Patient-specific induced pluripotent stem cell-derived macrophages for cell therapy

Abstract

This specification describes compositions, pharmaceutical compositions, methods, administration methods, and kits related to cell therapy for cancer, particularly glioblastoma. In certain embodiments, cells can be isolated from a patient, reprogrammed to a pluripotent state using the methods described herein, and then differentiated into cells having a macrophage or macrophage-like phenotype. The differentiated (or terminally differentiated) cells can then be re-administered to a patient in need, for example, a patient with cancer (e.g., glioblastoma). In other embodiments, the reprogrammed cells may be derived from the patient's blood, plasma, tissue (e.g., periosteal sample), or other cell source. In other embodiments, terminally differentiated cells can also be genetically modified for self-destruction or for the production (or enhanced production) of exogenous (or endogenous) cytokines, growth factors, or other peptide therapeutic agents.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 63 / 495,953, filed on April 13, 2023. The content of the same application is incorporated herein by reference as if fully set forth herein.

[0002] Sequence Listing An official copy of the sequence listing is electronically submitted through the Patent Center as a sequence listing in XML format, in a file named MDA22_120_1424935.xml, created on April 12, 2024, and having a size of 82 kb. The sequence listing contained in this XML-formatted document is part of this specification and is incorporated herein by reference in its entirety.

Background Art

[0003] Immunotherapies, including checkpoint inhibitors and chimeric antigen receptor (CAR) T cell therapies, have each achieved success in some cancers such as leukemia. However, these therapies have not been effective against other cancers, such as nervous system cancers like glioblastoma multiforme (GBM) in clinical trials. Different from CAR T cells, genetically engineered macrophages (GEMs) may be more effective against solid cancers considering their innate migratory ability to myeloid-enriched tumor microenvironments, persistence in immunosuppressive tumor microenvironments, resistance to tumor polarization signals, lack of antigen-specific antitumor strategies that inhibit tumor resistance due to antigen drift, phagocytic ability, and antigen presentation to T cells via major histocompatibility antigen complex (MHC) class I / II. Although GEMs offer significant advantages over CAR T cells, the generation of GEMs for clinical applications remains a technical challenge.

Summary of the Invention

[0004] This summary is provided to introduce some of the concepts that will be explained in more detail in the detailed description. This summary is not intended to identify the main or essential features of the subject matter described in the claims, nor is it intended to be used as an aid to limit the scope of the subject matter described in the claims.

[0005] This specification describes compositions and methods relating to patient-specific macrophage therapy. In certain embodiments, this specification describes a method for generating patient-specific macrophages, the method comprising reprogramming one or more somatic cells from a sample derived from a subject to a pluripotent state, culturing one or more reprogrammed cells, and differentiating one or more reprogrammed cells to a macrophage-like phenotype, the differentiation proceeding without embryoid body formation. Reprogramming may include delivering one or more somatic cells one or more reprogramming expression vectors containing nucleic acids encoding OCT3 / 4, SOX2, KFL4, L-MYC, and LIN28, as well as shRNA for p53. In certain embodiments, at least one of the one or more expression vectors may be an episomal expression vector. In certain embodiments, expansion culture may include expanding the culture of one or more reprogrammed cells for about 10 to about 25 passages. In certain embodiments, differentiation may include removing b-FGF from the culture medium and gradually adding IL-3 and M-CSF over a period of time. In certain embodiments, IL-3 may be added at a concentration of approximately 20 ng / ml to approximately 30 ng / ml. In certain embodiments, M-CSF may be added at a concentration of approximately 45 ng / ml to approximately 55 ng / ml. In certain embodiments, the duration may be approximately 15 to approximately 20 days. In certain embodiments, the subject may be a subject having or suspected of having glioblastoma. In certain embodiments, the sample may include the cranial membrane (also referred to herein as “periosteum”) from the subject. In certain embodiments, the sample may be enriched with periosteum-derived progenitor cells (PDPCs). In certain embodiments, the method may include, before reprogramming, the steps of subdividing the sample after sampling, culturing the subdivided sample for a certain period of time, and isolating one or more somatic cells from the subdivided sample. In certain embodiments, the method described herein may further include, after differentiation, the step of delivering one or more treatment expression vectors to one or more differentiated cells.One or more treatment expression vectors contain therapeutic nucleic acids, each independently encoding a cytokine, checkpoint protein, or self-destructive protein. In certain embodiments, the cytokine may be interleukin-12 (IL-12), IFN-γ, or IL-15, or any combination thereof. In certain embodiments, the cytokine is interleukin-12 (IL-12). In certain embodiments, the checkpoint protein may include one or more of PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. In certain embodiments, the self-destructive protein may be thymidine kinase. In certain embodiments, the expression vector may contain nucleic acids encoding proteins usable as markers for tracking cells after transplantation, such as enhanced green fluorescent protein or CD-19. In certain embodiments, the expression vector may be an inducible vector that expresses cytokines, checkpoint proteins, or self-destructive proteins only in the presence of an inducer (e.g., a doxycycline induction system).

[0006] Furthermore, this specification also describes modified cells and pharmaceutical compositions, including cells produced by the methods described herein.

[0007] Furthermore, this specification also describes methods for treating subjects in need of treatment. In certain embodiments, a method for treating subjects in need of treatment comprises administering one or more differentiated cells described herein to a subject in need of treatment. In certain embodiments, a subject in need of treatment is a subject having or suspected of having glioblastoma, neuroblastoma, or melanoma. In certain embodiments, a subject in need of treatment may be a subject having or suspected of having glioblastoma. In certain embodiments, administration may be intravenous or intra-arterial. In certain embodiments, administration is intra-arterial. [Brief explanation of the drawing]

[0008] This application includes the following figures. These figures are intended to illustrate specific embodiments and / or features of the compositions and methods and to supplement the description of the compositions and methods. The figures do not limit the scope of the compositions and methods unless expressly indicated so in the specification.

[0009] [Figure 1] Figure 1 is a schematic diagram illustrating one aspect of personalized cell therapy according to this disclosure, showing (1) reprogramming, (2) preclinical validation studies (flow cytometry, immunohistochemistry), and (3) clinical administration of MDA Hi-Mac.

[0010] [Figure 2] Figures 2A-2B show flow cytometry data indicating that macrophages reprogrammed from induced pluripotent stem cells (i.e., HiMac) express the standard macrophage marker CD68 and have very low expression rates of tumor-promoting markers (CD206+ and CD163+, 0.07% of the total population). The immunophenotype of macrophages derived from hiPSC strains was characterized by multicolor flow cytometry (Fortessa X-20). To distinguish between live and dead cells, live-and-dead fixable aqua staining was performed first. CD14+CD68+ macrophages were identified using the negative exclusion gating strategy previously reported by our group. CD66b+ neutrophils were excluded, and then T cells, B cells, and NK cells were excluded using a lineage (Lin) cocktail containing mAbs against CD3, CD19, CD20, and CD56, respectively (Lin->60%; Figure 2A). Further characterization revealed that up to 75% of Lin- cells expressed CD68+, a panmacrophage / phagocytic cell marker (Figure 2A). More than 97% of CD68+ macrophages lacked the classic tumor-promoting M2 macrophage marker CD206-CD163-, indicating that they were primarily M1 pro-inflammatory / antitumor macrophages (Figure 2B).

[0011] [Figure 3]Figures 3A-3B are photographs demonstrating that the HiMac described herein can be subjected to viral transduction (e.g., adenovirus transduction with delta-24RGD oncolytic virus expressing GFP). Antihexone staining was also performed to show viral replication within the HiMac.

[0012] [Figure 4] Figure 4 shows an embodiment of an expression cassette that can be introduced into HiMac. Such a construct enables HiMac (as soon as it is introduced into cells by methods such as transfection, transduction, or nucleofection) to express cytokines (such as IL-12) and a "kill switch" (in embodiment, a CD163 or CD206 self-driven HSV / TK kill switch, e.g., SEQ ID NO: 5) that causes the cell to self-destruct before evolving into a pro-tumor phenotype, and can be monitored in accordance with current FDA guidelines (<5 copies).

[0013] [Figure 5] Figure 5 is a 5x phase-contrast micrograph of cranial progenitor cells cultured from the cranial membrane of a brain tumor patient undergoing glioblastoma resection surgery.

[0014] [Figure 6] Figure 6 shows first-generation IL-12 constructs exhibiting Dox-induced IL-12 expression, as well as CD19t and HSV-TK expression (top panel). ELISA (bottom left) and Western blot (bottom right) results for IL-12 expression in HEK 293 T cells only after Dox treatment are also shown.

[0015] [Figure 7]Figure 7 shows schematic diagrams of a second-generation IL-12 construct constitutively expressing CD19t and HSV / TK (neomycin resistance genes, upper panel, e.g., SEQ ID NOs: 18 and 20 for mouse CD19t, and respectively for human CD19t) and another construct with doxycycline (Dox)-induced IL-12 expression (puromycin resistance genes, lower panel, e.g., SEQ ID NOs: 19 and 21 for mouse IL-12, and respectively for human IL-12). The second generation improves upon the safety of the first generation by enabling constitutive expression of genes that can be used to identify and eliminate iMac as needed, while simultaneously suppressing toxicity by maintaining controlled IL-12 expression using doxycycline. [Modes for carrying out the invention]

[0016] Detailed explanation The following description enumerates various aspects and embodiments of the composition and method. No particular embodiment is intended to define the scope of the composition and method. Rather, the embodiments merely illustrate non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed composition and method. This description should be interpreted from the perspective of those skilled in the art, and therefore does not necessarily contain information that is well known to those skilled in the art.

[0017] I. Terminology Unless otherwise defined, all technical terms, notations, and other scientific or medical terms used herein are intended to have meanings generally understood by those skilled in the art. In some cases, terms having generally understood meanings are defined herein for clarity and / or easy reference, and the inclusion of such definitions herein should not be construed as substantially different from the definitions of terms generally understood in the art.

[0018] As used herein, the articles "a" and "an" are used to refer to one or more of the grammatical objects thereof (i.e., at least one). For example, "an element" means at least one element and can include two or more elements.

[0019] As used herein, the terms "including", "comprising", or "having" and variations thereof mean including the elements listed thereafter and their equivalents, as well as additional elements. Embodiments described with a particular element "including", "comprising", or "having" are also considered "consisting essentially of" and "consisting of those certain elements". As used herein, "and / or" refers to all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted alternatively ("or"), and includes.

[0020] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be interpreted as including the recited materials or steps, as well as those that do not materially affect the basic and novel features or characteristics of the claims of the present disclosure. See, e.g., In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in original). See also MPEP§2111.03. Thus, as used herein, the term "consisting essentially of" should not be interpreted as synonymous with "comprising".

[0021] The description of a range of values in this specification is, unless otherwise specified herein, intended only as a shorthand way of referring individually to each separate value that falls within the range, and each separate value is incorporated herein as if it were individually recited herein. For example, if a concentration range is described as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3% are explicitly recited herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values between (and including) the recited minimum and maximum values are considered to be explicitly recited in this disclosure.

[0022] As used herein, the terms “about” and “approximately” generally mean an acceptable degree of error for the measured quantity, taking into account the nature or precision of the measurement. Exemplary degrees of error are within 20%, preferably within 10%. More preferably, within 5% of a given value or range of values. A reference to “about X” or “approximately X” specifically indicates values of at least X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, an expression such as “about X” or “approximately X” is intended to teach and provide written support for a claim limitation such as “0.98X”. Numerical values presented herein are approximate values unless otherwise specified, meaning that the term “about” or “approximately” may be inferred even if not explicitly stated. When “about” is applied to the beginning of a numerical range, it is applied to both ends of the range.

[0023] When used throughout this specification, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotide,” or other grammatically synonymous terms mean that at least two nucleotides (either deoxyribonucleotides or ribonucleotides) or analogs thereof are covalently linked. Polynucleotides are polymers of any length, such as 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. The polynucleotides described herein generally contain phosphodiester bonds, but may in some cases have at least one different bond (e.g., a phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bond), as well as peptide nucleic acid backbones and bonds. Mixtures of naturally occurring polynucleotides and analogs, or mixtures of different polynucleotide analogs, or mixtures of naturally occurring polynucleotides and analogs may also be prepared. Non-limiting examples of polynucleotides include genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, etc. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. If modified nucleotides are present, modifications to the nucleotide structure may be introduced before or after the assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by binding with labeling components. The term includes both double-stranded and single-stranded molecules. Unless otherwise specified or required, the term “polynucleotide” encompasses both the double-stranded form and two complementary single-stranded forms known or expected to constitute the double-stranded form.A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) (or uracil instead of thymine if the polynucleotide is RNA). Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. Unless otherwise specified, a particular polynucleotide sequence implicitly includes, in addition to the explicitly stated sequence, its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more (or all) selected codons is replaced with a mixed base and / or deoxyinosine residue.

[0024] Unless otherwise stated, a given nucleic acid sequence implicitly includes, in addition to the explicitly indicated sequence, its conservatively modified variants, alleles, homologous genes, SNPs, and complementary sequences.

[0025] The terms “transfection,” “transfer,” “to transfect,” or “to transfect” are interchangeable and are defined as the process of introducing nucleic acid molecules or proteins into cells. Nucleic acids are introduced into cells using non-viral or viral methods. Nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral transfection methods include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, heat shock transfection, magnetiffection, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation according to standard procedures well known in the art. For viral transfection methods, any useful viral vector may be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, and adeno-associated virus vectors. In some embodiments, nucleic acid molecules are introduced into cells using retroviral vectors according to standard procedures well known in the art. The term “transfection” also refers to the introduction of proteins into cells from an external environment. Typically, protein transfection or transfection relies on a peptide or protein that can cross the cell membrane attaching to the protein of interest. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.Examples of vector skeletons, in addition to other vector properties (i.e., Kozak sequences (e.g., GACACC), precoding sequences, signal peptides, cleavage domains (e.g., P2A domains), linkers, secretory signals, promoters, and induction systems), are described at least in the informal sequence listing on the last page of this application.

[0026] Where used herein in reference to genes, the terms “expressed” or “expressed” mean the transcript and / or translation product of the gene. The expression level of a DNA molecule in a cell may be determined based on the amount of corresponding mRNA present in the cell or the amount of the DNA-encoded protein produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0027] The expression of a transfected or transduced gene ("transfected" meaning a gene introduced into a cell by a viral vector, such as lentiviral infection) is transient or stable within the cell. During "transient expression," the transfected or transduced gene does not move to daughter cells during cell division. Because expression is limited to the transfected or transduced cell, gene expression is lost over time. In contrast, "stable" expression of a transfected or transduced gene can occur when the gene is co-transfected with another gene that gives the transfected or transduced cell a selective advantage. Such a selective advantage may be resistance to a particular toxin presented to the cell. The expression of a transfected or transduced gene can also be achieved by transposon-mediated insertion into the host genome. During transposon-mediated insertion, the gene is positioned predictably between two transposon linker sequences, allowing for insertion into the host genome and subsequent removal. "Inducible" expression means that the transfected or introduced gene is expressed only under specific conditions, and can be switched on and off in the presence of an introduction agent such as tetracycline or doxycycline.

[0028] The term "plasmid" refers to a nucleic acid molecule that encodes a gene and / or a regulator necessary for gene expression. Gene expression from a plasmid can occur in cis or trans configuration. When a gene is expressed in cis, the gene and its regulator are encoded by the same plasmid. Trans expression refers to the case where the gene and its regulator are encoded by separate plasmids.

[0029] The term "episome" refers to the extrachromosomal state of a plasmid within a cell. Episomal plasmids are nucleic acid molecules that replicate independently of chromosomal DNA, and are not part of chromosomal DNA.

[0030] The term "exogenous" refers to molecules or substances (e.g., nucleic acids or proteins) that originate from outside a particular cell or organism. Conversely, the term "endogenous" refers to molecules or substances that are specific to a particular cell or organism, or that originate from within that particular cell or organism.

[0031] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted and introduced into a host cell. In some embodiments, the vectors used in accordance with this disclosure are vectors capable of autonomous replication and / or expression of ligated nucleic acids. A vector capable of inducing the expression of a functionally ligated gene is referred to herein as an "expression vector." Thus, an "expression vector" is a special type of vector that contains the regulatory region necessary for the expression of a target gene in a host cell. In some embodiments, the target gene is functionally ligated to another sequence within the vector, such as a promoter. Vectors include plasmids and non-viral vectors such as viral vectors.

[0032] A "viral vector" is a nucleic acid derived from a virus that can transport another nucleic acid into a cell. When present in the right environment, a viral vector can induce the expression of a protein encoded by one or more genes contained within the vector. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, lentiviruses, and adeno-associated virus vectors.

[0033] The term "functionally linked" refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, where the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Functionally linked nucleic acid sequences do not need to be physically adjacent to each other. The term "functionally linked" also refers to a functional linkage between a nucleic acid expression regulatory sequence (such as a promoter or an array of transcription factor binding sites) and a transcribable nucleic acid sequence, in which case the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the transcribable sequence.

[0034] The terms “regulatory sequence” and “promoter” are used interchangeably herein and refer to nucleic acid sequences that induce or control the transcription of protein-coding sequences to which they are functionally linked, such as start signals, enhancers, and promoters. In some cases, the transcription of recombinant genes is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in the cell type in which expression is intended. It will also be understood that recombinant genes may be under the control of transcriptional regulatory sequences that are identical or different to the sequences that control the transcription of native proteins. In some cases, promoter sequences are recognized by the cell’s synthetic mechanisms, or introduced synthetic mechanisms, which are necessary to initiate the transcription of a particular gene.

[0035] An "expression cassette" refers to a polynucleotide containing a promoter or other regulatory sequence operably ligated to a protein-coding sequence.

[0036] The term "siRNA" refers to a nucleic acid that forms a double-stranded RNA. This double-stranded RNA has the ability to reduce or inhibit the expression of a gene or target gene when the siRNA is expressed in the same cell as the gene or target gene. In the context of this disclosure, the term "siRNA" includes miRNA. Therefore, "siRNA" refers to a double-stranded RNA formed by a complementary strand. The complementary portion of an siRNA that hybridizes to form a double-stranded molecule typically has substantial or complete identity. In one embodiment, siRNA refers to a nucleic acid that has substantial or complete identity with the target gene and forms a double-stranded siRNA. The sequence of the siRNA may correspond to the full length or a partial sequence of the target gene. Typically, siRNA is at least about 15–50 nucleotides long (for example, each complementary sequence in a double-stranded siRNA is 15–50 nucleotides long, and the length of a double-stranded siRNA is about 15–50 base pairs, preferably about 20–30 base pairs, preferably about 20–25 nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).

[0037] The term "shRNA" generally refers to siRNA introduced into cells as part of a larger DNA construct. Typically, such constructs enable the stable expression of siRNA within the cell after introduction, for example, by incorporating the construct into the host genome.

[0038] An "antisense" oligonucleotide or polynucleotide is a nucleotide sequence that is substantially complementary to a target polynucleotide or a portion thereof and has the ability to specifically hybridize with the target polynucleotide.

[0039] Ribozymes are enzyme RNA molecules capable of catalyzing the specific cleavage of RNA. The composition of a ribozyme molecule preferably includes one or more sequences complementary to the target mRNA and a well-known catalytic sequence or a functionally equivalent sequence responsible for mRNA cleavage (see, for example, U.S. Patent No. 5,093,246, which is incorporated herein by reference in its entirety). Ribozyme molecules designed to catalytically cleave a target mRNA transcript may also be used to inhibit the translation of the target mRNA in question.

[0040] In this specification, the terms “polypeptide” and “peptide” are used interchangeably to refer to polymers in which amino acid residues are linked together in a single chain. These terms apply to the following amino acid polymers: those in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as natural and non-natural amino acid polymers. Amino acid polymers may contain only L-amino acids, only D-amino acids, or mixtures of L-amino acids and D-amino acids. As used herein, the term “protein” refers to a polypeptide, or a dimer (i.e., two) or polymer (i.e., three or more) of a single-chain polypeptide. The single-chain polypeptides of a protein may be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions. The terms “part” and “fragment” are used interchangeably in this specification to refer to a part of a polypeptide, nucleic acid, or other molecular structure.

[0041] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids include not only those encoded by the genetic code, but also later modified amino acids such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids, such as hydrogen, carboxyl groups, amino groups, and α-carbons bonded to R groups, for example, homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide skeletons, but retain the same basic chemical structure as natural amino acids. Amino acid mimes are compounds that have a structure different from the general chemical structure of amino acids, but function similarly to natural amino acids.

[0042] In this specification, amino acids may be represented by either a commonly known three-letter code or a single-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be represented by a commonly accepted single-letter code.

[0043] In the context of peptides, the term "amino acid sequence recombination" refers to a change or mutation in the amino acid sequence of a reference peptide, where the biological properties of the reference peptide are maintained after the change in amino acid sequence. For example, amino acid sequence recombination may be a conservative amino acid substitution or an amino acid sequence modification (addition, deletion, or substitution) to generate a chimeric peptide.

[0044] The amino acids in the polypeptides described herein may be any of the 20 natural amino acids, D-stereoisomers of natural amino acids, unnatural amino acids, or chemically modified amino acids. Unnatural amino acids (i.e., amino acids that do not naturally exist in proteins) are also known in the art and are described, for example, in Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581-87 (2013); Xie et al. “Adding amino acids to the genetic repertoire,” Curr. Opin. Chem. Biol. 9(6): 548-54 (2005); and all references cited therein. Beta-amino acids and gamma-amino acids are also known in the art and are considered unnatural amino acids herein.

[0045] In this specification, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, the side chain may be modified to include a signaling site such as a phosphor or radiolabel. The side chain may also be modified to include a novel functional group such as a thiol group, a carboxylic acid group, or an amino group. Post-translational modified amino acids are also included in the definition of a chemically modified amino acid.

[0046] As used herein in the context of polynucleotide or polypeptide sequences, the terms “identity” or “substantial identity” refer to a sequence having at least 60% sequence identity with a reference sequence. Alternatively, the identity percentage can be any integer from 60% to 100%. Exemplary embodiments include, when compared with a reference sequence using the programming described herein, preferably BLAST with the standard parameters described below, at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Those skilled in the art will understand that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame position, etc.

[0047] In sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, subsequence coordinates are specified as needed, and sequence algorithm programming parameters are specified. Either default programming parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the relative sequence identity percentage between the test sequence and the reference sequence based on the programming parameters.

[0048] As used herein, the “comparison window” includes a reference to one segment of a number of consecutive positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, where two sequences may be optimally aligned in the segment and then compared with a reference sequence having the same number of consecutive positions. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be performed by the local homology algorithm of Smith & Waterman Add. APL. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), computer implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0049] Suitable algorithms for determining percent sequence identity and sequence similarity are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analysis is available on the National Center for Biotechnology Information (NCBI) website. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. These words either satisfy or match a positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighbor word score threshold (Altschul et al. (1977)). These initial neighbor word hits serve as a seed to initiate a search for longer HSPs containing them. Word hits are then extended bidirectionally along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The expansion of word hits in each direction stops when the cumulative alignment score decreases by X from the maximum value, when the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of alignment. BLASTN programming (for nucleotide sequences) uses a default word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, BLASTP programming uses a default word size (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0050] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which is an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.01, more preferably less than about 10⁻⁵, and most preferably less than about 10⁻², the nucleic acid is considered similar to the reference sequence.

[0051] The term "library" is used, in accordance with common usage in the relevant technical field, to refer to a collection of molecules that are, if desired, organized and / or cataloged so that their individual members can be identified. Libraries include, but are not limited to, combinatorial chemical libraries, natural product libraries, and peptide libraries.

[0052] When referring to cells or biological samples, the term "derived from" indicates that the cells or sample were obtained from a source described at a given time. For example, cells derived from an organism may refer to primary cells obtained directly from that organism (i.e., unmodified), or they may refer to cells that have been modified through the introduction of recombinant vectors, culture under specific conditions, immortalization, etc. In some cases, cells derived from a particular source may undergo cell division and / or differentiation, and the original cells may no longer exist, but the remaining cells are understood to have originated from the same source.

[0053] In the context of cells, the term "allogeneic" refers to donor cells introduced into a recipient that is not genetically identical to the donor.

[0054] In the context of cells, the term "autologous" refers to donor cells introduced into a recipient that is genetically identical to the donor.

[0055] A "cell culture" is a population of cells that exist outside the body of an organism. If desired, these cells may be primary cells isolated from a cell bank, animal bank, or blood bank, or secondary cells obtained from any of these sources and immortalized over a long period in in vitro culture.

[0056] When referring to cell culture itself or the culture process, terms such as “culturing,” “growing,” “maintaining,” “broadening,” and “broadening” can be used interchangeably because they mean that cells are maintained in a state suitable for survival outside the body (e.g., outside the body). Cultured cells can survive, and culture can result in cell proliferation, differentiation, or division. The terms do not mean that all cells in a culture will survive, proliferate, or divide; some cells may spontaneously age, for example. Cells are typically cultured in a modifiable medium during the culture process.

[0057] The terms “culture medium” and “culture solution” refer to the cell culture environment. Culture mediums are typically isotonic and can be liquid, gelatinous, or semi-solid, for example, to provide a matrix for cell adhesion or support. Culture mediums as used herein may contain components for the nutritional, chemical, and structural support necessary for culturing cells.

[0058] In the context of cells or tissues (e.g., normal cells, cancer cells, or cancer stem cells), the term “marker” means any gene product, such as non-coding RNA (non-messenger RNA), mRNA and polypeptides, antigens, molecules, or other chemical or biological entities that are specifically present within or on the cells of interest and can be used to identify cells affected by disease or injury.

[0059] When referring to cellular markers such as leukemia stem cell markers, the term "expression level" refers to the measurable amount of gene product produced by a gene in a patient's sample, and the gene product may be a transcript or a translated transcript. Therefore, expression levels can relate to nucleic acid gene products such as RNA and cDNA, or polypeptides. Expression levels originate from biological samples, control samples, cell culture samples, and / or control samples, and may, for example, be newly detected or consistent with previous measurement results.

[0060] The terms “detecting expression levels” or “expression levels are detected” as used in relation to genes mean applying a method to quantitatively, semi-quantitatively, or qualitatively determine the amount of gene expression products, such as RNA, mRNA, or polypeptide products, to a sample, such as a target sample, a biological sample, a cell culture sample, and a control sample. For example, gene expression levels can be measured by a variety of methods, including, but not limited to, array methods, other hybridization methods, and PCR protocols. In some cases, PCR methods include probes, primers, or primer sets used to determine the amount of nucleic acid in a gene. For example, gene expression levels can be measured using a probe set for a specific gene described herein, or one or more probes contained in a probe set. Furthermore, if multiple probe sets exist, they can also be used to measure gene expression levels.

[0061] Other methods for detecting the amount of gene expression products present in a sample include Nanostrin® technology, sequential gene expression analysis (SAGE), RNA sequencing, RNase protection assays, and Northern blotting. Polypeptide levels can be measured by immunoassays such as Western blotting, flow cytometry, immunohistochemistry, ELISA, and immunoprecipitation. In these methods, a gene or gene signature detector (e.g., a labeled antibody) specifically binds to the polypeptide product encoded by the gene, allowing for the determination of the relative or absolute amount of polypeptide in the sample.

[0062] When applied to proteins, the term “isolated” means that the protein essentially does not contain other cellular components associated with it in its native state. The protein may be in either a dry solution or an aqueous solution, but it is preferably in a homogeneous state. Purity and homogeneity are usually measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, which is the main species present in the preparation, is substantially purified. The term “purified” means that the protein essentially produces one band on the electrophoretic gel. In particular, it means that the purity of the protein is at least 85%, more preferably at least 95%, and most preferably at least 99%.

[0063] "Contact" is used according to its obvious and ordinary meaning and refers to a process that allows at least two different species (e.g., compounds containing biomolecules, or cells) to come into close enough proximity to react, interact, or physically come into contact. The two species may be cells as described herein (e.g., transfected non-totipotent cells, non-totipotent cells, totipotent cells) and inhibitors as described herein (e.g., zygote-specific gene repressor inhibitors, growth factors). In some embodiments, contact may involve transfected non-totipotent cells as described herein and zygote-specific gene repressor inhibitors. In other embodiments, contact may involve non-totipotent cells as described herein and zygote-specific gene repressor inhibitors. However, it should be understood that the reaction product obtained may be produced directly from the reaction between the added reagents or from intermediates from one or more of the added reagents that may be produced in the reaction mixture.

[0064] As used herein, “somatic cells” refers to cells that are differentiated or partially differentiated compared to embryonic stem cells. Therefore, the term includes, for example, cells such as fibroblasts derived from embryonic stem cells but differentiated. Somatic cells include cells that make up the organs, skin, blood, bone, and connective tissue of an organism, but do not include germ cells.

[0065] "Primary cells" are cells that are directly collected from living tissue (e.g., by biopsy) and established for in vitro growth. Such cells may represent the major functional components of the tissue from which they originate. Types of primary cells include, but are not limited to, fibroblasts (including mouse embryonic fibroblasts (MEFs)), keratinocytes, melanocytes, myoblasts, mesenchymal cells, endothelial cells, epithelial cells, adipocytes, and stromal cells.

[0066] Stem cells are cells characterized by their ability to self-replicate through mitosis and differentiate into tissues or organs. Mammalian stem cells are classified into embryonic stem cells and somatic stem cells. Embryonic stem cells exist in the blastocyst and give rise to embryonic tissue, while somatic stem cells exist in adult tissue and are intended for tissue regeneration and repair.

[0067] The term "reprogramming" refers to the process of dedifferentiating non-pluripotent or non-totipotent cells (typically somatic cells) into cells that exhibit the characteristics of pluripotent or totipotent stem cells.

[0068] "Self-renewal" refers to the ability of a cell to divide and produce at least one daughter cell that possesses the self-renewal properties of the parent cell. The second daughter cell may be determined to differentiate into a specific differentiation pathway. For example, a hematopoietic stem cell with self-renewal ability can divide to form one daughter stem cell and another daughter cell determined to differentiate into a myeloid or lymphoid pathway. The determined progenitor cell typically loses its self-renewal ability and, through cell division, produces two daughter cells exhibiting a more differentiated (i.e., limited) phenotype. Non-self-renewing cells refer to cells that divide and produce daughter cells. Neither cell possesses the differentiation ability of the parent cell type and instead produces differentiated daughter cells.

[0069] The term "pluripotency" refers to cells that, under appropriate conditions, have the ability to produce offspring that can differentiate into cell types exhibiting a comprehensive set of characteristics related to the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to the tissues of prenatal, postnatal, or adult organisms. Standard tests recognized in the art, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population. However, the identification of various characteristics of pluripotent stem cells can also be used to identify pluripotent cells.

[0070] "Pluripotent stem cell characteristics" refer to the cellular properties that distinguish pluripotent stem cells from other cells. The expression or non-expression of specific combinations of molecular markers is an example of pluripotent stem cell characteristics. More specifically, human pluripotent stem cells may express at least some, and preferably all, of the markers included in the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Lin28, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells.

[0071] "Induced pluripotent stem cells" refer to pluripotent stem cells artificially induced from non-pluripotent cells. Non-pluripotent cells may be cells with lower self-renewal and differentiation capabilities than pluripotent stem cells. Cells with low differentiation capabilities include, but are not limited to, somatic stem cells, tissue-specific progenitor cells, primary cultured cells, or secondary cultured cells.

[0072] The term "embryonic stem cell" is used to refer to pluripotent stem cells contained within the inner cell mass of a blastocyst (see U.S. Patents 5,843,780 and 6,200,806). Characteristic properties of embryonic stem cells define their phenotype. Therefore, if a cell possesses one or more unique characteristics of an embryonic stem cell, distinguishing it from other cells, then that cell has the embryonic stem cell phenotype. Examples of characteristic embryonic stem cell properties include, but are not limited to, gene expression profiles, proliferative capacity, differentiation capacity, normal karyotype, and responsiveness to specific culture conditions.

[0073] The term "exogenous" refers to substances present in cells or organisms that are not of their original origin. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to nucleic acids or proteins introduced through artificial processes into a biological system, such as cells or organisms, where they are not normally present or are only present in small amounts. If a substance is introduced into a cell that inherits it or into the ancestors of that cell, that substance is considered exogenous. On the other hand, the term "endogenous" refers to substances that are naturally present in a biological system.

[0074] As used herein, the term “isolated cells” refers to cells removed from an organism that originally existed, or to the descendants of such cells. “Isolated” cells may be cultured in vitro in the presence of other cells. If desired, the cells may be subsequently introduced into another organism, or the cells (or their ancestral cells) may be reintroduced into the organism from which they were isolated.

[0075] In this specification, “proliferation” and “growth” refer to an increase in the number of cells in a cell population through cell division (proliferation). Cell proliferation is generally understood to occur through the coordinated activation of multiple signaling pathways in response to an environment containing growth factors and other mitogens. Cell proliferation may also be promoted by liberation from the action of intracellular or extracellular signals or mechanisms that inhibit or inhibit cell proliferation.

[0076] An "Oct polypeptide" refers to any of the following: a natural member of the octamer transcription factor family, a variant thereof that maintains transcription factor activity (at least 50%, 80%, or 90% of activity) compared to the nearest natural family member, or a polypeptide that contains at least the DNA-binding domain of a natural family member and may also contain a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. For example, Oct3 / 4 (referred to herein as "Oct4") contains a POU domain, a 150-amino acid sequence conserved among Pit-1, Oct-1, Oct-2, and uric-86. See Ryan, AK & Rosenfeld, MG Genes Dev. 11, 1207-1225 (1997). In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity across its entire sequence compared to those listed above, or to naturally occurring Oct polypeptide family members such as those listed under GenBank accession numbers NP_002692.2 (human Oct4) or NP_038661.1 (mouse Oct4). The Oct polypeptide (e.g., Oct3 / 4) may be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, a protein from the same species as the cell type being manipulated is used.

[0077] "Klf polypeptide" or "KLF" refers to any of the following: a natural member of the Kruppel-like factor (Klf) family; a zinc finger protein containing an amino acid sequence similar to that of the Drosophila embryonic pattern regulator Kruppel; a variant of a natural member that maintains similar transcription factor activity (at least 50%, 80%, or 90%) compared to the nearest related natural family member; or a polypeptide containing at least a DNA-binding domain of a natural family member, and possibly also containing a transcriptional activation domain. See Dang, DT, Pevsner, J. & Yang, VW. Cell Biol. 32, 1103-1121 (2000). Members of the Klf family include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 have been confirmed to be factors that produce iPS cells in mice, and related genes Klf1 and Klf5 are also known to produce iPS cells, although the efficiency appears to be low. See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In some embodiments, the mutants have at least 85%, 90%, or 95% amino acid sequence identity across their entire sequence compared to those listed above, or naturally occurring Klf polypeptide family members such as those listed under GenBank accession numbers CAX16088 (mouse Klf4) or CAX14962 (human Klf4). The Klf polypeptides (e.g., Klf1, Klf4, and Klf5) may be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins of the same species as the cell type being manipulated are used.

[0078] "Myc polypeptide" or "Myc" refers to a polypeptide that contains at least a DNA-binding domain of a naturally occurring member of the Myc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. Cell Biol. 6:635-645 (2005)), or a variant thereof that maintains similar transcription factor activity (at least 50%, 80%, or 90%) to the nearest naturally occurring family member, or a polypeptide that may also contain a transcription-activating domain of at least a naturally occurring family member. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity across its entire sequence to a naturally occurring Myc polypeptide family member, such as those listed above or listed under GenBank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) may be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins of the same species as the cells being manipulated are used.

[0079] "Sox polypeptide" or "Sox" refers to a naturally occurring member of the SRY-related HMG box (Sox) transcription factor characterized by the presence of a high-mobility group (HMG) domain, or a variant thereof that maintains equivalent transcription factor activity (at least 50%, 80%, or 90%) compared to the nearest naturally occurring family member, or a polypeptide that contains at least the DNA-binding domain of a naturally occurring family member, and may also contain a transcriptional activation domain. See, for example, Dang, DT, et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000). Exemplary Sox polypeptides include, for example, Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to generate iPS cells with a similar efficiency to Sox2, and the Sox3, Sox15, and Sox18 genes have also been shown to generate iPS cells, although at a slightly lower efficiency than Sox2. See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity across their entire sequence compared to naturally occurring Sox polypeptide family members such as those listed above or those listed under Genbank accession number CAA83435 (human Sox2). The Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) may be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins from the same species as the cell type being manipulated are used.

[0080] The term "p53" generally refers to a protein with a molecular weight of approximately 55 kDa on SDS-PAGE that functions as a tumor suppressor element. Protein and nucleic acid sequences of p53 proteins from various species, from humans to Drosophila, are known and publicly available in databases. For example, accession numbers NM_000546, NP_000537, NM_011640, and NP_035770 are available for human and mouse sequences. Mammalian p53 sequences are highly conserved across species. Mouse and human p53 proteins are 85% identical. In humans, p53 is encoded by the TP53 gene located on the short arm of chromosome 17 (17p13.1). The p53 proteins in this disclosure include allelic variants, other functional variants, and homologous genes. In some embodiments, the mutants have at least 85%, at least 90%, or at least 95%, or more, amino acid sequence identity across their entire sequence compared to naturally occurring p53 family members, such as those described with accession number NP_000537 (human p53). Generally, proteins of the same species as the cell species being manipulated are used.

[0081] The term "LIN28" refers to an RNA-binding protein that binds to IGF-2 (insulin-like growth factor 2) mRNA and promotes its translation. Protein and nucleic acid sequences of LIN28 proteins from various organisms, from humans to Drosophila, are known, and human and mouse sequences are available in public databases such as accession numbers AAH28566.1, XP_011540450.1, Q8K3Y3.1, and XP_006539380.1. In the context of this disclosure, LIN28 proteins include allelic variants, other functional variants, and homologous genes. In some embodiments, the variants have at least 85%, at least 90%, or at least 95%, or more, amino acid sequence identity across the entire sequence compared to a native LIN28 family member, such as the one described in accession number AAH28566.1 (human LIN28). In some embodiments, the variant retains the functionality of LIN28 (e.g., at least 60%, 70%, 80%, 90%, or 95% of the activity of LIN28). Generally, a protein of the same species as the cell type being manipulated is used.

[0082] A "control" sample or control value refers to a sample that serves as a reference (usually a known reference) for comparison with a test sample or test condition. For example, a test sample may contain cells exposed to the test condition or test substance, while a control is not exposed to the test condition or test substance (e.g., a negative control). A control may also be a positive control, such as known primary cultured cells or cells exposed to known conditions or substances, for comparison with the test condition. A control may also represent an average value collected from multiple samples, for example, to obtain an average. In therapeutic applications, a sample taken from a patient suspected of having a particular disorder or deficiency may be compared to a sample from a known normal (non-deficient) individual. A control may also represent an average value collected from a population of similar individuals, e.g., patients with a particular deficiency, or healthy individuals with similar medical history, age, weight, etc. Control values ​​may also be obtained from the same individual, e.g., a sample taken before the onset of the disease or deficiency, or before treatment. Those skilled in the art will understand that controls can be designed for the evaluation of any number of parameters.

[0083] The term "biological sample" encompasses a wide variety of samples obtained from living organisms or cell lines. This includes liquid samples of blood and other biological origins, solid tissue samples such as biopsy specimens or tissue cultures, or cells and their offspring derived therefrom. It also includes samples that have been manipulated in any way after acquisition, such as by treatment with reagents, solubilization, or enrichment of specific components. Clinical samples include cells in cell culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples.

[0084] In this specification, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject, particularly human, for whom diagnosis, treatment, or therapy is desired.

[0085] When used in the context of "normal cells," the term "normal" refers to cells exhibiting an untransformed phenotype or the morphology of untransformed cells in the tissue type being examined. "Cancer cells" refer to cancer cells that can be identified, for example, by abnormalities in cell growth or proliferation (e.g., uncontrolled growth or proliferation), cell cycle regulation, cell motility, cell-cell interactions, or metastasis.

[0086] As used herein, the term “clinical health status” refers to a patient’s clinical or physiological health status or state or degree of health. A clinician may assess a patient’s clinical health status by performing a physical examination or one or more tests or assays.

[0087] The terms “inhibitor,” “activator,” and “modulator” of expression or activity are used to refer to inhibitory, activating, or modulating molecules identified by in vitro and in vivo assays for the expression or activity of the described target protein (or the polynucleotide encoding it), respectively. Examples include ligands, agonists, antagonists, and their homologs and mimetic compounds. The term “modulator” includes inhibitors and activators. An inhibitor is a drug that inhibits or binds to the expression of the described target protein, partially or completely blocks the activity of a stimulant or protease inhibitor, reduces, inhibits, delays, inactivates, desensitizes, or downmodulates its activity, such as an antagonist. An activator is, for example, a factor (e.g., an agonist) that induces or activates the expression of the target protein described, or binds to, stimulates, increases, releases, activates, promotes, enhances, enhances, or sensitizes or upmodulates the activation or protease inhibitory activity of the target protein (or the polynucleotide encoding it). Modulators include natural and synthetic ligands, antagonists, and agonists (e.g., small chemical molecules, antibodies, etc., that function as agonists or antagonists). Such assays of inhibitors and activators include, for example, applying a putative modulator compound to cells expressing the target protein described, and then determining the functional effect on the activity of the target protein described, as described above. Samples or assays containing the target protein described treated with a potential activator, inhibitor, or modulator are compared to a control sample without the inhibitor, activator, or modulator to examine the degree of the effect. The control sample (not treated with a modulator) is assigned a relative activity value of 100%. Inhibition of the target proteins described is achieved when the activity level is approximately 80%, preferably 50%, or 25%, 10%, 5%, or 1% compared to the control.Activation of the target proteins described is achieved when the activity level is 110%, preferably 150%, preferably 200%, 300%, 400%, 500%, or 1000–3000% (or more) higher than that of the control.

[0088] The terms “administer,” “deliver,” and “introduce” can be used interchangeably to indicate the introduction of a therapeutic composition or drug (e.g., cells) into the body of a subject. A therapeutic composition or drug may be administered by any suitable means that at least a portion of the composition or drug is delivered to a desired site of the subject, thereby maintaining the therapeutic capacity of the composition or drug. Useful methods for delivering therapeutic agents include, but are not limited to, intravenous delivery, subcutaneous delivery, intradermal delivery, intracoronary delivery, intracardiac delivery, oral delivery, or any combination thereof.

[0089] The term "continuous administration" refers to the continuous delivery of therapeutic drugs (e.g., compounds, molecules, peptides, biologics, chemicals, etc.) over a set period of time, such as 24 hours.

[0090] The term "therapeutic effective dose" refers to the amount of therapeutic agent that is effective in treating at least one symptom of a disease or disorder in a subject. In other words, such a dose is sufficient to produce a beneficial or desirable clinical effect. The "therapeutic effective dose" of an administered drug may vary based on the desired activity, the medical condition of the subject being treated, the dosage form, the method of administration, subject factors such as the sex, genotype, weight, and age of the subject, the underlying cause of the condition or disease being treated, the route of administration and bioavailability, the persistence of the administered drug in the body, evidence of natriuretic and / or diuretic effects, the type of formulation, and the potency of the drug.

[0091] In this specification, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” mean a composition that, when administered to a subject, does not substantially cause any adverse reaction, such as toxicity, allergy, or immunological reaction.

[0092] The terms “therapy,” “treatment,” and “improvement” refer to, for example, a reduction in the severity of symptoms of cancerous malignancies or neurological disorders. In this specification, the terms “treatment” and “prevention” are not intended to be absolute terms. Treatment may include delaying the onset of the disease, improving symptoms, improving patient survival rates, improving cognitive function or coordination, or increasing survival time or survival rates. The effects of treatment can be compared to an untreated individual or a pool of individuals, or to the same patient at different points in time before or during treatment. In some embodiments, the severity of the disease is reduced by at least 10% compared to, for example, an individual before administration or an untreated control individual. In some embodiments, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or, in some cases, becomes undetectable using standard diagnostic techniques.

[0093] The terms “cancer,” “neoplasm,” “tumor,” and “carcinoma” are used interchangeably herein and refer to cells that exhibit relatively autonomous growth, resulting in an abnormal proliferation phenotype characterized by a significant loss of control over cell proliferation. Generally, cells subject to detection or treatment in this application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Types of cancer that may be relevant to this disclosure include, but are not limited to, neurological malignancies, such as glioblastoma.

[0094] Glioblastoma, or glioblastoma multiforme (also known as grade IV astrocytoma), is a type of cancer with a low survival rate that forms from glial cells in the brain, particularly astrocytes, which provide essential support for the proper functioning of nerve cells. These are usually diagnosed at stage IV, not the prodromal stage. They can usually metastasize within the central nervous system (even beyond the corpus callosum), but generally do not metastasize outside the central nervous system. Treatment is difficult, and there is currently no cure. Even with aggressive treatment including surgery, chemotherapy, and radiation therapy, the survival period is approximately 14 months. Therefore, there is an urgent need for more effective treatments for GBM. Unlike other cancers such as melanoma, immunotherapy has been largely ineffective against GBM. This is partly due to the immunosuppressive environment of these tumors. Myeloid cells, such as macrophages, are the most abundant immune cells in GBM and contribute significantly to the immunosuppressive environment. This demonstrates that by targeting macrophages with drugs or immunostimulant decoy macrophages such as Hi-Mac, immunosuppression can be overcome and the immune system can act more effectively against GBM. The methods and compositions described herein are applicable to all solid tumors, including neuroblastoma and melanoma.

[0095] Neuroblastoma refers to tumors that originate from the sympathetic nervous system, such as the adrenal glands and sympathetic ganglia (Brodeur, Nat. Rev. Cancer, 2003, 3:203-216). It is one of the most common solid tumors in children. It is the most common malignant tumor diagnosed within the first year of life, and its clinical phenotype is diverse. While the tumor spontaneously regresses in some patients, the majority develop highly malignant metastatic disease (Maris et al., Lancet, 2007, 369:2106-20). The latter type of neuroblastoma has a survival rate of less than 40% despite intensive chemoradiotherapy and still accounts for 15% of childhood cancer mortality (Maris et al. (2007) Lancet, 369:2106-20; Matthay et al. (1999) N. Eng. J. Med., 341:1165-73). This cancer may originate from neuroblasts of the sympathetic nervous system (e.g., early nerve cells). The term neuroblastoma includes all stages of the cancer as determined, for example, according to the International Neuroblastoma Staging System (INSS) or the International Neuroblastoma Risk Group Staging System (INRGSS).

[0096] Those skilled in the art can understand which controls are useful in specific situations and analyze data based on comparison with control values. Controls are also useful in determining the significance of the data. For example, if the values ​​of a certain parameter differ significantly between control groups, the variation in the test samples is not considered significant.

[0097] In this specification, the following terms have their respective meanings unless otherwise specified. Other terms used in this specification in the fields of recombinant nucleic acid technology, microbiology, immunology, and molecular cell biology will be generally understood by those skilled in the art.

[0098] II. Introduction Glioblastoma is the most severe and common form of brain tumor in adults (Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352, 987-996 (2005). https: / / doi.org:10.1056 / NEJMoa043330). The median survival time is 14 months, and this has not changed significantly over the past decade. Immunotherapy, such as checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy, has shown success in certain cancers, including leukemia (Tawbi, HA et al. Relatlimab and Nivolumab versus Nivolumab in Untreatment Advanced Melanoma. N Engl J Med 386, 24-34 (2022). doi.org:10.1056 / NEJMoa2109970; Sheykhhasan, M., Manoochehri, H. & Dama, P. Use of CAR T-cell for acute lymphoblastic leukemia (ALL) treatment: a review study. Cancer Gene Ther 29, 1080-1096 (2022). doi.org:10.1038 / s41417-021-00418-1).

[0099] However, these treatments have been ineffective against glioblastoma (GBM) in clinical trials. This is partly due to the inability of CAR-T cells to migrate throughout the tumor tissue, antigen drift that occurs after CARs target specific epitopes, and the inability of T cells to survive in the immunosuppressive solid tumor microenvironment.

[0100] Unlike AR-T cells, genetically modified macrophages (GEMs) may be more effective against solid tumors, considering their ability to migrate to myeloid-enriched tumor microenvironments, persistence in immunosuppressive tumor microenvironments, resistance to tumor polarization signaling, lack of antigen-specific antitumor strategies that inhibit tumor resistance through antigen drift, phagocytic ability, and antigen presentation to T cells via major histocompatibility complex (MHC) class I / II. Although GEMs have significant advantages over CAR-T cells, the creation of GEMs for clinical application remains a technical challenge. The inventors generated GEMs from circulating monocytes (Brempelis, KJ et al. Genetically engineered macrophages persist in solid tumors and locally deliver therapy proteins to activate immunorespons. J Immunother Cancer 8 (2020). doi.org:10.1136 / jitc-2020-001356; Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947-953 (2020). doi.org:10.1038 / s41587-020-0462-y). Since monocytes make up only 2-8% of the total circulating white blood cells and have limited extracellular proliferation, obtaining enough starting monocytes to generate GEMs requires either administering monocyte expansion culture stimulants (e.g., filgrastim / Neurast / recombinant granulocyte colony-stimulating factor (G-CSF)) to outpatients daily until one week before monocyte collection (Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947-953 (2020). doi.org:10.1038 / s41587-020-0462-y) or combining multiple HLA-matched donor monocytes.

[0101] Although GEMs offer significant advantages over CAR-T cells, generating GEMs for clinical application remains a technical challenge. GEMs have so far been generated from circulating monocytes (Brempelis et al.). However, since monocytes account for only 2-8% of total circulating white blood cells and have limited in vitro proliferation, obtaining a sufficient quantity of starting monocytes to generate GEMs requires either administering monocyte expansion culture stimulants (Klichinsky et al., filgrastim / Neurasta / recombinant granulocyte colony-stimulating factor (G-CSF)) to outpatients daily for one week before monocyte collection, or combining multiple monocytes from HLA-matched donors.

[0102] This specification describes a method for inducing a clinically significant number of GEMs from patient-specific induced pluripotent stem cells. This eliminates the need for monocyte pooling or the administration of expensive monocyte-stimulating factors.

[0103] Administering stimulants before harvesting circulating monocytes to induce macrophage differentiation is too expensive. For example, Neulasta® costs $6,417 per dose and is administered daily for a week, but it may have side effects such as splenic rupture, acute respiratory distress syndrome, renal impairment, thrombocytopenia, myelodysplastic syndrome, or leukemia formation.

[0104] Several reports suggest that cancer alters the transcriptome of human circulating monocytes. Cassetta, L. et al. demonstrated that human endometrial cancer and breast cancer significantly alter the transcriptome of circulating monocytes and their progeny macrophages (Cassetta, L. et al., 2019, Cancer Cell 2019, 35(4): 588-602.E10, doi.org / 10.1016 / j.ccell.2019.02.009). Therefore, inducing GEMs from circulating monocytes whose transcription has been reprogrammed by tumors and which have acquired tumorigenic functions may not be ideal. This could not only inhibit the therapeutic effect of GEMs but also potentially increase the malignancy of the tumor.

[0105] The use of allogeneic (HLA-matched) donors presents challenges because, like organ transplantation, finding an HLA-matched donor is difficult and costly. Furthermore, there is a higher risk of rejection by the patient's immune system, which can affect the effectiveness of the treatment. To minimize systemic rejection, treated patients must be immunosuppressed before receiving HLA-matched gems, which can interfere with the effectiveness of immunotherapy. Additionally, to minimize systemic rejection, HLA-matched gems must be injected directly into the brain tumor, which requires a second surgical procedure.

[0106] Autologous human patient-specific induced pluripotent stem cell (iPSC)-derived GEMs (also known herein as “iMac,” “HiMac,” “hi-Mac,” or “MDA hi-Mac”) can be generated without stimulation from the patient’s own tissue (e.g., cranial tissue, also known herein as “periosteum” or “periosteal tissue”) and can be systemically administered (intra-arterial or intravenous) while minimizing rejection by the patient’s immune system. Furthermore, systemic administration of hi-Mac may be more effective because intravenous or intra-arterial administration allows for broader homing to invasive tumor areas that rely on vascular supply. This systemic administration approach makes hi-iMac applicable to other types of cancer as well.

[0107] Commercial aspects of this embodiment Obtaining MDA hi-Macs in high yield according to this disclosure eliminates the need for expensive recombinant stimulants. The high yield is evident from the presence of a macrophage stem cell source that can produce macrophages for more than four weeks (instead of patients with limited sources).

[0108] MDA hi-Macs can be administered systemically with reduced risk of immune rejection. According to some embodiments of this disclosure, MDA hi-Macs can be administered by direct intra-arterial delivery or intravenous systemic delivery. In either delivery method, MDA hi-Macs can be resuspended in plasma light supplemented with 0.5% human serum albumin (HSA or other isotonic solution). For intra-arterial delivery, MDA hi-Macs can be resuspended in 20 ml of plasma light supplemented with HSA. The entire volume can be administered at a rate of 1 ml / min into a tumor feeding vessel accessible from the angiography suite. For intravenous administration, MDA hi-Macs can be resuspended in 100 ml of plasma light containing HSA and administered intravenously. Therefore, it is not necessary to perform brain surgery again to deliver the drug directly to the tumor.

[0109] GEM derived from donors requires expensive HLA matching. HLA matching is not necessary for the production of MDA hi-Mac or for its administration to patients.

[0110] Method of cell line generation according to this disclosure Hi-Mac can be induced from any cell type in a patient's body that can be reprogrammed into iPSCs (e.g., blood cells or cutaneous fibroblasts). However, sources with a resident population of stem cell-like cells are thought to enhance the efficiency of reprogramming to iPSCs. This specification describes a protocol for harvesting cranial membrane (tissue covering the skull) enriched with stem cell-like pluripotent periosteal progenitor cells (PDPCs). In some embodiments of this disclosure, cranial membrane can be harvested from the skull of a patient undergoing brain tumor surgery to induce PDPCs. The harvested cranial membrane can be washed with Hanks' fluid, mechanically fragmented, and digested for 2 hours in DMEM / Ham's F12 medium containing collagenase, human serum, and antibiotics. The cells can be pelleted, resuspended in DMEM / Ham's F12 medium containing human serum, seeded in a petri dish, and allowed to adhere for at least one week. The adherent cells containing PDPCs (fibroblast-like morphology) can then be harvested from the petri dish and reprogrammed into iPSCs. Unlike hematopoietic cells and dermal fibroblasts, which are primarily terminally differentiated cells, the periosteum is enriched with PDPCs, allowing for more efficient reprogramming into iPSCs and, consequently, macrophages. Therefore, the method described herein is superior to other sources in that it uses a stem cell-like source (PDPC) for iPSCs. The method disclosed herein also avoids the administration of expensive stimulants required for peripheral blood sources of macrophages (i.e., patients are not administered additional stimulants for macrophage stimulation).

[0111] In certain embodiments, a cranial membrane for generating MDA hi-Macs is described when a patient undergoes initial surgery for a brain tumor (IRB-approved protocol; 2022-0943, "Induced pluripotent stem cell derived genetically modified macrophages (HiMacs) for personalized cell-based cancer therapy"). In some embodiments, a 2 cm × 2 cm cranial membrane flap can be harvested during brain tumor surgery, PDPCs can be induced, and then sent to a laboratory for reprogramming into iPSCs.

[0112] HiMacs can be induced and stored for the treatment of disease relapses in patients, which are unavoidable in diseases such as GBM, while the patient is undergoing standard treatment and follow-up imaging. In some embodiments, Hi-Macs can be stored in standard freezing medium containing 10% DMSO. Before use, these cells can be washed three times with PBS or plasma light before further study or clinical administration.

[0113] Differences between the literature and this method Brempelis et al.: In this preclinical study, circulating monocytes were used, but it was necessary to use a pool of HLA-matched monocytes from multiple donors or autologous monocytes from patients receiving stimulants. The protocol described herein differs from this disclosure in that iPSCs can be created using periosteum (or blood) and then hi-Macs can be created. In some embodiments of this disclosure, the cranial membrane can be harvested from the skull of a patient undergoing brain tumor surgery to create PDPCs. The cranial membrane can be washed with Hanks' solution, mechanically fragmented, and digested for 2 hours in DMEM / Ham's F12 medium containing collagenase, human serum, and antibiotics. The cells can be pelleted, resuspended in DMEM / Ham's F12 medium containing human serum, seeded in a dish, and allowed to adhere for at least one week. Adhering cells containing PDPCs can be harvested from a petri dish and reprogrammed into iPSCs. Furthermore, the differentiation process of hi-Mac requires a different set of cytokines than the method of Brempelis et al., setting it apart from other methods (e.g., the method of Ackermann et al. described below). This is because the method described herein has been improved to shorten the induction time of MDA hi-Mac. MDA hi-Mac is customized to the patient, and stimulant administration is not required.

[0114] Ackermann et al.: This group published a protocol for differentiation from iPSCs to macrophages that previously took up to four weeks. This protocol has been improved, for example, by eliminating the need for embryoid body formation, terminal differentiation, and bioreactors for mass production (with comparable yield). This allows for the production of MDA hi-Macs from iPSCs in two weeks, enabling rapid delivery to patients in need. Therefore, by using the protocol described herein, MDA hi-Macs can be produced and administered to patients in a shorter timeframe compared to studies by other groups, according to this disclosure.

[0115] Since there are reports that patient-derived monocyte-derived GEMs tend to revert to a tumor-promoting phenotype after administration, it is possible to introduce a gene switch that induces self-destruction when MDA hi-Mac develops a tumor-promoting phenotype (Figure 4). This approach has not been described before and will ensure the safety of the product after administration.

[0116] To overcome these obstacles and challenges, this specification describes the use of patient-specific induced pluripotent stem cell (iPSC)-derived macrophages (HiMacs). This specification describes their manufacturing methods, compositions containing them, administration methods, and related genetically modified cells. Furthermore, this specification describes modified cells for delivering novel therapeutic agents such as IL-12, IFN-γ (e.g., NCBI Accession: AAB59534.1), IL-15 (e.g., NCBI Accession: AAI00963.1), MCP-1 (e.g., Accession: AAB29926.1), and Delta-24RGD oncolytic adenovirus to patients from whom iPSCs originated. This makes it possible to generate enough cells for clinical trials and perform multiple treatments on the same patient, while minimizing immune-mediated rejection of GEMs.

[0117] Various methods in the art can be used to modify cells for the delivery of therapeutic drugs. In some embodiments, the delivery of therapeutic drugs may include the delivery of one or more nucleic acids encoding IL-12 subunits α and β (e.g., human or mouse IL-12 subunits, e.g., SEQ ID NOs: 8 and 10 for mouse IL-12β and IL-12α, respectively, and SEQ ID NOs: 16 and 17 for human IL-12β and IL-12α, respectively). The nucleic acids can be delivered by methods known in the art, e.g., transfection of a vector (e.g., plasmid) expressing the coding sequence of the target protein, electroporation, or viral transduction (e.g., by lentivirus or adeno-associated virus). In some embodiments, such coding sequences may be operably linked to a constitutive promoter (e.g., CAG or elongation factor 1α (EF1α)). In some embodiments, such coding sequences may be operably linked to an inducible promoter or other inducible factor (e.g., a repressor that requires removal for transcription) that requires the presence of an inducer (i.e., tetracycline or doxycycline, among others). In some embodiments, nucleic acids may be packaged into lentiviruses that can be used to transduce cells as described herein. Exemplary vector systems available include, for example, the backbone of SEQ ID NOs: 6 and 11. Exemplary IL-12 expression constructs include SEQ ID NOs: 19 and 21.

[0118] The methods and compositions described herein may provide a source for cell-based therapies that can overcome the problems associated with CAR-T cells. For example, MDA-HiMac can also deliver genes, cytokines, and viruses, phagocytosis, and antigens (the latter three of which cannot be performed by CAR-T cells). Other macrophage sources, such as monocytes, require pooling of donor samples or pre-harvest stimulation of patients to obtain sufficient cell numbers for clinical application. These strategies require the administration of expensive drugs before monocyte harvesting. The approach described herein overcomes these hurdles by generating high yields of clinically usable macrophages without pre-treatment, particularly the administration of additional cell stimulants to patients before sampling for cell separation, reprogramming, and differentiation.

[0119] MDA-HiMacs can be used to deliver immunostimulatory cytokines, full-length checkpoint antibodies, oncolytic viruses, exosomes, mRNA, and tumor antigens to any type of cancer. MDA-HiMacs can be used to study how macrophages transform the tumor microenvironment into an immunosuppressive microenvironment, thereby elucidating the mechanisms by which this phenomenon is overcome in patients. MDA-HiMacs can also be used to study the fundamental functions of macrophages in non-cancerous disease conditions such as inflammation and infection.

[0120] Currently, CAR-T cells are only approved by the FDA as a cell-based immunotherapy for cancer. While CAR-T cells are effective against many humoral tumors, most solid tumors are resistant to treatment. This is partly due to the fact that T cells cannot migrate within solid tumors or remain in the immunosuppressive microenvironment induced by macrophages.

[0121] This disclosure relates to the treatment of cancerous malignancies, particularly solid tumors (particularly glioblastoma, also known herein as glioblastoma multiforme (GBM)) that are refractory to CAR-T cell therapy, using the MDA-HiMac described herein. Any cancer, including solid tumors, may benefit from the compositions and methods described herein. Additional examples, but not limited to, include melanoma, breast cancer, and ovarian cancer.

[0122] III. In Vitro Methods This specification describes a method for reprogramming pluripotent cells into macrophages (or cells having a macrophage-like phenotype and lacking non-macrophage markers).

[0123] In certain embodiments, the Specified Description describes somatic cells or progenitor cells (or populations thereof) that can be reprogrammed into induced pluripotent stem cells (iPSCs). Such cells may originate from patient samples (i.e., autologous cell sources) of patients diagnosed with or suspected to have a cancerous malignancy as described herein, or from other subjects, such as subjects with HLA matching that of patients diagnosed with or suspected to have a cancerous malignancy (i.e., allogeneic cell sources).

[0124] Patient samples may be blood, saliva, plasma, urine, tissue, cerebrospinal fluid (CSF), or other sample types. In certain embodiments, tissue may be periosteum. In certain embodiments of the methods described herein, patient samples are collected without administering a cytostimulant to the patient or subject.

[0125] Cells for reprogramming can be isolated or enriched from patient samples before reprogramming. In some embodiments, to induce iPSCs from the cranial membrane, a 2 cm × 2 cm (or other size, e.g., 1 cm × 1 cm) cranial membrane can be taken from the skull of a patient undergoing brain tumor surgery. The cranial membrane can be washed with Hanks' fluid, mechanically fragmented, and digested for 2 hours in DMEM / Ham's F12 medium containing collagenase, human serum, and antibiotics. The cells can be pelleted, resuspended in DMEM / Ham's F12 medium containing human serum, seeded on a dish, and allowed to adhere for 1 week. The adherent cells containing PDPCs can be harvested from the dish and reprogrammed into iPSCs. In some embodiments, to induce iPSCs from blood, patient blood can be taken and PBMCs can be isolated using a Ficoll gradient. The PBMCs can then be reprogrammed into iPSCs.

[0126] After isolation from patient samples, cells can be reprogrammed into a pluripotent state (i.e., induced pluripotent stem cells) using established protocols. For example, a vector (particularly an episomal vector or a vector lacking chromosomal integration) can be delivered to cells by known methods (e.g., transfection, transduction, nucleofection, etc.) to overexpress reprogramming factors (OCT family proteins, Klf family proteins, Myc family proteins, Sox family proteins, and even LIN family proteins) while knocking down, knocking out, or reducing the expression of other cell cycle regulatory proteins (e.g., p53 family proteins). Other reprogramming methods, such as the use of Sendai virus vectors, can also be used in accordance with this disclosure.

[0127] After reprogramming the cells to a pluripotent state, they can be differentiated (or terminally differentiated) into iPSC-derived macrophages (or iPSC-derived cells exhibiting a macrophage-like phenotype, expressing macrophage markers, but lacking the expression of non-macrophage markers, which are markers specific to non-macrophage cells). Such differentiation can be achieved by culturing the cells for a certain period in a differentiation medium different from the reprogramming or maintenance medium used to maintain the cells in a pluripotent state. In some embodiments, first, the medium for iPSCs cultured in iPSC medium (at a concentration of approximately 80%) is replaced daily for four consecutive days with iPSC medium + 10 μM ROCK inhibitor and 25% reduced concentrations of bFGF (approximately 7.5 ng / ml, 5 ng / ml, 2.5 ng / ml, and 0 ng / ml, respectively). From day 10, the culture medium can be gradually switched to X-VIVO-15 (LONZA) medium containing 25 ng / ml IL3 and 50 ng / ml M-CSF. The culture medium composition on day 10 can consist of 75% iPSC medium containing 10 μM ROCK inhibitor, mixed with 25% X-VIVO-15 medium (containing IL3 and M-CSF). On day 11, the culture medium can consist of 50% iPSC medium containing 10 μM ROCK inhibitor, mixed with 50% X-VIVO-15 medium (containing IL3 and M-CSF). On day 12, the culture medium can consist of 25% iPSC medium containing 10 μM ROCK inhibitor, mixed with 75% X-VIVO-15 medium (containing IL3 and M-CSF). On day 13, the culture medium can be 100% X-VIVO-15 medium (containing IL3 and M-CSF). Incubation can be performed using an orbital shaker set to approximately 100 revolutions per minute. Subsequently, the supernatant / acclimatization medium containing hi-MACS is collected and cultured for 7 days in a medium containing 50 ng / ml of M-CSF to induce final differentiation.

[0128] After differentiating the cells into macrophages (or cells with a macrophage-like phenotype; hereafter referred to herein as iMac, hiMac, or hi-macs-MDA-hiMacs), the cells can be further phenotypically classified and isolated, if necessary, based on various cellular markers (e.g., IBA-1 or CD68, and the absence of tumor-promoting markers such as CD206 or CD163).

[0129] Such differentiated cells can undergo further genetic differentiation as desired. For example, vectors that express or knock down target genes (e.g., pLenti-EF1a-C-tGFP) can be introduced into the cells (e.g., viral vectors that overexpress cytokines, growth factors, or other therapeutic peptides). Vectors that increase or decrease the cells' susceptibility to cell disruption or cell death can also be added as needed (e.g., CD163 promoter-driven HSV / TK switches).

[0130] In one embodiment, a method for generating patient-specific macrophages comprises reprogramming one or more somatic cells (or progenitor cells) from a sample derived from a subject to a pluripotent state, and differentiating one or more of the reprogrammed cells into a macrophage-like phenotype, wherein the differentiation proceeds without the formation of embryoid bodies. Such cells must be positive for the Iba1 and CD68 markers and lack markers for T cells, NK cells, dendritic cells, and B cells, or tumor-promoting markers such as CD163 and CD206. The cells must also lack OCT3 / 4 expression, which can be verified by immunocytochemistry techniques known in the art. In other embodiments of the method herein, the method may further include culturing one or more of the reprogrammed cells and expanding the culture of one or more of the reprogrammed cells into a population of induced pluripotent stem cells.

[0131] In some embodiments of this disclosure, iPSCs can be induced / cultured / maintained under feeder-free conditions because they can yield a pure human-derived cell population. For each induced iPSC strain, a library of frozen vials of iPSCs (n=25-50, 1×10) is obtained at less than 10 passages. 6 Cells / vials can be prepared. By further passage the thawed iPSC vials 4-5 times (this expansion culture takes approximately 15-20 days), the number of starting iPSCs required for hi-MACS production can be reached.

[0132] According to further embodiments of the present disclosure, a method for producing patient-specific macrophages comprises reprogramming one or more somatic cells from a sample derived from a subject to a pluripotent state, culturing one or more of the reprogrammed cells, expanding the culture of one or more of the reprogrammed cells into a population of induced pluripotent stem cells, and differentiating one or more of the reprogrammed cells of the population into a macrophage-like phenotype, wherein the differentiation proceeds without embryoid body formation (or embryoid body selection).

[0133] In certain embodiments of this disclosure, reprogramming may involve delivering one or more reprogramming expression vectors containing nucleic acids encoding OCT3 / 4, SOX2, KFL4, L-MYC, and LIN28 to one or more somatic or progenitor cells. These vectors may be mixed in ratios that ensure all factors are induced with approximately equal efficiency, as routinely described in various publications. In certain embodiments, reprogramming may involve knocking down (or inhibiting) p53, for example, by introducing a vector containing shRNA for p53. In some embodiments of this disclosure, at least one of the one or more expression vectors may be an episomal expression vector (i.e., a vector that does not exhibit chromosomal integration or integration into the host genome).

[0134] In certain embodiments of this disclosure, iPSCs can be cultured in culture media for maintaining pluripotency as known in the literature (e.g., containing components such as Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, L-glutamine, antibiotics (e.g., penicillin-streptomycin), β-mercaptoethanol, and basic fibroblast growth factor (B-FGF)). In some embodiments, iPSCs can be cultured in media such as mTeSR1.

[0135] In certain aspects, iPSCs can be cultured for approximately 10 to 25 passages. Karyotyping can be performed to confirm the normal karyotype of these iPSCs after expansion culture / passaging.

[0136] In certain embodiments, differentiating iPSCs into macrophages (or macrophage-like cells) involves removing b-FGF from the culture medium and administering IL-3 and M-CSF over a period of time.

[0137] In certain embodiments, the sample used to reprogram and differentiate cells includes the cranial membrane of the subject. In some embodiments, a 2 cm × 2 cm cranial membrane can be harvested from the skull of a patient undergoing brain tumor surgery to induce iPSCs from the cranial membrane. The cranial membrane can be rinsed in Hanks' solution, mechanically fragmented, and digested for 2 hours in DMEM / Ham's F12 medium containing collagenase, human serum, and antibiotics. The cells can be pelleted, resuspended in DMEM / Ham's F12 medium containing human serum, seeded on a dish, and allowed to adhere for 1 week. Adhering cells containing PDPCs can be harvested from the dish and reprogrammed into iPSCs. In certain embodiments, the sample may be enriched with periosteal-derived progenitor cells (PDPCs) before reprogramming.

[0138] In certain embodiments, after differentiation, one or more therapeutic vectors can be introduced into differentiated cells along with one or more nucleic acids encoding cytokines (e.g., IL-12, IFN-γ, IL-15, MCP1), growth factors, or other suitable therapeutic proteins known in the art to be effective against the symptoms of cancerous malignancies. In certain embodiments, the therapeutic vectors may include therapeutic nucleic acids, each independently encoding a cytokine, checkpoint protein, or self-destructive protein. In additional embodiments, one or more therapeutic vectors may encode genes expressing proteins that can be used to track cells after transplantation, such as eGFP or CD19. In additional embodiments, one or more therapeutic vectors may be inducible vectors that require the presence of an inducer (e.g., tetracycline or doxycycline) to express the target gene, such as a cytokine. In some embodiments, one or more therapeutic vectors may be viral vectors that can be used to package the target gene into a virus (e.g., a lentivirus) that can be used for transduction of the cells described herein (e.g., iPSCs and HI-MACS).

[0139] IV. Pharmaceutical Compositions and Formulations This specification describes compositions comprising one or more reprogrammed and subsequently differentiated cells (i.e., iPSC-derived macrophages). The compositions may further comprise diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants used in the methods disclosed herein. Such compositions can be used in subjects having or suspected of having cancerous malignancies who would benefit from any of the compositions described herein. In some embodiments, the cells can be resuspended in plasma light or isotonic saline containing human serum albumin. Approximately 20 ml is typically required for an IA dose, and approximately 100 ml for an IV dose.

[0140] In certain embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dose and concentration used. In certain embodiments, the formulation materials are for subcutaneous and / or intravenous administration. Formulations suitable for administration (or intra-arterial, IA, formulation). In certain embodiments, the pharmaceutical composition may include, for example, formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or osmosis. In certain embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); fillers (such as mannitol, glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, hydroxypropyl-β-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, dextrin); proteins (such as serum albumin, gelatin, immunoglobulins); colorants and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (na Examples of such substances include, but are not limited to, thorium, preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, etc.), solvents (glycerin, propylene glycol, polyethylene glycol, etc.), sugar alcohols (mannitol, sorbitol, etc.), suspending agents, surfactants or wetting agents (pluronic acid, PEG, sorbitan esters, polysorbates (polysorbate 20, polysorbate 80, etc.), triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.), stability enhancers (sucrose or sorbitol, etc.), tonicity enhancers (alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, etc.), delivery media, diluents, excipients and / or pharmaceutical adjuvants.(Allen (2012) Remington - The Science and Practice of Pharmacy, 22nd Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). In particular embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended route of administration, the form of delivery, and the desired dose. See, for example, Allen (2012) Remington - The Science and Practice of Pharmacy, 22nd Edition, Lloyd V, Allen, ed., The Pharmaceutical Press. In particular embodiments, such compositions may affect the physical state, stability, bioavailability, and / or bioavailability of the cells or compositions described herein.

[0141] In certain embodiments, the primary medium or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, in certain embodiments, suitable mediums or carriers may be water for injection, saline, or artificial cerebrospinal fluid, and may be supplemented with other substances commonly found in parenteral administration compositions. In certain embodiments, saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary mediums. In certain embodiments, the pharmaceutical composition includes a pH-controlled buffer such as phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition containing macrophages (or HiMacs) prepared by the methods disclosed herein may be prepared for storage in the form of lyophilized cake or aqueous solution by mixing a selected composition having the desired purity with any formulation agent (see Allen (2012) Remington - The Science and Practice of Pharmacy, 22d Edition, Lloyd V, Allen, ed., The Pharmaceutical Press). Furthermore, in certain embodiments, compositions comprising macrophages (or HiMacs) prepared by the methods disclosed herein can be formulated as lyophilized products using appropriate excipients. In some cases, the appropriate excipients may include cryopreservatives, fillers, surfactants, or combinations thereof. Exemplary excipients include, for example, one or more polyols, disaccharides, or polysaccharides such as mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some cases, the cryopreservative may be sucrose or trehalose. In some cases, the filler may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as polysorbate-20 or polysorbate-80.

[0142] Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including physiological saline and buffer solutions). Parenteral bases include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixative oils. Intravenous bases include hydration and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives such as antibacterial agents, antioxidants, and chelating agents, and other additives may also be present.

[0143] In certain embodiments, the formulation components are present at the administration site at an acceptable concentration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically in the pH range of about 5 to about 8. For example, the pH is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some cases, the pH of the pharmaceutical composition may be in the range of 6.6 to 8.5, for example, 7.0 to 8.5, 6.6 to 7.2, 6.8 to 7.2, 6.8 to 7.4, 7.2 to 7.8, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 8.2, 7.6 to 8.5, or 7.8 to 8.3. In some cases, the pH of the pharmaceutical composition may be in the range of 5.5 to 7.5, for example, 5.5 to 5.8, 5.5 to 6.0, 5.7 to 6.2, 5.8 to 6.5, 6.0 to 6.5, 6.2 to 6.8, 6.5 to 7.0, 6.8 to 7.2, or 6.8 to 7.5. In some cases, the pH of the pharmaceutical composition may be in the range of 4.0 to 5.5, for example, 4.0 to 4.3, 4.0 to 4.5, 4.2 to 4.8, 4.5 to 4.8, 4.5 to 5.0, 4.8 to 5.2, or 5.0 to 5.5.

[0144] In certain embodiments, the pharmaceutical composition may be selected for parenteral administration. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In certain embodiments, when parenteral administration is considered, the therapeutic composition may be in the form of a pyrogenically-free parenterally acceptable aqueous solution containing macrophages (or HiMac) prepared by the method disclosed herein in a pharmaceutically acceptable medium. In certain embodiments, the medium for parenteral injection is sterile distilled water, appropriately stored, containing macrophages (or HiMac) prepared by the method disclosed herein as a sterile isotonic solution. In certain embodiments, the preparation involves compounding the desired molecule with the agent, such as injection microspheres, biodegradable particles, polymer compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. These agents allow for controlled or sustained release of the product, which can then be delivered by depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting sustained persistence in the circulating blood. In certain embodiments, the desired molecule can be introduced using an implantable drug delivery device.

[0145] Pharmaceutical compositions used for in vivo administration are typically sterilized. In certain embodiments, sterilization is achieved by filtration through a sterile filtration membrane. In certain embodiments, parenteral compositions are generally filled into containers having a sterile access port, such as intravenous solution bags or vials with stoppers that can be punctured with a subcutaneous needle.

[0146] In certain embodiments, the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, or emulsion after formulation.

[0147] In certain embodiments, the pharmaceutical composition may contain an effective amount (also referred to herein as a “therapeutic effective amount”) of macrophages (or hiMacs) prepared by the methods disclosed herein, as a mixture with a non-toxic excipient suitable for the manufacture of tablets. Such an effective amount may, in some embodiments, be about 1 × 10⁵, about 1 × 10⁶, or about 1 × 10⁷ per kg of body weight for intra-arterial administration, and is administered at a maximum of once for intra-arterial administration, or once a week for three weeks for intravenous infusion. In certain embodiments, the solution may be prepared in unit dose form by dissolving the tablets in sterile water or other suitable medium. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0148] In certain embodiments, the effective dose used for treatment of a pharmaceutical composition comprising macrophages (or HiMacs) prepared by the methods disclosed herein depends, for example, on the context and purpose of the treatment. Those skilled in the art will understand that the appropriate dose level for treatment according to a particular embodiment will vary in part on the molecules being delivered, the indications for which the cells or cell populations provided herein are used, the route of administration, and the patient's size (body weight, body surface area, or organ size) and / or condition (age and overall health). Clinicians may titrate the dose and modify the route of administration to obtain the optimal therapeutic effect. Dosages will be discussed further in the following sections of this disclosure.

[0149] In certain embodiments, the route of administration of the pharmaceutical composition follows known methods, such as injection, sustained-release system, or implantable device via intra-arterial, intra-venous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, subcutaneous, portal vein, or intralesional routes. In certain embodiments, the composition may be administered by bolus injection, continuous infusion, or implantable device. In certain embodiments, individual elements of the combination therapy may be administered via different routes.

[0150] In certain embodiments, the composition may be administered topically, for example, during surgery or locally. If desired, topical administration may be carried out via the implantation of a membrane, sponge, or other suitable material into which the desired cells are absorbed or encapsulated. In certain embodiments, if an implantable device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be carried out by diffusion, sustained-release bolus, or continuous administration.

[0151] In certain embodiments, it may be desirable to use a pharmaceutical composition containing macrophages (or HiMacs) prepared by the methods disclosed herein in vitro.

[0152] V. Treatment method As described herein, this disclosure provides a method for treating subjects having a cancerous malignancy, comprising administering a certain amount or therapeutically effective amount of macrophages (or HiMacs) prepared by the method disclosed herein. In some embodiments, the subject has, is determined to have, or is expected to have a cancerous malignancy, such as glioblastoma.

[0153] The compositions described herein are particularly useful in methods for treating cancerous malignancies in subjects. In this specification, the term “subject” means a mammalian subject. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, and sheep. In some embodiments, the subject is human. In some embodiments, the subject has or is suspected of having a cancerous malignancy. In some embodiments, the subject is diagnosed with a cancerous malignancy. In some embodiments, the subject is a human suspected of having a cancerous malignancy. In this disclosure, the terms “subject” and “patient” are used interchangeably.

[0154] The main symptoms of cancerous malignancies described herein may include headache, seizures, difficulty speaking, and paralysis.

[0155] Cells and compositions prepared according to the methods of this disclosure can also be used as prophylactic therapies for cancerous malignant tumor diseases. The antibodies and fragments provided may be used not only for prophylactic and therapeutic administration, but also by passive immunization using substantially purified polypeptide products, and gene therapy by introducing polynucleotide sequences encoding the product or a portion thereof. Thus, the antibodies and fragments provided can be administered to high-risk subjects to reduce the likelihood and / or severity of cancerous malignant tumors, or to subjects already exhibiting active cancerous malignancies. For example, in some embodiments, the compositions and methods described herein can be used as tumor antigens for priming macrophages, which can then be injected so that the macrophages present the antigen to T cells and generate an antitumor T cell population (see, for example, Figure 1).

[0156] In this specification, “administration” or “dosage” means the act of injecting or physically delivering an extracorporeal substance (e.g., cells or compositions containing such cells, prepared according to the methods of this disclosure) to a patient by means of mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery, and / or other physical delivery methods described herein or known in the art. When treating a disease or its symptoms, administration of the substance is usually performed after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance is usually performed before the onset of the disease or its symptoms. Additional information relating to administration is also provided in the preceding sections of this disclosure.

[0157] The compositions can be administered to subjects, such as human subjects, by various methods that depend in part on the route of administration. Routes of administration include, for example, intra-arterial injection or infusion (IA), intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavitary, oral, intracranial, or intrathecal injection (IT). Injections can be performed by bolus injection or continuous infusion. Techniques for preparing antibody-containing injectables or infusion delivery systems are well known to those skilled in the art. Generally, such systems should use components that do not significantly impair the biological properties of cells (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those skilled in the art can readily determine various parameters and conditions for producing the cell-based compositions provided in this disclosure without relying on excessive experimentation.

[0158] Administration can be carried out, for example, by local injection, injection, or implantation. Implants may consist of porous, non-porous, or gelatinous materials, including membranes or fibers such as cialastic membranes. Implants may be configured to continuously or periodically release the composition to a target. See, for example, U.S. Patent Application Publication No. 20080241223, U.S. Patents No. 5,501,856, 5,164,188, 4,863,457, and 3,710,795. The composition can be delivered to a target via implantable devices based on diffusion, erosion, or convection systems, such as osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, foaming pumps, piezoelectric pumps, erosion systems, or electromechanical systems. In some embodiments, the cells or compositions of this disclosure are therapeutically delivered to a target by local administration.

[0159] Treating a disease or disorder, or treatment, means improving the disease or disorder present in the subject, or the symptoms thereof. The term "improvement" means a therapeutically beneficial outcome, such as a reduction in severity or progression, remission or extension of remission, or cure, in the treatment of a disease condition such as a cancerous malignant tumor. Thus, treating or treatment involves improvement of at least one physical parameter or symptom. Treating or treatment involves modulating the disease or disorder physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Treating or treatment involves delaying or preventing metastasis. Thus, in the disclosed manner, treatment can mean a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition, or in the symptoms of the disease or condition. For example, a method of treating a target cancerous malignant tumor by administering the composition described herein is considered a treatment if one or more symptoms of the target cancer are reduced by 10% compared to a control group. Thus, reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between 10% and 100% compared to the natural or control group. It should be understood that a treatment does not necessarily mean a cure or complete disappearance of a disease, condition, or symptoms of a disease or condition.

[0160] As used in this disclosure, the term “therapeutic effective dose” refers to the amount of therapeutic agent effective in treating at least one symptom of a disease or disorder in a subject. In other words, such a dose is sufficient to produce a beneficial or desired clinical effect. As used herein, the term “preventive effective dose” refers to a dose of the cells or composition described herein that is sufficient to produce the desired effect in protecting an individual from symptoms of cancerous malignancy for a reasonable period, such as one to two months or longer after administration. However, a preventive effective dose is not a dose that causes adverse side effects such as hyperviscosity syndrome, pulmonary edema, or congestive heart failure. Generally, the preventive effective dose may vary depending on the age, condition, sex of the subject, and the severity of the disease in the subject, and may be determined by those skilled in the art. Other factors may include, for example, other medical conditions the subject has had at the time or in the past, the subject’s overall health status, the subject’s genetic predisposition, diet, timing of administration, excretion rate, combination of drugs, and other additional therapeutic agents administered to the subject. The preventive effective dose may be adjusted by the individual physician or veterinarian if complications occur. In some cases, the effective prophylactic dose may vary in the range of about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 20 mg / kg, and most preferably about 0.2 mg / kg to about 2 mg / kg, in one or more doses (priming and boosting).

[0161] The pharmaceutical composition may contain a therapeutic or prophylactic dose of the cell population described herein. In some embodiments, such a dose may be about 1 × 10⁵ cells, about 1 × 10⁶ cells, or about 1 × 10⁷ cells per kg of body weight. Further aspects of such a dose can be readily determined by those skilled in the art, as described above. Considerations include the effect of the administered cells or composition described herein, or the combined effect with two or more activators in or used in combination with the pharmaceutical composition.

[0162] Where described herein, the appropriate dose of cells or compositions for humans can be further evaluated, for example, in a Phase I dose-escalation study. See, for example, van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

[0163] Where described herein, the toxicity and therapeutic effects of cells and compositions can be determined by known pharmacological procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, for example, to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutic dose in 50% of the population). The dose-to-toxicity ratio is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Cells or compositions described herein are preferably found to exhibit a high therapeutic index. Formulations exhibiting toxic side effects may be used, but care should be taken to design a delivery system that targets such constructs to affected tissue, minimizes potential damage to normal cells, and thereby mitigates side effects.

[0164] In some embodiments, as described herein, cells or compositions may be administered to a subject as monotherapy. Alternatively, cells or compositions described herein may be administered in combination with other cancer therapies (combination therapy). In some embodiments, but not limited to, combination therapies that may be administered with embodiments of this disclosure include radiotherapy prior to systemic HiMacs administration, and immune checkpoint inhibitors such as nivolumab before or after HiMacs administration. For example, a composition may be administered to a subject simultaneously with, before, or after a second therapy. In some embodiments, cells or compositions described herein are administered simultaneously with one or more additional activators. If desired, cells or compositions described herein may be administered first, followed by one or more additional activators. In some embodiments, one or more additional activators may be administered first, followed by cells or compositions described herein. If desired, cells or compositions described herein and one or more additional activators may be administered simultaneously via the same or different routes. For example, a composition comprising cells or compositions described herein may optionally include one or more additional activators.

[0165] In some embodiments, the compositions and methods described herein are described in combination with radiotherapy prior to systemic iMac administration, or with immune checkpoint inhibitors such as nivolumab before or after iMac administration.

[0166] Where described in the specification, cells or compositions may replace or enhance previously or currently administered therapies. For example, when treating with cells or compositions described herein, the administration of one or more additional activators may be discontinued or reduced, for example, by administering them at lower levels or doses. In some embodiments, the administration of previous therapies may be maintained. In some embodiments, previous therapies are maintained until the level of cells or compositions described herein reaches a level sufficient to produce a therapeutic effect.

[0167] As defined herein, monitoring a subject (e.g., a human patient) for improvement of a cancerous malignancy means evaluating the subject for changes in disease parameters, e.g., reduction of one or more symptoms of the cancerous malignancy exhibited by the subject. In some embodiments, the evaluation is performed at least one hour after administration, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1, 2, 4, 10, 13, or 20 days, or at least 1, 2, 4, 10, 13, or 20 weeks. The subject may be evaluated at one or more of the following periods: before the start of treatment, during treatment, or after the administration of one or more elements of treatment. Evaluation may include evaluating the need for further treatment, e.g., whether the dose, frequency of administration, or duration of treatment should be changed. It may also include evaluating the need to add or discontinue a selected treatment, e.g., adding or discontinuing any of the treatments for cancerous malignancies described herein.

[0168] Clinicians also consider the pharmacokinetic parameters of macrophages (or HiMacs) prepared by the methods disclosed herein when selecting the dosage frequency in the formulation to be used. In certain embodiments, clinicians administer the composition until a dose is reached that achieves the desired effect. Thus, in certain embodiments, the composition can be administered as a single dose or over time as two or more doses (which may or may not contain the same amount of the desired molecule). Alternatively, it can be administered as a continuous infusion, for example, via an implantable device or catheter. Further refinement of appropriate dosages is routinely performed by those skilled in the art and is within the scope of routine practice. In certain embodiments, appropriate dosages can be confirmed using appropriate dose-response data.

[0169] Examples Example 1: Production of human-induced pluripotent stem cell-derived macrophages (HI-MACs) for brain tumor treatment. A. Establishment of induced pluripotent stem cells (iPSCs) from the peripheral blood of patients. Peripheral blood mononuclear cells (PBMCs) collected from patients were subjected to non-integrated (episome) expression of standard human OCT3 / 4 and p53 shRNA (pCXLE-hOCT3 / 4-shp53-F, Addgene catalog number 27077), non-integrated (episome) expression of human SOX2 and KLF4 (pCXLE-hSK, Addgene catalog number 27078), and non-integrated (episome) expression of human L-MYC and LIN28 (pCXLE-UL, Addgene catalog number 27080) (K. et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 8, 409-412 (2011). doi.org:10.1038 / nmeth.1591). Induced pluripotent stem cells were generated according to the guidelines of the Human Embryo and Induced Pluripotent Stem Cell Supervision Committee (HEIPSCRO). The claw cells were analyzed for karyotype using the MDACC's cytogenetics core to confirm chromosomal status and the presence or absence of a normal karyotype. The iPSC status was validated in vitro (self-renewal assay, karyotype analysis) and in vivo (teratoma formation assay) as previously described (for the purpose of describing the teratoma formation assay, Nelakanti, RV, Kooreman, NG & Wu, JC Teratoma formation: a tool for monitoring pluripotency in stem cell research. Curr Protoc Stem Cell Biol 32, 4A 8 1-4A 8 17 (2015). doi.org:10.1002 / 9780470151808.sc04a08s32 is incorporated by reference as if fully described herein for the purpose of describing the teratoma formation assay).

[0170] B. Differentiation of human iPSCs into macrophages or Hi-Macs: In this example, iPSCs were cultured in DMEM medium containing 20% ​​knockout serum replacement solution, 1 mM L-glutamine, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, and 10 ng / ml basic fibroblast growth factor (B-FGF) or mTeSR1 medium. The addition of B-FGF during subculturing was omitted, and differentiation into macrophages was directly induced in the culture dish by adding 10 μM Y-27632 (ROCK inhibitor) (no selection or other formation of embryoid bodies was performed) (Figure 1). After 5 days, X-VIVO 15 (Lonza), containing 1 mM L-glutamine, 1% penicillin-streptomycin, 0.05 mM β-mercaptoethanol, 25 ng / ml IL-3, and 50 ng / ml M-CSF, was added as described in Section 0118. The supernatant / conditioned medium containing hi-MACS was collected and cultured for 7 days in a medium containing 50 ng / ml M-CSF to achieve terminal differentiation (data not shown; see immunocytochemistry shown in Figure 1B of U.S. Provisional Patent Application No. 63 / 495,953). After 7 days, Hi-Macs were selectively detached by exposure to Ca++Mg+-free PBS for 10 minutes. The collected Hi-Macs were pelletized and sent for flow cytometry analysis. Cells can be pelletized by centrifugation at 300 g for 10 minutes at room temperature using a clinical centrifuge. Remaining adherent cells were cultured in X-VIVO 15 (Lonza) containing 1 mM L-glutamine, 1% penicillin-streptomycin, 0.05 mM β-mercaptoethanol, 25 ng / ml IL-3, and 50 ng / ml M-CSF in preparation for the next Hi-Macs harvest.

[0171] Characterization of C. Hi-Mac by flow cytometry The immunophenotype of Hi-Mac was characterized using multicolor flow cytometry (Fortessa X-20). To distinguish between live and dead cells, Live / Dead Fixable Aqua staining was performed first. CD14+CD68+ macrophages were identified using the negative exclusion gating strategy previously reported by our group of researchers (the negative exclusion gating strategy is incorporated herein by reference as if fully described herein: Banerjee, P. et al. Trabectedin Reveals a Strategy of Immunomodulation in Chronic Lymphocytic Leukemia. Cancer Immunol Res 7, 2036-2051 (2019). doi.org:10.1158 / 2326-6066.CIR-19-0152). After excluding CD66b-positive neutrophils, T cells, B cells, and NK cells were excluded using a phylogenetic (Lin) cocktail containing mAbs for CD3, CD19, CD20, and CD56, respectively (Lin positivity rate >60%). Further analysis revealed that up to 40% of Lin-positive cells expressed CD68 positivity, a panmacrophage / phagocytic cell marker (Figure 2A). More than 90% of CD68-positive macrophages lacked CD206-CD163 positivity, a classical tumor-promoting M2 macrophage marker, and were therefore mainly M1 type pro-inflammatory / antitumor macrophages (Figure 2B). Immunocytochemical fluorescence staining revealed that hi-Mac cells derived from iPSC Line 1 (male) and Line 2 (female) expressed the classical macrophage markers IBA-1 and CD68, with the majority of cells showing positivity for both markers (data not shown; see Figure 1B of U.S. Provisional Patent Application No. 63 / 495,953).

[0172] Example 2: IPSC-derived macrophage cell therapy for cancer and inflammatory diseases Procedure: To obtain a patient-specific iPSC source, skin cells, blood, or cranial membrane can be collected from the patient. Cells derived from these samples can be reprogrammed into iPSCs using a published protocol. The iPSCs can then be differentiated into macrophages (MDA-HiMac) using a published protocol. Up to 30 x 10⁶ MDA-HiMacs can be collected from the culture medium once a week for three weeks. Next, lentiviruses containing non-functional truncated human CD19t (for detection of exogenous HiMacs), cDNA encoding herpes simplex virus thymidine kinase (HSV / TK; a suicide gene for eliminating HiMacs, e.g., SEQ ID NO: 5), or a diphtheria toxin-related suicide gene, biologically active human IL-12 (hIL-12) subunits (hp35 and hp40 cDNAs, corresponding to IL-12α and IL-12β subunits, respectively), and cDNA encoding WPRE (woodchuck hepatitis virus post-transcriptional regulator) are introduced into the patient-derived iMacs. As shown in Figures 3A and 3B, these cells are suitable for viral transduction. Examples of vectors that can be used with these elements include pCMV-VSV-G enveloped and adenovirus type 5 vectors. WPRE can be used by real-time PCR to determine the copy number of the incorporated lentivirus. Viral transduction can be titrated to produce fewer than 5 incorporated lentiviral copies per genome (according to FDA guidelines). Biologically active IL-12 secretion can be confirmed by evaluating T cell activation based on IFN-γ release by incubation with autologous CD3+ T cells via ELISA and in vitro. The susceptibility of TK-expressing HiMac to ganciclovir (GCV; 1-10 μM) is also validated in vitro. Delta-24RGD oncolytic virus is also transduced into MDA-HiMac. The virus, including MDA-HiMac, is delivered to patient tumors obtained via iPSCs directly or via arterial access.

[0173] Characteristics: Two MDA-HiMac cell lines were previously generated from human induced pluripotent stem cells using macrophage colony-stimulating factor (M-CSF) and interleukin-3 (IL-3) (as shown in the figure). Immunophenotyping of both MDA-HiMac cells (Line 1 is male, Line 2 is female) was examined by multicolor flow cytometry (Fortessa X-20). Live / dead Fixed Aqua staining was performed first to distinguish between live and dead cells. CD14+CD68+ macrophages were identified by the previously described negative exclusion gating strategy. After excluding CD66b- neutrophils, T cells, B cells, and NK cells were excluded using a lineage (Lin) cocktail containing monoclonal antibodies against CD3, CD19, CD20, and CD56, respectively (>60% Lin-; unpublished data). Further characterization revealed that up to 40% of Lin- cells expressed CD68+ and Iba1, which are pan-macrophage / phagocytic cell markers. More than 90% of CD68+ macrophages lacked the classical tumor-promoting macrophage marker CD206-CD163, indicating that they were primarily pro-inflammatory / anti-tumor macrophages. Based on cell count, the protocol using 6-well plates yielded an average of 4 × 10⁶ cells. 6 This resulted in the generation of 10 HiMacs. This suggests the potential to scale up the protocol to a bioreactor and harvest up to 30 × 10 e6 HiMacs for cell therapy in patients.

[0174] Example 3: Collection and culture of human cranial membrane-derived cranial progenitor cells In short, a 1 x 1 inch human cranial membrane was collected during exposure for brain surgery. The tissue was brought to the laboratory and detached by mechanical and trypsin-assisted enzymatic degradation. The detached tissue was cultured in DMEM / Ham's F12 medium containing 10,000 U / ml collagenase II, 10% human alloserum, 2.5% Hepes, and 1% penicillin / streptomycin solution. Progenitor cells were identified and propagated from a monolayer cultured at the bottom of the culture dish (Figure 5). Progenitor cells were identified by their adherent and proliferative monolayer morphology.

[0175] Example 4: IMACS derived from primary human cranial progenitor cells Progenitor cells can be isolated, cultured, and expanded from the cranial membrane tissue of GBM patients undergoing brain surgery (Figure 5 and Example 3 above). These progenitor cells can be reprogrammed into induced pluripotent stem cells (iPSCs; for example, by the method described in at least Example 1 above). The iPSCs can then be differentiated into macrophages (iMacs). iMac can be transduced with viruses encoding the following cDNAs (e.g., lentivirus or Delta-24RGD oncolytic virus): non-functional cleaved CD19t (for detecting exogenous iMac; e.g., NCBI GenBank ID: AAL57719.1; SEQ ID NO: 3; or human CD19t (SEQ ID NO: 14)); herpes simplex virus thymidine kinase (HSV / TK; suicide gene for eliminating iMac); biologically active human IL-12 (hIL-12) subunits (hp35 and hp40 cDNA, e.g., NCBI GenBank accession numbers: AAD56385.1 and AAD56386.1, respectively; e.g., SEQ ID NOs: 16 and 17); and WPRE (woodchuck hepatitis virus post-transcriptional regulator). The top panel of Figure 6 shows a schematic diagram of the first generation of the construct, and Figure 7 shows a schematic diagram of the second generation of the IL-12 construct, in which only IL-12 expression is Dox-inducible, while CD19t and HSV / TK are constitutive. Biologically active IL-12 secretion can be verified by conventional methods such as ELISA and Western blotting. For example, the first generation IL-12 construct is shown in the lower left and lower right panels of Figure 6. Furthermore, cells can be incubated in vitro with autologous CD3+ T cells to evaluate T cell activation based on IFN-γ release. The sensitivity of TK-expressing iMacs to ganciclovir (GCV; 1-10 μM) can also be verified in vitro.

[0176] Materials and Method of Example 4 In the in vitro assay described above, peripheral-derived CD3+ sorting T cells were activated and cultured in vitro for 48 hours using Dynabeads (CD3 / CD28 costimulation). After activation, the CD3+ T cells were cultured for several days. Dynabeads were released from the T cells, and the activated / cultured T cells were ready for analysis. To test whether IL-12 secreted from the construct induces IFN-γ release from T cells, supernatant from doxycycline-treated cells supplemented with the IL-12 construct was added to T cells in 96-well plates in groups of three for each condition (recombinant IL-12 was added to T cells in a separate well as a positive control). Supernatant was collected from T cells at multiple time points after incubation. IFN-γ in the supernatant was measured using ELISA. In the TK assay, cells stably expressing IL-12-HSV TK were incubated with 0, 1, 5, and 10 μM ganciclovir in a 96-well plate (triple-row) for 24 hours. Cell viability and sensitivity were determined by the percentage of cells remaining in the HSV / TK-expressing wells compared to the untreated wells (0 μM).

[0177] Example 5: Evaluation of the efficacy of IMAC in vivo To replicate the human systemic immune compartment in vivo, for example, NSG-SGM3 immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ) can be used. Unlike conventional NSG mice, NSG-SGM3 mice express human IL-3, CSF2, and KITLG, which promote myeloid cell engraftment. After transplantation of human CD34+ HSCs, NSG-SGM3 mice were shown to have higher engraftment and persistence of dendritic cells, macrophages, B cells, and CD4 / CD8 / FoxP3 regulatory T cells (compared to NSG mice). After sublethal total body irradiation (4 Gy), patient-derived human CD34-positive HSCs (e.g., 5 x 10 per mouse) were transplanted. 4The success or failure of transplantation can be confirmed by injecting (1 x 10) cells into the tail vein of NSG-SGM3 mice and analyzing the immune profile (T cells, B cells, dendritic cells, monocytes / macrophages) using flow cytometry at post-injection (e.g., 6 and 10 weeks after injection). After confirming transplantation, luciferase-expressing human glioblastoma stem cells (GSCs) derived from each patient can be stereotactically transplanted into the cortex of NSG-SGM3 mice (n=20 per GSC strain) at post-HSC transplantation (e.g., 10 weeks later) (e.g., 1 x 10). 5 (individual). Self-iMacs with a constitutive CD19 / HSV TK construct and a Dox-inducible IL-12 construct (Figure 7) were implanted in each mouse (e.g., 16 weeks after HSC implantation and 6 weeks after GSC implantation; e.g., 2 × 10⁶). 5 It can be administered to cells. In this example, the iMac is of human origin, and the mouse is a humanized mouse that has the same human-derived immune system from which the iMac is derived; therefore, the iMac is autologous.

[0178] Subsequently, tumor growth can be monitored, for example, at various time points by weekly bioluminescence imaging (BLI). Changes in the circulating immune cell repertoire can also be monitored using flow cytometry for a standard immune population. To determine the extent to which IL-12 iMacs migrate, maintain antitumor polarity, and recruit activated T cells, animals may be sequentially euthanized, and immunohistochemical staining (e.g., two animals per week), single-cell RNA sequencing (scRNA seq), and peripheral organ (lung, liver, and spleen) can be collected for bioavailability testing. Ganciclovir (GCV) may be administered to animals a few days after treatment (e.g., 3 days post-treatment) (e.g., 100 mg / kg of GCV to 3 mice per group; those skilled in the art can determine doses above and below this range), and brain samples may be collected after a certain period (e.g., 5 days later) to confirm the elimination of iMacs based on anti-CD19 immunohistochemistry (compared to mice not administered GCV). reference TIFF2026515773000002.tif184164

[0179] Materials, compositions, and components that can be used for, in combination with, or in preparation thereof, the disclosed embodiments are disclosed. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and collective combinations and permutations of these compositions are understood to be specifically assumed and described herein, even if not expressly disclosed. For example, where a method is disclosed and discussed, and several modifications that can be made to several molecules included in that method are discussed, all combinations and permutations of the method, and possible modifications, are specifically assumed unless otherwise indicated. Similarly, these subsets or combinations are also specifically assumed and disclosed. This concept applies to all aspects of this disclosure, including but not limited to the steps of a method using the disclosed compositions. Therefore, where various additional steps are possible, each of these additional steps should be considered to be performable using any particular step or combination of steps of the disclosed method, and each of such combinations or subsets of combinations should be considered specifically assumed and disclosed.

[0180] Publications cited herein and materials from which they are cited are incorporated herein by reference in their entirety. The following description provides further non-limiting examples of the disclosed compositions and methods.

[0181] Unofficial sequence list [Table 1] [Table 2] [Table 3] [Table 4] Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21

Claims

1. A method for generating patient-specific macrophages, Reprogramming one or more somatic cells from a sample of the subject to a pluripotent state; Culturing one or more of the aforementioned reprogrammed cells; and Differentiation of one or more of the aforementioned reprogrammed cells into a macrophage-like phenotype, wherein the differentiation proceeds without the formation of embryoid bodies. Methods that include...

2. The method according to claim 1, wherein the reprogramming includes the step of delivering one or more reprogramming expression vectors, each containing nucleic acids encoding OCT3 / 4, SOX2, KFL4, L-MYC, and LIN28, to one or more somatic cells.

3. The method according to claim 2, wherein the reprogramming comprises delivering one or more reprogramming expression vectors containing a nucleic acid encoding shRNA for p53 to one or more somatic cells.

4. The method according to any one of claims 1 to 3, wherein at least one of the one or more expression vectors is an episome expression vector.

5. The method according to any one of claims 1 to 4, comprising expanding culture of one or more of the reprogrammed cells for about 10 to about 25 passages.

6. The method according to any one of claims 1 to 5, wherein the differentiation comprises removing b-FGF from the culture medium and gradually adding IL-3 and M-CSF over a certain period of time.

7. The method according to claim 6, wherein the IL-3 is added at a concentration of about 20 ng / ml to about 30 ng / ml.

8. The method according to claim 6, wherein the M-CSF is added at a concentration of about 45 ng / ml to about 55 ng / ml.

9. The method according to claim 6, wherein the aforementioned period is approximately 15 to approximately 20 days.

10. The method according to any one of claims 1 to 9, wherein the subject is a subject having or suspected of having glioblastoma.

11. The method according to claim 12, wherein the sample includes a cranial membrane from the subject.

12. The method according to claim 12, wherein the sample is enriched with periosteal progenitor cells (PDPCs).

13. Before reprogramming, Subdividing the sample after sampling; The subdivided sample is cultured for a certain period of time; and To isolate one or more somatic cells from the aforementioned subdivided sample. The method according to any one of claims 1 to 14, further comprising:

14. The method according to any one of claims 1 to 15, further comprising delivering one or more treatment expression vectors to one or more differentiated cells after differentiation, wherein the one or more treatment expression vectors comprise therapeutic nucleic acids, each therapeutic nucleic acid independently encoding a cytokine, a checkpoint protein, or a self-destructive protein.

15. The method according to claim 16, wherein the cytokine is any one of interleukin-12 (IL-12), IFN-gamma, or IL-15, or any combination thereof.

16. The method according to claim 17, wherein the cytokine is interleukin-12 (IL-12).

17. The method according to claim 16, wherein the checkpoint protein comprises one or more of PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2.

18. The method according to claim 16, wherein the self-destructing protein is thymidine kinase.

19. The method according to claim 13, wherein a nucleic acid encoding a cytokine is operably linked to an inducible promoter, or the expression of the cytokine requires an inducer or removal of an inhibitory element.

20. The method according to any one of claims 13 to 18, further comprising delivering one or more treatment expression vectors containing nucleic acids encoding detectable markers after differentiation.

21. The method according to claim 19, wherein the detectable marker is CD19t.

22. Manipulated cells comprising the cells described in any one of claims 1 to 20.

23. Manipulated cells comprising the cells described in any one of claims 16 to 20.

24. One or more differentiated cells according to any one of claims 1 to 20; and Pharmacologically acceptable excipients A pharmaceutical composition containing the following:

25. A method of treating an object that requires treatment, A method comprising administering one or more differentiated cells according to any one of claims 1 to 20 to a subject requiring treatment.

26. The method according to claim 24, wherein the subject requiring treatment is a subject who has or is suspected of having glioblastoma, neuroblastoma, or melanoma.

27. The method according to claim 25, wherein the subject requiring treatment is a subject who has or is suspected of having glioblastoma.

28. The method according to any one of claims 25 to 26, wherein the administration is intravenous or intra-arterial.

29. The method according to any one of claims 25 to 27, wherein the administration is intra-arterial administration.

30. The method according to any one of claims 24 to 28, further comprising administering ganciclovir (GCV) to the subject at a time after administration of one or more differentiated cells.

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