Engineering of immune cells for ex vivo cell therapy applications

Abstract

To provide compositions and methods that facilitate transfection of cells.SOLUTION: Methods and compositions containing ethanol and an isotonic salt solution are used for delivery of compounds and compositions to non-adherent cells, for example, T cells.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 897,250, filed September 6, 2019, U.S. Provisional Application No. 63 / 022,944, filed May 11, 2020, and U.S. Provisional Application No. 63 / 047,054, filed July 1, 2020, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to the delivery of drugs to mammalian cells for immunotherapy. [Background technology]

[0003] background Cell transfection efficiency varies among different cell types. Transfection of suspension cells, e.g., non-adherent cells, using conventional methods has proven difficult. Therefore, there is a need for compositions and methods that facilitate the transfection of such cells. Furthermore, there is a need for improved manufacturing strategies aimed at ensuring the efficacy of immune cells and cell therapy. Summary of the Invention

[0004] The present invention provides a solution to the engineering of immune cells for ex vivo cell therapy applications. The compositions and methods described herein facilitate cell engineering techniques that enable next-generation cell therapy products requiring complex modifications and high levels of cellular function. The SOLUPORE™ delivery method described herein is a non-viral means of delivering cargo to primary immune cells simply, quickly, and efficiently while preserving cell viability and function. Furthermore, the use of these engineered immune cells, e.g., T cells, reduces the likelihood of T cell exhaustion, thus enabling the use of these engineered immune cells for complex therapeutic needs.

[0005] Thus, provided herein are immune cells (or immune cell populations), e.g., T cells, natural killer (NK) cells, B cells, macrophages, or other immune cells, containing exogenous cargo, wherein the immune cells bearing the exogenous cargo have a molecular profile with a gene or protein expression level within a log2 fold change of 3 24 hours after cargo delivery compared to the gene or protein expression level in control immune cells 24 hours after cargo delivery. The gene or protein is a member of the activator protein 1 (AP-1) signaling pathway, and the molecular profile is independent of the type of cargo delivered. The control immune cells are immune cells that have not undergone a cell manipulation or activation process. For example, the control immune cells have not been manipulated using electroporation, viral transduction, or other methods of delivering cargo to cells (including the SOLUPORE™ method).

[0006] For example, the molecular profile of immune cells containing exogenous cargo has gene or protein expression within a log2 fold change of 3, or within a log2 fold change of 2, or within a log2 fold change of 1 compared to the level of the gene or protein in control immune cells. For example, the molecular profile (gene expression profile) is assessed 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours after cargo delivery, or about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours after cargo delivery. Alternatively, the molecular profile of immune cells containing the exogenous cargo has gene or protein expression within a log2 fold change of 3, or within a log2 fold change of 2, or within a log2 fold change of 1 relative to the level of the gene or protein in control immune cells at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 4 weeks, 1 month, 2 months, 3 months, or 4 months after cargo delivery.

[0007] The cell manipulation process may include electroporation (also called electrotransfer), which is a process of applying an electric field to cells to increase cell membrane permeability. Additionally, the cell manipulation process may refer to any known transfection method for intracellular delivery, including the SOLUPORE™ delivery method, membrane disruption methods (electroporation, sonoporation, magnetofection, optoporation), or carrier-based methods (e.g., lipid nanoparticles).

[0008] Molecular profile refers to the gene expression, genomic profile, protein expression, protein activity, or proteomic profile of an immune cell. In other examples, immune cells with exogenous cargo have a molecular profile with gene or protein expression levels within a log2 fold change of 2 relative to the expression levels of the genes or proteins in control immune cells. In further examples, immune cells with exogenous cargo have a molecular profile with gene or protein expression levels within a log2 fold change of 1 relative to the expression levels of the genes or proteins in control immune cells.

[0009] In embodiments, the exogenous cargo of the immune cell comprises a nucleic acid, a small molecule, a protein, a polypeptide, or a combination thereof. For example, the nucleic acid comprises a messenger ribonucleic acid (mRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a deoxyribonucleic acid (DNA), or any combination thereof.

[0010] Exogenous cargo, or "payload," is a term used to describe compounds, e.g., nucleic acids, including mRNA, or compositions, that are delivered to the interior of a cell across the cell plasma membrane via aqueous solution.

[0011] Immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos (v-fos FBJ murine osteosarcoma viral oncogene homolog, FBJ murine osteosarcoma viral oncogene homolog), Jun v-jun avian sarcoma viral 17 oncogene homolog), or a combination thereof, is expressed at a log2 fold change level within 3 relative to the level expressed in control immune cells. For example, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, or a combination thereof is expressed at a log2 fold change level of about -3 relative to the level expressed in control immune cells.

[0012] In other examples, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, or a combination thereof are expressed at a log2 fold change level within 2 relative to the level expressed in a control immune cell. In further examples, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, or a combination thereof are expressed at a log2 fold change level within 1 relative to the level expressed in a control immune cell.

[0013] In some embodiments, Fos includes human Fos, which comprises the exemplary nucleic acid sequence of SEQ ID NO: 1. In some embodiments, Jun includes human Jun, which comprises the exemplary nucleic acid sequence of SEQ ID NO: 2.

[0014] Immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, FosB (FBJ murine osteosarcoma viral oncogene homolog B; SEQ ID NO:3), BATF (basic leucine zipper transcription factor ATF-like), BATF (basic leucine zipper transcription factor ATF-like; SEQ ID NO:4), BATF3 (basic leucine zipper transcription factor ATF-like 3; SEQ ID NO:5), or combinations thereof are expressed at a log2 fold change level within 3, within a log2 fold change level within 2, or within a log2 fold change level within 1 compared to levels expressed in control immune cells (immune cells without exogenous cargo). In embodiments, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, FosB, BATF, or BATF3 are expressed at a log2 fold change level within about -3, within about -2, or within about -1 relative to the level expressed in control immune cells (immune cells that have not undergone a cell manipulation or activation process), e.g., (negative numbers), the gene is expressed at a level less than that of the control immune cells.

[0015] In examples, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, FosB, BATF, or BATF3 are expressed at a log2 fold change level within 1 relative to the levels expressed in control immune cells. In examples, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, FosB, BATF, or BATF3 are expressed at a log2 fold change level within 2 relative to the levels expressed in control immune cells. In examples, immune cells of the invention, e.g., immune cells bearing exogenous cargo, have a molecular profile in which Fos, Jun, FosB, BATF, or BATF3 are expressed at a log2 fold change level within 3 relative to the levels expressed in control immune cells.

[0016] In some embodiments, the exogenous cargo comprises a nucleic acid. For example, the cargo comprises a messenger ribonucleic acid (mRNA). For example, the mRNA encodes a chimeric antigen receptor (CAR). For example, the CAR targets CD19 (cluster of differentiation 19). An exemplary mRNA encoding a CD19 CAR comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:8.

[0017] In other embodiments, the mRNA encodes TRAIL-DR5 (TNF-related apoptosis-inducing ligand (TRAIL) death receptor 5) variant mRNA (SEQ ID NO:10), TRAIL DNA (SEQ ID NO:11). See, e.g., U.S. Patent No. 7,994,281, incorporated herein by reference in its entirety. In other embodiments, the mRNA encodes IL-15 (interleukin-15) mRNA or TCR (T cell receptor) mRNA.

[0018] In other examples, the exogenous cargo comprises a Cas9 (CRISPR-associated protein 9) protein and a guide RNA comprising, for example, TRAC (T-cell receptor alpha constant) or PD-1 (programmed death ligand 1). For example, the Cas9 protein sequence comprises SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The sequence of a human TRAC-targeting gRNA comprises SEQ ID NO: 25. The sequence of a human PDCD1-targeting gRNA comprises SEQ ID NO: 26.

[0019] In other examples, the exogenous cargo comprises a Cas12a protein (CRISPR-associated protein 12a) comprising a guide RNA comprising TRAC and PD-1. The Cas12a protein sequence comprises SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. In examples, the exogenous cargo comprises a MAD7 protein and a guide RNA comprising TRAC and PD-1. In examples, the exogenous cargo comprises an SgCas and a guide RNA comprising TRAC and PD-1. In examples, the exogenous cargo comprises a Cas13 and a guide RNA comprising TRAC and PD-1. Alternatively, the exogenous cargo comprises a base editor, e.g., Cas9n, or a zinc finger nuclease, or a MegaTAL.

[0020] In examples, the exogenous cargo comprises a Sleeping Beauty 100 transposon / transposase system, or a Sleeping Beauty 1000 transposon / transposase system, or a Piggy Bac transposon / transposase system, or a TcBuster transposon / transposase system.

[0021] In other examples, the exogenous cargo comprises DNA, for example, CD19 CAR DNA, TRAIL DNA, or IL-15 DNA.

[0022] In some embodiments, the exogenous cargo comprises a Yamanaka factor, which is used to generate stable induced pluripotent stem cells from adult human cells. For example, Yamanaka factors include c-Myc (MYC proto-oncogene, a bHLH transcription factor, SEQ ID NO: 13), Klf4 (Kruppel-like factor 4, SEQ ID NO: 14), Oct4 (octamer-binding transcription factor 4, SEQ ID NO: 15), or Sox2 (SRY (sex determining region Y)-box 2, SEQ ID NO: 16).

[0023] In a further example, the exogenous cargo comprises an siRNA (small interfering RNA), for example, against PD-1. In a further example, the exogenous cargo comprises an shRNA (short hairpin RNA), for example, against PD-1.

[0024] The term "exogenous" refers to a cargo (or payload) that originates or is extracellularly derived from outside a cell, e.g., an immune cell, as opposed to an endogenous agent that originates within the immune cell.

[0025] Various methods can be used to characterize the molecular profile of immune cells. For example, molecular profiling can be performed using DNA analysis, RNA analysis, protein analysis, cytokine analysis, or a combination thereof. For example, molecular profiling can be performed by RNA analysis. In some embodiments, RNA analysis includes RNA quantification. In some embodiments, RNA quantification is performed by reverse transcription quantitative PCR (RT-qPCR), multiplex qRT-PCR, fluorescent in situ hybridization (FISH), and combinations thereof. In some embodiments, molecular profiling is performed by DNA analysis. In some embodiments, DNA analysis includes amplification of DNA sequences from one or more identified cells. In some embodiments, amplification is performed by polymerase chain reaction (PCR). In some embodiments, molecular profiling is performed by RNA sequencing or DNA sequencing. In some embodiments, RNA sequencing or DNA sequencing is performed by methods including, but not limited to, whole transcriptome analysis, whole genome analysis, barcode sequencing of whole or targeted regions of the genome, and combinations thereof. In other examples, molecular profiling is performed by protein analysis, for example, protein analysis at the proteome level.

[0026] In embodiments, immune cells bearing exogenous cargo have a molecular profile with a gene or protein expression level (e.g., in the AP signaling pathway) that is a log2 fold change of about -3, a log2 fold change of about -2, or a log2 fold change of about -1 compared to the expression level of the gene or protein in a control immune cell. For example, immune cells bearing exogenous cargo have a molecular profile with a gene or protein expression level in the AP-1 signaling pathway that is a log2 fold change of about -1 compared to the expression level of the gene or protein in a control immune cell. For example, immune cells bearing exogenous cargo have a molecular profile with a gene or protein expression in the AP-1 signaling pathway that is a log2 fold change of about -2 compared to the level of the gene or protein in a control immune cell. In other examples, immune cells bearing exogenous cargo have a molecular profile with a gene or protein expression that is a log2 fold change of about -1 compared to the level of the gene or protein in a control immune cell.

[0027] In some embodiments, immune cells bearing exogenous cargo have a molecular profile with gene or protein expression levels within a log2 fold change of 3, within a log2 fold change of 2, or within a log2 fold change of 1 compared to the expression levels of the gene or protein in control immune cells, and genes or proteins in the AP-1 (activator protein 1) signaling pathway. AP-1 is a transcription factor that regulates gene expression in response to various stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. AP-1 controls numerous cellular processes, including differentiation, proliferation, and apoptosis. For example, exhausted T cells exhibit low expression of AP-1 factors, including Fos, Jun, and / or Fosb (FBJ murine osteosarcoma viral oncogene homolog B). For example, Fos has the nucleic acid sequence of human SEQ ID NO:1. In other examples, Jun comprises the nucleic acid sequence of SEQ ID NO:2.

[0028] In some embodiments, the immune cells of the present invention comprise at least two or more exogenous cargoes (e.g., three, four, five, six, seven, eight, nine, or ten exogenous cargoes). The exogenous cargoes include nucleic acids (e.g., RNA (ribonucleic acid), mRNA (messenger RNA), or DNA (deoxyribonucleic acid)), proteins or peptides, small chemical molecules, or any combination thereof.

[0029] The immune cells of the present invention (e.g., immune cells bearing exogenous cargo) are associated with numerous advantages. For example, immune cells processed using the SOLUPORE™ method exhibit little or no phenotypic characteristics of T cell exhaustion or T cell anergy. T cell anergy is a dysfunctional state of T cells stimulated in the absence of costimulatory signals. T cell exhaustion refers to the progressive loss of T cell effector function due to prolonged stimulation with an antigen. Furthermore, T cell stimulation refers to cell activation through the binding of the T cell receptor (TCR) / CD3 (cluster of differentiation 3) complex to costimulatory receptors, such as CD28 (cluster of differentiation 28). Because cells processed using the SOLUPORE™ method exhibit little or no phenotypic characteristics of T cell exhaustion or T cell anergy, they are more suitable for clinical use; i.e., their immune function is preserved, thus conferring superior clinical benefits compared to electroporated cells.

[0030] T cell "exhaustion" describes a state in which T cells do not respond adequately due to prolonged exposure to antigen during chronic viral infection, cancer, or other manipulation, for example, during prolonged binding of cell surface receptors, such as CD3 or CD28, to ligands, such as anti-CD3 or anti-CD28 antibodies. "T cell exhaustion" is characterized by the loss of T cell function. Exhausted T cells exhibit a transcriptional profile distinct from that of functional effector or memory T cells, characterized by the expression of inhibitory cell surface receptors, including PD-1, LAG-3 (lymphocyte activation gene 3), TIM-3 (T cell immunoglobulin mucin-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), and CTLA-4 (cytotoxic T lymphocyte-associated protein 4), and the production of IL-2 (interleukin 2), TNF (tumor necrosis factor), and IFN-γ (interferon gamma) cytokines. NFAT (nuclear factor of activated T cells) and AP-1 transcription factors synergistically play a central role in inducing hyporesponsive states such as anergy and exhaustion. Exhausted cells show low expression of AP-1 factors (FOS, FOSB, and Jun). Furthermore, T cell anergy can refer to a tolerance mechanism, in which leukocytes are intrinsically functionally inactivated after antigen encounter but persist in a hyporesponsive state for extended periods.

[0031] In some embodiments, the immune cells of the present invention (e.g., immune cells carrying exogenous cargo) include unstimulated immune cells. For example, the immune cells have not been stimulated by a ligand for CD3, CD28, or a combination thereof. In other words, the immune cells have not been contacted with a CD3 ligand or a CD28 ligand, for example, an antibody or antibody fragment that binds to CD3, CD28, or both.

[0032] In some aspects, the immune cells of the present invention (e.g., immune cells bearing exogenous cargo) secrete at least one cytokine at a log2 fold change level within 3 compared to the level of immune cells that have not undergone a cell engineering process. In other embodiments, the immune cells of the present invention (e.g., immune cells bearing exogenous cargo) secrete at least one cytokine at a log2 fold change level within 2 compared to the level of immune cells that have not undergone a cell engineering process. In other embodiments, the immune cells of the present invention (e.g., immune cells bearing exogenous cargo) secrete at least one cytokine at a log2 fold change level within 1 compared to the level of immune cells that have not undergone a cell engineering process. For example, the immune cells of the present invention do not cause non-specific secretion (also called "release" and refers to cytokine release from cells, e.g., immune cells) of cytokines compared to control immune cells.

[0033] In embodiments, immune cells of the invention (e.g., immune cells with exogenous cargo) secrete the cytokines IL-2 (interleukin 2) and / or IL-8 (interleukin 8) at a log2 fold change level within 3 compared to immune cells that have not undergone a cell engineering process. In embodiments, immune cells of the invention (e.g., immune cells with exogenous cargo) secrete the cytokines IL-2 or IL-8 at a log2 fold change level within 2 compared to immune cells that have not undergone a cell engineering process. In embodiments, immune cells of the invention (e.g., immune cells with exogenous cargo) secrete the cytokines IL-2 or IL-8 at a log2 fold change level within 1 compared to immune cells that have not undergone a cell engineering process.

[0034] In embodiments, immune cells of the present invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IL-2 or IL-8 at a log2 fold change level of about -3 compared to immune cells that have not undergone the cell engineering process.

[0035] In embodiments, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IL-2 or IL-8 at a log2 fold change level of about -2 compared to immune cells that have not undergone a cell engineering process. In embodiments, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IL-2 or IL-8 at a log2 fold change level of about -1 compared to immune cells that have not undergone a cell engineering process.

[0036] In embodiments, the IL-2 (e.g., human IL-2) comprises the nucleic acid sequence of SEQ ID NO: 17. In other embodiments, the IL-8 (e.g., human IL-8) comprises the nucleic acid sequence of SEQ ID NO: 18.

[0037] In embodiments, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete the cytokines IFN-γ (interferon gamma), IL-2, TNFα (tumor necrosis factor alpha), IL-8, GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1 alpha), MIP-1β (macrophage inflammatory protein 1 beta), IL-17A (interleukin 17A), fractalkine, or ITAC (interferon-inducible T-cell alpha chemoattractant) at a log2 fold change level within 3 compared to immune cells that have not undergone the cell engineering process. In other examples, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IFN-γ, IL-2, TNFα, IL-8, GM-CSF, IL-10, MIP-1α, MIP-1β, IL-17A, fractalkine, ITAC, or combinations thereof at a log2 fold change level within 2 compared to immune cells that have not undergone the cell engineering process. In other examples, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IFN-γ, IL-2, TNFα, IL-8, GM-CSF, IL-10, MIP-1α, MIP-1β, IL-17A, fractalkine, ITAC, or combinations thereof at a log2 fold change level within 1 compared to immune cells that have not undergone the cell engineering process.

[0038] In examples, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IFN-γ, IL-2, TNFα, IL-8, GM-CSF, IL-10, MIP-1α, MIP-1β, IL-17A, fractalkine, ITAC, or combinations thereof at a log2 fold change level of about -3 compared to immune cells that have not undergone the cell engineering process. In other examples, immune cells of the invention (e.g., immune cells bearing exogenous cargo) secrete cytokines IFN-γ, IL-2, TNFα, IL-8, GM-CSF, IL-10, MIP-1α, MIP-1β, IL-17A, fractalkine, ITAC, or combinations thereof at a log2 fold change level of about -2 compared to immune cells that have not undergone the cell engineering process. In other examples, immune cells of the invention (e.g., immune cells carrying exogenous cargo) secrete cytokines IFN-γ, IL-2, TNFα, IL-8, GM-CSF, IL-10, MIP-1α, MIP-1β, IL-17A, fractalkine, ITAC, or combinations thereof, at a log2 fold change level of about −1 compared to immune cells that have not undergone the cell engineering process.

[0039] Also provided herein is a method for delivering a cargo (or "exogenous cargo") across the plasma membrane of non-adherent immune cells. Thus, the method includes providing a population of non-adherent cells and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising a payload and an alcohol at a concentration greater than 0.2 percent (v / v), wherein the immune function of the non-adherent immune cells comprises a cell phenotype that has not undergone a cell engineering process, and the immune function is selected from (i) cytokine release; (ii) gene expression; and (iii) metabolic rate.

[0040] For example, the alcohol concentration is greater than or equal to about 0.2 percent (v / v), or greater than or equal to about 0.5 percent (v / v), or greater than or equal to about 2 percent (v / v), or greater than or equal to 5 percent (v / v). In other embodiments, the alcohol is greater than or equal to 10 percent (v / v).

[0041] For example, the alcohol comprises ethanol, e.g., 10% or more ethanol. In some embodiments, the aqueous solution comprises 20-30% ethanol, e.g., 27% ethanol.

[0042] Electroporation (a cell manipulation process) includes, for example, intracellular delivery methods (also called electrotransfer) that apply an electric field to cells to increase cell membrane permeability. As used herein, the term "cell manipulation process" can refer to any known transfection method for intracellular delivery, including the SOLUPORE™ delivery method, membrane disruption methods (electroporation, sonoporation, magnetofection, optoperation), or carrier-based methods (lipid nanoparticles). Exemplary forms of electroporation include bulk electroporation and flow-through electroporation. Electroporation suppliers and devices include Maxcyte, Lonza-Nucleofector, Cellectis-Pulse Agile, BioRad-Gene Pulser, Thermofisher-Neon, or Celetrix-Nanopulser.

[0043] Provided herein are methods for delivering a payload (or "exogenous cargo") across the plasma membrane of non-adherent cells. The methods include providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution comprising a payload and a percentage of alcohol greater than or equal to 0.1 percent (v / v), 0.5 percent (v / v), 1 percent (v / v), 2 percent (v / v), 2.5 percent (v / v), 5 percent (v / v) concentration.

[0044] In some embodiments, the aqueous solution includes an alcohol, which may include ethanol. In other embodiments, the aqueous solution includes greater than 10% ethanol, 20-30% ethanol, or about 27% ethanol. In some embodiments, the aqueous solution includes 12.5-500 mM potassium chloride (KCl), or about 106 mM KCl.

[0045] Aqueous solutions for delivering exogenous cargo to cells contain a salt, such as 12.5 to 500 mM potassium chloride (KCl). For example, the solution is isotonic relative to the cytoplasm of mammalian cells, such as human T cells. An exemplary such isotonic delivery solution is 106 mM KCl.

[0046] In other examples, the aqueous solution may include an ethanol concentration of 5-30% (e.g., 0.2%-30%). The aqueous solution may include one or more of 75-98% HO, 2-45% ethanol, 6-91 mM sucrose, 2-500 mM KCl, 2-35 mM ammonium acetate, and 1-14 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). For example, the delivery solution contains 106 mM KCl and 27% ethanol. For example, the delivery solution contains 106 mM KCl and 10% ethanol. For example, the delivery solution contains 106 mM KCl and 5% ethanol. For example, the delivery solution contains 106 mM KCl and 2% ethanol.

[0047] Exemplary non-adherent / suspension cells include primary hematopoietic stem cells (HSCs), T cells (e.g., CD3+ cells, CD4+ cells, CD8+ cells), natural killer (NK) cells, cytokine-induced killer (CIK) cells, human umbilical cord blood CD34+ cells, B cells, or cell lines such as Jurkat T cell lines. Non-adherent cells may be substantially confluent, e.g., greater than 75 percent confluent. Cell confluency refers to cells in contact with each other on a surface. For example, confluency can be expressed as an estimated (or counted) percentage. For example, 10% confluency means that 10% of a surface, e.g., the surface of a tissue culture vessel, is covered with cells, and 100% means complete coverage. For example, non-adherent cells may be spun down or pulled down by vacuum or tissue culture medium aspiration from the top of the cell mass, or removed by aspiration or vacuum removal from the bottom of the vessel. The cells may form a cell monolayer. For example, the cells are 10%, 25%, 50%, 75%, 90%, 95%, or 100% confluent.

[0048] In embodiments, the immune cells are not activated prior to cargo delivery. In other embodiments, the immune cells have not been contacted with a ligand for CD3, CD28, or a combination thereof prior to contacting the immune cells with the exogenous cargo.

[0049] In some embodiments, the non-adherent cells include peripheral blood mononuclear cells. In some embodiments, the non-adherent cells include immune cells, e.g., T lymphocytes, where the immune cells are not stimulated, e.g., by CD3 or CD28, or any combination thereof. In other embodiments, the non-adherent cells include immune cells, e.g., T lymphocytes.

[0050] In some embodiments, the immune cells include unstimulated immune cells. For example, the immune cells have not been stimulated by a ligand for CD3, CD28, or a combination thereof. In other words, the immune cells have not been contacted with a CD3 or CD28 ligand, for example, an antibody or antibody fragment that binds to CD3, CD28, or both. In some embodiments, the population of non-adherent cells forms a monolayer. For example, the monolayer is contacted with a spray of the aqueous solution.

[0051] The method involves delivering an exogenous cargo in a delivery solution to a population of non-adherent cells that constitute a monolayer. For example, the monolayer is contacted with a spray of an aqueous delivery solution. The method delivers the payload / cargo (compound or composition) into the cytoplasm of the cells, and this cell population has a greater percent survival rate compared to payload delivery by electroporation or nucleofection, which is a significant advantage of the solporation system.

[0052] In certain embodiments, a monolayer of non-adherent / suspension cells is on a membrane filter. In some embodiments, the membrane filter is vibrated after contacting the cell monolayer with the delivery solution spray. The membrane filter may be vibrated or vigorously shaken before, during, and / or after spraying the cells with the delivery solution.

[0053] The volume of solution delivered to the cells is a multiple of units, e.g., a spray, e.g., a multiple of droplets on the surface of a water particle. The volume is expressed relative to a single cell or relative to the exposed surface area of ​​a confluent or substantially confluent (e.g., at least 75%, at least 80% confluent, e.g., 85%, 90%, 95%, 97%, 98%, 100%) cell population. For example, the volume is 6.0 x 10 -7 microliters / cells ~7.4x10 -4 Microliters / cell is acceptable. Volume is 4.9x10 -6 microliters / cells ~2.2x10 -3 microliters / cell. Volume is 9.3x10 -6microliters / cells ~2.8x10 -5 The volume may be about 1.9x10 microliters / cell, which is within about 10 percent. The volume is 6.0x10 -7 microliters / cells ~2.2x10 -3 The volume is 2.6 x 10 per square micrometer of exposed surface area. -9 The volume may be between 5.3x10 microliters per square micrometer of exposed surface area and 1.6x10 microliters per square micrometer of exposed surface area. The volume may be about 1.1x10 microliters per square micrometer of exposed surface area.

[0054] Throughout this specification, the term "about" may mean within 10% of a stated amount or other measure.

[0055] Cell confluency refers to cells in contact with each other on a surface. For example, confluency can be expressed as an estimated (or counted) percentage. For example, 10% confluency means that 10% of a surface, such as the surface of a tissue culture vessel, is covered with cells, and 100% means complete coverage. For example, adherent cells can grow two-dimensionally on the surface of a tissue culture well, plate, or flask. Non-adherent cells can be spun down or pulled down by vacuum or tissue culture medium aspiration from the top of the cell mass, or removed by aspiration or vacuum removal from the bottom of the vessel.

[0056] The payload (exogenous cargo) may include a small chemical molecule, a peptide or protein, or a nucleic acid. The small chemical molecule may be less than 1,000 Da. The chemical molecule may include MitoTracker Red CMXRos, propidium iodide, methotrexate, and / or DAPI (4',6-diamidino-2-phenylindole). The peptide may be approximately 5,000 Da. The peptide may include ecallantide (brand name Kalbitor, a 60-amino acid polypeptide used to treat hereditary angioedema and prevent blood loss in cardiothoracic surgery), liraglutide (sold under the brand name Victoza for the treatment of type 2 diabetes and Saxenda for the treatment of obesity), and icatibant (brand name Firazyer, a peptidomimetic drug used to treat acute attacks of hereditary angioedema). Small interfering ribonucleic acid (siRNA) molecules can be approximately 20-25 base pairs in length and approximately 10,000-15,000 Da. siRNA molecules can reduce the expression of any gene product, for example, knocking down gene expression of clinically relevant target genes or model genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA, GAPDH siRNA-FITC cyclophilin B siRNA, and / or lamin siRNA. Protein therapeutics can include peptides, enzymes, structural proteins, receptors, cellular proteins, or circulating proteins, or fragments thereof. The protein or polypeptide can be approximately 100-500,000 Da, e.g., 1,000-150,000 Da. The protein can include any therapeutic, diagnostic, or research protein or peptide, such as β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and / or horseradish peroxidase. In other examples, the protein may include a cancer-specific apoptotic protein, such as tumor necrosis factor-related apoptosis-inducing protein (TRAIL).

[0057] The antibody generally has a molecular weight of about 150,000 Da. The antibody may include an anti-actin antibody, an anti-GAPDH antibody, an anti-Src antibody, an anti-Myc antibody, and / or an anti-Raf antibody. The antibody may include a green fluorescent protein (GFP) plasmid, a GLuc plasmid, and a BATEM plasmid. The DNA molecule may be greater than 5,000,000 Da. In some examples, the antibody may be a murine monoclonal antibody, such as ibritumomab, tiuxetin, muromomab-CD3, tositumomab, a human antibody, or a humanized murine (or other species of origin) antibody. In other examples, the antibody may be a chimeric monoclonal antibody, such as abciximab, basiliximab, cetuximab, infliximab, or rituximab. In yet other examples, the antibody may be a humanized monoclonal antibody, such as alemtuzamab, bevacizumab, certolizumab pegol, daclizumab, gentuzumab ozogamicin, trastuzumab, tocilizumab, ipilimumab, or panitumumab. The antibody may also include an antibody fragment, such as abatecept, aflibercept, alefacept, or etanercept. The present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineered single-chain Fv molecules; or chimeric molecules, such as antibodies containing the binding specificity of one antibody, e.g., a murine-derived antibody, and the remaining portion of another antibody, e.g., a human-derived antibody.

[0058] The payload (or "exogenous cargo") may include a therapeutic agent. A therapeutic agent, e.g., a drug or active agent, may refer to any compound useful for therapeutic or diagnostic purposes. This term may be understood to mean any compound administered to a patient for the treatment of a condition. Thus, therapeutic agents may include proteins, peptides, antibodies, antibody fragments, and small molecules. Therapeutic agents described in U.S. Pat. No. 7,667,004 (incorporated herein by reference) can be used in the methods described herein. The therapeutic agent may include at least one of cisplatin, aspirin, statins (e.g., pitavastatin, atorvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, promazine IICl, chloropromazine HO, thioridazine HCl, polymyxin B sulfate, chloroxine, benfluorex HCl, and phenazopyridine HCl), and fluoxetine. The payload may include a diagnostic agent. The diagnostic agent may include a detectable label or marker, such as at least one of methylene blue, patent blue V, and indocyanine green. The payload may include a fluorescent molecule. The payload may include a detectable nanoparticle. The nanoparticle may include a quantum dot.

[0059] The payload ("exogenous cargo") includes an alcohol. The term "alcohol" refers to a polyatomic organic compound containing a hydroxyl (-OH) functional group attached to at least one carbon atom. The alcohol may be a monohydric alcohol and may contain at least one carbon atom, such as methanol. The alcohol may contain at least two carbon atoms (e.g., ethanol). In other aspects, the alcohol may contain at least three carbon atoms (e.g., isopropyl alcohol). The alcohol may contain at least four carbon atoms (e.g., butanol) or at least seven carbon atoms (e.g., benzyl alcohol). For example, the payload may contain up to 50% (v / v) alcohol; more preferably, the payload contains 2-45% (v / v) alcohol, 5-40% alcohol, and 10-40% alcohol. The payload may contain 20-30% (v / v) alcohol.

[0060] Most preferably, the payload delivery solution contains 25% (v / v) alcohol. Alternatively, the payload may contain 2-8% (v / v) alcohol or 2% alcohol. The alcohol may include ethanol, and the payload may contain 5% (v / v), 10% (v / v), 20% (v / v), 25% (v / v), 30% (v / v), and up to 400 / 0 or 50% (v / v) ethanol, e.g., 27%. An exemplary method may include methanol as the alcohol, and the payload may contain 5% (v / v), 10% (v / v), 20% (v / v), 25% (v / v), 30% (v / v), or 40% (v / v) methanol. The payload may contain 2-45% (v / v) methanol, 20-30% (v / v), or 25% (v / v) methanol. Preferably, the payload comprises 20-30% (v / v) methanol. Alternatively, the alcohol is butanol and the payload comprises 2% (v / v), 4% (v / v), or 8% (v / v) butanol.

[0061] In some aspects of the present subject matter, the payload is in an isotonic solution or buffer.

[0062] According to the present invention, the payload may include at least one salt. The salt may be selected from NaCl, KCl, NaHPO, CHONH, and KHPO. For example, the KCl concentration is 2 mM to 500 mM. In some preferred embodiments, the concentration is greater than 100 mM, e.g., 106 mM. According to exemplary methods of the present invention, the payload may include a sugar (e.g., sucrose or a disaccharide). According to exemplary methods, the payload includes less than 121 mM sugar, 6 to 91 mM, or 26 to 39 mM sugar. Further, the payload includes 32 mM sugar (e.g., sucrose). Optionally, the sugar is sucrose, and the payload includes 6.4 mM, 12.8 mM, 19.2 mM, 25.6 mM, 32 mM, 64 mM, 76.8 mM, or 89.6 mM sucrose.

[0063] In embodiments, the method for delivering exogenous cargo across the plasma membrane of an immune cell further comprises delivering at least two exogenous cargoes (or "two payloads"). The exogenous cargoes include nucleic acids, small molecules, proteins, polypeptides, or combinations thereof. For example, the nucleic acids include messenger ribonucleic acid (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), deoxyribonucleic acid (DNA), or any combination thereof. In examples, the immune cell comprises two exogenous cargoes, three exogenous cargoes, four, five, six, seven, eight, nine, or ten exogenous cargoes.

[0064] In some embodiments, at least two exogenous cargoes are delivered simultaneously. This means that two exogenous cargoes are delivered at the same time (e.g., dual delivery). For example, the immune cells of the present invention (containing an exogenous cargo) may be delivered to contain a second exogenous cargo. The term "engineered" as used herein may refer to any known transfection method for intracellular delivery, including SOLUPORE™ delivery, membrane disruption methods (electroporation, sonoporation, magnetofection, optoporation), or carrier-based methods (lipid nanoparticles).

[0065] In some embodiments, at least two exogenous cargoes are delivered sequentially. For example, sequential delivery may refer to the delivery of one exogenous cargo followed by the delivery of a second, third, or fourth exogenous cargo. Then, for example, the immune cells of the present invention (containing an exogenous cargo) may be further engineered to contain a second exogenous cargo. As used herein, the term "engineered" may refer to any known transfection method for intracellular delivery, including SOLUPORE™ delivery method, membrane disruption methods (electroporation, sonoporation, magnetofection, optopellation), or carrier-based methods (lipid nanoparticles). Electroporation includes, for example, an intracellular delivery method (also called electrotransfer) that applies an electric field to cells to increase cell membrane permeability.

[0066] In another aspect, provided herein is a method for delivering exogenous cargoes across the plasma membrane of non-adherent cells, the method comprising: providing a population of non-adherent cells; (i) contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising the exogenous cargoes and an alcohol at a concentration greater than 0.5 percent (v / v); and using at least two intracellular delivery methods selected from (ii) viral transduction, (iii) electroporation, or (iv) nucleofection, thereby delivering the two exogenous cargoes to immune cells. For example, the aqueous solution comprises alcohol, and the alcohol comprises ethanol. The alcohol concentration in the aqueous solution is greater than 0.2 percent (v / v), or greater than 0.5 percent (v / v), or greater than 2 percent (v / v), or greater than 5 percent (v / v), or greater than 10 percent (v / v). In some examples, the aqueous solution comprises 20-30% ethanol, e.g., 27% ethanol.

[0067] In embodiments, the intracellular delivery method comprises contacting a population of cells with a volume of an isotonic aqueous solution, followed by viral transduction, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v). In other embodiments, the intracellular delivery method comprises viral transduction, followed by contacting a population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v).

[0068] [The present invention 1001] An immune cell comprising an exogenous cargo, the immune cells have a molecular profile comprising a gene or protein expression level within a log2 fold change of 3 relative to the expression level of the gene or protein in control immune cells 24 hours after cargo delivery, wherein the gene or protein is in the activator protein 1 (AP-1) signaling pathway. immune cells. [The present invention 1002] 1001. An immune cell of the present invention, wherein the expression level of a gene or protein is within a log2 fold change of 2 relative to the expression level of the gene or protein in a control immune cell, or wherein the expression level of a gene or protein is within a log2 fold change of 1 relative to the expression level of the gene or protein in a control immune cell. [The present invention 1003] 1001. The immune cell of the present invention, wherein the exogenous cargo comprises a nucleic acid, a small molecule, a protein, a polypeptide, or a combination thereof. [The present invention 1004] 1003. The immune cell of the present invention, wherein the nucleic acid comprises messenger ribonucleic acid (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), deoxyribonucleic acid (DNA), or any combination thereof. [The present invention 1005] 1001. The immune cell of the present invention, wherein the expression of a gene or protein in the AP-1 signaling pathway comprises Fos (v-fos FBJ murine osteosarcoma viral oncogene homolog, FBJ murine osteosarcoma viral oncogene homolog), Jun (v-jun avian sarcoma virus 17 oncogene homolog), or a combination thereof. [The present invention 1006] 1001. The immune cell of the present invention, wherein the gene or protein in the AP-1 signaling pathway comprises Fos, Jun, FosB (FBJ murine osteosarcoma viral oncogene homolog B), BATF (basic leucine zipper transcription factor ATF-like), BATF3 (basic leucine zipper transcription factor ATF-like 3), or a combination thereof. [The present invention 1007] 1005. The immune cell of the present invention, wherein Fos comprises human Fos comprising the nucleic acid sequence of SEQ ID NO:1, and Jun comprises human Jun comprising the nucleic acid sequence of SEQ ID NO:2. [The present invention 1008] 1001. The immune cell of the present invention, wherein the cargo comprises messenger ribonucleic acid (mRNA). [The present invention 1009] 1008. The immune cell of the present invention, wherein the mRNA encodes a chimeric antigen receptor (CAR). [The present invention 1010] The immune cell of the present invention 1009, wherein the CAR targets CD19 (cluster of differentiation 19) ("CD19 CAR"). [The present invention 1011] 1010. The immune cell of the present invention, wherein the CD19 CAR comprises the mRNA sequence of SEQ ID NO:6 or SEQ ID NO:8. [The present invention 1012] 1010. The immune cell of the present invention, wherein the CD19 CAR comprises the protein sequence of SEQ ID NO:7 or SEQ ID NO:9. [The present invention 1013] 1001. The immune cell of the present invention, wherein the expression of a gene or protein in the AP-1 signaling pathway in the immune cell containing the exogenous cargo is a log2 fold change of about -3 compared to the control immune cell. [The present invention 1014] 1001. The immune cell of the present invention, wherein the expression of a gene or protein in the AP-1 signaling pathway in the immune cell containing the exogenous cargo is a log2 fold change of about -2 compared to the control immune cell. [The present invention 1015] 1001. The immune cell of the present invention, wherein the expression of a gene or protein in the AP-1 signaling pathway in the immune cell containing the exogenous cargo is a log2 fold change of about -1 compared to the control immune cell. [The present invention 1016] 1001. An immune cell of the present invention comprising at least two exogenous cargoes. [The present invention 1017] 1001. The immune cell of the present invention, wherein the immune cell containing the exogenous cargo does not exhibit a T cell exhaustion or T cell anergy phenotype. [The present invention 1018] 1001. The immune cell of the present invention, wherein the immune cell comprising the exogenous cargo comprises an unstimulated immune cell. [The present invention 1019] Immune cells containing exogenous cargo that secrete at least one cytokine at a log2 fold change level within 3 compared to the level of immune cells that have not undergone a cell manipulation process. [The present invention 1020] 1019. The immune cells of the present invention, which secrete at least one cytokine at a log2 fold change level within 2 compared to the level of immune cells that have not undergone the cell manipulation process. [The present invention 1021] 1019. The immune cells of the present invention, which secrete at least one cytokine at a log2 fold change level within 1 compared to the level of immune cells that have not undergone the cell manipulation process. [The present invention 1022] The immune cell of the present invention 1019, wherein the cytokine comprises human IL-2 (interleukin 2) comprising the nucleic acid sequence of SEQ ID NO: 17, human IL-8 (interleukin 8) comprising the nucleic acid sequence of SEQ ID NO: 18, or a combination thereof. [The present invention 1023] The immune cell of the present invention 1019, wherein the cytokine comprises IFN-γ (interferon gamma), IL-2 (interleukin 2), TNFα (tumor necrosis factor alpha), IL-8 (interleukin 8), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), IL-17A (interleukin 17A), fractalkine, or ITAC (interferon-inducible T cell alpha chemoattractant). [The present invention 1024] 1. A method for delivering exogenous cargo across the plasma membrane of a non-adherent immune cell, comprising: providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.2 percent (v / v), wherein the immune function of the non-adherent immune cells comprises a phenotype of cells that have not undergone a cell manipulation process, the immune function being selected from: (i) cytokine release; (ii) gene expression; and (iii) metabolic rate. A method comprising: [The present invention 1025] 1024. The method of claim 1024, wherein the alcohol is greater than a 0.5 percent (v / v) concentration. [The present invention 1026] 1024. The method of claim 1024, wherein the alcohol is greater than a 2 percent (v / v) concentration. [The present invention 1027] 1024. The method of claim 1024, wherein the alcohol is greater than a 5 percent (v / v) concentration. [The present invention 1028] 1024. The method of claim 1024, wherein the alcohol is greater than a 10 percent (v / v) concentration. [The present invention 1029] The method of claim 1024, wherein the immune cells are not activated prior to cargo delivery. [The present invention 1030] The method of claim 1024, wherein the immune cells have not been contacted with a ligand for CD3, CD28, or a combination thereof prior to contacting the immune cells with the exogenous cargo. [The present invention 1031] The method of claim 1024, further comprising at least two exogenous cargoes. [The present invention 1032] The method of claim 1031, wherein the at least two exogenous cargoes are simultaneous. [The present invention 1033] The method of claim 1031, wherein at least two exogenous cargoes are delivered sequentially. [The present invention 1034] 1. A method for delivering at least two exogenous cargoes across the plasma membrane of a non-adherent immune cell, comprising: providing a population of non-adherent cells; and (i) contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v); (ii) using at least two intracellular delivery methods selected from viral transduction, (iii) electroporation, or (iv) nucleofection; and thereby delivering two types of exogenous cargo to immune cells. A method comprising: [This invention 1035] The intracellular delivery method is contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v); and subsequently, viral transduction. The method of the present invention 1034, comprising: [The present invention 1036] The intracellular delivery method is following viral transduction, contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising the exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v). The method of the present invention 1034, comprising: Other features and advantages of the present invention will be apparent from the following description of preferred embodiments of the invention and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI submissions identified by accession numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference. In the case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting. [Brief explanation of the drawings]

[0069] [Figure 1-1] Figures 1A-1D are bar graphs demonstrating efficient T cell engineering using the SOLUPORE™ delivery method. (Figure 1A) PBMC (peripheral blood mononuclear cell)-primed T cells and (Figure 1B) CD3+ (differentiation cluster 3) purified T cells show expression of the model cargo GFP (green fluorescent protein) and cell viability 24 hours after delivery of GFP mRNA. Figure 1C is a bar graph showing CD3 expression and cell viability on day 2 after delivery of TRAC (T cell receptor alpha constant) RNP (ribonucleoprotein). Figure 1D is a bar graph showing PD-1 (programmed cell death protein 1) indel (insertion or deletion) efficiency (insertion or deletion) and cell viability quantified by Sanger sequencing and TIDE (Tracking of Indels by Decomposition) analysis in cells collected on day 4 after delivery of PDCD1 (programmed cell death protein 1) RNP. Cells from three donors were used in all studies. n=2. [Figure 1-2]See description of Figure 1-1. [Figure 2A] Figures 2A-2D are graphs demonstrating that the SOLUPORE™ delivery method enables dual sequential cargo delivery due to multiple modifications. Figure 2A is a bar graph showing co-expression of CD19 (cluster of differentiation 19) CAR (chimeric antigen receptor) and GFP by flow cytometry and cell viability 24 hours after dual delivery of the corresponding mRNA (n=3). Figure 2B is a series of representative flow cytometry plots from a single donor showing CAR-only, GFP-only, and cell populations expressing both CAR and GFP. Figure 2C is a series of bar graphs showing CD19 CAR expression by flow cytometry, CD3 knockdown, and cell viability on day 3 after sequential delivery of TRAC RNP on day 0 and CD19 CAR mRNA on day 2 (n=3). Figure 2D is a representative flow plot from a single donor showing CD3-only, CAR-only, and CD3 and CAR expression in the populations. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3A]Figures 3A-3C are depictions of data showing that a comparison of intracellular delivery methods revealed minimal disruption of cytokine release and immune gene expression with the SOLUPORE™ delivery method. Figure 3A is a series of line graphs showing cytokine release from activated T cells after the SOLUPORE™ delivery method compared to electroporation delivery of GFP mRNA, as measured by Luminex multiplex assay. Five donors were included. Each donor included two technical replicates. Figure 3B is a series of Volcano plots. Each point represents a gene, and the point's location within the plot indicates the degree to which it is up- or down-regulated compared to control cells. The Volcano plots show results from a study in which non-activated T cells from three donors (Study 1) were mock-transfected, RNA was collected after 6 or 24 hours of treatment, and gene expression was compared to untreated control cells using a Nanostring CAR-T characterization panel. The Volcano plots show the fold change and p-value for individual genes 6 and 24 hours after transfection compared to untreated control cells. Figure 3C is a filtered heatmap showing gene expression log2 fold changes greater than 1 (>2-fold) with a statistical significance of p<0.05. Only genes that were changed in at least one of the groups are shown. Green, red, and black (indicated by shading and arrows) indicate down-regulated, up-regulated, and unchanged, respectively. See also Tables 7, 8, and 9. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4]Figures 4A and 4B are graphs showing that intracellular delivery methods differentially affect T cell proliferation rates and in vivo engraftment. Figure 4A is a line graph showing T cell proliferation after GFP mRNA delivery using either the SOLUPORE™ delivery method or electroporation. N=5 donors. n=5 technical replicates each were used. Figure 4B is a graph showing engraftment of human CD45+ (cluster of differentiation) cells in the spleens of NOD-scid IL-2Rγnull (non-obese diabetic (NOD) severe combined immunodeficiency (SCID)) mice 28 days after injection. [Figure 5A-1] Figure 5A is a graph showing CAR expression in T cells and in vitro killing of RAJI tumor cells 24 hours after delivery, as measured by impedance assay, from three different donors. Figure 5B is a schematic diagram of the protocol used to evaluate the ability of CD19 CAR T to kill RAJI cells in an established model. 2.5x105 luciferase-expressing RAJI cells were injected into NOD-scid IL-2Rγnull mice, followed three days later by injection of 1x106, 2x106, or 4x106 cells treated with SOLUPORE™ or electroporation. n=10 mice per group. Animals were euthanized on day 15, and bioluminescence was measured. Figure 5C is a photographic image showing bioluminescence imaging on day 15. Figure 5D is a series of dot plot graphs showing that human T cells were detected in the blood of mice on day 15 by flow cytometry analysis of human CD3 expression. Figure 5E is a bar graph showing that RAJI tumor cells were detected in the blood of mice by flow cytometry analysis of human CD20 (cluster of differentiation 20) expression on day 15. Figures 5A-5E demonstrate that CD19 CAR-T cells exhibited effective cytotoxicity in vitro and in vivo. CAR-T cells were generated by SOLUPORE™ delivery or electroporation-mediated delivery of CD19 CAR mRNA. [Figure 5A-2] See legend to Figure 5A-1. [Figure 5A-3] See legend to Figure 5A-1. [Figure 5B] See legend to Figure 5A-1. [Figure 5C] See legend to Figure 5A-1. [Figure 5D] See legend to Figure 5A-1. [Figure 5E] See legend to Figure 5A-1. [Figure 6-1] Figures 6A-6I are line graphs showing cytokine release from activated T cells after SOLUPORE™ or electroporation delivery of GFP mRNA, as measured by Luminex multiplex assay. Five donors were used, with each donor including two technical replicates. Figure 6A is a graph showing IFN-γ (interferon) release. Figure 6B is a graph showing IL-10 (interleukin 10) release. Figure 6C is a graph showing TNF-α (tumor necrosis factor α) release. Figure 6D is a graph showing GM-CSF (granulocyte-macrophage colony-stimulating factor) release. Figure 6E is a graph showing MIP-1a (macrophage inflammatory protein 1a) release. Figure 6F is a graph showing MIP-1b (macrophage inflammatory protein 1b) release. Figure 6G is a graph showing ITAC (interferon-inducible T cell α chemoattractant) release. Figure 6H is a graph showing fractalkine release. Figure 6I is a graph showing Il-17A (interleukin 17A) release. In summary, the cytokine release data in Figures 6A-6I demonstrate that the SOLUPORE™ delivery method causes minimal stress to T cells, as evidenced by the lack of difference in cytokine release compared to untreated control cells. In contrast, the electroporation process resulted in the release of IL-2 and IL-8 from T cells. [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 7]An image of an unfiltered heatmap showing log2 fold changes in gene expression greater than 1 (>2-fold) with a statistical significance of p<0.05, including all genes analyzed. Green, red, and black (indicated by shading and arrows) indicate downregulated, upregulated, and unchanged, respectively. [Figure 8] Image showing pathway analysis from immune-related gene profiling studies. Blue indicates gene downregulation 6 hours post-transfection, and red indicates gene upregulation 6 hours post-transfection. Beige or lighter shades of blue / red indicate gene expression similar to UT, with no change being beige. The farther the color is from the base color, the greater the fold change in gene expression; dark blue indicates a z-activation score of -15, and dark red indicates a z-activation score of 15. Gene names are shown on the y-axis. The x-axis, from left to right, indicates "Treated Mock N(F115) vs. UT" and "Treated Mock vs. UT." [Figure 9] 9A-9C are bar graphs showing the results of the area under the curve (AEC) in the in vitro RAJI cell killing assay calculated for each donor (FIG. 9A—donor 1; FIG. 9B—donor 2, and FIG. 9C—donor 3). [Figure 10] FIG. 1 shows the AP-1 (activator protein 1) signaling pathway. [Figure 11] Figure 1 shows AP-1 related genes from Study 1 and Study 2 (see Example 4). Cells in Study 1 and Study 2 were unstimulated cells. Study 2 was a repeat that included only the 24 hour analysis and EO-115 electroporation method. [Figure 12]This is a depiction of log2 fold change versus linear change. Tables show comparisons or calculations of gene expression differences that can be expressed by log2 fold change or linear fold change. Increases or decreases in gene or protein expression can be expressed as fold differences or log differences. The term "log2" or log2 was used to normalize results for up-regulated and down-regulated genes to equal values ​​along the axis. An example calculation is shown below: Gene A treated vs. control = 7.0 (overexpression); Gene B control vs. treatment = 7.0 or treated vs. control = 0.142 (underexpression). Although both genes are over- or under-expressed to the same degree, a linear scale would not reflect this change. Instead, gene A is up-regulated 7.0-fold and gene 2 is down-regulated 0.142-fold. Expressed in log2 format, gene A is up-regulated 2.81-fold and gene B is down-regulated -2.81-fold. [Figure 13] Illustrated are images of surface programmed cell death protein 1 (PD-1) staining performed on activated T cells after solporation or nucleofection. [Figure 14] 10 is an image depicting surface cluster of differentiation 69 (CD69) staining of activated T cells after solporation or nucleofection. [Figure 15] Bar graphs depict activated human T cells solporated or nucleofected (electroporation protocol EO115) with or without mRNA-GFP, with supernatants collected 6 hours post-transfection. ChromaDazzle lactate assays were performed. L-lactate concentrations were extrapolated from a standard curve using Microsoft Excel, and data are presented as fold change compared to control. Five donors, n=2. [Figure 16]Figures 16A and 16B illustrate a series of illustrations of calculations made from Seahorse traces: spare respiratory capacity (SRC), maximal respiration, and basal oxidative phosphorylation rate (OxPhos) calculations from the oxygen consumption rate (OCR) trace (Figure 16A), and glycolytic reserve, glycolytic capacity, and basal glycolytic rate from the extracellular acidification rate (ECAR) trace (Figure 16B). [Figure 17] A series of line graphs depicting activated human T cells (EO115) treated with mRNA-GFP or nucleofected with mRNA-GFP or in the absence of cargo (mock). Cells were harvested and subjected to a Seahorse assay. OCR and ECAR traces (n=1) are shown. [Figure 18] A series of bar graphs depicting glycolysis, OxPhos, glycolytic capacity, and maximum respiration of solporated or nucleofected T cells are shown. The data illustrate the metabolic activity of cells approximately 18 hours after transfection. [Figure 19] Figure 19A shows an image of GFP expression on CAR-positive cells analyzed by flow cytometry 24 hours after transfection. Figure 19B shows a bar graph of viability assessed using NC-3000 24 hours after transfection. n=1 for 3 donors. UT=untreated control. 3 days after activation, PBMC-primed T cells were transduced with CD19 CAR lentiviral vector (LV-CAR). 24 hours after viral delivery, cells were harvested and then transfected with GFP mRNA using SOLUPORE™ technology. DETAILED DESCRIPTION OF THE INVENTION

[0070] Detailed Description Provided herein are cell engineering technologies that enable next-generation cell therapy products requiring complex modifications and advanced cellular functions. Non-viral engineering techniques address the limitations associated with viral vectors. While electroporation is the most widely used non-viral modality, concerns about its impact on cellular function have led to the exploration of alternative approaches. The SOLUPORE™ delivery method described herein is a non-viral means of delivering cargo to primary immune cells simply, rapidly, and efficiently while preserving cell viability and function.

[0071] Issues with viral delivery methods Safe, versatile, and efficient intracellular delivery of exogenous materials is a critical requirement for many cell engineering applications. Examples of cell engineering applications include the treatment of hematological malignancies and disorders, where immune cells are modified ex vivo to replace, correct, or insert target genes. Viral transduction has been the most commonly used method for genetic engineering, but its limitations are well known.

[0072] The timeline from Good Manufacturing Practice (GMP) production initiation to batch release for cell therapy vectors, including obtaining plasmid DNA for transfection, can be lengthy and expensive. Challenges regarding the scalability and associated costs of vector production have driven interest in developing nonviral alternatives. Viral delivery systems are also susceptible to vector-mediated genotoxicity, such as random insertions that disrupt normal genes, accidental oncogene activation, or insertional mutagenesis that can lead to adverse immunogenicity and side effects. Beyond biosafety issues, limitations regarding the cargo packaging capacity of viral vectors have also motivated the development of intracellular delivery methods that can be used to deliver a wide range of bioactive constructs. The combination of extensive multiplexing capabilities, accelerated manufacturing timelines, and flexibility to accommodate variations between cell types and sizes, while avoiding the side effects associated with viral vectors, are attractive attributes of any single intracellular delivery method, making them safer and more economical.

[0073] CAR T cell therapy Autologous CAR T cell therapy has demonstrated unprecedented efficacy and durable responses in select cohorts of relapsed or refractory cancer patients with liquid tumors, resulting in the approval of two CAR T products to date. Proof-of-concept studies using these cell products have driven research, development, and commercial activity in the ex vivo cell therapy field. The success of these "living" drugs has been driven by complex manufacturing and logistical processes that have created a new paradigm for drug production. Engineering early breakthrough products was enabled by viral vectors. While this delivery modality remains important, issues related to availability, complexity, cost, safety, and efficiency mean that next-generation therapies require advanced gene transfer technologies. The present invention provides engineered cell populations containing exogenous cargo molecules to address the shortcomings and challenges of previous approaches for introducing nucleic acids and other molecules into cells. If the promise of success of engineered cell therapy is realized in patients with other earlier-stage liquid tumors and in patients with solid tumors, key focus areas will likely include optimizing cell therapy for liquid tumors, accelerating innovation cycle times to enable success in solid tumors, and transforming manufacturing processes. The virus-free protocols described herein play a key role in all of these aspects, ultimately improving patient access. The methods described herein do not rely on viruses or the application of electrical current to cells to mediate delivery of exogenous molecules to cells. The methods described herein do not rely on lipid nanoparticles to mediate delivery of exogenous molecules to cells.

[0074] Non-viral delivery Unlike traditional viral transduction, non-viral alternatives can deliver a wider range of constructs to a wider variety of cell types while avoiding the extensive biosafety and regulatory requirements for manufacturing vectors for cell therapy.

[0075] Intracellular delivery can be facilitated by a variety of techniques that broadly fall into two main categories: membrane disruptive or carrier-based.

[0076] Intracellular delivery methods can be broadly classified into two main categories: physical / mechanical methods such as electroporation, sonoporation, magnetofection, optopexy, gene guns, microinjection, and cell constriction / squeezing, and non-viral vectors such as lipid nanoparticles. Although electroporation platforms enable efficient cargo delivery, they present several challenges, including loss of proliferation capacity, reduced efficacy, and sustained intracellular calcium levels.

[0077] Chemical vectors, such as cationic polymers and lipids, can be used to deliver genetic material into cells without eliciting a significant immune response, however, to date the efficiency of chemical vectors does not compare to their viral counterparts.

[0078] The membrane disruption modality, SOLUPORE™ delivery method, has the potential to increase throughput, reduce manufacturing time, minimize processing steps, yet yield highly functional and potent cells.

[0079] Some physical transfection methods can affect the health of cells, leading to adverse effects on their proliferative capacity accompanied by altered gene expression profiles.

[0080] Good in vitro growth and effector function correlate with improved anti-tumor function in vivo, and therefore the SOLUPORE™ intracellular delivery method enables transfection of a diverse array of cargoes into multiple cell types with minimal disruption of normal cell function.

[0081] To address the consequences associated with disruption of normal cell function, the SOLUPORE™ delivery method was developed. The SOLUPORE™ delivery method is a non-viral, non-electrical (does not utilize the application of electrical current to cells) technology that transiently permeabilizes cell membranes to rapidly deliver cargoes of different compositions, properties, and sizes, such as macromolecules and nucleic acids, into cells. As demonstrated, the SOLUPORE™ delivery method successfully facilitates the delivery of gene editing tools, such as CRISPR / Cas9 and mRNA, into primary human immune cells, including human T cells, without adversely affecting cellular function.

[0082] Furthermore, the SOLUPORE™ delivery platform was developed as an advanced technology aimed at addressing the development and manufacturing needs of the cell therapy field. This technology is non-viral, meaning that many of the issues associated with viral vectors, such as availability, safety, process complexity, and associated costs, are not a concern. Continuing advances in genetic engineering tools also mean that genome targeting is now possible through non-viral approaches.

[0083] As described herein, the delivery efficiency of the SOLUPORE™ delivery method was evaluated using the flexibility to work with a wide range of cargo types and T cell populations cultured using diverse protocols. While other non-viral devices, such as electroporators, often require cell-specific programs and buffers, the same SOLUPORE™ delivery method program and buffer can be used for a wide range of cell types, with cell density being the primary parameter that is varied. A table of seeding densities is provided below (expected ranges were calculated using average cell size). A high degree of consistency was observed in the results obtained, creating a predictable process.

[0084] (Table 18) Cell seeding density TIFF2025156332000002.tif72128

[0085] The platform's ability to support dual and sequential gene editing without compromising cell viability is a key feature. When targeting and efficacy must be enhanced in autologous cell therapies for both liquid and solid tumors, cells require multiple modifications using steps consistent with the manufacturing process. This may involve multiplexed or sequential manipulation steps. Similar demands apply to allogeneic approaches. In this case, issues of cell rejection and GvHD mean that complex editing is likely to be required. Viral vector volume constraints and electroporation toxicity mean that these modalities may be inappropriate for certain complex manipulation methods. Furthermore, the long lead times required to design and produce even research-grade viral vectors mean that timelines may be longer than desired during development. This is particularly concerning in the context of advancing approaches to solid tumors. In this case, the challenges of targeting and efficacy mean that numerous candidate target antigens and cell efficacy enhancements must be tested. It is necessary to evaluate numerous cell compositions in a rapid, high-throughput manner, which would likely be highly limited if relying solely on viral vectors. The SOLUPORE™ delivery method therefore addresses these concerns and is compatible with optimizing CAR T for liquid tumors and accelerating the innovation cycle time required for impactful advances in addressing solid tumors.

[0086] Cells treated with SOLUPORE™ delivery preserve critical immune function and minimize exhaustion If cell manipulation must be useful, cell function must be maintained.Therefore, there is interest in this field to develop alternative non-viral delivery methods that can be efficient yet gentle to cells.The test reported herein demonstrates that SOLUPORE™ delivery method has minimal impact on protein expression and gene expression in T cells.Importantly, biological characteristics such as proliferation and gene expression profile are maintained.In addition, when using SOLUPORE™ delivery method, CAR T cells kill cells both in vitro and in vivo, thus proving the function of these cells.

[0087] Electroporation is the most widely used non-viral cargo delivery method, but nonspecific changes in protein and gene expression and reduced anti-tumor efficacy have previously been observed in T cells engineered by this method. The SOLUPORE™ delivery method altered the expression of only a few immune-related genes. Of the 10 genes identified in the 6-hour SOLUPORE™ delivery group (6 hours after the procedure), 8 were shared with the 6-hour electroporation group. This suggests that these may be genes related to cell membrane disruption or other aspects common to the two delivery methods.

[0088] The finding that electroporation dramatically affects gene expression in T cells is consistent with studies showing increased intracellular calcium levels and increased transcriptional activity in electroporated T cells in the absence of exogenous stimuli (see Zhang M, et al. J Immunol Methods 2014;408:123-131, incorporated herein by reference in its entirety). Calcium release from the endoplasmic reticulum activates the transcription factor NFAT (nuclear factor of activated T cells), one of the central regulators of exhaustion.

[0089] The observation that electroporated cells showed increased expression of genes involved in AP-1 (activator protein 1) signaling suggests that transcription factors such as AP-1 and NFAT may play a role in the cellular stress response to electroporation. The results described herein support previous studies showing that electroporation-induced disruption of these critical pathways contributes to cellular exhaustion, thereby rendering the cells unsuitable for mammalian cell therapy. In contrast, the SOLUPORE™ delivery method causes only minimal disruption of these pathways, and the profile of treated cells remains similar to that of control cells.

[0090] Electroporation, including nucleofection, reduces cell proliferation, which is suggested to be caused by activation of the DNA damage response pathway. This is a concern with gene editing approaches, and it is unclear how these effects of electroporation ultimately impact the efficacy of cell therapy products. In a 12-day RAJI tumor mouse model, electroporated CAR T cells performed similarly to cells delivered using the SOLUPORE™ delivery method, but in a 30-day in vivo engraftment model, electroporated cells did not engraft as expected, indicating that the health of the cells was adversely affected.

[0091] There has also been growing interest in the possibility of engineering resting T cells to reduce cell exhaustion. According to the present invention, resting cells are engineered and then expanded. This approach has the added advantage of requiring significantly less cargo. Findings herein and by others have demonstrated that electroporation induces disruption at the transcriptional level in resting T cells (signifying cell exhaustion), thus making nucleofection / electroporation a less desirable approach for this application. In contrast, the SOLUPORE™ delivery method has the potential to enable the engineering of these cells.

[0092] AP-1 signaling and T cell exhaustion Activator protein 1 (AP-1) is a transcription factor that regulates gene expression in response to various stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. AP-1 controls numerous cellular processes, including differentiation, proliferation, and apoptosis.

[0093] Activator protein-1 (AP-1) is a family of four subfamilies: (1) Jun ("v-jun avian sarcoma virus 17 oncogene homolog, jun oncogene" or "c-jun"), c-Jun (transcription factor AP1), JunB (transcription factor jun-B), JunD (transcription factor jun-D isoform deltaJunD)), (2) Fos (c-Fos (proto-oncogene)-FosB (also known as FosB and G0 / G1 switch regulatory protein 3 (G0S3)), Fra1 (Fos-related antigen 1 (FRA1)), Fra2 (Fos-related antigen 2 (FRA2)), (3) Maf (musculoaponeurotic fibrosarcoma) - (c-Maf (also known as the proto-oncogene c-Maf or V-maf musculoaponeurotic fibrosarcoma oncogene homolog), MafB (also known as V-maf musculoaponeurotic fibrosarcoma oncogene homolog B), MafA (transcription factor MafA), Mafg / f / k (bZip Maf transcription factor protein), Nrl (neural retina-specific leucine zipper protein)), and (4) ATF-activating transcription factors (ATF2 (activating transcription factor 2), LRF1 / ATF3 (cyclic AMP-dependent transcription factor ATF-3), BATF (basic leucine zipper transcription factor ATF-like), JDP1 (DnaJ (Hsp40) homolog, subfamily C), JDP2 (Jun dimerization protein 2)) This is a group of transcription factors consisting of:

[0094] These AP-1 transcription factors regulate a wide range of cellular processes, from cell proliferation and survival to tumor transformation, differentiation, and apoptosis. AP-1 transcription factors are proteins that form homodimers or heterodimers. Members of the AP-1 protein family differ significantly in their potential to transactivate AP-1-responsive genes and their ability to form dimers. For example, the Fos subfamily cannot homodimerize but can form stable heterodimers with Jun members. Fos and Jun proteins have high transactivation potency, whereas other proteins, such as JunB, JunD, Fra-1, and Fra-2, have weaker transactivation potency. Initial studies using mouse fibroblasts demonstrated that some AP-1 members have antagonistic properties relative to other members. For example, cJun transcriptional activity is attenuated by JunB, which is due to differences in their activation domains. Nevertheless, current knowledge suggests that differential expression of AP-1 components and the cellular context of their interactions determine the complex functions of AP-1 transcription factors.

[0095] In T cells, the AP-1 transcription factor is characterized by its pleiotropic effects and central role in various aspects of the immune system, including T cell activation, Th differentiation, T cell anergy, and exhaustion. The MAPK (MAP kinase) signaling cascade is important for regulating AP-1 transcriptional activation and DNA binding activity to various AP-1 target genes.

[0096] T cell anergy is a state of non-responsiveness of T cells, resulting in activation in the absence of positive costimulatory signals, whereas T cell exhaustion is a state in which CD8+ cells become inadequately responsive due to prolonged exposure to antigens during chronic viral infection or cancer. + This is called the T cell state. Some of the hallmarks of anergic T cells are their inhibited proliferation and inability to synthesize IL-2 in response to TCR (T cell receptor) engagement.

[0097] T cell exhaustion is characterized by high expression of inhibitory receptors and extensive transcriptional and epigenetic changes, but the mechanisms underlying the dysfunction of exhausted T cells are unknown. Blockade of PD-1 (programmed death protein 1) can reactivate some exhausted T cells but does not fully restore function. Studies combining PD-1 blockade with CAR T cells have failed to demonstrate efficacy. In a model in which healthy T cells are driven to exhaustion by expression of a persistently signaling CAR, exhausted human T cells exhibited widespread epigenomic dysregulation of AP-1 transcription factor binding motifs and increased expression of bZIP and IRF transcription factors, which have been implicated in the regulation of exhaustion-associated genes. See Lynn RC et al. Nature. 2019;576(7786):293-300, incorporated herein by reference in its entirety.

[0098] Gene expression of several members of the AP-1 signaling pathway is altered in resting T cells after nucleofection compared to untreated cells. The consequences for T cell activation in vivo are unclear. In contrast, cells treated with SOLUPORE™ exhibit a gene expression profile much closer to that of untreated cells, indicating that these cells are minimally perturbed and likely retain more normal activity in vivo.

[0099] The human amino acid sequence of AP-1 (SEQ ID NO:27) is presented herein and is publicly available under GenBank Accession No: P05412.2, which is incorporated herein by reference. TIFF2025156332000003.tif23142

[0100] Exemplary critical residues, domains, and fragments of AP-1 include, but are not limited to, residues 255-310 (helical region), residues 255-306 (helical region), and residues 8, 58, 63, 89, and 93 (phosphorylation). Fragments of AP-1 proteins are less than the length of the full-length protein, e.g., fragments are at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, or more residues in length, but are less than 331 residues in the case of AP-1 described above.

[0101] The human AP-1 nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No: NM_005354.6 (SEQ ID NO:28), which is incorporated herein by reference. TIFF2025156332000004.tif128144

[0102] Exemplary important residues, domains, and fragments of AP-1 include, but are not limited to, residues 139-1182 (coding region).

[0103] The AP-1 signaling pathway is illustrated in Figure 10. The relevant genes involved in the AP-1 signaling pathway include FOS, Jun, FOSB (Fos proto-oncogene), BATF (basic-leucine zipper transcription factor ATF-like), BATF3 (basic-leucine zipper transcription factor ATF-like 3), IRF4 (interferon regulatory factor 4), NFATc1 (nuclear factor of activated T cells, cytoplasmic 1), MAP2K2 (dual specificity mitogen-activated protein kinase kinase 2), MAPK3 (mitogen-activated protein kinase 3), MAP2K7 (dual specificity mitogen-activated protein kinase kinase 7), PLCG1 (phospholipase C, gamma 1), NFKB2 (nuclear factor NF-κB p100 subunit), and NFKB1A (nuclear factor NF-κB p105 subunit).

[0104] The human amino acid sequence of Fosb (FBJ murine osteosarcoma viral oncogene homolog B) (SEQ ID NO:3) is presented herein and is publicly available under GenBank Accession No: NP_001107643.1, which is incorporated herein by reference. TIFF2025156332000005.tif22142

[0105] Exemplary key residues, domains, and fragments of Fosb include, but are not limited to, residues 1-302 (coding region), residues 255-306 (helical region), and residues 8, 58, 63, 89, and 93 (phosphorylation). Fragments of the FosB protein are less than the length of the full-length protein, e.g., fragments at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, or more residues in length, but less than 302 residues, for example, in the case of FosB described above.

[0106] The human Fosb nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No: NM_006732.1 (SEQ ID NO:29), which is incorporated herein by reference. TIFF2025156332000006.tif198148TIFF2025156332000007.tif46144

[0107] Exemplary important residues, domains, and fragments of Fosb include, but are not limited to, residues 594-1610 (coding region), residues 3754-3759 (regulatory region), or residue 3775 (polyA site).

[0108] The human amino acid sequence of BATF (basic leucine zipper transcription factor ATF-like) (SEQ ID NO:4) is presented herein and is publicly available under GenBank Accession No: CH471061.1, which is incorporated herein by reference. TIFF2025156332000008.tif11142

[0109] Exemplary significant residues, domains, and fragments of BATF include, but are not limited to, residues 1-125 (coding region). Fragments of the BATF protein are less than the length of the full-length protein, e.g., fragments are at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more residues in length, but are, for example, less than 125 residues in the case of BATF described above.

[0110] The human BATF nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No. NM_006399.3 (SEQ ID NO:30), which is incorporated herein by reference. TIFF2025156332000009.tif62142

[0111] Exemplary important residues, domains, and fragments of BATF include, but are not limited to, residues 243-620 (coding region), residues 306-410 (exon), residues 411-941 (exon); residues 922-927 (polyA sequence); residue 941 (polyA site).

[0112] The human amino acid sequence of BATF3 (basic leucine zipper transcription factor ATF-like 3) (SEQ ID NO:5) is presented herein and is publicly available under GenBank Accession No: NP_061134.1, which is incorporated herein by reference. TIFF2025156332000010.tif11142

[0113] Exemplary critical residues, domains, and fragments of BATF3 include, but are not limited to, residues 1-127 (coding region), residues 2 or 31 (phosphorylation site), residues 37-62 (basic motif), or residues 63-91 (leucine zipper). Fragments of the BATF3 protein are less than the length of the full-length protein, e.g., fragments are at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more residues in length, but are, for example, less than 127 residues in the case of BATF3 described above.

[0114] The human BATF3 nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No. NM_018664.2 (SEQ ID NO:31), which is incorporated herein by reference. TIFF2025156332000011.tif66146

[0115] Exemplary critical residues, domains, and fragments of BATF3 include, but are not limited to, residues 224-607 (coding region), 314-418 (exon), 419-981 (exon), 908-913 (polyA signal sequence), or residues 926 or 981 (polyA site).

[0116] FOS ("c-Fos" or "v-fosFBJ murine osteosarcoma viral oncogene homolog, FBJ murine osteosarcoma viral oncogene homolog") c-Fos is a proto-oncogene that is the human homolog of the retroviral oncogene v-fos. cFos is part of the larger Fos transcription factor family, which includes c-Fos, FosB, Fra-1, and Fra-2. c-Fos encodes a 62-kDa protein that heterodimerizes with c-jun (a member of the Jun transcription factor family) to form the AP-1 (activator protein-1) complex, which binds to DNA at AP-1-specific sites in the promoter and enhancer regions of target genes, transducing extracellular signals into changes in gene expression. c-Fos plays an important role in many cellular functions and has been found to be overexpressed in a variety of cancers.

[0117] The human amino acid sequence of FOS (SEQ ID NO:32) is presented herein and is publicly available under GenBank Accession No: AY212879.1, which is incorporated herein by reference. TIFF2025156332000012.tif27142

[0118] Exemplary important residues, domains, and fragments of FOS include, but are not limited to, residues 147-199 (coil region). Fragments of the FOS protein are less than the length of the full-length protein, e.g., fragments at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, or more residues in length, but, for example, in the case of FOS described above, less than 380 residues.

[0119] The human FOS nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No: NM_005252.2 (SEQ ID NO: 1), which is incorporated herein by reference. TIFF2025156332000013.tif135144

[0120] Exemplary important residues, domains, and fragments of FOS include, but are not limited to, residues 156-1298 (coding region), 1803-1808 (polyA region), and 2079-2084 (polyA region).

[0121] Jun ("v-jun avian sarcoma virus 17 oncogene homolog, jun oncogene," or "c-jun") c-Jun is a protein encoded by the JUN gene in humans. c-Jun binds to c-Fos to form the AP-1 early response transcription factor. c-jun was the first oncogenic transcription factor discovered. The proto-oncogene c-Jun is the cellular homolog of the viral oncoprotein v-jun (P05411). Human JUN encodes a protein that is highly similar to this viral protein and interacts directly with specific target DNA sequences to regulate gene expression.

[0122] Both Jun and its dimerization partner in AP-1 formation are regulated by a wide variety of extracellular stimuli, including peptide growth factors, inflammatory cytokines, oxidative and other types of cellular stress, and UV irradiation. For example, UV irradiation is a potent inducer of c-jun expression. c-jun transcription is autoregulated by Jun, its own product. Jun transcription is induced when Jun (AP-1) binds to the high-affinity AP-1 binding site in the Jun promoter region. This positive autoregulation by stimulating its own transcription may be a mechanism for extending signals from extracellular stimuli. This mechanism may have biological significance for c-jun activity in cancer.

[0123] Phosphorylation of Jun at serines 63 and 73 and threonines 91 and 93 increases the transcription of c-Jun target genes. Therefore, c-Jun activity can be regulated by N-terminal phosphorylation by Jun N-terminal kinase (JNK). Jun activity (AP-1 activity) in stress-induced apoptosis and cell proliferation has been shown to be regulated by its N-terminal phosphorylation.

[0124] The human amino acid sequence of Jun (SEQ ID NO:33) is presented herein and is publicly available under GenBank Accession No: AAV38564.1, which is incorporated herein by reference. TIFF2025156332000014.tif23142

[0125] Exemplary important residues, domains, and fragments of Jun include, but are not limited to, residues 255-306 (the coiled-coil region). Fragments of the Jun protein are shorter than the length of the full-length protein, e.g., fragments are at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, or more residues in length, but are, for example, less than 331 residues in the case of Jun above.

[0126] The human Jun nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No: NM_002228.3 (SEQ ID NO:2), which is incorporated herein by reference. TIFF2025156332000015.tif217148

[0127] Exemplary important residues, domains, and fragments of Jun include, but are not limited to, residues 1044-2039 (coding region), 3302-3307 (regulatory region), 2624 (polyA region).

[0128] T cell exhaustion T cell "exhaustion" refers to a state in which T cells do not respond adequately due to prolonged exposure to antigens, such as during chronic viral infection or cancer. "T cell exhaustion" is characterized by the loss of T cell function, which can occur as a result of infection or disease. Exhausted T cells exhibit a transcriptional profile distinct from that of functional effector or memory T cells, characterized by the expression of inhibitory cell surface receptors, including PD-1 (programmed death protein 1), LAG-3 (lymphocyte activation gene 3), TIM-3 (T cell immunoglobulin mucin-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains), and CTLA-4 (cytotoxic T lymphocyte-associated protein 4), and impaired IL-2 (interleukin 2), TNF (tumor necrosis factor), and IFN-γ (interferon gamma) cytokine production. NFAT (nuclear factor of activated T cells) and AP-1 transcription factors synergistically play a central role in inducing hyporesponsive states such as anergy and exhaustion. Exhausted cells exhibit low expression of AP-1 factors (FOS, FOSB, and Jun). See, e.g., Wherry, J. and Kurachi, M. "Molecular and cellular insights into T cell exhaustion," Nat Rev Immunol. 2015 August;15(8):486-499, incorporated herein by reference in its entirety.

[0129] Effect of SOLUPORE™ Processed T Cell Function The data described herein provide an understanding of the SOLUPORE™ process for T cell function. Specifically, T cell function is compared to cells transfected by nucleofection and electroporation. In the examples, soluporation, nucleofection, and electroporation (both of which utilize the application of electrical current to cells) without cargo (e.g., mock) or with a model cargo (e.g., mRNA-GFP) are compared and evaluated. A number of functional assays were performed in conjunction with the above transfection methods, including 1) phenotypic analysis, 2) cytokine release, 3) gene expression profiling of over approximately 700 immune-related genes, and 4) metabolic rate.

[0130] Cytokine release upon immune cell transfection Viral delivery systems for cell manipulation are prone to vector-mediated genotoxicity, resulting in adverse immunogenicity and severe side effects. Electroporation is a commonly used tool for delivering exogenous materials into cells for therapeutic purposes, but electroporation-induced perturbations include nonspecific cytokine release. Using the SOLUPORE™ delivery method described herein, no significant differences were observed compared with untreated control cells. In contrast, significant differences were observed in electroporated immune cells, such as T cells.

[0131] For example, cytokines that were not perturbed using the methods described herein include IL-2 (interleukin 2), IFN-γ (interferon gamma), TNFα (tumor necrosis factor alpha), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-8 (interleukin 8), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), fractalkine, ITAC (interferon-inducible T-cell alpha chemoattractant), and IL-17A (interleukin 17A). In contrast, electroporated cells showed significant differences in IL-2 and IL-8.

[0132] The human amino acid sequence of IL-2 (SEQ ID NO:34) is presented herein and is publicly available under GenBank Accession No: NP_000577.2, which is incorporated herein by reference. TIFF2025156332000016.tif11142

[0133] Exemplary significant residues, domains, and fragments of IL-2 include, but are not limited to, fragments that are less than the length of the full-length protein, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 residues in length, or longer, but e.g., less than 153 residues in the case of IL-2 described above.

[0134] The human IL-2 nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No. NM_000586.2 (SEQ ID NO:17), which is incorporated herein by reference. TIFF2025156332000017.tif70145

[0135] Exemplary important residues, domains, and fragments of IL-2 include, but are not limited to, residues 295-756 (coding region), 295-354 (signal peptide), 355-753 (mature peptide).

[0136] The human amino acid sequence of IL-8 (SEQ ID NO:35) is presented herein and is publicly available under GenBank Accession No: NP_001341769.1, which is incorporated herein by reference. TIFF2025156332000018.tif7139

[0137] Exemplary critical residues, domains, and fragments of IL-8 include, but are not limited to, fragments that are less than the full-length protein, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 residues in length, or longer, but, e.g., for IL-8 described above, less than 95 residues. Exemplary critical residues, domains, and fragments of IL-8 include, but are not limited to, residues 1-95 (protein precursor); residues 1-20 (signal peptide).

[0138] The human IL-8 nucleic acid sequence (start and stop codons are underlined) is shown below and is available under GenBank Accession No: NM_001354840.3 (SEQ ID NO: 18), which is incorporated herein by reference. TIFF2025156332000019.tif136144

[0139] Exemplary important residues, domains, and fragments of IL-8 include, but are not limited to, residues 91-378 (coding region) or 91-150 (signal peptide).

[0140] CAR Plus Delivery CAR plus refers to (1) a cell population that has been transduced with a virus and then transduced with an additional intracellular delivery method (e.g., SOLUPORE™ delivery method, electroporation, or nucleofection, or any combination thereof), or (2) a cell population in which an exogenous cargo has been delivered using a SOLUPORE™ delivery method and the cells have then been subjected to an additional intracellular delivery method (e.g., viral transduction, SOLUPORE™ delivery method, electroporation, or nucleofection, or any combination thereof). Even if the cells have first been transduced with a virus and then subjected to intracellular delivery using a SOLUPORE™ delivery method, viral components may still be present.

[0141] SOLUPORE™ delivery method is used together with the cells that have undergone additional cargo delivery procedures.For example, SOLUPORE™ delivery method is used to deliver exogenous cargo, for example, mRNA, to cells that have already been transduced by virus (Figure 19A and Figure 19B).Or, first, SOLUPORE™ delivery method is used to deliver exogenous cargo, for example, mRNA, and then the cells are subjected to additional delivery procedures, for example, viral transduction.

[0142] Exemplary additional intracellular delivery methods include SOLUPORE™ delivery, viral transduction, electroporation, nucleofection, or any combination thereof. Exemplary viruses that can be used for intracellular delivery include lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV). In a preferred embodiment, the virus is a lentivirus.

[0143] Overview of viruses used in gene delivery applications TIFF2025156332000020.tif58150Pharmaceutics 2020, 12, 183, which is incorporated herein by reference in its entirety.

[0144] Gene editing and indel (insertion-deletion) analysis Gene editing is a form of genetic engineering in which DNA is inserted, deleted, modified, or replaced at site-specific locations within a cell's genome. A common method for such editing uses engineered nucleases to create site-specific double-strand breaks (DSBs) at desired locations within the genome. The induced double-strand breaks are then repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR), resulting in targeted mutations ("edits"). NHEJ can result in genetic disruption by introducing insertions, deletions, translocations, or other DNA rearrangements at the DSB site. Alternatively, precise DNA editing can occur by providing a donor DNA template encoding the desired DNA change, flanked by sequences homologous to regions upstream and downstream of the DSB. The cell then integrates sequences from the exogenous DNA template into the DSB site via homology-directed repair (HDR).

[0145] Electroporation can cause cell damage and stress, resulting in a decrease in cell proliferation rate.The effect of electroporation can make the DNA repair pathway required for gene editing inefficient, resulting in a decrease in gene editing efficiency.SOLUPORE™ delivery method does not cause cell damage or decrease in cell proliferation, and therefore is more suitable than electroporation for achieving efficient gene editing level.

[0146] Furthermore, complex editing is necessary to generate effector cells suitable for allogeneic applications or targeting solid tumors. However, when multiple nucleases and DNA templates are used simultaneously in cells, multiple DSBs occur, making it impossible to control the location in the genome where each template is inserted. Therefore, to ensure that a given exogenous DNA template is inserted into the desired region, it is desirable to perform multiple edits sequentially rather than simultaneously. However, electroporation is a harsh technique that causes cell damage, making it very difficult to perform multiple electroporations. In contrast, the SOLUPORE™ technology is gentle on cells and allows for multiple sequential transfections (see Example 2). This allows for better control of complex editing regimes in cells.

[0147] Exogenous cargo The exogenous cargo (or "payload") delivered to immune cells refers to a compound or composition that is delivered to the interior of the cell through an aqueous solution across the cell plasma membrane. The exogenous cargo may include nucleic acids (e.g., RNA (ribonucleic acid), mRNA (messenger RNA), or DNA (deoxyribonucleic acid)), proteins or peptides, small chemical molecules, or any combination thereof. Small chemical molecules may be less than 1,000 Da. Small molecules are compounds with a mass of less than 2,000 Daltons. The molecular weight of small molecules is preferably less than 1,000 Daltons, more preferably less than 600 Daltons; for example, the compound is less than 500 Daltons, 400 Daltons, 300 Daltons, 200 Daltons, or 100 Daltons.

[0148] In a preferred embodiment, the exogenous cargo comprises a nucleic acid, such as messenger RNA (mRNA). Exogenous cargo comprising mRNA includes CD19 CAR-second generation mRNA (SEQ ID NO:6), CD19 CAR-third generation mRNA (SEQ ID NO:8), TRAIL-DR5 (TNF-related apoptosis-inducing ligand (TRAIL) death receptor 5) variant mRNA (SEQ ID NO:10), TRAIL (SEQ ID NO:11), IL-15 (interleukin 15) mRNA, and TCR (T cell receptor) mRNA.

[0149] In other examples, the exogenous cargo comprises a Cas9 (CRISPR-associated protein 9) protein with a guide RNA comprising, for example, TRAC (T-cell receptor alpha constant SEQ ID NO:25) or PD-1 (programmed death ligand 1 SEQ ID NO:26). In other examples, the exogenous cargo comprises a Cas12a protein (CRISPR-associated protein 12a) with a guide RNA comprising TRAC and PD-1. In examples, the exogenous cargo comprises a MAD7 protein (see Price MA, et al, Rosser SJ. Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7—Biotechnol Bioeng. 2020;117(6):1805-1816, incorporated herein by reference) with a guide RNA comprising TRAC or PD-1. In examples, the exogenous cargo comprises SgCas (see Petris G, et al. Hit and go CAS9 delivered through a lentiviral based self-limiting circuit. Nat Commun. 2017;8:15334, published May 22, 2017, incorporated herein by reference) with a guide RNA comprising TRAC or PD-1. In examples, the exogenous cargo comprises Cas13 with a guide RNA comprising TRAC and PD-1. Alternatively, the exogenous cargo comprises a base editor such as Cas9n, or a zinc finger nuclease, or MegaTAL.

[0150] In examples, the exogenous cargo comprises a Sleeping Beauty 100 transposon / transposase system, or a Sleeping Beauty 1000 transposon / transposase system, or a Piggy Bac transposon / transposase system, or a TcBuster transposon / transposase system.

[0151] In other examples, the exogenous cargo comprises DNA, for example, CD19 CAR DNA, TRAIL DNA, or IL-15 DNA.

[0152] In some embodiments, the exogenous cargo comprises Yamanaka factors used to generate stable induced pluripotent stem cells from adult human cells, such as c-Myc (a MYC proto-oncogene, a bHLH transcription factor), Klf4 (Kruppel-like factor 4), Oct4 (octamer-binding transcription factor 4), or Sox2 (SRY (sex determining region Y)-box 2).

[0153] In a further example, the exogenous cargo comprises an siRNA (small interfering RNA), for example, against PD-1. In a further example, the exogenous cargo comprises an shRNA (short hairpin RNA), for example, an shRNA against PD-1.

[0154] The CD19 CAR mRNA sequence is provided below (SEQ ID NO:6) TIFF2025156332000021.tif158159

[0155] The CD19 CAR protein sequence is provided below (SEQ ID NO:7): TIFF2025156332000022.tif39159

[0156] The Avectas CD19 CAR mRNA sequence is provided below (SEQ ID NO:8) TIFF2025156332000023.tif119159

[0157] The Avectas CD19 CAR protein sequence is provided below (SEQ ID NO:9) TIFF2025156332000024.tif39159

[0158] The full-length E195R / D269H TRAIL(DR5)-variant: (846 nucleotides) mRNA is provided below (SEQ ID NO: 10). TIFF2025156332000025.tif84155

[0159] The full-length E195R / D269H TRAIL (DR5 variant) protein sequence is provided below (SEQ ID NO:36). Note that E195R / D269H is bolded and underlined. TIFF2025156332000026.tif21136

[0160] The full-length TRAIL protein sequence is provided below (SEQ ID NO:11): TIFF2025156332000027.tif20136

[0161] RNP The sequence of the gRNA targeting human TRAC is TIFF2025156332000028.tif4128, and the sequence of the gRNA targeting human PDCD1 is The file was TIFF2025156332000029.tif4128.

[0162] Human cDNAs for Oct4, Sox2, Klf4, and c-Myc were amplified using the following primers: for human Oct4, TIFF2025156332000030.tif18158For human Sox2, TIFF2025156332000031.tif18163For human Klf4, TIFF2025156332000032.tif26159For human c-Myc, TIFF2025156332000033.tif18161 was amplified by RT-PCR from human ES poly(A+) RNA.

[0163] Streptococcus pyogenes Cas9 NCBI Reference Sequence: NZ_CP010450.1 (SEQ ID NO: 19), incorporated herein by reference. TIFF2025156332000034.tif102159

[0164] Staphylococcus agnetis Cas9 NCBI Reference Sequence: NZ_CP045927.1 (SEQ ID NO:20), incorporated herein by reference. TIFF2025156332000035.tif50159

[0165] Synthetic construct Cas9 from Staphylococcus aureus NCBI Reference Sequence: MN548085.1 (SEQ ID NO:21), incorporated herein by reference TIFF2025156332000036.tif57159

[0166] Candidatus Methanomethylophilus alvus Mx1201 Cas12a NCBI Reference Sequence: NC_020913.1 (SEQ ID NO:22), incorporated herein by reference. TIFF2025156332000037.tif65159

[0167] Candidatus Methanomethylophilus albus isolate MGYG-HGUT-02456 Cas12a NCBI Reference Sequence: NZ_LR699000.1 (SEQ ID NO:23), incorporated herein by reference. TIFF2025156332000038.tif61159

[0168] Candidatus Methanoplasma termitum strain MpT1 chromosome Cas12a NCBI Reference Sequence: NZ_CP010070.1 (SEQ ID NO:24), incorporated by reference. TIFF2025156332000039.tif65159

[0169] definition The following definitions are included for purposes of understanding the subject matter of the present invention and for purposes of framing the appended claims: The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0170] While various embodiments and aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that in practicing the present invention, various alternatives can be used to replace the embodiments of the invention described herein.

[0171] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, literature, books, manuals, and articles, are expressly incorporated herein by reference in their entirety for any purpose.

[0172] A "patient" or "subject in need thereof" refers to a living member of the animal kingdom that is afflicted with or is susceptible to the indicated disorder. In embodiments, the subject is a member of a species that includes individuals that are naturally susceptible to the disease. In embodiments, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs, e.g., police dogs, military dogs, racing dogs, or show dogs), horses (e.g., race horses and working horses), cats (e.g., domestic cats), livestock (e.g., pigs, cows, donkeys, mules, bison, goats, camels, and sheep), and deer. In embodiments, the subject is a human.

[0173] The terms "subject," "patient," "individual," etc. are not intended to be limiting and are generally interchangeable; that is, an individual described as a "patient" does not necessarily have a particular disease, but may simply be seeking medical advice.

[0174] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claim to the specified materials or steps and to materials or steps that do not materially affect the basic and novel characteristics" of the invention.

[0175] In the description and claims herein, phrases such as "at least one of" or "one or more of" may be followed by a conjunctive list of elements or features. The term "and / or" may appear in a list of two or more elements or features. Unless implicitly or explicitly contradicted by the context in which the term is used, such a phrase is intended to mean any of the listed elements or features individually, or any of the listed elements or features in combination with any of the other listed elements or features. For example, the phrases "at least one of A and B," "one or more of A and B," and "A and / or B" are intended to mean "A only, B only, or A and B," respectively. A similar interpretation is intended for lists containing more than two items. For example, the phrases "at least one of A, B, and C," "one or more of A, B, and C," and "A, B, and / or C" are intended to mean "A only, B only, C only, A and B, A and C, B and C, or A, B, and C," respectively. Furthermore, use of the term "based on" above and in the claims is intended to mean "based at least in part on," such that unrecited features or elements are also permitted.

[0176] As used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein is substantially free from other cellular materials, substantially free from culture medium when produced by recombinant methods, and free from chemical precursors or other chemicals when chemically synthesized. For example, a purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound. Purity is measured by any appropriate standard method, such as column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) or polypeptide is free from naturally occurring adjacent amino acid or nucleic acid sequences. Purified also defines a degree of sterility that makes it safe for administration to a human subject, eg, free from infectious or toxic agents.

[0177] The level ascertained compared to the control level can be an increased level. As used herein, the term "increased" with respect to a level (e.g., T cell cytokine release, gene regulation, or metabolic rate following the described SOLUPORE™ method) refers to any percent increase above the control level. In various embodiments, an increased level can be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, or at least or about a 95% increase compared to the control level.

[0178] The level ascertained compared to the control level can be a decreased level. As used herein, the term "decreased" with respect to a level (e.g., T cell cytokine release, gene regulation, or metabolic rate following the described SOLUPORE™ method) refers to any percent decrease below the control level. In various embodiments, a decreased level can be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, or at least or about a 95% decrease compared to the control level.

[0179] Increases or decreases may also be expressed as fold differences or log differences (see, e.g., Figure 12 for correlation). For example, Log base 2 (or log2) was used to normalize results along an axis with the same values ​​for up-regulated and down-regulated genes. An exemplary calculation is shown below: Gene A treatment vs. control = 7.0 (overexpression); Gene B control vs treated = 7.0 or treated vs control = 0.142 (underexpressed).

[0180] Both are over- or under-expressed with the same intensity, but a linear scale does not reflect this. Alternatively, gene A is up-regulated 7.0-fold and gene 2 is down-regulated 0.142-fold. Expressed in log2, gene A is up-regulated 2.81-fold and gene B is down-regulated -2.81-fold. [Example]

[0181] The following examples illustrate certain specific aspects of the present invention and are not intended to limit the scope of the invention.

[0182] The embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are merely intended to illustrate the embodiments and should not be construed as limiting the scope of the present specification. The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

[0183] Example 1: Efficient and versatile manipulation of primary human immune cells We evaluated the ability of the SOLUPORE™ delivery method to deliver a model cargo, GFP (green fluorescent protein) mRNA, to primary human T cells. Because T cell therapy manufacturing processes vary widely and involve various cell culture regimes, we used both PBMC (peripheral blood mononuclear cell)-initiated T cell cultures and CD3+ (cluster of differentiation 3) purified T cell cultures isolated from three human donors. At 24 hours, GFP expression in PBMC-initiated and CD3+ purified T cells was 65-75% and 40-50%, respectively, with cell viability exceeding 70% (Figure 1A and Figure 1B).

[0184] Next, we evaluated the efficiency of the SOLUPORE™ delivery method using functional cargo with Cas9 (CRISPR-associated endonuclease Cas9 (Cas9)) protein-gRNA ribonucleoprotein (RNP) complexes designed to target the TRAC (T cell receptor alpha) and PDCD1 (programmed death cell protein 1) genes. RNPs were delivered to T cells isolated from three donors. For TRAC RNPs, CD3 expression decreased from 90% to 35%, with corresponding cell viability of >90% (Figure 1C). For PDCD1, an indel (insertion or deletion of bases) efficiency of 25% was reached, with cell viability of >90% (Figure 1D).

[0185] Example 2: Dual sequential delivery of multiple cargoes Next-generation immune cell therapy products will need to be somewhat modified. This means that transfection techniques are needed to deliver multiple cargoes. However, such manipulations are only useful if cell health and function are not adversely affected by the delivery method. Therefore, the SOLUPORE™ delivery method was evaluated for simultaneous or sequential delivery of two cargoes. Maintenance of cell viability was also evaluated.

[0186] Dual Cargo Delivery To test the concept of dual cargo delivery, we co-delivered CD19 (cluster of differentiation 19) CAR (chimeric antigen receptor) mRNA and GFP mRNA to stimulated T cells from three donors using either the SOLUPORE™ delivery method or electroporation. Twenty-four hours after transfection, 68.7 ± 4.1% of the cell population using the SOLUPORE™ delivery method was CD3+ CAR-positive, and cell viability remained high (Figure 2A). Representative flow cytometry plots are shown in Figure 2B.

[0187] Therapeutic benefits can be achieved by delivering multiple cargoes, and multiple or complex cargoes are required for effective treatment.

[0188] Delivery of multiple cargoes allows for complex editing at the cell surface, with each cargo imparting a specific function or characteristic to the cell. For autologous cell therapies targeting both liquid and solid tumors, where enhanced targeting and efficacy are required, cells require multiple modifications, often in tandem with the manufacturing process. This may involve multiplexed or sequential engineering steps. Similar demands apply to allogeneic approaches. In this case, issues of cell rejection and GvHD mean that complex editing is likely required. Viral vector volume constraints and electroporation toxicity mean that these modalities may be inappropriate for certain complex engineering methods. Furthermore, the long lead times required to design and generate even research-grade viral vectors mean that development timelines may be longer than desired. This is particularly concerning when advancing approaches to solid tumors. In this case, the challenges of targeting and efficacy mean that numerous candidate target antigens and cell enhancements must be tested. There is a need to assess a myriad of cellular compositions in a rapid, high-throughput manner that would likely be highly limited if reliant entirely on viral vectors.

[0189] Continuous cargo delivery To evaluate sequential delivery, TRAC (T cell receptor alpha) RNP (ribonucleoprotein) was delivered to T cells, and two days later, CD19 CAR mRNA was delivered to the same cell population. The following day, cells were collected and analyzed for CD3 and CAR expression. Mean CAR expression was 67.5 ± 8.4%, CD3 knockdown was 79.7 ± 2.4%, and 56.7 ± 3.4% of cells were CAR-positive and CD3-negative (Figure 2C). Mean cell viability was 76.7 ± 10.9%, compared with 94.74 ± 4.5% for untreated control cells. Representative flow cytometry plots are shown in Figure 2D. Similar to dual / multiplex delivery, sequential delivery of cargoes offers therapeutic benefits, allowing cells to be engineered with multiple novel features that enhance their ability to target or kill tumor cells effectively. For example, targeting multiple tumor antigens may be necessary to enhance targeting. Additionally, there is interest in expressing chemokine receptors or cytokines on CAR-T cells to increase T cell trafficking, or stromal degrading enzymes to increase CAR-T cell migration through the tumor, or enhancing persistence by expressing dominant / negative CAR-T inhibitors, e.g., PD-1 and TGFβ, as well as many other strategies.

[0190] Example 3: Cytokine release demonstrated minimal cellular disruption The cargo delivery study in Example 2 above demonstrated that transfection using the SOLUPORE™ delivery method was efficient with minimal impact on cell viability. However, it has been reported that while delivery methods such as electroporation may have minimal impact on T cell viability, they can cause stress to the cells, which can lead to unintended changes in gene and protein expression and ultimately cell function. Therefore, the effect of transfection on cytokine release and immune gene expression in T cells (Example 4) was evaluated.

[0191] We first investigated whether the SOLUPORE™ delivery method nonspecifically releases cytokines from T cells using a multiplex assay (Luminex). The panel included 11 human analytes: IFN-γ (interferon gamma), IL-2 (interleukin 2), TNFα (tumor necrosis factor alpha), IL-8 (interleukin 8), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), IL-17A (interleukin 17A), fractalkine, and ITAC (interferon-inducible T cell alpha chemoattractant).

[0192] GFP mRNA was delivered by soluporation to stimulated T cells from five donors. Two technical replicates were included for each donor. A mock transfection (no cargo) was also included and cytokine release was measured over a five-day time course. No significant differences were observed for any of the cytokines analyzed when comparing the SOLUPORE™ delivery GFP mRNA and mock transfection groups with untreated control cells, indicating that the cells were not perturbed in a way that would nonspecifically release these cytokines.

[0193] In contrast, when electroporation was used to transfect the cells, significant differences in IL-2 and IL-8 secretion were evident. This suggests that the electroporation process induced cellular stress, which led to the release of cytokines from these cells (Figure 3A and Figures 6A-6I). T cells release cytokines either in response to specific stimulatory ligands or nonspecifically in response to stress. No specific stimulatory ligands were used in these experiments. This indicates that stress was imposed on the electroporated cells.

[0194] Example 4: Immune gene profiling demonstrates minimal cellular perturbation The experiments described herein were performed cargo-independently. This means that they were conducted to demonstrate that the SOLUPORE™ delivery method has minimal impact on protein and gene expression in T cells and, importantly, preserves biological characteristics such as proliferation. Furthermore, the addition of exogenous cargo to immune cells using the SOLUPORE™ delivery method only minimally impacts protein and gene expression, while maintaining biological function and activity. The Nanostring CAR-T characterization panel, which measures the gene expression of up to 780 immune-related genes, including genes associated with immune cell exhaustion, activation, and persistence, was used to assess the impact of the transfection process on gene expression in T cells. It has been reported that electroporation can dramatically affect gene expression in T cells. To avoid the potential confounding effects of cargo on gene expression, mock-transfected unstimulated T cells (or "rested T cells") were used. The "highly efficient for T cells" FI-115 electroporation program recommended by the manufacturer (Lonza) was used.

[0195] In the first of these studies (Study 1), resting T cells from three donors, each with two technical replicates, were mock-transfected using either the SOLUPORE™ delivery method or electroporation. Gene expression was analyzed 6 and 24 hours after transfection. In the 6-hour SOLUPORE™ delivery group, 1.7% of genes were identified as differentially expressed compared to untreated control cells (10 / 582 genes, 1 log2 fold (>2-fold) change, p<0.05, Tables 7-8). No changes in gene expression were identified 24 hours after transfection (SOLUPORE™ delivery method) (0 / 582 genes).

[0196] In contrast, 265 / 582 genes were found to be altered in the 6-hour electroporation group, representing 45.5% of the genes detected (Tables 7-9). In the 24-hour electroporation group, 11.3% of the genes were differentially expressed (66 / 582, Table 9). Comparing the 6-hour and 24-hour electroporation groups, 37 genes were found to be differentially expressed at both time points (Table 4 below).

[0197] Table 4. Study 1 - Electroporation: 37 genes were common at 6 and 24 hours. TIFF2025156332000040.tif187128

[0198] Of the 10 genes identified in the SOLUPORE™ delivery method 6 hour group, 8 genes were in common with the electroporation 6 hour group (Table 5 below).

[0199] Table 5. Genes common at 6 hours in the electroporation and SOLUPORE™ delivery groups of Study 1 TIFF2025156332000041.tif45128

[0200] Volcano plots (Figure 3B) and heat maps (Figure 3C and Figure 7) were generated to obtain a complete picture of differentially expressed genes. Pathway analysis was also completed (Table 1 below and Figure 8). The majority of genes identified in the 6-hour electroporation group were located in pathways related to T cell activation, metabolism, and exhaustion.

[0201] Table 1. Pathway analysis of genes identified in the CAR-T characterization panel at 6 hours TIFF2025156332000042.tif74160

[0202] Given the large number of gene changes in the electroporation group, a second study was conducted to verify this finding. In Study 2, gene expression was analyzed 24 hours after transfection. Each group included unstimulated T cells from two donors and two technical replicates, with a third donor performed once, all mock-transfected. The results were similar to those observed in the first study, with only 9 / 597 (1.5%) genes identified with the SOLUPORE™ delivery method and 43 / 597 (7.2%) genes identified in the FI-115 electroporation group (Tables 11 and 12). This demonstrates consistency with Study 1. In this study, four genes were identified as common between the SOLUPORE™ delivery method and the 24-hour electroporation group (Table 6 below).

[0203] Table 6. Genes common at 24 hours in the electroporation and SOLUPORE™ delivery groups of Study 2 TIFF2025156332000043.tif30128

[0204] Thirty-eight genes were found to be common in the 24-hour electroporation groups when comparing Study 1 and Study 2, further demonstrating consistency between studies (Table 2 below).

[0205] Table 2. Comparison of Test 1 and Test 2 electroporation groups at 24 hours showing common genes identified by a log2 fold (>2-fold) change of 1, p<0.05 TIFF2025156332000044.tif19276

[0206] An additional nucleofection program, EO-115, was also included in Study 2. This program is described by the manufacturer as "highly cellular functional" and is likely less stringent than FI-115. Using the EO-115 program, 16 / 597 (2.7%) genes were differentially expressed (Tables 11 and 12). There was a high degree of overlap in the genes identified in the two nucleofection programs, with 12 of the 16 genes in the EO-115 group also present in the FI-115 group (Tables 11 and 12). The small number of genes identified in the EO-115 group compared to FI-115 coincided, indicating this was a less stringent electroporation program.

[0207] Characterization and phenotype of T cell exhaustion For next-generation CAR T therapy, several issues have been investigated with the aim of achieving long-term disease control in a larger number of patients. In this regard, improving responses in solid tumors and T cell exhaustion have received increasing attention. The T cell exhaustion phenotype naturally occurs after prolonged exposure to antigens during chronic viral infections or cancer and is characterized by the expression of inhibitory receptors, metabolic dysfunction, and reduced expression of effector functions such as cytokine secretion. Exhaustion involves a significant rewiring of metabolic processes via TCR (T cell receptor) signaling, and transcription factors, including the AP-1 (activator protein 1) complex, IRF4 (interferon regulatory factor 4), BATF (basic leucine zipper transcription factor ATF-like), and NFAT (nuclear factor of activated T cells), have been suggested to play important roles in this process.

[0208] Antigen signaling through the T cell receptor activates these signaling pathways, but cellular stress can also stimulate these pathways in T cells. Therefore, exhaustion-related genes in the CAR-T characterization panel were evaluated. In the electroporation group, expression of FOSB (Fos proto-oncogene), FOS (proto-oncogene), JUN, BATF (basic leucine zipper transcription factor ATF-like), BATF3 (basic leucine zipper transcription factor ATF-like 3), and IRF4 (interferon regulatory factor 4) genes was consistently upregulated between both studies. In contrast, perturbation of these genes was minimal when using the SOLUPORE™ delivery method compared to untreated control cells (Table 3 below).

[0209] (Table 3) AP-1 (activator protein 1)-related genes identified with a log2 fold (>2-fold) change of 1 in Study 1 and Study 2, p<0.05 TIFF2025156332000045.tif77161n.i.=Not specified

[0210] In other aspects, the immune cells of the present invention (immune cells bearing exogenous cargo) have a molecular profile in which programmed death protein 1 (PD1) is expressed at a log2 fold change level of 3, a log2 fold change level of 2, or a log2 fold change level of 1 relative to the level expressed in control immune cells.

[0211] Exhausted T cells exhibit a transcriptional profile distinct from functional effector T cells or memory T cells, characterized by the expression of inhibitory cell surface receptors, including PD-1. For example, immune cells of the present invention (immune cells carrying exogenous cargo) have a molecular profile in which PD-1 is expressed at a log2 fold change level of about 1 compared to the level expressed in control immune cells. For example, immune cells of the present invention (immune cells carrying exogenous cargo) have a molecular profile in which PD-1 is expressed at a log2 fold change level of about 2 compared to the level expressed in control immune cells. For example, immune cells of the present invention (immune cells carrying exogenous cargo) have a molecular profile in which PD-1 is expressed at a log2 fold change level of about 3 compared to the level expressed in control immune cells. In some embodiments, immune cells of the present invention (immune cells carrying exogenous cargo) have a molecular profile in which PD1 is expressed at a log2 fold change of about -3, about -2, or about -1 compared to the level expressed in control immune cells.

[0212] Example 5: Growth and in vivo engraftment of transfected cells Taken together, the cargo delivery and gene and protein expression studies described above demonstrate that the SOLUPORE™ delivery method can efficiently deliver cargo to modify T cells while minimizing cellular stress and nonspecific perturbations of protein and gene expression. However, for cell therapy manufacturing applications, it is also necessary to confirm that the modified cells retain desirable biological attributes, such as vigorous proliferation and in vivo engraftment. Therefore, these characteristics were examined in transfected T cells.

[0213] To examine the effect of transfection on T cell proliferation, cells isolated from five random donors were transfected with GFP mRNA. Each donor contained five independent technical replicates, and cell proliferation was measured over a seven-day period. The proliferation rate of cells transfected using the SOLUPORE™ delivery method was similar to that of untreated control cells (Figure 4A). In contrast, cells transfected using electroporation proliferated at a slower rate than untreated control cells.

[0214] To further evaluate the impact of transfection on T cell health and function, an in vivo engraftment mouse model was used. The xeno-GvHD humanized mouse model, based on an immunodeficient strain injected with human peripheral blood mononuclear cells (hu-PBMCs), is an important tool for examining human immune function. This model is characterized by engraftment of hu-PBMCs into the blood of injected mice and ultimately into the spleen, lymph nodes, and bone marrow. These cells are derived from immunodeficient NOD / SCID / γ mice lacking T, B, and NK cells and harboring a targeted mutation in the IL-2 receptor γ chain (IL-2Rγnull), which allows them to recognize human cells and tissues. - / - They readily engraft after intravenous injection into non-steroidal anti-inflammatory (NSG) mice. Successful engraftment and the development of GvHD depend on hu-PBMC reactivity with mouse MHC class I and II, and therefore on highly viable and functional donor cells.

[0215] Human PBMCs were transfected with 3 kDa dextran-Alexa Fluor 488 using the SOLUPORE™ delivery method or electroporation and injected into irradiated NOD-scid IL-2Rγnull mice. At 28 days post-injection, cells delivered using the SOLUPORE™ delivery method were found to have engrafted in the spleen at levels similar to those of untreated control cells (Figure 4B). In contrast, electroporated cells showed low levels of engraftment, indicating reduced functional potency of these cells.

[0216] Example 6: Generation of CD19 CAR-T cells and in vitro and in vivo cytotoxicity Having demonstrated that the SOLUPORE™ delivery method efficiently modifies T cells while preserving their proliferation and engraftment capacity (Examples 2-5 above), CAR-T cells were generated and evaluated for their ability to kill cancer cells in vitro and in vivo.

[0217] CD19 CAR mRNA was delivered to T cells derived from three donors using either the SOLUPORE™ delivery method or electroporation. CD19 CAR expression using the SOLUPORE™ delivery method was slightly lower than that of electroporated cells, ranging from 72% to 76% and 74% to 81%, respectively, across the three donors (Figure 5A). In vitro cytotoxicity against CD19-expressing RAJI cells was confirmed using a real-time cell impedance assay. CAR-T cells delivered using the SOLUPORE™ delivery method exhibited comparable cytotoxicity against target RAJI cells as electroporated CAR-T cells, despite their low levels of CAR expression (Figure 5A).

[0218] The in vivo therapeutic potential of CAR T cells generated by the SOLUPORE™ delivery method was evaluated using a luciferase-expressing RAJI tumor model in NSG mice (Figure 5B). CD19 CAR T cells were generated using the SOLUPORE™ method, and electroporation was used as a positive control. The average CAR expression was 73% and 85% CAR, respectively. Mice were injected with 1x10 6 pieces, 2x10 6 pieces, or 4x10 6 Doses of CAR T cells were administered, and disease progression was monitored by bioluminescence imaging. Twelve days after CAR T cell dosing, a dose-dependent reduction in tumor growth was evident using the SOLUPORE™ delivery method as well as the positive control electroporation cohort (Figure 5D). Tumor burden reduction was similar between each SOLUPORE™ delivery method and electroporation dose, with the highest dose of 4x106 It was notable that 3 / 10 mice in the CAR T cell group appeared disease-free. This observation correlated with the significantly higher presence of human T cells in the blood of mice receiving SOLUPORE™ delivery CAR-T cells compared to the electroporation control group, as confirmed by flow cytometry analysis (Figure 5D). Similarly, at each dose tested, tumor engraftment, as determined by CD20 (cluster of differentiation 20) expression, was lower in mice receiving SOLUPORE™ delivery-prepared CAR-T cells (t-test) (Figure 5E).

[0219] Example 7: Phenotypic analysis of activated human T cells after transfection Activated CD3+ T cells from three donors were solporated or nucleofected (using the program EO115-"highly functional for T cells") with mRNA-GFP or in the absence of cargo (mock). Cells were analyzed using a panel of monoclonal antibodies (mAbs) specific for T cell-associated activation / exhaustion surface markers (PD-1 and CD69 are primary targets) (Figures 13 and 14).

[0220] Among the three donors, CD4 + The CD4:CD8 population accounts for 65% of the T cell population in both naive and activated UT cells, while cluster of differentiation 8 (CD8) (CD4-negative staining) accounts for 35%. The CD4:CD8 ratio is 65:35. The CD4:CD8 ratio of T cells is maintained after GFP nucleofection (67:33) or mock nucleofection (63:37). The CD4:CD8 ratio of T cells remains unchanged after GFP nucleofection (65:35) or mock nucleofection (69:31) (Figures 13 and 14).

[0221] In three donors, PD1 expression on naive CD4+ T cells was 2%. Upon activation, this increased to 18% ± 5%. PD1 expression after sorbulation with GFP or mock sorbulation was 16% ± 4% or 14% ± 5%, respectively. PD1 expression on CD4+ cells after nucleofection with GFP or mock nucleofection was 14% ± 5% or 17% ± 5%, respectively. In the same three donors, PD1 expression on naive CD8+ T cells was 1%. Upon activation, PD1 expression on CD8+ T cells after sorbulation with GFP or mock sorbulation was 6% ± 1% or 7% ± 1%, respectively. PD1 expression on CD8+ cells after nucleofection with GFP or mock nucleofection was 5% ± 2% or 6% ± 2%, respectively (Figure 13).

[0222] In three donors, CD69 expression on naive CD4+ T cells was 2% ± 4%. Upon activation, CD69 expression was upregulated to 61% ± 1%. After sorbulation with GFP or mock sorbulation, CD69 expression was 66% ± 1% or 62% ± 1%, respectively. After nucleofection with GFP or mock nucleofection, CD69 expression on CD4+ cells was 69% ± 2% or 62% ± 2%, respectively. In the same three donors, CD69 expression on naive CD8+ T cells was 4% ± 8%. Upon activation, CD69 on CD8+ T cells increased to 29% ± 3%. After sorbulation with GFP or mock sorbulation, CD69 expression was 32% ± 1% or 64% ± 2%, respectively. CD69 expression on CD8+ cells after nucleofection with GFP or mock nucleofection was 30%±2% or 32%±1%, respectively (FIG. 14).

[0223] Thus, neither PD-1 nor CD69 expression changes after solporation or nucleofection.

[0224] Example 8: Metabolic studies The metabolic rates of transfected T cells were assessed in three ways: (1) lactate production, (2) oxygen consumption rate, and (3) extracellular acidification rate. Activated T cells release lactate as they undergo metabolic remodeling from oxidative phosphorylation to aerobic glycolysis, which is required for energy-demanding proliferation and effector function. Extracellular lactate correlates well with T cell proliferation. Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) provides insight into the important cellular functions of mitochondrial respiration and glycolysis.

[0225] Lactate production in activated human T cells after transfection The ChromaDazzle lactate assay was used to assess lactate production in cells after transfection. Activated CD3+ T cells from five donors were solporated or nucleofected with mRNA-GFP (using the program EO115-"Highly functional for T cells") or in the absence of cargo (mock). Supernatants were collected 6 hours after transfection and stored at -20°C. The supernatants were subjected to the ChromaDazzle lactate assay (a kinetic reaction catalyzed by an enzyme), and lactate production compared to the control is shown in Figure 15.

[0226] Lactate production from UT cells was set to 1. Compared to UT, solporation- or nucleofection-treated cells produced slightly less lactate, and all cells produced 0.8–0.9 times the amount of lactate compared to UT (Figure 15).

[0227] Metabolism of activated T cells after transfection The Seahorse instrument measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indicators of mitochondrial respiration and glycolysis, respectively. A diagram of the Seahorse data analysis method is shown below (Figures 16A and 16B). Raw data traces from a single donor treated with solporation or nucleofection are shown in Figure 17. Using the calculations in Figures 16A and 16B, glycolysis, oxidative phosphorylation, glycolytic capacity, and maximum respiration are shown in Figure 18.

[0228] Oxidative phosphorylation (OCR data) The Seahorse experimental setup is shown in Figures 16A and 16B. For OCR data focused on UT, the OCR rate slightly decreased upon oligo addition, increased upon FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) stimulation, and decreased again upon the addition of Rot / AA. The modulators included in this assay kit are oligomycin, carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), rotenone, and antimycin A, which should stimulate the oxygen consumption rate pattern seen in Figures 16A and 16B. In the experiment shown, mock sorption followed a similar profile to UT, indicating that the sorption process itself does not interfere with normal cellular oxidative phosphorylation (Figure 17). Sorption with mRNA-GFP showed a slight increase in OCR rate compared to UT after FCCP stimulation, from approximately 60 pmol / min to around 100 pmol / min. However, the increase in OCR rate after FCCP stimulation is more pronounced in nucleofected cells, with the OCR rate increasing to nearly 150 pmol / min (Figures 17 and 18). Thus, in this experiment, the spare respiratory capacity (SRC) of nucleofected cells is more distant from the UT than the SRC of solporated cells. It is important to note that this is a snapshot of the metabolism of these cells at 18 hours post-transfection.

[0229] Glycolysis measurement (ECAR) The Seahorse experimental setup is shown in Figures 16A and 16B. For ECAR data focusing on UT, the ECAR rate increases slightly upon oligo addition and then dramatically decreases after 2DG. This exactly reproduces the pattern seen in Figures 16A and 16B. Similar to the OCR rate, the mock solporation ECAR rate follows a similar trajectory to UT, indicating that the solporation process itself does not interfere with normal cellular glycolysis (Figure 17). Solporation with mRNA-GFP shows a slight increase in ECAR rate compared to UT, from approximately 50 mpH / min basally to around 70 mpH / min, and from approximately 60 mpH / min to around 80 mpH / min after oligo addition. However, the OCR rate increase after FCCP stimulation is more pronounced in nucleofected cells, with the OCR rate increasing to nearly 150 pmol / min (Figures 17 and 18). This data suggests that there is no large or significant difference in the cellular glycolysis (ECAR) of T cells subjected to the SOLUPORE™ process compared to untreated T cells or T cells subjected to nucleofection. This applies to both basal glycolysis measurements and glycolytic capacity measurements of T cells. Glycolytic capacity is the maximum glycolytic rate that cells can reach when forced to glycolyze, and is a measure of the glycolytic machinery available to the cells.

[0230] Example 9: CAR Plus Data SOLUPORE™ delivery method is used together with the cells that have undergone additional cargo delivery engineering methods.For example, SOLUPORE™ delivery method is used to deliver exogenous cargo, such as mRNA, to cells that have already been transduced by virus.Or, first, SOLUPORE™ delivery method is used to deliver exogenous cargo, such as mRNA, and then the cells are subjected to additional delivery engineering methods, such as viral transduction.

[0231] The term "CAR plus" refers to either (1) a cell population that has been transduced with a virus and then transduced with an additional intracellular delivery method (e.g., SOLUPORE™ delivery method, electroporation, or nucleofection, or any combination thereof), or (2) a cell population in which an exogenous cargo has been delivered using a SOLUPORE™ delivery method and the cells have then been subjected to an additional intracellular delivery method (e.g., viral transduction, SOLUPORE™ delivery method, electroporation, or nucleofection, or any combination thereof). Even though the cells have first been transduced with a virus and then subjected to intracellular delivery using a SOLUPORE™ delivery method, viral components may still be present.

[0232] The feasibility of the SOLUPORE™ delivery method for virally transduced CAR T cells was evaluated. + GFP expression and viability were assessed in T cells (3 donors x n=1). Figures 19A and 19B demonstrate the feasibility of the SOLUPORE™ delivery method for generating cells with multiple modifications. Using the SOLUPORE™ delivery method, a 65% transfection efficiency (Figure 19A) was observed in virally transduced CAR T cells (63%, 60%, and 67% GFP+ in CAR+ T cells across the three donors), with a viability of over 80% at 24 hours (Figure 19B).

[0233] The following materials and methods were used in the studies described herein.

[0234] Cell isolation and culture PBMCs were isolated from fresh Leukopaks using lymphoprep density gradient medium (StemCell) and cryopreserved using standard methods. Once thawed, PBMCs were primed (i.e., stimulated or activated) into T cells using antibodies specific for cell surface markers on T cells, such as soluble CD3 (clone: ​​OKT3) and CD28 (clone: ​​15E8) antibodies at 100 ng / ml each (both Miltenyi Biotech). Cells were initiated for 3 days in complete culture medium consisting of CTS OpTimizer+ supplement (Gibco), 5% Physiologix serum replacement (Nucleus Biologics), 1% L-glutamine, and 250 IU / ml IL-2 (CellGenix). Twenty-four hours after collection, human CD3+ T cells were isolated directly from the Leukopak using a MultiMACS 24 (Miltenyi Biotech) and Straightfrom Leukopak CD4 and CD8 T cell reagent (Milteyni Biotech) according to the manufacturer's instructions. 1 × 10 cells were cultured in CTS culture medium (Gibco) supplemented with 2 mM L-glutamine and 250 U / ml IL-2 (CellGenix). 6 T cells were cultured at a density of 1 / ml and activated with anti-CD3 / CD28 coated beads (Cell Therapy Systems (CTS) Dynabeads) at a 2:1 bead:cell ratio.

[0235] SOLUPORE(TM) Delivery Method The SOLUPORE™ delivery method was modified from a previously described method. 5 cells / well in a 96-well filter-bottom plate (Agilent), or 6x10 6Cells were transferred to pods (Avectas) at cell / pod. Culture medium was removed from the 96-well plate by centrifugation at 350 × g for 120 seconds and from the pod by gravity flow. Cargo was combined with a delivery solution (32.5 mM sucrose, 106 mM potassium chloride, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) dissolved in water), and 1 μl or 50 μl was delivered to cells in the 96-well plate and pod, respectively. For mRNA delivery, the delivery solution also contained 12% v / v ethanol. For ribonucleoprotein (RNP) delivery, the delivery solution also contained 25 mM ammonium acetate and 10% v / v ethanol. After a 30-second incubation at room temperature, 50–2000 μl of 0.5X phosphate-buffered saline solution (68.4 mM sodium chloride, 1.3 mM potassium chloride, 4.0 mM sodium dihydrogen phosphate, 0.7 mM potassium dihydrogen phosphate) was added, followed 30 seconds later by the addition of complete culture medium containing CTS OpTimizer+ supplement (Gibco), 5% Physiologix serum replacement (Nucleus Biologics), 1% L-glutamine, and 250 IU / ml IL-2 (CellGenix).

[0236] Electroporation Cells were electroporated using the standard method 4D-Nucleofector system (Lonza) (20-μl nucleocuvette or 100-μl nucleocuvette format) with P3 primary cell 4-D Nucleofector solution and preloaded FI-115 and EO-115 pulse programs according to the manufacturer's protocol.

[0237] GFP mRNA and CAR mRNA delivery For both the SOLUPORE™ delivery method and electroporation, GFP mRNA (model cargo) and CD19 CAR mRNA (functional cargo) (both from TriLink Biotechnologies) were delivered at 2 μg and 3.3 μg / 1x10 cells per million cells, respectively. 6The cells were delivered to a final concentration of 1000. CD19 CAR expression was assessed using a biotin-conjugated CD19 CAR detection reagent (Miltenyi Biotec) followed by streptavidin-PE and 7-aminoactimycin D (7AAD) as a viability stain.

[0238] CD19 CAR sequence (SEQ ID NO:8) TIFF2025156332000046.tif110161

[0239] RNP complex For the SOLUPORE™ delivery method and electroporation, Cas9 protein (Integrated DNA Technologies) precombined with a 2 molar excess of guide RNA (gRNA) (CRISPR-associated endonuclease Cas9 (Cas9) - 2.48 μM and gRNA 4.96 μM; Integrated DNA Technologies) was used at 3.3 μg / 1x10 6 The sequence of the gRNA targeting human TRAC (T cell receptor alpha) was: TIFF2025156332000047.tif4128, and the sequence of the gRNA targeting human PDCD1 (programmed cell death protein 1) is TIFF2025156332000048.tif4128. CD3 (cluster of differentiation 3) expression was analyzed by flow cytometry on day 2 post-transfection. Cells were harvested on day 4 post-transfection for PDCD1 gene indel (insertion or deletion) analysis.

[0240] Flow cytometry analysis Flow cytometry was performed using a NovoCyte 3000. Data were examined using NovoExpress software (Acea Biosciences).

[0241] PDCD1 gene indel analysis Genomic DNA was extracted from cells using the MagNA Pure Compact Nucleic Acid Isolation Kit 1 (Roche). PCR was performed to amplify a 305-bp region surrounding the editing site. TIFF2025156332000049.tif11162; Integrated DNA Technologies). PCR products were sequenced by Sanger sequencing (Eurofins Genomics), and Tracking of Indels by Decomposition (TIDE) analysis was performed on the above sequences in TIDE (https: / / tide.nki.nl / ).

[0242] Cytokine release analysis GFP mRNA was delivered to activated human T cells from five healthy donors using either the SOLUPORE™ delivery method or nucleofection. Four hours after treatment, cells were plated at 1x10 in 96-well plates. 6 The cells were replated at 1x10 / ml and supernatants were collected daily for 5 days. Cells were counted and plated daily at 1x10 6 Cell proliferation assays were performed using a similar method, with cells reseeded at 1000 μg / ml. A custom Luminex assay panel (Merck Millipore) was designed to measure 11 human analytes: IL-2 (interleukin 2), IFN-γ (interferon gamma), TNFα (tumor necrosis factor α), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-8 (interleukin 8), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), fractalkine, ITAC (interferon-inducible T cell α chemoattractant), and IL-17A (interleukin 17A). Supernatant samples were analyzed in duplicate using the protocol for the human high-sensitivity T cell magnetic bead panel on a Luminex 200™ system (Merck Millipore).

[0243] Genetic profiling RNA was isolated from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Transcripts were analyzed using the NanoString nCounter Human CAR-T Characterization Panel (NanoString). Differential expression was displayed using log2 fold change and a table filtered for -1 ≥ log2 ≤ 1. The NanoString panel is a comprehensive immune panel containing 770 genes from 14 different immune cell types, common checkpoint inhibitors, CT (cancer / testis) antigens, and genes covering both adaptive and innate immune responses.

[0244] A table of immune cell type gene coverage for the NanoString panel is provided below. TIFF2025156332000050.tif223159

[0245] In vivo mouse engraftment study Human PBMCs were transfected with 3 μM Alexa Fluor™-labeled 3 kDa dextran-Alexa488 using the SOLUPORE™ delivery method or nucleofection. Non-obese diabetic / severe combined immunodeficiency (NOD / SCID) IL-2Rγ null (NSG) mice were irradiated (2.4 Gy) on day 0. Four hours later, mice were intravenously injected with PBMCs (1 x 10^6 / g). Mice were closely observed for signs of illness, particularly the development of GvHD, throughout the study (28 days). On day 14, peripheral blood was collected from mice for analysis of human (CD45 (clone HI30, Biolegend), CD3 (clone UCHT1, Biolegend), CD4 (clone SK3, Biolegend), and CD8 (clone SK1, Biolegend)) cell engraftment by flow cytometry. Spleens were collected during the study or at the time of sacrifice at the end of the study (day 28) for analysis of human (CD45, CD3, CD4, CD8) cell engraftment by flow cytometry.

[0246] In vitro cytotoxicity assay CD19 CAR mRNA was delivered to T cells using the SOLUPORE™ delivery method or electroporation. 24 hours after transfection, cells were cryopreserved in CryoStor CS10 (Sigma Aldrich). In vitro cytotoxicity was measured by impedance assay using an xCELLigence® Real-Time Cell Analyzer Single Plate (RTCA SP) instrument (ACEA Biosciences). Wells of an electronic microtiter plate were coated with 4 μg / ml CD40 (cluster of differentiation 40) (ACEA Biosciences) for 3 hours. RAJI cells (ATCC®) were cultured at 5x10 4 Cells were seeded at 1000 cells / well and allowed to adhere overnight. The following day, 19-21 hours later, CAR T cells were thawed, counted, and added to RAJI cells at the following effector:target ratios: 2.5:1, 1.25:1, 0.6:1, 0.3:1, and 0.15:1. Impedance was monitored every minute for 4 hours, every 5 minutes for 8 hours, and then every 15 minutes for at least 92 hours. Cell index (CI) was normalized to the CI at the time of CAR-T cell addition, and specific lysis was calculated relative to control effector cell-only cultures.

[0247] In vivo mouse CAR T cell efficacy testing CD19 CAR mRNA was delivered to T cells using the SOLUPORE™ delivery method or nucleofection, and the cells were cryopreserved. On day 0, NSG™ mice were injected with CD19+RAJI-luciferase tumor cells (2.5x10 5 Mice were randomized between treatment groups based on body weight. On day 3, CAR T cells were thawed and administered at 1x10 per animal. 6 pieces, 2x10 6 pieces, or 4x10 6 Bioluminescence imaging was performed on day 15, and the animals were euthanized by CO2 asphyxiation.

[0248] Statistics The significance of comparative tumor or CAR T cell engraftment in vivo was assessed using an unpaired Student's t-test. 95% confidence intervals were used to compare the mean average of each replicate analyzed by Luminex. Two-way ANOVA (analysis of variance) was used to compare the mean of each group with untreated controls at each time point. **P<0.01; *P<0.05. All statistical analyses were performed using GraphPad Prism 8.0.

[0249] Cell phenotyping and metabolic assays Surface expression of T cell activation markers and glycolytic activity of Avectas-generated T cells were assessed using flow cytometry and Seahorse analysis, respectively. Briefly, T cells were activated with Dynabeads and IL-2 for 19 hours, after which cells were either untreated (UT), mock-transfected using solporation (Sol Mock) or nucleofection (NF Mock), or transfected with GFP mRNA using solporation (Sol) or nucleofection (NF). Cells were analyzed using a panel of mAbs specific for T cell-associated activation / exhaustion surface markers (PD-1 and CD69 were primary targets). During analysis, GFP+ cells were gated for solporation and nucleofection and compared with untreated activated cells (UT). For extracellular flux analysis, 2x10^5 T cells were plated in quadruplicate onto Seahorse culture plates and rested overnight in IL-2 medium. The next day, cells were attached to Seahorse culture plates using CellTak and resuspended in Seahorse culture medium (adjusted for pH and nutrients). Cells were then analyzed using a Seahorse analyzer, with four measurements taken for each time point. Oxygen consumption (OCR) was also measured in addition to extracellular acidification rate (ECAR), which represents the oxidative phosphorylation rate (OxPhos). Resting T cells utilize OxPhos, but after activation, they "switch" to glycolytic metabolism.

[0250] Lactate assay L-lactate production in activated T cells transfected with mRNA-GFP using solubility or nucleofection (program EO115) was analyzed using the ChromaDazzle lactate assay kit (AssayGenie). Supernatants were collected 6 hours after transfection and stored at -20°C.

[0251] Table 7. Dataset S1 Test at 6 hours (Electroporation FI-115 at 6 hours) TIFF2025156332000051.tif94146TIFF2025156332000052.tif221146TIFF2025156332000053.tif221146TIFF2025156332000054.tif221146TIFF2025156332000055.tif221146TIFF2025156332000056.tif221146TIFF2025156332000057.tif118146Filter = log2 fold change of >1 and <-1 (2-fold linear change)

[0252] Table 8. Dataset S1 Study at 6 Hours (SOLUPORE™ Delivery Method at 6 Hours) TIFF2025156332000058.tif65140

[0253] Table 9: Dataset S2 Study 1 at 24 hours, Electroporation FI-115 at 24 hours TIFF2025156332000059.tif221143TIFF2025156332000060.tif118143Filter = log2 fold change for >1 and <-1 (2-fold linear change) *No genes identified at 24 hours in the SOLUPORE™ delivery group.

[0254] Table 10: Dataset S3 Study 2 at 24 hours, Electroporation FI-115 at 24 hours TIFF2025156332000061.tif75130TIFF2025156332000062.tif153130Filter = log2 fold change for >1 and <-1 (2-fold linear change)

[0255] Table 11. Dataset S3 Study 2 using SOLUPORE™ delivery method at 24 hours TIFF2025156332000063.tif65128 filter = log2 fold change for >1 and <-1 (2-fold linear change)

[0256] Table 12. Dataset S3 Study 2, Comparison of FI-115 Electroporation with EO-115 Electroporation at 24 Hours TIFF2025156332000064.tif85128 filter = log2 fold change for >1 and <-1 (2-fold linear change)

[0257] Table 13. T cells 6 hours after delivery (using SOLUPORE™ delivery method) TIFF2025156332000065.tif82138TIFF2025156332000066.tif219138TIFF2025156332000067.tif219138TIFF2025156332000068.ti f219138TIFF2025156332000069.tif219138TIFF2025156332000070.tif219138TIFF2025156332000071.tif219138TIFF20251563320 00072.tif219138TIFF2025156332000073.tif219138TIFF2025156332000074.tif219138TIFF2025156332000075.tif219138TIFF202 5156332000076.tif219138TIFF2025156332000077.tif219138TIFF2025156332000078.tif219138TIFF2025156332000079.tif139138

[0258] Table 14. T cells 24 hours after delivery (using SOLUPORE™ delivery method) TIFF2025156332000080.tif66128TIFF2025156332000081.tif219118TIFF2025156332000082.tif219118TIFF2025156332000083.ti f219118TIFF2025156332000084.tif219118TIFF2025156332000085.tif219118TIFF2025156332000086.tif219118TIFF20251563320 00087.tif219118TIFF2025156332000088.tif219118TIFF2025156332000089.tif219118TIFF2025156332000090.tif219118TIFF202 5156332000091.tif219118TIFF2025156332000092.tif219118TIFF2025156332000093.tif219118TIFF2025156332000094.tif208118

[0259] Table 15. T cells 6 hours after nucleofection (FI-115) delivery TIFF2025156332000095.tif218123TIFF2025156332000096.tif219123TIFF2025156332000097.tif219123TIFF2025156332000098.tif219123 TIFF2025156332000099.tif219123TIFF2025156332000100.tif219123TIFF2025156332000101.tif219123TIFF2025156332000102.tif171128

[0260] Table 16. T cells 24 hours after nucleofection (FI-115) delivery TIFF2025156332000103.tif33128TIFF2025156332000104.tif219123TIFF2025156332000105.tif219123TIFF2025156332000106.tif219123TIFF2025156332000107.tif219123TIFF2025156332000108.tif219123TIFF2025156332000109.tif219123TIFF2025156332000110.tif219123TIFF2025156332000111.tif219123TIFF2025156332000112.tif219123TIFF2025156332000113.tif219123TIFF2025156332000114.tif219123TIFF2025156332000115.tif219123TIFF2025156332000116.tif219123TIFF2025156332000117.tif38128

[0261] (Table 17) T cells 24 hours after nucleofection (EO-115) delivery TIFF2025156332000118.tif167128TIFF2025156332000119.tif219120TIFF2025156332000120.tif219120TIFF2025156332000121.tif219120TIFF2025156332000122.tif219120TIFF2025156332000123.tif219120TIFF2025156332000124.tif219120TIFF2025156332000125.tif219120TIFF2025156332000126.tif219120TIFF2025156332000127.tif219120TIFF2025156332000128.tif219120TIFF2025156332000129.tif219120TIFF2025156332000130.tif219120TIFF2025156332000131.tif219120TIFF2025156332000132.tif27128

[0262] Other Aspects While the present invention has been described with reference to the detailed description of the invention, the foregoing description is intended for purposes of illustration and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0263] The patents and scientific literature referred to in this specification define the knowledge available to those skilled in the art.All references cited in this specification, such as U.S. patents, U.S. patent application publications, PCT patent applications designating the U.S., and published foreign patents and patent applications, are incorporated herein by reference in their entirety.The GenBank and NCBI submissions indicated by the accession numbers cited in this specification are incorporated herein by reference.All other published references, documents, manuscripts, and scientific literature cited in this specification are incorporated herein by reference.In the case of conflict, the present specification, including definitions, shall prevail.In addition, the above-mentioned materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0264] While the present invention has been shown and described in detail, particularly with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0265] Sequence information SEQUENCE LISTING <110> Avectas Limited <120> ENGINEERING OF IMMUNE CELLS FOR EX VIVO CELL THERAPY APPLICATIONS <150> US 62 / 897,250 <151> 2019-09-06 <150> US 63 / 022,944 <151> 2020-05-11 <150> US 63 / 047,054 <151> 2020-07-01 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 2084 <212> DNA <213> Homo sapiens <400> 1 aaccgcatct gcagcgagca actgagaagc caagactgag ccggcggccg cggcgcagcg 60 aacgagcagt gaccgtgctc ctacccagct ctgcttcaca gcgcccacct gtctccgccc 120 ctcggcccct cgcccggctt tgcctaaccg ccacgatgat gttctcgggc ttcaacgcag 180 actacgaggc gtcatcctcc cgctgcagca gcgcgtcccc ggccggggat agcctctctt 240 actaccactc acccgcagac tccttctcca gcatgggctc gcctgtcaac gcgcaggact 300 tctgcacgga cctggccgtc tccagtgcca acttcattcc cacggtcact gccatctcga 360 ccagtccgga cctgcagtgg ctggtgcagc ccgccctcgt ctcctctgtg gccccatcgc 420 agaccagagc ccctcaccct ttcggagtcc ccgccccctc cgctggggct tactccaggg 480 ctggcgttgt gaagaccatg acaggaggcc gagcgcagag cattggcagg aggggcaagg 540 tggaacagtt atctccagaa gaagaaga aaaggaagaat ccgaagggaa agaataaga 600 tgggctcagc caaatgccgc aaccggagga gggagctgac tgatacactc caagcggaga 660 cagaccaact agaagatgag aagtctgctt tgcagaccga gattgccaac ctgctgaagg 720 agaaggaaa actagagttc atcctggcag ctcaccgacc tgcctgcaag atccctgatg 780 acctgggctt cccagaagag atgtctgtgg cttcccttga tctgactggg ggcctgccag 840 aggttgccac cccggagtct gaggaggcct tcaccctgcc tctcctcaat gaccctgagc 900 ccaagccctc agtggaacct gtcaagagca tcagcagcat ggagctgaag accgagccct 960 ttgatgactt cctgttccca gcatcatcca ggcccagtgg ctctgagaca gcccgctccg 1020 tgccagacat ggacctatct gggtccttct atgcagcaga ctgggagcct ctgcacagtg 1080 gctccctggg gatggggccc atggccacag agctggagcc cctgtgcact ccggtggtca 1140 cctgtactcc cagctgcact gcttacacgt cttccttcgt cttcacctac cccgaggctg 1200 actctcttcc cagctgtgca gctgcccacc gcaagggcag cagcagcaat gagccttcct 1260 ctgactcgct cagctcaccc acgctgctgg ccctgtgagg gggcagggaa ggggaggcag 1320 ccggcaccca caagtgccac tgcccgagct ggtgcattac agagaggaga aacacatctt 1380 ccctagaggg ttcctgtaga cctagggagg accttatctg tgcgtgaaac acaccaggct 1440 gtgggcctca aggacttgaa agcatccatg tgtggactca agtccttacc tcttccggag 1500 atgtagcaaa acgcatggag tgtgtattgt tcccagtgac acttcagaga gctggtagtt 1560 agtagcatgt tgagccaggc ctgggtctgt gtctcttttc tctttctcct tagtcttctc 1620 atagcattaa ctaatctatt gggttcatta ttggaattaa cctggtgctg gatattttca 1680 aattgtatct agtgcagctg attttaacaa taactactgt gttcctggca atagtgtgtt 1740 ctgattagaa atgaccaata ttatactaag aaaagatacg actttatttt ctggtagata 1800 gaaataaata gctatatcca tgtactgtag tttttcttca acatcaatgt tcattgtaat 1860 gttactgatc atgcattgtt gaggtggtct gaatgttctg acattaacag ttttccatga 1920 aaacgtttta ttgtgttttt aatttattta ttaagatgga ttctcagata tttatatttt 1980 tattttatt tttctacct tgaggtctt tgacatgtgg aaagtgaatt tgaatgaaaa 2040 atttaagcat tgtttgctta ttgttccaag acatgtca taaa 2084 <210> 2 <211> 3338 <212> DNA <213> Homo sapiens <400> 2 gatacatgg gctattttta ggggttgact ggtagcagat aagtgttgag ctcggggctgg 60 ataagggctc agagttgcac tgagtgtggc tgagcagcg agggcgggt ggaggtgcgc 120 ggagtcaggc agacagacag accagccag ccagccaggt cggcagtata gtccgaactg 180 caatcttat tttctttca ccttctctct aactgcccag agctagcgcc tgtggctccc 240 gggctggtgt tcgggagtg tccagagagc ctggtctcca gccgcccccg ggaggagagc 300 cctgctgccc aggcgctgtt gaagcggcg gaagcagcg gtacccacgc gcccgccggg 360 ggaagtcggc gagcggctgc agcagcaag aactttcccg gctgggagga ccggagacaa 420 gtggcagagt cccggagcga actttgcaa gccttttcctg cgtcttaggc ttctccacgg 480 cggtaaagac cagaaggcgg cggagagcca cgcaagagaa gaaggacgtg cgctcagctt 540 cgctcgcacc ggttgttgaa cttgggcgag cgcgagccgc ggctgccggg cgccccctcc 600 ccctagcagc ggaggagggg acaagtcgtc ggagtccggg cggccaagac ccgccgccgg 660 ccggccactg cagggtccgc actgatccgc tccgcgggga gagccgctgc tctgggaagt 720 gagttcgcct gcggactccg aggaaccgct gcgcccgaag agcgctcagt gagtgaccgc 780 gacttttcaa agccgggtag cgcgcgcgag tcgacaagta agagtgcggg aggcatctta 840 attaaccctg cgctccctgg agcgagctgg tgaggagggc gcagcgggga cgacagccag 900 cgggtgcgtg cgctcttaga gaaactttcc ctgtcaaagg ctccgggggg cgcgggtgtc 960 ccccgcttgc cagagccctg ttgcggcccc gaaacttgtg cgcgcagccc aaactaacct 1020 cacgtgaagt gacggactgt tctatgactg caaagatgga aacgaccttc tatgacgatg 1080 ccctcaacgc ctcgttcctc ccgtccgaga gcggacctta tggctacagt aaccccaaga 1140 tcctgaaaca gagcatgacc ctgaacctgg ccgacccagt ggggagcctg aagccgcacc 1200 tccgcgccaa gaactcggac ctcctcacct cgcccgacgt ggggctgctc aagctggcgt 1260 cgcccgagct ggagcgcctg ataatccagt ccagcaacgg gcacatcacc accacgccga 1320 cccccaccca gttcctgtgc cccaagaacg tgacagatga gcaggagggc ttcgccgagg 1380 gcttcgtgcg cgccctggcc gaactgcaca gccagaacac gctgcccagc gtcacgtcgg 1440 cggcgcagcc ggtcaacggg gcaggcatgg tggctcccgc ggtagcctcg gtggcagggg 1500 gcagcggcag cggcggcttc agcgccagcc tgcacagcga gccgccggtc tacgcaaacc 1560 tcagcaactt caacccaggc gcgctgagca gcggcggcgg ggcgccctcc tacggcgcgg 1620 ccggcctggc ctttcccgcg caaccccagc agcagcagca gccgccgcac cacctgcccc 1680 agcagatgcc cgtgcagcac ccgcggctgc aggccctgaa ggaggagcct cagacagtgc 1740 ccgagatgcc cggcgagaca ccgcccctgt cccccatcga catggagtcc caggagcgga 1800 tcaaggcgga gaggaagcgc atgaggaacc gcatcgctgc ctccaagtgc cgaaaaagga 1860 agctggagag aatcgcccgg ctggaggaaa aagtgaaaac cttgaaagct cagaactcgg 1920 agctggcgtc cacggccaac atgctcaggg aacaggtggc acagcttaaa cagaaagtca 1980 tgaaccacgt taacagtggg tgccaactca tgctaacgca gcagttgcaa acattttgaa 2040 gagagaccgt cggggctga ggggcaacga agaaaaaaaa taacacagag agacagactt 2100 gagaacttga caagttgcga cggagagaaa aaagaagtgt ccgagaacta aagccaaggg 2160 tatccaagtt ggactgggtt gcgtcctgac ggcgccccca gtgtgcacga gtgggaagga 2220 cttggcgcgc cctcccttgg cgtggagcca gggagcggcc gcctgcgggc tgccccgctt 2280 tgcggacggg ctgtccccgc gcgaacggaa cgttggactt ttcgttaaca ttgaccaaga 2340 actgcatgga cctaacattc gatctcattc agtattaaag gggggagggg gagggggtta 2400 caaactgcaa tagagactgt agattgcttc tgtagtactc cttaagaaca caaagcgggg 2460 ggagggttgg ggaggggcgg caggagggag gtttgtgaga gcgaggctga gcctacagat 2520 gaactctttc tggcctgcct tcgttaactg tgtatgtaca tatatatatt ttttaatttg 2580 atgaaagctg attactgtca ataaacagct tcatgccttt gtaagttatt tcttgtttgt 2640 ttgtttgggt atcctgccca gtgttgtttg taaataagag atttggagca ctctgagttt 2700 accatttgta ataaagtata taattttttt atgttttgtt tctgaaaatt ccagaaagga 2760 tatttaagaa aatacaataa actattggaa agtactcccc taacctcttt tctgcatcat 2820 ctgtagatac tagctatcta ggtggagttg aaagagttaa gaatgtcgat taaaatcact 2880 ctcagtgctt cttactatta agcagtaaaa actgttctct attagacttt agaaataaat 2940 gtacctgatg tacctgatgc tatggtcagg ttatactcct cctcccccag ctatctatat 3000 ggaattgctt accaaaggat agtgcgatgt ttcaggaggc tggaggaagg ggggttgcag 3060 tggagaggga cagccccactg agaagtcaaa catttcaaag tttggattgt atcaagtggc 3120 atgtgctgtg accatttata atgttagtag aaattttaca ataggtgctt attctcaaag 3180 caggaattgg tggcagattt tacaaaagat gtatccttcc aatttggaat cttctctttg 3240 acaattccta gataaaaaga tggcctttgc ttatgaatat ttataacagc attcttgtca 3300 caataaatgt attcaaatac caaaaaaaaa aaaaaaaa 3338 <210> 3 <211> 302 <212> PRT <213> Homo sapiens <400> 3 Met Phe Gln Ala Phe Pro Gly Asp Tyr Asp Ser Gly Ser Arg Cys Ser 1 5 10 15 Ser Ser Pro Ser Ala Glu Ser Gln Tyr Leu Ser Ser Val Asp Ser Phe 20 25 30 Gly Ser Pro Pro Thr Ala Ala Ala Ser Gln Glu Cys Ala Gly Leu Gly 35 40 45 Glu Met Pro Gly Ser Phe Val Pro Thr Val Thr Ala Ile Thr Thr Ser 50 55 60 Gln Asp Leu Gln Trp Leu Val Gln Pro Thr Leu Ile Ser Ser Met Ala 65 70 75 80 Gln Ser Gln Gly Gln Pro Leu Ala Ser Gln Pro Pro Val Val Asp Pro 85 90 95 Tyr Asp Met Pro Gly Thr Ser Tyr Ser Thr Pro Gly Met Ser Gly Tyr 100 105 110 Ser Ser Gly Gly Ala Ser Gly Ser Gly Gly Pro Ser Thr Ser Gly Thr 115 120 125 Thr Ser Gly Pro Gly Pro Ala Arg Pro Ala Arg Ala Arg Pro Arg Arg 130 135 140 Pro Arg Glu Glu Thr Glu Thr Asp Gln Leu Glu Glu Glu Lys Ala Glu 145 150 155 160 Leu Glu Ser Glu Ile Ala Glu Leu Gln Lys Glu Lys Glu Arg Leu Glu 165 170 175 Phe Val Leu Val Ala His Lys Pro Gly Cys Lys Ile Pro Tyr Glu Glu 180 185 190 Gly Pro Gly Pro Gly Pro Leu Ala Glu Val Arg Asp Leu Pro Gly Ser 195 200 205 Ala Pro Ala Lys Glu Asp Gly Phe Ser Trp Leu Leu Pro Pro Pro Pro 210 215 220 Pro Pro Pro Leu Pro Phe Gln Thr Ser Gln Asp Ala Pro Pro Asn Leu 225 230 235 240 Thr Ala Ser Leu Phe Thr His Ser Glu Val Gln Val Leu Gly Asp Pro 245 250 255 Phe Pro Val Val Asn Pro Ser Tyr Thr Ser Ser Phe Val Leu Thr Cys 260 265 270 Pro Glu Val Ser Ala Phe Ala Gly Ala Gln Arg Thr Ser Gly Ser Asp 275 280 285 Gln Pro Ser Asp Pro Leu Asn Ser Pro Ser Leu Leu Ala Leu 290 295 300 <210> 4 <211> 125 <212> PRT <213> Homo sapiens <400> 4 Met Pro His Ser Ser Asp Ser Ser Asp Ser Ser Phe Ser Arg Ser Pro 1 5 10 15 Pro Pro Gly Lys Gln Asp Ser Ser Asp Asp Val Arg Arg Val Gln Arg 20 25 30 Arg Glu Lys Asn Arg Ile Ala Ala Gln Lys Ser Arg Gln Arg Gln Thr 35 40 45 Gln Lys Ala Asp Thr Leu His Leu Glu Ser Glu Asp Leu Glu Lys Gln 50 55 60 Asn Ala Ala Leu Arg Lys Glu Ile Lys Gln Leu Thr Glu Glu Leu Lys 65 70 75 80 Tyr Phe Thr Ser Val Leu Asn Ser His Glu Pro Leu Cys Ser Val Leu 85 90 95 Ala Ala Ser Thr Pro Ser Pro Pro Glu Val Val Tyr Ser Ala His Ala 100 105 110 Phe His Gln Pro His Val Ser Ser Pro Arg Phe Gln Pro 115 120 125 <210> 5 <211> 127 <212> PRT <213> Homo sapiens <400> 5 Met Ser Gln Gly Leu Pro Ala Ala Gly Ser Val Leu Gln Arg Ser Val 1 5 10 15 Ala Ala Pro Gly Asn Gln Pro Gln Pro Gln Pro Gln Gln Gln Ser Pro 20 25 30 Glu Asp Asp Asp Arg Lys Val Arg Arg Arg Glu Lys Asn Arg Val Ala 35 40 45 Ala Gln Arg Ser Arg Lys Lys Gln Thr Gln Lys Ala Asp Lys Leu His 50 55 60 Glu Glu Tyr Glu Ser Leu Glu Gln Glu Asn Thr Met Leu Arg Arg Glu 65 70 75 80 Ile Gly Lys Leu Thr Glu Glu Leu Lys His Leu Thr Glu Ala Leu Lys 85 90 95 Glu His Glu Lys Met Cys Pro Leu Leu Leu Cys Pro Met Asn Phe Val 100 105 110 Pro Val Pro Pro Arg Pro Asp Pro Val Ala Gly Cys Leu Pro Arg 115 120 125 <210> 6 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> CD19 CAR mRNA <400> 6 atggctctcc cagtgactgc cctactgctt cccctagcgc tgctgctgca tgcggcgcgc ccggacatcc agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg 120 accatcagct gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag cccgacggca ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc 240 agccggttta gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctgga caggagata tcgccaccta cttttgccag cagggcaaca cactgcccta cactttggc ggcggaacaa agctggaaat caccggcgga ggcggatctg gcggcggagg atctggggga ggcggctctg aggtgaagct gcaggaagc ggccctggcc tggtggcccc cagccagagc 480 ctgagcgtga cctgcaccgt gagcggcgtg agcctgcccg actacggcgt gagctggatc 540 cggcagcccc cggcagcc cctggaatgg ctggggcgtga tctggggcag cgagaccacc tactacaaca gcgccctgaa gagccggctg accatcatca aggacaacag caagagccag gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ctgcgccaag 720 cactact acggcggcag ctacgccatg gactactggg gccagggcac cagcgtgacc 780 gtgagcgcgg ccgcaattga agttatgtat cctcctcctt acctagacaa tgagaagagc 840 aatggaacca ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 900 ccttctaagc ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1020 agtgactaca tgaacatgac tcccgccgc cccgggccca cccgcaagca ttaccagccc 1080 tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1140 gacgccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1200 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1260 ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1320 gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1380 cttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1440 ctgccccctc gctaa 1455 <210> 7 <211> 484 <212> PRT <213> Artificial Sequence <220> <223> CD19 CAR protein <400> 7 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 Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ala Ala Ala Ile Glu Val Met Tyr Pro Pro 260 265 270 Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys 275 280 285 Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro 290 295 300 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 305 310 315 320 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 325 330 335 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 340 345 350 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 355 360 365 Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 420 425 430 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 435 440 445 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 450 455 460 Leu Ser Thr Wing Thr Lys Asp Thr Tyr Asp Wing Leu His Met Gln Wing 465,470,475,480 Leu Pro Pro Arg <210> 8 <211> 1552 <212> DNA <213> Artificial Sequence <220> <223> CD19 CAR mRNA <400> 8 tgatatccag atgacccaga ccaccagcag cctgtctgcc tctctgggcg atagagtgac 60 catcagctgt agagccagcc aggacatcag caagtacctg aactggtatc agcagaaacc 120 cgacggcacc gtgaagctgc tgatctacca caccagcaga ctgcacagcg gcgtgccaag 180 cagatttct ggcagcggct ctggcaccga ctacagcctg acaatcagca acctggaaca 240 agaggatatc gctacctact tctgccagca aggcaacacc ctgccttaca cctttggcgg 300 aggcaccaag ctggaaatca ccggctctac aagcggcagc ggcaaacctg gatctggcga 360 gggatctacc aagggcgaag tgaaactgca agagtctggc cctggactgg tggccccatc 420 tcagtctctg agcgtgacct gtacagtcag cggagtgtcc ctgcctgatt acggcgtgtc 480 ctggatcaga cagcctcctc ggaaaggcct ggaatggctg ggagtgatct ggggcagcga 540 600. ccctgaagtc ccggctgacc atcatcaagg acaactccaa 660. gagccaggtg ttcctgaaga tgaacagcct gcagaccgac gacaccgcca tctactattg cgccaagcac tactactacg gcggcagcta cgccatggat tattggggcc agggcaccag 720 cgtgaccgtg tctagtacaa caacccctgc tcctcggcct cctacaccag ctcctacaat tgccagccag ccactgtctc tgaggcccga agcttgtag cctgctgctg gcggagccgt 840 gcatacaaga ggactggatt tcgcctgcga cttctgggtg ctcgtggttg ttggcggagt 900 gctggcctgt tacagcctgc tggttaccgt ggccttcatc atcttttggg tccgaagcaa 960 gcggagccgg ctgctgcact ccgactacat gaacatgacc cctagacggc ccggacctac 1080. cagaaagcac taccagcctt acgctcctcc tagagacttc gccgcctaca gatccaagcg gggcagaaag aactgctct acatctcaa gcagccctc atgcggcccg tgcagaccac 1140 acagaggaa gatggctgct cctgcagatt ccccgaggaa gagaaggcg gctgcgagct 1200 gagagtgaag ttcagcagat ccgccgacgc tcccgcctat aagcagggac agaaccagct 1260 gtacaacgag ctgaacctgg ggagagaga agagtacgac gtgctggaca agcggagagg 1320 cagggatcct gaatgggcg gcaagcccag acggagat cctcaagagg gcctgtataa 1380 tgagctgcag aagacaga tggccgaggc ctacagcgag atcggaatg agggcgagcg 1440 cagaagaggc aagggacacg atggactgta ccagggactg agcaccgcca ccaggatac 1500 ctatgacgcc ctgcacatgc agccctgcc tccagataa gtcgacaatc aa 1552 <210> 9 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> CD19 CAR protein <400> 9 Asp Ile Gln Met Thr Gln Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 245 250 255 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 260 265 270 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 275 280 285 Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 290 295 300 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 305 310 315 320 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 325 330 335 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 340 345 350 Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 355 360 365 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 370 375 380 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 385 390 395 400 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 405 410 415 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 420 425 430 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 435 440 445 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 450 455 460 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 465 470 475 480 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 485 490 495 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 510 <210> 10 <211> 846 <212> DNA <213> Artificial Sequence <220> <223> E195R / D269H TRAIL (DR5)-variant <400> 10 atggctatga tggaggtcca ggggggaccc agcctgggac agacctgcgt gctgatcgtg 60 atcttcacag tgctcctgca gtctctctgt gtggctgtaa cttacgtgta cttaccaac 120 gagctgaagc agatgcagga caagtactcc aaaagtggca ttgcttgttt cttaaaagaa 180 gatgacagtt attgggacccc caatgacgaa gagagtatga acagcccctg ctggcaagtc 240 aagtggcaac tccgtcagct cgttagaaag atgatttga gaacctctga ggaaaccatt 300 tctacagttc aagaaaagca acaaaatatt tctcccctag tgagagaaag aggtcctcag 360 agagtagcag ctcacataac tgggaccaga ggaagaagca acacattgtc ttctccaaac 420 tccaagaatg aaaaggctct gggccgcaaa ataaactcct gggaatcatc aaggagtggg 480 cattcattcc tgagcaactt gcacttgagg aatggtgaac tggtcatcca tgaaaaaggg 540 ttttactaca tctattccca aacatacttt cgatttcagg agcgaataaa agaaaacaca 600 aagaacgaca aacaaatggt ccaatatatt tacaaataca caagttatcc tgaccctata 660 ttgttgatga aaagtgctag aaatagttgt tggtctaaag atgcagaata tggactctat 720 tccatctatc aagggggaat atttgagctt aaggaaaatg acagaatttt tgtttctgta 780 acaaatgagc acttgataga catgcaccat gaagccagtt ttttcggggc ctttttagtt 840 ggctaa 846 <210> 11 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> TRAIL protein <400> 11 Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 1 5 10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 65 70 75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95 Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105 110 Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 115 120 125 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 130 135 140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly 145 150 155 160 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175 His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190 Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205 Tyre Ile Tyr Lys Tyre Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220 Only Arg Asn Only Cys Trp Only Lys Asp Only Glu Tyr Gly Leu Tyr 225 230 235 240 Tyr Gln Gly Gly and Phe Glu Leu Glu Asn Asp Arg Ile 245 250 255 Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 260 265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly 275,280 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PDCD1 reverse primer <400> 12 late 21st century <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> c-Myc primer <400> 13 ggatccgaat tcatgcccct caacgttagc ttcac 35 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Klf4 primer <400> 14 ggatccgaat tcatggctgt cagcgacgcg ctgc 34 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Oct4 primer <400> 15 ggatccgaat tcatggcggg acacctggct tcgg 34 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Sox2 primer <400> 16 ggatccgaat tcatgtacaa catgatggag acgg 34 <210> 17 <211> 1047 <212> DNA <213> Homo sapiens <400> 17 cgaattcccc tatcacctaa gtgtgggcta atgtaacaaa gagggatttc acctacatcc 60 attcagtcag tctttggggg tttaaagaaa ttccaaagag tcatcagaag aggaaaaatg 120 aaggtaatgt ttttcagac aggtaaagtc tttgaaaata tgtgtaatat gtaaaacatt 180 ttgacacccc cataatatttt ttccagaatt aacagtataa attgcatctc ttgttcaaga 240 gttccctatc actcttta atcactactc acagtaacct caactcctgc cacaatgtac 300 aggatgcaac tcctgtcttg cattgcacta agtcttgcac ttgtcacaaa cagtgcacct 360 acttcaagtt ctacaaagaa aacacagcta caactggagc atttactgct ggatttacag 420 atgatttga atggaattaa taattacaag aatcccaaac tcaccaggat gctcacattt 480 aagttttaca tgcccaagaa ggccacagaa ctgaaacatc ttcagtgtct agaagaagaa 540 ctcaaacctc tggaggaagt gctaaattta gctcaaagca aaaactttca cttaagaccc 600 agggacttaa tcagcaatat caacgtaata gttctggaac taaagggatc tgaaacaaca 660 ttcatgtgtg aattgctga tgagacagca accattgtag aatttctgaa cagatggatt 720 accttttgtc aaagcatcat ctcaacactg acttgataat taagtgcttc ccacttaaaa 780 catatcaggc cttctattta tttaaatatt taaattttat atttattgtt gaatgtatgg 840 tttgctacct attgtaacta ttattcttta tcttaaact aataatatgg atcttttatg 900 attcttttg taagccctag gggctctaaa atggttttcac ttattttacc caaatattt 960 attattatgt tgaatgttaa atatagtac tatgtagat ggttagtaaa actatttaat 1020 Aatttgata Attadaaaaaaaaa 1047 <210> 18 <211> 2058 <212> DNA <213> Homo sapiens <400> 18 aaaactttc aaactttc xctagg xctagga xctagga xctagg xcxc 60 ccggaaggaa ccatctcact gtgtgtaac atgacttcca agctggccgt ggctcttg 120 gcagccttcc tgatttctgc agctctgtgt gaagtgcag tttgccaag gagtgctaaa 180 gaacttagat gtcagtgcat aaagacatac tccaacctt tccaccccaa atttatcaa 240 gaactgagag tgattgagag tggaccacac tgcgccaca cagaattat tgtaaagctt 300 tctgatggaa gagagctctg tctggacccc aaggaaact gggtgcagag ggttgtggag 360 aagttttga agaggtaagt tatatatttt ttaatttaaa ttttcattt atcctgagac 420 atataatcca aagtcagcct aataatttct ttctgttgct aaaatcgtc attaggtatc 480 tgccttttg gttaaaaaaaaaggaatag catcaatagt gagtttgttg tactcatgac 540 cagaaagacc atacatagtt tgcccaggaa attctggtt taagcttgtg tcctatactc 600 ttagtaagt tctttgtcac tcccagtagt gtcctatttt agatgataat ttctttgatc 660 tccctattta tagttgagaa tatagagcat ttctacaca tgaatgtca agactatatt 720 gactttca gaaccctact ttccttctta ttaaacatag ctcatcttta tattttaat 780 tttatttag ggctgagaat tcataaaaaa attcattc tgtggtatcc aagaatcagt 840 gaagatgcca gtgaaacttc aagcaatct acttcacac ttcatgtatt gtgtgggtct 900 gttgtaggt tgccagatgc atacagat tcctggttaa atttgaattt cagtaaacaa 960 tgaatagtttt ttcattgtac catgaaatat ccagaacata cttatatgta aagtattatt 1020 tatttgaatc tacaaaaac aaaataat ttttaatt aaggatttc ctagatattg 1080 cacgggagaa tatacaata gcaaattga ggccaagggc cagagaata tccgaacttt 1140 aatttcagga attgaatggg tttgctagaa tgtgatattt gaagcatcac ataaaaatga 1200 tgggacaata aattttgcca taaagtcaaa tttagctgga aatcctggat tttttctgt 1260 taaatctggc aaccctagtc tgctagccag gatccacaag tccttgttcc actgtgcctt 1320 ggtttctcct ttatttctaa gtggaaaaag tattagccac catcttacct cacagtgatg 1380 ttgtgaggac atgtggaagc actttaagtt ttttcatcat aacataaatt attttcaagt 1440 gtaacttatt aacctattta ttatttatgt atttatttaa gcatcaaata tttgtgcaag 1500 aatttggaaa aatagaagat gaatcattga ttgaatagtt ataaagatgt tatagtaaat 1560 ttattttatt ttagatatta aatgatgttt tattagataa atttcaatca gggtttttag 1620 attaaacaaa caaacaattg ggtacccagt taaattttca tttcagataa acaacaaata 1680 atttttagt ataagtacat tattgtttat ctgaaatttt aattgaacta acaatcctag 1740 tttgatactc ccagtcttgt cattgccagc tgtgttggta gtgctgtgtt gaattacgga 1800 ataatgagtt agaactatta aaacagccaa aactccacag tcaatattag taatttcttg 1860 ctggttgaaa cttgttttatt atgtacaat agattcttattt aatgactgc 1920 atttttaaat acaggcttt atatttttaa ctttaagattg tttttagtg ctctccaaat 1980 ttttttact gtttctgatt gtatggaat aaaagtaa atatgaaca tttaaaatat 2040 aatttgttgt Caagtha 2058 <210> 19 <211> 1368 <212> PRT <213> Streptococcus pyogenes <400> 19 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Ala Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Arg Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Leu Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Ser Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Ala Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Lys Glu Met Ile Glu Glu Arg Leu Lys Lys Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Asp Ile Leu Lys Glu Tyr Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Lys Gln Leu Asn Ala Lys With Thr Gln Arg Lys 885,890,895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys Tyr Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys With Arg Link Glu Val Arg With Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Arg Lys Asp Leu Ile Ile Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Leu Glu Gln Lys Gly Asn Leu Glu Wing Pro Ser Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser is Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Leu Valley Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 20 <211> 994 <212> PRT <213> Staphylococcus agnetis <400> 20 Met Asn Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly 1 5 10 15 Tyr Gly Ile Val Asp Ser Asp Thr Arg Glu Ile Lys Asp Ala Gly Val 20 25 30 Arg Leu Phe Pro Glu Ala Asn Val Asp Asn Asn Glu Gly Arg Arg Ser 35 40 45 Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Ile His Arg Leu Asp 50 55 60 Arg Val Lys His Leu Leu Ala Glu Tyr Asn Leu Leu Asp Leu Thr Asn 65 70 75 80 Ile Pro Lys Ser Thr Asn Pro Tyr Gln Ile Arg Val Lys Gly Leu Asn 85 90 95 Glu Lys Leu Ser Lys Asp Glu Leu Val Ile Ala Leu Leu His Ile Ala 100 105 110 Lys Arg Arg Gly Ile His Asn Val Asn Val Met Met Asp Asp Asn Asp 115 120 125 Ser Gly Asn Glu Leu Ser Thr Lys Asp Gln Leu Lys Lys Asn Ala Lys 130 135 140 Ala Leu Ser Asp Lys Tyr Val Cys Glu Leu Gln Leu Glu Arg Phe Glu 145 150 155 160 Gln Asp Tyr Lys Val Arg Gly Glu Lys Asn Arg Phe Lys Thr Glu Asp 165 170 175 Phe Val Arg Glu Ala Arg Lys Leu Leu Glu Thr Gln Ser Lys Phe Phe 180 185 190 Glu Ile Asp Gln Thr Phe Ile Met Arg Tyr Ile Asp Leu Val Glu Thr 195 200 205 Arg Arg Glu Tyr Phe Glu Gly Pro Gly Lys Gly Ser Pro Phe Gly Trp 210 215 220 Glu Gly Asn Ile Lys Lys Trp Phe Glu Gln Met Met Gly His Cys Thr 225 230 235 240 Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Ser Ala Glu 245 250 255 Leu Phe Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp 260 265 270 Glu Glu Ala Lys Leu Asn Tyr Gly Glu Lys Phe Gln Ile Ile Glu Asn 275 280 285 Val Phe Lys Gln Lys Lys Thr Pro Asn Leu Lys Gln Ile Ala Lys Glu 290 295 300 Ile Gly Val Ser Glu Thr Asp Ile Lys Gly Tyr Arg Val Asn Lys Ser 305 310 315 320 Gly Lys Pro Glu Phe Thr Gln Phe Lys Leu Tyr His Asp Leu Lys Asn 325 330 335 Ile Phe Glu Asp Ser Lys Tyr Leu Asn Asp Val Gln Leu Met Asp Asn 340 345 350 Ile Ala Glu Ile Ile Thr Ile Tyr Gln Asp Pro Glu Ser Ile Ile Lys 355 360 365 Glu Leu Asn Gln Leu Pro Glu Leu Leu Ser Glu Lys Glu Lys Glu Lys 370 375 380 Ile Ser Ala Leu Ser Gly Tyr Ala Gly Thr His Arg Leu Ser Leu Lys 385 390 395 400 Cys Ile Asn Leu Leu Leu Asp Asp Leu Trp Glu Ser Ser Leu Asn Gln 405 410 415 Met Glu Leu Phe Thr Lys Leu Asn Leu Lys Pro Lys Lys Ile Asp Leu 420 425 430 Ser Gln Gln His Lys Ile Pro Ile Lys Leu Val Asp Asp Phe Ile Leu 435 440 445 Ser Pro Val Val Lys Arg Ala Phe Ile Gln Ser Ile Gln Val Val Asn 450 455 460 Ala Ile Ile Asp Lys Tyr Gly Leu Pro Glu Asp Ile Ile Ile Glu Leu 465 470 475 480 Ala Arg Glu Asn Asn Ser Asp Asp Arg Arg Lys Phe Leu Asn Gln Leu 485 490 495 Gln Lys Gln Asn Ala Glu Thr Arg Lys Gln Val Glu Lys Val Leu Arg 500 505 510 Glu Tyr Gly Asn Asp Asn Ala Lys Arg Ile Val Gln Lys Ile Lys Leu 515 520 525 His Asn Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Lys Asp Ile Pro 530 535 540 Leu Glu Asp Leu Leu Lys Asn Pro Asn His Tyr Glu Val Asp His Ile 545 550 555 560 Ile Pro Arg Ser Val Ala Phe Asp Asn Ser Met His Asn Lys Val Leu 565,570,575 Val Arg Ala Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Tyr Gln 580,585,590 Tyr Leu Asn Ser Ser Glu Ser Leu Ser Tyr Asn Glu Phe Lys Gln 595,600,605 His Ile Leu Asn Leu Ser Lys Lys Asp Arg Ile Thr Lys Lys 610 615 620 Arg Glu Tyr Leu Glu Glu Arg Asp With Asn Lys Tyr Asp Val Gln 625 630 635 640 Lys Glu Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg 645,650,655 Glu Thr Ser Ser Leu Lys To Tyr Phe Ser To Asn Asn To Asp 660,665,670 Val Lys Val Lys Thr Ile Asn Gly Ser Phe Thr Asn Tyr Leu Arg Lys 675,680,685 Val Trp Lys Phe Asp Lys Asp Arg Asn Lys Gly Tyr Lys His Ala 690,695,700 Glu Asp Ala Leu Ile Ala Asn Ala Asp Phe Leu Phe Lys His Asn 705 710 715 720 Lys Lys Leu Arg Asn Ile Asn Lys Val Leu Asp Ala Pro Ser Lys Glu 725 730 735 Val Asp Lys Lys Arg Val Thr Val Gln Ser Glu Asp Glu Tyr Asn Gln 740 745 750 Met Phe Glu Asp Thr Gln Lys Ala Gln Ala Ile Lys Lys Phe Glu Ile 755 760 765 Arg Lys Phe Ser His Arg Val Asp Lys Lys Pro Asn Arg Gln Leu Ile 770 775 780 Lys Asp Thr Leu Tyr Ser Thr Arg Asn Ile Asp Gly Ile Glu Tyr Val 785 790 795 800 Val Glu Ser Ile Lys Asp Ile Tyr Ser Val Asn Asn Asp Lys Val Lys 805 810 815 Thr Lys Phe Lys Lys Asp Pro His Arg Leu Leu Met Tyr Arg Asn Asp 820 825 830 Pro Gln Thr Phe Glu Lys Phe Glu Lys Val Phe Lys Gln Tyr Glu Ser 835 840 845 Glu Lys Asn Pro Phe Ala Lys Tyr Tyr Glu Glu Thr Gly Glu Lys Ile 850 855 860 Arg Lys Phe Ser Lys Thr Gly Gln Gly Pro Tyr Ile Asn Lys Ile Lys 865 870 875 880 Tyr Leu Arg Glu Arg Leu Gly Arg His Cys Asp Val Thr Asn Lys Tyr 885,890,895 With Arg Asn Lys With Val Gln Leu Lys With Tyr Ser Tyr Arg 900 905 910 Phe Asp With Tyr Gln Tyr Gly Asn Asn Tyr Lys Met With Thr Ser 915,920,925 Tyr Ile Asp Leu Glu Gln Lys Ser Asn Tyr Tyr Ile Ser Arg Glu 930,935,940 Lys Tyr Glu Gln Lys Lys Asp Lys Gln Ile Asp Asp Ser Tyr Lys 945 950 955 960 Phe Ile Gly Ser Phe Tyr Lys Asn Asp Ile Asn Tyr Asn Gly Glu 965,970,975 Met Tyr Arg Val Ile Gly Val Asn Asp Ser Glu Lys Ile Lys Phe Ser 980,985,990 Leu Ile <210> 21 <211> 1110 <212> PRT <213> Artificial Sequence <220> <223> Staphylococcus aureus Cas9 sequence <400> 21 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val 20 25 30 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 35 40 45 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 50 55 60 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 65 70 75 80 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 85 90 95 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 100 105 110 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu 115 120 125 Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 130 135 140 Gly Asn Glu Leu Ser Thr Arg Glu Gln Ile Ser Arg Asn Ser Lys Ala 145 150 155 160 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 165 170 175 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 180 185 190 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 195 200 205 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 210 215 220 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 225 230 235 240 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 245 250 255 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 260 265 270 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 275 280 285 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 290 295 300 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 305 310 315 320 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 325 330 335 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 340 345 350 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 355 360 365 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 370 375 380 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser 385 390 395 400 Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 405 410 415 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 420 425 430 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 435 440 445 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 450 455 460 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 465 470 475 480 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 485 490 495 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 500 505 510 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 515 520 525 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 530 535 540 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 545 550 555 560 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro 565 570 575 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 580 585 590 Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 595 600 605 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 610 615 620 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 625 630 635 640 Tyr Leu Leu Glu Glu Arg Asp With Asn Arg Phe Serving Val Gln Lys Asp 645,650,655 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu 660,665,670 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 675,680,685 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 690,695,700 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His Ala Glu Asp 705 710 715 720 Only Leu Ile On Asn Only Asp Phe Ile Phe Lys Glu Trp Lys Lys 725 730 735 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Arg 740,745,750 Gln Ala Glu Ser Met Pro Glue Ile Glu Thr Glu Gln Glu Tyr Lys Glu 755,760,765 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 770,775,780 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 785 790 795 800 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 805 810 815 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 820 825 830 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 835 840 845 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 850 855 860 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 865 870 875 880 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 885 890 895 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 900 905 910 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 915 920 925 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 930 935 940 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 945 950 955 960 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 965 970 975 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 980 985 990 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 995 1000 1005 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn 1010 1015 1020 Met Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser 1025 1030 1035 Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn 1040 1045 1050 Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys 1055 1060 1065 Gly Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys 1070 1075 1080 Light Light Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Gly 1085 1090 1095 Phe Ala Asn Glu Leu Gly Pro Arg Leu Met Gly Lys 1100 1105 1110 <210> 22 <211> 1235 <212> PRT <213> Artificial Sequence <220> <223> Candidatus Methanomethylophilus alvus Mx1201 Cas12a <400> 22 Met His Thr Gly Gly Leu Leu Ser Met Asp Ala Lys Glu Phe Thr Gly 1 5 10 15 Gln Tyr Pro Leu Ser Lys Thr Leu Arg Phe Glu Leu Arg Pro Ile Gly 20 25 30 Arg Thr Trp Asp Asn Leu Glu Ala Ser Gly Tyr Leu Ala Glu Asp Arg 35 40 45 His Arg Ala Glu Cys Tyr Pro Arg Ala Lys Glu Leu Leu Asp Asp Asn 50 55 60 His Arg Ala Phe Leu Asn Arg Val Leu Pro Gln Ile Asp Met Asp Trp 65 70 75 80 His Pro Ile Ala Glu Ala Phe Cys Lys Val His Lys Asn Pro Gly Asn 85 90 95 Lys Glu Leu Ala Gln Asp Tyr Asn Leu Gln Leu Ser Lys Arg Arg Lys 100 105 110 Glu Ile Ser Ala Tyr Leu Gln Asp Ala Asp Gly Tyr Lys Gly Leu Phe 115 120 125 Ala Lys Pro Ala Leu Asp Glu Ala Met Lys Ile Ala Lys Glu Asn Gly 130 135 140 Asn Glu Ser Asp Ile Glu Val Leu Glu Ala Phe Asn Gly Phe Ser Val 145 150 155 160 Tyr Phe Thr Gly Tyr His Glu Ser Arg Glu Asn Ile Tyr Ser Asp Glu 165 170 175 Asp Met Val Ser Val Ala Tyr Arg Ile Thr Glu Asp Asn Phe Pro Arg 180 185 190 Phe Val Ser Asn Ala Leu Ile Phe Asp Lys Leu Asn Glu Ser His Pro 195 200 205 Asp Ile Ile Ser Glu Val Ser Gly Asn Leu Gly Val Asp Asp Ile Gly 210 215 220 Lys Tyr Phe Asp Val Ser Asn Tyr Asn Asn Phe Leu Ser Gln Ala Gly 225 230 235 240 Ile Asp Asp Tyr Asn His Ile Ile Gly Gly His Thr Thr Glu Asp Gly 245 250 255 Leu Ile Gln Ala Phe Asn Val Val Leu Asn Leu Arg His Gln Lys Asp 260 265 270 Pro Gly Phe Glu Lys Ile Gln Phe Lys Gln Leu Tyr Lys Gln Ile Leu 275 280 285 Ser Val Arg Thr Ser Lys Ser Tyr Ile Pro Lys Gln Phe Asp Asn Ser 290,295,300 Lys Glu Met Val Asp Cys Ile Cys Asp Tyr Val Ser Lys Ile Glu Lys 305 310 315 320 Ser Glu Thr Val Glu Arg Ala Leu Lys Leu Val Arg Asn Ile Ser Ser 325 330 335 Phe Asp Leu Arg Gly Ile Phe Val Asn Lys Lys Asn Leu Arg Ile Leu 340 345 350 Ser Asn Lys Leu Ile Gly Asp Trp Asp Ala Ile Glu Thr Ala Leu Met 355 360 365 His Ser Ser Ser Ser Glu Asn Asp Lys Lys Ser Val Tyr Asp Ser Ala 370 375 380 Glu Ala Phe Thr Leu Asp Asp Ile Phe Ser Val Lys Lys Phe Ser 385 390 395 400 Asp Only Ser Only Only Glu Asp With Gly Asn Arg Only Glu Asp With Cys Arg 405 410 415 Val Ile Ser Glu Thr Ala Pro Phe Ile Asn Asp Leu Arg Ala Val Asp 420 425 430 Leu Asp Ser Leu Asn Asp Gly Tyr Glu Ala Ala Val Ser Lys Ile 435 440 445 Arg Glu Ser Leu Glu Pro Tyr Met Asp Leu Phe His Glu Leu Glu Ile 450 455 460 Phe Ser Val Gly Asp Glu Phe Pro Lys Cys Ala Ala Phe Tyr Ser Glu 465 470 475 480 Leu Glu Glu Will Be Glu Gln Leu Ile Glu Ile Ile Pro Leu Phe Asn 485,490,495 Lys Ala Arg Ser Phe Cys Thr Arg Lys Arg Tyr Ser Thr Asp Lys Ile 500 505 510 Lys Val Asn Leu Lys Phe Pro Thr Leu Ala Asp Gly Trp Asp Leu Asn 515,520,525 Lys Glu Arg Asp Asn Lys Ala Ala Ile Leu Arg Lys Asp Gly Lys Tyr 530 535 540 Tyr Leu - I'll Be There For You (Official Music Video) 545 550 555 560 Ser Asp Glu Asp Glu Ser Phe Glu Lys Met Glu Tyr Lys Leu Leu 565,570,575 Pro Ser Pro Val Lys Met Leu Pro Lys Ile Phe Val Lys Ser Lys Ala 580 585 590 Ala Lys Glu Lys Tyr Gly Leu Thr Asp Arg Met Leu Glu Cys Tyr Asp 595 600 605 Lys Gly Met His Lys Ser Gly Ser Ala Phe Asp Leu Gly Phe Cys His 610 615 620 Glu Leu Ile Asp Tyr Tyr Lys Arg Cys Ile Ala Glu Tyr Pro Gly Trp 625 630 635 640 Asp Val Phe Asp Phe Lys Phe Arg Glu Thr Ser Asp Tyr Gly Ser Met 645 650 655 Lys Glu Phe Asn Glu Asp Val Ala Gly Ala Gly Tyr Tyr Met Ser Leu 660 665 670 Arg Lys Ile Pro Cys Ser Glu Val Tyr Arg Leu Leu Asp Glu Lys Ser 675 680 685 Ile Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Tyr Ser Glu Asn Ala His 690 695 700 Gly Asn Lys Asn Met His Thr Met Tyr Trp Glu Gly Leu Phe Ser Pro 705 710 715 720 Gln Asn Leu Glu Ser Pro Val Phe Lys Leu Ser Gly Gly Ala Glu Leu 725 730 735 Phe Phe Arg Lys Ser Ser Ile Pro Asn Asp Ala Lys Thr Val His Pro 740 745 750 Lys Gly Ser Val Leu Val Pro Arg Asn Asp Val Asn Gly Arg Arg Ile 755 760 765 Pro Asp Ser Ile Tyr Arg Glu Leu Thr Arg Tyr Phe Asn Arg Gly Asp 770 775 780 Cys Arg Ile Ser Asp Glu Ala Lys Ser Tyr Leu Asp Lys Val Lys Thr 785 790 795 800 Lys Lys Ala Asp His Asp Ile Val Lys Asp Arg Arg Phe Thr Val Asp 805 810 815 Lys Met Met Phe His Val Pro Ile Ala Met Asn Phe Lys Ala Ile Ser 820 825 830 Lys Pro Asn Leu Asn Lys Lys Val Ile Asp Gly Ile Ile Asp Asp Gln 835 840 845 Asp Leu Lys Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr 850 855 860 Val Thr Met Val Asp Arg Lys Gly Asn Ile Leu Tyr Gln Asp Ser Leu 865 870 875 880 Asn Ile Leu Asn Gly Tyr Asp Tyr Arg Lys Ala Leu Asp Val Arg Glu 885 890 895 Tyr Asp Asn Lys Glu Ala Arg Arg Asn Trp Thr Lys Val Glu Gly Ile 900 905 910 Arg Lys Met Lys Glu Gly Tyr Leu Ser Leu Ala Val Ser Lys Leu Ala 915 920 925 Asp Met Ile Ile Glu Asn Asn Ala Ile Ile Val Met Glu Asp Leu Asn 930 935 940 His Gly Phe Lys Ala Gly Arg Ser Lys Ile Glu Lys Gln Val Tyr Gln 945 950 955 960 Lys Phe Glu Ser Met Leu Ile Asn Lys Leu Gly Tyr Met Val Leu Lys 965 970 975 Asp Lys Ser Ile Asp Gln Ser Gly Gly Ala Leu His Gly Tyr Gln Leu 980 985 990 Ala Asn His Val Thr Thr Leu Ala Ser Val Gly Lys Gln Cys Gly Val 995 1000 1005 Ile Phe Tyr Ile Pro Ala Ala Phe Thr Ser Lys Ile Asp Pro Thr 1010 1015 1020 Thr Gly Phe Ala Asp Leu Phe Ala Leu Ser Asn Val Lys Asn Val 1025 1030 1035 Ala Ser Met Arg Glu Phe Phe Ser Lys Met Lys Ser Val Ile Tyr 1040 1045 1050 Asp Lys Ala Glu Gly Lys Phe Ala Phe Thr Phe Asp Tyr Leu Asp 1055 1060 1065 Tyr Asn Val Lys Ser Glu Cys Gly Arg Thr Leu Trp Thr Val Tyr 1070 1075 1080 Thr Val Gly Glu Arg Phe Thr Tyr Ser Arg Val Asn Arg Glu Tyr 1085 1090 1095 Val Arg Lys Val Pro Thr Asp Ile Ile Tyr Asp Ala Leu Gln Lys 1100 1105 1110 Ala Gly Ile Ser Val Glu Gly Asp Leu Arg Asp Arg Ile Ala Glu 1115 1120 1125 Ser Asp Gly Asp Thr Leu Lys Ser Ile Phe Tyr Ala Phe Lys Tyr 1130 1135 1140 Ala Leu Asp Met Arg Val Glu Asn Arg Glu Glu Asp Tyr Ile Gln 1145 1150 1155 Ser Pro Val Lys Asn Ala Ser Gly Glu Phe Phe Cys Ser Lys Asn 1160 1165 1170 Ala Gly Lys Ser Leu Pro Gln Asp Ser Asp Ala Asn Gly Ala Tyr 1175 1180 1185 Asn Ile Ala Leu Lys Gly Ile Leu Gln Leu Arg Met Leu Ser Glu 1190 1195 1200 Gln Tyr Asp Pro Asn Ala Glu Ser Ile Arg Leu Pro Leu Ile Thr 1205 1210 1215 Asn Lys Ala Trp Leu Thr Phe Met Gln Ser Gly Met Lys Thr Trp 1220 1225 1230 Lys Asn 1235 <210> 23 <211> 1227 <212> PRT <213> Artificial Sequence <220> <223> Candidatus Methanomethylophilus alvus isolate MGYG-HGUT-02456 Cas12a <400> 23 Met Asp Ala Lys Glu Phe Thr Gly Gln Tyr Pro Leu Ser Lys Thr Leu 1 5 10 15 Arg Phe Glu Leu Arg Pro Ile Gly Arg Thr Trp Asp Asn Leu Glu Ala 20 25 30 Ser Gly Tyr Leu Ala Glu Asp Arg His Arg Ala Glu Cys Tyr Pro Arg 35 40 45 Ala Lys Glu Leu Leu Asp Asp Asn His Arg Ala Phe Leu Asn Arg Val 50 55 60 Leu Pro Gln Ile Asp Met Asp Trp His Pro Ile Ala Glu Ala Phe Cys 65 70 75 80 Lys Val His Lys Asn Pro Gly Asn Lys Glu Leu Ala Gln Asp Tyr Asn 85 90 95 Leu Gln Leu Ser Lys Arg Arg Lys Glu Ile Ser Ala Tyr Leu Gln Asp 100 105 110 Ala Asp Gly Tyr Lys Gly Leu Phe Ala Lys Pro Ala Leu Asp Glu Ala 115 120 125 Met Lys Ile Ala Lys Glu Asn Gly Asn Glu Ser Asp Ile Glu Val Leu 130 135 140 Glu Ala Phe Asn Gly Phe Ser Val Tyr Phe Thr Gly Tyr His Glu Ser 145 150 155 160 Arg Glu Asn Ile Tyr Ser Asp Glu Asp Met Val Ser Val Ala Tyr Arg 165 170 175 Ile Thr Glu Asp Asn Phe Pro Arg Phe Val Ser Asn Ala Leu Ile Phe 180 185 190 Asp Lys Leu Asn Glu Ser His Pro Asp Ile Ile Ser Glu Val Ser Gly 195 200 205 Asn Leu Gly Val Asp Asp Ile Gly Lys Tyr Phe Asp Val Ser Asn Tyr 210 215 220 Asn Asn Phe Leu Ser Gln Ala Gly Ile Asp Asp Tyr Asn His Ile Ile 225 230 235 240 Gly Gly His Thr Thr Glu Asp Gly Leu Ile Gln Ala Phe Asn Val Val 245 250 255 Leu Asn Leu Arg His Gln Lys Asp Pro Gly Phe Glu Lys Ile Gln Phe 260 265 270 Lys Gln Leu Tyr Lys Gln Ile Leu Ser Val Arg Thr Ser Lys Ser Tyr 275 280 285 Ile Pro Lys Gln Phe Asp Asn Ser Lys Glu Met Val Asp Cys Ile Cys 290 295 300 Asp Tyr Val Ser Lys Ile Glu Lys Ser Glu Thr Val Glu Arg Ala Leu 305 310 315 320 Lys Leu Val Arg Asn Ile Ser Ser Phe Asp Leu Arg Gly Ile Phe Val 325 330 335 Asn Lys Lys Asn Leu Arg Ile Leu Ser Asn Lys Leu Ile Gly Asp Trp 340 345 350 Asp Ala Ile Glu Thr Ala Leu Met His Ser Ser Ser Ser Glu Asn Asp 355 360 365 Lys Lys Ser Val Tyr Asp Ser Ala Glu Ala Phe Thr Leu Asp Asp Ile 370 375 380 Phe Ser Ser Val Lys Lys Phe Ser Asp Ala Ser Ala Glu Asp Ile Gly 385 390 395 400 Asn Arg Ala Glu Asp With Cys Arg Val Ser Glu Thr Ala Pro Phe 405 410 415 I have Asp Asp Leu Arg Ala Val Asp Leu Asp Ser Leu Asp Asp Gly 420 425 430 Tyr Glu Wing Wing Val Ser Lys Ile Arg Glu Ser Leu Glu Pro Tyr Met 435 440 445 Asp Leu Phe His Glu Leu Glu Ile Phe Ser Val Gly Asp Glu Phe Pro 450 455 460 Lys Cys Ala Ala Phe Tyr Ser Glu Leu Glu Glu Val Ser Glu Gln Leu 465 470 475 480 Ile Glu Ile Ile Pro Leu Phe Asn Lys Ala Arg Ser Phe Cys Thr Arg 485,490,495 Lys Arg Tyr Ser Thr Asp Lys Ile Lys Val Asn Leu Lys Phe Pro Thr 500 505 510 Leu Ala Asp Gly Trp Asp Leu Asn Lys Glu Arg Asp Asn Lys Ala Ala 515,520,525 The Arg Lys Asp Gly Lys Tyr Tyr Ala Ile The Asp Met Lys 530 535 540 Lys Asp Leu Ser Ser Ile Arg Thr Ser Asp Glu Asp Glu Ser Ser Phe 545 550 555 560 Glu Lys Met Glu Tyr Lys Leu Leu Pro Ser Pro Val Lys Met Leu Pro 565,570,575 Lys Ile Phe Val Lys Ser Lys Ala Ala Lys Glu Lys Tyr Gly Leu Thr 580,585,590 Asp Arg Met With Glu Cys Tyr Asp Lys Gly Met His Lys Ser Gly Ser 595,600,605 Ala Phe Asp Leu Gly Phe Cys His Glu Leu Ile Asp Tyr Tyr Lys Arg 610 615 620 Cys Ile Ala Glu Tyr Pro Gly Trp Asp Val Phe Asp Phe Lys Phe Arg 625 630 635 640 Glu Thr Ser Asp Tyr Gly Ser Met Lys Glu Phe Asn Glu Asp Val Ala 645,650,655 Gly Ala Gly Tyr Tyr Met Ser Leu Arg Lys Ile Pro Cys Ser Glu Val 660,665,670 Tyr Arg to Asp Glu Lys Ser and Tyr to Phe Gln to Tyr Asn 675,680,685 Lys Asp Tyr Ser Glu Asn Ala His Gly Asn Lys Asn Met His Thr Met 690,695,700 Tyr Trp Glu Gly Leu Phe Ser Pro Gln Asn Leu Glu Ser Pro Val Phe 705 710 715 720 Lys Leu Ser Gly Gly Ala Glu Leu Phe Phe Arg Lys Ser Ser Ile Pro 725 730 735 Asn Asp Ala Lys Thr Val His Pro Lys Gly Ser Val Leu Val Pro Arg 740 745 750 Asn Asp Val Asn Gly Arg Arg Ile Pro Asp Ser Ile Tyr Arg Glu Leu 755 760 765 Thr Arg Tyr Phe Asn Arg Gly Asp Cys Arg Ile Ser Asp Glu Ala Lys 770 775 780 Ser Tyr Leu Asp Lys Val Lys Thr Lys Lys Ala Asp His Asp Ile Val 785 790 795 800 Lys Asp Arg Arg Phe Thr Val Asp Lys Met Met Phe His Val Pro Ile 805 810 815 Ala Met Asn Phe Lys Ala Ile Ser Lys Pro Asn Leu Asn Lys Lys Val 820 825 830 Ile Asp Gly Ile Ile Asp Asp Gln Asp Leu Lys Ile Ile Gly Ile Asp 835 840 845 Arg Gly Glu Arg Asn Leu Ile Tyr Val Thr Met Val Asp Arg Lys Gly 850 855 860 Asn Ile Leu Tyr Gln Asp Ser Leu Asn Ile Leu Asn Gly Tyr Asp Tyr 865 870 875 880 Arg Lys Ala Leu Asp Val Arg Glu Tyr Asp Asn Lys Glu Ala Arg Arg 885 890 895 Asn Trp Thr Lys Val Glu Gly Ile Arg Lys Met Lys Glu Gly Tyr Leu 900 905 910 Ser Leu Ala Val Ser Lys Leu Ala Asp Met Ile Ile Glu Asn Asn Ala 915 920 925 Ile Ile Val Met Glu Asp Leu Asn His Gly Phe Lys Ala Gly Arg Ser 930 935 940 Lys Ile Glu Lys Gln Val Tyr Gln Lys Phe Glu Ser Met Leu Ile Asn 945 950 955 960 Lys Leu Gly Tyr Met Val Leu Lys Asp Lys Ser Ile Asp Gln Ser Gly 965 970 975 Gly Ala Leu His Gly Tyr Gln Leu Ala Asn His Val Thr Thr Leu Ala 980 985 990 Ser Val Gly Lys Gln Cys Gly Val Ile Phe Tyr Ile Pro Ala Ala Phe 995 1000 1005 Thr Ser Lys Ile Asp Pro Thr Thr Gly Phe Ala Asp Leu Phe Ala 1010 1015 1020 Leu Ser Asn Val Lys Asn Val Ala Ser Met Arg Glu Phe Phe Ser 1025 1030 1035 Lys Met Lys Ser Val Ile Tyr Asp Lys Ala Glu Gly Lys Phe Ala 1040 1045 1050 Phe Thr Phe Asp Tyr Leu Asp Tyr Asn Val Lys Ser Glu Cys Gly 1055 1060 1065 Arg Thr Leu Trp Thr Val Tyr Thr Val Gly Glu Arg Phe Thr Tyr 1070 1075 1080 Ser Arg Val Asn Arg Glu Tyr Val Arg Lys Val Pro Thr Asp Ile 1085 1090 1095 Tyr Asp Has Gln Lys Has Gly Has Ser Val Glu Gly Asp 1100 1105 1110 Leu Arg Asp Arg Ile Ala Glu Ser Asp Gly Asp Thr Leu Lys Ser 1115 1120 1125 Ile Phe Tyr Ala Phe Lys Tyr Ala Leu Asp Met Arg Val Glu Asn 1130 1135 1140 Arg Glu Glu Asp Tyr Ile Gln Ser Pro Val Lys Asn Ala Ser Gly 1145 1150 1155 Glu Phe Phe Cys Ser Lys Asn Ala Gly Lys Ser Leu Pro Gln Asp 1160 1165 1170 Ser Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Lys Gly Ile Leu 1175 1180 1185 Gln Leu Arg Met Leu Ser Glu Gln Tyr Asp Pro Asn Ala Glu Ser 1190 1195 1200 Ile Arg Leu Pro Leu Ile Thr Asn Lys Ala Trp Leu Thr Phe Met 1205 1210 1215 Gln Ser Gly Met Lys Thr Trp Lys Asn 1220 1225 <210> 24 <211> 1238 <212> PRT <213> Artificial Sequence <220> <223> Candidatus Methanoplasma termitum strain MpT1 chromosome Cas12a <400> 24 Met Asn Asn Tyr Asp Glu Phe Thr Lys Leu Tyr Pro Ile Gln Lys Thr 1 5 10 15 Ile Arg Phe Glu Leu Lys Pro Gln Gly Arg Thr Met Glu His Leu Glu 20 25 30 Thr Phe Asn Phe Phe Glu Glu Asp Arg Asp Arg Ala Glu Lys Tyr Lys 35 40 45 Ile Leu Lys Glu Ala Ile Asp Glu Tyr His Lys Lys Phe Ile Asp Glu 50 55 60 His Thr Thr Asn Met Ser Leu Asp Trp Asn Ser Leu Lys Gln Ile Ser 65 70 75 80 Glu Glu Lys Tyr Lys Ser Arg Glu Glu Lys Asp Lys Val Phe Leu 85 90 95 Ser Glu Gln Lys Arg Met Arg Gln Glu Ile Val Ser Glu Phe Lys Lys 100 105 110 Asp Asp Arg Phe Lys Asp Leu Phe Ser Lys Lys Leu Phe Ser Glu Leu 115 120 125 Leu Lys Glu Glu Ile Tyr Lys Gly Asn His Gln Glu Ile Asp Ala 130 135 140 Leu Lys Ser Phe Asp Lys Phe Ser Gly Tyr Phe Ile Gly Leu His Glu 145 150 155 160 Asn Arg Lys Asn Met Tyr Ser Asp Gly Asp Glu Ile Thr Ala Ile Ser 165 170 175 Asn Arg Ile Val Asn Glu Asn Phe Pro Lys Phe Leu Asp Asn Leu Gln 180 185 190 Lys Tyr Gln Glu Ala Arg Lys Tyr Pro Glu Trp Ile Lys Ala 195 200 205 Glu Ser Ala Leu Val Ala His Asn Ile Lys Met Asp Glu Val Phe Ser 210 215 220 Leu Glu Tyr Phe Asn Lys Val Leu Asn Gln Glu Gly Ile Gln Arg Tyr 225 230 235 240 Asn Leu Ala Leu Gly Gly Tyr Val Thr Lys Ser Gly Glu Lys Met Met 245 250 255 Gly Leu Asn Asp Ala Leu Asn Leu Ala His Gln Ser Glu Lys Ser Ser 260 265 270 Lys Gly Arg Ile His Met Thr Pro Leu Phe Lys Gln Ile Leu Ser Glu 275 280 285 Lys Glu Ser Phe Ser Tyr Ile Pro Asp Val Phe Thr Glu Asp Ser Gln 290 295 300 Leu Leu Pro Ser Ile Gly Gly Phe Phe Ala Gln Ile Glu Asn Asp Lys 305 310 315 320 Asp Gly Asn Ile Phe Asp Arg Ala Leu Glu Leu Ile Ser Ser Tyr Ala 325 330 335 Glu Tyr Asp Thr Glu Arg Ile Tyr Ile Arg Gln Ala Asp Ile Asn Arg 340 345 350 Val Ser Asn Val Ile Phe Gly Glu Trp Gly Thr Leu Gly Gly Leu Met 355 360 365 Arg Glu Tyr Lys Ala Asp Ser Ile Asn Asp Ile Asn Leu Glu Arg Thr 370 375 380 Cys Lys Val Asp Lys Trp Leu Asp Ser Lys Glu Phe Ala Leu Ser 385 390 395 400 Asp Val Leu Glu Ala Ile Lys Arg Thr Gly Asn Asn Asp Ala Phe Asn 405 410 415 Glu Tyr With Lys Ser Met Arg Thr Ala Arg Glu Lys With Asp Ala Ala 420 425 430 Arg Lys Glu Met Lys Phe Ile Ser Glu Lys Ile Ser Gly Asp Glu Glu 435 440 445 I'm Not Afraid I'm Not Afraid I'm Not Afraid Asp I'm Val Gln Gln Phe Leu 450 455 460 His Phe Phe Asn Leu Phe Lys Ala Arg Gln Asp Ile Pro Leu Asp Gly 465 470 475 480 Ala Phe Tyr Ala Glu Phe Asp Glu Val His Ser Lys Leu Phe Ala Ile 485,490,495 Val Pro Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Lys Asn Leu 500 505 510 Asn Thr Lys Lys Ile Lys Leu Asn Phe Lys Asn Pro Thr Leu Ala Asn 515,520,525 Gly Trp Asp Gln Asn Lys Val Tyr Asp Tyr Ala Ser Leu Ile Phe Leu 530 535 540 Arg Asp Gly Asn Tyr Tyr Leu Gly Ile Ile Asn Pro Lys Arg Lys Lys 545 550 555 560 Asn Ile Lys Phe Glu Gln Gly Ser Gly Asn Gly Pro Phe Tyr Arg Lys 565 570 575 Met Val Tyr Lys Gln Ile Pro Gly Pro Asn Lys Asn Leu Pro Arg Val 580 585 590 Phe Leu Thr Ser Thr Lys Gly Lys Lys Glu Tyr Lys Pro Ser Lys Glu 595 600 605 Ile Ile Glu Gly Tyr Glu Ala Asp Lys His Ile Arg Gly Asp Lys Phe 610 615 620 Asp Leu Asp Phe Cys His Lys Leu Ile Asp Phe Phe Lys Glu Ser Ile 625 630 635 640 Glu Lys His Lys Asp Trp Ser Lys Phe Asn Phe Tyr Phe Ser Pro Thr 645 650 655 Glu Ser Tyr Gly Asp Ile Ser Glu Phe Tyr Leu Asp Val Glu Lys Gln 660 665 670 Gly Tyr Arg Met His Phe Glu Asn Ile Ser Ala Glu Thr Ile Asp Glu 675 680 685 Tyr Val Glu Lys Gly Asp Leu Phe Leu Phe Gln Ile Tyr Asn Lys Asp 690 695 700 Phe Val Lys Ala Ala Thr Gly Lys Lys Asp Met His Thr Ile Tyr Trp 705 710 715 720 Asn Ala Ala Phe Ser Pro Glu Asn Leu Gln Asp Val Val Val Lys Leu 725 730 735 Asn Gly Glu Ala Glu Leu Phe Tyr Arg Asp Lys Ser Asp Ile Lys Glu 740 745 750 Ile Val His Arg Glu Gly Glu Ile Leu Val Asn Arg Thr Tyr Asn Gly 755 760 765 Arg Thr Pro Val Pro Asp Lys Ile His Lys Lys Leu Thr Asp Tyr His 770 775 780 Asn Gly Arg Thr Lys Asp Leu Gly Glu Ala Lys Glu Tyr Leu Asp Lys 785 790 795 800 Val Arg Tyr Phe Lys Ala His Tyr Asp Ile Thr Lys Asp Arg Arg Tyr 805 810 815 Leu Asn Asp Lys Ile Tyr Phe His Val Pro Leu Thr Leu Asn Phe Lys 820 825 830 Ala Asn Gly Lys Lys Asn Leu Asn Lys Met Val Ile Glu Lys Phe Leu 835 840 845 Ser Asp Glu Lys Ala His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn 850 855 860 Leu Leu Tyr Tyr Ser Ile Ile Asp Arg Ser Gly Lys Ile Ile Asp Gln 865 870 875 880 Gln Ser Leu Asn Val Ile Asp Gly Phe Asp Tyr Arg Glu Lys Leu Asn 885 890 895 Gln Arg Glu Ile Glu Met Lys Asp Ala Arg Gln Ser Trp Asn Ala Ile 900 905 910 Gly Lys Ile Lys Asp Leu Lys Glu Gly Tyr Leu Ser Lys Ala Val His 915 920 925 Glu Ile Thr Lys Met Ala Ile Gln Tyr Asn Ala Ile Val Val Met Glu 930 935 940 Glu Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln 945 950 955 960 Ile Tyr Gln Lys Phe Glu Asn Met Leu Ile Asp Lys Met Asn Tyr Leu 965 970 975 Val Phe Lys Asp Ala Pro Asp Glu Ser Pro Gly Gly Val Leu Asn Ala 980 985 990 Tyr Gln Leu Thr Asn Pro Leu Glu Ser Phe Ala Lys Leu Gly Lys Gln 995 1000 1005 Thr Gly Ile Leu Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys Ile 1010 1015 1020 Asp Pro Thr Thr Gly Phe Val Asn Leu Phe Asn Thr Ser Ser Lys 1025 1030 1035 Thr Asn Ala Gln Glu Arg Lys Glu Phe Leu Gln Lys Phe Glu Ser 1040 1045 1050 Ile Ser Tyr Ser Ala Lys Asp Gly Gly Ile Phe Ala Phe Ala Phe 1055 1060 1065 Asp Tyr Arg Lys Phe Gly Thr Ser Lys Thr Asp His Lys Asn Val 1070 1075 1080 Trp Thr Ala Tyr Thr Asn Gly Glu Arg Met Arg Tyr Ile Lys Glu 1085 1090 1095 Lys Lys Arg Asn Glu Leu Phe Asp Pro Ser Lys Glu Ile Lys Glu 1100 1105 1110 Ala Leu Thr Ser Ser Gly Ile Lys Tyr Asp Gly Gly Gln Asn Ile 1115 1120 1125 Leu Pro Asp Ile Leu Arg Ser Asn Asn Asn Gly Leu Ile Tyr Thr 1130 1135 1140 Met Tyr Ser Ser Phe Ile Ala Ala Ile Gln Met Arg Val Tyr Asp 1145 1150 1155 Gly Lys Glu Asp Tyr Ile Ile Ser Pro Ile Lys Asn Ser Lys Gly 1160 1165 1170 Glu Phe Phe Arg Thr Asp Pro Lys Arg Arg Glu Leu Pro Ile Asp 1175 1180 1185 Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Arg Gly Glu Leu 1190 1195 1200 Thr Met Arg Ala Ile Ala Glu Lys Phe Asp Pro Asp Ser Glu Lys 1205 1210 1215 Met Ala Lys Leu Glu Leu Lys His Lys Asp Trp Phe Glu Phe Met 1220 1225 1230 Gln Thr Arg Gly Asp 1235 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRAC gRNA <400> 25 agagtctctc agctggtaca 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDCD1 targeting gRNA <400> 26 gtctgggcgg tgctacaact 20 <210> 27 <211> 331 <212> PRT <213> Homo sapiens <400> 27 Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala 1 5 10 15 Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys 20 25 30 Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser 35 40 45 Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro 50 55 60 Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile 65 70 75 80 Ile Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln 85 90 95 Phe Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu 100 105 110 Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro 115 120 125 Ser Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala 130 135 140 Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser 145 150 155 160 Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe 165 170 175 Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala 180 185 190 Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln Gln Pro Pro 195 200 205 His His Leu Pro Gln Gln Met Pro Val Gln His Pro Arg Leu Gln Ala 210 215 220 Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu Thr Pro 225 230 235 240 Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Ala Glu 245 250 255 Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg 260 265 270 Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys 275 280 285 Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln 290 295 300 Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser Gly Cys 305 310 315 320 Gln Leu Met Leu Thr Gln Gln Leu Gln Thr Phe 325 330 <210> 28 <211> 1929 <212> DNA <213> Homo sapiens <400> 28 aggagccgcc gccagtggag ggccgggcgc tgcggccgcg gccggggcgg gcgcagggcc 60 gagcggacgg gggggcgcgg gcccccggg aggccgcggc cactcccccc cgggccggcg 120 cggcggggga ggcggaggat ggaaaccc ttctacggcg atgaggcgct gagcggcctg 180 ggcggcggcg ccagtggcag cggcggcagc ttcgcgtccc cgggccgct gttccccggg 240 gcgccccga cggccgcggc cggcagcatg atgaagaagg acgcgctgac gctgagcctg 300 agtgagcagg tgcggcagc gctcaagcct gcggccgcgc cgcctcctac ccccctgcgc 360 gccgacggcg cccccagcgc ggcacccccc gacggcctgc tcgcctctc cgacctgggg 420 ctgctgaagc tggcctcccc cgagctcgag cgcctcatca tccagtccaa cgggctggtc 480 accaccacgc cgacgagctc acagttcctc taccccaagg tggcggccag cgaggagcag 540 gagttcgccg agggcttcgt caaggccctg gaggatttac acaagcagaa ccagctcggc 600 gcgggcgcgg ccgctgccgc cgccgccgcc gccgccgggg ggccctcggg cacggccacg 660 ggctccgcgc cccccggcga gctggccccg gcggcggccg cgcccgaagc gcctgtctac 720 gcgaacctga gcagctacgc gggcggcgcc gggggcgcgg ggggcgccgc gacggtcgcc 780 ttcgctgccg aacctgtgcc cttcccgccg ccgccacccc caggcgcgtt ggggccgccg 840 cgcctggctg cgctcaagga cgagccacag acggtgcccg acgtgccgag cttcggcgag 900 agcccgccgt tgtcgcccat cgacatggac acgcaggagc gcatcaaggc ggagcgcaag 960 cggctgcgca accgcatcgc cgcctccaag tgccgcaagc gcaagctgga gcgcatctcg 1020 cgcctggaag agaaagtgaa gaccctcaag agtcagaaca cggagctggc gtccacggcg 1080 agcctgctgc gcgagcaggt ggcgcagctc aagcagaaag tcctcagcca cgtcaacagc 1140 ggctgccagc tgctgcccca gcaccaggtg cccgcgtact gagtccgcgc gcggggcgca 1200 tgcgcggcca ccctccccaa ggggcgggct cgcggggggg tgtcgtgggc gccccggact 1260 tggagagggt gcggccctgg ggaccccccc tccccgagtg tgcccaggaa ctcagagagg 1320 gcgcggcccc cggggattcc ccccccccga gggtgcccag gactcgacaa gctggacccc 1380 ctgctcccgg gggggcgagc gcatgacccc cccgccctcg cgctgcctct ttcccccgcg 1440 cggccgcccc gtgttgcaca aacccgcgcg tctcggctgc ccctttgtac accgcgccgc 1500 ggaagggggc tccgaggggg cgcagcctca aaccctgcct ttcctttact tttacttttt 1560 tttttttttc tttggaagag agaagaacag agtgttcgat tctgccctat ttatgtttct 1620 actcgggaac aaacgttggt tgtgtgtgtg tgtgttttct tgtgttggtt ttttaaagaa 1680 atgggaagaa gaaaaaaaaaa ttctccgccc ctttcctcga tctcgctccc cccttcggtt 1740 ctttcgaccg gtcccccctc ccttttttgt tctgttttgt tttgtttttgc tacgagtcca 1800 cattcctgtt tgtaatcctt ggttcgcccg gttttctgtt ttcagtaaag tctcgttacg 1860 ccagctcggc tctccgcctc cttcttcccc cgccggggcc tggcgggctg ggcggggcct 1920 ggttcgctt 1929 <210> 29 <211> 3775 <212> DNA <213> Homo sapiens <400> 29 cattcataag actcagagct acggccacgg cagggacacg cggaaccaag acttggaaac 60 ttgattgttg tggttcttct tgggggttat gaaatttcat taatcttttt tttttccggg 120 gagaaagttt ttggaaagat tcttccagat atttcttcat tttcttttgg aggaccgact 180 tacttttttt ggtcttcttt attactcccc tccccccgtg ggacccgccg gacgcgtgga 240 ggagaccgta gctgaagctg attctgtaca gcgggacagc gctttctgcc cctgggggag 300 caacccctcc ctcgcccctg ggtcctacgg agcctgcact ttcaagaggt acagcggcat 360 cctgtggggg cctgggcacc gcaggaagac tgcacagaaa ctttgccatt gttggaacgg 420 gacgttgctc cttccccgag cttccccgga cagcgtactt tgaggactcg ctcagctcac 480 cggggactcc cacggctcac cccggacttg caccttactt ccccaacccg gccatagcct 540 tggcttcccg gcgacctcag cgtggtcaca ggggcccccc tgtgcccagg gaaatgtttc 600 aggctttccc cggagactac gactccggct cccggtgcag ctcctcaccc tctgccgagt 660 ctcaatatct gtcttcggtg gactccttcg gcagtccacc caccgccgcg gcctcccagg 720 agtgcgccgg tctcggggaa atgcccggtt ccttcgtgcc cacggtcacc gcgatcacaa 780 ccagccagga cctccagtgg cttgtgcaac ccaccctcat ctcttccatg gcccagtccc 840 aggggcagcc actggcctcc cagcccccgg tcgtcgaccc ctacgacatg ccgggaacca 900 gctactccac accaggcatg agtggctaca gcagtggcgg agcgagtggc agtggtgggc 960 cttccaccag cggaactacc agtgggcctg ggcctgcccg cccagcccga gcccggccta 1020 ggagaccccg agaggagacg ctcaccccag aggaagagga gaagcgaagg gtgcgccggg 1080 aacgaaataa actagcagca gctaaatgca ggaaccggcg gagggagctg accgaccgac 1140 tccaggcgga gacagatcag ttggaggaag aaaaagcaga gctggagtcg gagatcgccg 1200 agctccaaaa ggagaaggaa cgtctggagt ttgtgctggt ggcccacaaa ccgggctgca 1260 agatccccta cgaagagggg cccgggccgg gcccgctggc ggaggtgaga gatttgccgg 1320 gctcagcacc ggctaaggaa gatggcttca gctggctgct gccgcccccg ccaccaccgc 1380 ccctgccctt ccagaccagc caagacgcac cccccaacct gacggcttct ctctttacac 1440 acagtgaagt tcaagtcctc ggcgacccct tccccgttgt taacccttcg tacacttctt 1500 cgtttgtcct cacctgcccg gaggtctccg cgttcgccgg cgcccaacgc accagcggca 1560 gtgaccagcc ttccgatccc ctgaactcgc cctccctcct cgctcggtga actctttaga 1620 cacacaaaac aaacaaacac atggggaga gagacttgga agaggagag gaggaggaga 1680 aggaggag agagggaag agacaaagtg ggtgtgtggc ctccctggct cctccgtctg 1740 accctctgcg gccactgcgc cactgccatc ggacaggagg attccttgtg ttttgtcctg 1800 cctcttgttt ctgtgccccg gcgaggccgg agagctggtg actttggggga cagggggtgg 1860 gaaggggatg gacaccccca gctgactgtt ggctctctga cgtcaaccca agctctgggg 1920 atgggtgggg aggggggcgg gtgacgccca ccttcgggca gtcctgtgtg aggatgaagg 1980 gacgggggtg ggaggtaggc tgtggggtgg gctggagtcc tctccagaga ggctcaacaa 2040 ggaaaaatgc cactccctac ccaatgtctc ccacacccac cctttttttg gggtgcccag 2100 gttggtttcc cctgcactcc cgaccttagc ttattgatcc cacatttcca tggtgtgaga 2160 tcctctttac tctgggcaga agtgagcccc cccttaaagg gaattcgatg cccccctaga 2220 ataatctcat ccccccaccc gacttctttt gaaatgtgaa cgtccttcct tgactgtcta 2280 gccactccct cccagaaaaa ctggctctga ttggaatttc tggcctccta aggctcccca 2340 ccccgaaatc agcccccagc cttgtttctg atgacagtgt tatcccaaga ccctgccccc 2400 tgccagccga ccctcctggc cttcctcgtt gggccgctct gatttcaggc agcaggggct 2460 gctgtgatgc cgtcctgctg gagtgattta tactgtgaaa tgagttggcc agattgtggg 2520 gtgcagctgg gtggggcagc acacctctgg ggggataatg tccccactcc cgaaagcctt 2580 tcctcggtct cccttccgtc catccccctt cttcctcccc tcaacagtga gttagactca 2640 agggggtgac agaaccgaga agggggtgac agtcctccat ccacgtggcc tctctctctc 2700 tcctcaggac cctcagccct ggcctttttc tttaaggtcc cccgaccaat ccccagccta 2760 ggacgccaac ttctcccacc ccttggcccc tcacatcctc tccaggaagg cagtgagggg 2820 ctgtgacatt tttccggaga agatttcaga gctgaggctt tggtaccccc aaacccccaa 2880 tatttttgga ctggcagact caaggggctg gaatctcatg attccatgcc cgagtccgcc 2940 catccctgac catggttttg gctctcccac cccgccgttc cctgcgcttc atctcatgag 3000 gatttcttta tgaggcaaat ttatattttt taatatcggg gggtggacca cgccgccctc 3060 catccgtgct gcatgaaaaa cattccacgt gccccttgtc gcgcgtctcc catcctgatc 3120 ccagacccat tccttagcta tttatccctt tcctggtttc cgaaaggcaa ttatatctat 3180 tatgtataag taaatatatt atatatggat gtgtgtgtgt gcgtgcgcgt gagtgtgtga 3240 gcgcttctgc agcctcggcc taggtcacgt tggccctcaa agcgagccgt tgaattggaa 3300 actgcttcta gaaactctgg ctcagcctgt ctcgggctga cccttttctg atcgtctcgg 3360 cccctctgat tgttcccgat ggtctctctc cctctgtctt ttctcctccg cctgtgtcca 3420 tctgaccgtt ttcacttgtc tcctttctga ctgtccctgc caatgctcca gctgtcgtct 3480 gactctgggt tcgttgggga catgagattt tattttttgt gagtgagact gagggatcgt 3540 agattttac aatctgtatc tttgacaatt ctgggtgcga gtgtgagagt gtgagcaggg 3600 cttgctcctg ccaaccacaa ttcaatgaat ccccgacccc cctaccccat gctgtacttg 3660 tggttctcttt ttgtatttt gcatctgacc ccggggggct gggacagatt ggcaatgggc 3720 cgtcccctct ccccttggtt ctgcactgtt gccaataaaa agctcttaaa aacgc 3775 <210> 30 <211> 953 <212> DNA <213> Homo sapiens <400> 30 caagagagag agagagcgtg caagccccaa agcgagcgac atgtcccttt ggggagcagt 60 ccctctgcac cccagagtga ggaggacgca ggggtcagag gtggctacag ggcaggcaga 120 ggaggcacct gtagggggtg gtgggctggt ggcccaggag aagtcaggaa gggagcccag 180 ctggtgacaa gagagcccag aggtgcctgg ggctgagtgt gagagcccgg aagatttcag 240 ccatgcctca cagctccgac agcagtgact ccagctcag ccgctctcct ccccctggca 300 aacaggactc atctgatgat gtgagaagag ttcaggag ggagaaaaat cgtattgccg 360 cccagaagag ccgacagagg cagacacaga agccgacac cctgcacctg gagagcgaag 420 acctggagaa acagaacgcg gcttacgca aggagatcaa gcagctcaca gaggaactga 480 agtactcac gtcggtgctg aacagccacg agcccctgtg ctcggtgctg gccgccagca 540 cgccctcgcc cccgaggtg gtgtacagcg cccaccatt cccaacct catgtcagct 600 ccccgcgctt ccagccctga gcttccgatg cggggagagc agagcctcgg gaggggcaca 660 cagactgtgg cagagctgcg cccatcccgc agaggcccct gtccacctgg agacccggag 720 acagaggcct ggacaggag tgacacggg aactgtcacg actggaaggg cgtgaggcct 780 cccagcagtg ccgcagcgtt tcgaggggcg tgtgctggac cccaccactg tggttgcag 840 gcccaatgca gaagagtatt aagaagatg ctcaagtccc atggcacaga gcaaggcggg 900 cagggaacgg ttatttttct aaaaatgc tttaaaaga aaaaaaaaa aaa 953 <210> 31 <211> 992 <212> DNA <213> Homo sapiens <400> 31 ggggcagacg tgggacggga aggacggctg ccgggactgg cggcgcgggga cactgggccg acgcgtggag tagcggggag agcgggagc ctgaggggggc ggggccggcg cgaggccgtg 120 ggtgcggcac gaggatgccg gcggcgggac agcgcccgta ggcagcccca cgggcagggc 180 gcgcgggcgg ggcggggcgg gccgggccag aggagcgcc ggcatgtcgc aagggctccc 240 ggccgccggc agcgtcctgc agaggagcgt cgcggcgccc gggaccagc cgcagccgca 300. gccgcagcag cagagccctg aggatgatga caggaaggtc cgaaggagag aaaaaaaccg agttgctgct cagagaagtc ggaagaagca gacccagaag gctgacaagc tccatgagga atatgagagc ctggagcaag aaaacaccat gctgcggaga gagatcggga agctgacaga 480 ggagctgaag cacctgacag aggcactgaa ggagcacgag aagatgtgcc cgctgctgct 540 ctgccctatg aactttgtgc cagtgcctcc ccggccggac cctgtggccg gctgcttgcc 600 ccgatgaagc cggggacact cctctgccca gcaaggagcc ttggtcattt tcatacctgg 660 gaggaaggct ttccttcac aattgtatac agggggcacc tgtggccagg cctcctcctg 720 ggagctccag gaccagccag ctgtgttccc tgcagactgg gctcagcccg acatccaaca 780 ggcgccaac tcacagagcc cttgtgcaga tccagcatgg agccaccct caggagtgac 840 ttctcatcca ccctggcagc tagtaggttc tgctgttatg cagagccatt tcctctagaa 900 tttggataat aagatgctt attgtctc ccttctccag tttgggaat ttacaggcac 960 atacacttc cttttcctgg aaaaaaaaa aa 992 <210> 32 <211> 380 <212> PRT <213> Homo sapiens <400> 32 Met Met Phe Ser Gly Phe Asn Ala Asp Tyr Glu Ala Ser Ser Arg 1 5 10 15 Cys Ser Ser Ala Ser Pro Ala Asp Ser Leu Ser Tyr Tyr His Ser Ser 20 25 30 Pro Ala Asp Ser Phe Ser Ser Met Gly Ser Pro Val Asn Ala Gln Asp 35 40 45 Phe Cys Thr Asp Leu Ala Val Ser Ser Ala Asn Phe Ile Pro Thr Val 50 55 60 Thr Ala Ile Ser Thr Ser Pro Asp Leu Gln Trp Leu Val Gln Pro Ala 65 70 75 80 Leu Val Ser Ser Val Ala Pro Ser Gln Thr Arg Ala Pro His Pro Phe 85 90 95 Gly Val Pro Ala Pro Ser Ala Gly Ala Tyr Ser Arg Ala Gly Val Val 100 105 110 Lys Thr Met Thr Gly Gly Arg Ala Gln Ser Ile Gly Arg Arg Gly Lys 115 120 125 Val Glu Gln Leu Ser Pro Glu Glu Glu Glu Lys Arg Arg Ile Arg Arg 130 135 140 Glu Arg Asn Lys Met Ala Ala Ala Lys Cys Arg Asn Arg Arg Arg Glu 145 150 155 160 Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Leu Glu Asp Glu Lys 165 170 175 Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys 180 185 190 Leu Glu Phe Ile Leu Ala Ala His Arg Pro Ala Cys Lys Ile Pro Asp 195 200 205 Asp Leu Gly Phe Pro Glu Glu Met Ser Val Ala Ser Leu Asp Leu Thr 210 215 220 Gly Gly Leu Pro Glu Val Ala Thr Pro Glu Ser Glu Glu Ala Phe Thr 225 230 235 240 Leu Pro Leu Leu Asn Asp Pro Glu Pro Lys Pro Ser Val Glu Pro Val 245 250 255 Lys Ser Ile Ser Ser Met Glu Leu Lys Thr Glu Pro Phe Asp Asp Phe 260 265 270 Leu Phe Pro Ala Ser Ser Arg Pro Ser Gly Ser Glu Thr Ala Arg Ser 275 280 285 Val Pro Asp Met Asp Leu Ser Gly Ser Phe Tyr Ala Ala Asp Trp Glu 290 295 300 Pro Leu His Ser Gly Ser Leu Gly Met Gly Pro Met Ala Thr Glu Leu 305 310 315 320 Glu Pro Leu Cys Thr Pro Val Val Thr Cys Thr Pro Ser Cys Thr Ala 325 330 335 Tyr Thr Ser Ser Phe Val Phe Thr Tyr Pro Glu Ala Asp Ser Phe Pro 340 345 350 Ser Cys Ala Ala Ala His Arg Lys Gly Ser Ser Ser Asn Glu Pro Ser 355 360 365 Ser Asp Ser Leu Ser Ser Pro Thr Leu Leu Ala Leu 370 375 380 <210> 33 <211> 331 <212> PRT <213> Homo sapiens <400> 33 Met Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala 1 5 10 15 Ser Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys 20 25 30 Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser 35 40 45 Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro 50 55 60 Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile 65 70 75 80 Ile Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln 85 90 95 Phe Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu 100 105 110 Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro 115 120 125 Ser Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala 130 135 140 Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser 145 150 155 160 Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe 165 170 175 Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala 180 185 190 Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln Gln Pro Pro 195 200 205 His His Leu Pro Gln Gln Met Pro Val Gln His Pro Arg Leu Gln Ala 210 215 220 Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu Thr Pro 225 230 235 240 Pro Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Ala Glu 245 250 255 Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys Arg 260 265 270 Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys 275 280 285 Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln 290 295 300 Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser Gly Cys 305 310 315 320 Gln Leu Met Leu Thr Gln Gln Leu Gln Thr Phe 325 330 <210> 34 <211> 153 <212> PRT <213> Homo sapiens <400> 34 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 35 <211> 95 <212> PRT <213> Homo sapiens <400> 35 Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Ile Ser 1 5 10 15 Ala Ala Leu Cys Glu Gly Ala Val Leu Pro Arg Ser Ala Lys Glu Leu 20 25 30 Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys Phe 35 40 45 Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr 50 55 60 Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro 65 70 75 80 Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys Phe Leu Lys Arg 85 90 95 <210> 36 <211> 281 <212> PRT <213> Artificial Sequence <220> <223> E195R / D269H TRAIL (DR5 Variant) <400> 36 Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 1 5 10 15 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 65 70 75 80 Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95 Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105 110 Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 115 120 125 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 130 135 140 Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly 145 150 155 160 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175 His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190 Gln Glu Arg Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205 Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220 Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 225 230 235 240 Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 245 250 255 Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met His His Glu Ala 260 265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly 275 280 <210> 37 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Klf4 primer <400> 37 aaaaaactcg aggcggcgtc tgcgtctgcg gcgtctgcga aagtgcctct tcatgtgtaa 60 ggc 63 <210> 38 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Oct4 primer <400> 38 aaaaaagtcg acgcggcgtc tgcgtctgcg gcgtctgcgg tttgaatgca tgggagagcc 60 <210> 39 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Sox2 primer <400> 39 aaaaaactcg aggcggcgtc tgcgtctgcg gcgtctgcgc atgtgcgaca ggggcagtg 59 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PDCD1 forward primer <400> 40 agcactgcct ctgtcactct cg 22 <210> 41 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> human c-Myc primer <400> 41 aaaaaactcg aggcggcgtc tgcgtctgcg gcgtctgcgc gcacaagagt tccgtagctg 60 ttc 63

Claims

1. An immune cell comprising an exogenous cargo, The immune cells show a log 24-hour increase in gene or protein expression levels relative to control immune cells after cargo delivery. 2 have a molecular profile comprising gene or protein expression levels with a fold change within 3, wherein the gene or protein is in the activator protein 1 (AP-1) signaling pathway; immune cells.

2. The expression level of the gene or protein is within 2 log of the expression level of the gene or protein in control immune cells 2 Fold change, or the expression level of a gene or protein is within 1 log of the expression level of the gene or protein in control immune cells 2 The immune cells of claim 1, wherein the fold change is

3. The immune cell of claim 1, wherein the exogenous cargo comprises a nucleic acid, a small molecule, a protein, a polypeptide, or a combination thereof.

4. The immune cell of claim 3, wherein the nucleic acid comprises messenger ribonucleic acid (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), deoxyribonucleic acid (DNA), or any combination thereof.

5. The immune cell of claim 1, wherein the expression of a gene or protein in the AP-1 signaling pathway comprises Fos (v-fos FBJ murine osteosarcoma viral oncogene homolog, FBJ murine osteosarcoma viral oncogene homolog), Jun (v-jun avian sarcoma virus 17 oncogene homolog), or a combination thereof.

6. The immune cell of claim 1, wherein the genes or proteins in the AP-1 signaling pathway include Fos, Jun, FosB (FBJ murine osteosarcoma viral oncogene homolog B), BATF (basic leucine zipper transcription factor ATF-like), BATF3 (basic leucine zipper transcription factor ATF-like 3), or a combination thereof.

7. The immune cell of claim 5, wherein Fos comprises human Fos comprising the nucleic acid sequence of SEQ ID NO: 1, and Jun comprises human Jun comprising the nucleic acid sequence of SEQ ID NO:

2.

8. The immune cell of claim 1, wherein the cargo comprises messenger ribonucleic acid (mRNA).

9. The immune cell of claim 8, wherein the mRNA encodes a chimeric antigen receptor (CAR).

10. The immune cell of claim 9, wherein the CAR targets CD19 (cluster of differentiation 19) ("CD19 CAR").

11. The immune cell of claim 10, wherein the CD19 CAR comprises the mRNA sequence of SEQ ID NO: 6 or SEQ ID NO:

8.

12. The immune cell of claim 10, wherein the CD19 CAR comprises the protein sequence of SEQ ID NO: 7 or SEQ ID NO:

9.

13. The expression of genes or proteins in the AP-1 signaling pathway in immune cells containing exogenous cargo is reduced by approximately -3 log compared to control immune cells. 2 The immune cells of claim 1, wherein the fold change is

14. The expression of genes or proteins in the AP-1 signaling pathway in immune cells containing exogenous cargo is reduced by approximately -2 log compared to control immune cells. 2 The immune cells of claim 1, wherein the fold change is

15. The expression of genes or proteins in the AP-1 signaling pathway in immune cells containing exogenous cargo is reduced by approximately -1 log compared to control immune cells. 2 The immune cells of claim 1, wherein the fold change is

16. The immune cell of claim 1, comprising at least two types of exogenous cargo.

17. The immune cell of claim 1, wherein the immune cell comprising the exogenous cargo does not exhibit a T cell exhaustion or T cell anergy phenotype.

18. The immune cell of claim 1, wherein the immune cell comprising the exogenous cargo comprises an unstimulated immune cell.

19. within 3 logs of the level of immune cells containing exogenous cargo that have not undergone the cell engineering process. 2 Immune cells secreting at least one cytokine at the fold change level.

20. Within 2 logs of levels in immune cells that have not undergone the cell manipulation process 2 20. The immune cell of claim 19, wherein the immune cell secretes at least one cytokine at a fold change level.

21. Within 1 log of levels in immune cells that have not undergone the cell manipulation process 2 20. The immune cell of claim 19, wherein the immune cell secretes at least one cytokine at a fold change level.

22. The immune cell of claim 19, wherein the cytokine comprises human IL-2 (interleukin 2) comprising the nucleic acid sequence of SEQ ID NO: 17, human IL-8 (interleukin 8) comprising the nucleic acid sequence of SEQ ID NO: 18, or a combination thereof.

23. 20. The immune cell of claim 19, wherein the cytokine comprises IFN-γ (interferon gamma), IL-2 (interleukin 2), TNFα (tumor necrosis factor alpha), IL-8 (interleukin 8), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10 (interleukin 10), MIP-1α (macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β), IL-17A (interleukin 17A), fractalkine, or ITAC (interferon-inducible T-cell alpha chemoattractant).

24. 1. A method for delivering exogenous cargo across the plasma membrane of a non-adherent immune cell, comprising: providing a population of non-adherent cells; and contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.2 percent (v / v), wherein the immune function of the non-adherent immune cells comprises a phenotype of cells that have not undergone a cell manipulation process, the immune function being selected from (i) cytokine release; (ii) gene expression; and (iii) metabolic rate. A method comprising:

25. 25. The method of claim 24, wherein the alcohol is greater than a 0.5 percent (v / v) concentration.

26. 25. The method of claim 24, wherein the alcohol is greater than a 2 percent (v / v) concentration.

27. 25. The method of claim 24, wherein the alcohol is greater than a 5 percent (v / v) concentration.

28. 25. The method of claim 24, wherein the alcohol is greater than a 10 percent (v / v) concentration.

29. 25. The method of claim 24, wherein the immune cells are not activated prior to cargo delivery.

30. 25. The method of claim 24, wherein the immune cells have not been contacted with a ligand for CD3, CD28, or a combination thereof prior to contacting the immune cells with the exogenous cargo.

31. 25. The method of claim 24, further comprising at least two exogenous cargoes.

32. 32. The method of claim 31, wherein the at least two exogenous cargoes are simultaneous.

33. 32. The method of claim 31, wherein the at least two exogenous cargoes are delivered sequentially.

34. 1. A method for delivering at least two exogenous cargoes across the plasma membrane of a non-adherent immune cell, comprising: providing a population of non-adherent cells; and (i) contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v); (ii) using at least two intracellular delivery methods selected from viral transduction, (iii) electroporation, or (iv) nucleofection; and thereby delivering two types of exogenous cargo to immune cells. A method comprising:

35. Intracellular delivery methods include: contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising an exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v); and subsequently, viral transduction.

35. The method of claim 34, comprising:

36. Intracellular delivery methods include: Following viral transduction, contacting the population of cells with a volume of an isotonic aqueous solution, the aqueous solution comprising the exogenous cargo and an alcohol at a concentration greater than 0.5 percent (v / v).

35. The method of claim 34, comprising:

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