TW202300644A - Chimeric antigen receptors in immune cells - Google Patents

Abstract

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.

Description

Chimeric antigen receptors in immune cells

Cancer (eg, neoplasm, tumor) is the leading cause of death worldwide, accounting for approximately 10 million deaths per year. Cancer continues to place an increasing health, economic and emotional burden on individuals, families, communities and nations. Increased knowledge of cancer biology (eg, cancer immunobiology in particular) and genetic engineering has facilitated the development of adoptive cell therapies (eg, cellular immunotherapy) with the goal of treating or controlling many different cancers.

The present disclosure provides methods and systems for treating cancer. Aspects of the disclosure provide engineered immune cells, such as engineered natural killer (NK) cells, and methods for their use in treating cancer, such as hematological malignancies or solid tumors.

In one aspect, the present disclosure provides engineered NK cells comprising: a chimeric polypeptide receptor comprising an antigen-binding portion capable of binding to an antigen, wherein the antigen-binding portion Includes an ankyrin repeat domain.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises an immune cell antigen of an immune cell that is not an NK cell. In some embodiments, the immune cells are T cells. In some embodiments, the antigen is selected from CD3, CD4, CD8, CCR7, CD45RO, CR45RA, and fragments thereof. In some embodiments, the antigen is CD3. In some embodiments, the antigen is CD4. In some embodiments, the antigen is CD8. In some embodiments, the antigen is CCR7. In some embodiments, the antigen is CD45RO. In some embodiments, the antigen is CR45RA.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises NY-ESO-1 or a fragment thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises CD33 or a fragment thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises CD123 or a fragment thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises CD4 or a fragment thereof. In some embodiments, the antigen binding portion comprises at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to the polypeptide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 amino acid sequence.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises HER2 or a fragment thereof. In some embodiments, the antigen binding portion comprises an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the polypeptide sequence of SEQ ID NO:5.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises EGFR or a fragment thereof. In some embodiments of any one of the engineered NK cells disclosed herein, the antigen binding portion comprises a polypeptide with a member selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 The sequences are amino acid sequences that are at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least 80%, at least 90%, at least 95%, at least 98%, at least 99% of the polypeptide sequence of SEQ ID NO: 10 Amino acid sequences with % or 100% identity.

In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least 80%, at least 90%, at least 95%, at least 98%, at least 99% of the polypeptide sequence of SEQ ID NO: 11 % or 100% identity to the leader sequence.

In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a linker between the antigen-binding portion and the transmembrane domain, wherein the linker has at least 80° with the polypeptide sequence of SEQ ID NO: 12 %, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity.

In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least 80%, at least 90%, at least 95%, at least 98%, at least 99% of the polypeptide sequence of SEQ ID NO: 13 % or 100% identity transmembrane domain.

In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least 80%, at least 90%, at least 95%, At least one signaling domain that is at least 98%, at least 99%, or 100% identical. In some embodiments of any one of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises at least 80%, at least 90%, at least 95%, at least 98%, at least 99% of the polypeptide sequence of SEQ ID NO: 14 A first signaling domain of % or 100% identity and a first signaling domain having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity with the polypeptide sequence of SEQ ID NO: 15 Two signaling domains.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises a cytokine or a fragment thereof. In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises a cytokine receptor or fragment thereof. In some embodiments of any of the engineered NK cells disclosed herein, the cytokine is an interleukin. In some embodiments, the interleukin is selected from IL2, IL3, IL7, IL12, IL15, IL18, and IL21. In some embodiments, the interleukin is IL2. In some embodiments, the interleukin is IL3. In some embodiments, the interleukin is IL7. In some embodiments, the interleukin is IL12. In some embodiments, the interleukin is IL15. In some embodiments, the interleukin is IL18. In some embodiments, the interleukin is IL21.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises an intracellular antigenic peptide. In some embodiments, the intracellular antigenic peptide is presented by the major histocompatibility complex.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen binding portion is part of the extracellular domain of a chimeric polypeptide receptor.

In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises multiple different antigen binding moieties capable of binding to multiple different antigens.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell further comprises an additional chimeric polypeptide receptor comprising an additional antigen binding moiety capable of binding to an additional antigen , wherein said additional antigen is different from said antigen.

In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor or one or more chimeric polypeptide receptors comprises a chimeric antigen receptor.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells are derived from hematopoietic cells obtained from a peripheral blood sample of a subject.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells are derived from NK cells obtained from a peripheral blood sample of a subject.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells are derived from isolated stem cells. In some embodiments, the isolated stem cells are embryonic stem cells. In some embodiments, the isolated stem cells are induced pluripotent stem cells.

In one aspect, the present disclosure provides compositions comprising any of the engineered NK cells disclosed herein.

In some embodiments, the composition further includes a single therapeutic agent. In some embodiments, separate therapeutic agents include different antigen binding moieties. In some embodiments, the sole therapeutic agent includes a chemotherapeutic agent.

In one aspect, the present disclosure provides methods comprising administering any one of the compositions disclosed herein to a subject in need thereof.

Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be realized, the disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and descriptions are to be regarded as illustrative and not restrictive. [incorporated by reference]

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material.

[cross reference]

This application claims the benefit of International Application No. PCT/CN2021/077674, filed February 24, 2021, which is hereby incorporated by reference in its entirety.

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

As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor" includes a plurality of chimeric transmembrane receptors.

The terms "about" or "approximately" generally mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., measuring System limitations. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5 times the value, more preferably within 2 times the value. Where specific values are described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range for the specific value.

The use of an alternative (eg, "or") should be understood to mean either, both, or any combination of the alternatives. The term "and/or" should be understood to mean one or both of the alternatives.

The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., from plant crops, fruits, vegetables, grains, Soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, squash, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodium, hornworts, liverworts , moss cells), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh et al), seaweed (e.g. kelp), fungal cells (e.g. yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g. Drosophila, cnidaria, echinoderms, nematodes, etc.), cells from vertebrates Cells from animals (e.g., fish, amphibians, reptiles, birds, mammals), from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, human etc.) cells, etc. Sometimes cells are not derived from natural organisms (for example, cells can be made synthetically, sometimes called artificial cells).

As used interchangeably herein, the terms "reprogramming", "dedifferentiation", "increasing cell potential" or "increasing developmental potential" generally refer to methods of increasing cell potential or dedifferentiating cells to a less differentiated state. For example, a cell with increased cellular potential has more developmental plasticity (ie, can differentiate into more cell types) than the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell at a lower state of differentiation than the same cell in a non-reprogrammed state.

The term "differentiation" generally refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (eg, immune cells). A differentiated or differentiation-induced cell is a cell that occupies a more specialized ("committed") position in a cell lineage. The term "committed" generally refers to a cell that has progressed to a point in the differentiation pathway where it would normally continue to differentiate into a particular cell type or subclass of cell type and would not normally be able to differentiate to 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 a subject or somatic cell (ie, an embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers (ectoderm, mesoderm and endoderm). Pluripotency can be a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (such as ectodermal stem cells), which cannot give rise to complete organisms, to more primitive, more potent cells, which are able to give rise to Whole organisms (such as embryonic stem cells)).

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

The term "embryonic stem cell" (ESC) generally refers to the naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent, producing all derivatives of the three main germ layers (ectoderm, endoderm, and mesoderm) during development. In some cases, ESCs can be engineered to differentiate directly into committed cells (eg, NK cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (eg, HSCs), which can then be further induced to differentiate into committed cells (eg, NK cells).

The term "isolated stem cell" generally refers to any type of stem cell disclosed herein (eg, ESC, HSC, mesenchymal stem cell (MSC), etc.) isolated from a multicellular organism. For example, HSCs can be isolated from the body of a mammal (eg, a human). In another example, embryonic stem cells can be isolated from embryos.

The term "isolated" generally refers to a cell or population of cells 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 exists. An isolated cell can be a cell that has been removed from some or all components found in its natural environment, such as a cell isolated from a tissue or biopsy sample. The term also includes cells that have had at least one, some or all components removed from cells found in non-naturally occurring environments, such as cells isolated from cell culture or cell suspensions. Thus, an isolated cell is partly or completely separated from at least one component (including other substances, cells or populations of cells) found in nature or grown, stored or subsisted in a non-naturally occurring environment.

As used interchangeably herein, the terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g. differentiate into NK cells), and include multipotent hematopoietic stem cells (hemoblasts), myeloid progenitors, megakaryoline progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes , megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). In some instances, HSCs may be CD34+ hematopoietic cells capable of giving rise to mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells.

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.

The term "NK cells" or "natural killer cells" 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 instances, the NK cell, which is phenotypically CD3- and CD56+, expresses at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both to the same or different extent), and optionally expresses CD16, But lack expression of one or more of: PLZF, SYK, FceRγ and EAT-2. In some instances, the isolated subpopulation 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.

As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. A nucleotide may be the monomeric unit of a nucleic acid sequence such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may include ribonucleoside triphosphates (adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP)), and deoxyribonucleoside triphosphate , such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may denote dideoxyribonucleoside triphosphate (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well known techniques. Labeling can also be done with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Fluorescent labels for nucleotides can include, but are not limited to, luciferin, 5-carboxyluciferin (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyluciferin ( JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'Dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-Aminoethyl)aminonaphthalene- 1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides may include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [ JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP; FluoroLink Deoxynucleotide, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP from Amersham, Arlington Heights, Ill.; available from Boehringer Mannheim, Indianapolis , Luciferin-15-dATP, Luciferin-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Luciferin-12-ddUTP, Luciferin-12 from Ind. -UTP and luciferin-15-2'-dATP; and chromosomally labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY available from Molecular Probes, Eugene, Oreg. -TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Luciferin -12-UTP, Luciferin-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine Rhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and Texas Red-12-dUTP. Nucleotides can also be labeled or labeled by chemical modification. The chemically modified mononucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin -14-dCTP) and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

The terms "polynucleotide", "oligonucleotide" or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleosides Acids, or analogs thereof, whether in single-, double-, or multiple-chain form. A polynucleotide can be exogenous or endogenous to the cell. Polynucleotides can be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide may include one or more analogs (eg, altered backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, can be imparted before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, diol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7 - deaza-GTP, fluorophores (such as rhodamine or luciferin linked to sugars), thiol-containing nucleotides, nucleotides linked to biotin, fluorescent base analogs, CpG islands, formazan -7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braidin and wyoside. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), translocation RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, Vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

The term "gene" generally refers to a nucleic acid encoding an RNA transcript (eg, DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence. As used herein with reference to genomic DNA, the term includes intervening non-coding regions as well as regulatory regions, and may include both 5' and 3' ends. In some uses, 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 will include an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequence necessary to encode a polypeptide (eg, an "open reading frame" or "coding region"). In some instances, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some instances, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene" or a non-native gene. A non-native gene may refer to a gene that is not normally present 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 that is 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 (eg, a non-native sequence) that includes mutations, insertions, and/or deletions.

The term "expression" generally refers to the process or processes by which a polynucleotide is transcribed (e.g., into mRNA or other RNA transcript) from a DNA template and/or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein process. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. With respect to expression, "up-regulation" 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 the wild-type state, while "down-regulation" generally refers to Reduced expression levels of a polynucleotide (eg, RNA such as mRNA) and/or polypeptide sequence relative to its expression in the wild-type state. Expression of the transfected gene can occur transiently or stably in the cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Since its expression is restricted to transfected cells, gene expression 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 selective advantage on the transfected cells. Such a selective advantage may be resistance to a certain toxin presented to the cell.

As used interchangeably herein, the terms "peptide", "polypeptide" or "protein" generally refer to a polymer of at least two amino acid residues linked by peptide bond(s). The term does not denote a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide was produced using recombinant techniques, chemical or enzymatic synthesis, or occurs naturally. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, polymers may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (eg, domains). The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" generally refers to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

As used herein with respect to polypeptides, the term "derivative", "variant" or "fragment" generally refers to, for example, by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) ) and/or a polypeptide functionally related to the wild-type polypeptide. Derivatives, variants, and fragments of a polypeptide may include one or more amino acid changes (eg, mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

As used herein with respect to polypeptide molecules (e.g., proteins), the terms "engineered", "chimeric" or "recombinant" generally refer to genetic engineering techniques applied to a nucleic acid encoding a polypeptide molecule and to expressing the polypeptide molecule. A polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence in a cell or organism. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the term "engineered" or "recombinant" generally refers to a polynucleotide having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques Acid molecule. 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 (eg, genomic DNA sequences) can be modified or altered in part by gene editing.

The term "gene editing portion" generally refers to a portion that can edit a nucleic acid sequence, whether exogenous or endogenous to the cell comprising the nucleic acid sequence. In some embodiments, the gene editing moiety regulates the expression of a gene by editing a nucleic acid sequence. In some cases, gene editing moieties can regulate gene expression by editing genomic DNA sequences. In some cases, gene editing moieties can regulate gene expression by editing mRNA templates. In some cases, editing a nucleic acid sequence can alter the underlying template for gene expression.

Alternatively or additionally, the gene editing moiety may be capable of modulating the production of DNA (e.g., chromosomal DNA or cDNA) by specifically binding to a target sequence operably coupled to the gene (or a target sequence within the gene). mRNA to regulate gene expression or activity. In some cases, the gene editing moiety can recruit or include 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. The gene-editing moiety itself can bind to the DNA and regulate transcription through physical barriers, such as preventing the assembly of proteins such as RNA polymerase and other related proteins on the DNA template. Gene editing moieties can regulate gene expression at the translational level, for example, by modulating protein production from mRNA templates. In some cases, gene editing moieties can regulate gene expression by affecting the stability of mRNA transcripts.

The term "antibody" generally refers to a protein binding molecule with immunoglobulin-like function. The term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, etc.). Derivatives, variants or fragments thereof may refer to functional derivatives or fragments that retain (eg, complete and/or partial) binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibody, diabody, and single domain antibody ("sdAb"). " or "Nanobodies" or "Camelid antibodies"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).

The term "chimeric polypeptide receptor" generally refers to a non-native polypeptide receptor that includes one or more antigen-binding moieties, each antigen-binding moiety capable of binding a specific antigen. Chimeric polypeptide receptors may be monospecific (ie, capable of binding one type of specific antigen). Alternatively, chimeric polypeptide receptors may be multispecific (ie, capable of binding two or more different types of specific antigens). Chimeric polypeptide receptors can be monovalent (ie, include a single antigen-binding portion). Alternatively, chimeric polypeptide receptors can be multivalent (ie, include 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).

The term "antigen binding domain" generally refers to a construct that exhibits preferential binding to a particular target antigen. An antigen binding domain may be a polypeptide construct such as an antibody, a functional variant thereof (eg, a designed ankyrin repeat protein (DARPin)), a modification thereof, a fragment thereof, or a combination thereof. The antigen binding domain can be any antibody disclosed herein or a functional variant thereof. Non-limiting examples of antigen binding domains can include murine antibodies, human antibodies, humanized antibodies, camelid Ig, shark heavy-chain-only antibody (VNAR), Ig NAR, 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 bond stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAbs, nanobodies), recombinant heavy chain-only antibodies (VHH) and whole antibody binding specificity maintained other antibody fragments.

The term "designed ankyrin repeat protein" or "DARPin" generally refers to a synthetic polypeptide comprising one or more ankyrin repeat domains, wherein the one or more ankyrin repeat domains are capable of binding one or more antigens. An ankyrin repeat domain described herein typically includes at least one ankyrin repeat motif. In some examples, the ankyrin repeat motif consists of two antiparallel α-helices followed by a loop containing a β-protrusion and β-hairpin that connects it to the next repeat, each repeat having about 33 residues. A recombinant protein comprising a designed ankyrin repeat motif or its binding domain may be referred to as a DARPin protein or a DARPin polypeptide.

In some cases, an ankyrin repeat domain described herein can include (i) a core backbone that provides structure and (ii) target binding residues that bind a target (eg, a target antigen). The structural core may include conserved amino acid residues, and the target binding surface may include amino acid residues that vary according to the target. In another example, the ankyrin repeat motif can comprise the sequence: DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx (SEQ ID NO: 1), where "x" represents any amino acid. In another example, the ankyrin repeat motif can comprise the sequence: DxxGxTPLHLAxxxGxxxIVxVLLxxGADVNAx (SEQ ID NO: 2), wherein "x" represents any amino acid.

In some cases, multiple ankyrin repeat domains can be linked (by covalent bonding or non-covalent association) to form bispecific or multispecific molecules (e.g., bispecific or multispecific chimeric polypeptides subject to body).

Examples and details of DARPin constructs are provided, for example, in International Patent Application No. PCT/EP2001/010454, which is incorporated herein by reference in its entirety.

The term "safety switch" generally refers to an engineered polypeptide construct designed to prevent potential toxicity or other adverse effects of a cell therapy. When expressed in a cell, a safety switch can induce host cell death, thereby inactivating the activity of cells in the host (eg, within an object). Therefore, the safety switch can be the suicide part. In some cases, a cell can be programmed to express a suicide moiety at a certain stage of its life cycle (eg, time-programmed). In some cases, expression of the suicide moiety in the cell can be conditional or inducible. In some examples, conditional regulation (eg, expression) of a suicide moiety can include control by small molecule-mediated post-translational initiation and tissue-specific and/or temporal transcriptional regulation. Thus, the safety switch could be part of the inducible suicide. Safety switches 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 initiated by an exogenous molecule (e.g., a drug or prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., an engineered NK cell disclosed herein) .

The term "immunomodulatory polypeptide" generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secreted polypeptide, cleavable polypeptide, non-cleavable polypeptide) capable of modulating or controlling one or more properties of immune cells (such as NK cells). peptides, etc.). One or more properties of an immune cell can include differentiation of the immune cell, morphology of the immune cell, expression of polynucleotide or polypeptide constructs within the immune cell, or activity of the immune cell (e.g., engineered NK cells against diseased cells (e.g., cancer cells) ) cytotoxic activity). The immunomodulatory polypeptide may be endogenous to the host cell. Alternatively or additionally, the immunomodulatory polypeptide may be heterologous to the host cell. In some instances, control of one or more properties of immune cells can be mediated by down-regulating expression (eg, inhibition, knockdown, or knockout) of an immunomodulatory polypeptide. Alternatively or additionally, control of one or more properties of immune cells can be mediated by upregulation of expression of an immunomodulatory polypeptide (eg, upregulation of an endogenous gene or knock-in of a heterologous gene encoding an immunomodulatory polypeptide). In another alternative or in addition, control of one or more properties of an immune cell may be mediated by maintaining expression of an immunomodulatory polypeptide for a period of time longer than the native or normal expression profile of the immunomodulatory polypeptide in the host cell. In some instances, an immunomodulatory polypeptide can include a hypoimmune modulator. In some instances, an immune modulating polypeptide can include an immune checkpoint inhibitor.

The term "modulator of hypoimmunity" generally refers to a polypeptide construct in a cell wherein there is either enhanced expression (e.g., by knock-in of a heterologous gene) or decreased expression (e.g., by Knockout or knockdown of an endogenous gene) can help the cell reduce or avoid an immune response (eg, an immune attack, such as adaptive immune rejection) from the host's body after administration to the host's body. In some cases, cells (e.g., engineered NK cells disclosed herein) can be modified to exhibit enhanced expression or reduced expression of a modulator of immunity, so that the cells can be used after a second or further infusion of the cells into the host (i.e., receptors) can evade host immune attack. Thus, the cell (i) will not be rejected by the host's immune system and/or (ii) will be more aggressively rejected by the host's immune system compared to a control cell without enhanced expression or reduced expression of the immunomodulator. Slow rate rejection. Cells exhibiting low enhanced expression or reduced expression of a modulator of immunity can be said to exhibit "hypoimmunity" or "immune privilege."

The term "immune checkpoint inhibitor" generally refers to a group of molecules present on the cell surface of immune cells (eg, T cells, myeloid cells, NK cells, B cells, etc.) Targeting the immune response of cells such as cancer cells (ie, an anti-cancer or anti-tumor immune response) modulates the cellular immune response. Target cells may express receptors or ligands for immune checkpoint inhibitors present on the surface of immune cells to engage the immune checkpoint inhibitor and downregulate or suppress the immune cell's immune response to the target cell. Thus, in some cases, down-regulating or inhibiting the expression of immune checkpoint inhibitors in immune cells can enhance or prolong the immune response of immune cells to target cells.

The term "immune response" generally refers to a T-cell-mediated and/or B-cell-mediated immune response from a host's immune system to an object (eg, a foreign body). Examples of immune responses include T cell responses such as cytokine production and cellular cytotoxicity. In some cases, the immune response can be achieved indirectly through T cell priming, such as antibody production (humoral response) and cytokine responsive cells such as macrophages.

The term "enhanced expression", "increased expression" or "upregulated expression" generally refers to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) to a level higher than that of the moiety of interest in a host strain (e.g., a host cell). level of normal expression. The normal expression level can be substantially zero (or null) or higher than zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. A portion of interest may include a heterologous gene or polypeptide construct introduced into the host strain 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 a host strain for enhanced expression of the polypeptide of interest in the host strain.

The term "enhanced activity", "increased activity" or "up-regulated activity" generally refers to the modification of the activity of the moiety of interest (e.g., polynucleotide or polypeptide) to a level higher than that sensed in the host strain (e.g., host cell). The level of the normal activity level of the part of interest. A normal activity level may be substantially zero (or null) or above zero. Portions of interest may include polypeptide constructs of the host strain. A portion of interest may include a heterologous polypeptide construct introduced into a host strain or introduced into a host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked in (KI) into the genome of a host strain for enhanced activity of the polypeptide of interest in the host strain.

The terms "reduced expression", "decreased expression" or "down-regulated expression" generally refer to production of a moiety of interest (e.g., a polynucleotide or polypeptide) to a level lower than that of the moiety of interest in a host strain (e.g., a host cell). level of normal expression. Normal expression levels are above zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. In some cases, a 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 complete suppression of such expression in the host strain.

The term "reduced activity", "reduced activity" or "down-regulated activity" generally refers to the modification of the activity of the moiety of interest (e.g., polynucleotide or polypeptide) to be lower than that in the host strain (e.g., host cell). The level of the normal activity level of the part of interest. Normal activity levels are above zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. In some cases, a moiety of interest can be knocked out or knocked down in the host strain. In some examples, reduced activity of a moiety of interest can include complete inhibition of such activity in a host strain.

As used interchangeably herein, the term "item", "individual" or "patient" generally refers to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

The terms "therapy" or "treatment" generally refer to means of obtaining a beneficial or desired result, including but not limited to therapeutic and/or prophylactic benefits. For example, treatment can include administration of a system or population of cells disclosed herein. A therapeutic benefit refers to any therapeutically relevant improvement or effect on one or more diseases, conditions or symptoms being treated. For prophylactic benefit, compositions may be administered to subjects at risk of developing a particular disease, condition or symptom, or to subjects reporting one or more physiological symptoms of a disease, even though the disease, condition or symptom may not have manifested.

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

T cells are part of the adaptive immune system and can be primed to recognize specific threats by recognizing immune proteins (ie, antigens) on the surface of foreign cells. In contrast, NK cells are part of the innate immune response and can respond to various objects considered "non-self". Generally, unlike T cells, NK cells can attack their target cells without sensitization (ie, antigen-specific sensitization) to eliminate foreign substances.

Unmodified NK cells derived from a subject (eg, HSCs derived from a subject) can be cultured and expanded ex vivo, and then administered to a subject as a therapy to attack its target cells (eg, cancer cells). However, NK cell-based therapies may be limited due to their short ex vivo or in vivo half-life and/or poor proliferation. In addition, unmodified NK cells may not effectively target more difficult-to-treat cancers, such as myeloma or solid tumors. Furthermore, the ex vivo production of NK cells based on blood-derived stem cells (eg, HSCs) can generate a limited supply of NK cells for effective adoptive immunotherapy.

Therefore, there remains a large unmet need for NK cells that are sourced and engineered to exhibit, for example, enhanced proliferation, half-life and cytotoxic activity against target cells. B. Engineered Immune Cells

The present disclosure describes systems and methods for immunotherapy. Immune cells can be engineered to exhibit increased half-life compared to control cells (eg, non-engineered immune cells). Immune cells can be engineered to exhibit enhanced proliferation compared to control cells. Immune cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered immune cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. Engineered immune cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). 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 (eg, engineered NK cells) disclosed herein can be derived from isolated stem cells (eg, isolated ESCs). In some cases, engineered immune cells disclosed herein can be derived from induced stem cells (eg, iPSCs).

In some cases, the stem cells (eg, isolated stem cells, induced stem cells) disclosed herein can be autologous cells or derived from autologous cells. Autologous cells can be obtained from a subject having or suspected of having a condition. Alternatively, autologous cells may be obtained from the subject before the subject is found to have the condition. In some cases, autologous cells may be allogeneic cells, such as universal stem cells that have reduced immunogenicity, reduced numbers, or do not require immunosuppressive drugs. Autologous cells can be obtained from healthy donors.

In some cases, engineered immune cells (eg, engineered NK cells) can be autologous cells. Engineered immune cells can be obtained from a subject having the condition or suspected of having the condition. Alternatively, engineered immune cells can be obtained from the subject before the subject is found to have the condition. In some cases, engineered immune cells can be allogeneic cells, eg, for general allogeneic immunotherapy with reduced immunogenicity and requiring reduced or no immunosuppressive drugs. Engineered immune cells can be obtained from healthy donors.

In some aspects, T cells can be engineered to exhibit an increased half-life compared to control cells (eg, non-engineered T cells). T cells can be engineered to exhibit enhanced proliferation compared to control cells. T cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered T cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. Engineered T cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). The engineered T cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

In some aspects, NK cells can be engineered to exhibit increased half-life compared to control cells (eg, non-engineered NK cells). NK cells can be engineered to exhibit enhanced proliferation compared to control cells. NK cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. The engineered NK cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). The engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can include chimeric polypeptide receptors (e.g., at least 1, 2, 3, 4, 5, or more chimeric polypeptide receptors) or include genes encoding chimeric polypeptide receptors (e.g., heterologous gene ). A chimeric polypeptide receptor can include at least one antigen-binding portion capable of binding to an antigen. The antigen binding portion may include an ankyrin repeat domain. The antigen binding moiety can be a designed ankyrin repeat protein (DARPin) configured to specifically bind one or more antigens.

In some embodiments, a chimeric polypeptide receptor can include an antigen binding portion capable of binding to an antigen. A chimeric polypeptide receptor can comprise multiple antigen binding moieties (eg, at least 2, 3, 4, 5 or more antigen binding moieties capable of binding different antigens).

In some embodiments, the DARPins disclosed herein can exhibit specific binding affinity for (or against) a target antigen, and the binding affinity (e.g., the equilibrium dissociation constant (K _D ) between the DARPin and the target antigen, as measured by a surface As determined by plasmon resonance (SPR) or isothermal titration calorimetry (ITC)) compared to the binding affinity between the control antibody or fragment thereof (e.g., scFv) and the target antigen can be at least or at most about 0.1 fold, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or At most about 0.9 times, at least or at most about 1 times, at least or at most about 1.1 times, at least or at most about 1.2 times, at least or at most about 1.3 times, at least or at most about 1.4 times, at least or at most about 1.5 times, at least or at most About 1.6 times, at least or at most about 1.7 times, at least or at most about 1.8 times, at least or at most about 1.9 times, at least or at most about 2 times, at least or at most about 2.5 times, at least or at most about 3 times, at least or at most about 3 times 3.5 times, at least or at most about 4 times, at least or at most about 4.5 times, at least or at most about 5 times, at least or at most about 5.5 times, at least or at most about 6 times, at least or at most about 6.5 times, at least or at most about 7 times times, at least or at most about 7.5 times, at least or at most about 8 times, at least or at most about 8.5 times, at least or at most about 9 times, at least or at most about 9.5 times or at least or at most about 10 times.

In some embodiments, the equilibrium dissociation constant (K _D ) (e.g., determined by SPR or ITC) between a DARPin disclosed herein and a target antigen can be at least or at most about 1 x 10 ^-12 molar (M), at least or At most about 2 x 10 ^-12 M, at least or at most about 3 x 10 ^-12 M, at least or at most about 4 x 10 ^-12 M, at least or at most about 5 x 10 ^-12 M, at least or at most about 6 x 10 ^-12 M, at least or At most about 7 x 10 ^-12 M, at least or at most about 8 x 10 ^-12 M, at least or at most about 9 x 10 ^-12 M, at least or at most about 1 x 10 ^-11 M, at least or at most about 2 x 10 ^-11 M, at least or At most about 3 x 10 ^-11 M, at least or at most about 4 x ^{10 -11} M, at least or at most about 5 x 10 ^-11 M, at least or at most about 6 x 10 ^-11 M, at least or at most about 7 x 10 ^-11 M, at least or At most about 8 x 10 ^-11 M, at least or at most about 9 x 10 ^-11 M, at least or at most about 1 x 10 ^-10 M, at least or at most about 2 x 10 ^-10 M, at least or at most about 3 x 10 ^-10 M, at least or At most about 4 x 10 ^-10 M, at least or at most about 5 x ^{10 -10} M, at least or at most about 6 x 10 ^-10 M, at least or at most about 7 x 10 ^-10 M, at least or at most about 8 x 10 ^-10 M, at least or At most about 9 x 10 ^-10 M, at least or at most about 1 x 10 ^-9 M, at least or at most about 2 x 10 ^-9 M, at least or at most about 3 x 10 ^-9 M, at least or at most about 4 x 10 ^-9 M, at least or At most about 5 x 10 ^-9 M, at least or at most about 6 x 10 ^-9 M, at least or at most about 7 x 10 ^-9 M, at least or at most about 8 x 10 ^-9 M, at least or at most about 9 x 10 ^-9 M, at least or At most about 1 x 10 ^-8 M, at least or at most about 2 x 10 ^-8 M, at least or at most about 3 x 10 ^-8 M, at least or at most about 4 x 10 ^-8 M, at least or at most about 5 x 10 ^-8 M, at least or At most about 6 x 10 ^-8 M, at least or at most about 7 x 10 ^-8 M, at least or at most about 8 x 10 ^-8 M, or at least or at most about 9 x 10 ^-8 M.

In some embodiments, an ankyrin repeat domain (eg, DARPin) can be configured to specifically bind an immune cell antigen. Immune cell antigens can be presented by or derived from immune cells. In some instances, the immune cells may not be immune cells. The engineered immune cell can be an engineered NK cell, and the ankyrin repeat domain of the chimeric polypeptide receptor of the engineered NK cell can be designed to specifically bind to an immune cell antigen of a T cell, so that the engineered NK cell can recruit T cell. Non-limiting examples of immune cell antigens for T cells may include CD3, CD4, CD8, CCR7, CD45RO, CR45RA, CD45RA, T cell receptor (TCR), modifications thereof, and fragments thereof. In some examples, engineered immune cells (e.g., NK cells) can include one or more chimeric antigen receptors, and the one or more chimeric antigen receptors can include two different DARPins for specific binding (i) immune cell antigens of different immune cells (eg T cells) and (ii) different antigens (eg disease specific antigens eg NY-ESO01 , CD33 and/or CD123).

Engineered immune cells can include a CD16 variant for enhanced CD16 signaling compared to control cells.

Engineered immune cells can include cytokines (eg, secreted cytokines) that are heterologous to the immune cells. In some instances, a heterologous cytokine can include a heterologous interleukin (IL) (eg, a heterologous secreted IL-15).

The engineered immune cells disclosed herein can include a heterologous receptor or a heterologous gene encoding a heterologous receptor. In some cases, the heterologous receptor can be the corresponding receptor for a heterologous cytokine disclosed herein (e.g., a heterologous IL-15 receptor (IL-15R, such as IL-15α or IL-15 receptor for heterologous IL-15). -15β)). Alternatively, engineered immune cells may not, and need not, include any heterologous receptors that are corresponding receptors for heterologous cytokines. For example, engineered immune cells that include heterologous IL (eg, IL-15) lack a heterologous receptor for heterologous IL (eg, IL-15R).

A heterologous gene or heterologous polynucleotide disclosed herein can be integrated into the chromosome (eg, nuclear chromosome) of the engineered immune cell. Alternatively, the heterologous gene or polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell. In one example, mRNAs encoding heterologous cytokines can be introduced (or inserted) into engineered immune cells.

In some cases, a heterologous cytokine disclosed herein can be a heterologous IL. The heterologous IL disclosed herein may comprise at least 1, 2, 3, 4, 5 or more different types of heterologous IL. The heterologous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of heterologous IL. Alternatively, the heterologous IL may be a single type of heterologous IL. Non-limiting examples of heterologous IL may include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22 , IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL -35, IL-36, IL-37 and IL-38. In some examples, the heterologous IL may include one or more members selected from IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and functional modifications thereof. For example, the engineered immune cells (eg, engineered NK cells) disclosed herein can include at least a portion of heterologous human IL-15 (or the gene encoding it).

The heterologous cytokines (eg, heterologous IL) disclosed herein can be secreted cytokines. Alternatively, heterologous cytokines may not and need not be secreted by engineered immune cells. In this case, for example, heterologous cytokines can be bound to the cell surface of engineered immune cells.

In some instances, a heterologous cytokine (eg, heterologous IL) disclosed herein can be a secreted cytokine. Expression cassettes encoding heterologous cytokines can be introduced into engineered immune cells. The expression cassette may further encode additional heterologous polypeptides, such as heterologous receptors. Within the expression cassette, a first polynucleotide sequence encoding a heterologous cytokine and a second polynucleotide sequence encoding an additional heterologous polypeptide (e.g., a heterologous receptor) can be identified by a polynucleotide encoding a cleavage linker. Linkers are coupled to each other. In some examples, the heterologous receptor can be the corresponding receptor for a heterologous cytokine (eg, heterologous IL-15α or IL-15β of heterologous IL-15). Alternatively, the expression cassette may not and need not encode any other heterologous polypeptide other than a heterologous cytokine.

A cleavable linker disclosed herein may include a self-cleaving peptide, such as a self-cleaving 2A peptide. Self-cleaving peptides can be found in members of the picornaviridae family of viruses, including aphthviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis virus (ERAV), thosea asigna virus ) (TaV) and porcine Czech virus-1 (PTV-1), as well as cardioviruses such as Theileria virus (eg Theileria murine encephalomyelitis) and encephalomyocarditis virus. Non-limiting examples of self-cleaving 2A peptides may include "F2A", "E2A", "P2A", "T2A" and functional variants thereof.

In some cases, a heterologous cytokine (eg, heterologous IL) disclosed herein can bind to the cell surface of an engineered immune cell (eg, engineered NK cell). In some examples, engineered immune cells can be genetically modified to couple a heterologous polynucleotide sequence encoding a heterologous cytokine to a gene encoding an endogenous transmembrane protein of the engineered immune cell. In one example, an endogenous transmembrane protein can be the corresponding receptor for a heterologous cytokine (eg, heterologous IL-15α or IL-15β of heterologous IL-15). In some examples, an expression cassette encoding a heterologous fusion polypeptide comprising (i) a heterologous cytokine (e.g., including IL15) coupled to (ii) a heterologous receptor can be introduced into engineered immune cells. A fusion of at least a portion of IL15Ra and at least a portion of IL15Ra). Here, the heterologous cytokine may not and need not be cleavable from the heterologous receptor. Non-limiting examples of heterologous receptors may include corresponding receptors for heterologous cytokines (e.g., heterologous IL-15α or IL-15β for heterologous IL-15), or different receptors, such as a common gamma chain (γ _C ) receptors or modifications thereof.

The engineered immune cells disclosed herein (e.g., engineered NK cells) can exhibit the expression of heterologous cytokines (e.g., heterologous IL, such as heterologous IL-15) and/or heterologous receptors (e.g., heterologous IL-15) disclosed herein. Enhanced signaling of the endogenous signaling pathway of heterologous IL receptors, such as heterologous IL-15R). Enhanced signaling of the endogenous signaling pathways disclosed herein can be determined by a number of methods, including but not limited to: (i) downstream signaling proteins (e.g., for IL-15/IL-15R, JAK3, STAT3, STAT5, etc. ) or (ii) downstream genes by Western blot or polymerase chain reaction (PCR) techniques (e.g., for IL-15/IL-15R, Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng etc.) expression.

In some cases, in engineered immune cells of the present disclosure induced by heterologous cytokines and/or heterologous receptors (e.g., by heterologous cytokines and/or heterologous receptors disclosed herein, such as IL -15/IL-15R-induced) enhanced signaling of endogenous signaling pathways can be characterized by an increase in phosphorylation of downstream signaling proteins to at least or at most about 0.1-fold, at least or at most about 0.2-fold compared to control cells , at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, At least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or At most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most About 5,000 times, or at least or at most about 10,000 times.

In some cases, in engineered immune cells of the present disclosure induced by heterologous cytokines and/or heterologous receptors (e.g., by heterologous cytokines and/or heterologous receptors disclosed herein, such as IL -15/IL-15R-induced) enhanced signaling of endogenous signaling pathways can be characterized by an increase in the expression of downstream genes by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most, compared to control cells At most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most About 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 7 times 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times , or at least or at most about 10,000 times.

Enhanced CD16 signaling (eg, constitutively enabled signaling of CD16) of engineered immune cells (eg, engineered NK cells) disclosed herein can be achieved by having non-cleavable CD16 variants in the subject cells. CD16 (eg, CD16a) is a transmembrane protein expressed by immune cells (eg, NK cells) that binds to monomeric IgG attached to target cells to prime immune cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC) . In non-engineered immune cells, binding between CD16 and monomeric IgG induces cleavage of CD16 protein at the cleavage site near the transmembrane domain, thereby modulating the cell surface density of CD16 upon immune cell priming. Therefore, endogenous CD16 of engineered immune cells can be modified to enhance their signaling. Alternatively, enhanced signaling variants of CD16 can be artificially introduced into engineered immune cells.

In some cases, the endogenous gene encoding CD16 of engineered immune cells can be genetically modified in its extracellular domain (e.g., F176V) by the action of the gene editing moieties disclosed herein, thereby modifying CD16 exhibits higher binding affinity to its targets (eg, monomeric IgG). In some cases, a heterologous gene encoding such modified CD16 can be introduced into the cell.

In some cases, the endogenous gene encoding CD16 of the engineered immune cells can be genetically modified by the action of the gene editing moieties disclosed herein such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling. In some examples, a cleavage site (e.g., positions 195-198) in the membrane proximal region (positions 189-212) of CD16 can be modified or eliminated (e.g., a CD16 S197P variant that is a non-cleavable CD16 variant) . In some cases, a heterologous gene encoding such modified CD16 can be introduced into the cell.

In some cases, a heterologous gene encoding a heterologous CD16 variant (i) that exhibits a higher sensitivity to its target (e.g., monomeric IgG) can be introduced into cells (i.e., hnCD16). binding affinity, and (ii) are non-cleavable. In some examples, the heterologous CD16 variant can be a modified CD16 including, for example, F176V and S197P, as disclosed herein. In some examples, the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) a different cell surface protein, such as a glycoprotein (eg, CD64). The extracellular domain, which exhibits enhanced binding to the target (eg, monomeric IgG) compared to unmodified CD16.

Enhanced CD16 signaling of the engineered immune cells (e.g., engineered NK cells) disclosed herein can be determined by a variety of methods, including but not limited to (i) phosphorylation of downstream signaling proteins (e.g., SHP-1 ) by Western blotting or (ii) expression of downstream genes (such as CD25, IFN-γ, TNF, etc.) by Western blot or PCR techniques.

In some cases, the CD16 signaling of the engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.2 times, the CD16 signaling of control cells. At least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least Or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or At most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most About 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times larger.

In some instances, enhanced CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be characterized by increased phosphorylation of downstream signaling proteins by at least or at most About 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.5 times 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times , at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, At least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, enhanced CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be characterized by an increase in the expression of downstream genes by at least or at most about 0.1-fold compared to control cells , at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, At least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or At most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most About 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some instances, CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be prolonged to (e.g., longer than CD16 signaling initiated) compared to CD16 signaling of control cells duration) at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times , at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, At least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

The engineered immune cells disclosed herein can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. The expression or activity of endogenous immunomodulatory polypeptides can be reduced in engineered immune cells (eg, engineered NK cells), eg, by the action of the gene editing moieties disclosed herein.

Reduced expression or activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be determined by a variety of methods, including but not limited to (i) downstream signaling proteins (e.g., for PD1/ PDL1 signaling, phosphorylation or dephosphorylation of SHP2, Igα/β, Syk, etc.); or (ii) expression of endogenous immunomodulatory polypeptides (eg, PD1) by Western blot or PCR techniques.

In some instances, the reduced expression of an endogenous immunomodulatory polypeptide in an engineered immune cell disclosed herein (e.g., an engineered NK cell) can be characterized by expression of an endogenous immunomodulatory polypeptide (e.g., PD1 ) as compared to a control cell Reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, At least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or At most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/ 70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, Or at least or at most about 1/10,000.

In some cases, reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be characterized by downstream signaling proteins (e.g., for PD1/PDL1 signaling) as compared to control cells. conduction, SHP2) phosphorylation is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most About 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1 /9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60 , at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least Or at most about 1/5,000, or at least or at most about 1/10,000.

In some cases, reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be characterized by downstream target signaling proteins (e.g., for PD1/PDL1 ) as compared to control cells. Signaling, phosphorylation of Igα/β or Syk) is increased by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least Or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or At most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most About 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times. A downstream target signaling protein may be a protein that is normally inhibited by the functional signaling pathway of an endogenous immunomodulatory polypeptide.

The engineered immune cells disclosed herein can include reduced activity of endogenous cytokine signaling (eg, endogenous IL signaling, eg, endogenous IL-17 signaling).

In some instances, engineered NK cells can be treated with inhibitors of endogenous cytokine signaling (eg, small molecule inhibitors). In some cases, engineered NK cells can include reduced expression of endogenous IL-17 or its endogenous receptor (eg, by indel or transgenic mutation, by transient or permanent suppression, etc.). In some instances, engineered NK cells can include reduced expression of endogenous IL-17. In some instances, engineered NK cells can include reduced expression of endogenous IL-17R. In some instances, engineered NK cells can include reduced expression of endogenous IL-17 and endogenous IL-17R.

In some instances, an endogenous cytokine disclosed herein can be endogenous IL. The endogenous IL disclosed herein may comprise at least 1, 2, 3, 4, 5 or more different types of endogenous IL. The endogenous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of endogenous IL. Alternatively, the endogenous IL may be a single type of endogenous IL. Non-limiting examples of endogenous ILs may include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22 , IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL -35 and IL-36. In some examples, the endogenous IL can be IL-17. Non-limiting examples of endogenous IL-17 can include IL-17A, IL-17F, and natural mutations thereof. For example, engineered immune cells (eg, engineered NK cells) disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F.

In some cases, an endogenous gene encoding an endogenous cytokine disclosed herein (eg, endogenous IL, such as IL-17) can be modified through the action of the gene editing moiety disclosed herein.

The endogenous receptor can be the corresponding receptor for any cytokine disclosed herein (eg, the corresponding receptor for any IL disclosed herein). In some cases, the endogenous receptor can be the corresponding receptor for IL (eg, IL-17R for IL-7 signaling). Non-limiting examples of IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof. In one example, the endogenous IL-17R comprises IL-17RA.

In some instances, reduced expression or activity of an endogenous cytokine disclosed herein (e.g., endogenous IL, such as IL-17) or its endogenous receptor can be determined by a variety of methods, including but not limited to: (i) downstream Phosphorylation of signaling proteins (e.g. for IL-17, PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc.) or (ii) expression of downstream genes (by Western blot or PCT technique). In some examples, genes downstream of IL cytokines such as IL-17 may include chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.), cytokines (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.), acute phase response molecules (eg, SAA, CRP, lipocalin 2/24p3, etc.) and/or enzymes (eg, metalloproteases, eg, MMP1, MMP3, MMP9, MMP13).

In some instances, reduced expression or activity of an endogenous cytokine (e.g., endogenous IL, such as IL-17) or its endogenous receptor in engineered immune cells (e.g., engineered NK cells) disclosed herein can characterize For, compared with control cells, the phosphorylation of downstream signaling proteins of endogenous cytokines is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/0. 1. At least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, At least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or At most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000.

In some instances, reduced expression or activity of an endogenous cytokine (e.g., endogenous IL, such as IL-17) or its endogenous receptor in engineered immune cells (e.g., engineered NK cells) disclosed herein can characterize is, compared with control cells, the expression of downstream genes of endogenous cytokines is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4 , at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most About 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1 /50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500 , at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000.

The engineered immune cells disclosed herein can exhibit Enhanced expression profiles of specific cellular markers (eg, NK cell markers) for committed immune cells. For example, non-limiting examples of specific cellular markers committed to NK cells may include CD57 or killer immunoglobulin-like receptor (KIR). KIRs can include KIR2D and/or KIR3D. KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4 and/or KIR2DS5. KIR3Ds can include KIR3DL1, KIR3DL2, KIR3DL3 and/or KIR3DS1.

As disclosed herein, enhanced expression profiles of specific cellular markers for committed immune cells (eg, CD57 or KIRs for NK cells) can be determined by a variety of methods including, but not limited to, Western blot or PCR techniques.

In some cases, the engineered immune cells of the disclosure may express at least or at most about 0.1-fold, or at most about 0.1-fold, a specific cellular marker for committed immune cells (e.g., NK cell CD57 or KIR) in the engineered immune cells of the disclosure, At least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least Or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or At most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most About 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times higher, or at least or at most about 10,000 times higher.

The engineered immune cells disclosed herein may exhibit one or more of: (i) heterologous transcription factors (e.g., heterologous STATs), and (ii) endogenous enzymes (e.g., ligases such as CBL-B) decreased expression or activity.

The heterologous transcription factors may include at least 1, 2, 3, 4, 5 or more different types of heterologous transcription factors. Heterologous transcription factors may include up to 5, 4, 3 or 2 different types of transcription factors. Alternatively, the heterologous transcription factors can have a single type of transcription factor. Transcription factors may be involved in the immune activity, proliferation, apoptosis and/or differentiation of engineered immune cells. In some cases, the heterologous transcription factor of engineered immune cells (eg, engineered NK cells) can be a STAT. Non-limiting examples of STATs may include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof. In one example, STAT may include STAT3. In another example, STAT may include STAT5B.

In some instances, compared to control cells without reduced activity of (i) heterologous transcription factors (e.g., heterologous STATs) or (ii) endogenous cytokine signaling (e.g., endogenous IL-17 signaling) , the engineered immune cells (eg, engineered NK cells) disclosed herein can exhibit enhanced survival in the presence of tumor cells. In some cases, the engineered immune cells may survive at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.4-fold, at least or at most about 0.4-fold, At least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or At most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most About 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times longer.

The engineered immune cells disclosed herein can exhibit reduced expression or activity of endogenous cell markers (eg, NK cell markers such as KIRs) specific for the committed immune cells disclosed herein as compared to control cells. In some examples, the specific endogenous cell marker is a KIR. Reduced expression or activity of specific endogenous cellular markers for committed immune cells disclosed herein (eg, KIRs for NK cells) can be determined by a number of methods, including but not limited to Western blot or PCR techniques.

In some cases, the expression of endogenous cell markers (eg, KIRs of NK cells) specific for committed immune cells in the engineered immune cells of the present disclosure can be at least or at most about 1/3 of the expression thereof by control cells. 0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, At least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or At most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/ 80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 Low.

The engineered immune cells disclosed herein can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (eg, CD94, CD96, TGFβ receptor, SHIP2, etc.). In some instances, engineered immune cells may exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGFβ receptor, and (iv) ) endogenous SHIP (eg SHIP2).

In some instances, the engineered immune cell can exhibit reduced expression or activity of endogenous CD94, and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: ( ii) endogenous CD96, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some instances, the engineered immune cell can exhibit reduced expression or activity of endogenous CD96, and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: ( i) endogenous CD94, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some instances, engineered immune cells may exhibit reduced expression or activity of endogenous TGFβ receptors and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of : (i) endogenous CD94, (ii) endogenous CD96 and (iv) endogenous SHIP (eg SHIP2).

In some instances, engineered immune cells may exhibit reduced expression or activity of an endogenous SHIP (e.g., SHIP2) and also exhibit reduced expression of one or more (e.g., 1, 2, or all) of Or active: (i) endogenous CD94, (ii) endogenous CD96 and (iii) endogenous TGFβ receptor.

In some cases, reduced expression or activity of an immune checkpoint inhibitor (e.g., CD94, CD96, TGFβ receptor, SHIP2, etc.) in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be a response to a control cell. Its expression is at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0. 0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or At most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/1/ 5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD94 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD96 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous TGFβ receptors in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most About 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1 /5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20 , at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least Or at least about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of an endogenous SHIP (e.g., SHIP2) in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be at least or at most about 1/0.1 of its expression by a control cell , at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most About 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1 /20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80 , at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower .

The engineered immune cells disclosed herein can exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide as disclosed herein. In some instances, an endogenous immunomodulatory polypeptide includes one or more hypoimmune modulators. In some instances, the engineered immune cells exhibit reduced expression or activity from one or more of the following immunomodulators: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL , (iv) endogenous CD40L, (v) endogenous MICA or MICB or (vi) endogenous NKG2DL.

In some cases, as disclosed herein, endogenous modulators of hypoimmunity (e.g., CD80, CD86, CD80, CD86, The reduced expression or activity of ICOSL, CD40L, MICA, MICB, NKG2DL, etc.) can be at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, At least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or At most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/ 40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, At least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD80 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD86 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous ICOSL in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of the endogenous hypoimmunity modulator CD40L in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/3 of its expression by control cells. 0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, At least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or At most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/ 80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 Low.

In some cases, the reduced expression or activity of endogenous MICA or MICB in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most About 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1 /5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20 , at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least Or at least about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous NKG2DL in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some instances, endogenous immunomodulatory polypeptides include hypoimmunity modulators (eg, ICAM1).

In some cases, as disclosed herein, reduced expression or activity of endogenous ICAM1 in engineered immune cells of the present disclosure (e.g., engineered NK cells) may be an increase in response to control cells as determined by Western blot or PCT techniques. Its expression is at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0. 0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, At least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or At most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/1/ 5,000, or at least or at most about 1/10,000 lower.

An engineered immune cell can include an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some instances, an immunomodulatory polypeptide includes a hypoimmune modulator. The hypoimmune modulator can be PDL2. Alternatively or additionally, the hypoimmune modulator may be TGF-β.

An engineered immune cell can include an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some instances, an immunomodulatory polypeptide includes a hypoimmune modulator. Hypoimmune modulators may include one or more members of: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous Source CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CCL21 and one or more (e.g., 1, 2, 3, or all) of the following: (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous IL-10 and one or more (e.g., 1, 2, 3, or all) of the following: (i) Heterologous CCL21, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD46 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD55 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD59 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, and (iv) heterologous CD55. C. Additional Aspects of Engineered Immune Cells

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can include a heterologous cytokine disclosed herein (eg, heterologous IL, such as IL-15). In some instances, engineered immune cells (eg, engineered NK cells) include heterologous receptors that are corresponding receptors for heterologous cytokines (eg, heterologous IL-15R), as disclosed herein. Thus, engineered immune cells (e.g., engineered NK cells) can exhibit induction by heterologous cytokines and/or heterologous receptors disclosed herein (e.g., by heterologous cytokines and/or heterologous receptors such as IL -15/IL-15R induced) enhanced signaling of the endogenous signaling pathway.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can exhibit reduced expression or activity of endogenous CD38 as compared to a control cell. Such engineered immune cells can be used to treat subjects with or suspected of having a leukocyte cancer, such as multiple myeloma (MM).

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the expression or activity of endogenous CD38 of the engineered immune cell may and need not be modified. Such engineered immune cells can be used to treat a subject having or suspected of having a disease (eg, cancer, tumor) other than multiple myeloma.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can include heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or fragment thereof can be obtained from Engineered immune cell secretion.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can include heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or fragment thereof can bind to the cell surface membrane of engineered immune cells.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise at least one chimeric polypeptide receptor comprising an antigen-binding portion capable of binding an antigen, as in provided in this disclosure. In some examples, engineered immune cells can include multiple different chimeric polypeptide receptors to specifically bind multiple different antigens. In some examples, engineered immune cells can include at least one chimeric polypeptide receptor that includes multiple antigen binding portions to specifically bind multiple different antigens.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can include a safety switch capable of affecting the death of the engineered immune cell. Engineered immune cells can include genes encoding safety switches by the effect of gene editing in part (eg, integrated into the genome of the immune cell), as disclosed herein. In some cases, prodrugs can be introduced into engineered immune cells (e.g., administered to an object comprising engineered immune cells) and can be passed through the safety switch molecule in the event of an adverse event or when adaptive immunotherapy is no longer needed. to activate the prodrug to kill the subject immune cells. In some cases, the safety switch may comprise a protein selected from the group consisting of caspases (e.g., caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR, B cell CD20, and functional variants thereof. One or more members of . In some cases, a safety switch can be initiated by an initiator (e.g., a small molecule or protein, such as an antibody) for post-translational, temporal, and/or site-specific regulation of death (or exhaustion) of the subject engineered immune cells . Non-limiting examples of safety switches and their initiators may include caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); and cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Alternatively or additionally, a modified epidermal growth factor receptor (EGFR) comprising an epitope recognized by an antibody (e.g., an anti-EGFR Ab, such as cetuximab) can be used for depletion engineering when the subject cells are exposed to the antibody immune cells. In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein may comprise a protein selected from the group consisting of caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR and safety switch proteins in CD20.3 of B cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can include a heterologous immune receptor polypeptide. The immunomodulatory polypeptide may include one or more members selected from HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide, as disclosed herein. Endogenous immunomodulatory polypeptides include immune checkpoint inhibitors or hypoimmunity modulators (or both).

In some cases, the immune checkpoint inhibitors disclosed herein can include an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGFβ receptor, and 2B4 one or more members. In some instances, the immune checkpoint inhibitor can include SHIP2.

In some cases, a hypoimmune modulator disclosed herein may comprise a protein selected from the group consisting of B2M, CIITA, TAP1, TAP2, tap-related protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB One or more members of , ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise a CD16 variant for enhanced CD16 signaling compared to a control cell, wherein CD16 variants are heterologous to engineered immune cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can exhibit enhanced cytotoxicity against target cells compared to control cells. In some cases, an engineered immune cell disclosed herein may exhibit cytotoxicity (e.g., in vitro, ex vivo, or in vivo) toward a target cell or target cell population, as determined, for example, by tracking changes in the number of the target cell population At least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times the cytotoxicity of control cells , at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, At least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least Or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or At most about 100 times larger, at least or at most about 500 times larger, at least or at most about 1,000 times larger, at least or at most about 5,000 times larger, or at least or at most about 10,000 times larger.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can induce T cells from isolated immune cells (e.g., isolated T cells in vitro and and/or B cells, or the immune response of the host after administration of the engineered immune cells to the host). In some cases, as by, for example, measuring (i) the change in the number of the initial population of engineered immune cells after exposure to the isolated immune cells, or (ii) the number of isolated immune cells after exposure to the initial population of engineered immune cells The engineered immune cells disclosed herein can reduce the immune response from the isolated immune cells by at least or at most about 1 compared to the immune response from the isolated immune cells when exposed to control cells, as determined by changes in cytokine release. /0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7 , at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most About 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1 /80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/1/ 10,000.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can be compared to control cells after exposure to isolated immune cells (e.g., isolated T cells in vitro). and/or B cells, or when the engineered immune cells are administered to the host, the host's immune cells) exhibit increased half-life. In some cases, when exposed to isolated immune cells (e.g., in vitro or in vivo), the half-life of the engineered immune cells can be At least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most the half-life of the control cells About 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 3 times 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times , at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve an enhancement of a function or pathological condition of a body tissue of a subject as compared to a control cell. In some cases, treatment with engineered immune cells can achieve at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times , at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, At least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times enhancement of the function or pathological condition of bodily tissues.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve delayed degradation of a functional or pathological condition of a body tissue of the subject as compared to control cells. In some cases, treatment with engineered immune cells can achieve at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times , at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, At least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times Delayed degeneration of body tissues in functional or pathological conditions.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the bodily tissue may comprise a tissue selected from the group consisting of blood, plasma, serum, urine, perilymph, feces, saliva, semen, amniotic fluid, Cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bones, heart, thymus, arteries, blood vessels, lungs, muscles, stomach, intestines, liver, pancreas, spleen, kidneys, gallbladder, thyroid, adrenals, One or more members of breast, ovary, prostate, testis, skin, fat, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can induce an immune response against the target cell. Targets can be, for example, diseased cells, cancer cells, tumor cells, and the like.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the heterologous gene can be operably associated with constitutive, inducible, temporal, tissue-specific and/or cellular Type-specific promoter coupling (eg, for knock-in). Non-limiting examples of promoters of interest may include CMV, EF1a, PGK, CAG, and UBC. Non-limiting examples of insertion sites may include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tap-related protein, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 and TIGIT.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more of the following endogenous genes may exhibit reduced expression or activity to enhance the performance of the engineered immune cells in the tumor microenvironment (ie, tumor microenvironment genes or "TME"). In some instances, having reduced expression or activity of the TME can enhance the immune activity of the engineered immune cell against the target cell. In some instances, the TME gene can be an immune checkpoint inhibitor. Non-limiting examples of TMEs may include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGFβ receptor, 2B4, and SHIP2.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more heterologous genes (e.g., knocked in) may be expressed for enhanced function, e.g., CD137, CD80, CD86 , DAP10 (eg, with or without point mutations).

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, may exhibit reduced expression or activity of one or more of the following endogenous genes for, e.g., low immunity: B2M, CIITA, TAP1, TAP2, tap-related proteins, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and NKG2DL (eg, ULBP1).

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more heterologous genes (e.g., knocked in) may be expressed for, e.g., hypoimmunity: HLA-E, CD47 , CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55, and CD59.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, one or more heterologous genes (eg, knocked in): CD3, CD4, CD80, 41BBL, and CD131 may be expressed. D. Chimeric Antigen Receptor

Engineered immune cells of the present disclosure (e.g., engineered NK cells) can include chimeric polypeptide receptors disclosed herein (e.g., at least 1, 2, 3, 4, 5 or more different types of chimeric polypeptide receptors) . Engineered immune cells can be engineered to express chimeric polypeptide receptors temporarily or permanently. In some cases, recombinant chimeric polypeptide receptors can be delivered to engineered immune cells by, for example, liposomes and incorporated into engineered immune cells by membrane fusion. In some cases, a heterologous polynucleotide construct (eg, DNA or RNA) encoding a chimeric polypeptide receptor can be delivered to engineered immune cells. The heterologous polynucleotide construct (i.e., gene) encoding the heterologous polynucleotide construct may be incorporated into the chromosome (i.e., chromosomal gene) of the engineered immune cell, or may not or need not be integrated into In the chromosomes of the engineered immune cells disclosed herein.

A chimeric polypeptide receptor can include a T cell receptor fusion protein (TFP). The term "T cell receptor fusion protein" or "TFP" generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of the TCR extracellular domain, (iii) at least a portion of the TCR transmembrane domain, and (iv) at least a portion of the TCR intracellular domain.

In some cases, the endogenous T cell receptor (TCR) of the engineered immune cells (eg, engineered NK cells) of the present disclosure can be inactivated. In some examples, the function of the endogenous TCR of the engineered immune cell can be inhibited by an inhibitor. In some examples, a gene encoding a subunit of an endogenous TCR can be inactivated (eg, edited by the action of the gene editing moieties disclosed herein) such that the endogenous TCR is inactivated. The gene encoding an endogenous TCR subunit may be one or more of the following: TCRα, TCRβ, CD3ε, CD3δ, CD3γ, and CD3ζ.

Chimeric polypeptide receptors can include chimeric antigen receptors (CARs). The term "chimeric antigen receptor" or "CAR" generally refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and including a functional signaling structure derived from a stimulatory molecule domain (also referred to herein as an "intracellular or intrinsic signaling domain"). In some instances, the stimulatory molecule can be the zeta chain associated with the T cell receptor complex. In some cases, the intracellular signaling domain also includes one or more functional signaling domains derived from at least one co-stimulatory molecule. In some instances, co-stimulatory molecules can include 4-1BB (ie, CD137), CD27, and/or CD28. In one aspect, the CAR includes an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally removed from the antigen recognition domain (e.g., scFv, scFv, DARPin, etc.) cutting.

The CAR can be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. First-generation CARs (e.g., CD19R or CD19CAR) include: an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof, such as a DARPin, scFv, Fab fragment, VHH domain, or heavy chain-only antibody) specific for a specific antigen VH domain of a self-regulatory immune receptor), a transmembrane domain derived from a self-regulatory immune receptor (such as a transmembrane domain from a CD28 receptor), and a signaling domain derived from a self-regulatory immune receptor (such as one or more (eg three) ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ). Second-generation CARs work by adding costimulatory domains (e.g., derived from costimulatory receptors that co-act with T cell receptors (e.g., CD28, CD137/4-1BB, and CD134/OX40) to the intracellular signaling domain portion of the CAR. body) to modify first-generation CARs, which eliminates the need to administer cofactors (such as IL-2) with the first-generation CARs. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (eg, CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB). Fourth-generation CARs initiate cytokines (e.g., IL-12, IL-23, or IL-27) by adding the intracellular signaling portion of the CAR (e.g., between one or more co-stimulatory domains and the CD3ζ ITAM domain). ) or modify second- or third-generation CARs under the control of a CAR-inducible promoter (eg, the NFAT/IL-2 minimal promoter). In some cases, the CAR may be a new generation CAR system other than the first, second, third, or fourth generation CAR systems disclosed herein.

The hinge domain (e.g., the linker between the extracellular antigen-binding domain and the transmembrane domain) of the CAR in the engineered immune cells disclosed herein (e.g., engineered NK cells) may include CD3D, CD3E, CD3G, CD3c, CD4 , CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12 , IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or the full length or at least a portion of the native or modified transmembrane region of a T cell receptor polypeptide.

The transmembrane domain of the CAR in the engineered immune cells disclosed herein (eg, engineered NK cells) may include CD3D, CD3E, CD3G, CD3c, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4 -1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T The full length or at least a portion of a native or modified transmembrane region of a cell receptor polypeptide. In some examples, the transmembrane domain of the CAR disclosed herein may include CD3d, CD3e, CD3g, CD3c, CD4, CD8a, CD8b, CD27, CD28, CD40, CD80, CD86, 4-1BB, OX40, ICOS, ICAM- 1. At least a portion of the transmembrane region of CTLA-4, PD-1, 2B4, CD16, NKp30, NKp44, NKp46, NKG2C and/or NKG2D (or modified variants thereof).

The hinge domain and the transmembrane domain of the CARs disclosed herein (eg, for engineering immune cells, eg, engineered NK cells) can be derived from the same protein (eg, CD8). Alternatively, the hinge and transmembrane domains of the CARs disclosed herein can be derived from different proteins.

The signaling domain of the CAR may comprise at least or at most about 1 signaling domain, at least or at most about 2 signaling domains, at least or at most about 3 signaling domains, at least or at most about 4 signaling domains domain, at least or at most about 5 signaling domains, at least or at most about 6 signaling domains, at least or at most about 7 signaling domains, at least or at most about 8 signaling domains, at least or at most about 9 signaling domains or at least or at most about 10 signaling domains.

The signaling domain (e.g., the signaling peptide of the intracellular signaling domain) of the CAR in the engineered immune cells disclosed herein (e.g., engineered NK cells) may include CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB ), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D or the full length or at least a portion of a polypeptide of any combination thereof. In some examples, the signaling domain of a CAR disclosed herein can include that of CD3ζ, 2B4, DAP10, DAP12, DNAM1, 4-1BB, NKp30, NKp44, NKp46, NKG2C, and/or NKG2D (or modified variants thereof). One or more immunoreceptor tyrosine activation motif (ITAM) sequences.

Alternatively or in addition (i.e., co-stimulatory domains), the signaling domain CARs in engineered immune cells (e.g., engineered NK cells) disclosed herein may include CD27, CD28, 4-1BB, OX40 , ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4 or NKG2D or any combination of the full length or at least a portion of the polypeptide.

In some cases, an engineered immune cell disclosed herein (e.g., an engineered NK cell) includes a chimeric polypeptide receptor (e.g., a CAR) that includes at least a CD8 transmembrane domain and one or more of Two: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some cases, an engineered cell disclosed herein (e.g., an engineered NK cell) includes a chimeric polypeptide receptor (e.g., TFP or CAR) that includes at least (i) a CD8 transmembrane domain, (ii) 2B4 signaling domain and (iii) DAP10 signaling domain. The 2B4 signaling domain may be flanked by a CD8 transmembrane domain and a DAP10 signaling domain. Alternatively, the DAP10 signaling domain can be flanked by a CD8 transmembrane domain and a 2B4 signaling domain. In some cases, the chimeric polypeptide receptors disclosed herein can further comprise an additional signaling domain derived from CD3ζ.

The antigen (ie, target antigen) of the antigen-binding portion of a chimeric polypeptide receptor (eg, TFP or CAR) disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.

Non-limiting examples of antigens (i.e., target antigens) of the antigen-binding portion of a chimeric polypeptide receptor (e.g., TFP or CAR) disclosed herein may include ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, carcinoembryonic Antigen (CEA), CD3ζ, CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD133, CD138, CD269 (BCMA ), CD S, CLEC12A, antigens of cytomegalovirus (CMV)-infected cells (such as cell surface antigens), epithelial glycoprotein 2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule ( EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), gp100, human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrins B7, Interleukin 13 receptor subunit alpha-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), kappa, Lewis A (CA19.9), Lewis Y (LeY) , L1 Cell Adhesion Molecule (L1-CAM), LILRB2, MART-1, Melanoma Antigen Family A 1 (MAGE-A1), MICA/B, Mucin 1 (Muc-1), Mucin 16 (Muc-16) , mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, carcinoembryonic antigen (h5T4), PRAIVIE, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1) and various pathogen antigens (eg, those derived from viruses, bacteria, fungi, parasites, and protozoa capable of causing disease). In some examples, the pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse virus, influenza virus, or coronavirus.

Other examples of antigens for the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include: 1-40-beta-amyloid, 4-1BB, 5AC, 5T4, Activin receptor-like kinase 1, ACVR2B, adenocarcinoma Antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF, beta-amyloid, B lymphoma cells , C242 antigen, C5, CA-125, domestic dog (Canis lupus familiaris) IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR4, CCR5 , CD11, CD18, CD125, CD140a, CD147 (basicin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD25 (α of IL-2 receptor chain), CD27, CD274, CD28, CD3, CD3ε, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-associated antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, agglutination factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B. Dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, E. coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, gangliosides GD2, GD2, GD3 Ganglioside, Glypican 3, GMCSF receptor alpha chain, GPNMB, Growth differentiation factor 8, GUCY2C, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, Histone Complex, HIV-1, HLA-DR, HNGF, Hsp90, Human Scatter Factor Receptor Kinase, Human TNF, Human Beta Amyloid, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-1 7. IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, A Influenza virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α/β receptor, interferon γ Inducible protein, ITGA2, ITGB2 (CD18), KIR2D, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, Mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neuronal apoptosis-regulating protease 1, NGF, N-hydroxyethyl Acylneuraminic acid, NOGO-A, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, sodium phosphate cotransporter, phosphatidylserine, Platelet-derived growth factor receptor beta, prostate cancer cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, Sclerostin, SDC1, Selectin P, SLAMF7, SOST, Sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, Tenascin C, TFPI, TGF-β1, TGF-β2, TGF- β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA , VEGFR1, VEGFR2, vimentin and VWF.

Other examples of antigens for the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include: 707-AP, biotinylated molecules, a-actinin-4, abl-bcr alb-b3 (b2a2), abl- bcr alb-b4 (b3a2), adipophilin, AFP, AIM-2, annexin II, ART-4, BAGE, b-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210 (b3a2), BING-4, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE- 3. GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR/ FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic Enzyme, Mammaglobin-A, MART-1/Melan-A, MART-2, MC1R, M-CSF, Mesothelin, MUC1, MUC16, MUC2 , MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, carcinoembryonic antigen (h5T4 ), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK , RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin (Survivin), Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ - light chain.

Other examples of antigens of the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include antibodies, fragments or variants thereof. Such antibodies may be natural antibodies (eg, naturally secreted by immune cells of a subject, such as B cells), synthetic antibodies, or modified antibodies. In some cases, the antigen of the antigen binding portion of a chimeric polypeptide receptor disclosed herein may comprise an Fc domain of an antibody from the group consisting of: 20-(74)-(74) (milatuzumab (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abavolumab (abagovomab), abciximab (abciximab), abituzumab (abituzumab), lintuzumab (zlintuzumab)), aktomumab (actoxumab), adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab ), AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, aletuzumab (alirocumab), altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, Leixing-anetuzumab ( anetumab ravtansine), anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab), APX005M, aspirin Arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also known as tocilizumab), attolimumab, avelumab, B-701, bapineuzumab, baciximab Anti (basiliximab), bavituximab (bavituximab), BAY1129980, BA Y1187982, betumomab, begelomab, belimumab, benralizumab, bertilimumab Besilesomab, Betalutin (177Lu-tetraxetan-tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brodalumab Brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine ), caplacizumab, caprumab pendetide, carlumab, catumaxomab, CBR96-doxorubicin Immunoconjugates, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab Monoclonal antibody (cetuximab), CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, citatuzumab bogatox, cetuzumab (cixutumumab), clarizumab (clazakizumab), clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab ), Concizumab, Cotara (iodine I-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab ( trastuzumab biosimilars), dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, dapirolizumab pegol, Daratumumab, Daratumumab Enhanze (daratumumab), Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depa Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, Denutuximab (dinutuximab), diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, trazitumab, DS-1123, DS-8895, dugortuzumab Duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab ), edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, efalizumab Elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enakinumab avatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, western apilimomab ( epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144 , fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, plus Gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK317943998, 1lk 18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab , IGN002, IGN523, igovomab (igovomab), IMAB362, IMAB362 (claudiximab), imalumumab (imaluma b), IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan), IMMU-114, ImmuTune IMP701 antagonistic antibody, INCAGN1876, inclacumab, INCSHR1210 , indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, ipilimumab ( iratumumab), isatuximab, istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, Keleximab Monoclonal antibody (keliximab), KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab (lulizumab pegol), lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI- 3617, MEDI-551 (inebilizumab), M EDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine , mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, motuximab Moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natal Natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab ), obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, entuzumab (onartuzumab), ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab ), otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab ), palivizumab, panitumumab, panko mab), PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, Perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin , pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, ranibizumab Quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, Rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-GN-CD19A CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab , stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab), Sym015, TAB08, tabalumab, tacatuzumab tetraxetan), tadocizumab, talizumab, tanezumab, tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin , telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, Tetulomab (tetulomab), TG-1303, TGN1412, thorium-227-epratuzumab conjugate, ticilimumab, tigatuzumab, tiratuzumab Tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, Tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuviruma b. U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, vadastuximab Talirine, Vitin-vandotuzumab (vandortuzumab vedotin), vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB -101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen The domain may be capable of specifically and preferentially binding an antigen comprising a ligand selected from BCMA, CD20, CD22, CD30, CD33, CD38, CD70, kappa, Lewis Y, NKG2D, ROR1, NY-ESO-1, One or more members of NY-ESO-2, MART-1 and gp100. Non-limiting examples of NKG2D ligands include one or more members selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In some cases, engineered immune cells disclosed herein (e.g., engineered NK cells) can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding receptor capable of specifically binding a target cell antigen. domain, and the engineered immune cell may exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor. Thus, populations of engineered immune cells can avoid targeting and killing each other, eg, when administered to a subject in need.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen A domain may be capable of specifically and preferentially binding CD38. In some instances, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38. In some instances, a subject engineered immune cell comprising a chimeric polypeptide receptor for CD38 may be capable of targeting and effecting the death (or degradation) of plasma cells.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen A domain may be capable of specifically and preferentially binding CD38. In some instances, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38. E. Stem cells

Any of the engineered immune cells (eg, engineered NK cells) disclosed herein can be derived from isolated stem cells (eg, ESCs) or induced stem cells (iPSCs). Isolated stem cells or induced stem cells can be modified (eg, genetically modified) to generate engineered immune cells.

In some cases, the pluripotency of a stem cell (eg, ESC or iPSC) can be determined in part by assessing the pluripotency characteristics of the cell. Pluripotency characteristics may include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential; (iii) pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3/4 , SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or expression of CD50; (iv) ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation consisting of all three somatic lineages; and (vi) formation by Formation of embryoid bodies composed of cells of three somatic lineages.

In some cases, stem cells (eg, ESCs or iPSCs) can be genetically modified to generate (eg, induce differentiation into) CD34+ hematopoietic stem cells. Stem cells can be genetically modified to express any of the heterologous polypeptides disclosed herein (eg, cytokines, receptors, etc.) before, after, or during induced differentiation of hematopoietic stem cells. Stem cells can be genetically modified to reduce the expression or activity of any of the endogenous genes or polypeptides (eg, cytokines, receptors, etc.) disclosed herein before, after, or during induced hematopoietic stem cell differentiation. In some instances, such genetically modified CD34+ hematopoietic stem cells are any of the engineered immune cells of the disclosure or a source thereof.

In some examples, stem cells disclosed herein can be cultured in media with ROCKi (Y-27632) (e.g., at about 10 micromolar (μM)), SCF (e.g., at about 40 nanogram/milliliter (ng/mL)) , VEGF (for example, in a medium of about 20 ng/mL) and BMP-4 (for example, in a medium of about 20 ng/mL) in APEL medium to differentiate into CD34+ hematopoietic stem cells.

In some cases, CD34+ hematopoietic stem cells can be induced (eg, genetically modified with one or more characteristics of any of the engineered immune cells of the disclosure) to differentiate into committed immune cells, such as T cells or NK cells. Thus, in some cases, the induced differentiation process produces any of the engineered NK cells of the disclosure.

In some examples, IL-3 (e.g., about 5 ng/mL), IL-7 (e.g., about 20 ng/mL), IL-15 (e.g., about 10 ng/mL), SCF (e.g., about 20 ng/mL) and Flt3L (eg, about 10 ng/mL) to culture genetically modified CD34+ hematopoietic stem cells to differentiate into CD45+ NK cells.

In some cases, CD45+ NK cells can be expanded in culture using a gas permeable rapid expansion (G-Rex) platform, eg, in media including IL-2, mbIL-21 aAPC.

In some cases, the iPSC-derived NK cells disclosed herein can be cultured with one or more heterologous cytokines including Il-2, IL-15, or IL-21. In some cases, the iPSC-derived NK cells disclosed herein can be cultured (eg, for cell expansion) with one or more heterologous cytokines selected from IL-2, IL-15, and IL-21. In some cases, the iPSC-derived NK cells disclosed herein can be combined with two or more heterologous cytokines selected from IL-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-21 -2 and IL-21, or IL-15 and IL-21) were cultured together simultaneously or sequentially in any order. In some cases, the iPSC-derived NK cells disclosed herein can be cultured with all of II-2, IL-15, and IL-21 simultaneously or sequentially in any order. F. Gene editing or delivery of genetic material

The gene editing moieties disclosed herein may include CRISPR-associated polypeptides (Cas), zinc finger nucleases (ZFNs), zinc finger-associated gene regulatory polypeptides, transcriptional initiation factor-like effector nucleases (TALENs), transcriptional initiation factor-like effector-associated genes Regulatory polypeptides, meganucleases, natural master transcription factors, epigenetic modifying enzymes, recombinases, flippases, transposases, RNA binding proteins (RBPs), Argonaute proteins, any derivatives thereof, any variants thereof, or any fragment. In some embodiments, the actuator moiety includes a Cas protein, and the system further includes a guide RNA (gRNA) complexed with the Cas protein. In some embodiments, the actuator moiety comprises a RBP complexed with a gRNA capable of forming a complex with the Cas protein. In some embodiments, the gRNA includes a targeting segment that exhibits at least 80% sequence identity to the target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

In some cases, suitable gene editing moieties include CRISPR-associated (Cas) proteins or Cas nucleases, including Type I CRISPR-associated (Cas) polypeptides, Type II CRISPR-associated (Cas) polypeptides, Type III CRISPR-associated (Cas) polypeptides, Type III CRISPR-associated (Cas) polypeptides, (Cas) polypeptides, Type IV CRISPR-associated (Cas) polypeptides, Type V CRISPR-associated (Cas) polypeptides, and Type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcriptional initiation factor-like effector nucleic acids Enzymes (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; (aAgo) and eukaryotic Argonaute (eAgo)); any derivatives thereof, any variants thereof; and any fragments thereof.

Non-limiting examples of Cas proteins include: c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1 , Csf2, Csf3, Csf4 and Cul966 and homologues or modified forms thereof.

In some cases, the gene editing moieties disclosed herein can be fused with additional functional moieties (e.g., to form fusion moieties), and non-limiting examples of the functions of the additional functional moieties can include: methyltransferase activity, demethylation Base enzyme activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, unwinding Enzyme activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, Hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity and demyristylation activity. For example, a fusion protein can be a fusion of a Cas protein with part of an effector or repressor function.

Alternatively or additionally, gene editing (eg knock-in) or delivery of heterologous genetic material can be achieved. Other viral and non-viral based gene transfer methods can be used to introduce nucleic acids into host cells (eg, stem cells, hematopoietic stem cells, etc. disclosed herein). Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism). Viral vector delivery systems can include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells. Non-viral vector delivery systems can include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes.

Systems based on RNA or DNA viruses can be used to target specific cells and transport viral payloads to the cell's nucleus. Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo). Alternatively, the viral vector can be administered directly (in vivo) to the subject. Viral-based systems can include retroviral, lentiviral, adenoviral, adeno-associated viral and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur by retroviral, lentiviral, and adeno-associated viral gene transfer methods, which can result in long-term expression of the inserted transgene.

Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, artificial virions, and Reagent Enhanced Absorption of DNA. Cationic and neutral lipids for lipofection can be recognized using efficient receptors suitable for polynucleotides.

Alternatively or additionally, antisense oligonucleotides can be used to inhibit or silence target gene expression. Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA), microRNA (miRNA), and small interfering RNA (siRNA). G. Combination therapy

The engineered immune cells (eg, engineered NK cells) of the disclosure can be combined with co-therapeutic agents to treat a subject in need. In some cases, the engineered immune cells can be administered to the subject before, simultaneously with, or after the co-therapeutic agent is administered to the subject.

In one aspect, the present disclosure provides a composition comprising (a) any of the engineered immune cells disclosed herein (e.g., engineered NK cells) and (b) a combination therapeutic agent (i.e., a single therapeutic agent) ( For example, an antibody, such as an anti-CD20 antibody or an anti-PD1 antibody). In some cases, engineered immune cells may include one or more of: (i) a heterologous cytokine disclosed herein (eg, heterologous IL, such as IL-15), (ii) a heterologous cytokine disclosed herein for enhancing and (iii) a chimeric polypeptide receptor disclosed herein comprising an antigen-binding portion capable of binding an antigen. In some examples, the combination therapy includes an anti-CD20 antibody.

In some cases, engineered immune cells may include a heterologous cytokine disclosed herein (eg, IL-15) and one or both of: (ii) a CD16 variant for enhanced CD16 signaling, and (iii) ) A chimeric polypeptide receptor comprising an antigen binding portion.

In some cases, engineered immune cells can include CD16 variants for enhanced CD16 signaling and one or both of: (i) a heterologous cytokine (e.g., IL-15), and (iii) include Chimeric Polypeptide Receptors of Antigen Binding Portions.

In some cases, engineered immune cells can include a chimeric polypeptide receptor that includes an antigen binding portion and one or both of: (i) a heterologous cytokine (e.g., IL-15), and (ii) CD16 variants for enhanced CD16 signaling.

Non-limiting examples of combination therapeutic agents may include: cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for the treatment of cancer, For example, anti-CD20 antibodies, anti-PD1 antibodies (e.g. pembrolizumab) platelet-derived growth factor inhibitors (e.g. GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g. celecoxib), Interferons, cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets PDGFR-β, BlyS, APRIL, BCMA receptors, TRAIL/Apo2, other bioactive agents and organic chemical agents, etc. .

The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. Non-limiting examples of cytotoxic agents can include: radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu), chemotherapeutic agents such as methotrexate, doxorubicin Adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other embedded agents, enzymes and fragments thereof (such as nucleolytic enzymes), antibiotics and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin).

Non-limiting examples of chemotherapeutic agents may include: alkylating agents such as thiotegua and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and pipoxafan; aziridine Pyridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine , triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone )); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolamine lectin, and 9-aminocamptothecin); bryostatin; callystatin; CC- 1065 (including its adolaisine, kizellesine and bizelesine synthetic analogues); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (specifically nostocin 1 and candocin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; (pancratistatin); sarcodictyn; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, clophosphamide, estramustine, ifosfamide amine, dichloromethyldiethylamine, methoxymustine hydrochloride, melphalan, new enbixing, benzyl mustard cholesterol, prednimustine, cyclophosphamide, uramustine; nitroso Ureas such as carmustine, chlorurecin, formustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics; danemycin ( dynemicins), including danemycin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarithromycin (aclacinomysins), actinomycin, athramycin, azaserine, bleomycins, cactinomycin, carabicin, carmine, carcinophilus Carzinophilin, Chromomycin dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanogen Morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ethorubicin, idarubicin, macillomycin, silk Mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivine, pelomycin, potfiromycin, puromycin, quelamycin , rhodorubicin, streptoglobin, streptozotocin, tubercidin, ubenimex, netastatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercapto Purines, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azuridine, carmofur, arabinose Cytidine, dideoxyuridine, doxifluridine, enoxitabine, floxuridine; androgens such as calusterone, drostanolone propionate, cyclic Thiandrol, metandrostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trolosteine; folic acid supplements such as folinic acid; aceglatone; Aidophosphamide glycoside; aminolevulinic acid; eniluracil; amsacridine; bestrabucil; bisantrene; edatraxate; defofamine; Demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguanidine hydrazone; mitoxantrone; mopidanmol; Nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Pr oducts, Eugene, Oreg.); razoxane; rhizocin; ,2″-Trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine )); Urethane (urethan); Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Dibromomannitol; Dibromodulcitol; Neo (gacytosine); arabinoside ("Ara-C"); thiotepa; taxanes, such as taxanes, including TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE without Cremophor ™, albumin-engineered nanoparticles of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE® docetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine ( GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosf amide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; formyltetrahydrofolate; vinorelbine (NAVELBINE®); daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid ; capecitabine (XELODA®); any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP (cyclophosphamide, doxorubicin, vinca Neospine and Prednisolone Combination Therapy), and FOLFOX (short for Oxaliplatin (ELOXATIN™) Combination of 5-FU and Leucovorin). Additional chemotherapeutic agents include cytotoxic agents useful as antibody drug conjugates such as the maytansinoids (eg DM1 ) and the auristatins MMAE and MMAF.

Examples of chemotherapeutic agents may also include "antihormonal agents" or "endocrine therapeutics" that act to modulate, reduce, block or inhibit the action of hormones that can promote cancer growth, and are usually in systemic form, or systemic therapy. They may be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4- Hydroxytamoxifen, travoxifene, raloxifene hydrochloride (keoxifene), LY117018, onapristone, and FARESTON® toremifene; antiprogestins; estrogen receptor down-regulators (ERDs) ; agents that suppress or shut down ovarian function, such as luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate, and triptorelin (tripterelin); other antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal gland), For example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® trazole and ARIMIDEX® anastrozole. Additionally, this definition of a chemotherapeutic agent includes bisphosphonates such as clodronate (e.g. BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronic acid salt, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; and troxacitabine (1,3-di oxolane nucleoside cytosine analogs); antisense oligonucleotides, especially those that inhibit genes in signaling pathways associated with abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor Antisense oligonucleotides expressed by the receptor (EGFR); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and any of the pharmaceutically acceptable salts, acids, or derivatives of the foregoing.

Examples of chemotherapeutic agents may also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab ( Bexxar, Corixia) and an antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Other humanized monoclonal antibodies that have therapeutic potential as agents in combination with the compounds of the invention include: apratuzumab, atezolizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cilizumab, Tocilizumab pegylated, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozo Mixing, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motuzumab, motovizumab, natalizumab, niger Tocilizumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, peckizumab, ralivizumab, ranibizumab, reslivizumab, reslivizumab, resyvizumab, Rovetuzumab, ruplizumab, cilostuzumab, siplizumab, sortuzumab, tacatuzumab tetraxetan, tacatuzumab, tacatuzumab, tefibazumab, tocilizumab, toralizumab , tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin 12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) (this is a genetically modified to recognize interleukin 12 p40 recombinant full-length IgG1λ antibody with only human sequences of the protein).

Examples of chemotherapeutic agents may also include "tyrosine kinase inhibitors" such as EGFR targeting agents (e.g. small molecules, antibodies, etc.); small molecule HER2 tyrosine kinase inhibitors such as TAK165 (available from Takeda); CP -724,714 (oral selective inhibitors of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 that preferentially binds EGFR but inhibits cells overexpressing both HER2 and EGFR (available from Wyeth); lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (canertinib) (CI-1033; Pharmacia); Raf-1 inhibitors, such as the antisense reagent ISIS-5132 (available from ISIS Pharmaceuticals) that inhibits Raf-1 signaling; non-HER-targeted TK inhibitors, such as formazan Imatinib sulfonate (GLEEVEC®, available from Glaxo SmithKline); multitargeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors , such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-( 3-Chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H - pyrrolo[2,3-d]pyrimidines; curcumin (diferulic methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing nitrothiophene moiety Tyrphostins; PD-0183805 (Warner-Lamber); antisense molecules (eg, those that bind to HER-encoding nucleic acids); quinoxalines (US Pat. No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS3521; Isis/Lilly); Ni (GLEEVEC®); PKI166 (Novartis); G W2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Sematinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); Mycin (sirolimus, RAPAMUNE®).

Examples of chemotherapeutic agents may also include: dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol , amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, Denileukin, dextropropylimide, epoetin alfa, erlotinib, filgrastim, histrelin acetate, imomodane Anti (ibritumomab), interferon α-2a, interferon α-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, norfetumomab, o Preleukin, palivermin, pamidronate, pegasin, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, Porfimer sodium, quinacrine, rasburicase, sargragrastim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate and zoledronic acid, and pharmaceutically acceptable salts thereof.

Examples of chemotherapeutic agents may also include hydrocortisone, hydrocortisone acetate, cortisone acetate, ticortisone pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide , budesonide, desonide, fluocinolone, fluocinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocorolone, hydrocortisone-17-butyl hydrocortisone-17-pentanoate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone- 17-butyrate, clobetasol-17-propionate, fluocorone hexanoate, flucorone pivalate, and flupredidine acetate: Immunoselective anti-inflammatory peptides (ImSAIDs) such as Phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC); antirheumatic agents such as azathioprine, cyclosporine (cyclosporine A ), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers (such as etanercept (ENBREL®)), Infliximab (REMICADE®), adalimumab (HUMIRA®), beselizumab (CIMZIA®), golimumab (SIMPONI®), interleukin 1 (IL-1) inhibitor Blockers (such as anakinra (KINERET®)), T cell co-stimulation blockers (such as abatacept (ORENCIA®)), interleukin 6 (IL-6) blockers (such as tocilizumab anti (ACTEMERA®)); interleukin 13 (IL-13) blockers (e.g. lebrikizumab); interferon alpha (IFN) blockers (e.g. rontalizumab); β7 integrin blockers blockers (e.g. rhuMAbβ7); IgE pathway blockers (e.g. anti-M1 Prime); secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa/β2 blockers (e.g. anti-lymphotoxin alpha (LTa )); miscellaneous investigational agents (e.g., thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832)); polyphenols , such as quercetin, resveratrol, picatanol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid and its derivatives; phagocytosis inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolamine Lectins and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTOR AL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, azole Ledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or Risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; guarifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tipifamib (R11577); orafenib, ABT510; oblimersen sodium) (GENASENSE®); picogenin; farnesyltransferase inhibitors, such as lonafamib (SCH 6636, SARASAR™); and any pharmaceutically acceptable salt, acid, or derivative thereof; or more combinations.

The term "growth inhibitory agent" generally refers to a compound or composition that inhibits the growth and/or proliferation of cells (eg, cells whose growth is dependent on PD-L1 expression) in vitro or in vivo. A growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase. Non-limiting examples of growth inhibitory agents include agents that block cell cycle progression (outside of S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as the anthracycline doxorubicin ((8S-cis)- 10-[(3-Amino-2,3,6-trideoxy-α-L-lyxosyl-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6 ,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthonaphthalenedione), epirubicin, daunorubicin, etoposide and bleomycin . Those agents that block G1 also spill over into S-phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and Cytarabine (Ara-C). Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from taxanes. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly of tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of mitosis of cells. H. How to use

Engineered immune cells (eg, engineered NK cells) of the disclosure can be used (eg, administered) to treat a subject in need. A subject may have or may be suspected of having a condition such as a disease (eg, cancer, tumor, tissue degeneration, fibrosis, etc.). Cells (eg, stem cells or committed adult cells) can be obtained from a subject, and such cells can be cultured ex vivo and genetically modified to produce any of the subject engineered immune cells (eg, any engineered NK cell) disclosed herein. The engineered immune cells can then be administered to the subject for adaptive immunotherapy.

At least or at most about 1 dose, at least or at most about 2 doses, at least or at most about 3 doses, at least or at most about 4 doses, at least or at most about 5 doses, at least or at most about 6 doses, At least or at most about 7 doses, at least or at most about 8 doses, at least or at most about 9 doses, or at least or at most about 10 doses of the population of engineered immune cells (e.g., engineered NK cells) of the present disclosure The object is treated (eg administered).

In one aspect, the present disclosure provides a method comprising: (a) obtaining a cell from a subject; and (b) producing any of the engineered immune cells disclosed herein (eg, engineered NK cells) from the cell. In some cases, the cells obtained from the article are ESCs. In some cases, cells (eg, fibroblasts, eg, adult dermal fibroblasts) obtained from the subject are modified and converted into iPSCs.

In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising any of the engineered immune cells (eg, engineered NK cells) disclosed herein. In some cases, the method can further comprise administering to the subject a co-therapeutic agent (eg, a chemotherapeutic agent, an anti-CD20 antibody, etc.).

In one aspect, the present disclosure provides a method comprising administering any one of the compositions disclosed herein to a subject in need thereof. In some cases, a composition can include (i) any of the engineered immune cells disclosed herein (eg, engineered NK cells), and (ii) a combination therapeutic agent (eg, a chemotherapeutic agent, an anti-CD20 antibody, etc.).

Any of the methods disclosed herein can be used to treat a target cell, target tissue, target condition, or target disease of a subject.

The disease of interest may be a viral, bacterial, and/or parasitic infection; an inflammatory and/or autoimmune disease; or a neoplasm, such as cancer and/or tumor.

Target cells can be diseased cells. Diseased cells may have altered metabolism, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. A diseased cell can be a cell from a diseased object. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, and the like.

A variety of target cells can be killed using any of the engineered immune cells (eg, engineered NK cells) disclosed herein. Target cells can include a variety of cell types. Target cells can be in vitro. Target cells can be in vivo. Target cells can be ex vivo. Target cells can be isolated cells. A target cell may be a cell in an organism. A target cell can be an organism. Target cells can be cells in cell culture. The target cell can be one of a collection of cells. Target cells can be mammalian cells or derived from mammalian cells. The target cell can be a rodent cell or be derived from a rodent cell. Target cells can be human cells or derived from human cells. Target cells can be prokaryotic cells or derived from prokaryotic cells. The target cell may be a bacterial cell or may be derived from a bacterial cell. The target cell may be an archaeal cell or be derived from an archaeal cell. Target cells can be eukaryotic cells or derived from eukaryotic cells. Target cells can be pluripotent stem cells. The target cell can be a plant cell or be derived from a plant cell. Target cells can be animal cells or derived from animal cells. The target cell may be an invertebrate cell or be derived from an invertebrate cell. The target cell can be a vertebrate cell or be derived from a vertebrate cell. The target cell may be a microbial cell or be derived from a microbial cell. The target cell may be a fungal cell or be derived from a fungal cell. Target cells can be from specific organs or tissues.

Target cells can be stem cells or progenitor cells. Target cells can include stem cells (eg, adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (eg, cardiac progenitor cells, neural progenitor cells, etc.). Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, and the like. Clonal cells can include progeny of the cells. Target cells can include target nucleic acids. Target cells can be in living organisms. Target cells can be genetically modified cells. A target cell can be a host cell.

Target cells may be totipotent stem cells, however, in some embodiments of the present disclosure, the term "cell" may be used but may not denote totipotent stem cells. The target cell may be a plant cell, but in some embodiments of the present disclosure, the term "cell" may be used but may not refer to a plant cell. Target cells can be pluripotent cells. For example, a target cell may be a multipotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage, but may not be able to differentiate into any other non-hematopoietic cells. Target cells may be capable of developing into whole organisms. A target cell may or may not be able to develop into a whole organism. Target cells can be whole organisms.

Target cells can be primary cells. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15 or more times. A cell may be a unicellular organism. Cells can be grown in culture.

Target cells can be diseased cells. Diseased cells may have altered metabolism, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. A diseased cell can be a cell from a diseased object. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, and the like.

If the target cells are primary cells, they can be harvested from the individual by any means. For example, leukocytes can be harvested by apheresis, leukapheresis, density gradient separation, and the like. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Appropriate solutions may be used to disperse or suspend harvested cells. Such a solution may typically be a balanced salt solution (e.g., physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors and low concentrations of acceptable buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, and the like. Cells can be used immediately, or they can be stored (eg, by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, media buffer (eg, 10% DMSO, 50% serum, 40% buffered media), and/or some other such common solution for preserving cells at freezing temperatures.

Non-limiting examples of cells that may be target cells include, but are not limited to: lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells , cytokine-induced killer (CIK) cells (see for example US20080241194); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils/multilobed neutrophils), Monocytes/macrophages, erythrocytes (reticulocytes), mast cells, platelets/megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid (thyroid epithelium, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (chromaffin cells), pineal (pineal cells) cells; cells of the nervous system, including glial cells (astrocytes, microglia), giant cell neurosecretory cells, stellate cells, Bertscher cells, and pituitary gland (gonadotrophs, corticotropin-secreting cells, thyrotrophs, somatotrophs, prolactin-secreting cells); cells of the respiratory system, Including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including stomach (gastric chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage/bone/muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); chondrocytes, including chondroblasts, chondrocytes; skin cells , including trichocytes, keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, and mesangial/renal cells Extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells; reproductive system cells including spermatozoa, Sertoli cells, Leydig cells, eggs; and other cells including adipocytes, fibroblasts cells, tenocytes, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of fingernails and toenails, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, surface Hair shaft cells, epidermal root sheath cells, Huxley layer root sheath cells, Henle layer root sheath cells, outer root sheath cells, hair matrix cells (stem cells), moisture stratified barrier epithelial cells, (cornea, tongue, oral cavity, Surface epithelium of stratified squamous epithelium (of esophagus, anal canal, distal urethra, and vagina), basal cells (stem cells) of epithelium (of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina), urethra Epithelial cells (lining the bladder and urethra), exocrine epithelial cells, salivary gland mucous cells (polysaccharide-rich secretions), salivary gland serous cells (glycoproteinase-rich secretion), von Ebner gland cells in the tongue (taste bud wash), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumen gland cells in the ear (wax secretion Eccrine cells), eccrine dark cells (glycoprotein secretions), eccrine clear cells (small molecule secretions), apocrine sweat gland cells (odourous secretions, sensitive to sex hormones), eyelid mole gland cells (specialized sweat glands ), sebocytes (lipid-rich sebum secretion), nasal Bowman's glands (olfactory epithelium washings), duodenal Bruner's glands (enzymes and alkaline mucus), seminal vesicles (secrete semen components, including for sperm motility fructose), prostate cells (secrete semen components), bulbourethral gland cells (mucus secretion), Bartholin cells (vaginal lubricating secretion), Littley gland cells (mucus secretion), endometrial cells (carbohydrate secretion) secretions), isolated goblet cells of the respiratory and gastrointestinal tracts (mucus secretions), mucous cells of the gastric mucosa (mucus secretions), gastric gland zymogen cells (pepsinogen ), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), intestinal Paneth cells (lysozyme secretion), lung type II pneumocytes (surfactant secretion), lung Clara cells, hormone secretion cells, anterior pituitary cells, somatotroph cells, prolactin hormone secreting cells, thyrotroph cells, gonadotroph cells, corticotroph cells, middle pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid principal cells, eosinophils, adrenal cells, chromaffin cells, Leydig cells, follicular endometrial cells, luteal cells of ruptured follicles, Granular luteal cells, alveolar luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and genitourinary tract), kidney, Type I pneumocytes (lined air spaces in the lungs), pancreatic duct cells (alveolar heart cells), non-striated duct cells (of sweat glands, salivary glands, breast, etc.), duct cells (of seminal vesicles, prostate, etc.), duct cells that close internal body cavities Epithelial cells, ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes , connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in central nervous system), blood and immune system ( Various types) of neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells , reticulocytes, stem cells and committed progenitor cells, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory sensor cells , autonomic neuronal cells, sensory organs and peripheral neuronal support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelium, germ cells, oogonia/oocytes , spermatids, spermatocytes, spermatogonia (spermatocyte stem cells), spermatozoa, feeder cells, follicular cells, Sertoli cells (in the testis), thymic epithelial cells, mesenchymal cells, and mesenchymal kidney cells.

Of particular interest are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include cancer cells including: acanthoma, acinar cell carcinoma, acoustic neuroma, acral melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, enamel epithelioma, adenocarcinoma, adenoid cyst Sexual carcinoma, adenoma, odontogenic adenomatoid tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal Carcinoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix carcinoma, astrocytoma, atypical teratoid rhabdoid tumor, basal Cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract carcinoma, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner's tumor, bronchial neoplasm, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, carcinoma of unknown primary (cancer), carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, unknown primary Carcinoma, carcinosarcoma, giant lymph node hyperplasia, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, Choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, chronic neutrophil leukemia, clear cell neoplasm, colon cancer, colorectal cancer, craniopharynx cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, hyperplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma , endometrial sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymal blastoma, ependymoma, epithelioid sarcoma, erythroleukemia , Esophageal cancer, Olfactory neuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer , Fetus in Fetus, Fibroma, Fibrosarcoma, Follicular Lymphoma, Follicular Thyroid Cancer, Gallbladder Cancer, Gallbladder Cancer, Ganglioglioma, Gangliocytoma, Gastric Cancer, Gastric Lymphoma, Gastrointestinal Cancer, Gastrointestinal Tract carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma , glioma, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma , angiosarcoma, hematologic malignancies Tumors, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal carcinoma, hypothalamic glioma, inflammatory breast cancer, eye Intrinsic melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, kidney cancer, Kratskin's tumor, Krucken Berger's tumor, laryngeal cancer, laryngeal cancer, lentiginous melanoma, leukemia, leukaemia, lip and mouth cancer, liposarcoma, lung cancer, luteinoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia , lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant salamander Newt tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meninges tumor, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck carcinoma, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous Sexual neoplasms, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, Myeloproliferative disorders, myxoma, nasal cavity carcinoma, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, neuronoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgies Gold lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumors, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer , oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, breast Paget's disease, lung superior sulcus tumor, pancreatic cancer, pancreatic cancer , papillary thyroid carcinoma, papillomatosis, paraganglioma, paranasal sinus carcinoma, parathyroid carcinoma, penile carcinoma, perivascular epithelioid cell tumor, pharyngeal carcinoma, pheochromocytoma, pineal differentiation Solid tumors, pinealoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, multiple embryonal tumor, precursor T lymphoblastic lymphoma, primary central nervous system Lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, Respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannoma, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous adenoma, branch Aid - Stromal cell tumor, sex cord stromal cell tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small bowel cancer, soft tissue sarcoma , somatostatoma, sooty warts, spinal cord tumors, spinal cord tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoma, testicular cancer, alveoli Cell tumor, laryngeal carcinoma, thymic carcinoma, thymoma, thyroid carcinoma, renal pelvis and ureter transitional cell carcinoma, transitional cell carcinoma, urachal carcinoma, urethral carcinoma, genitourinary tract neoplasm, uterine sarcoma, uveal melanoma, vaginal carcinoma, Verner Morrison Syndrome, Verrucous Carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenström's Macroglobulinemia, Warthin's Tumor, Wilms' Tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a population of cancer cells, such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, such as lymphoma. The antigen may be a tumor-associated antigen.

In some instances, a target cell (eg, a B cell) disclosed herein is associated or suspected of being associated with an autoimmune disease. A subject treated with any one of the engineered immune cells (eg, engineered NK cells) of the present disclosure may have or may be suspected of having an autoimmune disease.

Non-limiting examples of autoimmune diseases may include: acute diffuse encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic Rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant rejection, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, Autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune Retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Barrow's disease, Behcet's disease, bullous pemphigoid, Cardiomyopathy, giant lymphadenopathy, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome, Cicatricial Pemphigoid/Benign Mucosal Pemphigoid, Crohn's Disease, Cogans Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie's Myocarditis, CREST Disease, Idiopathic Mixed Cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devick's disease (neuromyelitis optica), discoid lupus, Dresler syndrome, endometriosis , eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, pulmonary Hemorrhage-nephritic syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Bali syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes of pregnancy, hypogamma Proteinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent Diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis , linear IgA disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Murren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis , recurrent rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disease associated with streptococcal bacteria), paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, pars plana (peripheral uveitis), pemphigus, peripheral nerve Lesions, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyinflammation nodosa, autoimmune polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, gangrenous pus Dermatosis, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis , sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susak's syndrome, Sympathetic ophthalmia, Taurus arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease ( UCTD), uveitis, vasculitis, vesicular bullous dermatosis, vitiligo, Waldenström macroglobulinemia (WM), and Wegener's granulomatosis (Granuloma with polyangiitis (GPA)).

In some instances, the autoimmune disease comprises one or more members selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple Sclerosis, scleroderma, Addison's disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergamma Proteinemia, monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia (WM), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Hashimoto's encephalopathy (HE), Hashimoto's Thyroiditis, Graves' disease, Wegener's granulomatosis, and antibody-mediated transplant rejection (eg, for tissue transplants, such as kidney transplants). In examples, the autoimmune disease may be type 1 diabetes, lupus or rheumatoid arthritis.

In some instances, the target disease is acute myeloid leukemia (AML). For example, any engineered immune cell (for example, engineered NK cell) disclosed herein can be administered to an object in need to treat AML, and the engineered immune cell includes one or more of the following: (i) comprising: A chimeric polypeptide receptor of an antigen-binding domain that binds an antigen disclosed herein (eg, CD33), (ii) a heterologous cytokine disclosed herein (eg, IL-15), and (iii) a method disclosed herein for enhancing CD16 variants of CD16 signaling.

In some instances, the target disease is non-Hodgkin's lymphoma (NHL).

In some instances, the target disease is chronic lymphocytic leukemia (CLL).

In some instances, the disease of interest is B-cell leukemia (BCL). For example, any engineered immune cell (for example, engineered NK cell) disclosed herein can be administered to an object in need to treat BCL, and the engineered immune cell includes one or more of the following: (i) A chimeric polypeptide receptor comprising an antigen binding domain capable of binding CD19, (ii) a heterologous cytokine disclosed herein (e.g., IL-15), and (iii) a CD16 disclosed herein for enhanced CD16 signaling Variants.

In some instances, the target disease is non-small cell lung cancer (NSCLC).

In some instances, target cells form tumors (ie, solid tumors). Tumors treated with the methods herein can result in stable tumor growth (eg, one or more tumors that do not increase in size by more than 1%, 5%, 10%, 15%, or 20%, and/or do not metastasize). In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some instances, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some instances, tumors were completely eliminated or reduced below detection levels. In some instances, the subject remains tumor-free (eg, in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after treatment. In some instances, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after treatment. In some instances, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment. [Example] Example 1 : Engineered immune cells configured to target immune cell antigens

The engineered immune cells (eg, engineered NK cells) of the present disclosure can be configured to specifically target and bind immune cells (eg, diseased immune cells) of a subject, eg, to treat leukemia or lymphoma. In some embodiments, engineered immune cells can include one or more chimeric polypeptide receptors (e.g., chimeric antigen receptors) that exhibit association with at least one immune cell antigen, e.g., CD3, CD4, CD8, CCR7, CD45RO , CR45RA, CD45RA, T cell receptor (TCR), modifications thereof and specific binding of fragments thereof. In some embodiments, one or more chimeric polypeptide receptors can include an antigen binding domain directed against at least one immune cell antigen, and the antigen binding domain can include an ankyrin repeat domain. In some cases, an antigen binding domain can be based on a DARPIn polypeptide or a modification thereof.

For example, engineered NK cells can include a CAR that includes an anti-CD4 DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (eg, 100%) sequence identity.

SEQ ID NO: 3 : MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAMDHFGFTPLHLAAKVGHLEIVEVLLKYGADVNADDMDGETPLHLAAAIGHLEIVEVLLKNGADVNAHDTWGFTPLHLAASYGHLEIVEVLRKYGADVNAXDKFGETTFDISIDNGNEDLXEILQKLN

In another example, engineered NK cells can include a CAR that includes an anti-CD4 DARPin that exhibits at least about 60%, at least about 65%, at least about 70% of the polynucleotide sequence of SEQ ID NO: 4 %, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% %, at least about 97%, at least about 98%, at least about 99% or more (eg, 100%) sequence identity.

SEQ ID NO: 4 : MRGSHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAIDLYGKTPLHLAAQWGHLEIVEVLLKYCADVNAADGDGMTPLHLAAWNGHLEIVDVLLKHGADVNAQDKFGKTAFDISINNNGNEDLAEILQKLN Example 2 : Engineered Immune Cells Configured to Target Cancer Cell Antigens

The engineered immune cells (eg, engineered NK cells) of the present disclosure can be configured to specifically target and bind immune cells (eg, diseased immune cells) of a subject, eg, to treat leukemia or lymphoma. In some embodiments, engineered immune cells can include one or more chimeric polypeptide receptors (eg, chimeric antigen receptors) that exhibit specific binding to at least one cancer cell antigen, eg, CD19, HER2, EGFR, etc. In some embodiments, one or more chimeric polypeptide receptors can include an antigen binding domain directed against at least one immune cell antigen, and the antigen binding domain can include an ankyrin repeat domain. In some cases, an antigen binding domain can be based on a DARPIn polypeptide or a modification thereof.

For example, engineered NK cells can include a CAR that includes an anti-HER2 DARPin that exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (eg, 100%) sequence identity.

SEQ ID NO: 5 : MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYLATAHGHLEIVEVLLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKTAFDISIGNGNEDLAEILQKLN

In another example, engineered NK cells can include a CAR that includes an anti-EGFR DARPin that exhibits the same expression as selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: The polynucleotide sequence of a member of 9 has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least About 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more (e.g., 100%) sequence identity .

SEQ ID NO: 6 : DLGKKLLEAARAGQDDEVRILMANGADVNADDTWGWTPLHLAAYQGHLEIVEVLLKNGADVNAYDYIGWTPLHLAADGHLEIVEVLLKNGADVNASDYIGDTPLHLAAHNGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNNGNEDLAEILQ

SEQ ID NO: 7 : DLGKKLLEAARAGQDDEVRILMANGADVNATDNDGNTPLHLSAWIGHLEIVEVLLKHGADVNADDLLGMTPLHLAADTGHLEIVEVLLKYGADVNARDTRGKTPLHLAARDGHLEIVEVLLKHDADVNAQDKFGKTAFDISIDNGNEDLAEILQ

SEQ ID NO: 8 : DLGKKLLEAARAGQDDEVRILMANGADVNAFDYWGMTPLHLAADNGHLEIVEVLLKHGADVNASDNFGFTPLHLAAFYGHLEIVEVLLKHGADVNAFDMWGNTPLHLAAQNGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQ

SEQ ID NO: 9 ： DLGKKLLEAARAGQDDEVRILMANGADVNADDNAGRTPLHLAANFGHLEIVEVLLKNGADVNAKGHHENTPLHLAAWAGHLEIVEVLLKYGADVNADDDEGYTPLHLAADIGDLEIVEVLLKYGADVNAWDMYGRTPLHLAASAGHLEIVEVLLKYGADVNAQDKFGKTAFDISIDNGNEDLAEILQ實施例 3 ：工程化 NK 細胞 通過基因敲入生成工程化 NK 細胞

Human iPSC cells can be engineered by knock-in gene edits such as chimeric antigen receptors (CARs) that include DARPin domains (eg, anti-HER2 DARPins). This engineered iPSC cells can be differentiated into NK cells. Alternatively, peripheral blood (PB)-NK cells (eg, human PB-NK cells) can be engineered with an AAV system encoding a CAR including at least a DARPin domain. Cells disclosed herein can be engineered to further include (i) heterologous CD16 variants (eg, hnCD16) and/or (ii) heterologous cytokines and/or their receptors (eg, IL15-IL15Ra fusions). In vitro antitumor activity of HER2- targeted CAR

A variety of breast cancer cell lines, including HER2 negative cell lines (LCL lymphoma, MDA-MB-468, U87 glioma), low HER2 expression cell lines (MDA-MB-361, 231BR) and high HER2 expression cell lines (SKBR3 , BT474, BBM1), which can be used to characterize the in vitro anti-tumor activity of engineered NK cells expressing CARs including anti-HER2 DARPins. Flow cytometry can be used to assess the effect of co-culture of engineered NK cells including anti-HER2 DARPin CAR, or control NK cells without CAR, or control NK cells with CAR including anti-HER2 scFv, with tumor targets (e.g., 72 hours). Tumor cell killing after co-culture). For the CAR NK cells disclosed herein, tumor cell killing can be measured at effector:HER2 positive cells (i.e., tumor cells) (E:T) ratios of 0.25:1 to 2:1. Engineered NK cells including an anti-HER2 DARPin CAR could be more effective in tumor cell killing in vitro compared to control NK cells without CAR. Engineered NK cells comprising an anti-HER2 DARPin CAR could be as effective (if not more effective) in tumor cell killing in vitro than control NK cells with a CAR comprising an anti-HER2 scFv. In vivo antitumor activity of HER2- targeted CAR

The activity of engineered NK cells including an anti-HER2 DARPin CAR can be assessed in a patient-derived breast-to-brain metastasis model. Mice can be treated by administering engineered NK cells comprising an anti-HER2 DARPin CAR, or control NK cells without a CAR, or control NK cells with a CAR comprising an anti-HER2 scFv. Optical imaging of tumors and Kaplan-Meier survival curves can be used to analyze treated mice (eg, topical or intravenous treatment, at days 3, 8, or 14 after engineered NK cell administration).

To assess antitumor efficacy in a human xenograft model of breast to brain metastasis, NSG mice can be injected (eg, intracranially) with BBM1 cells (eg, 0.2 M) or BT474 (eg, 0.15 M). On day 8 after tumor injection, the engineered NK cells herein (or control NK cells) can be administered (eg, intratumoral administration). BBM1 and/or BT474 tumors can be monitored by luciferase-based optical imaging. Kaplan Meier curves can be used to represent optical imaging data. Embodiment 4 : Engineered NK cells generate engineered NK cells by gene knock-in

NK cells (e.g., NK92 cell line) can be modified by knocking in a gene edit (e.g., delivered via AAV) (e.g., including a DARPin domain (e.g., anti-EGFR DARPin, e.g., the polynucleotide sequence of SEQ ID NO: 6). Synthetic Antigen Receptor (CAR)) to engineer. Cells disclosed herein can be engineered to further include (i) heterologous CD16 variants (eg, hnCD16) and/or (ii) heterologous cytokines and/or their receptors (eg, IL15-IL15Ra fusions).

The ability of engineered NK cells to induce target cell killing (eg, killing in vitro) can be assayed. Target cells for killing assays can include K562 cells expressing the target antigen (eg, EGFR+ K562 cells). The E:T (effector:target) ratio of the killing assay can range from 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 to 10. Following incubation (eg, for 4 hours) of target cells with engineered NK cells, the corresponding percentage of specific killing (target cell lysis) can be calculated. CAR construct

For example, NK92 cells are engineered to express a CAR comprising an extracellular domain comprising an anti-EGFR DARPin comprising the polynucleotide sequence of SEQ ID NO: 6 (i.e. NK92-E01), so that Specifically target and kill cells expressing EGFR, such as EGFR+ K562 cells. See illustration in Figure 2.

NK92 cells were engineered by knocking in genetic constructs (shown as "DNA Construct" in Figure 3). The genetic construct encodes a CAR under the control of a constitutive promoter in mammalian systems (eg, SV40, CMV, UBC, EF1a, PGK, and CAGG), such as the human EF1a promoter. The genetic construct further encodes a leader sequence (e.g., CD8 leader sequence), an anti-EGFR DARpin (i.e., E01-DARPin), a linker (e.g., a GGGS _e linker, such as 2xGGGS), a transmembrane domain (e.g., a CD8 transmembrane structure domain) and two intracellular signaling domains (e.g., 2B4 and CD3ζ) CAR. This genetic construct further encodes a reporter gene (eg, EGFP reporter) under the control of an internal ribosome entry site (IRES), such that the reporter will be cleaved from the CAR after transcription and translation.

The polynucleotide sequence comprising the CAR against EGFR DARPin is shown in SEQ ID NO: 10. The polynucleotide sequence of SEQ ID NO: 10 includes CD8 leader sequence (SEQ ID NO: 11), anti-EGFR DARPin (SEQ ID NO: 6), linker (SEQ ID NO: 12), CD8 transmembrane domain (SEQ ID NO::13), 2B4 cytoplasmic signaling domain (SEQ ID NO: 14) and CD3ζ signaling domain (SEQ ID NO: 15).

SEQ ID NO: 10 ： MALPVTALLLPLALLLHAARPDLGKKLLEAARAGQDDEVRILMANGADVNADDTWGWTPLHLAAYQGHLEIVEVLLKNGADVNAYDYIGWTPLHLAADGHLEIVEVLLKNGADVNASDYIGDTPLHLAAHNGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQGGGGSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO: 11 : MALPVTALLLLPLALLLLHAARP

SEQ ID NO: 12 : GGGGSGGGGS

SEQ ID NO: 13 : TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLLSVITLYC

SEQ ID NO: 14 : WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS

SEQ ID NO: 15 : Cytotoxicity of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR to EGFR+ target cells

The in vitro cell killing efficacy of engineered NK92-E01 cells was evaluated, as shown in Figure 4. Engineered NK92-E01 cells were cultured with EGFR+ K562 cancer cells (ie NK92-E01:K562-EGFR). For controls, engineered NK92-E01 cells were cultured with K562 cells that had essentially no EGFR expression (ie, NK92-E01:K562). For another control, NK92 cells that do not express the anti-EGFR DARPin CAR were cultured with EGFR+ K562 cells (ie, NK92:K562-EGFR) or with K562 cells that did not substantially express EGFR (ie, NK92:K562). The E:T (effector:target) ratios tested for the killing assay were 4:1, 2:1, 1:1, 0.5:1 and 0.25:1. As shown in FIG. 5 , there were multiple E:T ratios (e.g., 4:1, 2 :1, 1:1, and 0.5:1), the highest degree of target cell killing (i.e., % of cells lysed) was observed, suggesting that (i) NK cells expressing CARs including EGFR-specific DARPins can efficiently kill EGFR-expressing cancers/ Tumor cells, and (ii) NK cells expressing CARs including DARPins targeting specific antigens can effectively bind and kill cancer cells/tumor cells expressing the specific antigens.

While preferred embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of example only. It is intended that the invention not be limited to the specific examples provided in the specification. While the invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations or relative proportions set forth herein which are subject to various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be better understood by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, together with the accompanying drawings (also referred to herein as "the Figures") in which:

Figure 1 schematically illustrates an exemplary chimeric polypeptide receptor comprising an antigen binding portion comprising one or more ankyrin repeat domains (e.g. DARPins), wherein the one or more ankyrin repeat Domains are designed to specifically target (eg, bind) one or more antigens.

Figure 2 schematically illustrates the killing of antigen-expressing target cells by NK cells expressing a CAR that includes a designed antigen-specific ankyrin repeat protein (DARPin) domain.

Figure 3 schematically illustrates an exemplary polynucleotide construct encoding a CAR comprising a DARPin domain directed against epidermal growth factor receptor (EGFR).

Figure 4 schematically illustrates an exemplary cell killing assay between various NK cells and various target cells.

Figure 5 shows the in vitro killing efficacy of engineered NK cells on target cancer cells.

            <![CDATA[<110> HANGZHOU QIHAN BIOTECHNOLOGY CO., LTD.)]]> <![CDATA[<120> Chimeric antigen receptors in immune cells] ]> <![CDATA[<130> 54809-715.851]]> <![CDATA[<140> TW 111106837]]> <![CDATA[<141> 2022-02-24]]> <![CDATA[ <150> PCT/CN2021/077674]]> <![CDATA[<151> 2021-02-24]]> <![CDATA[<160> 17 ]]> <![CDATA[<170> PatentIn version 3.5 ]]> <![CDATA[<210> 1]]> <![CDATA[<211> 33]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence] ]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptides]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (2)..(3)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> < ![CDATA[<221> MOD_RES]]> <![CDATA[<222> (5)..(5)]]> <![CDATA[<223> any amino acid]]> <![CDATA[ <220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (12)..(14)]]> <![CDATA[<223> any amino acid] ]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (16)..(18)]]> <![CDATA[< 223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (21)..(21)]] > <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222 > (25)..(26)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (33)..(33)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<400> 1]]> Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Xaa Xaa Xaa Gly Xaa 1 5 10 15 Xaa Xaa Val Val Xaa Leu Leu Leu Xaa Xaa Gly Ala Asp Val Asn Ala 20 25 30 Xaa <![CDATA[<210> 2]]> <! [CDATA[<211> 33]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA [<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (2).. (3)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[< 222> (5)..(5)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]] > <![CDATA[<222> (12)..(14)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA [<221> MOD_RES]]> <![CDATA[<222> (16)..(18)]]> <![CDATA[<223> any amino acid]]> <![CDATA[<220> ]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (21)..(21)]]> <![CDATA[<223> any amino acid]]> < ![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (25)..(26)]]> <![ CDATA[<223> any amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (33)..(33 )]]> <![CDATA[<223> any amino acid]]> <![CDATA[<400> 2]]> Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Xaa Xaa Xaa Gly Xaa 1 5 10 15 Xaa Xaa Ile Val Xaa Val Leu Leu Xaa Xaa Gly Ala Asp Val Asn Ala 20 25 30 Xaa <![CDATA[<210> 3]]> <![CDATA[<211> 169]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> < ![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (142)..(142)]]> <![CDATA[<223> any Amino acid]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES]]> <![CDATA[<222> (162)..(162)]]> <! [CDATA[<223> any amino acid]]> <![CDATA[<400> 3]]> Met Arg Gly Ser His His His His His His His Gly Ser Asp Leu Gly Lys 1 5 10 15 Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile 20 25 30 Leu Met Ala Asn Gly Ala Asp Val Asn Ala Met Asp His Phe Gly Phe 35 40 45 Thr Pro Leu His Leu Ala Ala Lys Val Gly His Leu Glu Ile Val Glu 50 55 60 Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala Asp Asp Met Asp Gly 65 70 75 80 Glu Thr Pro Leu His Leu Ala Ala Ala Ile Gly His Leu Glu Ile Val 85 90 95 Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala His Asp Thr Trp 100 105 110 Gly Phe Thr Pro Leu His Leu Ala Ala Ser Tyr Gly His Leu Glu Ile 115 120 125 Val Glu Val Leu Arg Lys Tyr Gly Ala Asp Val Asn Ala Xaa Asp Lys 130 135 140 Phe Gly Glu Thr Thr Phe Asp Ile Ser Ile Asp Asn Gly Asn Glu Asp 145 150 155 160 Leu Xaa Glu Ile Leu Gln Lys Leu Asn 165 <![CDATA[<210> 4]]> <![CDATA[<211> 136]]> <![CDATA[<212> PRT]]> <![CDATA [<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 4]]> Met Arg Gly Ser His His His His His His Gly Ser Asp Leu Gly Lys 1 5 10 15 Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile 20 25 30 Leu Met Ala Asn Gly Ala Asp Val Asn Ala Ile Asp Leu Tyr Gly Lys 35 40 45 Thr Pro Leu His Leu Ala Ala Gln Trp Gly His Leu Glu Ile Val Glu 50 55 60 Val Leu Leu Lys Tyr Cys Ala Asp Val Asn Ala Ala Asp Gly Asp Gly 65 70 75 80 Met Thr Pro Leu His Leu Ala Ala Trp Asn Gly His Leu Glu Ile Val 85 90 95 Asp Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe 100 105 110 Gly Lys Thr Ala Phe Asp Ile Ser Ile Asn Asn Gly Asn Glu Asp Leu 115 120 125 Ala Glu Ile Leu Gln Lys Leu Asn 130 135 <![CDATA[< 210> 5]]> <![CDATA[<211> 136]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 5]]> Met Arg Gly Ser His His His His His His His Gly Ser Asp Leu Gly Lys 1 5 10 15 Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Ile 20 25 30 Leu Met Ala Asn Gly Ala Asp Val Asn Ala Lys Asp Glu Tyr Gly Leu 35 40 45 Thr Pro Leu Tyr Leu Ala Thr Ala His Gly His Leu Glu Ile Val Glu 50 55 60 Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Val Asp Ala Ile Gly 65 70 75 80 Phe Thr Pro Leu His Leu Ala Ala Phe Ile Gly His Leu Glu Ile Ala 85 90 95 Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Gln Asp Lys Phe 100 105 110 Gly Lys Thr Ala Phe Asp Ile Ser Ile Gly Asn Gly Asn Glu Asp Leu 115 120 125 Ala Glu Ile Leu Gln Lys Leu Asn 130 135 <![CDATA[<210> 6]]> <![CDATA[<211> 153]]> <![CDATA[<212> PRT]]> <![CDATA [<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 6]]> Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asp Asp 20 25 30 Thr Trp Gly Trp Thr Pro Leu His Leu Ala Ala Tyr Gln Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Tyr 50 55 60 Asp Tyr Ile Gly Trp Thr Pro Leu His Leu Ala Ala Asp Gly His Leu 65 70 75 80 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ser 85 90 95 Asp Tyr Ile Gly Asp Thr Pro Leu His Leu Ala Ala His Asn Gly His 100 1 05 110 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 115 120 125 Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn Gly 130 135 140 Asn Glu Asp Leu Ala Glu Ile Leu Gln 145 150 <![CDATA[<210> 7]]> <![CDATA[<211> 154]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptides]]> <![CDATA[<400> 7]]> Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Thr Asp 20 25 30 Asn Asp Gly Asn Thr Pro Leu His Leu Ser Ala Trp Ile Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Asp 50 55 60 Asp Leu Leu Gly Met Thr Pro Leu His Leu Ala Ala Asp Thr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Arg Asp Thr Arg Gly Lys Thr Pro Leu His Leu Ala Ala Arg Asp Gly 100 105 110 His Leu Glu Ile Val Glu Val Leu Leu Lys His Asp Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln 145 150 <![CDATA[<210> 8]]> <![CDATA[<211> 154]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 8]]> Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Phe Asp 20 25 30 Tyr Trp Gly Met Thr Pro Leu His Leu Ala Ala Asp Asn Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala Ser 50 55 60 Asp Asn Phe Gly Phe Thr Pro Leu His Leu Ala Ala Phe Tyr Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys His Gly Ala Asp Val Asn Ala 85 90 95 Phe Asp Met Trp Gly Asn Thr Pro Leu His Leu Ala Ala Gln Asn Gly 100 105 110 His Leu G lu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn 115 120 125 Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn 130 135 140 Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln 145 150 <! [CDATA[<210> 9]]> <![CDATA[<211> 187]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![ CDATA[<220>]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 9]]> Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp 1 5 10 15 Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Asp Asp 20 25 30 Asn Ala Gly Arg Thr Pro Leu His Leu Ala Ala Asn Phe Gly His Leu 35 40 45 Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Lys 50 55 60 Gly His His Glu Asn Thr Pro Leu His Leu Ala Ala Trp Ala Gly His 65 70 75 80 Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn Ala 85 90 95 Asp Asp Asp Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Asp Ile Gly 100 105 110 Asp Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val Asn 115 120 125 Ala Trp Asp Met Tyr Gly Arg Thr Pro Leu His Leu Ala Ala Ser Ala 130 135 140 Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly Ala Asp Val 145 150 155 160 Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp 165 170 175 Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln 180 185 <![CDATA[<210> 10]]> <![CDATA [<211> 485]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 10]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg 20 25 30 Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp 35 40 45 Val Asn Ala Asp Asp Thr Trp Gly Trp Thr Pro Leu His Leu Ala Ala 50 55 60 Tyr Gln Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala 65 70 75 80 Asp Val Asn Ala Tyr Asp Tyr Ile Gly Trp Thr Pro Leu His Leu Ala 85 90 95 Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala 100 105 110 Asp Val Asn Ala Ser Asp Tyr Ile Gly Asp Thr Pro Leu His Leu Ala 115 120 125 Ala His Asn Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His Gly 130 135 140 Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile 145 150 155 160 Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Gly Gly 165 170 175 Gly Gly Ser Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro 180 185 190 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 195 200 205 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 210 215 220 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 225 230 235 240 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Trp Arg Arg 245 250 255 Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu Phe Leu Thr 260 265 270 Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn His Glu Gln 275 280 285 Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser Met Ile Gln 290 295 300 Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr Thr Leu Tyr 305 310 315 320 Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys Arg Asn His 325 330 335 Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly Lys Ser Gln 340 345 350 Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu Leu Glu Asn 355 360 365 Phe Asp Val Tyr Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <![CDATA[<210> 11]] > <![CDATA[<211> 21]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> < ![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 11]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <![CDATA[<210> 12]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA [<400> 12]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <![CDATA[<210> 13]]> <![CDATA[<211> 69]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> < ![CDATA[<400> 13]]> Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys 65 <![CDATA[< 210> 14]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptides]]> <![CDATA[<400> 14]]> Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu 1 5 10 15 Phe Leu Thr Ile Tyr Glu As p Val Lys Asp Leu Lys Thr Arg Arg Asn 20 25 30 His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser 35 40 45 Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr 50 55 60 Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys 65 70 75 80 Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly 85 90 95 Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu 100 105 110 Leu Glu Asn Phe Asp Val Tyr Ser 115 120 <![CDATA[<210> 15]]> <![CDATA[<211> 112]]> <![CDATA[<212 > PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![ CDATA[<400> 15]]> Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <![ CDATA[<210> 16]]> <![CDATA[<211> 4]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptides]]> <![CDATA[<220>]]> <![CDATA[<223> About Substitutions and Best Practices For example, see the submitted instructions]]> <![CDATA[<400> 16]]> Gly Gly Gly Ser 1 <![CDATA[<210> 17]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 17]] > Gly Gly Gly Ser Gly Gly Gly Ser 1 5
      

Claims (33)

A kind of engineering NK cell, it comprises: A chimeric polypeptide receptor comprising an antigen binding portion capable of binding to an antigen, wherein the antigen binding portion comprises an ankyrin repeat domain. The engineered NK cell according to claim 1, wherein the antigen comprises EGFR or a fragment thereof. The engineered NK cell according to claim 2, wherein the antigen-binding portion comprises an amino acid sequence having at least 80% identity to the polypeptide sequence of SEQ ID NO: 6. The engineered NK cell according to claim 2, wherein the antigen-binding portion comprises an amino acid sequence having at least 90% identity to the polypeptide sequence of SEQ ID NO: 6. The engineered NK cell according to claim 2, wherein the chimeric polypeptide receptor comprises an amino acid sequence having at least 80% identity to the polypeptide sequence of SEQ ID NO: 10. The engineered NK cell according to claim 2, wherein the chimeric polypeptide receptor comprises an amino acid sequence having at least 90% identity to the polypeptide sequence of SEQ ID NO: 10. The engineered NK cell according to claim 1, wherein the antigen includes immune cell antigens of immune cells other than NK cells. The engineered NK cell according to claim 7, wherein the immune cell is a T cell. The engineered NK cell according to claim 7, wherein the antigen is selected from CD3, CD4, CD8, CCR7, CD45RO, CR45RA and fragments thereof. The engineered NK cell according to claim 9, wherein the antigen is CD3. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises NY-ESO-1 or a fragment thereof. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises CD33 or a fragment thereof. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises CD123 or a fragment thereof. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises a cytokine or a fragment thereof. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises cytokine receptors or fragments thereof. The engineered NK cell according to any one of the preceding claims, wherein the cytokine is an interleukin. The engineered NK cell according to claim 16, wherein the interleukin is selected from IL2, IL3, IL7, IL12, IL15, IL18 and IL21. The engineered NK cell according to any one of the preceding claims, wherein the antigen comprises an intracellular antigenic peptide. The engineered NK cell according to claim 18, wherein the intracellular antigen peptide is presented by major histocompatibility complex. The engineered NK cell according to any one of the preceding claims, wherein said antigen binding portion is part of the extracellular domain of said chimeric polypeptide receptor. The engineered NK cell of any one of the preceding claims, wherein the chimeric polypeptide receptor comprises a plurality of different antigen binding moieties capable of binding to a plurality of different antigens. The engineered NK cell according to any one of the preceding claims, further comprising an additional chimeric polypeptide receptor comprising an additional antigen-binding moiety capable of binding to an additional antigen, wherein the additional The antigen is different from said antigen. The engineered NK cell according to any one of the preceding claims, wherein said chimeric polypeptide receptor or said one or more chimeric polypeptide receptors comprises a chimeric antigen receptor. The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is derived from hematopoietic cells obtained from a peripheral blood sample of the subject. The engineered NK cell of any one of the preceding claims, wherein the engineered NK cell is derived from NK cells obtained from a peripheral blood sample of a subject. The engineered NK cell according to any one of the preceding claims, wherein said engineered NK cell is derived from an isolated stem cell. The engineered NK cell according to claim 26, wherein the isolated stem cells are embryonic stem cells. The engineered NK cell according to claim 26, wherein the isolated stem cells are induced pluripotent stem cells. A composition comprising the engineered NK cells according to any one of the preceding claims. The composition according to claim 29, further comprising a single therapeutic agent. The composition of claim 30, wherein said separate therapeutic agents comprise different antigen binding moieties. The composition of claim 30, wherein said separate therapeutic agent comprises a chemotherapeutic agent. A method comprising applying the composition of any one of the preceding claims to an object in need thereof.

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