WO2024199407A1

WO2024199407A1 - Cancer immunotherapies using engineered cells

Info

Publication number: WO2024199407A1
Application number: PCT/CN2024/084663
Authority: WO
Inventors: Jiabiao HU; Xiaomeng BIAN; Lina ZHOU; Yixuan ZHOU; Luhan Yang
Original assignee: Qihan Hong Kong Ltd
Current assignee: Qihan Hong Kong Ltd
Priority date: 2023-03-31
Filing date: 2024-03-29
Publication date: 2024-10-03
Anticipated expiration: 2025-09-30
Also published as: CN120826463A

Abstract

The disclosure provides hematopoietic cells wherein one or more selected second signaling molecule (s) are knocked out, such that stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced.

Description

CANCER IMMUNOTHERAPIES USING ENGINEERED CELLS

FIELD

This disclosure relates to immunotherapies to treat cancer using engineered hematopoietic cells, e.g., natural killer (NK) cells or T-cells.

BACKGROUND

At present, the vast majority of cell therapy products are autologous CAR-T cells. The preparation process of autologous CAR-T cells is time-consuming and labor-intensive with high cost.

Natural killer (NK) cells are cytotoxic lymphocytes of innate immune system whose natural function is to kill microbial infected and/or cancerous cells. NK cell-mediated immunotherapy has been tried in patients with leukemia and other cancers. Initially, autologous NK cells were used, by isolating hematopoietic cells from the patient, expanding the NK cells, and reintroducing the NK cells back to the patient. NK cells comprising transgenes to enhance activity and/or reduce immunogenicity have been described, e.g., to provide enhanced IL-15 expression, or CD16 signaling using CD64/CD16A fusion protein, or to express a hypo-immunity regulator polypeptide, e.g., comprising one or more members selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, or engineered to comprise a heterologous transcription factor (e.g., STAT) coupled with reduced activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) , e.g., as described in WO2022095902A1, the contents of which are incorporated herein by reference, to the fullest extent permitted by law. Using autologous NK cells, rather than allogeneic NK cells, avoids the need for immunosuppressive therapies to prevent rejection of the engineered cells. But, perhaps due to their compatibility with the patient’s immune system, the autologous NK cells are also often ineffective against the cancers, e.g., due to inhibitory interactions between the autologous NK cells and self-MHC I molecules.

An allogeneic NK cell product that could be used “off-the-shelf” for patients has been a goal for many years, but engineering allogeneic NK cells for reduced immunogenicity (hypoimmunity) has proved challenging. Currently, gene editing for hypoimmunity typically involves knocking out MHC-I and MHC-II to escape targeting and killing of the allogeneic NK cells by host T cells. The problem is that knocking out the MHC-I may lead to “missing self” -induced killing by the other NK cells, a phenomenon sometimes referred to as fratricide. To overcome this fratricide, NK inhibitory molecules such as HLA-E/G are introduced to inhibit the killing by the other allogeneic NK cells. Due to the heterogeneity of NK cells, however, it is difficult to suppress all allogeneic NK cells by expressing these NK cell suppressor molecules. So while the allogeneic NK cells are protected from the host’s immune system by the absence of MHC-I and MHC-II, they will nevertheless rapidly dwindle due to ineffective suppression of the fraternal NK cells.

Allogenic T-cells, for example allogenic CAR T cells, may also encounter CD8 T cell rejection. Current approaches to address this problem include blocking or reducing expression of HLA class I molecules on the T cells. For example, deletion of the conserved gene beta 2-microglobulin completely removes surface expression of HLA class. However, while immunogenic recognition by CD8 T cells will be reduced by this approach, the complete loss of HLA class I molecules will increase the risk of recognition of the allogeneic CAR T cells by NK cells, due to the ‘missing self’ mechanism. CD4 T cells can also contribute to rejection of allogeneic T cells through recognition of HLA class II molecules.

Better approaches to engineering hypoimmune allogeneic hematopoietic cells are needed.

SUMMARY

We have discovered that hematopoietic cells, e.g. NK cells or T cells, in which the MHC-I molecules is preserved, but by knocking out selected second signaling molecules, e.g., one or more of CD58, CD86, or ICAM1; these cells can avoid stimulation of CD8+ T cells. So, by knocking out only these second signaling molecule, we can make the cells hypoimmunogenic.

Induced pluripotent stem cells (iPSC) can provide hypo-immunogenic cell products, including NK cells or T-cells, which can be prepared in large quantities, with high homogeneity and low cost. The hypo-immunogenic cell products derived from iPSC differentiation can overcome the limitations of autologous CAR-T and can be prepared in large quantities in advance, with high homogeneity and low cost. Hypo-immunogenic cell products can resist the killing of immune cells in the patient's body, which can improve the survival of the product in the body, so as to make the efficacy of the product better. Since the hypo-immunogenic cell products do not provoke an immune rejection, multiple on-demand administrations can be performed. Current cell therapy products need to be infused after lymphodepletion. The lymphodepletion itself has great toxic side effects on the patient and makes the patient susceptible to infection. Hypo-immunogenic cell products can reduce or even eliminate these complex procedures, making the administration of cell therapy products easier and more convenient. In summary, the hypo-immunogenic cell products as described herein can become an off-the-shelf product.

The disclosure thus provides induced pluripotent stem cells (iPSC) and hematopoietic cells, e.g. NK cells or T cells, derived therefrom, wherein a second signaling molecule is knocked out, rendering the cells hypo-immunogenic, as well as methods of producing such cells, and methods of treating cancer using such cells.

Further more specific embodiments are set forth in the detailed description below, and in the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts flow cytometry analysis of CD86, ICAM1, and CD58 knock-outs, showing that the CRISPR-Cas9 constructs effectively knock-out protein expression of these genes.

Figure 2 shows the killing capacity of the NK cells having CD86, ICAM1, or CD58 knocked out, demonstrating that the knockouts do not affect the ability of the NK cells to kill cancer cells (Raji cells in this case) .

Figure 3 shows that the CD58, CD86, and ICAM1 knockouts do not affect MHC-1 expression in the eNK cells.

Figure 4 shows that the CD58, CD86, and ICAM1 knockouts exhibit significantly lowered stimulation of CD8+ cells.

Figure 5 shows the effect of knocking out various genes and combinations of genes on CD8+ stimulation.

Figure 6 depicts the effect of QN-019 eNK cells, containing CD19-CAR, CD16 and IL15 transgenes, with and without ICAM1, CD86, and CD58 knockouts, showing that these knockouts also reduce the stimulation by QN-019 eNK of CD8+ T cells,

DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

The term “reprogramming, ” “dedifferentiation, ” “increasing cell potency, ” or “increasing developmental potency, ” as used interchangeable herein, generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.

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

The term “pluripotent” generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) . For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .

The term “induced pluripotent stem cells” (iPSCs) generally refers to stem cells that are derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature. In some cases, iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells) . In some cases, iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .

The term “embryonic stem cell” (ESCs) generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In some cases, ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) . In some cases, ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .

The term “isolated stem cells” generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism. For example, HSCs can be isolated from a mammal’s body, such as a human body. In another example, an embryonic stem cells can be isolated from an embryo.

The term “isolated” generally refers to a cell or a population of cells, which has been separated from its original environment. For example, a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist. An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample. The term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated from a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.

The term “hematopoietic stem and progenitor cells, ” “hematopoietic stem cells, ” “hematopoietic progenitor cells, ” or “hematopoietic precursor cells, ” as used interchangeably herein, generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells or T cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the 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, HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.

The term “immune cell” generally refers to a differentiated hematopoietic cell. Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.

The term “NK cell” or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) . In some cases, NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some cases, isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.

The term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′and 3′ends. In some uses, the term encompasses the transcribed sequences, including 5′and 3′untranslated regions (5′-UTR and 3′-UTR) , exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .

The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product. ” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated, ” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

The term “peptide, ” “polypeptide, ” or “protein, ” as used interchangeably herein, generally refers to a polymer of at least two amino acid residues joined by peptide bond (s) . This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) . The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids, ” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

The term “derivative, ” “variant, ” or “fragment, ” as used herein with reference to a polypeptide, generally refers to a polypeptide related to a wild type polypeptide, for example either 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 can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.

The term “engineered, ” “chimeric, ” or “recombinant, ” as used herein with respect to a polypeptide molecule (e.g., a protein) , generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule. The term “engineered” or “recombinant, ” as used herein with respect to a polynucleotide molecule (e.g., a DNA or RNA molecule) , generally refers to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

The term “gene editing moiety” generally refers to a moiety which can edit a nucleic acid sequence, whether exogenous or endogenous to a cell comprising the nucleic acid sequence. In some embodiments, a gene editing moiety regulates expression of a gene by editing a nucleic acid sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing genomic DNA sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.

Alternatively or in addition to, a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operatively coupled to the gene (or a target sequence within the gene) , and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. In some cases, a gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. A gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template. A gene editing moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template. In some cases, a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript.

The term “antibody” generally refers to a proteinaceous 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, immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc. ) . A derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab′, F (ab′) 2, variable fragment (Fv) , single chain variable fragment (scFv) , minibodies, diabodies, and single-domain antibodies ( “sdAb” or “nanobodies” or “camelids” ) . The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies) .

The term “chimeric polypeptide receptor” generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each antigen binding moiety 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 multi-specific (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 a plurality of 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) .

The term “antigen binding domain” generally refers to a construct exhibiting 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) ) , modification thereof, fragment thereof, or a combination thereof. The antigen binding domain can be any antibody as disclosed herein, or a functional variant thereof. Non-limiting examples of an antigen binding domain can include a murine antibody, a human antibody, a humanized antibody, a camel Ig, a shark heavy-chain-only antibody (VNAR) , Ig NAR, a chimeric antibody, a recombinant antibody, or antibody fragment thereof. Non-limiting examples of antibody fragment include Fab, Fab′, F (ab) ′2, F (ab) ′3, Fv, single chain antigen binding fragment (scFv) , (scFv) 2, disulfide stabilized Fv (dsFv) , minibody, diabody, triabody, tetrabody, single-domain antigen binding fragments (sdAb, Nanobody) , recombinant heavy-chain-only antibody (VHH) , and other antibody fragments that maintain the binding specificity of the whole antibody.

The term “safety switch” generally refers to an engineered polypeptide construct designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. When expressed in a cell, the safety switch can induce death of the host cell, thereby inactivating activity of the cell in a host (e.g., in a subject’s body) . Thus, the safety switch can be a suicide moiety. In some cases, the cell can be programmed to express the suicide moiety at certain stage of its life-cycle (e.g., time-programmed) . In some cases, expression of the suicide moiety in a cell can be conditional or inducible. In some examples, conditional regulation (e.g., expression) of a suicide moiety can include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation. Thus, the safety switch can be an inducible suicide moiety. A safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion. In some cases, a safety switch can be activated by an exogenous molecule (e.g., a drug or a prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., engineered NK cell as disclosed herein) .

The term “immune regulator polypeptide” generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secretory polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc. ) capable of regulating or controlling one or more attributes of an immune cell, such as a NK cell. One or more attributes of an immune cell can comprise differentiation of the immune cell, immune cell morphology, expression of a polynucleotide or polypeptide construct within the immune cell, or activity of the immune cell (e.g., cytotoxic activity of an engineered NK cell against a diseased cell, such as a cancer cell) . An immune regulator polypeptide can be endogenous to a host cell. Alternatively or in addition to, an immune regulator polypeptide can be heterologous to a host cell. In some cases, controlling the one or more attributes of the immune cell can be mediated by downregulating expression of the immune regulator polypeptide (e.g., suppression, knock-down or knock-out) . Alternatively or in addition to, controlling the one or more attributes of the immune cell can be mediated by upregulating expression of the immune regulator polypeptide (e.g., upregulation of an endogenous gene or knock-in of a heterologous gene encoding the immune regulator polypeptide) . Yet in another alternative or additionally, controlling the one or more attributes of the immune cell can be mediated by maintaining expression of the immune regulator polypeptide for time period that is longer than a natural or normal expression profile of the immune regulator polypeptide in a host cell. In some cases, an immune regulator polypeptide can comprise a hypo-immunity regulator. In some case, an immune regulator polypeptide can comprise an immune checkpoint inhibitor.

The term “hypo-immunity regulator” generally refers to a polypeptide construct in a cell, wherein either enhanced expression (e.g., via knock-in of a heterologous gene) or reduced expression (e.g., via knock-out or knock-down of an endogenous gene) of the hypo-immunity regulator in the cell can help the cell to reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration to the host’s body. In some cases, cells (e.g., engineered NK cells as disclosed herein) can be modified to exhibit either enhanced expression or reduced expression of the hypo-immunity regulator, such that the cells can evade the host immune attack upon second or further infusion of the cells into the host (i.e., recipient) . As such, the cells (i) would not be rejected by the host’s immune system (e.g., antibody-mediated complement cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC) ) and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator. A cell exhibiting the enhanced expression or reduced expression of the hypo-immunity regulator can be referred to as exhibiting “hypo-immunity” or being “immune-privileged. ” For example, enhanced hypo-immunity (e.g., enhanced resistance against ADCC) of a population of engineered immune cells (e.g., a population of engineered NK cells) as disclosed herein can be ascertained in vitro (e.g., in the presence of human serum or human complement and an antibody (e.g., SSEA-4 antibody) ) or in vivo (e.g., upon administration to a subject’s bloodstream) .

In some cases, engineering of an immune cell (e.g., NK cells) as disclosed herein can enhance the immune cell’s resistance against 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%.

In some cases, the enhanced resistance against immune rejection (e.g., ADCC) can be ascertained in vitro in a medium comprising 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%, or at least or up to about 80%.

The term “immune checkpoint inhibitor” generally refers to a group of molecules presented on a cell surface of an immune cell (e.g., T cells, myeloid cells, NK cells, B cells, etc. ) that can modulate immune response of the cell by down-regulating or inhibiting the immune response of the immune cell against a target cell, such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) . The target cell can express a receptor or a ligand of the immune checkpoint inhibitor presented on the surface of the immune cell, to engage with the immune checkpoint inhibitor and down-regulate or inhibit the immune response of the immune cells against the target cell. As such, down-regulating or inhibiting expression of the immune checkpoint inhibitor in the immune cell can, in some cases, enhance or prolong the immune response of the immune cell against a target cell.

The term “immune response” generally refers to T cell mediated and/or B cell mediated immune responses from a host’s immune system to an object (e.g., a foreign object) . An example of an immune response includes T cell responses, e.g., cytokine production and cellular cytotoxicity. In some cases, an immune response can be indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, such as macrophages.

The term “enhanced expression, ” “increased expression, ” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.

The term “enhanced activity, ” “increased activity, ” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

The term “reduced expression, ” “decreased expression, ” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.

The term “reduced activity, ” “decreased activity, ” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.

The term “subject, ” “individual, ” or “patient, ” as used interchangeably herein, generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

The term “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

The present disclosure describes systems and methods for immunotherapies. Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell) . Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell. Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can be derived from an isolated stem cell (e.g., isolated ESCs) . In some cases, engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSCs) . In some cases, engineered immune cells disclosed herein are hematopoietic stem cells or hematopoietic cells (e.g., NK cells, T cells, B cells, NKT cells, macrophages, monocytes, e.g., NK cells or T cells) . In some cases, the engineered immune cells are the progeny of any of the above cells.

In some cases, the stem cell disclosed herein (e.g., isolated stem cell, induced stem cell) can be an autologous cell or derived from the autologous cell. The autologous cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the autologous cell can be obtained from the subject before the subject is found to have the condition. In some cases, the autologous cell can be an allogeneic cell, e.g., a universal stem cell with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The autologous cell can be obtained from a healthy donor.

In some cases, the engineered immune cell (e.g., engineered NK cell) can be an autologous cell. The engineered immune cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the engineered immune cell can be obtained from the subject before the subject is found to have the condition. In some cases, the engineered immune cell can be an allogeneic cell, e.g., for a universal allogenic immunotherapy with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The engineered immune cell can be obtained from a healthy donor.

In some aspects, NK cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., an isogenic non-engineered NK cell) . NK cells can be engineered to exhibit enhanced proliferation as compared to a control cell. NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered NK cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.

In a first embodiment, the disclosure provides a population of hematopoietic cells (Cell 1) wherein one or more selected second signaling molecule (s) are knocked out, such that stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced.

For example, the disclosure provides:

1.1. Cell 1 wherein the cells are derived from induced pluripotent stem cells (iPSC) wherein one or more selected second signaling molecule (s) are knocked out.

1.2. Cell 1 or 1.1 wherein the cells are hematapoietic stem cells.

1.3. Cell 1 –1.2 wherein the cells are natural killer (NK) cells.

1.4. Cell 1 –1.2 wherein the cells are T cells.

1.5. Any foregoing cell population wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days.

1.6. Any foregoing cell population wherein stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced at least 20%, e.g., at least 30%, e.g. at least 50%, reduced relative to isogenic cells without the one or more selected second signaling molecule (s) are knocked out.

1.7. Any foregoing cell population wherein the cells or their progeny do not exhibit significant levels of fratricide, e.g., due to “missing self” -induced killing.

1.8. Any foregoing cell population which expresses normal levels of MHC-I.

1.9. Any foregoing cell population, wherein the one or more second signaling molecule (s) are selected from one or more of CD48, CD80, CD86, LAF-1, ICAM1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.

1.10. Any foregoing cell population, wherein the one or more second signaling molecule (s) are selected from at least two of CD48, CD80, CD86, LAF-1, ICAM1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.

1.11. Any foregoing cell population, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58.

1.12. Any foregoing cell population, wherein the one or more second signaling molecule (s) comprise two or more of CD86, CD58 and ICAM1.

1.13. Any foregoing cell population, wherein the one or more second signaling molecule (s) comprise CD86 and CD58.

1.14. Any foregoing cell population wherein the one or more second signaling molecule (s) comprise CD58, and ICAM1.

1.15. Any foregoing cell population wherein the one or more second signaling molecule (s) comprise CD86, and ICAM1.

1.16. Any foregoing cell population wherein the one or more second signaling molecule (s) comprise CD58, CD86, and ICAM1.

1.17. Any foregoing cell population wherein the one or more second signaling molecule (s) are knocked out by means of disruption of the gene or genes encoding the one or more second signaling molecule (s) .

1.18. Any foregoing cell population wherein the one or more second signaling molecule (s) are knocked out by means of CRISPR/Cas 9 targeted disruption of the gene or genes encoding the one or more second signaling molecule (s) .

1.19. Any foregoing cell population wherein the cells further comprise one or more transgenes, e.g., transgenes expressing CAR constructs and/or hypoinflammatory antigens or cytokines, e.g., transgenes selected from one or more of CD19-CAR, CD16 and IL15 transgenes.

1.20. Any foregoing cell population wherein the cells are CAR-T cells or CAR-NK cells.

1.21. Any foregoing cell population wherein the cells exhibit reduced stimulation of heterologous CD8+ T cells of at least 10%, preferably at least 20%, at least 30%, at least 40%, more preferably at least 50%reduction relative to a control, e.g., in an in vitro assay as described in the Examples below.

1.22. Any foregoing cell population for use in a method of treating cancer, comprising administering a composition comprising any foregoing cell to a patient in need thereof.

1.23. Any foregoing cell population for use in a method of treating cancer, e.g., blood cancer, lung cancer, colorectal cancer, pancreatic cancer, renal cell cancer, or breast cancer, e.g., acute myeloid leukemia, acute lymphoblastic leukemia, Burkitt lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia.

1.24. Any foregoing cell population for use in a method of treating an auto-immune disease or condition, e.g., comprising administering a composition comprising any foregoing cells to a patient in need thereof.

1.25. Any foregoing cell population for use in a method of treating an auto-immune disease or condition, e.g., lupus, systemic lupus erythematosus, multiple sclerosis, systemic sclerosis, vasculitis, Crohn’s disease, Myasthenia Gravis, Stiff-Person syndrome, Sjorgen’s syndrome, rheumatoid arthritis, and/or a pulmonary condition.

1.26. Any foregoing cell population for use in a method of treating a disease or condition with multiple on-demand administrations.

1.27. Any foregoing cell population for use in a method of treating blood cancer or a solid tumor, e.g., myeloma or lymphoma, e.g., 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 said iPSCs.

1.28. Any foregoing cell population for use in a method of treating an auto-immune disease, e.g., 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 said iPSCs.

1.29. Any foregoing cell population for use in a method of treating diabetes, e.g., comprising administering a composition comprising iPSCs, islet cells, or islet cells derived from said iPSCs.

1.30. Any foregoing cell population for use in regenerative medicine treatments, e.g., cardiomyocyte transplantation for heart injury or failure, islet cell transplantation for diabetes, neural progenitor cell transplantation for stroke or central nervous system disorders/injury, endothelial cells for treatment of ischemia or ischemic damage, e.g., comprising administering a composition comprising iPSCs, NK cells, T cells, B cells, macrophages, monocytes, cardiomyocytes, islet cells, neural cells, neural progenitor cells, endothelial cells, mesenchymal cells, or are said cells derived from said iPSCs.

1.31. Any foregoing cell population for use in the manufacture of a medicament for use in treating cancer or an auto-immune disease or condition, e.g., comprising administering a composition comprising any foregoing cell to a patient in need thereof.

1.32. The progeny of any foregoing cell population.

1.33. A pharmaceutical composition comprising engineered cells 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 auto-immune disease or condition.

In a further embodiment, the disclosure provides induced pluripotent stem cells (iPSC) wherein one or more selected second signaling molecule (s) are knocked out, e.g. such that stimulation of heterologous CD8+ T cells by the IPSCs or their progeny is substantially reduced, e.g., wherein the progeny of the iPSCs comprise a cell population according to any of Cell 1, et seq.

In a further embodiment, the disclosure provides a pharmaceutical composition comprising any of Cell 1, et seq., in a pharmaceutically acceptable carrier suitable for intravenous infusion, e.g., selected from saline solution, e.g., 0.9%w/v saline solution, and lactated Ringer’s solution; e.g., wherein the cell population is allogeneic with respect to the patient and wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days; e.g., wherein the population comprises NK cells or T cells wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58.

In a further embodiment, the disclosure provides a method of treating cancer, comprising administering any of Cell 1, et seq., or a pharmaceutical composition comprising any of Cell 1, et seq., to a patient in need thereof, e.g. wherein the cells are allogeneic with respect to the patient and wherein the patient’s CD8+ T cells are not significantly stimulated by the administration; e.g., wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days; e.g., wherein the population comprises NK cells wherein the one or more second signaling molecule (s) which 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 population comprises T cells wherein the one or more second signaling molecule (s) which 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.

In a further embodiment, the disclosure provides a method of making hematopoietic cells wherein one or more selected second signaling molecule (s) are knocked out, such that stimulation of heterologous CD8+ T cells by the cell is substantially reduced, comprising culturing a population of induced pluripotent stem cells (iPSC) , wherein one or more selected second signaling molecule (s) are knocked out, under conditions which induce differentiation of the iPSCs into hematopoietic cells, e.g., wherein the hematopoietic cells are a population according to any of Cell 1, et seq.; e.g., wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58; e.g., wherein the hematopoietic cells are NK cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into NK cells; or wherein the hematopoietic cells are T cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into T cells.

EXAMPLES

Example 1: Second Signaling Molecule Knockout Cells

eNK cells are derived from iPSCs and cultured in NK medium including 95%Lymphocyte Serum-Free Medium KBM 581 (Corning, Cat# 88581CM) , 5%human AB serum (Access Bio, Cat#515) , 1x Non-Essential Amino Acids Solution (Gibco, Cat# 11140050) , 2mM L-Glutamine (Gibco, Cat# 25030081) and 200 IU/ml IL2 (R&D, Cat#202-GMP) . The CD86, ICAM1 and CD58 genes in eNK cells are knocked out by a CRISPR/Cas9 system. CRISPR/Cas9 includes two components, Cas9 and sgRNA. Cas9 protein is purchased from Thermo Fisher Scientific (Cat# A36499) and sgRNA is synthesized from Genescript Biotech Corp. Cas9 protein and sgRNA are mixed together to form ribonucleoprotein complex (RNP) and electroporated into eNK cells using P3 Primary Cell 4D-Nucleofector solution (Lonza Biosciences, Catalog #: V4XP-3032) along with the 4D-Nucleofector program CM137, according the manufacturer’s instructions. The sgRNA is selected from the following sequences:

Flow cytometry analysis performed 7 days after electroporation shows that these genes are effectively knocked out at the protein level. hCD86-sg3, ICAM1-sg1 and hCD58-sg3 have the highest efficiency, with 85.95%, 88.83%and 90.32%, respectively. See Figure 1, showing that CD86, ICAM1, and CD58 are effectively knocked using the CRISPR-Cas9 constructs.

The eNK cells with indicated gene knockout are mixed with a cancer cell line (Raji) labeled with GFP at a ratio of 0.3: 1, and the GFP signal is monitored continuously by using IncucyteLive-Cell Analysis System. We see that the killing ability of these edited eNKs against Raji cells is comparable to that of wild type (WT) eNK, indicating that gene knockout would not affect the killing capacity of eNK cells against the tumor cells. See Figure 2, showing that knocking out CD86, ICAM1, or CD58 does not affect the killing capacity of the eNK against the cancer cells.

After knocking out the ICAM1/CD86/CD58 genes, MHC-I is tested in these eNKs, and we find that the expression of MHC-I is not affected. MHC-I is a key inhibitory ligand for NK cells, and the presence of MHC-I protects eNK from allogeneic NK cells in patients. See Figure 3, showing that the CD58/CD86/ICAM1 knockouts did not affect MHC-I expression in the eNK cells.

T cell proliferation is used to evaluate the stimulation of T cells by the edited eNK cells. More specifically, frozen human PBMC, purchased from SAILY Bio (Shanghai, China) , is thawed, labeled with Cell Trace Dye Carboxyfluorescein succinimidyl ester (CFSE, Cat# C34554 from Thermo Fisher Scientific) and mixed with edited eNK in NK medium with 20IU/ml IL2 instead of 200IU/ml for 6 days, medium is refreshed every other day. At day 6, the percentage of proliferative CD3+ CD8+ T cells that become negative for CFSE labeling is measured by flow cytometry. The stimulation of CD8+ T cells by NK after knocking out the ICAM1/CD86/CD58 gene is significantly lower than that of WT and SH sg1 (sgRNA targeting to safe harbor, which does not knock out any functional proteins) , where CD58 knockout is comparable to B2M knockout (MHC-I stimulates CD8+ T cells by binding to TCR on CD8+ T cells) . B2M is a component of MHC-I, and B2M knockout causes loss of MHC-I, thereby avoiding stimulation of CD8+ T cells. See Figure 4, showing that the CD58, CD86, and ICAM 1 knockouts exhibit significantly lowered stimulation of CD8+ cells.

Different combinations of ICAM1, CD86, and CD58 knockouts are tested. As seen in Figure 5, the CD58/CD86/ICAM1 triple knockout exhibited significantly lower stimulation of CD8+ T cells and CD58+CD86 knockout had the lowest stimulation of the CD8+T cells.

Example 2 –Second signal knockout in cells expressing transgenes

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

Claims

A population of hematopoietic cells wherein one or more selected second signaling molecule (s) are knocked out, such that stimulation of heterologous CD8+ T cells by the hematopoietic cells is substantially reduced.
The population according to claim 1 wherein the cells are derived from induced pluripotent stem cells (iPSC) .
The population according to claim 1 or claim 2 wherein the cells are natural killer (NK) cells.
The population according to claim 1 or claim 2 wherein the cells are T cells.
The population according to any foregoing claim wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days.
The population according to any foregoing claim wherein the cells express normal levels of MHC-I.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) are selected from one or more of CD48, CD80, CD86, LAF-1, ICAM 1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) are selected from at least two of CD48, CD80, CD86, LAF-1, ICAM 1, VLA4, VCAM 1, CD2, CD58, B7, CD155, and CD122.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) are selected from one or more of CD86, ICAM1 and CD58.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) comprise two or more of CD86, ICAM1 and CD58.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) comprise CD86 and CD58.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) comprise CD86 and ICAM1.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) comprise CD58 and ICAM1.
The population according to any foregoing claim wherein the one or more second signaling molecule (s) comprise CD86, CD58, and ICAM1.
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) are knocked out by any means of disruption of the gene or genes encoding the one or more second signaling molecule (s) .
The population according to any foregoing claim, wherein the one or more second signaling molecule (s) are knocked out by means of CRISPR/Cas 9 targeted disruption of the gene or genes encoding the one or more second signaling molecule (s) .
The population according to any foregoing claim, wherein the cells further comprise one or more transgenes, e.g., transgenes expressing CAR constructs and/or hypoinflammatory antigens or cytokines, e.g., transgenes selected from one or more of CD19-CAR, CD16 and IL15 transgenes.
The population according to any foregoing claim, wherein the cells are induced pluripotent stem cells (iPSC) or stem cells.
The population according to any foregoing claim, wherein the cells are immune cells, cardiomyocyte, islet cell, neural cells, hematopoietic cells, e.g., hematopoietic stem cells, natural killer (NK) cells, T-cells, Macrophage, or Monocyte; e.g., wherein the said cells are derived from the iPSCs or stem cells of claim 18.
The population according to any foregoing claim, for use in a method of treating cancer, auto-immune disease or use in regenerative medicine treatments, comprising administering a composition comprising any foregoing cell to a patient in need thereof.
A pharmaceutical composition comprising a population according to any foregoing claim in a pharmaceutically acceptable carrier suitable for intravenous infusion.
The pharmaceutical composition according to claim 21 wherein the pharmaceutically acceptable carrier suitable for intravenous infusion is selected from saline solution, e.g., 0.9%saline solution, and lactated Ringer’s solution.
The pharmaceutical composition according to claim 21 or 22 wherein the population is allogeneic with respect to the patient and wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days.
The pharmaceutical composition according to any of claim 21-23 wherein the population comprises NK cells wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58.
The pharmaceutical composition according to any of claim 21-24 wherein the population comprises NK cells wherein the one or more second signaling molecule (s) which are knocked out comprise CD86 and CD58, or CD86, ICAM1 and CD58.
The pharmaceutical composition according to any of claims 21-25 wherein the population comprises T cells wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58; e.g., CD86 and CD58, or CD86 and ICAM1, or ICAM1 and CD58.
A method of treating a disease or condition, comprising administering a population of hematopoietic cells wherein one or more selected second signaling molecule (s) are knocked out, such that the cell does not stimulate heterologous CD8+ T cells, e.g., according to claims 1-20, or a pharmaceutical composition according to claim 21 –26, to a patient in need thereof; e.g., wherein the administering comprises multiple, on-demand administrations; e.g., wherein the disease or condition comprises cancer, an auto-immune disease or condition, diabetes, heart injury or failure, a stroke or central nervous system disorder, ischemia or ischemic damage, e.g., Myeloma, lymphoma, solid tumors, blood cancer.
The method according to claim 27 wherein the population is allogeneic with respect to the patient and wherein the cells or their progeny, when engrafted into a recipient, remain in circulation for at least 30 days, e.g., for at least 60 days.
The method according to claim 27 or 28 wherein the population comprises NK cells wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58.
The method according to any of claims 27–29 wherein the population comprises NK cells wherein the one or more second signaling molecule (s) which are knocked out comprise CD86 and CD58, or CD86, ICAM1 and CD58.
The method according to claim 27 or 28 wherein the population comprises T cells wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58, wherein the one or more second signaling molecule (s) which are knocked out comprise CD86 and CD58, or CD86, ICAM1 and CD58.
A method of making hematopoietic cells wherein one or more selected second signaling molecule (s) are knocked out, such that stimulation of heterologous CD8+ T cells by the cell is substantially reduced, comprising culturing a population of induced pluripotent stem cells (iPSC) , wherein one or more selected second signaling molecule (s) are knocked out, under conditions which induce differentiation of the iPSCs into hematopoietic cells.
The method of claim 32 wherein the hematopoietic cells are a population according to any of claims 1-18.
The method of claims 32 or 33 wherein the one or more second signaling molecule (s) which are knocked out comprise two or more of CD86, ICAM1 and CD58.
The method of any of claim 32 –34 wherein the hematopoietic cells are NK cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into NK cells.
The method of any of claim 32 –34 wherein the hematopoietic cells are T cells and the conditions which induce differentiation of the iPSCs into hematopoietic cells are conditions which further induce differentiation into T cells.