26. A pharmaceutical composition according to any one of claims 21-25, wherein the cell population comprises T cells, and the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1 and CD58; for example, CD86 and CD58, or CD86 and ICAM1, or ICAM1 and CD58.

27. A method for treating a disease or condition, the method comprising administering to a patient in need thereof a population of hematopoietic cells, for example, according to claims 1-20, wherein one or more selected second signaling molecules are knocked out such that the cells do not stimulate allogeneic CD8+ T cells, or a pharmaceutical composition according to claims 21-26; for example, wherein the administration comprises multiple on-demand administrations; for example, wherein the disease or condition comprises cancer, an autoimmune disease or condition, diabetes, heart damage or failure, stroke or central nervous system condition, ischemia or ischemic injury, for example, myeloma, lymphoma, solid tumor, blood cancer.

28. The method of claim 27, wherein the cell population is allogeneic to the patient, and wherein the cells or their progeny persist in the circulation for at least 30 days, such as at least 60 days, after being implanted into the recipient.

29. The method of claim 27 or 28, wherein the cell population comprises NK cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58.

30. The method of any one of claims 27-29, wherein the cell population comprises NK cells, wherein the one or more second signaling molecules that are knocked out comprise CD86 and CD58, or CD86, ICAM1, and CD58.

31. The method of claim 27 or 28, wherein the cell population comprises T cells, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58, wherein the one or more second signaling molecules that are knocked out comprise CD86 and CD58, or CD86, ICAM1, and CD58.

32. A method for preparing hematopoietic cells, wherein one or more selected second signal molecules are knocked out, so that the stimulation of the cells to allogeneic CD8+T cells is significantly reduced, the method comprising culturing a population of induced pluripotent stem cells (iPSCs) under conditions that induce iPSCs to differentiate into hematopoietic cells, wherein the one or more selected second signal molecules are knocked out.

33. The method of claim 32, wherein the hematopoietic cells are a cell population according to any one of claims 1-18.

34. The method of claim 32 or 33, wherein the one or more second signaling molecules that are knocked out comprise two or more of CD86, ICAM1, and CD58. 35 . The method according to any one of claims 32 to 34 , wherein the hematopoietic cells are NK cells, and the conditions for inducing iPSCs to differentiate into hematopoietic cells are conditions that further induce differentiation into NK cells.

36. The method according to any one of claims 32 to 34, wherein the hematopoietic cells are T cells, and the conditions for inducing iPSCs to differentiate into hematopoietic cells are conditions that further induce differentiation into T cells.

Images