TW202227636A - Methods and compositions of infecting, activating, and expanding immune cells - Google Patents

Abstract

Provided are methods and compositions for effectively infect, activate and expanded immune cells, such as natural killer (NK) cells and [gamma][delta] T cells.

Description

Methods and compositions for infecting, activating and expanding immune cells

NK cells have great potential in tumor immunotherapy because they can kill tumor cells directly and rapidly. Redirecting the function of NK cells (CAR-NK) using genetic modification is an effective strategy to overcome the multiple inhibitory receptors expressed on NK cells and enhance the use of targeted therapy. Currently, preclinical studies and clinical studies have shown that NK cells expressing chimeric antigen receptor (CAR) can exert significant anti-tumor effects and their safety is higher than CAR-T cell therapy. See, eg, Wang et al., Int. Immunopharmacol. 2019;74:105695.

However, CAR-NK cell therapy still faces some challenges, such as in vitro expansion and activation of primary NK cells, difficulty in storing and transporting NK cell products, and low transduction efficiency. Accordingly, there remains a need for compositions and methods for efficiently producing NK cells for use in therapy. The present invention satisfies these needs and also provides related advantages.

In one aspect, provided herein are pseudotyped gamma retroviral particles comprising modified RD114 feline endogenous retroviral envelope glycoprotein (RD114TR) and modified baboon envelope glycoprotein (BaEVTR). In some embodiments, the RD114TR glycoprotein comprises, consists essentially of, or further consists of the extracellular and transmembrane domains of the RD114 glycoprotein and the cytoplasmic domain of the amphiphilic murine leukemia virus (MLV-A) glycoprotein consists of it. Additionally or alternatively, the BaEVTR glycoprotein comprises, consists essentially of, or further consists of the extracellular and transmembrane domains of the baboon envelope glycoprotein (BaEV) and the cytoplasmic domain of the MLV-A glycoprotein. In some embodiments, RD114TR and BaEVTR are incorporated into the particle envelope as membrane proteins. In some embodiments, the pseudotyped gamma retroviral particle is selected from the group consisting of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried Murine Embryonic Stem Cell Virus (friend murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus species.

In another aspect, methods of preparing (including but not limited to, infecting, activating or expanding) populations of natural killer (NK) cells are provided. The method comprises culturing, consisting essentially of, or further consisting of one or more immune cell activating agents (eg, NK cell activating agents), the cell population comprising one or more or substantially Consists of or further consists of: NK cells, progenitor cells capable of derivation of NK cells, or stem cells capable of derivation of NK cells. In some embodiments, this culturing step is repeated one, two, three or more times using the same or different one or more immune cell activating agents (eg, NK cell activating agents), or a combination thereof. In some embodiments, the cell population is depleted in the cell population for cells expressing one or more of CD3, CD4, CD8, T cell receptor (TCR) alpha chain, TCR beta chain, or αβTCR.

In some embodiments, the one or more immune cell activators (eg, NK cell activators) are selected from one or more of the following: artificial antigen presenting cells (aAPCs), which express tumor-associated antigens (TAAs), and and/or viral antigens and optionally activate and/or stimulate immune cell growth; one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind stimulatory receptors on one or more of NK cells, progenitor cells or stem cells , thereby activating or proliferating NK cells; one or more interkines thereby activating or proliferating NK cells; or one or more chemical moieties thereby activating or proliferating NK cells.

In one aspect, there is provided a method of preparing a population of γδ T cells, the method comprising culturing a population of cells comprising one or more of the following with one or more immune cell activators (e.g., γδ T cell activators) Either consist essentially of or further consist of: γδ T cells, progenitor cells capable of deriving γδ T cells, or stem cells capable of deriving γδ T cells. In some embodiments, the cell population is depleted in the cell population for cells expressing one or more of the T cell receptor (TCR) alpha chain, TCR beta chain, or alpha beta TCR. In some embodiments, this culturing step is repeated one, two, three or more times using the same or different one or more immune cell activating agents (eg, γδ T cell activating agents), or a combination thereof.

In some embodiments, the one or more immune cell activators (eg, γδ T cell activators) are selected from one or more of the following: artificial antigen presenting cells (aAPCs) that express tumor associated antigens (TAAs) and/or or viral antigens and optionally activate and/or stimulate immune cell growth; one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind to stimulating receptors on one or more of γδ T cells, progenitor cells or stem cells , thereby activating or proliferating γδ T cells; one or more interkines thereby activating or proliferating γδ T cells; or one or more chemical moieties thereby activating or proliferating γδ T cells.

In some embodiments that may relate to any of the aspects of the invention herein, the aAPC further expresses one or more of the following: 4-1BBL, membrane bound (mb) IL-15, mb IL-21, CD64, CD80 , CD83, CD86, OX40L, ICOSL (inducible T cell costimulatory ligand, B7H2, B7RP1), MICA (MHC class I polypeptide-related sequence A), CD 40L, CD137L, mb IL-2, mb IL-18, mbIL -12. mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D superagonist (IL-15SA/IL-15RαSu-Fc; ALT-803) complexed with IL-15RαSushi-Fc fusion protein, or modulating Cell surface markers mediating CD122/CD132 signaling. In one embodiment, the aAPC further expresses mb IL-21 and 4-1BBL.

In some embodiments that may relate to any of the aspects of the invention herein, the interleukin is selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL -2. Low toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D superagonist complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu- Fc; ALT-803 soluble), IL-15, IL-18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1) or MICA. In some embodiments, the cell population is cultured with any one or any two or all three of 100-500 IU/ml IL-2, 20 ng/ml IL-15, or 25 ng/mL IL-21 . In some embodiments, the cell population is cultured with either or both of 50 IU/ml IL-2 and 0.5 ng/ml IL-15. In some embodiments, the cell population is cultured with 50 IU/ml IL-2.

Some embodiments of any of the methods as disclosed herein further include, for example, introducing a polynucleotide into a population of cultured cells for expression before and/or after one or more of the culturing steps as disclosed herein. In some embodiments, the polynucleotide encodes a CAR and/or another therapeutic protein or polypeptide (eg, an antibody or fragment thereof, enzyme, ligand, or receptor).

Some embodiments of any of the methods as disclosed herein further comprise introducing pseudotyped gamma retroviral particles into a population of cultured cells, thereby introducing a polynucleotide as disclosed herein into the cultured cells. In some embodiments, the particle includes RD114TR and BaEVTR as envelope proteins. In some embodiments, the pseudotyped gamma retroviral particle is selected from the group consisting of Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried Murine Embryonic Stem Cell Virus (FMEV), heterophilic MuLB Types of related viruses, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus.

Some embodiments of any of the methods as disclosed herein further include the use of RetroNectin to facilitate the introduction of lentiviral or retroviral vectors (eg, pseudotyped gamma retroviral particles as disclosed herein) comprising polynucleotides as disclosed herein and the proposed introduction Cellular co-localization of polynucleotides. One example is to coat RetroNectin on the inner surface of a container into which a polynucleotide as disclosed herein is introduced into a cell population. RetroNectin is a 63 kD recombinant human fibronectin fragment that enhances lentivirus- and retrovirus-mediated gene transduction. It is available from TaKaRa www.takarabio.com/products/gene-function/t-cell-transduction-and-culture/retronectin-reagent, last accessed September 4, 2020.

Additionally, methods of producing retroviral particles, such as gamma retroviral particles, are provided. The method comprises, consists essentially of, or further consists of: (i) introducing a vector expressing the vector genome into a first packaging cell line suitable for packaging the vector genome into a first retroviral particle , (ii) transducing the first retroviral particle into a second packaging cell line suitable for replicating the first retroviral particle; and (iii) isolating the replicated retroviral particle. In some embodiments, the method further comprises culturing the vector-introduced first packaging cell line. In other embodiments, the method further comprises isolating the first retroviral particle from the culture of the first packaging cell line into which the vector was introduced. Additionally or alternatively, the method further comprises culturing the transduced second packaging cell line. Accordingly, retroviral particles produced by this method and the use of the produced retroviral particles in the production of engineered immune cells, such as immune cells engineered to express a chimeric antigen receptor (CAR), are provided.

In some embodiments that may relate to any of the aspects of the invention herein, the cell population is cultured for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 6 days before the introducing step About 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Additionally or alternatively, the cell population is cultured prior to the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 13 days 14 days, no more than 15 days, no more than 3 weeks or no more than 1 month. In one embodiment, the cell population is cultured for about 5 days to about 10 days prior to the introducing step.

In some embodiments that may relate to any aspect of the invention herein, the cell population comprises, consists essentially of or further consists of any one or more of the following: NK cells, γδ T cells, stem cells, hematopoietic cells Stem cells (HSCs), induced pluripotent stem cells (iPSCs), NK cells derived from any one or more of stem cells, HSCs or iPSCs or γδ derived from any one or more of stem cells, HSCs or iPSCs T cells. Additionally or alternatively, the cell population system is isolated from the individual's umbilical cord blood, the individual's peripheral blood, or the individual's bone marrow.

In one aspect, a method of inhibiting the growth of cancer cells is provided. The method comprises contacting, consisting essentially of, or further consisting of a population of CAR expressing cells made by the methods as disclosed herein with cancer cells. In some embodiments, the antigen recognized by the CAR is expressed by TAA on cancer cells. The contacting step can be performed in vivo or in vitro.

In another aspect, a method of treating cancer in an individual is provided. The method comprises administering, consisting essentially of, or further consisting of a population of CAR expressing cells made by the methods as disclosed herein. In some embodiments, the antigen recognized by the CAR is a TAA expressed by cancer cells.

In yet another aspect, engineered aAPCs expressing an antigen and one or more of the following cell surface markers are provided: 4-1BBL, membrane bound (mb) IL-15, mb IL-21, CD64, CD80, CD83 , CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, and IL-15RαSushi-Fc Fusion protein complexed mb IL-15-N72D superagonist (IL-15SA/IL-15RαSu-Fc; ALT-803) or cell surface markers that mediate CD122/CD132 signaling.

In another aspect, cell populations of NK cells, γδ T cells, or either or both, made by the methods as disclosed herein are provided. In some embodiments, the cells and/or cell populations express a CAR and/or another therapeutic protein or polypeptide (eg, an antibody or fragment thereof, enzyme, ligand, or receptor).

In one aspect, there is provided a composition comprising, consisting essentially of, or further consisting of, a cell as disclosed herein or a population thereof and a carrier, an optionally pharmaceutically acceptable carrier.

In another aspect, there is provided a kit comprising, consisting essentially of, or further consisting of, one or more agents suitable for use in the methods as disclosed herein and optional instructions. In some embodiments, the agent is selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting a cellular phenotype, a Antibodies for the isolation or enrichment or purification of immune cells, primers for the detection of polynucleotides, cytokines and aAPC.

In yet another aspect, viral packaging systems for producing pseudotyped gamma retroviral particles and methods of producing pseudotyped gamma retroviral particles are provided. The system comprises, consists essentially of, or further consists of: (a) a plastid expressing a vector gene body; (b) a packaging plastid; and (c) one or more sets expressing RD114TR and BaEVTR membranoplasts, and the method comprises introducing or consisting essentially of or further consisting of the system into a packaging cell line under conditions suitable for packaging pseudotyped gamma retroviral particles.

The foregoing general description and the following detailed description are exemplary and explanatory and are intended to further explain the claimed disclosure. Other objects, advantages and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and embodiments of the present invention.

Cross-references to related applications

This application claims priority under 35 USC § 119(e) to U.S. Provisional Application Nos. 63/075,651, filed on September 8, 2020, and 63/075,747, filed on September 8, 2020, The contents of each application are incorporated by reference into this application in their entirety. definition

It should be understood that the section or subsection headings used herein are for organizational purposes only and should not be construed to limit and/or isolate the subject matter set forth.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure in light of prior invention.

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA well known to those skilled in the art. See, eg, Sambrook and Russell, eds., (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition; Ausubel et al., eds., (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., N.Y.) series; MacPherson et al, (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al, (1995) PCR 2: A Practical Approach; Harlow and Lane, eds., (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed.; Gait, ed., (1984) Oligonucleotide Synthesis, US Patent No. 4,683,195; Hames and Higgins, eds., (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Edited by Hames and Higgins, (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Edited by Miller and Calos, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); edited by Makrides, (2003) Gene Transfer and Expression in Mammalian Cells; edited by Mayer and Walker, (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Pr ess, London); Herzenberg et al., ed., (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press).

All specified values (eg, pH, temperature, time, concentration, and molecular weight, inclusive) are approximations that vary (+ or -) in increments of 0.1 or 1.0, as appropriate. It should be understood, but not always explicitly stated, that all specified numerical values are preceded by the term "about." It should also be understood, but not always explicitly stated, that the reagents set forth herein are exemplary only and that equivalents of such reagents are known in the art.

As used herein, the term "about" when referring to measurable values such as amounts or concentrations and the like is intended to encompass a change in the specified amount of 20%, 10%, 5%, 1%, 0.5%, or even 0.1%.

As used in the specification and claims, the singular forms "a (a)," "an (an)," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

As will be understood by those skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Additionally, ranges include each individual member, as understood by those skilled in the art.

As used herein, the term "comprising or comprises" means that the compositions and methods include the recited elements, but do not exclude other elements. When using "consisting essentially of" to define compositions and methods, it should be meant to exclude other elements of any essence to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding other ingredients and elements in excess of trace amounts of substantial process steps for administering the compositions of the present invention or process steps for producing the compositions or achieving the desired results. Embodiments defined by each of these transition terms are within the scope of this disclosure.

"Optional" or "optionally" means that the circumstance described subsequently may or may not occur, such that the description includes instances in which the circumstance occurs and instances in which the circumstance does not occur.

As used herein, "and/or" means and encompasses any and all possible combinations of one or more of the associated listed items, and when interpreted in an alternate sense ("or") means no combination .

"Substantially" or "substantially" means almost all or completely, such as 95% or greater of some given amount. In some embodiments, "substantially" or "substantially" means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

In some embodiments, the terms "first," "second," "third," "fourth," or the like in component names are used to distinguish and identify more than one species that share a certain attribute in their names. components. For example, "first cell line" and "second cell line" are used to distinguish two cell lines.

The term "isolated" as used herein with respect to nucleic acids (eg, DNA or RNA) refers to the separation of molecules from other DNA or RNA, respectively, that are present in the source of natural macromolecules. The term "isolated nucleic acid" is intended to encompass nucleic acid fragments that are in the form of non-native fragments and are not found in their natural state. The term "isolated" is also used herein to refer to polypeptides, proteins, viruses, and/or host cells that are separated from other cellular proteins and is intended to encompass purified and recombinant polypeptides, proteins, viruses, and/or host cells. In other embodiments, the term "isolated" is meant to mean components, cells, and other components normally associated with cells, tissues, polynucleotides, peptides, polypeptides, proteins, viruses, antibodies or fragments thereof in nature Component separation. For example, isolated cell lines are cells isolated from tissues or cells with different phenotypes or genotypes. As will be appreciated by those skilled in the art, a non-natural polynucleotide, peptide, polypeptide, protein, virus or antibody or fragment thereof does not necessarily have to be "isolated" to distinguish it from its natural counterpart.

In some embodiments, the term "engineered" or "recombinant" refers to having at least one modification not normally found in native proteins, polypeptides, polynucleotides, strains, wild-type strains, or parental host strains of the mentioned species . In some embodiments, the terms "engineered" or "recombinant" refer to synthesis by human intervention. As used herein, the term "recombinant protein" refers to a polypeptide produced by recombinant DNA techniques, wherein the DNA encoding the polypeptide is typically inserted into a suitable expression vector, which is then used to transform a host cell to produce a heterologous protein.

The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to polymeric nucleotide forms of any length (deoxyribonucleotides or ribonucleotides or their analogs). A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (eg, probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plastids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. Non-nucleotide components can interrupt the nucleotide sequence. Polynucleotides can be further modified after polymerization by, for example, conjugation to labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any polynucleotide embodiment of the invention encompasses the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

A polynucleotide consists of a specific sequence of the following 4 nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and when the polynucleotide is RNA Thymine is replaced by uracil (U). Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This letter indicates that it can be entered into a database of a computer with a central processing unit and used for bioinformatics applications (eg functional genomics and homology searches).

Expression "polynucleotide amplification" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR), and amplification methods. Such methods are known and widely practiced in the industry. See, eg, US Patent Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, PCR procedures describe a method of gene amplification that includes: (i) specific hybridization of primer sequences to a particular gene within a DNA sample (or library), (ii) subsequent rounds involving multiple rounds of DNA polymerase Amplification of annealing, elongation and denaturation, and (iii) screening of PCR products for bands of appropriate size. The primers used are oligonucleotides of sufficient length and appropriate sequence to initiate polymerization, ie, each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

Reagents and hardware for performing PCR are commercially available. Sequences that can be used to amplify from a particular gene region are preferably complementary to, and specifically hybridize to, sequences in the target region or its flanking regions. The nucleic acid sequences generated by amplification can be sequenced directly. Alternatively, amplified sequences can be cloned prior to sequence analysis. Methods for direct selection and sequence analysis of enzymatically amplified gene body segments are known in the art.

"Gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein after transcription and translation.

The term "expression" refers to the production of a gene product, such as mRNA, peptide, polypeptide or protein. As used herein, "expression" refers to the process of transcribing a polynucleotide into mRNA and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, the expression may comprise splicing of mRNA in eukaryotic cells.

"Gene product" or alternatively "gene expression product" refers to an amino acid (eg, a peptide or polypeptide) that is produced when a gene is transcribed and translated. In some embodiments, a gene product can refer to mRNA produced when a gene is transcribed.

As applied to a polynucleotide, the term "encode" means that if the polynucleotide in its native state or when manipulated by methods well known to those skilled in the art can undergo transcription and/or translation to produce a polypeptide and The mRNA of/or a fragment thereof can be considered to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from it.

"Under transcriptional control" (which is also used herein as "directed expression") is a term well understood in the art and indicates that transcription of a polynucleotide sequence (usually a DNA sequence) relies on its operative linkage to facilitate initiation Elements that transcribe or promote transcription. "Operably linked" means that the polynucleotide is configured in a manner that allows it to function in a cell.

The term "regulatory sequence", "expression control element" or "promoter" as used herein means a polynucleus operably linked to a target polynucleotide to be transcribed and/or replicated and which facilitates expression and/or replication of the target polynucleotide Glycosides. A promoter line represents one example of a control element or regulatory sequence. A promoter can be located 5' or upstream of a gene or other polynucleotide to provide a point of control for regulated gene transcription. Polymerase II and III are examples of promoters.

The term "promoter" as used herein refers to any sequence that regulates the expression of a coding sequence (eg, a gene). A promoter can be, for example, constitutive, inducible, repressive, or tissue-specific. A "promoter" is a control sequence and is a region of a polynucleotide sequence that controls the initiation and rate of transcription. It may contain genetic elements that can bind regulatory proteins and molecules such as RNA polymerase and other transcription factors. Non-limiting examples of promoters include the EF1α promoter and the CMV promoter. EF1α sequences are known in the art (see, eg, addgene.org/11154/sequences/, ncbi.nlm.nih.gov/nuccore/J04617, each last accessed March 13, 2019; and Zheng and Baum (2014) Int'l. J. Med. Sci. 11(5):404-408). CMV promoter sequences are known in the art (see, e.g., snapgene.com/resources/plasmid-files/?set=basic_cloning_vectors&plasmid=CMV_promoter, last accessed March 13, 2019; and Zheng and Baum (2014) (see above) arts)). An example is: EF1 alpha promoter sequence: SEQ ID NO: 148 and equivalents thereof as appropriate.

Enhancers are regulatory elements that increase the expression of target sequences. A "promoter/enhancer" is a polynucleotide containing sequences capable of providing both promoter function and enhancer function. For example, the long terminal repeats of retroviruses contain promoter functions and enhancer functions. An enhancer/promoter can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer/promoter is the natural linker to a given gene in the gene body. An "exogenous" or "heterologous" enhancer/promoter is one that is juxtaposed to a gene by means of genetic manipulation (ie, molecular biotechnology) so that transcription of the gene is directed by the linked enhancer/promoter.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence specific manner. The complex can include two strands (forming a double helix), three or more strands (forming a multi-stranded complex), a single self-hybridizing strand, or any combination thereof. The hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be carried out under conditions of varying "stringency". Generally, low stringency hybridization reactions are performed at about 40°C in 10 x SSC or a solution of equivalent ionic strength/temperature. Moderate stringency hybridizations are typically performed in 6 x SSC at about 50°C, and high stringency hybridization reactions are typically performed in 1 x SSC at about 60°C. Hybridization reactions can also be carried out under "physiological conditions" well known to those skilled in the art. A non-limiting example of physiological conditions is temperature, ionic strength, pH, and Mg ²⁺ concentration commonly found in cells.

When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, the reaction is called "annealing" and the polynucleotides are described as "complementary." A double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide provided that hybridization can occur between one strand of the first polynucleotide and the second polynucleotide. "Complementarity" or "homology" (the degree to which one polynucleotide is complementary to another) can be quantified based on the proportion of bases in opposing strands that are expected to hydrogen bond to each other according to well-established base pairing rules .

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in the sequences, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, molecules are homologous at that position. The degree of homology between sequences varies with the number of matches or homologous positions shared by the sequences. "Non-related" or "non-homologous" sequences share less than 40% identity or alternatively less than 25% identity with a sequence of the invention.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (eg 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of another sequence "Sequence identity" means the percentage of bases (or amino acids) that are identical when the two sequences being compared are aligned. This alignment and determination of percent homology or sequence identity can be performed using software programs known in the art, such as those described in Ausubel et al., eds., (2007) Current Protocols in Molecular Biology. Preferably, the comparison is performed using preset parameters. One alignment program uses BLAST with default parameters. Specifically, the formulas are BLASTN and BLASTP using the following preset parameters: genetic code=standard; screen=none; strand=two; cutoff=60; expected=10; matrix=BLOSUM62; description=50 sequences; Classification by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: www.ncbi.nlm.nih.gov/cgi-bin/BLAST. In another embodiment, the program is any of the following: Clustal Omega available at www.ebi.ac.uk/Tools/msa/clustalo/, available at www.ebi.ac.uk Needle (EMBOSS) at /Tools/psa/emboss_needle/, available at www.ebi.ac.uk/Tools/psa/emboss_stretcher/ at Stretcher (EMBOSS), available at www.ebi.ac.uk/Tools Water (EMBOSS) at /psa/emboss_water/, available at www.ebi.ac.uk/Tools/psa/emboss_matcher/ at Matcher (EMBOSS), available at www.ebi.ac.uk/Tools/psa LALIGN at /lalign/. In other embodiments, preset settings are used.

In some embodiments, the polynucleotides as disclosed herein are RNAs. In some embodiments, the polynucleotides as disclosed herein are DNA. In some embodiments, the polynucleotides as disclosed herein are hybrids of DNA and RNA.

In some embodiments, equivalents of the reference nucleic acid, polynucleotide or oligonucleotide encode the same sequence encoded by the reference. In some embodiments, equivalents of the reference nucleic acid, polynucleotide, or oligonucleotide hybridize to a reference, complementary reference, reverse reference, and/or reverse complementary reference, as appropriate, under conditions of high stringency.

Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide has at least 70% or at least 75% or at least 80% sequence identity or alternatively at least 85% to the reference nucleic acid, polynucleotide or oligonucleotide % sequence identity or alternatively at least 90% sequence identity or alternatively at least 92% sequence identity or alternatively at least 95% sequence identity or alternatively at least 97% sequence identity or alternatively at least 98% sequence identity , or alternatively, an equivalent nucleic acid hybridizes to a reference polynucleotide or its complement under high stringency conditions. In one aspect, the equivalents necessarily encode functional proteins that are optionally identifiable by one or more of the assays described herein. Additionally or alternatively, a polynucleotide equivalent encodes a protein or polypeptide having the same or similar function as the reference or parent polynucleotide.

As applied to the production of engineered cells (eg, chimeric antigen receptor cells), the term "transduce or transduction" refers to the process of introducing foreign nucleotide sequences into cells. In some embodiments, this transduction is performed via a vector (eg, a viral or non-viral vector).

As used herein, a restriction enzyme is an enzyme that cleaves DNA into fragments at or near specific recognition sites (referred to as restriction sites) within a molecule. It is used to aid in the insertion of polynucleotides (eg, genes) into plastid vectors during gene cloning and protein production experiments. For optimal application, plastids typically used for genetic selection (such as those encoding viral vector genes) are modified to include short polymeric linker sequences rich in restriction enzyme recognition sequences (called multiple selection sites or MCS) . This provides flexibility when inserting gene fragments into plastid vectors; restriction sites naturally contained within the gene influence the choice of endonucleases for digestion of DNA due to the need to avoid restriction while intentionally cutting DNA ends Expect DNA. To clone the gene fragment into the vector, the plastid DNA and the gene insert are typically cleaved with the same restriction enzymes and then glued together with the aid of enzymes called DNA ligases.

The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to refer to two or more subunit amino acid, amino acid analog, or peptidomimetic compounds. The subunits can be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds (eg, esters, ethers, etc.). A protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids (including glycine and D and L optical isomers), amino acid analogs, and peptidomimetics.

As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and any vertebrate Similar molecules are produced during immune responses in vertebrates, eg, mammals (eg, humans, goats, rabbits, and mice) and non-mammalian species (eg, shark immunoglobulins). Unless specifically stated otherwise, the term "antibody" includes whole immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) and do not substantially Binds to other molecules (for example, antibodies and antibody fragments have binding constants for molecules of interest greater than binding constants to other molecules in the biological sample by at least 10 ³ M ^-1 , greater than at least 10 ⁴ M ^-1 , or greater than at least 10 ^{5M -1} ). The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (eg, murine or humanized non-primate antibodies), heteroconjugated antibodies (eg, bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Owen et al., Kuby Immunology, 7th ed., WH Freeman & Co., 2013; Murphy, Janeway's Immunobiology, 8th ed., Garland Science, 2014; Male et al., Immunology (Roitt), 8th edition, Saunders, 2012; Parham, The Immune System, 4th edition, Garland Science, 2014.

As used herein, the term "monoclonal antibody" refers to an antibody produced by a single B-lymphocyte clone or by cells that have been transfected with the light and heavy chain genes of a single antibody. Monoclonal antibody systems are produced by methods known to those skilled in the art, such as by fusing myeloma cells with immune spleen cells to produce hybrid antibody-forming cells. Monoclonal antibodies include humanized monoclonal antibodies.

In terms of antibody structure, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains constant and variable regions (these regions are also referred to as "domains"). The combination of heavy chain variable region and light chain variable region specifically binds antigen. The light and heavy chain variable regions contain "framework" regions interspersed with three hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The ranges of framework regions and CDRs have been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is now maintained online. Framework region sequences of different light or heavy chains are relatively conserved within species. The framework regions of antibodies (ie, the combined framework regions that make up the light and heavy chains) predominantly adopt a beta-sheet configuration and the CDRs form loops that connect and in some cases form part of the beta-sheet structure. Thus, the framework regions serve to form a framework for positioning the CDRs in the proper orientation through interchain, non-covalent interactions.

The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3 (numbered sequentially from the N-terminus), and are also commonly identified by the chain on which a particular CDR is located (heavy chain regions are labeled CDRH, such as CDRH1, CDRH2, and CDRH3 and light chain regions are labeled CDRLs, such as CDRL1, CDRL2, and CDRL3). Thus, CDRH3 is the CDR3 from which the variable domain of the antibody heavy chain is found, and CDRL1 is the CDR1 from which the variable domain of the antibody light chain is found. For example, TNT antibodies have _VH and _VL region sequences specific to TNT-associated antigens, and thus have specific CDR sequences. Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. Although CDRs vary between antibodies, only a limited number of amino acid positions within CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

As used herein, a crystallizable fragment (Fc) region refers to the tail region of an antibody, which in some embodiments can be used to stabilize the antibody and optionally interact with (eg, bind to) Fc receptors on immune cells or platelets or Binds complement proteins. In some embodiments, Fc mutants can be used, eg, in Fc or its equivalent at a position corresponding to a position in a human IgG4 Fc region (eg, for SEQ ID NO: 81, the corresponding position is SEQ ID NO: One or both or all three of the human IgG4 Fc region mutations F234A, L235A and N297Q are included at amino acids (aa) 16, aa 17 and aa 79) of 81.

Polypeptides or their respective equivalents may be followed by an additional 50 amino acids or alternatively about 40 amino acids or alternatively about 30 amino acids or alternatively about 20 amino acids or alternatively at the carboxy terminus About 10 amino acids or alternatively about 5 amino acids or alternatively about 4 or 3 or 2 or 1 amino acid.

Equivalents thereof include polypeptides having at least 80% amino acid identity with CAR or polypeptides encoded by polynucleotides that hybridize to the complement of a polynucleotide encoding a CAR under high stringency conditions, wherein high stringency conditions include Incubation temperature of about 55°C to about 68°C; buffer concentration of about 1×SSC to about 0.1×SSC; formamide concentration of about 55% to about 75%; and about 1×SSC, 0.1×SSC, or deionized water wash solution.

Alternative embodiments include one or more CDRs of the LC variable region with appropriate CDRs from other antibody CDRs (eg, CDRl, CDR2, CDR3). and includes their respective equivalents. Thus and as an example, CDR1 and CDR2 from the LC variable region can be combined with the CDR3 of the LC variable region of another antibody, and in some aspects, can include an additional 50 amino acids at the carboxy terminus or substitute about 40 amino acids or alternatively about 30 amino acids or alternatively about 20 amino acids or alternatively about 10 amino acids or alternatively about 5 amino acids or alternatively about 4 or 3 or 2 or 1 amino acid. In another aspect, the EGFR CAR is the CAR disclosed in WO 2016/164370.

In one aspect, the term "equivalent" or "bioequivalent" of an antibody means the ability of an antibody to selectively bind its epitope protein or fragment thereof, as measured by ELISA or other suitable method. Bioequivalent antibodies include, but are not limited to, those antibodies, peptides, antibody fragments, antibody variants, antibody derivatives, and antibody mimetics that bind to the same epitope as the reference antibody.

It can be inferred that not expressly recited and unless otherwise desired, when the invention recites polypeptides, proteins, polynucleotides or antibodies, equivalents or bioequivalents of those substances are intended to be within the scope of the invention. As used herein, when referring to a reference protein, antibody, polypeptide or nucleic acid, the term "bioequivalent thereof" is intended to be synonymous with "equivalent thereof" and is intended to mean having minimal homology while still maintaining the desired structure or functional ones. Unless specifically recited herein, any polynucleotide, polypeptide or protein mentioned herein is also considered to include equivalents thereof. For example, equivalents are intended to have at least about 70% homology or identity or at least 80% homology or identity and alternatively or at least about 85% or alternatively at least about 90% to the reference protein, polypeptide or nucleic acid % or alternatively at least about 95% or alternatively 98% percent homology or identity and exhibit substantially equivalent biological activity. Alternatively, when referring to a polynucleotide, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

The term "antibody variant" is intended to encompass antibodies produced in species other than mice. It also includes antibodies that contain post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.

The term "antibody derivative" is intended to encompass molecules that bind an epitope as defined above and are modifications or derivatives of the natural monoclonal antibodies of the invention. Derivatives include, but are not limited to, for example, bispecific antibodies, multispecific antibodies, xenospecific antibodies, trispecific antibodies, tetraspecific antibodies, multispecific antibodies, bivalent antibodies, chimeric antibodies, recombinant antibodies and Humanized Antibodies.

As used herein, the term "specifically binds" means contact between an antibody and an antigen with a binding affinity of at least 10 ⁻⁶ M. In certain aspects, the antibody binds with an affinity of at least about 10 ⁻⁷ M, and preferably 10 ⁻⁸ M, 10 ⁻⁹ M, 10 ⁻¹⁰ M, 10 ⁻¹¹ M or 10 ⁻¹² M.

As used herein, the term "antigen" refers to a compound, composition or substance that can be specifically bound by a particular product of humoral or cellular immunity, such as an antibody molecule or T cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (eg, oligosaccharides), lipids and hormones, and macromolecules (eg, complex carbohydrates (eg, polysaccharides), phospholipids, and proteins). Common antigen classes include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasite antigens, tumor antigens, antigens involved in autoimmune diseases, allergies and graft rejection, toxins, and various other antigens.

In some embodiments, an antigen for a binding moiety (eg, an antibody, antigen-binding fragment thereof, or CAR) may be provided herein as an "antigen" followed by a binding moiety (eg, a BCMA CAR), or with an antigen preceding the antigen "Anti" and have a binding moiety after the antigen (eg, an anti-BCMA antibody), or a binding moiety, then "on" or "against" and then the antigen (eg, an antibody for CS1).

As used herein, the terms tumor-associated antigen (TAA), cancer antigen, tumor antigen, cancer-associated antigen and tumor-associated antigen are used interchangeably herein and refer to antigenic substances of cancer or tumor cells. In some embodiments, TAA is present on some tumor or cancer cells and also optionally on some normal cells at lower levels. In some embodiments, TAA is only presented on tumor or cancer cells, but not normal cells. In some embodiments, the TAA line is selected from the group consisting of G protein coupled receptor class C family 5 member D (GPRC5D), B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild type epidermal growth factor receptor ( EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2 /neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanylinyl cyclase C (GUCY2C), Tumor-Associated Glycoprotein-72 (TAG-72), Thymidine Kinase 1 (TK1), Hypoxanthine Guanine Phosphoribosyltransferase (HPRT1), Cancer/Testis (CT), CD33, Ganglioside G2 (GD2 ), GD3, Tn Ag, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3 , KIT, IL-13Ra2, IL-11Rα, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, Mucin 1 (Muc1), NCAM, Prostatin, PAP, ELF2M, Ephrin B2, Fibroblast Activation Protein Alpha (FAP), IGF-I Receptor, CAIX, LMP2, gp100, bcr-abl, Tyrosinase, Ephrin type A receptor 2 precursor (EphA2), Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO- 1. LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate Survivin, survivin and telomerase, PCTA-1/galectin 8, MelanA/MAR T1, Ras mutant, hTERT, sarcoma translocation cut point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, Human Telomerase Reverse Transcriptase, RU1, RU2, Aspartame Endopeptidase, HPV E6, E7, Enteric Carboxyl Ester Enzyme, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, Glypican 3 (GPC3), FCRL5 or IGLL1.

As used herein, a viral antigen refers to an antigen expressed in a virus and/or encoded by the viral genome. Non-limiting examples include hemagglutinin (HA) and neuraminidase (NA) of influenza virus and the spike, S1, S2, nucleocapsid envelope proteins of COVID-19.

As used herein, the term "antigen binding domain" refers to any protein or polypeptide domain that can specifically bind to an antigen target.

As used herein, the term "autologous" in reference to cells refers to cells that have been isolated and infused back into the same individual (recipient or host). "Allogeneic" refers to non-autologous cells.

The term "chimeric antigen receptor" (CAR), as used herein, refers to a fusion protein comprising moieties: a transmembrane structure that is capable of binding to the extracellular domain of an antigen, a polypeptide derived from a polypeptide other than the polypeptide from which the extracellular domain is derived domain and at least one intracellular domain. A "chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," "T body," or "chimeric immune receptor (CIR)." "Capable of binding to an extracellular domain of an antigen" means any oligopeptide or polypeptide that can bind to an antigen. "Intracellular domain" or "intracellular signaling domain" means any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes in a cell. In certain embodiments, the intracellular domain may also include, consist essentially of, or further include one or more costimulatory signaling domains in addition to the primary signaling domain. "Transmembrane domain" means any oligopeptide or polypeptide that is known to span cell membranes and that can be used to link the extracellular and signaling domains. A chimeric antigen receptor may optionally include a "hinge domain" that serves as a linker between the extracellular domain and the transmembrane domain. Non-limiting examples of such domains are provided herein, eg: Hinge Domain: IgGl Heavy Chain Hinge Coding Sequence: SEQ ID NO: 112. Other non-limiting examples include the IgG4 hinge region, IgD and CD8 domains known in the art. Additional exemplary sequences are provided in the Sequence Listing, for example: Transmembrane Domain: CD28 Transmembrane Region Coding Sequence: SEQ ID NO: 113; Intracellular Domain: 4-1BB Costimulatory Signaling Region Coding Sequence: SEQ ID NO: 114 ; intracellular domain: CD28 costimulatory signaling region coding sequence: SEQ ID NO: 115; and intracellular domain: CD3 zeta signaling region coding sequence: SEQ ID NO: 116.

Other embodiments of each exemplary domain component include other proteins with similar biological functions that share at least 70%, or alternatively at least 80%, of the amino acid sequence with the protein encoded by the nucleic acid sequences disclosed above Identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Additionally, non-limiting examples of such domains are provided herein.

As used herein, the term "CD8 alpha hinge domain" refers to the specific protein fragment associated with this designation and any other molecule with similar biological function that is identical to the CD8 alpha hinge domain sequence as shown herein There is at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Exemplary sequences of CD8 alpha hinge domains of human, mouse and other species are provided in Pinto, R.D. et al. (2006) Vet. Immunol. Immunopathol. 110:169-177. Sequences related to the CD8 alpha hinge domain are provided in Pinto, R.D. et al. (2006) Vet. Immunol. Immunopathol. 110:169-177. Non-limiting examples of such sequences include: human CD8 alpha hinge domain: SEQ ID NO: 117; mouse CD8 alpha hinge domain: SEQ ID NO: 118; and feline CD8 alpha hinge domain: SEQ ID NO: 119 .

As used herein, the term "CD8 alpha transmembrane domain" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that is similar to the CD8 alpha transmembrane structure as shown herein The domain sequences share at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. with amino acid positions 183 to 203 of human T cell surface glycoprotein CD8 alpha chain (GenBank Accession No.: NP_001759.3) or with mouse T cell surface glycoprotein CD8 alpha chain (GenBank Accession No: NP_001074579.1) Additional examples of CD8 alpha transmembrane domains are provided by the fragment sequence associated with amino acid positions 197 to 217 and amino acid positions 190 to 210 of the rat T cell surface glycoprotein CD8 alpha chain (GenBank Accession No.: NP_113726.1) sexual sequence. The sequences associated with each of the listed accession numbers are provided as follows: human CD8 alpha transmembrane domain: SEQ ID NO: 120; mouse CD8 alpha transmembrane domain: SEQ ID NO: 121; and rat CD8 Alpha transmembrane domain: SEQ ID NO: 122.

As used herein, the term "CD28 transmembrane domain" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that shares the CD28 transmembrane domain sequence as shown herein At least 70% or alternatively at least 80% amino acid sequence identity, at least 90% sequence identity or alternatively at least 95% sequence identity. The fragment sequences associated with GenBank Accession Nos: XM_006712862.2 and XM_009444056.1 provide additional non-limiting example sequences for the CD28 transmembrane domain. Sequences associated with each of the listed accession numbers are provided herein.

As used herein, the term "4-1BB co-stimulatory signaling region" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that co-exists with 4-1BB as shown herein The stimulatory signaling region sequences share at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Non-limiting example sequences of the 4-1BB costimulatory signaling region are provided in US Publication 20130266551A1, such as the following example sequence provided: 4-1BB costimulatory signaling region: SEQ ID NO: 123.

As used herein, the term "CD28 costimulatory signaling domain" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that is identical to the CD28 costimulatory signaling domain sequences shown herein There is at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Exemplary sequences of CD28 costimulatory signaling domains are provided in U.S. Patent No. 5,686,281; Geiger, T.L. et al., Blood 98: 2364-2371 (2001); Hombach, A. et al., J Immunol 167: 6123-6131 (2001); Maher, J. et al, Nat Biotechnol 20: 70-75 (2002); Haynes, N.M. et al, J Immunol 169: 5780-5786 (2002); Haynes, N.M. et al, Blood 100 : 3155-3163 (2002). Non-limiting examples include residues 114-220 of the following CD28 sequence: SEQ ID NO: 124 and equivalents thereof.

As used herein, the term "ICOS costimulatory signaling region" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that is associated with the ICOS costimulatory signaling region as shown herein The sequences share at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Non-limiting example sequences of ICOS costimulatory signaling regions are provided in US Publication 2015/0017141A1, and exemplary polynucleotide sequences are provided as follows: ICOS costimulatory signaling region coding sequence: SEQ ID NO: 125.

As used herein, the term "OX40 costimulatory signaling domain" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that is associated with the OX40 costimulatory signaling domain as shown herein The sequences share at least 70% or alternatively at least 80% amino acid sequence identity or alternatively 90% sequence identity or alternatively at least 95% sequence identity. Non-limiting example sequences of OX40 co-stimulatory signaling regions are disclosed in US Publication 2012/20148552A1 and include exemplary sequences provided below: OX40 co-stimulatory signaling regions Coding sequences: ID NO: 126 and equivalents thereof .

Other costimulatory signaling regions can be used, such as those of CD27, CD40, CD40L and/or TLRs. See, eg, those disclosed in US Publication 20160340406A1.

As used herein, the term "CD3 zeta signaling domain" refers to the specific protein fragment associated with this designation and any other molecule with a similar biological function that is similar to the CD3 zeta signaling domain as shown herein The sequences share at least 70% or alternatively at least 80% amino acid sequence identity, preferably 90% sequence identity, more preferably at least 95% sequence identity. Non-limiting example sequences of CD3 zeta signaling domains are provided in US Publication 20130266551A1, eg, SEQ ID NO: 3.

As used herein, a signal peptide (sometimes referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide) refers to the presence at the N-terminus of most newly synthesized proteins that travel to the secretory pathway short peptides (usually 16-30 amino acids long). In one embodiment, the signal peptide is a secretion signal.

Secretory signal means a secretory signal peptide that allows export of proteins from the cytosol into the secretory pathway. Proteins can exhibit varying degrees of successful secretion and generally certain signal peptides can elicit lower or higher degrees when paired with a particular protein. In eukaryotes, signal peptides are strings of hydrophobic amino acids recognized by signal recognition particles (SRPs) in the cytosol of eukaryotic cells. After generation of the signal peptide from the mRNA-ribosome complex, SRP binds the peptide and stops protein translation. SRP then transports the mRNA/ribosome complex into the rough endoplasmic reticulum, where proteins are translated into the ER lumen. The signal peptide is then cleaved from the protein to generate a soluble or membrane-tagged (if the transmembrane region is also present) protein in the endoplasmic reticulum. Such substances are known in the art and are available from suppliers such as Oxford Genetics.

As used herein, a cleavable peptide is also referred to as a cleavable linker, which means a peptide that can be cleaved, for example, by an enzyme. A translated polypeptide that includes the cleavable peptide can yield two final products, thereby allowing expression of more than one polypeptide from one open reading frame. An example of a cleavable peptide is a self-cleaving peptide, such as the 2A self-cleaving peptide. 2A self-lysing peptides are a class of 18-22 aa long peptides that induce lysis of recombinant proteins in cells. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, T2A, E2A, F2A, and BmCPV2A. See, eg, Wang Y et al., 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep. 2015;5:16273 (published on Nov 5, 2015).

As used herein, the terms "T2A" and "2A peptide" are used interchangeably and refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or artificial peptides including those containing the consensus polypeptide motif D-V/ An essential amino acid in the relatively short peptide sequence of I-E-X-N-P-G-P (SEQ ID NO: 177) (approximately 20 amino acids in length for endopretoviruses), where X refers to any amino acid that would normally be considered self-cleaving.

"Detectable label," "label," "detectable label," or "label" are used interchangeably and include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). Detectable labels can also be attached to the polynucleotides, polypeptides, antibodies or compositions described herein.

As used herein, the term "label" or detectable label means a directly or indirectly detectable compound or composition, such as the N-terminus, that is coupled directly or indirectly to the composition to be detected to generate a "labeled" composition Histidine tags (N-His), magnetically active isotopes (eg ¹¹⁵ Sn, ¹¹⁷ Sn and ¹¹⁹ Sn), non-radioactive isotopes (eg ¹³ C and ¹⁵ N), polynucleotides or proteins (eg antibodies). The term also includes sequences coupled to polynucleotides that will provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP) and the like. The label can be detectable by itself (eg, a radioisotope label or a fluorescent label), or in the case of an enzymatic label, the label can catalyze a detectable chemical change in the substrate compound or composition. Labeling may be suitable for small-scale detection or more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). Labels can be easily detected or they can be quantified. Simple-detected responses typically include responses that merely demonstrate presence, while quantified responses typically include responses that have quantifiable (eg, can be reported numerically) values (eg, intensity, polarization, and/or other properties). In a luminescence or fluorescence assay, the luminophore or fluorophore associated with the assay component that actually participates in binding may be used directly or indirectly with another component (eg, a reporter gene or indicator) or fluorophores to generate a detectable reaction. Examples of signal-generating luminescent labels include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response typically includes a change or appearance of a luminescent signal. Suitable methods and luminophores for luminescently labeling analytical components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

As used herein, the term "immunoconjugate" includes combination with a second agent (eg, cytotoxic agent, detectable agent, radioactive agent, targeting agent, human antibody, humanized antibody, chimeric antibody, synthetic antibody, half- Synthetic antibodies or multispecific antibodies) associated or linked antibodies or antibody derivatives.

Examples of suitable fluorescent labels include, but are not limited to, luciferin, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarin Vitamins, Pyrene, Malacite Green, Stilbene, Lucifer Yellow, Cascade Blue™ and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th edition).

In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to cellular components (eg, cell surface markers) present in or on the surface of a cell or tissue. Suitable functional groups include, but are not limited to, isothiocyanate groups, amine groups, haloacetyl groups, maleimide, succinimidyl esters, and sulfonyl halides, all of which can be used to A fluorescent label is attached to the second molecule. The choice of functional group for the fluorescent label depends on the site of attachment to the linker, reagent, label or secondary labeling agent.

As used herein, a purification tag or label refers to a tag that can be used to purify a conjugated molecule or component of the tag, such as an epitope tag (including but not limited to a Myc tag, human influenza hemagglutinin (HA) ) tag, FLAG tag), affinity tag (including but not limited to) glutathione-S transferase (GST), polyhistidine (His) tag, calmodulin binding protein (CBP) or maltose binding protein ( MBP)) or fluorescent labels.

As used herein, a cell surface marker that mediates CD122/CD132 signaling refers to a protein expressed on cells (eg, immune cells) and capable of activating an IL2 pathway (eg, CD122 signaling pathway and/or CD132 signaling pathway) or a polypeptide or another part. CD122 is also known as interleukin-2 receptor subunit beta, and CD132 is also known as interleukin-2 receptor subunit gamma. The ability of the interleukin 2 receptor, which is involved in T cell-mediated immune responses, to bind interleukin 2 exists in three forms. The low affinity form is a monomer of the alpha subunit (also known as CD25) and is not involved in signal transduction. The intermediate affinity form consists of gamma/beta subunit heterodimers, while the high affinity form consists of alpha/beta/gamma subunit heterotrimers. Both the intermediate and high affinity forms of the receptor are involved in receptor-mediated endocytosis and transduction of mitogenic signals from interleukin-2. These proteins also form one of three IL-15 receptor subunits, and CD132 pairs with other ligand-specific receptors to direct lymphocyte responses to interleukins, including IL4, IL7, IL9, and IL21. Activated receptors increase the proliferation of CD8+ effector T cells. See, eg, Noguch et al., Science. 262(5141): 1877-80. Thus, non-limiting examples of cell surface markers that mediate CD122/CD132 signaling include IL2 receptors, IL4 receptors, IL7 receptors, IL9 receptors, IL15 receptors, IL21 receptors, membrane bound IL2, membrane Bound IL4, Membrane bound IL7, Membrane bound IL9, Membrane bound IL15 or Membrane bound IL21. Other recombinant proteins can be used, such as any cell fused to any transmembrane domain and the cytoplasmic domain of CD122, CD132, IL2 receptor, IL4 receptor, IL7 receptor, IL9 receptor, IL15 receptor or IL21 receptor outer domain.

As used herein, interleukin (IL) refers to the interleukin that is first expressed by white blood cells (leukocytes). The function of the immune system depends mainly on interleukins. Most interleukins are synthesized by helper CD4 T lymphocytes and by monocytes, macrophages, and endothelial cells. It promotes the development and differentiation of T and B lymphocytes and hematopoietic cells. As used herein, an interleukin can be a soluble interleukin secreted from a cell and/or a membrane-bound (mb) interleukin expressed on the cell surface. One skilled in the art can transform the soluble and membrane-bound forms of the interleukin, eg, by engineering the interleukin's transmembrane domain and/or signal peptide.

Interleukin-2 (IL-2) is an interleukin, a class of interleukin signaling molecules in the immune system. It is a 15.5-16 kDa protein that regulates the activity of white blood cells (leukocytes, usually lymphocytes) responsible for immunity. In some embodiments, L-2 is human IL-2. In some embodiments, the IL-2 is derived from other species, such as chimpanzee IL-2 with the NCBI reference sequence of XP_517425.1. Non-limiting exemplary sequences of this protein or potential gene can be found in the following Gene Card IDs: GC04M122451, HGNC (6001), NCBI Entrez Gene (3558), Ensembl (ENSG00000109471), OMIM® (147680), UniProtKB/Swiss-Prot ( P60568) and Open Targets form (ENSG00000109471), each of which is incorporated herein by reference in its entirety.

Thus, in some embodiments, IL-2 comprises or consists essentially of or further consists of MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 178). In some embodiments, IL-2 comprises, consists essentially of, or further consists of amino acids (aa) 21 to aa 153 of SEQ ID NO: 178.

In some embodiments, the IL-2 used herein is wild-type IL-2 or an equivalent thereof. In some embodiments, IL-2 as used herein is produced in vitro by host cells (eg, HEK 293 cells or CHO cells or E. coli or Pichia pastoris) Recombinant IL-2. See, eg, human IL-2 sold by Sigma-Aldrich (SRP3085, SRP6170, I7908, I2644, H7041 or I17002) and STEMCELL ^™ Technologies (78036). In other embodiments, the IL-2 equivalent stimulates the proliferation of NK cells or activates their cytotoxic function or both, which is significantly similar to wild-type IL-2. Assays for assessing the proliferative and cytotoxic function of NK cells, such as ex vivo culture and cell counting, are available to those skilled in the art (see, eg, Choi et al, J Immunother Cancer. 2019 Jul 5;7(1):168) , ⁵¹ Chromium release analysis (for example, see Dong et al., Cancer Discov. 2019 Oct; 9(10): 1422-1437. doi: 10.1158/2159-8290. CD-18-1259. Epub 2019 Jul 24), based on colorimetry Measured cytotoxicity assays (see, eg, Chava et al., J Vis Exp. 2020 Feb 22;(156):10.3791/60714) or flow cytometry-based cytotoxicity assays (see, eg, Kim et al., Front Immunol. 2020 Aug 14;11:1851). In some embodiments, IL-2 equivalents comprise, consist essentially of, or further consist of fragments of wild-type IL-2 (eg, aa 22 to aa 153 of SEQ ID NO: 179). Additionally or alternatively, IL-2 equivalents include or consist essentially of or further consist of variants of wild-type IL-2 or a fragment thereof, the variant being, for example, adding additional methylsulfide at the N-terminus The amino acid may have one or more of the following mutations: the amino acid residue corresponding to aa 38 of SEQ ID NO: 178 optionally mutated to methionine, the amino acid corresponding to aa 39 of SEQ ID NO: 178 The residue is optionally mutated to serine, the amino acid residue corresponding to aa 58 of SEQ ID NO: 178 is optionally mutated to alanine or lysine, the amino acid corresponding to aa 62 of SEQ ID NO: 178 The acid residue is mutated to lysine or isoleucine or alanine or glutamic acid as appropriate, the amino acid residue corresponding to aa 65 of SEQ ID NO: 178 is mutated to aspartic acid or Glutamic acid or alanine or arginine, the amino acid residue corresponding to aa 82 of SEQ ID NO: 178 optionally mutated to leucine or alanine, the amine corresponding to aa 88 of SEQ ID NO: 178 The amino acid residue is optionally mutated to valine, the amino acid residue corresponding to aa 145 of SEQ ID NO: 178 is optionally mutated to serine or alanine, or any combination thereof.非限制性實例包含由PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 179)組成之阿地介白素(Aldesleukin)、由MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 180)組成之替西介白素(Teceleukin)、Neoleukin 2/15 ( See, eg, Silva et al., Nature. 2019 Jan;565(7738):186-191) and those disclosed in US9206243B2 or US8012465B2. Additionally or alternatively, IL-2 equivalents include, consist essentially of, or further consist of IL-2 derivatives (eg, modified by glycosylation, acetylation or phosphorylation).

Interleukin-15 (IL-15) is an interleukin that is structurally similar to interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2/IL-15 receptor beta chain (CD122) and a common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by the virus. This cytokine induces the proliferation of natural killer cells. In some embodiments, the IL-15 is human IL-15. Non-limiting exemplary sequences of this protein or potential gene can be found in the following Gene Card IDs: GC04P141636, HGNC: 5977, NCBI Entrez Gene: 3600, Ensembl: ENSG00000164136, OMIM®: 600554 or UniProtKB/Swiss-Prot: P40933, among others The entire contents of each are incorporated herein by reference. In some embodiments, the IL-15 is human IL-15 isoform 1. Thus, in some embodiments, IL-15 comprises, consists essentially of, or consists further of, MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 181). In some embodiments, IL-15 comprises, consists essentially of, or further consists of amino acids (aa) 30 to aa 162 of SEQ ID NO: 181. In some embodiments, the IL-15 is human IL-15 isoform 2. Thus, in some embodiments, IL-15 comprises, consists essentially of, or further consists of MVLGTIDLCSCFSAGLPKTEANWVNVISDLKKIELDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 182). In some embodiments, IL-15 is derived from other species, such as rhesus IL-15 with UniProtKB reference ID: P48092.

In some embodiments, the IL-15 used herein is wild-type IL-15 or an equivalent thereof. In some embodiments, IL-15 as used herein is recombinant IL-15 produced in vitro by host cells (eg, HEK 293 cells or E. coli). See, eg, human IL-15 sold by Sigma-Aldrich (SRP6293 or SRP3077) and STEMCELL ^™ Technologies (78031). In other embodiments, the IL-15 equivalent stimulates the proliferation of NK cells or activates their cytotoxic function or both, which is significantly similar to wild-type IL-15. Assays for assessing the proliferative and cytotoxic function of NK cells are available to those skilled in the art, and are described herein. Non-limiting examples of IL-15 equivalents include those disclosed in US20190263877A1, US10450359B2, and US10537615B2. Additionally or alternatively, IL-15 equivalents include, consist essentially of, or further consist of IL-15 derivatives (eg, modified by glycosylation, acetylation or phosphorylation).

Interleukin-21 (IL-21) (also referred to herein as the IL-21 polypeptide) is an inhibitor of immune system cells, including natural killer (NK) cells and cytotoxic T cells that can destroy virally infected or cancerous cells. ) interferon with potent regulatory effect. This cytokine induces cell division/proliferation in its target cells. In some embodiments, the IL-21 is human IL-21. Non-limiting exemplary sequences of this protein or potential gene can be found in the following Gene Card IDs: GC04M122612, HGNC: 6005, NCBI Entrez Gene: 59067, Ensembl: ENSG00000138684, ^OMIM® : 605384 and UniProtKB/Swiss-Prot: Q9HBE4, among others The entire contents of each are incorporated herein by reference.

In other embodiments, the IL-21 equivalent stimulates the proliferation of immune cells (eg, NK cells) or maintains their viability or both, which is significantly similar to wild-type IL-21. Those skilled in the art can utilize assays for assessing cell proliferation and viability, such as ex vivo culture and cell counting or live/dead cell staining (eg, tetrazolium reduction assay, resazurin reduction assay, protease viability markers). analysis, ATP analysis or live cell real-time analysis). See, eg, Choi et al, J Immunother Cancer. 2019 Jul 5;7(1):168; and Riss et al, Cell Viability Assays. 2013 May 1; Edited by Markossian et al, Assay Guidance Manual. Bethesda (MD) : Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004; available May 14, 2021 at www.ncbi.nlm.nih.gov/books/NBK144065/.

CD122 is a receptor for interleukin-2 and is also known as interleukin-2 receptor subunit beta. This beta subunit is involved in receptor-mediated endocytosis and transduces the mitogenic signal of IL2. It most likely associates with IL15RA, which is involved in the stimulation of neutrophil phagocytosis by IL15. Non-limiting exemplary sequences of this protein or potential genes or suitable antibodies for detection of the protein can be found in the following Gene Card IDs: GC22M037125, HGNC: 6009, NCBI Entrez Gene: 3560, Ensembl: ENSG00000100385, OMIM®: 146710 or UniProtKB /Swiss-Prot: P14784, each of which is incorporated herein by reference in its entirety.

As used herein, a position of an amino acid (aa) or nucleotide (nt) residue in a sequence of interest that "corresponds to" an identified position in a reference sequence refers to a process between the sequence of interest and the reference sequence. This residue position aligns with the identified position when the sequences are aligned. Such sequence alignments can be performed using various programs, such as Clustal Omega and BLAST.

In some embodiments, the term "vector" means a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly dividing cells and integrate into the genome of a target cell.

A "plastid" is an extrachromosomal DNA molecule isolated from chromosomal DNA that is capable of replicating independently of chromosomal DNA. In many cases it is round and double stranded. Plastids provide a mechanism for horizontal gene transfer within a microbial population and generally provide selectivity advantages under given environmental conditions. The plastids can carry genes for resistance to natural antibiotics in competing environmental niches, or alternatively the produced proteins can be used as toxins in similar circumstances. Many plastids are commercially available for these uses. Insertion of the to-be-replicable gene into a copy of the plastid containing the gene that makes the cell resistant to a specific antibiotic and a multivariate site (MCS or polylinker) that contains a number of genes that allow easy insertion at this location A shorter region of commonly used restriction sites for DNA fragments. Another major use of plastids is in the production of large quantities of proteins. In this case, the investigators grow bacteria containing plastids with the gene of interest. Just as bacteria produce proteins to confer antibiotic resistance on them, they can also be induced to produce large amounts of proteins from inserted genes. This is a cheap and easy way to mass produce the gene or the protein that it then codes for.

A "viral vector" is defined as a recombinantly produced virus or viral particle that includes a polynucleotide intended for delivery into a host cell in vivo , ex vivo, or in vitro. As known to those skilled in the art, there are 6 types of viruses. DNA viruses constitute class I and class II. RNA viruses and retroviruses constitute the remaining species. Class III viruses have double-stranded RNA genomes. Class IV viruses have a positive single-stranded RNA gene body, which itself serves as mRNA. Class V viruses have a negative single-stranded RNA gene body that serves as a template for mRNA synthesis. Class VI viruses have positive single-stranded RNA genomes, but DNA intermediates in both replication and mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, upon virus infection of a cell, the RNA is reverse transcribed into a form of DNA that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

In several embodiments, the vector system is derived from or based on a wild-type virus. In other embodiments, the vector system is derived from or based on wild-type adenovirus, adeno-associated virus, or retrovirus (eg, gamma retrovirus and/or lentivirus). Examples of retroviruses include, but are not limited to, Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV) or Fried Murine Embryonic Stem Cell Virus (FMEV), Human Immunodeficiency Virus (HIV), Equine Infectious Anemia Virus (EIAV), Simian Immunodeficiency Virus (SIV) and Feline Immunodeficiency Virus (FIV). Viral vectors may include components derived from two or more different viruses, and may also include synthetic components. Vector components can be manipulated to achieve desired properties, such as target cell specificity.

The recombinant vectors of the present invention can be derived from primates and non-primates. Examples of primate lentiviruses include human immunodeficiency virus (HIV), human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "lentivirus" Visna/maedi virus (VMV) and related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia Virus (EIAV) and recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV). Recombinant lentiviral vectors of the prior art are known in the art, eg, see US Pat. Nos. 6,924,123, 7,056,699, 7,419,829, and 7,442,551, which are incorporated herein by reference. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. In other embodiments, the lentiviral vector has the U3 region lacking the TATA box. Additionally or alternatively, the lentiviral vector has a U3 region lacking one or more transcription factor binding sites.

Retroviruses (eg, gamma retroviruses and/or lentiviruses) include (a) envelopes, which include lipids and glycoproteins; (b) vector genomes, which are RNAs (usually at 5 A dimer RNA including a cap at the ' end and a poly A tail at the 3' end flanked by an LTR); (c) a capsid; and (d) a protein such as a protease. US Patent No. 6,924,123 discloses that certain retroviral sequences facilitate integration into target cell genomes. This patent teaches that each retroviral genome includes genes called gag, pol, and env, which encode virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). LTR is responsible for proviral integration and transcription. It also serves as an enhancer-promoter sequence. In other words, LTR can control the expression of viral genes. Retroviral RNA is encapsidated by means of a psi sequence located at the 5' end of the viral genome. The LTR itself is an identical sequence that can be divided into three elements, referred to as U3, R, and U5. U3 is a unique sequence derived from the 3' end of RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a unique sequence at the 5' end of the RNA. The size of the three elements can vary widely among different retroviruses. For the viral genome, the poly(A) addition (termination) site is located at the border between R and U5 in the right hand LTR. U3 contains most of the transcriptional control elements of the provirus, including a promoter and multiple enhancer sequences responsive to cellular and (in some cases) viral transcriptional activating proteins.

In terms of the structural genes gag, pol and env themselves, gag encodes an internal structural protein of the virus. Gag proteins are proteolytically processed into mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes a reverse transcriptase (RT) that contains DNA polymerase, the related RNase H, and integrase (IN) that mediate gene body replication. In some embodiments, one or more of the structural genes are provided by the packaging cell (also referred to herein as a host cell) that produces the viral vector, rather than in the vector genome itself.

To generate viral vector particles, the vector RNA genome is expressed in a host cell from the DNA construct encoding it. Particle components not encoded by the vector genome are provided in trans by other nucleic acid sequences expressed in the host cell ("packaging systems", which typically include either or both of the gag/pol and env genes). The set of sequences required for the production of viral vector particles can be introduced into a host cell by transient transfection, or it can be integrated into the host cell genome, or it can be provided in a mixed fashion. The techniques involved are known to those skilled in the art.

Gamma retrovirus is a genus of the family Retroviridae and can be used in the disclosure herein. Exemplary Species Lines Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried Murine Embryonic Stem Cell Virus (FMEV), Heterophilic MuLB Associated Virus, Feline Sarcoma Virus, Heterophilic Murine Leukemia virus-related virus (XMRV) and feline leukemia virus. Gamma retroviruses are spherical, enveloped virions between 80-100 nm in diameter. It contains nucleocapsid, reverse transcriptase, integrase, capsid, protease, envelope and surface units. The nucleocapsid is the nucleic acid-protein assembly within the virion and is a substructure of the virion. Reverse transcriptase is the enzyme responsible for converting RNA to DNA during the virion replication cycle. Integrase is used with reverse transcriptase to convert RNA to DNA. The gene system of gamma retrovirus is a single-stranded RNA (+) gene body of approximately 8.3 kb in size. It has a 5' cap and a 3' poly A tail, and it contains two long terminal repeats at the 5' and 3' ends. The isometric terminal repeats have U5, R and U3 regions as well as a polypurine region (at the 3' end) and a primer binding site (at the 5' end). A typical gamma retrovirus genome contains the gag, pol, and env genes, all of which can be omitted from the gene therapy vector. The capsid is the protein capsid that surrounds the gene body of a virus particle and whose primary function is to protect the gene body and deliver it to the host cell. The viral envelope is the membrane surrounding the viral capsid, which is a lipid bilayer derived from the host cell. As a potential vector for gene therapy, gamma retrovirus has several advantages over HIV as a lentiviral vector. In particular, the gamma reverse transcription packaging system need not incorporate any sequences that overlap with the coding sequences of gag, pol or helper genes. See, eg, Tobias Maetzig et al., Viruses. 2011 Jun;3(6):677-713. Epub 2011 Jun 3.

As used herein, pseudotyping is the process of producing a virus or a combination of a viral vector and a foreign viral envelope protein. This produces pseudotyped virus particles (also known as pseudoviruses). Using this approach, foreign viral envelope proteins can be used to alter host tropism or increase or decrease the stability of viral particles. Pseudotyped particles do not carry the genetic material for the production of other viral envelope proteins, so that phenotypic changes cannot be transmitted to progeny viral particles. In some cases, the inability to produce the viral envelope protein results in the inability of the pseudovirus to replicate. In this way, the properties of dangerous viruses can be studied in lower risk settings. Pseudotyping allows control over the expression of envelope proteins. A commonly used protein is glycoprotein G (VSV-G) from Vesicular stomatitis virus (VSV), which mediates entry through the LDL receptor. Envelope proteins incorporated into pseudoviruses allow the virus to readily enter into different cell types with corresponding host receptors.

Retroviruses use specific receptors for binding and entry into cells; both RD114-containing retroviruses and BaEV envelope-containing retroviruses use the neutral amino acid (aa) transporter (ASCT2). The RD114 pseudotyped viral vector uses only the sodium-dependent neutral amino acid transporter (ASCT2) as its entry receptor, whereas BaEV uses both receptors (ASCT1 and ASCT2) for cell entry. ASCT1 and ASCT2 mRNA were more abundant in both IL-15-NK cells and IL-21-NK cells than in freshly isolated NK and this may be the reason why the BaEV line is a better retroviral pseudotype for NK transduction (Colmartino et al. , Front Immunol. 2019 Dec 16;10:2873). Here, Applicants develop retroviral production cell lines expressing the BaEV and RD114 envelopes that can produce viruses with two envelopes on each virion, thereby facilitating access to DNA on target cells by two receptors to increase transduction efficiency.

As disclosed herein, two foreign recombinant envelope proteins of gamma retroviruses, the modified RD114 feline endogenous retroviral envelope glycoprotein (RD114TR) and the modified baboon envelope glycoprotein (BaEVTR), were used . The transduction efficiency of human primary lymphocytes depends on the type of envelope protein used to coat the retroviral vector, and activated NK cells highly express the receptor neutral amino acid transporter A (SLC1A4, also referred to herein as ASCT-1) and neutral amino acid transporter B(0) (SLC1A5, also referred to herein as ASCT-2), these receptors are used to enter the target baboon envelope glycoprotein (BaEV-TR) . The ASCT-2 receptor is used to enter the feline endogenous retroviral envelope glycoprotein (RD114TR) into cells.

The term "entry receptor" as used herein means a receptor between a virus (eg, a retrovirus) and a cell that causes membrane fusion by binding to the virus (eg, the viral envelope glycoprotein). In some embodiments, the retroviral entry receptors ASCT1, ASCT2, and Pit1 are expressed on cells of the human hematopoietic system. In other embodiments, any one or any two or any three of ASCT1, ASCT2 or Pit1 are expressed on activated and proliferating NK and T cells, which greatly improves the protection of viruses comprising the corresponding envelope glycoproteins Transduction efficiency.

Alanine/serine/cysteine/threonine transporter 1 (ASCT1) is also known as solute carrier family 1 member 4 (SLC1A4) or neutral amino acid transporter A. In some embodiments, ASCT1 is human ASCT1. Non-limiting exemplary sequences of this protein or potential gene can be found in Gene Card ID: GC02P064988, HGNC: 10942, NCBI Entrez Gene: 6509, Ensembl: ENSG00000115902, OMIM®: 600229 or UniProtKB/Swiss-Prot: P43007, each of which The entire contents of one are incorporated herein by reference.

Type 2 sodium-dependent neutral amino acid transporter (ASCT2) is also known as solute carrier family 1 member 5 (SLC1A5) or neutral amino acid transporter B(0). It is RD114/simian D-type retrovirus receptor, baboon M7 virus receptor and RD114 virus receptor. In some embodiments, the ASCT2 is human ASCT2. Non-limiting exemplary sequences of this protein or potential gene can be found in Gene Card IDs: GC19M051483, HGNC: 10943, NCBI Entrez Gene: 6510, Ensembl: ENSG00000105281, OMIM®: 109190 or UniProtKB/Swiss-Prot: Q15758, each of which The entire contents of one are incorporated herein by reference.

Phosphate transporter 1 (Pit1 or PiT-1 or PiT1) is also known as solute carrier family 20 member 1 (SLC20A1) or sodium-dependent phosphate transporter 1 or gibbon leukemia virus receptor 1 or leukemia virus receptor 1 homolog. It is a retroviral receptor that renders human cells susceptible to infection by gibbon leukemia virus, simian sarcoma-associated virus, feline leukemia virus subgroup B, and 10A1 murine leukemia virus. In some embodiments, the Pit1 is human Pit1. Non-limiting exemplary sequences of this protein or potential gene can be found in Gene Card ID: GC02P118061, HGNC: 10946, NCBI Entrez Gene: 6574, Ensembl: ENSG00000144136, OMIM®: 137570 or UniProtKB/Swiss-Prot: Q8WUM9, each of which The entire contents of one are incorporated herein by reference.

The terms "4-1BBL", "tumor necrosis factor superfamily member 9", "TNFSF9" or "4-1BBL polypeptide" are found on APCs (antigen presenting cells) and bind to 4-1BB (also known as CD137). Type 2 transmembrane glycoprotein receptor. The 4-1BB/4-1BBL complex belongs to the TNFR:TNF superfamily and is expressed on activated T lymphocytes. In some embodiments, the 4-1BBL is human 4-1BBL. Non-limiting exemplary sequences of this protein or potential gene can be found in Gene Card ID: GC19P006531, HGNC: 11939, NCBI Entrez Gene: 8744, Ensembl: ENSG00000125657, OMIM®: 606182 or UniProtKB/Swiss-Prot: P41273, each of which The entire contents of one are incorporated herein by reference.

CD56 (also known as neural cell adhesion molecule 1 (NCAM)) is a cell adhesion protein and member of the immunoglobulin superfamily that is involved in cell-cell and cell-matrix interactions during development and differentiation. It has been used as an NK cell marker. See, eg, Freud et al., Immunity. 2017 Nov 21;47(5):820-833. Non-limiting exemplary sequences of this protein or potential genes or suitable antibodies for detection of the protein can be found in Gene Card IDs: GC11P112961, HGNC: 7656, NCBI Entrez Gene: 4684, Ensembl: ENSG00000149294, OMIM®: 116930 or UniProtKB/Swiss - Prot: P13591, each of which is incorporated herein by reference in its entirety. In humans, two major subgroups of NK cells can be distinguished and characterized by distinct expressions of the adhesion molecule CD56 and the low affinity Fc receptor CD16 (FcyRIIIa). They are commonly referred to as CD56 ^bright and CD56 ^dim NK cells. CD56 ^dim NK cells predominate in peripheral blood, while CD56 ^bright NK cells constitute the majority of NK cells in secondary lymphoid tissues (eg, lymph nodes) and in several organ tissues (eg, liver, uterus, and kidney).

Non-limiting exemplary sequences of proteins or potential genes referred to herein or suitable antibodies for detection of proteins can be found in publicly available databases such as gene cards available at www.genecards.org/ or available at UniProtKB at www.uniprot.org/uniprot.

In some embodiments, RD114TR comprises, or instead consists essentially of or further consists of the extracellular and transmembrane domains of the RD114 glycoprotein and the cytoplasmic domain of the amphiphilic murine leukemia virus (MLV-A) glycoprotein consists of it. See, eg, Sandrin et al., Blood (2002) 100(3): 823-832.在一些其他實施例中，RD114TR包括MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLATNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFNRLVQFVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 172)或其等效物或替代地基本上由其組成或進一步由其組成。

In some embodiments, the BaEVTR comprises or alternatively consists essentially of or further consists of the extracellular and transmembrane domains of baboon envelope glycoprotein (BaEV) and the cytoplasmic domain of MLV-A glycoprotein. See, eg, Girard-Gagnepain, Blood (2014) 124(8): 1221-1231.在其他實施例中，BaEVTR包括MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSN TSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLPYLLPFLGPLLTLLLLLTIGPCIFNRLVQ FVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 173)或其等效物或替代地基本上由其組成或進一步由其組成。

在一些實施例中，野生型MLV-A包括MAARSTLSKPPQDKINPWKPLIVMGVLLGVGMAESPHQVFNVTWRVTNLMTGRTANATSLLGTVQDAFPKLYFDLCDLVGEEWDPSDQEPYVGYGCKYPAGRQRTRTFDFYVCPGHTVKSGCGGPGEGYCGKWGCETTGQAYWKPTSSWDLISLKRGNTPWDTGCSKVACGPCYDLSKVSNSFQGATRGGRCNPLVLEFTDAGKKANWDGPKSWGLRLYRTGTDPITMFSLTRQVLNVGPRVPIGPNPVLPDQRLPSSPIEIVPAPQPPSPLNTSYPPSTTSTPSTSPTSPSVPQPPPGTGDRLLALVKGAYQALNLTNPDKTQECWLCLVSGPPYYEGVAVVGTYTNHSTAPANCTATSQHKLTLSEVTGQGLCMGAVPKTHQALCNTTQSAGSGSYYLAAPAGTMWACSTGLTPCLSTTVLNLTTDYCVLVELWPRVIYHSPDYMYGQLEQRTKYKREPVSLTLALLLGGLTMGGIAAGIGTGTTALIKTQQFEQLHAAIQTDLNEVEKSITNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYADHTGLVRDSMAKLRERLNQRQKLFETGQGWFEGLFNRSPWFTTLISTIMGPLIVLLLILLFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKPLEYEP (SEQ ID NO: 174)或替代地基本上由其組成或進一步由其組成。 In other embodiments, the signal peptide of wild-type MLV-A comprises or alternatively consists essentially of or further consists of MAARSTLSKPPQDKINPWKPLIVMGVLLGVGMA (amino acids (aa) 1 to aa 33 of SEQ ID NO: 174). In some embodiments, the ectodomain domain of wild-type MLV-A comprises or alternatively consists essentially of or further consists of aa 1 to aa 599 of SEQ ID NO: 174. In some embodiments, the transmembrane region of wild-type MLV-A comprises or alternatively consists essentially of or further consists of LISTIMGPLIVLLLILLFGPCIL (aa 600 to aa 622 of SEQ ID NO: 174). In some embodiments, the cytoplasmic domain of wild-type MLV-A comprises or alternatively consists essentially of or further consists of NRLVQFVKDRISVVQAL (aa 623 to aa 639 of SEQ ID NO: 174). In some embodiments, the cytoplasmic domain wild-type of MLV-A comprises or alternatively consists essentially of or further consists of NRLVQFVKDRISVVQALVLTQQYHQLKPLEYEP (aa 623 to aa 655 of SEQ ID NO: 174).

In some embodiments, when referring to a protein, BaEV means the envelope glycoprotein of the baboon endogenous virus. Non-limiting exemplary sequences of this protein can be found in UniProtKB - P10269 or NCBI Reference Sequence: YP_009109691.1, each of which is incorporated herein by reference in its entirety.在一些實施例中，野生型BaEV包括MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLPYLLPFLGPLLTLLLLLTIGPCIFNRLTAFINDKLNIIHAMVLTQQYQVLRTDEEAQD (SEQ ID NO: 175)或替代地基本上由其組成或進一步由其組成。 In other embodiments, the signal peptide of wild-type BaEV comprises or alternatively consists essentially of or further consists of MGFTTKIIFLYNLVLVYA (amino acids (aa) 1 to aa 18 of SEQ ID NO: 175). In some embodiments, the ectodomain domain of wild-type BaEV comprises or alternatively consists essentially of or further consists of aa 1 to aa 506 of SEQ ID NO: 175. In some embodiments, the transmembrane region of wild-type BaEV comprises or alternatively consists essentially of or further consists of YLLPFLGPLLTLLLLLTIGPCIF (aa 507 to aa 529 of SEQ ID NO: 175). In some embodiments, the cytoplasmic domain of wild-type BaEV comprises or alternatively consists essentially of or further consists of NRLTAFINDKLNIIHAM (aa 530 to aa 546 of SEQ ID NO: 175). In some embodiments, the cytoplasmic domain of wild-type BaEV comprises or alternatively consists essentially of or further consists of NRLTAFINDKLNIIHAMVLTQQYQVLRTDEEAQD (aa 530 to aa 563 of SEQ ID NO: 175).

In some embodiments, when referring to a protein, RD114 means the envelope glycoprotein of the RD114 retrovirus. Non-limiting exemplary sequences of this protein can be found in GenBank: CAA61093.1, CBI Reference Sequence: YP_001497149.1 or GenBank: BAM17308.1, each of which is incorporated herein by reference in its entirety.在一些實施例中，野生型RD114包括MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLATNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFSRLMAFINDRLNVVHAMVLAQQYQALKAEEEAQD (SEQ ID NO: 176)或替代地基本上由其組成或進一步由其組成。 In other embodiments, the signal peptide of wild-type RD114 comprises or alternatively consists essentially of or further consists of MKLPTGMVILCSLIIVRA (amino acids (aa) 1 to aa 18 of SEQ ID NO: 176). In some embodiments, the ectodomain domain of wild-type RD114 comprises or alternatively consists essentially of or further consists of aa 1 to aa 504 of SEQ ID NO: 176. In some embodiments, the extracellular domain domain of wild-type RD114 comprises or alternatively consists essentially of or further consists of aa 1 to aa 507 of SEQ ID NO: 176. In some embodiments, the transmembrane region of wild-type RD114 comprises or alternatively consists essentially of or further consists of FLPYLLPLLGPLLTLLLILTIGPCVF (aa 505 to aa 530 of SEQ ID NO: 176). In some embodiments, the transmembrane region of wild-type RD114 comprises or alternatively consists essentially of or further consists of YLLPLLGPLLTLLLILTIGPCVF (aa 508 to aa 530 of SEQ ID NO: 176). In some embodiments, the cytoplasmic domain of wild-type RD114 comprises or alternatively consists essentially of or further consists of SRLMAFINDRLNVVHAM (aa 531 to aa 547 of SEQ ID NO: 176). In some embodiments, the cytoplasmic domain of wild-type RD114 comprises or alternatively consists essentially of or further consists of SRLMAFINDRLNVVHAMVLAQQYQALKAEEEAQD (aa 531 to aa 564 of SEQ ID NO: 176).

In some embodiments, when referring to a protein, GALV means the envelope glycoprotein of the gibbon leukemia virus envelope glycoprotein. A non-limiting exemplary sequence of this protein can be found in UniProtKB - P21415 (ENV_GALV), which is incorporated herein by reference in its entirety.在一些實施例中，野生型GALV包括MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLLSTIAGPLLLLLLLLILGPCIINKLVQFINDRISAVKILVLRQKYQALENEGNL (SEQ ID NO: 183)或替代地基本上由其組成或進一步由其組成。 In some embodiments, the wild-type GALV comprises or alternatively consists essentially of or further consists of aa 42 to aa 685 of SEQ ID NO: 183. In other embodiments, the signal peptide wild type of GALV comprises or alternatively consists essentially of or further consists of aa 1 to aa 41 of SEQ ID NO: 183. In some embodiments, the extracellular domain of wild-type GALV comprises or alternatively consists essentially of or further consists of aa 1 to aa 489 of SEQ ID NO: 183 or aa 42 to aa 489 of SEQ ID NO: 183 composition. In some embodiments, the transmembrane region of wild-type GALV comprises or alternatively consists essentially of or further consists of aa 490 to aa 670 of SEQ ID NO: 183. In some embodiments, the cytoplasmic domain of wild-type GALV comprises or alternatively consists essentially of or further consists of aa 671 to aa 685 of SEQ ID NO: 183.

As used herein, the multiplicity of infection (MOI) refers to the number of viral particles added to each cell during infection.

RetroNectin Reagent is a 63 kD recombinant human fibronectin fragment (also known as rFN-CH-296) that enhances the efficiency of lentiviral and retroviral mediated gene transduction. This is especially important for hematopoietic cells and other difficult-to-transfect cell types. It is believed that the enhancement of transduction results from the co-localization of viral particles and target cells. This is achieved by direct binding of viral particles to sequences in the heparin binding domain and interaction of target cell integrins with two other domains in rFN-CH-296. RetroNectin can be used highly efficiently in cells expressing integrin α4β1 (VLA-4) and/or integrin α5β1 (VLA-5). VLA-4 expressing cells include T cells, B cells, monocytes, NK cells, eosinophils, myelomonocytic cells and lymphoid progenitor cells. Thymocytes, activated T cells and mast cells express VLA-5.

In the aspect where gene transfer is mediated by a lentiviral vector, the vector construct refers to a polynucleotide comprising a lentiviral gene body or a portion thereof and a therapeutic gene. As used herein, "lentiviral mediated gene transfer" or "lentiviral transduction" encompasses the same meaning and refers to the transfer of a gene or nucleic acid by means of a virus that enters a cell and integrates its genome into the host cell genome The process of stable transfer of a sequence into a host cell. Viruses can enter host cells via their normal infection mechanisms or be modified so that they bind to different host cell surface receptors or ligands for cell entry. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse transcribed into the form of DNA and integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, a lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell via a viral or virus-like entry mechanism. "Lentiviral vectors" are a well-known class of retroviral vectors that have certain advantages over other retroviral vectors in transducing non-dividing cells. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

The lentiviral vector systems of the present invention are based on or derived from oncogenic retroviruses (subgroup of MLV-containing retroviruses) and lentiviruses (subgroup of HIV-containing retroviruses). Examples include ASLV, SNV and RSV, all of which are divided into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particles of the present invention can be altered in form based on specific retrovirus genes or otherwise (eg, by specific selection of packaging cell systems).

As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the virus species associated with this name and belonging to the Parvoviridae genus of the family Parvoviridae. Various serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of 11 serotypes, such as AAV2, AAV8, AAV9, or variant or synthetic serotypes (such as AAV-DJ and AAV PHP.B) . AAV particles include, consist essentially of, or further consist of, three major viral proteins: VP1, VP2 and VP3. In one embodiment, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, or AAV rh74. Such vectors are commercially available or described in patent or technical literature.

The vector particles of the present invention are "based on" a particular retrovirus, meaning that the vector system is derived from that particular retrovirus. The gene body of the vector particle includes components from the retrovirus as backbone. The vector particles contain the necessary vector components compatible with the RNA genome, including reverse transcription and integration systems. Typically, these components comprise gag and pol proteins derived from specific retroviruses. Thus, most of the structural components of the vector particle are typically derived from the retrovirus, but may have been genetically or otherwise altered to provide the desired useful properties. However, certain structural components and especially env proteins can be derived from different viruses. The vector host range and cell type infected or transduced can be varied by conferring different specificities on the vector particles using different env genes in the vector particle production system.

As used herein, cells can be prokaryotic or eukaryotic cells. In other embodiments, the cell line is an immune cell.

As used herein, "immune cells" include, for example, white blood cells (leukocytes, such as granulocytes (neutrophils, eosinophils, and basophils) that can be derived from hematopoietic stem cells (HSCs) produced in the bone marrow , monocytes and lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells), lymphocytes (T cells, B cells, natural killer (NK) cells and NKT cells) and myeloid-derived Cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). In some embodiments, the immune cell line is derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells , pluripotent stem cells, induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC) or immortalized cells. In some embodiments, the HSC line is derived from the individual's umbilical cord blood, the individual's peripheral blood, or the individual's bone marrow.

"Host cell" refers not only to a particular test cell, but to the progeny or potential progeny of such a cell. Such progeny may actually differ from the parent cell due to mutation or environmental influences that can cause certain changes to occur in subsequent generations but are still encompassed by the term as used herein.

"Packaging cell" refers to a host cell having a nucleic acid molecule including a viral helper construct by stable or transient transfection or transduction using a heterologous nucleotide sequence, wherein the construct is capable of stable or transient expression that can be expressed in trans Packaging functions (eg, proteins required for replication and encapsidation) are provided for the production of viral particles. The expression of the viral helper can be constitutive or inducible, eg, when the helper is under the control of an inducible promoter.

An "enriched population" of cells means a substantially homogeneous population of cells having certain specified properties. Cells are greater than 70% or alternatively greater than 75% or alternatively greater than 80% or alternatively greater than 85% or alternatively greater than 90% or alternatively greater than 95% or alternatively greater than 98% for a given property.

The term "propagation" means growing a cell or population of cells. The term "growth" also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, supporting cells, or any chemical or biological compound required to obtain a desired number of cells or cell types.

The term "culturing" refers to the in vitro propagation of cells or organisms on or in various media. It is understood that the progeny of cells grown in culture may not be identical (ie, morphologically, genetically, or phenotypically) to the parental cells.

Unmodified cells are sometimes referred to as "origin cells" or "origin stem cells." Cells can be prokaryotic or eukaryotic, and include, but are not limited to, bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells (eg, feline, canine, equine, murine, rat , ape, bovine, porcine and human cells).

In one embodiment, an "immature cell" refers to a cell that does not possess the desired (mature) phenotype or genotype. For example, in one embodiment, a mature cell line is a substituted cell. Techniques, including physical, biological, or chemical processes, can be performed on immature cells to alter, initiate (change), or change the cell's phenotype or genotype into "mature cells." A "mature cell" refers to a cell that possesses a desired phenotype or genotype.

As used herein, the term "NK cells" (also known as natural killer cells) refers to lymphocytes that originate in the bone marrow and play a key role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other responsive cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface. NK cells can be isolated or obtained from commercial sources. Non-limiting examples of commercial NK cell lines include cell lines NK-92 (ATCC® CRL-2407™), NK-92MI (ATCC® CRL-2408™). Other examples include, but are not limited to, the NK cell lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting example sources of such commercially available cell lines include the American Type Culture Collection or ATCC (www.atcc.org/) and the German Collection of Microorganisms and Cell Cultures) (www.dsmz.de/).

As used herein, the efficacy of a cell (eg, NK cells, NKT cells, cytotoxic T cells, or γδ T cells) includes cellular cytotoxicity (eg, in killing target cells) and/or cellular interleukin release ( For example, IFN-γ, also referred to herein as IFNγ or IFN-gamma).

Native Antigen Presenting Cells (APCs) refer to the processing and presentation of antigens by certain lymphocytes (eg T cells, classical APCs (including dendritic cells), macrophages, Langerhans cells ) and B cells) recognize immune cells that mediate cellular immune responses. As used herein, artificial antigen presenting cells (aAPCs) are synthetic forms of these APCs and attach specific immune cell (eg, T and/or NK cell) stimulatory signals to various macroscopic and microscopic biocompatible surfaces and / or cells to prepare. This can potentially reduce costs while controlling the production of large quantities of functional pathogen-specific immune cells for therapy.

The term "stem cell" refers to a cell in an undifferentiated or partially differentiated state and capable of self-renewal and/or generation of differentiated progeny. Self-renewal is defined as the ability of stem cells to proliferate and produce more of them, while maintaining their developmental potential (ie, totipotent, pluripotent, pluripotent, etc.). The term "somatic stem cell" is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Native somatic stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary natural somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural or neuronal stem cells (NSCs). In some embodiments, the stem cell progenitor cells can be embryonic stem cells or induced pluripotent stem cells (iPSCs). As used herein, "embryonic stem cells" refers to stem cells derived from tissue formed after fertilization but before the end of pregnancy, including at any time during pregnancy (usually but not necessarily before about 10-12 weeks of pregnancy) Pre-embryonic tissue (eg blastocyst), embryonic tissue or fetal tissue is obtained. Most commonly, embryonic stem cell lines are derived from the pluripotent cells of an early embryo or blastocyst. Embryonic stem cells can be obtained directly from suitable tissues (including, but not limited to, human tissues) or from established embryonic cell lines. An "embryonic stem cell" refers to a cell that shares one or more, but not necessarily all, of the properties of an embryonic stem cell.

"Differentiation" describes the process by which non-specialized cells acquire the characteristics of specialized cells, such as heart cells, liver cells, or muscle cells. "Directed differentiation" refers to manipulation of stem cell culture conditions to induce differentiation into a particular cell type. "Dedifferentiation" defines cells that revert to less directed positions within the cell lineage. As used herein, the term "differentiated or differentiated" defines a cell that is presented in a more directed ("differentiated") position within a cell lineage. As used herein, "a cell differentiated into a mesodermal (or ectodermal or endodermal) lineage" defines a cell belonging to a particular mesodermal, ectodermal, or endodermal lineage, respectively. Examples of cells that differentiate into the mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, adipogenic cells, smooth muscle cells, chondrogenic cells, cardiogenic cells, epidermal cells, hematopoietic cells, Angiogenic cells, myogenic cells, nephrogenic cells, genital cells, osteogenic cells, pericardiogenic cells or stromal cells.

As used herein, the term "differentiated or differentiated" defines a cell that is presented in a more directed ("differentiated") position within a cell lineage. "Dedifferentiation" defines cells that revert to less directed positions within the cell lineage. Examples of induced pluripotent stem cell line dedifferentiated cells.

In some embodiments, a first cell line derived from a second cell refers to a first cell differentiated from a second cell. Additionally or alternatively, a first cell derived from a second cell refers to a first cell engineered from a second cell.

As used herein, the "lineage" of a cell defines the heritability of a cell, that is, its predecessors and progeny. A cell lineage places cells within a genetic program for development and differentiation.

"Multilineage stem cells" or "pluripotent stem cells" refer to stem cells that propagate themselves and at least two further differentiated progeny cells from different developmental lineages. The lineages can be from the same germ layer (ie, mesoderm, ectoderm or endoderm) or from different germ layers. An example of two progeny cells with distinct developmental lineages from multilineage stem cell differentiation are myogenic cells and adipogenic cells (both derived from mesoderm but give rise to different tissues). Another example is neurogenic cells (ectoderm derived) and adipogenic cells (mesoderm derived).

A "precursor" or "progenitor cell" is intended to refer to a cell capable of differentiating into a particular type of cell. Progenitor cells can be stem cells. Progenitor cells can also be more specific than stem cells. Progenitor cells can be unipotent or pluripotent. In contrast to mature stem cells, progenitor cells can be in a later stage of cellular differentiation. An example of a progenitor cell includes, but is not limited to, progenitor neural cells.

As used herein, a "pluripotent cell" defines a smaller differentiated cell that produces at least two different (genotypic and/or phenotypic) further differentiated progeny cells. In another aspect, "pluripotent cells" include induced pluripotent stem cells (iPSCs), which are artificially derived stem cells from non-pluripotent cells (usually mature somatic cells) that have been borrowed in the past Produced by inducing the expression of one or more stem cell-specific genes. These stem cell-specific genes include, but are not limited to, the octameric transcription factor family, namely Oct-3/4; the Sox gene family, namely Sox1, Sox2, Sox3, Sox 15 and Sox 18; the Klf gene family, also Namely Klf1, Klf2, Klf4 and Klf5; Myc gene family, namely c-myc and L-myc; Nanog gene family, namely OCT4, NANOG and REX1; or LIN28. Examples of iPSCs are described in: Takahashi et al. (2007) Cell advance online publication, Nov. 20, 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260 -262; Yu et al. (2007) Science advance online publication, Nov. 20, 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication, Nov. 30, 2007.

"Embryoid bodies or EBs" are three-dimensional (3D) embryonic stem cell aggregates formed during culture that promote subsequent differentiation. When grown in suspension culture, EB cells form small cell aggregates surrounded by an outer layer of visceral endoderm. During growth and differentiation, EBs develop into sac-like embryoid bodies with fluid-filled cavities and an inner layer of ectoderm-like cells.

"Induced pluripotent cells" means embryonic-like cells that are reprogrammed from mature cells to an immature phenotype. Various methods are known in the art, such as "A simple new way to induce pluripotency: Acid." Nature, 29 Jan 2014 (and available at sciencedaily.com/releases/2014/01/140129184445, last accessed 2014 February 5, 2010) and US Patent Application Publication No. 2010/0041054. Human iPSCs also express stem cell markers and are able to generate cells characteristic of all three germ layers .

"Parthenogenetic stem cells" refers to stem cells derived from the activation of parthenogenetic eggs. Methods for generating parthenogenetic stem cells are known in the art. See, eg, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100 (Suppl) 11911-6.

As used herein, the term "pluripotency gene or marker" means an expressed gene or protein associated with an immature or undifferentiated phenotype, such as Oct28, Sox2, Nanog, c-Myc and LIN-28. Systems for identifying such species using methods known in the art to identify such species are commercially available, for example, from EMD Millipore (MILLIPLEX® Map Kit).

The term "phenotype" describes an individual characteristic or characteristic that is measurable and manifested only in a subgroup of individuals within a population. In one aspect of the invention, the phenotype of an individual comprises the phenotype of a single cell, a substantially homogeneous population of cells, a population of differentiated cells, or a tissue comprising a population of cells.

In some embodiments, a population of cells means a collection of more than one cells that are phenotypically and/or genotypically identical (clones) or not identical. A population can be purified, highly purified, substantially homogeneous or heterogeneous, as described herein.

The terms effective period (or time) and effective conditions refer to the desired or preferred period of time or other controllable conditions (such as for temperature, humidity for in vitro methods).

"Substantially homogeneous" describes wherein more than about 50% or alternatively more than about 60% or alternatively more than 70% or alternatively more than 75% or alternatively more than 80% or alternatively more than 85% or alternatively More than 90% or alternatively more than 95% of the cells belong to a population of cells of the same or similar phenotype. Phenotypes can be determined by preselecting cell surface markers or other markers.

As used herein, the term "depleted" means substantially depleted. For example, a cell population system depleted of CD3+ cells is meant to include less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than About 0.8%, less than about 0.9%, less than about 1.0%, less than about 1.1%, less than about 1.2%, less than about 1.3%, less than about 1.4%, less than about 1.5%, less than about 1.6%, less than about 1.7%, less than A cell population of about 1.8%, less than about 1.9%, or less than about 2.0% CD3+ cells.

The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any component, range, dosage form, etc. disclosed herein are intended to mean that the component, range, dosage form, etc., is suitable for the disclosed purpose.

As used herein, the terms "treating, treatment," and the like are used herein to mean obtaining a desired pharmacological and/or physiological effect. In some embodiments, the effect may be prophylactic (with respect to complete or partial prevention of the disorder or a sign or symptom thereof), and/or may be therapeutic (with respect to partial or complete cure of the disorder and/or adverse effects attributable to the disorder) ). Examples of "treating" include, but are not limited to: preventing the occurrence of a disorder in individuals susceptible to the disorder but not yet diagnosed with the disorder; inhibiting the disorder, ie, preventing its occurrence; and/or reducing or ameliorating the symptoms of the disorder. In one aspect, treatment is preventing the development of symptoms of a disease or disorder (eg, cancer). In some embodiments, it refers to (1) preventing the occurrence of a symptom or disease in individuals susceptible to the disease or not yet showing symptoms; (2) inhibiting the disease or preventing its occurrence; or (3) ameliorating the disease or disease symptoms or make it subside. As understood in the art, "treatment" is a means of obtaining beneficial or desired results, including clinical results. For the purposes of the present techniques, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, reduction in the extent of a condition (including disease), stabilization ( i.e. not worsening) the state of a condition (including a disease), delaying or slowing the progression of a condition (including a disease), ameliorating or alleviating the state of a condition (including a disease), and remission (whether partial or complete), whether such results may be detectable or undetectable. Where the disease is cancer, the following clinical endpoints are non-limiting examples of treatment: reduction in tumor burden, reduction in tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis, or reduction in tumor metastasis. In one aspect, treatment does not include prevention. In one aspect, treatment does not include prevention.

As used herein, a therapeutic protein or polypeptide refers to a protein and/or polypeptide suitable for use in therapy, including but not limited to antibodies or fragments thereof, enzymes, ligands or receptors. The therapeutic protein or polypeptide can be selected by a physician or one skilled in the art based on the disease to be treated. For example, to treat cancer, antibodies to immune checkpoint receptors or their ligands, such as anti-PD-1 antibodies and/or anti-PD-L1 antibodies, can be used.

In one embodiment, the term "disease" or "disorder" as used herein refers to cancer, a state diagnosed with cancer, a state suspected of having cancer, or a state at high risk of having cancer.

As used herein, "cancer" is a disease state characterized by the presence in an individual of cells that exhibit abnormal uncontrolled replication, and in some aspects the term is used interchangeably with the term "tumor". The term "cancer or tumor antigen" refers to an antigen that is known to associate with cancer cells or tumor cells or tissues and is expressed on its surface, and the term "cancer or tumor targeting antibody" refers to an antibody that targets such an antigen .

A "solid tumor" is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant, metastatic or non-metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.

"Composition" is intended to mean an active agent and another inert (eg, detectable agent or label) or active compound or ingredient (eg, adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives) , adjuvant or the like and including a pharmaceutically acceptable carrier).

The carrier also includes the following pharmaceutical excipients and additives: proteins, peptides, amino acids, lipids, and carbohydrates (eg, sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, such as aldoses) alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present individually or in combination and comprise 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin (eg, human serum albumin (HSA)), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components that may also exercise buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteamine acid, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame and the like. Carbohydrate excipients are also intended to be within the scope of this technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose , sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, raffinose, maltodextrin, dextran, starch and the like; and alditols such as mannitol, xylitol, maltitol, Lactitol, Xylitol Sorbitol (sorbitol) and Inositol.

A "pharmaceutical composition" is intended to comprise an active polypeptide, polynucleotide or antibody in combination with an inert or active carrier (eg, a solid carrier) that renders the composition suitable for use in in vitro, in vivo or ex vivo diagnosis or therapy sexual application.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions (eg, oil/water or water/oil emulsions) and various types the wetting agent. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th ed. (Mack Publ. Co., Easton). The term pharmaceutically acceptable carrier (or medium) (which is used interchangeably with the term biocompatible carrier or medium) refers to agents, cells, compounds, materials, compositions and/or dosage forms that are not only compatible with the intended Therapeutic administration is compatible with its cells and other agents and is also suitable, within the scope of sound medical judgment, for contact with human and animal tissues without undue toxicity, irritation, allergic reactions or other complications, and with reasonable benefit/risk more commensurate. Pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solids (eg, gels), and solid materials (eg, cellular structures and matrices, tubes, sheets and as known in the art and described in more detail herein). other such materials). These semi-solid and solid materials can be designed to prevent degradation in the body (non-biodegradable) or they can be designed to degrade in the body (biodegradable, bioerodible). Biodegradable materials can further be bioresorbable or bioabsorbable, that is, they can be dissolved and absorbed into body fluids (an example of a water-soluble implant system), or degraded in the body and eventually eliminated (by conversion to other materials may be decomposed and eliminated via natural pathways).

"Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg, phosphates), glycine, sorbic acid, sorbic acid Potassium, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, Polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, polyethylene glycols and lanolin . Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, which is a standard reference in the field. It can be administered according to the intended form (ie, oral lozenges, capsules, elixirs, syrups, and the like). Selections are made and should be consistent with conventional medical practice.

Compositions for use in the present invention can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage form" refers to a physically discrete unit suitable for use in an individual, each unit containing a predetermined quantity of the composition calculated to produce a dose associated with administration (ie, the appropriate route and regimen). Expect a response. The amount to be administered (in terms of therapeutic quantities and unit doses) will depend on the desired result and/or protection. The precise amount of composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the subject's physical and clinical state, route of administration, intended therapeutic goals (relief of symptoms versus curing), and efficacy, stability, and toxicity of a particular composition. After formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically or prophylactically effective amount. The formulations are readily administered in a variety of dosage forms, eg, injectable solutions of the type described herein.

As used herein, the term "contacting" means direct or indirect binding or interaction between two or more molecules. A specific example of direct interaction is binding. A specific example of an indirect interaction is where one entity acts on an intermediate molecule and that intermediate molecule in turn acts on the second mentioned entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in cells, and in vivo. In vivo contact may be referred to as administration or administration.

The "administration" or "delivery" of cells or vectors or other agents and compositions containing the same can be carried out in one dose continuously or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art, and will vary with the composition used for therapy, the purpose of therapy, the target cells being treated, and the individual being treated. Single or multiple administrations can be carried out using dose values and patterns selected by the treating physician or, in the case of animals, by the treating veterinarian. Suitable dosage formulations and methods of administration of the pharmaceutical agents are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will depend on the composition of the treatment, the purpose of the treatment, the state of health or disease stage of the individual being treated, and the target cells or organization. Non-limiting examples of routes of administration include oral administration, intraperitoneal administration, infusion, nasal administration, inhalation, injection, and topical application.

The term administration shall include, but is not limited to, oral, parenteral (eg, intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection or implantation) , by inhalation spray, nasal, vaginal, rectal, sublingual, transurethral (eg, urethral suppository) or topical routes of administration (eg, gels, ointments, creams, aerosols, etc.), and They may be formulated individually or together in suitable dosage unit formulations containing conventional pharmaceutically acceptable non-toxic carriers, adjuvants, excipients and vehicles suitable for each route of administration. The present invention is not limited by route of administration, formulation or schedule of administration.

"Administration" can be carried out in one dose continuously or intermittently throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art, and will vary with the composition used for therapy, the purpose of therapy, the target cells being treated, and the individual being treated. Single or multiple administrations can be carried out using dose values and patterns selected by the treating physician. Suitable dosage formulations and methods of administration of the pharmaceutical agents are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and will depend on the composition of the treatment, the purpose of the treatment, the state of health or disease stage of the individual being treated, and the target cells or organization.

The agents of the invention can be administered into therapy by any suitable route of administration. It should also be understood that the optimal route will vary with the condition and age of the recipient and the disease being treated.

"Subject", "individual" or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include (but are not limited to) rodents, rats, rabbits, apes, bovines, sheep, pigs, dogs, cats, farm animals, sport animals, pets, horses and primates, Especially humans. In addition to being used for human treatment, the present invention can also be used for veterinary medical treatment of companion mammals, foreign animals and domestic animals (including mammals and rodents). In one embodiment, mammals include horses, dogs, and cats. In another embodiment of the invention, the human is a fetus, an infant, a prepubertal individual, an adolescent, a pediatric patient, or an adult. In one aspect, the system is presymptomatic mammals or humans. In another aspect, the individual has minimal clinical symptoms of disease. The individual can be male or female, adult, infant or pediatric. In another aspect, a system is an adult. In some instances, the adult is an adult human, eg, an adult human greater than 18 years of age.

The term "suffering" in relation to the term "treating" means that a patient or individual has been diagnosed with or is susceptible to a syndrome cancer or viral infection. A patient may also be said to be "at risk of developing a disease" because it carries one or more genetic mutations. This patient has not developed a characteristic disease condition.

An "effective amount" is an amount sufficient to achieve beneficial or desired results. An effective amount can be administered in one or more administrations, applications or doses. This delivery depends on a number of variables, including the time period for which the individual dosage unit is intended to be administered, the bioavailability of the therapeutic agent, the route of administration, and the like. It is to be understood, however, that the specific dosage value of the therapeutic agents of the present invention for any particular individual will depend on a variety of factors, including the activity of the particular compound employed, the age, weight, general health, sex and diet, time of administration, excretion of the individual The rate, combination of drugs and severity of the particular condition being treated and the form of administration. Treatment doses can generally be escalated to optimize safety and efficacy. In general, dose-response relationships from in vitro and/or in vivo testing can first be used to guide appropriate doses for patients. In general, the desired administered amount of the compound is effective to achieve serum levels commensurate with the in vitro effective concentrations found. Determination of these parameters is well known in the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks.

A "therapeutically effective amount" of a drug or medicament means an amount of the drug or medicament sufficient to obtain a pharmacological response (eg, passive immunity); or alternatively, a drug or medicament sufficient to have the desired effect when administered to a patient with a specified condition or disease The amount of an effect (eg, treating, alleviating, ameliorating, alleviating, or eliminating one or more manifestations of a specified disorder or disease in a patient). A therapeutic effect need not be present by administration of one dose, and may only be present after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.

As used herein, "monotherapy" includes, but is not limited to, surgical resection, chemotherapy, cryotherapy, radiation therapy, immunotherapy, and targeted therapy. Agents for reducing cell proliferation are known in the art and widely used. Chemotherapy drugs that kill cancer cells only when they divide are called cell cycle specific. These drugs include agents that act in S phase, including topoisomerase inhibitors and antimetabolites.

Topoisomerase inhibitors are drugs that interfere with the action of topoisomerases (topoisomerases I and II). During the course of chemotherapy, topoisomerases control the manipulation of DNA structure required for replication and are thus cell cycle specific. Examples of topoisomerase I inhibitors include the camptothecan analogs listed above, irinotecan and topotecan. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide.

Antimetabolites are generally analogs of normal metabolic substrates that normally interfere with the processes involved in chromosome replication. It attacks cells at very specific times in the cycle. Antimetabolites include folate antagonists, such as methotrexate; pyrimidine antagonists, such as 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine (gemcitabine); purine antagonists such as 6-mercaptopurine and 6-thioguanine; adenosine deaminase inhibitors such as cladribine, fludarabine, nelarabine ) and pentostatin; and the like.

Phytoalkalis are derived from certain types of plants. Vinca alkaloids are prepared from periwinkle plants (Catharanthus rosea). Taxanes are obtained from the bark of the Pacific Yew tree (Yew). Vinca alkaloids and taxanes are also known as anti-microtubule agents. Podophyllotoxin is derived from the May apple plant. Camptothecin analogs are derived from the Asian "happy tree" (Camptotheca acuminata). Podophyllotoxin and camptothecin analogs are also classified as topoisomerase inhibitors. Plant alkaloids are generally cell cycle specific.

Examples of such agents include vinca alkaloids such as vincristine, vinblastine and vinorelbine; taxanes such as paclitaxel and docetaxel ; podophyllotoxins such as etoposide and teniposide; and camptothecin analogs such as irinotecan and topotecan.

Cryotherapy includes, but is not limited to, therapy involving lowering of temperature, such as hypothermia therapy.

Radiation therapy includes, but is not limited to, exposure to radiation (eg, ionizing radiation, UV radiation), as known in the art. Exemplary doses include, but are not limited to, ionizing radiation doses in the range of at least about 2 Gy to no more than about 10 Gy and/or in the range of at least about 5 J/m to no more than about 50 J/m (typically UV radiation dose of about 10 J/m2).

The phrases "first-line" or "second-line" or "third-line" refer to the order in which a patient receives treatment. The first-line regimen is the treatment that is given first, while the second- or third-line therapy is given after the first-line therapy or after the second-line therapy, respectively. The National Cancer Institute defines first-line therapy as the "first treatment" for a disease or condition. In cancer patients, the primary treatment may be surgery, chemotherapy, radiation therapy, or a combination of these. First-line therapy is also referred to as "primary therapy and primary therapy" by those skilled in the art. See National Cancer Institute website www.cancer.gov (last accessed May 1, 2008). Typically, a follow-up chemotherapy regimen is given to the patient because the patient does not show a positive clinical or subclinical response to the first-line therapy or because the first-line therapy has been discontinued. MODES OF IMPLEMENTING THE INVENTION

Several advantages of the compositions and/or methods as disclosed herein were observed and exemplified in the Figures and Experimental Methods. These advantages include, but are not limited to, efficient delivery of genes to immune cells (eg, NK cells), stability of delivered genes, successful expansion of cells, and high viability of transduced cells, thereby promoting and improving transduction Development and manufacture of immune cells, such as CAR-expressing immune cells.

In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 45%, at least about 50%, at least about about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cells Expression of CAR and/or another therapeutic protein or polypeptide. This performance can be assessed using various methods, such as fluorescence-activated cell sorting (FACS) or another immunostaining method utilizing antibodies or antigen-binding fragments thereof that specifically recognize and bind to the CAR and/or therapeutic protein or polypeptide.

In some embodiments, cell populations made by the methods as disclosed herein, for example, express the CAR and/or another therapeutic protein or polypeptide in cell culture for at least about 3 days, at least about 5 days, at least about 7 days days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 1 month, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, at least about 150 days, at least about 180 days or more. In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 45%, at least about 50%, at least about about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the cells (for example) expressing the CAR and/or another therapeutic protein or polypeptide in cell culture for at least about 3 days, at least about 5 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 1 month, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, at least about 150 days , at least about 180 days or more.

In some embodiments, the methods as disclosed herein generate more than 1 x 10 ⁹ cells, more than 2 x 10 ⁹ cells, more than 3 x 10 ⁹ cells, more than 4 x 10 ⁹ cells, more than More than 5 x 10 ⁹ cells, more than 6 x 10 ⁹ cells, more than 7 x 10 ⁹ cells, more than 8 x 10 ⁹ cells, more than 9 x 10 ⁹ cells, more than 1 x 10 ¹⁰ cells, more than 2 × 10 ¹⁰ cells, more than 3 × 10 ¹⁰ cells, more than 4 × 10 ¹⁰ cells, more than 5 × 10 ¹⁰ cells, more than 6 × 10 ¹⁰ cells, more than 7 × 10 ¹⁰ cells, more than 8 × 10 ¹⁰ cells, more than 9 × 10 ¹⁰ cells, more than 1 × 10 ¹¹ cells, more than 2 × 10 ¹¹ cells, more than 3 × 10 ¹¹ cells cells, more than 4 × 10 ¹¹ cells, more than 5 × 10 ¹¹ cells, more than 6 × 10 ¹¹ cells, more than 7 × 10 ¹¹ cells, more than 8 × 10 ¹¹ cells, more than 9 × 10 ¹¹ cells, more than 1 × 10 ¹² cells, more than 2 × 10 ¹² cells, more than 3 × 10 ¹² cells, more than 4 × 10 ¹² cells, more than 5 × 10 ¹² cells , more than 6 × 10 ¹² cells, more than 7 × 10 ¹² cells, more than 8 × 10 ¹² cells, more than 9 × 10 ¹² cells or more than 1 × 10 ¹³ cells, 1 × 10 ⁸ cells or their equivalents. Additionally or alternatively, the methods as disclosed herein generate up to 5 x 10 ¹⁰ cells, up to 6 x 10 ¹⁰ cells, up to 7 x 10 ¹⁰ cells, up to 8 x 10 ¹⁰ cells, up to 9 x 10 ¹⁰ cells cells, up to 1 × 10 ¹¹ cells, up to 2 × 10 ¹¹ cells, up to 3 × 10 ¹¹ cells, up to 4 × 10 ¹¹ cells, up to 5 × 10 ¹¹ cells, up to 6 × 10 ¹¹ cells, up to 7 × 10 ¹¹ cells, up to 8 × 10 ¹¹ cells, up to 9 × 10 ¹¹ cells, up to 1 × 10 ¹² cells, up to 2 × 10 ¹² cells, up to 3 × 10 ¹² cells, up to 4 × 10 ¹² cells, up to 5 × 10 ¹² cells, up to 6 × 10 ¹² cells, up to 7 × 10 ¹² cells, up to 8 × 10 ¹² cells, up to 9 × 10 ¹² cells, or up to 1 × 10 ¹³ cells, 1 x ¹⁰⁸ cells, or their equivalents. In some embodiments, the methods as disclosed herein generate about 5×10 ¹⁰ cells, about 6×10 ¹⁰ cells, about 7×10 ¹⁰ cells, about 8×10 ¹⁰ cells, about 9×10 ¹⁰ cells cells, about 1 × 10 ¹¹ cells, about 2 × 10 ¹¹ cells, about 3 × 10 ¹¹ cells, about 4 × 10 ¹¹ cells, about 5 × 10 ¹¹ cells, or about 6 × 10 ¹¹ cells , 1 × 10 ⁸ cells or its equivalent. The equivalent can be calculated by one skilled in the art by dividing or multiplying the number of cells produced and the number of initial cells by the same positive number.

In some embodiments, the viability of a population of cells as disclosed herein can be at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%. Various methods for testing cell viability are available, such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay or APT measurement assay. Pseudotyped gamma retroviral particle

In one aspect, pseudotyped gamma retroviral particles are provided. In some embodiments, the gamma retroviral particle comprises or instead consists essentially of or further consists of the modified RD114 feline endogenous retroviral envelope glycoprotein (RD114TR). Additionally or alternatively, the gamma retroviral particle comprises, consists essentially of, or further consists of a modified baboon envelope glycoprotein (BaEVTR). In other embodiments, the RD114TR glycoprotein comprises or alternatively consists essentially of the extracellular and transmembrane domains of the RD114 glycoprotein and the cytoplasmic domain of the amphiphilic murine leukemia virus (MLV-A) glycoprotein or further composed of it. In other embodiments, the BaEVTR glycoprotein comprises or alternatively consists essentially of or further consists of the extracellular and transmembrane domains of baboon envelope glycoprotein (BaEV) and the cytoplasmic domain of MLV-A glycoprotein composition. In some embodiments, RD114TR and BaEVTR are incorporated into the particle envelope as membrane proteins.

In some embodiments, the pseudotyped gamma retroviral particles as disclosed herein further comprise a vector gene body encapsulated in an envelope. In other embodiments, the vector genome comprises or alternatively consists essentially of or further consists of one or more of the following flanked by two long terminal repeats (LTRs): (A) encodes a chimeric antigen A polynucleotide of a receptor (CAR) and/or another therapeutic protein or polypeptide; (B) the reverse complement of (A); or (C) a polynucleotide that includes one or more recognition sites. In some embodiments, the therapeutic protein or polypeptide is selected from antibodies or fragments thereof, enzymes, ligands or receptors. In some embodiments, the recognition site is recognized and cleaved by a restriction enzyme suitable for insertion of the sequence of interest (eg, either or both of (A) and (B)) into the polynucleotide.

In some embodiments, the vector genome further comprises one or more of the following: a 5' LTR, a 5' cap, a 3' poly-A tail, and a 3' LTR.

In some embodiments, pseudotyped gamma retroviral particles as disclosed herein further comprise either or both of reverse transcriptase or integrase.

In some embodiments, the pseudotyped gamma retroviral particle is based on or derived from or is selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Reed murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV), or feline leukemia virus. Artificial Antigen Presenting Cells (aAPCs)

In another aspect, provided herein are artificial antigen presenting cells (aAPCs). In some embodiments, the aAPCs express one or more tumor-associated antigens (TAAs) or viral antigens, or both, as disclosed herein. In other embodiments, the aAPCs express one or more of the following: 4-1BBL, membrane-bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15 complexed with IL-15RαSushi-Fc fusion protein N72D superagonists (IL-15SA/IL-15RαSu-Fc; ALT-803) or cell surface markers that mediate CD122/CD132 signaling. In one embodiment, the aAPC further expresses mb IL-21 and 4-1BBL. In some embodiments, aAPCs as disclosed herein express tumor-associated antigens (TAAs) and/or viral antigens that activate and/or stimulate the growth of immune cells (eg, NK cells or γδ T cells).

In some embodiments, the aAPCs are engineered K562 cells.

In some embodiments, the aAPCs lack cell proliferation and/or lack long-term survival. Cell proliferation and long-term survival can be assessed by those skilled in the art, for example, by culturing and counting viable cells. In some embodiments, aAPCs as disclosed herein do not substantially survive for more than about 5 days, about 7 days, about 10 days, about 14 days, about 15 days, about 21 days, or about 30 days.

In some embodiments, the aAPCs are irradiated to inhibit cell proliferation and/or reduce long-term survival. In other embodiments, at 50 Gy or higher, 60 Gy or higher, 70 Gy or higher, 75 Gy or higher, 80 Gy or higher, 90 Gy or higher, 100 Gy or higher, 110 Gy or higher, 120 Gy or higher, 130 Gy or higher, 140 Gy or higher, 150 Gy or higher, 160 Gy or higher, 170 Gy or higher, 180 Gy or higher, 190 Gy or higher Irradiated aAPC at higher, 200 Gy or higher, 210 Gy or higher, 220 Gy or higher, 230 Gy or higher, 240 Gy or higher, 250 Gy or higher, and/or 300 Gy or higher . Additionally or alternatively, at 1000 Gy or less, 900 Gy or less, 800 Gy or less, 700 Gy or less, 600 Gy or less, 500 Gy or less, 400 Gy or less, 350 Gy or less, 300 Gy or less, 250 Gy or less, 240 Gy or less, 230 Gy or less, 220 Gy or less, 210 Gy or less, 200 Gy or less, 190 Gy or less Low, 180 Gy or less, 170 Gy or less, 160 Gy or less, 150 Gy or less, 140 Gy or less, 130 Gy or less, 120 Gy or less, 110 Gy or less, The aAPC is irradiated at 100 Gy or less, 90 Gy or less, 80 Gy or less, 70 Gy or less, or 60 Gy or less. In some embodiments, at about 50 Gy to about 300 Gy (including but not limited to, about 50 Gy to about 100 Gy, about 50 Gy to about 150 Gy, about 50 Gy to about 200 Gy, about 50 Gy to about 250 Gy, about 100 Gy to about 150 Gy, about 100 Gy to about 200 Gy, about 100 Gy to about 150 Gy, about 150 Gy to about 200 Gy, about 150 Gy to about 250 Gy, about 200 Gy to about 250 Gy ) irradiated aAPC. In other embodiments, the aAPC is irradiated at about 50 Gy, about 100 Gy, about 150 Gy, or about 200 Gy. In some embodiments, irradiation is performed prior to culturing aAPCs together with immune cells (eg, one or more of NK cells, NKT cells, and/or γδ T cells) or a population of cells thereof. NK infection and expansion

In another aspect, methods of making populations of natural killer (NK) cells are provided. The method comprises culturing or alternatively consisting essentially of or further consisting of an immune cell activating agent (eg, NK cell activating agent) comprising a population of cells comprising one or more of the following: NK cells, capable of deriving Progenitor cells of NK cells or stem cells from which NK cells can be derived. In some embodiments, the cell population is depleted in the cell population for cells expressing one or more of CD3, CD4, CD8, T cell receptor (TCR) alpha chain, TCR beta chain, or αβTCR.

In some embodiments related to any of the disclosures herein, the one or more immune cell activators (eg, NK cell activators) are selected from one or more of the following: artificial antigen presenting cells (aAPCs), which Express tumor-associated antigens (TAAs) and/or viral antigens and optionally activate and/or stimulate immune cell growth; one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind to one of NK cells, progenitor cells or stem cells Stimulatory receptors above, thereby activating or proliferating NK cells; one or more cytokines thereby activating or proliferating NK cells; and/or one or more chemical moieties thereby activating or proliferating NK cells, which Optionally selected from mTOR inhibitors, PI3K inhibitors or STING activating cyclic dinucleotides (CDNs).

In some embodiments, the stimulating receptor is one or more of the following: CD2, NKp46, CD16, NKG2D, DNAM-1 (CD226), 2B4 (natural killer cell receptor 2B4, CD244), NTB-A ( SLAM family member 6, SLAMF6) and/or NKp46. See, eg, Zamai L, Del Zotto G, Buccella F, et al. Understanding the Synergy of NKp46 and Co Activation Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy. Cells. 2020;9(3):753. Published 2020 Mar 19. doi:10.3390/ cells9030753.

In some embodiments, the aAPC is an aAPC as disclosed herein. In some embodiments, the aAPCs are compared to the cell population at the following cell number ratios (aAPC cell number: cell population cell number and/or aAPC cell number: immune cells in the cell population (e.g., NK cells, NKT cells and/or One or more of the γδ T cells) are cultured together: about 100:1 or more, about 50:1 or more, about 20:1 or more, about 10:1 or more, about 9:1 or greater, about 9:1 or greater, about 8:1 or greater, about 7:1 or greater, about 6:1 or greater, about 5:1 or greater, about 4:1 1 or greater, about 3:1 or greater, about 2:1 or greater, about 1:1 or greater, about 1:2 or greater, about 1:3 or greater, about 1:4 or greater greater, about 1:5 or greater, about 1:6 or greater, about 1:7 or greater, about 1:8 or greater, about 1:9 or greater, about 1:10 or greater , about 1:20 or more, about 1:50 or more, or about 1:100 or more. Additionally or alternatively, the aAPCs are compared to the cell population at the following cell number ratios (aAPCs:cells in a population and/or aAPCs:immune cells (e.g., NK cells, NKT cells and/or γδ) in the cell population One or more of the T cells) are cultured together: about 100:1 or less, about 50:1 or less, about 20:1 or less, about 10:1 or less, about 9 : 1 or less, about 9:1 or less, about 8:1 or less, about 7:1 or less, about 6:1 or less, about 5:1 or less, about 4:1 or less, about 3:1 or less, about 2:1 or less, about 1:1 or less, about 1:2 or less, about 1:3 or less, about 1:4 or less Small, about 1:5 or less, about 1:6 or less, about 1:7 or less, about 1:8 or less, about 1:9 or less, about 1:10 or less, About 1:20 or less, about 1:50 or less, or about 1:100 or less. In some embodiments, the aAPCs are combined with the cell population at a ratio of about 10:1 to about 1:10, about 5:1 to about 1:5, about 3:1 to about 1:3, about 2:1 to about 1:1 The ratio of the number of cells of 2 (the number of cells in aAPC: the number of cells in the cell population and/or the number of cells in aAPC: the ratio of immune cells (such as one or more of NK cells, NKT cells and/or γδ T cells) in the cell population. number of cells) were cultured together. In some embodiments, the aAPCs are combined with the cell population at about 10:1, about 5:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:1: A cell number ratio of 5 or about 1:10 (cell number of aAPC:cell number of cell population and/or cell number of aAPC:immune cells (e.g., one of NK cells, NKT cells and/or γδ T cells) in the cell population or more of) the number of cells) were cultured together.

In some embodiments, the interleukin is selected from the group consisting of B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low toxicity IL-2, and others IL-2 mutant, IL-7, IL-15-N72D superagonist complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803 soluble), IL-15, IL -18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1) or MICA.

In some embodiments, the interferon comprises, or instead consists essentially of or further consists of IL-2. In other embodiments, the cell population is cultured with about 1 IU/mL to about 1000 IU/ml IL2. In some embodiments, the cell population is combined with about 1 IU/mL or more, about 2 IU/mL or more, about 3 IU/mL or more, about 5 IU/mL or more, about 10 IU/mL mL or more, about 20 IU/mL or more, about 30 IU/mL or more, about 40 IU/mL or more, about 50 IU/mL or more, about 60 IU/mL or more, About 70 IU/mL or more, about 80 IU/mL or more, about 90 IU/mL or more, about 100 IU/mL or more, about 110 IU/mL or more, about 120 IU/mL or more, about 130 IU/mL or more, about 140 IU/mL or more, about 150 IU/mL or more, about 160 IU/mL or more, about 170 IU/mL or more, about 180 IU/mL or more, about 190 IU/mL or more, about 200 IU/mL or more, about 250 IU/mL or more, about 300 IU/mL or more, about 400 IU/mL or more more, about 500 IU/mL or more, about 600 IU/mL or more, about 700 IU/mL or more, about 800 IU/mL or more, about 900 IU/mL or more, or about 1000 IU/mL or more IL2 were incubated together. Additionally or alternatively, the cell population is combined with about 2000 IU/mL or less, about 1500 IU/mL or less, about 1000 IU/mL or less, about 900 IU/mL or less, about 800 IU/mL or less, about 700 IU/mL or less, about 600 IU/mL or less, about 500 IU/mL or less, about 400 IU/mL or less, about 300 IU/mL or less, about 250 IU/mL or less, about 200 IU/mL or less, about 100 IU/mL or less, about 90 IU/mL or less, about 80 IU/mL or less, about 70 IU/mL or less less, about 60 IU/mL or less, about 50 IU/mL or less, about 40 IU/mL or less, about 30 IU/mL or less, about 20 IU/mL or less, about 10 IU/mL or less, about 5 IU/mL or less, IL2 co-cultured. In some embodiments, the interferon includes IL-2. In other embodiments, the cell population is combined with about 1 IU/mL to about 1000 IU/ml IL2 (including but not limited to, about 10 IU/ml to about 100 IU/ml, about 100 IU/ml to 200 IU/ml) ml, about 200 IU/ml to about 300 IU/ml, about 300 IU/ml to about 400 IU/ml, about 400 to about 500 IU/ml, about 100 IU/ml to about 500 IU/ml IL2) were incubated together . In some embodiments, the cell population is combined with about 10 IU/mL, about 20 IU/mL, about 30 IU/mL, about 40 IU/mL, about 50 IU/mL, about 60 IU/mL, about 70 IU/mL mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 110 IU/mL, about 120 IU/mL, about 130 IU/mL, about 140 IU/mL, about 150 IU/mL, about 160 IU/mL, about 170 IU/mL, about 180 IU/mL, about 190 IU/mL, about 200 IU/mL, about 250 IU/mL, about 300 IU/mL, about 400 IU/mL, about 500 IU/mL, about 600 IU/mL, about 700 IU/mL, about 800 IU/mL, about 900 IU/mL, or about 1000 IU/mL IL2 were incubated together.

In some embodiments, the interleukin comprises, or instead consists essentially of or further consists of IL-15. In other embodiments, the cell population is cultured with about 0.1 ng/mL to about 500 ng/mL IL15, including any range and/or value falling therein. In some embodiments, the cell population is mixed with about 1 ng/mL to about 100 ng/ml IL15 (including but not limited to about 1 ng/ml to about 10 ng/ml, about 10 ng/ml to about 20 ng /ml, about 20 ng/ml to about 30 ng/ml, about 30 ng/ml to about 40 ng/ml, about 40 ng/ml to about 50 ng/ml IL 15) together. In some embodiments, the cell population is combined with about 1 ng/ml, about 5 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml or about 100 ng/ml IL15 were incubated together.

In some embodiments, the interleukin comprises or instead consists essentially of or further consists of IL-21. In other embodiments, the cell population is cultured with about 0.1 ng/mL to about 500 ng/mL IL21, including any range and/or value falling therein. In some embodiments, the cell population is mixed with about 1 ng/mL to about 100 ng/ml IL21 (including but not limited to about 1 ng/ml to about 10 ng/ml, about 10 ng/ml to about 20 ng /ml, about 20 ng/ml to about 30 ng/ml, about 30 ng/ml to about 40 ng/ml, about 40 ng/ml to about 50 ng/ml IL21) together. In some embodiments, the cell population is combined with about 1 ng/mL, about 5 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, About 75 ng/ml, about 80 ng/ml, about 90 ng/ml or about 100 ng/ml IL 21 were incubated together.

In some embodiments, the cell population is cultured with more than one interleukin (eg, a combination of interleukins as disclosed herein). In some embodiments, the cell population is cultured with any one or any two or all three of 100-500 IU/ml IL-2, 20 ng/ml IL-15, or 25 ng/mL IL-21 . In other embodiments, the cell population is cultured with either or both of 50 IU/ml IL-2 and 0.5 ng/ml IL-15.

In some embodiments, chemical moieties can also be used as immune cell activators (eg, NK activators), for example, treatment with ADAM17 inhibitors has been shown to increase NK cell ADCC by preventing CD16 receptor shedding , and treatment of NK cells with nicotinamide enhanced their L-selectin (which is known to be essential for cellular transport) performance. See, eg, Peled et al, Blood (2017) 130(Suppl 1): 657 and Childs RW, Carlsten M. Nat Rev Drug Discov. 2015;14(7):487-498. Other examples are mTOR inhibitors, PI3K inhibitors and/or STING-activated cyclic dinucleotides (CDNs).

In some embodiments, the methods as disclosed herein further comprise introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide into the cultured cell population for expression. In some embodiments, the CAR specifically recognizes and binds tumor-associated antigens (TAAs) and/or viral antigens. In other embodiments, tumor-associated antigens (TAAs) and/or viral antigens recognized and bound by the CAR are those expressed by aAPC. In some embodiments, the therapeutic protein or polypeptide is selected from antibodies or fragments thereof, enzymes, ligands or receptors.

In some embodiments, the methods as disclosed herein further comprise culturing the cell population with an immune cell activator (eg, an NK cell activator) before and/or after the introducing step. In other embodiments, the culturing step is repeated one, two, three or more times using the same or different immune cell activating agents (eg, NK cell activating agents), or a combination thereof.

In some embodiments, the activating agent is incubated with the cell population before and after the introducing step. In other embodiments, the two activators that are incubated with the cell population before and after the introducing step are the same. In some embodiments, the two activating agents incubated with the cell population before and after the introducing step are different from each other.

Some method embodiments include introducing pseudotyped gamma retroviral particles into a population of cultured cells, thereby introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide as disclosed herein into the population of cultured cells . In some embodiments, pseudotyped gamma retroviral particles are disclosed herein. In some embodiments, a pseudotyped gamma retroviral particle includes a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide as disclosed herein and/or its reverse complement. In other embodiments, the encoding polynucleotide and/or its reverse complement is flanked by two long terminal repeats (LTRs). Additionally or alternatively, pseudotyped gamma retroviral particles include RD114TR and/or BaEVTR as disclosed herein. In some embodiments, the pseudotyped gamma retroviral particle comprises a vector gene body comprising a 5' LTR, a 5' cap, an encoding polynucleotide or its reverse complement, a 3' poly-A tail, and a 3' The LTR or alternatively consists essentially of or further consists of. In other embodiments, the components as disclosed herein are located in the vector genome from 5' to 3'. In some embodiments, the pseudotyped gamma retroviral particle further comprises either or both of reverse transcriptase and integrase. In some embodiments, pseudotyped gamma retroviral particles are based on and/or derived from and/or are selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Reed murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoid virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus.

In some embodiments, pseudotyped gamma retroviral particles are introduced into the cultured cell population at a multiplicity of infection (MOI) of about 0.01 to about 100, including any range and/or value falling therein. In some embodiments, pseudotyped gamma retroviral particles are introduced into the cultured cell population at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5. In some embodiments, the pseudotype is at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Gamma retroviral particles are introduced into the cultured cell population.

In some embodiments, the cell population is cultured for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about the introducing step about 10 days. Additionally or alternatively, the cell population is cultured prior to the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 13 days 14 days, no more than 15 days, no more than 3 weeks or no more than 1 month. In some embodiments, the cell population is cultured for about 1 day to about 180 days (including any range and/or value falling therein) prior to the introducing step. In some embodiments, the cell population is cultured for about 5 days to about 10 days, eg, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days, prior to the introducing step.

In some embodiments, the encoding polynucleotide is introduced into a population of cells via transduction of a viral vector comprising the encoding polynucleotide or its reverse complement in the presence of RetroNectin. In some embodiments, RetroNectin is coated on the inner surface of the container of the transduced cell population. In other embodiments, the container is a bag suitable for culturing cells. In other embodiments, the container is a plate suitable for culturing cells. In another embodiment, the container is a flask suitable for culturing cells. In some embodiments, the cell population expresses either or both of integrin α4β1 (VLA-4) and integrin α5β1 (VLA-5).

In some embodiments, the cell population system is isolated from a biological sample (eg, a blood sample) of an individual. In some embodiments, the cell population system is isolated from one or more of the following: umbilical cord blood of an individual, peripheral blood of an individual, or bone marrow of an individual. In some embodiments, the cell population comprises or instead consists essentially of or further consists of: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells , stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC) or immortalized cells.

In some embodiments, the methods as disclosed herein further comprise enriching the population of cells for expression of any one or more of CD56, CD25, CD122, CD212, CD215, CD218, CD360, TGF-βR or IL-10R cell. In some embodiments, the methods as disclosed herein further comprise enriching, deriving and/or generating CD56 ^dim cells. In other embodiments, CD56 ^dim cells express CD56 surface antigen at low density and have specific cytotoxic functions. Additionally or alternatively, the methods as disclosed herein further comprise enriching, deriving and/or generating CD56 ^bright cells. In other embodiments, CD56 ^bright cells express CD56 surface antigen at high density and secrete specific cytokines. See, eg, Jacobs et al., Eur J Immunol. 2001 Oct;31(10):3121-7. In some embodiments, the cell population comprises or consists essentially of or further consists of peripheral blood NK cells and/or umbilical cord blood NK cells. In other embodiments, peripheral blood NK cells and/or cord blood NK cells express NCR, CD56, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2D, NKG2C and/or NKG2A any one or more of them. In other embodiments, the expression levels of peripheral blood NK cells and/or cord blood NK cells differ. In some embodiments, the methods as disclosed herein further comprise enriching the population of cells expressing NCR, CD56, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2D, NKG2C and/or or cells of any one or more of NKG2A. In some embodiments, the methods as disclosed herein further comprise enriching the cell population for peripheral blood NK cells and/or cord blood NK cells.

In some embodiments, the cell population of any of the steps and/or embodiments and/or aspects may comprise or alternatively consist essentially of or further consist of natural killer (NK) cells or a substantially purified composition thereof .

In some embodiments, the cell population (eg, depleted cell population) of any step and/or embodiment and/or aspect can comprise or alternatively consist essentially of or further Consists of: NK cells, progenitor cells, HSCs, iPSCs or substantially purified populations of each.

In some embodiments, NK cells may comprise or alternatively consist essentially of or further consist of cells derived from one or more of the following: progenitor cells, embryonic stem cells, cells derived from embryonic stem cells, embryos Germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or immortalized cells.

In some embodiments, one or more of the progenitor cells, HSCs or iPSCs are capable of deriving NK cells.

In some embodiments, the cell population of any step and/or embodiment and/or aspect is substantially free of T cells. In some embodiments, the cell population of any step and/or embodiment and/or aspect is substantially free of T regulatory cells.

Some embodiments of the methods as disclosed herein may further comprise isolating, enriching and/or purifying the cell population. In some embodiments, the cell populations of any of the steps and/or examples and/or aspects are isolated, enriched or purified.

In some embodiments, the cell population and aAPC are cultured together in a cell culture medium selected from the group consisting of StemSpan (Stemcell #09960), NK MACS® medium (Miltenyi research 30-114-429; GMP: 170- 076-356), TexMACS (170-076-306 GMP medium), Cellgenix serum-free stem cell growth medium (SCGM, #20806-0500), or ImmunoCult™-XF medium (from Stemcell technologies).

Some embodiments of the methods as disclosed herein further comprise either or both of the steps of: formulating a population of cells as disclosed herein (eg, expressing a CAR composition); and/or cryopreserving as disclosed herein Cell populations (eg, those expressing CAR compositions).

Some embodiments of the methods as disclosed herein further comprise either or both of the following steps: washing the cell population before or after one or more steps, and/or before or during or after one or more steps Test for one or more of the following: (i) viability of the cell population; (ii) sterility of the cell population; (iii) mycoplasma in the cell population; (iv) human leukocyte antigen (HLA) in the cell population (v) the number of cells of the cell population; (vi) the cell phenotype of the cell population; (vii) the HHV6 or HHV7 or both in the cell population or composition comprising the cell population; (viii) the cell population or comprising the cell Human immunodeficiency virus (HIV) types 1 and 2, human T-lymphotropic virus (HTLV) types I and II, hepatitis B virus (HBV), hepatitis C virus (HCV), One or more of CMV, Zika, West Nile, or Treponema pallidum; (ix) cellular phenotypes of aAPCs; (x) in compositions comprising cell populations IL-15 content; (xi) CAR performance in cell populations; (xii) endotoxins in cell populations or compositions comprising cell populations; (xiii) residual tumor burden and/or aAPC contamination of compositions comprising cell populations (xiv) Efficacy of Cell Population; (xv) IFN-γ, IL-15 and TNF-alpha Release of Cell Population; (xvi) Cytotoxic Activity of Cell Population; (xvii) Degranulation of Cell Population; and (xviii) ) monitor or measure one or more of the following in the culture: cell aggregation, glucose or lactate. In other embodiments, phenotypic detection includes detection of cellular expression and/or expression levels of one or more of the following: CD3, CD56, antigen (eg, CD19), CD45, HLA, NKp46, NKG2D, NKG2A, NCR , DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2C, CD107a, CAR or cell surface markers expressed by cell populations such as aAPC and/or immune cells. γδ T cell infection and expansion

In yet another aspect, a method of making a population of γδ T cells is provided. The method comprises culturing or alternatively consisting essentially of or further consisting of a population of cells comprising one or more of the following with one or more immune cell activators (eg, γδ T cell activators): γδ T cells, progenitor cells capable of deriving γδ T cells, or stem cells capable of deriving γδ T cells. In some embodiments, the cell population is depleted of cells in the cell population that express one or more of: a T cell receptor (TCR) alpha chain, a TCR beta chain, or an alpha beta TCR.

In some embodiments, the one or more immune cell activators (eg, γδ T cell activators) are selected from one or more of the following: artificial antigen presenting cells (aAPCs) that express tumor associated antigens (TAAs) and/or or viral antigens and optionally activate and/or stimulate immune cell growth; one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind to stimulating receptors on one or more of γδ T cells, progenitor cells or stem cells , thereby activating or proliferating γδ T cells; one or more cytokines thereby activating or proliferating γδ T cells; or one or more chemical moieties thereby activating or proliferating γδ T cells, optionally selected from mTOR inhibitors , PI3K inhibitors, or STING-activated cyclic dinucleotides (CDNs).

Some embodiments of the methods as disclosed herein further comprise introducing a polynucleotide encoding a CAR and/or another therapeutic protein or polypeptide into the cultured cell population for expression. In some embodiments, the therapeutic protein or polypeptide is selected from antibodies or fragments thereof, enzymes, ligands or receptors. In some embodiments, the CAR specifically recognizes and binds tumor-associated antigens (TAAs) and/or viral antigens. In other embodiments, tumor-associated antigens (TAAs) and/or viral antigens recognized and bound by the CAR are expressed on aAPCs.

Some embodiments of the methods as disclosed herein further comprise culturing the cell population with an immune cell activator (eg, a γδ T cell activator) or a combination thereof after the introducing step. In some embodiments, the incubation step is repeated one, two, three or more times using the same or different activating agents.

In some embodiments, the cell population of any step and/or embodiment and/or aspect may comprise or alternatively consist essentially of or further consist of one or more of the following: γδ T cells or It is a substantially purified composition. In some embodiments, the cell population of any step and/or embodiment and/or aspect (eg, a depleted cell population as disclosed herein) can comprise one or more of the following or alternatively substantially Consists of or further consists of: γδ T cells, HSCs, iPSCs, or substantially purified populations of each.

In some embodiments, one or more of the progenitor cells, HSCs or iPSCs are capable of deriving γδ T cells. In some embodiments, γδ T cells comprise or alternatively consist essentially of or further consist of cells derived from one or more of the following: progenitor cells, embryonic stem cells, cells derived from embryonic stem cells, embryos Germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or immortalized cells. In some embodiments, the cell population is substantially free of cells expressing TCR alpha chains or TCR beta chains. Antigens and Chimeric Antigen Receptors (CARs)

In some embodiments of any aspect as disclosed herein, the tumor-associated antigen is selected from one or more of the following, or fragments thereof, of each: G protein-coupled receptor class C family 5 member D (GPRC5D) , B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild-type epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD123 , CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanosyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanosine Purine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan Receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT, IL-13Ra2, IL-11Rα, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor Receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, mucin 1 (Muc1), NCAM, prostatase, PAP, ELF2M, ephrin B2, fibroblasts Activated protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephrin A receptor type 2 precursor (EphA2), fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1 , UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD -CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostaglandins, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation cut-off point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, asparagine Endopeptidase, HPV E6, E7, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, Glypican 3 ( GPC3), FCRL5 or IGLL1.

In some embodiments of any aspect as disclosed herein, a CAR as disclosed herein comprises or alternatively consists essentially of or further consists of (1) an antibody that specifically recognizes and binds an antigen. (2) hinge domain; (3) transmembrane domain; and (4) intracellular domain including signaling domain. In some embodiments, the CAR further comprises a signal peptide.

In some embodiments of any aspect as disclosed herein, the antigen binding domain of the CAR specifically recognizes and binds one or more of the following: G protein coupled receptor class C family 5 member D (GPRC5D) , B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild-type epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD123 , CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanosyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 (TK1), hypoxanthine guanosine Purine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan Receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT, IL-13Ra2, IL-11Rα, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor Receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, mucin 1 (Muc1), NCAM, prostatase, PAP, ELF2M, ephrin B2, fibroblasts Activated protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephrin A receptor type 2 precursor (EphA2), fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1 , UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD - CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostaglandins, survivin and terminal Granzyme, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation cut-off point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor , Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, Human Telomerase Reverse Transcriptase, RU1, RU2, Tianmen Paraparagine endopeptidase, HPV E6, E7, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, phosphatidylinositol Sugar 3 (GPC3), FCRL5 or IGLL1.

In some embodiments of any aspect as disclosed herein, the antigen binding domain of the CAR includes 6 CDRs of an antibody that specifically recognizes and binds to one or more of: G protein-coupled receptor class C Family 5 member D (GPRC5D), B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild-type epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variant III (EGFRVIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase-activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanosyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72), thymidine kinase 1 ( TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate-specific membrane antigen (PSMA), receptor Somatic tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT, IL-13Ra2, IL-11Rα, prostate stem cell antigen (PSCA) , PRSS21, Vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, mucin 1 (Muc1), NCAM, prostatase, PAP, ELF2M, liver Ligand B2, Fibroblast activating protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephrin A receptor type 2 precursor (EphA2), rock Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17 , XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants , prostaglandin, survivin and telomerase, PCTA-1/galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation cut point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase inverse Transcriptase, RU1, RU2, Aspartame Endopeptidase, HPV E6, E7, Enteric Carboxylesterase, Mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, Glypican 3 (GPC3), FCRL5 or IGLL1.

In some embodiments of any aspect as disclosed herein, the antigen binding domain of the CAR comprises 6 CDRs selected from the group consisting of antibodies or fragments thereof of anti-EGFRwt and anti-EGFRVIII, anti-FLT3 antibody, anti-BCMA Antibodies and/or anti-CS1 antibodies.

In some embodiments, the hinge domain includes a CD8 alpha hinge domain.

In some embodiments, the transmembrane domain comprises a CD8α transmembrane domain.

In some embodiments, the intracellular domain further comprises (1) one or two or more costimulatory signaling regions or (2) IL2Rβ or a fragment thereof comprising a JAK-STAT activation domain or (1) and ( 2). In other embodiments, the costimulatory signaling region comprises a CD28 costimulatory signaling region or a 4-1BB costimulatory signaling region or both.

In some embodiments, the signaling domain comprises or alternatively consists essentially of or further consists of the CD3 zeta signaling domain.

In some embodiments, the introduced polynucleotide further expresses one or more of an interleukin and/or an antibody, or wherein the method further comprises another that will express one or more of an interleukin and/or an antibody A polynucleotide is introduced into the depleted cell population.

In other embodiments, the interferon expressed by the polynucleotide is selected from one or more of the following: B7.1 (soluble or membrane-bound), CCL19 (soluble or membrane-bound), CCL21 ( soluble or membrane bound), CD40L (soluble or membrane bound), CD137L (soluble or membrane bound), GITRL (soluble or membrane bound), GM-CSF (soluble or membrane bound), IL-12 (soluble or membrane-bound), IL-2 (soluble or membrane-bound), low toxicity IL-2 (soluble or membrane-bound), IL-2 analogs lacking cd25 binding (soluble or membrane-bound), IL-15 -N72D superagonist and IL-15RαSushi-Fc fusion protein (soluble or membrane-bound), IL-15 (soluble or membrane-bound), IL-18 (soluble or membrane-bound), IL-21 (soluble or membrane-bound) bound), LEC (soluble or membrane bound), OX40L (soluble or membrane bound), IL-7 (soluble or membrane bound), ICOSL (B7H2, B7RP1, soluble or membrane bound) or MICA (soluble or membrane bound) membrane binding).

Additionally or alternatively, the antibodies expressed by the polynucleotides are monospecific or bispecific or multispecific antibodies. Additionally or alternatively, antibodies expressed by the polynucleotides are immune cell activators, such as NK cell activators.

In some embodiments, the resulting CAR-expressing cell population is suitable for inhibiting cancer cell growth and wherein the antigen is a tumor-associated antigen (TAA) expressed by the cancer cell. Encoding polynucleotides and vectors

In some embodiments, the polynucleotide encoding a CAR and/or a therapeutic protein or polypeptide (which is also referred to herein as an encoding polynucleotide) further encodes a signal peptide. Additionally or alternatively, the polynucleotide encoding the CAR and/or therapeutic protein or polypeptide further includes a suicide gene. In other embodiments, the suicide gene product is selected from one or more of the following: HSV-TK (herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, carboxylesterase, cytochrome P450 or PNP (purine nucleoside phosphorylase), truncated EGFR or inducible caspases (“iCasp”). In some embodiments, the encoding polynucleotide further comprises regulatory sequences that direct expression of the suicide gene. In other embodiments, the regulatory sequences are inducible.

In some embodiments, the encoding polynucleotide further comprises regulatory sequences that direct expression of the CAR or therapeutic protein or polypeptide. In other embodiments, the regulatory sequences directing expression of the CAR or therapeutic protein are inducible or constitutive activity.

In some embodiments, the encoding polynucleotide is introduced into the cell population via a vector. In other embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the non-viral vector is plastid. In some embodiments, the viral vector system is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a herpes virus vector. In another embodiment, the viral backbone contains the necessary nucleic acids or sequences for integration of the encoding polynucleotide into the gene body of the target cell. In some embodiments, the essential nucleic acids required for integration into the genome of the target cell comprise at the 5' and 3' ends the minimal LTR regions required for vector integration.

The present invention also provides vectors comprising or alternatively consisting essentially of or further consisting of a polynucleotide (eg, an encoding polynucleotide) as disclosed herein, optionally inserted into a viral backbone. In some embodiments, the vector is selected for expression in prokaryotic or eukaryotic cells. In some embodiments, the vector includes or instead consists essentially of or further consists of a polynucleotide encoding a modified protein as described herein. In some embodiments, the vector comprises or instead consists essentially of or further consists of a polynucleotide that allows replication of the polynucleotide as described herein. In other embodiments, the vector further comprises regulatory sequences operably linked to the polynucleotide and directing replication of the polynucleotide. In yet another embodiment, the regulatory sequence comprises or alternatively consists essentially of or further consists of one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a termination sub-, silencer, TATA box or Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE).

In some embodiments, the coding sequences are introduced into target cells via pseudotyped gamma retroviral particles as disclosed herein. packaging system

The present invention also provides a viral packaging system comprising: a vector as described herein, optionally wherein the backbone is derived from a virus; packaging plastids; and enveloped plastids. Packaging plastids contain polynucleotides encoding nucleosides, matrix proteins, capsids, and other components required to package the vector genome into viral particles. Packaging plastids are described in the patent literature, eg, US Pat. Nos. 7,262,049, 6,995,258, 7,252,991, and 5,710,037, which are incorporated herein by reference.

The system may also contain plastids encoding pseudotyped envelope proteins provided by envelope plastids. Pseudotyped viral vectors consist of vector particles with glycoproteins derived from other enveloped viruses or alternatively containing functional moieties. See, eg, US Patent No. 7,262,049, which is incorporated herein by reference. In some embodiments, the envelope plastid encodes an envelope protein that optionally does not result in non-specific binding of viral particles to a cell or population of cells. Viral particle specificity can be conferred by proteins or polypeptides (eg, antibody binding domains) inserted into the particle envelope. Examples of suitable envelope proteins include, but are not limited to, those containing VSVG or RD114 domains. In some embodiments, the envelope proteins used herein include, but are not limited to, RD114TR and/or BaEVTR.

The present invention also provides suitable packaging cell lines. In one aspect, the packaging cell line is the HEK-293 cell line. Other suitable cell lines are known in the art, such as those in US Pat. Nos. 7,070,994, 6,995,919, 6,475,786, 6,372,502, 6,365,150, and 5,591,624 (each incorporated herein by reference) explained in. In some embodiments, the packaging cell line is selected from or derived from one or more of the following: 293Vec-Galv, 293Vec-Ampho, 293Vec-RD114, or 293Vec-BaEV. See, eg, Ghani et al, Gene Ther. 2007 Dec;14(24):1705-11; Dakiw Piaceski A et al, Eur Cell Mater. 2018 Feb 14;35:73-86; Ghani et al, Hum Gene Ther. 2009 Sep;20(9):966-74; US Patent Publication No. US20060270042; and US Patent No. 8034335.

The present invention further provides methods of producing viral particles as disclosed herein, comprising transducing or alternatively consisting essentially of or further consisting of a packaging cell line using a viral system as set forth above under conditions suitable for packaging viral vectors its composition. These conditions are known in the art and are briefly described herein. Viral particles can be isolated from cell supernatants using methods known to those skilled in the art, such as centrifugation. Such isolated particles are further provided in the present invention.

The present invention further provides isolated viral particles produced by this method. Viral particles include or alternatively consist essentially of or further consist of a polynucleotide as disclosed herein.

The present invention also provides methods of making viral particles (eg, gamma retroviral particles) comprising a polynucleotide as disclosed herein (eg, an encoding polynucleotide as disclosed herein) by packaging the vector in an environment that facilitates This is achieved by transduction of a packaging cell line as described herein using the vector, envelope plastids and packaging plastids under conditions into enveloped particles. In some embodiments, the viral particles are pseudotyped viral particles. In other embodiments, the particles are isolated from cell supernatants and conjugated to antibodies for cell-specific targeting.

Methods of producing retroviral particles, such as gamma retroviral particles, are also provided. The method comprises, consists essentially of, or further consists of: (i) introducing a vector expressing the vector genome into a first packaging cell line suitable for packaging the vector genome into a first retroviral particle , (ii) transducing the first retroviral particle into a second packaging cell line suitable for replicating the first retroviral particle; and (iii) isolating the replicated retroviral particle.

In some embodiments, the vector is a non-viral vector. In other embodiments, the carrier is a plastid.

In some embodiments, the method further comprises culturing the vector-introduced first packaging cell line. In other embodiments, the method further comprises isolating the first retroviral particle from the culture of the first packaging cell line into which the vector was introduced (eg, from the supernatant).

In some embodiments, the method further comprises culturing the transduced second packaging cell line.

In some embodiments, the following two steps are used to generate the vector: Step 1: Plasmids (which express the retroviral vector gene body) are transfected into 293Vec-GALV cells to generate the retroviral vector. The resulting retroviral vector is enveloped by a lipid membrane comprising GALV. Additionally, since 293Vec-GALV cells were transiently transfected at this step, the retroviral vector produced was also referred to as a transient vector and was tested in Figure 10 . Step 2: The generated retroviral vector was transduced into 293Vec-BaEV cells to replicate the retroviral vector. The replicated retroviral vector has the same vector genome as the parental vector, but is enveloped by a lipid membrane including BaEV. In addition, since 293Vec-BaEV cells were stably transduced in this step, the replicated retroviral vector here is also referred to as a stable vector and tested in Figure 10 .

Surprisingly, T cells (transduced with the RQR8-encoded retroviral vector gene) expressed RQR8 at higher levels than the transient vector using the stable vector. Additionally, these stable virus-producing cells (also referred to as producers) that are generated are relatively stable and integrated with the transgene of interest. In some embodiments, the protein product encoded by the transgene is nontoxic. Therefore, without wishing to be bound by theory, as with the parental packaging cell line, producer cells can be continuously cultured ex vivo for as many generations as they were in logarithmic growth phase. In some embodiments, the producer may maintain the in vitro culture for no more than 30 passages.

In some embodiments, the cells are cultured for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about About 9 days or at least about 10 days. Additionally or alternatively, the cells are cultured for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 14 days 15 days, no more than 3 weeks or no more than 1 month. In some embodiments, the cells are cultured for about 1 day to about 180 days (including any range and/or value falling therein). In some embodiments, the cells are cultured for about 5 days to about 10 days, eg, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

In some embodiments, the first packaging cell line and the second packaging cell line express components required for packaging retroviral particles. In other embodiments, the components required for packaging retroviral particles are selected from one or more of the following: retroviral gag, retroviral pol, retroviral env, fragments thereof, or any combination thereof .

In some embodiments, the second packaging cell line includes a retroviral envelope protein in the cell membrane, but does not include an entry receptor for the retroviral envelope protein in the cell membrane.

Additionally, transduced second packaging cell lines, cells thereof, or cell populations thereof are provided. In some embodiments, the cells stably produce retroviral particles.

In some embodiments, the retroviral envelope protein is BaEV and its entry receptor is ASCT1 or ASCT2. In some embodiments, the retroviral envelope protein is RD114, and it enters the receptor system ASCT2. In some embodiments, the retroviral envelope protein is GALV and it enters the receptor Pit1.

In some embodiments, the first packaging cell line includes an entry receptor for the retroviral envelope protein of the first retroviral particle. In other embodiments, the first packaging cell line does include an entry receptor for the retroviral envelope protein of the first retroviral particle.

In some embodiments, the vector is introduced into the first packaging cell line at a multiplicity of infection (MOI) of about 0.01 to about 100, including any range and/or value falling therein. In some embodiments, the vector is introduced into the first packaging cell line at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5. In some embodiments, the vector is introduced at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 in the first packaging cell line.

In some embodiments, the first retroviral particle is introduced into (eg, transduced into) the second packaging cell line at a multiplicity of infection (MOI) of about 0.01 to about 100 (including any range and/or value falling therein) middle. In some embodiments, the first retroviral particle is introduced into (eg, transduced into) the second packaging cell at an MOI of about 0.1 to about 10, about 0.2 to about 5, about 1 to about 10, or about 1 to about 5 in the department. In some embodiments, the first is compounded at an MOI of about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. Retroviral particles are introduced (eg, transduced) into a second packaging cell line.

Accordingly, retroviral particles produced by the methods as disclosed herein are provided.

In another aspect, methods of producing engineered immune cells (eg, CAR-expressing immune cells) are provided. The method includes introducing, eg, transducing, into, consisting essentially of, or further consisting of, an immune cell or a precursor thereof, retroviral particles produced using the methods as disclosed herein.

In some embodiments, the genetic information of the viral vector particle (which is also referred to herein as the vector genome or viral genome) is RNA, which includes at the 5' and 3' ends the minimal LTR regions required for vector integration and A polynucleotide located between two LTR regions as disclosed herein or alternatively consists essentially of or further consists of. In some embodiments, an encapsidation signal (psi region) required to package the carrier RNA into the particle is further included between the two LTR regions. In some embodiments, the psi region is followed by a Rev-responsive element (RRE) and a central polypurine region sequence (cPPT), which are used for efficient packaging into vector particles by transporting full-length vector transcripts out of the nucleus to enhance vector production.

In some embodiments, the listed genetic elements are transcribed into full-length RNA molecules that are packaged into vector particles and contain all the genetic information to be integrated into the transduced cells.

In some embodiments, the full-length RNA transcript is packaged within the capsid of a carrier particle containing the nucleocapsid, capsid, and matrix proteins generated from packaging plastids. In some embodiments, the reverse transcriptase polymerase generated from the packaging plastids is also located within the capsid with the RNA transcript. In some embodiments, the capsid encapsulates and protects the full-length RNA transcript.

In some embodiments, cells of the packaging cell line (eg, HEK-293T cells) are plated at 75% confluency in complete DMEM medium 24 hours prior to transfection. Transfection mixtures were prepared at least 24 hours after plating cells. Incubate 3 mL of serum-free medium with 150 µl of lipofection reagent for 20 min at room temperature. Plasmids were then added to the medium/lipofection reagent mixture at a ratio (packaging plastids:viral vector plastids:envelope plastids) for 30 minutes without incubation. During this final incubation period, the medium/lipofection reagent/DNA mixture was then added to HEK-293T cells overnight for transfection to occur. The next day, the transfection medium was removed and fresh complete DMEM was added. After 72 hours, the cell culture supernatant can be collected and concentrated by ultracentrifugation at 20,000 rpm for 1.5 hours.

After the carrier particles are germinated from the packaging cells and released into the supernatant, they can be isolated and/or purified by antibodies that specifically recognize or bind to the particles and/or by conjugation of antibodies on the particle envelope as defined herein this carrier particle.

Accordingly, in one aspect, provided herein is a viral packaging system for the production of pseudotyped gamma retroviral particles comprising: (a) a plastid expressing a vector gene body; (b) a packaging plastid; and (c) One or more mantle plastids expressing RD114TR and BaEVTR. In some embodiments, the packaging system further comprises a packaging cell line. In other embodiments, the packaging cell line is the 293T cell line.

In some embodiments, the vector genome includes one or more of the following flanked by two long terminal repeats (LTRs): (A) encoding a chimeric antigen receptor (CAR) and/or another therapeutic protein or a polynucleotide of a polypeptide; (B) the reverse complement of (A); or (C) comprising one or more recognition sites recognized by restriction enzymes suitable for insertion of the coding sequence or its reverse complement into the polynucleotide point polynucleotide. In some embodiments, the therapeutic protein or polypeptide is selected from antibodies or fragments thereof, enzymes, ligands or receptors. In some embodiments, the vector genome further comprises one or more of the following: a 5' LTR, a 5' cap, a 3' poly-A tail, and a 3' LTR. In other embodiments, the encoded polypeptide is located between the 5' cap and the 3' poly-A tail.

In some embodiments, pseudotyped gamma retroviral particles are based on and/or derived from and/or are selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Reed murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoid virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus.

In another aspect, methods of producing pseudotyped gamma retroviral particles are provided. The method comprises transducing or alternatively consisting essentially of or further consisting of a packaging cell line using a packaging system as disclosed herein under conditions suitable for packaging pseudotyped gamma retroviral particles. In other embodiments, the packaging cell line is the 293T cell line.

In some embodiments, at about 5:5:1, 5:4:1, 5:3:1, 5:2:1, 5:1:1, 4:5:1, 3:5:1, 2:5:1, 1:5:1; 4:4:1, 4:3:1, 4:2:1, 4:1:1, 3:4:1, 2:4:1, 1: 4:1; 3:3:1, 3:2:1, 3:1:1, 2:3:1, 1:3:1; 2:2:1, 2:1:1, 1:2: 1; 1.5:1.5:1, 1.5:1:1, 1:1.5:1 or 1:1:1 ratios using plastids of (a), (b) and (c) (i.e. viral vector plastids: Packaging plastids: enveloped plastids) to transduce cell lines. In other embodiments, the packaging system includes at least two mantle plastids, one expressing RD114TR and the other expressing BaEVTR. In other embodiments, RD114TR-expressing plastids and BaEVTR-expressing plastids are transduced at ratios of 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, or 1:3. In some embodiments, cell lines are transduced with plastids of (a), plastids of (b), RD114TR-expressing plastids, and BaEVTR-expressing plastids at the following ratios: 3:3:1:1, 2:2 :1:1, 1.5:1.5:1:1, 1:1:1:1, 5:5:1:1, 5:4:1:1, 5:3:1:1, 5:2:1 :1, 5:1:1:1, 4:5:1:1, 3:5:1:1, 2:5:1:1, 1:5:1:1, 4:4:1:1 , 4:3:1:1, 4:2:1:1, 4:1:1:1, 3:4:1:1, 2:4:1:1, 1:4:1:1; 3 :3:1:1, 3:2:1:1, 3:1:1:1, 2:3:1:1, 1:3:1:1, 2:2:1:1, 2:1 :1:1, 1:2:1:1, 1.5:1.5:1:1, 1.5:1:1:1, 1:1.5:1:1 or 1:1:1:1, 2.5:2.5:1 :1:1, 2.5:2:1:1, 2.5:1.5:1:1, 2.5:1:1:1, 2.5:0.5:1:1, 2:2.5:1:1, 1.5:2.5:1 :1, 1:2.5:1:1, 0.5:2.5:1:1, 2:2:1:1, 2:1.5:1:1, 2:1:1:1, 2:0.5:1:1 , 1.5:2:1:1, 1:2:1:1, 0.5:2:1:1, 1.5:1.5:1:1, 1.5:1:1:1, 1.5:0.5:1:1, 1 :1.5:1:1, 0.5:1.5:1:1, 1:1:1:1, 1:0.5:1:1, 0.5:1:1:1, 0.75:0.75:1:1, 0.75:0.5 :1:1, 0.5:0.75:1:1 or 0.5:0.5:1:1. cells and cell populations

In one aspect, a population of cells and/or progeny thereof as disclosed herein is provided. In some embodiments, the cell population is clonal. In some embodiments, cell populations are isolated and/or enriched and/or engineered.

In one aspect, immune cells (eg, NK cells and/or γδ T cells) or populations thereof produced or prepared by methods as disclosed herein are provided. Further provided are compositions comprising cells or populations thereof and a carrier, optionally a pharmaceutically acceptable carrier. In some embodiments, immune cells or populations thereof are isolated and/or enriched and/or engineered.

Additionally or alternatively, the cell lines as disclosed herein are derived or differentiated from stem cells. In some embodiments, the derived and/or differentiated cells and/or cell populations comprise, consist essentially of, or further consist of immune cells. In some cases, the immune cell line is selected from B cells, T cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, cells of myeloid lineage, and/or neutrophils. In some embodiments, the T cells do not express CD3, ie are CD3- T cells. In some embodiments, the T cells do not express CD4, ie are CD4- T cells. In some embodiments, the T cells do not express CD8, ie are CD8- T cells. In some embodiments, the T cells do not express any one or more of the following: T cell receptor (TCR) alpha chain, TCR beta chain, or αβ TCR. In some embodiments, the T cells express TCR gamma chains. Additionally or alternatively, T cells express TCR delta chains. In some embodiments, the T cells are γδ T cells. In some cases, the isolated or enriched population of immune cells comprises, consists essentially of, or further consists of monocytes, macrophages, and/or microglia.

In some embodiments, the cell population substantially comprises immune cells, optionally derived from stem cells (eg, HSCs and/or induced pluripotent stem cells (iPSCs)). In some embodiments, the cell population consists essentially of stem cells (eg, HSCs and/or iPSCs) that are optionally derived into immune cells.

In some embodiments, the cell population is substantially homogeneous, for example, at least about 60% or at least about 70% or at least about 80% or at least about 85% or at least about 90% or at least about 91% or at least About 92% or at least about 93% or at least about 94% or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% of the cell lines are identical.

In another aspect, NK cells or populations thereof produced or prepared by methods as disclosed herein are provided. Further provided are compositions comprising cells or populations thereof and a carrier, optionally a pharmaceutically acceptable carrier.

In another aspect, γδ T cells or populations thereof produced or prepared by methods as disclosed herein are provided. Further provided are compositions comprising cells or populations thereof and a carrier, optionally a pharmaceutically acceptable carrier.

In some embodiments, cells and/or populations and/or compositions as disclosed herein comprise less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1.0%, less than about 1.1%, less than about 1.2%, less than about 1.3%, less than about 1.4%, less than about 1.5%, less than about 1.6%, less than about 1.7%, less than about 1.8%, less than about 1.9%, or less than about 2.0% aAPC.

Additionally or alternatively, cells and/or populations and/or compositions as disclosed herein have no less than 30%, no less than 40%, no less than 50%, no less than 55%, no less than 60%, no less than 65% %, not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90% or not less than 95% survival rate.

In some embodiments, cells and/or populations and/or compositions as disclosed herein are sterile.

In some embodiments, cells and/or populations and/or compositions as disclosed herein comprise less than a threshold pyrogenic dose (TPD) of endotoxin that is statistically evaluated in test individuals (eg rabbit, see eg Wachtel and Tsuji, 1976; Dabbah, et al., 1980) the extent of endotoxin activity required to induce fever. The TPD can be determined based on the route of administration. In one embodiment, the TPD is a 5 EU Kg ⁻¹ h ⁻¹ endotoxin, eg, for intravenous or intramuscular administration. In one embodiment, the TPD is a 0.2 EU Kg ⁻¹ h ⁻¹ endotoxin, eg, for intrathecal administration.

In some embodiments, the disclosed cells and/or populations and/or compositions are negative for one or more of the following: mycoplasma, foreign viruses, or HHV (eg, HHV6 and/or HHV7).

In some embodiments, the cell populations and/or compositions as disclosed herein comprise greater than about 25%, greater than about 30%, greater than 40%, greater than about 45%, greater than about 50%, greater than about 55% in total cells %, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of CAR-expressing cells. In other embodiments, the cell populations and/or compositions as disclosed herein comprise about 25%, about 30%, 40%, about 45%, about 50%, about 55%, about 60%, About 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the cells expressing the CAR.

In some embodiments, the cell populations and/or compositions as disclosed herein comprise greater than about 25%, greater than about 30%, greater than 40%, greater than about 45%, greater than about 50%, greater than about 55% in total cells %, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99% of expressed CD56 cell. In other embodiments, the cell populations and/or compositions as disclosed herein comprise about 25%, about 30%, 40%, about 45%, about 50%, about 55%, about 60%, About 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the cells expressing CD56.

In some embodiments, cell populations and/or compositions as disclosed herein comprise less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, less than about 1.0%, less than about 1.1%, less than about 1.2%, less than about 1.3%, less than about 1.4%, less than about 1.5%, less than about 1.6%, less than about 1.7%, less than about 1.8%, less than about 1.9%, or less than about 2.0% of cells expressing CD3.

In some embodiments, cells and/or populations and/or compositions as disclosed herein comprise less than about 2, less than about 3, less than about 4, less than about 5, less than about 6, less than about 7, less than about 8 , less than about 9, or less than about 10 polynucleotide copies per cell.

In some embodiments, cells and/or populations and/or compositions as disclosed herein lack proliferation in the absence of IL-2 and/or IL-21.

In some embodiments, cells and/or populations and/or compositions thereof as disclosed herein have potent potency (ie, efficacy), eg, not by any culturing step and/or introducing step as disclosed herein More than 1 times, more than 2 times, more than 3 times, more than 4 times, more than 10 times, more than 15 times, more than 20 times, or more than the prepared immune cells. Such efficacy can be measured as the secretion of IFNγ and/or other pro-inflammatory interleukins and/or the expression of CD107 (eg, CD107a). See, eg, Orange et al, J Exp Med. 1995 Oct 1;182(4):1045-56; and McElroy et al, J Immunol Methods. 2007 Dec 1;328(1-2):45-52.

In some embodiments, cells and/or populations and/or compositions thereof as disclosed herein express one or more antigens (eg, CD19, NKp46, or NKG2D).

In other embodiments, the cells, cell populations, and/or compositions can be used to treat and/or prevent cancer or to test new therapies in individuals in need.

In some embodiments, cells and/or populations and/or compositions as disclosed herein (eg, for administration to a subject in need thereof) comprise greater than 1 x ¹⁰ cells/kg of body weight of the subject to be treated (including (but not limited to) not limited to) greater than 2 x 10 ⁶ cells/kg, greater than 3 x 10 ⁶ cells/kg, greater than 4 x 10 ⁶ cells/kg, greater than 5 x 10 ⁶ cells/kg, greater than 6 x 10 ⁶ cells/kg /kg, greater than 7 × 10 ⁶ cells/kg, greater than 8 × 10 ⁶ cells/kg, greater than 9 × 10 ⁶ cells/kg, greater than 1 × 10 ⁷ cells/kg, greater than 2 × 10 ⁷ cells/kg /kg, >3 × ¹⁰⁷ cells/kg, > ⁴ × 107 cells/kg, > ⁵ × 107 cells/kg, >6 × 107 cells/kg, > ⁷ × ¹⁰⁷ cells/kg /kg, greater than ⁸ x 107 cells/kg, greater than ⁹ x 107 cells/kg or greater than 1 x ¹⁰⁸ cells/kg) or alternatively consist essentially of or further consist thereof. treatment method

In one aspect, methods of inhibiting the growth of cancer cells are provided. The method comprises contacting, for example, an effective amount of a body of CAR-expressing cells made by the methods as disclosed herein with or alternatively consisting essentially of or further consisting of a cancer cell. In some embodiments, the antigen recognized by the CAR is a tumor associated antigen (TAA) expressed by cancer cells. In some embodiments, the contacting is in vivo or in vitro.

In another embodiment, a method of treating cancer in an individual is provided. The method comprises administering, for example, an effective amount of a population of CAR-expressing immune cells made by the methods as disclosed herein to the individual or alternatively consists essentially of or further consists of the same. In some embodiments, the antigen recognized by the CAR is a TAA expressed by cancer cells. In some embodiments, the administration is first-line therapy, second-line therapy, third-line therapy, or fourth-line therapy.

In some embodiments of the methods as disclosed herein, the cell population comprises, or instead consists essentially of, or further consists of, NK cells. Additionally or alternatively, the cell population comprises less than or equal to about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0% , about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2.0% of CD3+ cells.

In some embodiments, less than 1 x 10 ² , 2 x 10 ² , 3 x 10 ² , 4 x 10 ² , 5 x 10 ² , 6 x 10 ² , 7 x 10 ² , 8 x 10 ² are administered to the individual , 9 × 10 ² , 1 × 10 ³ , 2 × 10 ³ , 3 × 10 ³ , 4 × 10 ³ , 5 × 10 ³ , 6 × 10 ³ , 7 × 10 ³ , 8 × 10 ³ , 9 × 10 ³ , 1 × 10 ⁴ T cells/kg body weight.

In some embodiments, the cancer cell line is selected from cancer cells of the circulatory system, such as the heart (sarcomas [angiosarcomas, fibrosarcomas, rhabdomyosarcomas, liposarcoma], myxomas, rhabdomyomas, fibroids, and lipomas) , mediastinum and pleura and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, such as nasal cavity and middle ear, paranasal sinuses, larynx, trachea, bronchi, and lungs, such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchial proto-carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchial) carcinoma, bronchial adenoma, sarcoma, lymphoma, cartilaginous hamartoma, mesothelioma; Gastrointestinal system such as esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (cancer, lymphoma, leiomyosarcoma), stomach, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma , gastrinoma, carcinoid tumor, pancreatic tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroids), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising anywhere; genitourinary tract such as kidney (adenocarcinoma) , Wilm's tumor [Wilm's tumor], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), Testicular (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, mesenchymal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); liver, e.g. hepatoma (liver cell carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (e.g., pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor, and glucagon tumor); bone, such as osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, Malignant giant cell tumor, chordoma, osteochondroma (osteochondral exostoses), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor; nervous system, eg, central Nervous system (CNS) neoplasms, primary CNS lymphomas, skull cancers (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meningeal carcinomas (meningiomas, meningosarcoma, hyperglial hyperplasia) ), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pineal tumor], glioblastoma pleomorphic, oligodendroglioma, neural Schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas); reproductive systems such as gynecology, uterus (endometrial cancer), cervix (cervical cancer, tumors) early cervical dysplasia), ovaries ( Ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], mesothelial cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma tumor), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, grape sarcoma (embryonic rhabdomyosarcoma), fallopian tube (cancer ) and other parts related to the female reproductive organs; penis, prostate, testis and other parts related to the male reproductive organs; hematological systems such as blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, eg, lips, tongue , gums, floor of mouth, upper jaw and other parts of the oral cavity, parotid and other salivary glands, tonsils, oropharynx, nasopharynx, piriform recess, hypopharynx and lips, other parts of the mouth and pharynx; skin, malignant melanoma, Cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, dermatofibroma and keloid; adrenal gland: neuroblastoma; and others Tissue, including connective and soft tissue, retroperitoneal cavity and peritoneum, eye, intraocular melanoma and appendages, breast, head or/and neck, anal area, thyroid, parathyroid, adrenal and other endocrine glands and related Structure, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of respiratory and digestive system and secondary malignant neoplasms of other parts.

In some embodiments, the cancer cell line is a solid tumor cell. In other embodiments, the cancer cells are not solid tumor cells. In other embodiments, the cancer cell line is a leukemia cancer cell. In some embodiments, the cancer cells are primary cancer cells or metastatic cancer cells. In some embodiments, the cancer cell line is from a carcinoma, sarcoma, myeloma, leukemia or lymphoma.

Other effective therapies may be combined with and/or added to the present invention as desired. Some embodiments of the methods as disclosed herein further comprise contacting the cells with or administering the monotherapy. In some embodiments, the monotherapy comprises or instead consists essentially of or further consists of surgical resection, chemotherapy, radiation therapy, immunotherapy, and targeted therapy. In some embodiments, the monotherapy is first-line therapy, second-line therapy, third-line therapy, or fourth-line therapy.

In some embodiments, an "effective amount" is delivered, that is, an amount sufficient to achieve beneficial or desired results. An effective amount can be administered in one or more administrations, applications or doses. This delivery depends on a number of variables, including the time period for which the individual dosage unit is intended to be administered, the bioavailability of the therapeutic agent, the route of administration, and the like. It is to be understood, however, that the specific dosage value of the therapeutic agents of the present invention for any particular individual will depend on a variety of factors, including the activity of the particular compound employed, the age, weight, general health, sex and diet, time of administration, excretion of the individual The rate, combination of drugs and severity of the particular condition being treated and the form of administration. Treatment doses can generally be escalated to optimize safety and efficacy. In general, dose-response relationships from in vitro and/or in vivo testing can first be used to guide appropriate doses for patients. In general, the desired administered amount of the gene or protein is effective to achieve serum levels commensurate with the effective in vitro concentrations found. Determination of these parameters is well known in the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit.

The term administration shall include, but is not limited to, oral, parenteral (eg, intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection or implantation) , Topical or systemic administration by inhalation spray, nasal, vaginal, rectal, sublingual, transurethral (e.g., urethral suppositories), intracranial or topical routes of administration (e.g., gels, ointments, creams) creams, aerosols, etc.), and may be formulated individually or together in suitable dosage unit formulations containing conventional pharmaceutically acceptable non-toxic carriers, adjuvants, excipients, and vehicles suitable for each route of administration. The present invention is not limited by route of administration, formulation or schedule of administration. In some embodiments, the administration is performed locally, eg, into the bone marrow or brain. In some embodiments, administration is performed systemically. In some embodiments, the administration is, for example, over about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours or about 1 day of infusion.

Further provided is a kit comprising or alternatively consisting essentially of or further consisting of one or more agents suitable for use in the methods as disclosed herein and optional instructions. In some embodiments, the agent is selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, an antibody for detecting a cellular phenotype, a Antibodies for isolation or enrichment or purification of immune cells, primers for detection of polynucleotides as disclosed herein, cytokines and aAPCs.

The following examples are intended to illustrate, and not to limit, the embodiments disclosed herein. Experimental Methods Experiment No. 1 : Unique Use of Gamma Retroviral Vector (PCIR) Mediated Gene Transfer to Deliver and Test Function of Chimeric Antigen Receptors in Human Primary NK Cells

Lack of efficient methods for gene delivery into human primary NK cells, transgene instability, and low viability of transduced CAR-NK cells are major obstacles to successful CAR-NK development and manufacturing. Vein-to-vein time is critical because undesired wait times in patients are associated with poorer prognosis and not all eligible patients receive therapy (due to poorer T-cell function, time to preparation, and/or time to relapse). Currently available therapies have variable efficacy and/or toxicity. In short, damaged T cells in a patient can affect product efficacy. This variability can lead to unpredictable treatment outcomes. In addition, it is not uncommon for grade 3-4 cytokine release to cause syndromes and neurotoxicity. Production costs are high due to complex logistics, scale inefficiencies and limited availability. Manufacturing failures thus include, but are not limited to, the inability to generate inventories in individualized therapy and the possible difficulty of re-treatment due to limited patient starting material.

To address these issues, provided herein is the unique use of gamma retroviral vector (PCIR)-mediated gene transfer for delivery and testing of function of chimeric antigen receptors (CARs) in human primary NK cells.

Lentiviral and retroviral vector-mediated gene delivery has proven to be a safe method for genetic modification. However, in many reports and even in applicants' first-hand experiments, conventional lentiviral vector-mediated transduction is less efficient for NK cells due to significantly reduced transgene performance during CAR-NK expansion. See, for example , Figures 5A and 5C . Accordingly, many attempts by the applicant have been directed to finding alternative gene delivery methods that overcome the inefficiencies of lentiviral vectors.

First, the Moloney murine leukemia virus-derived SFG gamma retroviral vector was tested for gene delivery to peripheral blood (PB)-derived human primary NK cells.

As shown in Figure 1 , the primary human NK cell line used in the present invention was derived from peripheral blood (PB) and isolated using the MACSxpress Human Whole Blood NK Cell Isolation Kit (Miltenyi Biotec, 0.130-098-185). Purity was determined by staining cells with CD56 and CD3 antibodies ( FIG. 2A ), and NK cells were defined as the CD56+/CD3- population. NK cells were cultured with NK MACS medium (130-114-429) in the presence of 50 IU/ml human IL-2 and irradiated K562-mb21-41BBL feeder cells (1:1 ratio). All gamma retroviral plastids (PCIR) were packaged using PEQ-PEM3(-E) and enveloped using BaEVTR or RD114TR plastids (at a 1.5:1.5:1 ratio) and via lipofectamine 3000 reagent (Invitrogen™ L3000075) or PEI MAX 40K (Polysciences, Inc, catalog number: 24765) was transiently transfected into 293T cells (ATCC CRL-3216). Compared to ASCT-2 recognized by RD114TR, BaEVTR plastids are practically beneficial for delivering larger inserts into activated NK cells expressing both viral entry receptors ASCT-1 and ASCT-2. Using non-tissue culture plates coated with retronectin reagent (TakaRa, T100A/B), Applicants directly performed titration assays by infecting the above retroviral particles into activated NK cells (7 days in culture) and using MOI 3 Generate CAR-NK. It was evident that after isolation and culture for 5 days, the primary NK cells entered a state of significant proliferation, whereby the activated NK cells on days 6-10 were used for gene delivery ( Fig. 2B ).

Transduction efficiency was then assessed on day 3 post-infection. Implement enrichment, expansion, functional and other therapeutic applications.

The experimental results show that there are NK cells with significantly higher survival rates (between 73.51% - 82.81%) after infection (which can be obtained by excluding low forward scatter (FSC) and high side scatter (SSC) events. Obtained roughly live cell gate) ( Fig. 3B ), the transduction efficiency of EGFR CAR gene (Fab-AF647 positive) in the three donors was between 65.11% - 72.75% ( Fig. 3C ), and at day 8 post-infection The high proliferative state yields in the three donors were 25, 34 and 45 times the initial infected NK numbers ( Fig. 3D ). This extensive optimization enables continuous transduction of CAR constructs in primary human NK cells at 50% - 80% efficiency; CAR NK cells from 10 to 20 million are expanded 25-45-fold ex vivo in 8 days to Hundreds of millions; and achieve CAR fll retention and NK cell ex vivo expansion.

Test longer cultures. See Figure 7 for example. This extensive optimization as disclosed herein enables expansion from tens of millions of NK cells to billions of NK cells within 17-24 days and provides a true off-the-shelf with excellent viability upon thawing of frozen products. Commercial manufacturing costs are at least about $2000 per dose and have greater than 500 doses per batch.

Retroviral titration based on direct infection of activated NK cells of the same proliferative state using the same delivery strategy is provided in Figure 4 .

In addition, on day 10 after sorting, GFP-positive cells were significantly reduced from about 100% to 39% for PCIL-EGFR-CAR-NK and PCIL-GFP empty vector controls ( Figures 5A and 5C ). However, flow analysis confirmed that PCIR-EGFR-CAR-NK had a highly stable expression of the transduced gene at days 10, 14 and 25 after sorting ( Figures 5B and 5C ).

It was also observed that the transduction rates of the empty vector truncated CD19 (EV-Tcd19) and EGFR transgenes were on average 85% (positive for CD19-PE) and 79.6% (positive for goat anti-mouse Fab-AF647), respectively ( FIG. 6A ). In addition, the engineered EGFR-CAR-NK had dynamic real-time killing compared to the empty vector mock-transduced group and the non-transduced NK group with the lowest viable cell index ( FIG. 6B ).

Second, Applicants optimized the time point of retroviral transduction of the expanded primary NK cells. Because gamma-retroviruses are capable of efficiently infecting dividing cells, Applicants determined the growth curve and the time point when primary NK cells enter a state of significant proliferation for gene delivery.

Third, several studies have clearly shown that the transduction efficiency of human primary lymphocytes depends on the type of envelope protein used to coat the retroviral vector, and activated NK cells highly express the receptors ASCT-1 and ASCT- 2 (which is the baboon envelope glycoprotein for entry into the target (BaEV-TR)). The ASCT-2 receptor was used to enter the feline endogenous retroviral envelope glycoprotein (RD114TR) into cells, thereby testing BaEVTR and RD114TR. BaEVTR plastids are used in practice for larger insert delivery, such as tandem CAR, dual CAR, and BiTE CAR.

Next, it was demonstrated that RetroNectin reagents can enhance retrovirus-mediated gene transduction by helping to co-localize target cells and virions. Specifically, viral particles bind RetroNectin reagents via interaction with the H domain, and target cells primarily via the cell surface integrin receptors VLA-5 and VLA-4 with fibronectin C domain and CS-1 position, respectively point interaction for binding. By promoting proximity, RetroNectin reagents can enhance retrovirus-mediated gene transfer to target cells expressing the integrin receptors VLA-4 and/or VLA-5.

In addition, Applicants have determined retroviral titrations based on direct infection of activated NK cells of the same proliferative state using the same delivery strategy, which aims to avoid over- or underestimation from titrations calculated from methods based on indirect measurement of different cell types.

As the findings disclosed herein demonstrate, the combined use of RD114-TR or BaEV-TR pseudotyped gamma retroviral particles with RetroNectin is a successful strategy for delivering therapeutic genes into human primary NK cells with high transduction Efficiency, sustained transgene performance and greater cell viability and expansion rate.

In some embodiments, NK cells are collected from umbilical cord blood of healthy neonates. The best NK cell candidates were selected as revealed. The NK cells are then expanded using feeder cells (eg, aAPCs as disclosed herein). The expanded NK cells are transduced to express the CAR and/or other components (eg, suicide switches, antibodies, and interferons). Sustained ex vivo expansion was also driven by exogenous IL-2. The final cell product is then harvested, cryopreserved, and thawed with high viability for ready administration to patients.

In some embodiments, the methods allow selection of the optimal cord blood product for NK cell expansion. Co-culture with aAPCs as disclosed herein can achieve large scale NK cell expansion. The production method significantly reduces the amount of retrovirus required and reduces NK cell expansion time by 20%. Achieving high transduction efficiency for expressing CAR. Experiment 2 : Cell Therapy : Process Development Research

Test media and/or supplements that support NK growth and/or expansion, including (but not limited to) StemSpan (Stemcell #09960), NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076-356) , TexMACS (170-076-306 GMP medium) and Cellgenix serum-free stem cell growth medium (SCGM, #20806-0500).

Compare interferon supplements to optimize yield, eg, compare one or more of the following: 100-500 U/ml IL-2, 20 ng/ml IL-15, 25 ng/mL IL-21 or any combination thereof.

NK cell enrichment was performed. The following commercially available kits were tested and compared: Stemcell NKdepletion EasySep NK Isolation Kit (17955RF), RosetteSep NK Enrichment Mix (#15065) and Miltenyi NK Isolation Kit (130-092-657) and CD56 ⁺ NK Cell Isolation kit (130-092-660).

Various CD3-depleted panels were tested that reduced CD3+ cells to <0.3% (ie, less than 0.3% of the depleted cell population). Such kits include, for example, Miltenti 130-096-535.

K562 engineered cells (eg, those expressing membrane-bound IL-15 (sushi domain) and/or IL-21) are generated. A primary cell bank (MCB) and/or a working cell bank (WCB) of engineered cells are developed according to current good manufacturing practices.

In addition, complete release testing of K562 MCB and WCB pools, including (but not limited to) testing one or more of the following: sterility, endotoxin, mycoplasma, foreign virus testing (in vitro and in vivo), PCR-based Virus testing, isozyme analysis, karyotyping, S ⁺ L ^- , etc. In addition, K562 marker co-expression (eg CD64/FcyRI, CD86/B7-2, CD137L/4-1BBL, truncated CD19, and membrane-bound IL-21) was determined during a 4-week culture period and during irradiation (21-day culture). ) after the stability. K562 cell irradiation was tested to ensure that the irradiator was qualified/effective. Kinetics of growth and/or viability were tested after tiling on days 1-14 post-irradiation with 50, 100, 150 Gy irradiation.

Expansion of cord blood-derived NK cells was tested using irradiated K562 cells at various NK:K562 ratios (eg, 1:2, 1:1, 1:3, 1:5 ratios).

Transduction efficiencies were compared using RetroNectin-coated AFC bags with RetroNectin-coated plates.

The process is then scaled up, eg by comparing G-Rex, Xuri, Bioflow and Xcellerex bioreactor systems.

More than ¹ x 1011 CD56+ cells were generated from ¹ x 108 CD56+ cells. Tests were performed first with fewer cultures to define growth and expansion parameters and to demonstrate functionality. Without wishing to be bound by theory, cells lose efficacy due to high expansion, whereby the balance between cell expansion and function is a major consideration for scale-up.

Test antibodies/reagents used include (but are not limited to): anti-CD56 Brilliant Violet 605 (BioLegend, San Diego, CA), anti-CD3 APC-H7, Granzyme B-PE-CF594, CCR4-BV421, CXCR3- PerCP Cy5.5, NKp46-BV711 (BD Biosciences, San Jose CA), CD57-PerCP (Bioss Woburn MA), anti-CD16 Brilliant Violet 650 and anti-CD19 PE (Miltenyi Biotec Inc.), CD44-BV785, CXCR4-BV605, 2B4-PE, NKG2D-PE, DNAM-FITC, TbetBV711, CD16-BV650, CX3CR1-PE-Cy7, CD62L-PE-Cy7, CXCR1-PE, CCR7-FITC, PD-1-BV421, NKp30-biotin (from Biolegend), NKp44-PerCP-eflour 710, Demesomorphin-efluor-660, KLRG-1-PE (eBiosciences San Diego CA), DAP12-PE, CD158-FITC (R&D Minneapolis MN), DAP10-FITC (Antibodies- Online Atlanta GA), NKG2A-PE-Cy7 (Beckman Coulter Irving, TX). Table 1 Example Tests Process steps test 1. Donor Eligibility Review CoAs for pathogen screening. Repeat viability, cell number, sterility, phenotype, myco, HLA, HHV 6, HHV 7 2. after ficoll Viability, cell number, sterility, phenotype 3. After CD56 enrichment Viability, cell number, sterility, phenotype 4. After CD3 depletion Viability, cell number, sterility, phenotype 5. After 1st amplification on K562 Viability, cell number, sterility, phenotype, K562 phenotype, mycoplasma 6. After RRV transduction Viability, cell number, sterility, phenotype, mycoplasma 7 a, b After amplification Viability, cell number, sterility, phenotype, mycoplasma, IL-15, CAR 8. after harvest Viability, cell number, sterility, phenotype, K562 phenotype, mycoplasma, IL-15, CAR 9. After provision/release Viability, cell count, sterility, phenotype, mycoplasma, product release testing, IL-15, CAR 10. Stability test Appearance, Viability, Cell#, Sterility, Mycoplasma, CAR 11. clinical material On-site review of container integrity and patient attributes

As stated above, K562 testing was performed, including, but not limited to, generating a pool of cloned K562-genetically modified cells (re-selection if the cells were not cloned); testing the pool for viability, sterility, mycoplasma, endotoxin, HLA and/or genetically modified surface markers (eg mb IL-15, mb IL-21, CD64, CD86, CD137L, etc.); development of certificates of analysis for K562-genetically modified cells; ensuring irradiators are calibrated and dosed Validated and tested 100 Gy, 200 Gy now cells irradiated and tested for proliferation/survival within 14 days. Table 2 illustrates the phenotypic analysis Process steps test 1. Donor Eligibility CD3, CD56, CD19, CD45, HLA 2. after ficoll CD3, CD56, CD19, CD45 3. After CD56 enrichment CD3, CD56, CD19, CD45, NKp46, NKG2D 4. After CD3 depletion CD3, CD56, CD19, CD45 5. After 1st amplification on K562 CD3, CD56, CD45, K562 markers 6. After RRV transduction CD56 7 a, b After amplification CD56, K562 markers, NKp46, NKG2D, CAR 8. after harvest CD56, K562 markers, NKp46, NKG2D, CAR 9. After provision/release CD3, CD56, CAR 10. Stability test CD56 11. clinical material On-site review of container integrity and patient attributes

Perform donor eligibility screening and testing of allogeneic products, including, but not limited to, testing for one or more of the following: Human Immunodeficiency Virus (HIV) types 1 and 2; Human T-lymphotropic virus ( HTLV) types I and II; hepatitis B virus (HBV); hepatitis C virus (HCV); CMV, Zika, West Nile; Treponema pallidum; HHV6/HHV7.

Umbilical cord blood contains approximately 5 x 10 ⁸ total nucleated cells (TNC), 30% of which are NK cells. Therefore, each cord blood unit (60 mL) contains about 1~2 × 10 ⁸ NK cells. Table 3 illustrates the characterization test method Viability, cell count 7-AAD staining, flow cytometry Sterility Gram, 21CFR610.12 endotoxin Limulus amebocyte lysate, Endosafe Mycoplasma MycoAlert Rapid Test Kit HHV test Test HHV6, HHV7 residual tumor burden Flow Cytometry number of cells Flow Cytometry CAR performance % of CAR+ cells, flow cytometry Purity, Phenotype % CD56+ cells, flow cytometry transgene copy number Taqman PCR effect Cytotoxicity against target antigen ⁺ cells Attributes HLA matching of donor and final product (low resolution class I) Table 4 illustrates in-process testing test method Recommended Specifications Viability, cell count 7-AAD staining, flow cytometry ⩾70% Survival Sterility Gram, 21CFR610.12 Negative on day 7 (automated test) endotoxin Limulus amoeba-like cell lysate, Endosafe <5 EU Kg ⁻¹ h ⁻¹ Mycoplasma MycoAlert Rapid Test Kit feminine residual tumor burden Flow cytometry, PCR < 0.3% K562 cells, K562 cell DNA number of cells Flow Cytometry 1ee7 cells/kg CAR performance % of CAR+ cells, flow cytometry > 50% CAR ⁺ cells purity, phenotype % CD56+ cells, flow cytometry > 95% CD56 ⁺ cells, < 0.3% CD3 ⁺ transgene copy number Taqman PCR < 4 copies/cell effect Cytotoxicity against target antigen ⁺ cells >10-fold increase in IFN-γ secretion or CD107 analysis Attributes HLA matching of donor and final product (low resolution class I) Confirmation mismatch information only Flow Cytometry CD19, NKp46, NKG2D, cell aggregates, glucose, lactate Table 5 illustrates the release test : NK-FLT3-CAR test method Recommended Specifications Exterior Visual inspection cloudy liquid suspension survivability Trypan blue exclusion, 7-AAD staining ⩾70% Survival Sterility 21CFR610.12 (14 day test) Negative on day 14 (aerobic and anaerobic) endotoxin Limulus amoeba-like cell lysate, Endosafe <5 EU Kg ⁻¹ h ⁻¹ Mycoplasma Culture method USP/EU feminine foreign virus PCR feminine HHV test Test HHV6, HHV7 feminine residual tumor burden Flow Cytometry ＜0.3% K562 cells number of cells Flow Cytometry ¹ x 107 cells/kg CAR performance % of CAR+ cells, flow cytometry > 50% FLT3 ⁺ cells Purity, Phenotype % CD56+ cells, flow cytometry > 95% CD56 ⁺ cells, < 0.3% CD3+ transgene copy number PCR ≤ 4 copies/cell effect Cytotoxicity against target antigen ⁺ cells ≥10-fold increase in IFN-γ secretion or CD107 analysis RCR S+/ L- Analysis The absence of replication-competent retroviral EOP cells change Test for growth of +/- IL-2 or IL-21 Ensuring proliferation in the absence of interferons Attributes HLA matching of donor and final product (low resolution class I) Confirmation mismatch

Test for NK purity. Without wishing to be bound by theory, it is critical that T cells derived from NK production should be reduced as patients develop ≥ grade II acute graft-versus-host disease ( ^GRHD ) while receiving ≥0.5 x 105 T cells/kg body weight ( GvHD), and the NK cell dose was well tolerated at a T cell contamination of 0.03 x ¹⁰⁵ T cells/kg body weight, thus defining an acceptance criterion of ≤ 0.3% CD3+ cells. See, for example, Stern M, Passweg JR, Meyer-Monard S, et al., Preemptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective Phase II study in two centers. Bone Marrow Transplant 2013;48:433-438.

Perform an efficacy analysis. Using CAR-NK cells using ELISA for IFN-γ, IL-15 and TNF-α and/or assays for cytotoxic activity (eg killing assays, degranulation assays (eg FACS for CD107a degranulation assays) ) and FACS for the intracellular cytokines IFN-γ and/or TNF-α) to test CAR+ and CAR- target cells. Experiment No. 3 : Stable Retrovirus Producer -293VEC-BaE for Generation of Vectors with Large Size Transgenes

Retroviral entry receptor performance was assessed on 293Vec-GalV and 293Vec-BaEV packaging cells and titrated using two cell lines Jurkat T and HT1080.

Among retroviruses, baboon endogenous virus (BaEV) and feline endogenous retrovirus (RD114) use a common cell surface receptor ASCT2 (sodium-dependent neutral amino acid transporter) to enter cells. In addition to ASCT2, BaEV also uses ASCT1 as a cell entry receptor. Gibbon leukemia virus (GALV) uses the sodium-dependent phosphate transporter (Pit1) as its entry receptor. See Figure 8A . Immunostaining for ASCT1, ASCT2 and Pit1 was performed on two packaging cell lines, 293Vec-GalV and 293Vec-BaEV, and two titer cell lines, Jurkat T and HT1080. See the results shown in Figure 8B . Two cell lines, Jurkat T and HT1080, can be used to titrate the three retroviral envelope proteins BaEV, RD114 and GALV (due to their ubiquitous expression of entry receptors). For the 293-based packaging cell lines 293Vec GALV and 293Vec BaEV, only the GALV pseudotyped retrovirus can infect them using the entry receptor Pit1.

A workflow for the transduction strategy used to generate the stable retroviral producer-293VEC-BaEV is provided herein.

A Moloney murine leukemia virus (Mo-MuLV) based retroviral vector (PCIR) was used as the target transgene delivery vehicle. The BaEV-pseudotyped 293Vec-BaEV packaging cell line was used to generate vehicle to infect cord blood-derived NK cells.

This production was performed using the following two packaging cell lines: 293Vec-GALV (to generate transient Gibbon Leukemia Virus (GALV) pseudotyped supernatant) and 293Vec-BaEV (to generate final vector). Both cell lines were supplied by BioVec Pharma.

First, GALV pseudotyped retroviral supernatants were generated by transiently transfecting the packaging cell line 293Vec-GALV using the reagent PEI MAX 40K (Polysciences, Inc, catalog number: 24765). Briefly, 0.6 million 293Vec-GALVs were seeded in 3 ml of complete DMEM medium (containing 10% heat-inactivated fetal bovine serum) on the wells of a 6-well plate one day before transfection. On the day of transfection, 293Vec-GALV should be 80% confluent. A total of 2.5 μg of a single Mo-MuLV-based retroviral vector containing the target transgene was diluted in 150 μl Opti-MEM™ reduced serum medium. At the same time, a total of 10 μg/10 μl of PEI MAX 40K was also diluted in 150 μl Opti-MEM™ Reduced Serum Medium. The ratio of plastid/DNA to PEI MAX 40K was 1:4. Diluted DNA was then added to PEI 40K, wells mixed, and incubated in a fume hood for 12 minutes at room temperature. After incubation, 300 μl of DNA-PEI MAX 40K complex was added gently dropwise to cells in 3 ml of culture.

This supernatant was then used to transduce the BaEV pseudotyped packaging cell line 293Vec-BaEV to generate bulk producers. For high titer clone generation, this 293Vec-BaEV bulk producer can be further single cell cloned by limiting dilution and screened for high titer clones selected by titrating the supernatant from each clone generation.

The supernatant can then be continuously produced from the high titer stable retrovirus producer-293VEC-BaEV.

Improved titers of the original supernatant were observed from the stable retroviral producer-293VEC-BaEV used for the vector with the large size transgene. See Figure 10 .

"Multiple targets in one" is a unique therapeutic strategy developed by the applicant. The BaEV pseudotyped 293Vec-BaEV packaging cell line was used to generate vehicle. A representative retroviral line for plastids with larger transgene inserts (>11 kb) was transiently and stably produced from 293Vec-BaEV packaging cells. A total of 3.5 million 293Vec-BaEV packaging cells were seeded in 100 mm tissue culture dishes and incubated at 37 °C for 48-72 h. The board should be 90-100% full. The original supernatant was then collected and spun at 1500 g for 5 min. The above supernatant was titered on Jurkat T cells. A series of viral supernatants were loaded onto RetroNectin precoated non-tissue culture treated 24-well plates and made up to 500 μl using complete DMEM medium including 10% heat-inactivated fetal bovine serum. The plate was spun at 32°C, 2000 g for 2 hours, and 0.1 million Jurkat T cells were added and spun at 1000 g for 5 min. Plates were incubated at 37°C for 48h, and then flow cytometry analysis was performed to determine titers (transduction units/ml) according to the following formula: Transduction units (TU)/mL = [(Number of target cells) × (Positive cells) %)]. In this example, the reporter gene RQR8 was detected by a flow cytometry antibody against human CD34 antibody (QBEnd/10) (Allophycocyanin) (Novus Biologicals, LLC, #FAB7227A). B). The original supernatant from the stable retroviral producer-293VEC-BaEV (TU=7.2E5) for vectors with large size transgenes has an improvement compared to the transiently produced virus (TU=2.2E5) potency. equivalent content

It should be understood that while the invention has been described in conjunction with the above-described embodiments, the foregoing descriptions and examples are intended to be illustrative and not limiting of the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will become apparent to those skilled in the art to which the invention pertains.

It should be understood that although the invention has been specifically disclosed in terms of specific embodiments and optional features, modifications, improvements and variations of the embodiments disclosed herein may be employed by those skilled in the art, and that such modifications, improvements and variations considered to be within the scope of the present invention. The materials, methods, and examples provided herein represent specific embodiments, are illustrative, and are not intended to limit the scope of the invention.

The scope of the invention has been described broadly and generically herein. Each of the narrower species and subgenus groups within the general disclosure also form part of this invention. This inclusion generally states that a condition or negative limitation removes any subject matter from this genus, regardless of whether the removed material is specifically recited herein.

In addition, if features or aspects of the invention are described with reference to the Markush group, those skilled in the art will recognize that the invention may thus also be mediated with reference to any individual member of the Markush group or subgroups of members.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. Partial sequence listing

IgG1 hinge domain: LEPKSCDKTHTCPPCPDPKGT (SEQ ID NO: 1)

CD28 transmembrane and cytoplasmic domain: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2)

CD3 zeta signaling domain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 3)

IL 2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 4),

IgG1 signal peptide: MGWSSIILFLVATATGVH (SEQ ID NO: 5)

Anti-NKG2D antigen binding domain CDRs: CDRL1:SGSSSNIGNNAVN (SEQ ID NO: 6) CDRL2: YDDLLPS (SEQ ID NO: 7) CDRL3: AAWDDSLNGPV (SEQ ID NO: 8) CDRH1:GFTFSSY (SEQ ID NO: 9) CDRH2:RYDGSN (SEQ ID NO: 10) CDRH3: DRGLGDGTYFDY (SEQ ID NO: 11)

Anti-NKG2D Light Chain Variable Region: QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL (SEQ ID NO: 12)

Anti-NKG2D heavy chain variable region: QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS (SEQ ID NO: 13)

Peptide linker: GGGGSGGGGSGGGGS (SEQ ID NO: 14)

人類IgG4 Fc區：ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 81)

具有F234A、L235A及N297Q突變(亦即分別係SEQ ID NO: 81之aa 16、aa 17及aa 79處之突變)之人類IgG4 Fc區等效物ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 82)

Human muscle aldolase (HMA) peptide linker: PSGQAGAAAASESLFVSNHAY (SEQ ID NO: 83)

Detectable label: YPYDVPDYA (SEQ ID NO: 84)

T2A peptide: HVGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 85)

Nucleotide sequence encoding signal peptide: ATGGGGTGGTCAAGCATTATTCTGTTTCTGGTCGCTACCGCTACAGGCGTCCAT (SEQ ID NO: 86)

Nucleotide sequence encoding signal peptide: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT (SEQ ID NO: 87)

Nucleotide sequence encoding linker peptide: GGTGGGGGCGGCTCTGGTGGCGGTGGCAGCGGCGGAGGTGGCAGT (SEQ ID NO: 88)

Nucleotide sequences encoding transmembrane and cytoplasmic domains: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 89)

編碼信號傳導結構域之核苷酸序列：AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 90)或其等效物；

Nucleotide sequence encoding cleavable peptide: CACGTGGGTTCTGGAGAAGGACGCGGTTCCTTGTTGACGTGTGGCGATGTAGAGGAAAATCCGGGTCCA (SEQ ID NO: 91)

Nucleotide sequence encoding linker: CCGAGCGGCCAGGCGGGCGCGGCGGCATCGGAGTCCCTGTTTGTGTCAAATCACGCCTAC (SEQ ID NO: 92)

Nucleotide sequence encoding hinge domain: CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAAGGTACC (SEQ ID NO: 109)

Peptide linker: (glycine-serine)n, where n is an integer from 1 to 6, (SEQ ID NO: 110)

6×His Tag: His His His His His His His (SEQ ID NO: 111)

Hinge Domain: IgG1 Heavy Chain Hinge Coding Sequence: CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG (SEQ ID NO: 112)

CD28 transmembrane region coding sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 113)

4-1BB costimulatory signaling region coding sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 114)

CD28 costimulatory signaling region coding sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 115)

CD3 zeta signaling region coding sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 116)

Human CD8 alpha hinge domain: PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 117).

Mouse CD8 alpha hinge domain: KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 118).

Feline CD8 alpha hinge domain: PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY (SEQ ID NO: 119).

Human CD8 alpha transmembrane domain: IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 120).

Mouse CD8 alpha transmembrane domain: IWAPLAGICVALLLSLIITLI (SEQ ID NO: 121).

Rat CD8 alpha transmembrane domain: IWAPLAGICAVLLLSLVITLI (SEQ ID NO: 122).

4-1BB costimulatory signaling region: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 123)

CD28序列：MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLDSAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO: 124)

ICOS costimulatory signaling region coding sequence: acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta (SEQ ID NO: 125)

OX40 costimulatory signaling region coding sequence: AGGGACCAG AGGCTGCCCC CCGATGCCCA CAAGCCCCCT GGGGGAGGCA GTTTCCGGAC CCCCATCCAA GAGGAGCAGG CCGACGCCCA CTCCACCCTG GCCAAGATC (SEQ ID NO: 126)

Linker: GGGGS (SEQ ID NO: 134).

Linker: (GGGGS)n, where n can be an integer 1 (SEQ ID NO: 134) or 2 (SEQ ID NO: 135) or 3 (SEQ ID NO: 14) or 4 (SEQ ID NO: 136) or 5 (SEQ ID NO: 137) or 6 (SEQ ID NO: 138) or 7 (SEQ ID NO: 139) or 8 (SEQ ID NO: 140) or 9 (SEQ ID NO: 141) or 10 (SEQ ID NO: 140) 142) or 11 (SEQ ID NO: 143) or 12 (SEQ ID NO: 144) or 13 (SEQ ID NO: 145) or 14 (SEQ ID NO: 146) or 15 (SEQ ID NO: 147) or greater .

EF1 alpha promoter sequence: AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC (SEQ ID NO: 148)

Reverse sequence of SEQ ID NO: 71: AVGTIVDQSAK (SEQ ID NO: 151)

IgG1 heavy chain signal peptide DNA ATGGAATTTGGGCTGCGCTGGGTTTTCCTTGTTGCTATTTTAAAAGATGTCCAGTGT (SEQ ID NO: 168)

protein MEFGLRWVFLVAILKDVQC (SEQ ID NO: 169)

IgG1 FcDNA GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGAGCTGCAACTGGAGGAGAGCTGTGCGGAGGCGCAGGACGGGGAGCTGGACGGGCTGTGGACGACCATCACCATCTTCATCACACTCTTCCTGTTAAGCGTGTGCTACAGTGCCACCGTCACCTTCTTCAAGGTGAAGTGGATCTTCTCCTCGGTGGTGGACCTGAAGCAGACCATCATCCCCGACTACAGGAACATGATCGGACAGGGGGCC (SEQ ID NO: 170)

由IgG1 FcDNA編碼之蛋白質EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIIPDYRNMIGQGA (SEQ ID NO: 171)本發明之例示實施例

Example 1. A pseudotyped gamma retroviral particle comprising a modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and a modified baboon envelope glycoprotein (BaEVTR), wherein: a. the RD114TR glycoprotein includes the extracellular and transmembrane domains of the RD114 glycoprotein and the cytoplasmic domain of the amphiphilic murine leukemia virus (MLV-A) glycoprotein; and b. wherein the BaEVTR glycoprotein comprises the extracellular and transmembrane domains of baboon envelope glycoprotein (BaEV) and the cytoplasmic domain of MLV-A glycoprotein.

Example 2. The pseudotyped gamma retroviral particle of Example 1, wherein the RD114TR and the BaEVTR are incorporated into the envelope of the gamma retroviral particle as membrane proteins.

Embodiment 3. The pseudotyped gamma retroviral particle of embodiment 1 or 2, further comprising a vector gene body encapsulated in the envelope, wherein the vector gene body comprises the following flanked by two long terminal repeats ( LTR) one or more of: (A) a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein or polypeptide optionally selected from antibodies or fragments thereof, enzymes, ligands or receptors, (B) the reverse complement of (A), or (C) A polynucleotide comprising one or more recognition sites that are optionally recognized and cleaved by restriction enzymes suitable for insertion of a CAR coding sequence or its reverse complement into the polynucleotide.

Embodiment 4. The pseudotyped gamma retroviral particle of any one of embodiments 1 to 3, wherein the vector genome further comprises one or more of the following: 5' LTR, 5' cap, 3' Poly-A tail and 3' LTR.

Embodiment 5. The pseudotyped gamma retroviral particle of any one of embodiments 1 to 4, further comprising either or both of reverse transcriptase or integrase.

Embodiment 6. The pseudotyped gamma retroviral particle of any one of embodiments 1 to 5, which is selected from any one of the following species: Moloney Murine Leukemia Virus (MMLV) , Murine stem cell virus (MSCV), Fried murine embryonic stem cell virus (friend murine embryonic stem cell virus, FMEV), heterophilic MuLB-related virus, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus.

Example 7. A method of preparing a population of natural killer (NK) cells comprising culturing a population of cells comprising one or more of the following with an immune cell activator (eg, an NK cell activator): NK cells , progenitor cells capable of deriving NK cells or stem cells capable of deriving NK cells, wherein the cell population is depleted in the cell population of cells expressing one or more of the following: CD3, CD4, CD8, T cell receptors ( TCR) alpha chain, TCR beta chain or alpha beta TCR, and wherein the immune cell activator is selected from one or more of the following (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of NK cells, progenitor cells or stem cells, thereby activating or proliferating NK cells, as appropriate wherein the The antibody system is selected from one or more of the following: anti-CD2 antibody, anti-CD16 antibody, anti-NKG2D antibody, anti-DNAM-1 antibody, anti-2B4 antibody, anti-NTB-A antibody or anti-NKp46 (natural cytotoxicity receptor 1 ( NCR1)) antibody, (iii) one or more cytokines thereby activating or proliferating NK cells, or (iv) one or more chemical moieties thereby activating or proliferating NK cells, optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs).

Embodiment 8. The method of Embodiment 7, further comprising introducing into the cultured cell a polynucleotide encoding a CAR or another therapeutic protein or polypeptide selected from the group consisting of an antibody or fragment thereof, enzyme, ligand or receptor as appropriate In a population for expression, wherein the CAR specifically recognizes and binds to the tumor-associated antigen (TAA) or viral antigen.

Embodiment 9. The method of embodiment 8, further comprising culturing the cell population with an immune cell activator after the introducing step of embodiment 8, wherein the immune cell activator is selected from the following (i) to ( iv) one or more of: (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind to stimulatory receptors on one or more of NK cells, progenitor cells or stem cells, thereby activating or proliferating NK cells, (iii) one or more cytokines thereby activating or proliferating NK cells, or (iv) one or more chemical moieties thereby activating or proliferating NK cells, optionally selected from mTOR inhibitors, PI3K inhibitors or STING activating cyclic dinucleotides (CDNs), Where appropriate, the culturing step is repeated one, two, three or more times with the same or different immune cell activating agents or a combination thereof.

Embodiment 10. The method of any one of embodiments 7-9, wherein the aAPCs further express one or more of the following: 4-1BBL, membrane bound (mb) IL-15, mb IL-21 , CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL-15-N72D superagonist (IL-15SA/IL-15RαSu-Fc; ALT-803) complexed with IL-15RαSushi-Fc fusion protein or a cell surface marker that mediates CD122/CD132 signaling.

Embodiment 11. The method of any one of embodiments 7-10, wherein the aAPCs further express mb IL-21 and 4-1BBL.

Embodiment 12. The method of any one of embodiments 7-11, wherein the aAPCs are engineered K562 cells.

Embodiment 13. The method of any one of embodiments 7-12, wherein the aAPCs are irradiated, thereby lacking cell proliferation or lacking long-term survival.

Embodiment 14. The method of Embodiment 13, wherein the aAPCs are irradiated at 50 Gy, 100 Gy, 150 Gy, or 200 Gy.

Embodiment 15. The method of any one of embodiments 7 to 14, wherein about 10:1, about 5:1, about 3:1, about 2:1, about 1:1, about 1:2, about A cell number ratio of 1:3, about 1:5, or about 1:10 is used to culture the aAPC with the cell population.

Embodiment 16. The method of any one of embodiments 7 to 15, wherein the interleukins are selected from the group consisting of: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL -12, IL-2, low toxicity IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D superagonist complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/ IL-15RαSu-Fc; ALT-803 soluble), IL-15, IL-18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1) or MICA.

Embodiment 17. The method of embodiment 16, wherein the cell population is treated with any one or both of 100-500 IU/ml IL-2, 20 ng/ml IL-15 or 25 ng/mL IL-21 or all three together.

Embodiment 18. The method of embodiment 16, wherein the cell population is cultured with either or both of 50 IU/ml IL-2 and 0.5 ng/ml IL-15.

Embodiment 19. The method of embodiment 16, wherein the cell population is cultured with 50 IU/ml IL-2.

Embodiment 20. The method of any one of embodiments 9 to 19, wherein the activator that is cultured with the cell population before and after the introducing step is the same.

Embodiment 21. The method of any one of embodiments 9 to 19, wherein the activating agents cultured with the cell population before and after the introducing step are different from each other.

Embodiment 22. The method of any one of embodiments 8 to 21, comprising introducing pseudotyped gamma retroviral particles into the cultured cell population, thereby introducing the CAR-encoding polynucleotide into the cultured cells , wherein the particle comprises the CAR-encoding polynucleotide or its reverse complement, RD114TR and BaEVTR, flanked by two long terminal repeats (LTRs).

Embodiment 23. The method of embodiment 22, wherein the pseudotyped gamma retroviral particle comprises a vector gene body comprising a 5' LTR, a 5' cap, the CAR encoding polynucleotide or its reverse complement , 3' poly-A tail and 3' LTR.

Embodiment 24. The method of embodiment 22 or 23, wherein the pseudotyped gamma retroviral particle further comprises either or both of reverse transcriptase and integrase.

Embodiment 25. The method of any one of embodiments 22 to 24, wherein the pseudotyped gamma retroviral particle is selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV) ), Fried murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoid virus, heterophilic murine leukemia virus-related virus (XMRV), and feline leukemia virus.

Embodiment 26. The method of any one of embodiments 22 to 25, wherein about 0.1, about 0.2, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8. A multiplicity of infection (MOI) of about 9 or about 10 to introduce the pseudotyped gamma retroviral particle into the cultured cell population.

Embodiment 27. The method of any one of embodiments 8 to 26, wherein the cell population is cultured prior to the introducing step for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days.

Embodiment 28. The method of any one of embodiments 8 to 27, wherein the cell population is cultured prior to the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 10 days 11 days, no more than 12 days, no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks or no more than 1 month.

Embodiment 29. The method of any one of embodiments 8 to 27, wherein the cell population is cultured for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days before the introducing step sky.

Embodiment 30. The method of any one of embodiments 8 to 29, wherein the CAR-encoding polynucleotide is introduced into the CAR-encoding polynucleotide via transduction of a viral vector comprising the CAR-encoding polynucleotide or its reverse complement in the presence of RetroNectin in the cell population.

Embodiment 31. The method of embodiment 30, wherein the RetroNectin is coated on the inner surface of the vessel in which the cell population is transduced.

Embodiment 32. The method of embodiment 30 or 31, wherein the cell population expresses either or both of integrin α4β1 (VLA-4) and integrin α5β1 (VLA-5).

Embodiment 33. The method of any one of embodiments 7 to 32, wherein the cell population system is isolated from a blood sample of an individual.

Embodiment 34. The method of any one of embodiments 7-33, wherein the cell population system is isolated from one or more of the following: umbilical cord blood of an individual, peripheral blood of an individual, or bone marrow of an individual.

Embodiment 35. The method of any one of embodiments 7 to 34, wherein the cell population comprises progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or immortalized cells.

Embodiment 36. The method of any one of embodiments 7-35, further comprising enriching the cell population for cells expressing any one or more of the following: CD56, CD25, CD122, CD212, CD215 , CD218, CD360, TGF-βR, or IL-10R, and further including enrichment of either or both of CD56 ^dim cells and CD56 ^bright cells as appropriate.

Embodiment 37. The method of any one of embodiments 7-36, wherein any cell population comprises natural killer (NK) cells or a substantially purified composition thereof.

Embodiment 38. The method of any one of embodiments 7 to 37, wherein the depleted cell population comprises one or more of the following: NK cells, progenitor cells, HSCs, iPSCs, or substantially each of them Purified population.

Embodiment 39. The method of embodiment 37 or 38, wherein the NK cells comprise cells derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryos Germ cell derived cells, stem cells, stem cell derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC) or immortalized cells.

Embodiment 40. The method of embodiment 38, wherein one or more of the progenitor cells, HSCs or iPSCs are capable of deriving NK cells.

Embodiment 41. The method of any one of embodiments 7-40, wherein any cell population is substantially free of T cells.

Embodiment 42. The method of any one of embodiments 7-41, wherein any cell population is substantially free of T regulatory cells.

Embodiment 43. The method of any one of embodiments 7-42, wherein any cell population system is isolated, enriched or purified.

Embodiment 44. The method of any one of embodiments 7 to 43, wherein in the culturing step the cell population and the aAPCs are cultured together in a cell culture medium selected from the group consisting of: StemSpan (Stemcell #09960), NK MACS® Medium (Miltenyi research 30-114-429; GMP: 170-076-356), TexMACS (170-076-306 GMP Medium), Cellgenix Serum-Free Stem Cell Growth Medium (SCGM, #20806-0500) or ImmunoCult™- XF medium (from Stemcell technologies).

Embodiment 45. The method of any one of embodiments 7 to 44, further comprising any one or both of the following steps: formulating the CAR-expressing population into a composition; and The CAR-expressing population is cryopreserved.

Embodiment 46. The method of any one of Embodiments 7-45, which generates greater than ¹ x 1011 cells from ¹ x 108 cells.

Embodiment 47. The method of any one of embodiments 7 to 46, further comprising any one or both of the following steps: washing the cell population before or after one or more steps, and Detect one or more of the following before, during or after one or more steps: (i) the viability of the cell population; (ii) the sterility of the cell population; (iii) Mycoplasma in the cell population; (iv) the human leukocyte antigen (HLA) type of the cell population; (v) the number of cells in the cell population; (vi) the cellular phenotype of the cell population; (vii) HHV6 or HHV7 or both in the cell population or a composition comprising the cell population; (viii) one or more of the following in the population of cells or a composition comprising the population of cells: human immunodeficiency virus (HIV) types 1 and 2, human T-lymphotropic virus (HTLV) type 1 and type II, hepatitis B virus (HBV), hepatitis C virus (HCV), CMV, Zika, West Nile or Treponema pallidum; (ix) the cellular phenotype of such aAPCs; (x) the IL-15 content in the composition comprising the cell population; (xi) CAR performance in the cell population; (xii) endotoxin in the cell population or a composition comprising the cell population; (xiii) residual tumor burden and/or aAPC contamination of the composition comprising the cell population; (xiv) progeny of the cell population; (xv) IFN-γ, IL-15 and TNF-α released by the cell population; (xvi) the cytotoxic activity of the cell population; (xvii) degranulation of the cell population; and (xviii) Monitor or measure the culture for one or more of the following: cell aggregation, glucose or lactate.

Embodiment 48. The method of embodiment 47, wherein the phenotypic detection comprises detection of cellular expression or expression levels of one or more of the following: CD3, CD56, antigen (eg CD19), CD45, HLA, NKp46, NKG2D, NKG2A, NCRs, DNAM-1, CD16, IL-2R, CXCR4, KIRS, CD8, CD57, adhesion molecules, NKG2C, CD107a, CAR or cell surface markers expressed by the cell population.

Embodiment 49. The method of embodiment 48, wherein the phenotypic detection comprises detecting the cellular expression level of CD56 in the cell population.

Embodiment 50. The method of any one of embodiments 8 to 49, wherein the antigen binding domain of the CAR specifically recognizes and binds one or more of the following: G protein-coupled receptor class C family 5 member D (GPRC5D), B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild-type epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variants III (EGFRVIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanosyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72 ), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific Membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT group, IL-13Ra2, IL- 11Rα, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, mucin 1 (Muc1), NCAM, Prostate enzyme, PAP, ELF2M, Ephrin B2, Fibroblast activating protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephrin A Type receptor 2 precursor (EphA2), fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97 , CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1 , MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostaglandins, survivin and telomerase, PCTA-1/galectin-8, MelanA/MART1, Ras mutations body, hTERT, sarcoma translocation cut point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, asparagine endopeptidase, HPV E6, E7, intestinal carboxylesterase, mut hsp70 -2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, Glypican 3 (GPC3), FCRL5 or IGLL1.

Embodiment 51. The method of any one of embodiments 8 to 50, wherein the antigen is selected from one or more of the following or fragments thereof: G protein-coupled receptor class C family 5 member D (GPRC5D), B cell maturation antigen (BCMA), SLAMF7 (CS1 or CD319), EGFR, wild-type epidermal growth factor receptor (EGFRwt), epidermal growth factor receptor variants III (EGFRVIII), FLT3, CD70, mesothelin, CD123, CD19, carcinoembryonic antigen (CEA), CD133, human epidermal growth factor receptor 2 (HER2), ERBB2 (Her2/neu), CD22, CD30, CD171, CLL-1 (CLECL1), GTPase activating protein (GAP), CD5, interleukin 13 receptor alpha 2 (IL13Ra2), guanosyl cyclase C (GUCY2C), tumor-associated glycoprotein-72 (TAG-72 ), thymidine kinase 1 (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT1), cancer/testis (CT), CD33, ganglioside G2 (GD2), GD3, Tn Ag, prostate specific Membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), TAG72, CD38, CD44v6, epithelial cell adhesion molecule precursor (EpCam or EPCAM), B7H3, KIT group, IL-13Ra2, IL- 11Rα, prostate stem cell antigen (PSCA), PRSS21, vascular endothelial growth factor receptor 2 (VEGFR2), LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, mucin 1 (Muc1), NCAM, Prostate enzyme, PAP, ELF2M, Ephrin B2, Fibroblast activating protein alpha (FAP), IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, ephrin A Type receptor 2 precursor (EphA2), fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, CXORF61, CD97 , CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1 , MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostaglandins, survivin and telomerase, PCTA-1/galectin-8, MelanA/MART1, Ras mutations body, hTERT, sarcoma translocation cut point, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, asparagine endopeptidase, HPV E6, E7, intestinal carboxylesterase, mut hsp70 -2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, Glypican 3 (GPC3), FCRL5 or IGLL1.

Embodiment 52. The method of any one of embodiments 8 to 51, wherein the CAR comprises (1) an antigen binding domain of an antibody that specifically recognizes and binds an antigen; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular domain, which includes a signaling domain.

Embodiment 53. The method of any one of embodiments 8 to 52, wherein the CAR comprises (1) an antigen binding domain of an antibody that specifically recognizes and binds an antigen; (2) a hinge domain; (3) a transmembrane domain; and (4) an intracellular domain, which includes a signaling domain and a costimulatory domain.

Embodiment 54. The method of any one of embodiments 8 to 53, wherein the CAR further comprises a signal peptide.

Embodiment 55. The method of embodiment 53 or 54, wherein the hinge domain comprises a CD8 alpha hinge domain.

Embodiment 56. The method of any one of embodiments 53 to 55, wherein the transmembrane domain comprises a CD8α transmembrane domain.

Embodiment 57. The method of any one of embodiments 53-56, wherein the intracellular domain further comprises (1) one or two or more costimulatory signaling regions.

Embodiment 58. The method of embodiment 57, wherein the costimulatory signaling region comprises a CD28 costimulatory signaling region or a 4-1BB costimulatory signaling region or both.

Embodiment 59. The method of any one of embodiments 53-58, wherein the signaling domain comprises a CD3 zeta signaling domain.

Embodiment 60. The method of any one of embodiments 8-59, wherein the introduced polynucleotide further expresses one or more of an interleukin and an antibody, or wherein the method further comprises expressing an interleukin and an antibody. Another polynucleotide of one or more of the antibodies is introduced into the depleted cell population.

Embodiment 61. The method of embodiment 60, wherein the interferon expressed by the polynucleotide is selected from one or more of the following: B7.1 (soluble or membrane bound), CCL19 (soluble or membrane-bound), CCL21 (soluble or membrane-bound), CD40L (soluble or membrane-bound), CD137L (soluble or membrane-bound), GITRL (soluble or membrane-bound), GM-CSF (soluble or membrane-bound) IL-2 (soluble or membrane-bound), IL-2 (soluble or membrane-bound), low toxicity IL-2 (soluble or membrane-bound), IL-2 analogs lacking cd25 binding (soluble or membrane-bound) membrane-bound), IL-15-N72D superagonist, and IL-15RαSushi-Fc fusion protein (soluble or membrane-bound), IL-15 (soluble or membrane-bound), IL-18 (soluble or membrane-bound) , IL-21 (soluble or membrane-bound), LEC (soluble or membrane-bound), OX40L (soluble or membrane-bound), IL-7 (soluble or membrane-bound), ICOSL (B7H2, B7RP1, soluble or membrane-bound) binding) or MICA (soluble or membrane bound).

Embodiment 62. The method of embodiment 60 or 61, wherein the antibody expressed by the polynucleotide is a monospecific antibody or a bispecific antibody or a multispecific antibody.

Embodiment 63. The method of any one of embodiments 60-62, wherein the antibody expressed by the polynucleotide is an activator of immune cells.

Embodiment 64. The method of any one of embodiments 8-63, wherein the polynucleotide further encodes a signal peptide.

Embodiment 65. The method of any one of embodiments 8-64, wherein the polynucleotide further comprises a suicide gene.

Embodiment 66. The method of embodiment 65, wherein the suicide gene product is selected from one or more of the following: HSV-TK (herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, Carboxylesterase, cytochrome P450 or PNP (purine nucleoside phosphorylase), truncated EGFR or inducible caspases ("iCasp").

Embodiment 67. The method of embodiment 65 or 66, wherein the polynucleotide further comprises a regulatory sequence directing expression of the suicide gene and wherein the regulatory sequence is inducible.

Embodiment 68. The method of any one of embodiments 8-67, wherein the polynucleotide further comprises a regulatory sequence directing expression of the CAR or the therapeutic protein.

Embodiment 69. The method of embodiment 68, wherein the regulatory sequence directing expression of the CAR or the therapeutic protein is inducible or constitutive activity.

Embodiment 70. The method of any one of embodiments 8-69, wherein the polynucleotide is introduced into the cell population via a vector.

Embodiment 71. The method of embodiment 70, wherein the vector is a viral vector or a non-viral vector.

Embodiment 72. The method of embodiment 71, wherein the non-viral vector is plastid.

Embodiment 73. The method of embodiment 71, wherein the viral vector system is selected from retroviral vector, lentiviral vector, adenoviral vector, adeno-associated virus vector or herpes virus vector.

Embodiment 74. The method of any one of embodiments 8-73, wherein the prepared CAR-expressing cell population is suitable for inhibiting cancer cell growth and wherein the antigen is a tumor-associated antigen (TAA) expressed by the cancer cell.

Embodiment 75. A method of inhibiting the growth of cancer cells, comprising contacting a population of CAR expressing cells prepared by the method of any one of embodiments 8 to 74 with the cancer cells, wherein the antigen recognized by the CAR is the TAA expressed by the cancer cells.

Embodiment 76. A method of treating cancer in an individual comprising administering to the individual a population of CAR-expressing immune cells made by the method of any one of embodiments 8-74, wherein the CAR-expressing immune cells are recognized by the CAR. The antigen is a TAA expressed by cells of the cancer.

Embodiment 77. The method of embodiment 76, wherein the cell population comprises NK cells and less than or equal to 0.3% CD3+ cells.

Embodiment 78. The method of embodiment 76 or 77, wherein less than ³ x 103 T cells/kg body weight are administered to the individual.

Embodiment 79. The method of any one of embodiments 74 to 78, wherein the cancer cell line is selected from cancer cells of the following organs: circulatory system, such as heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma] , myxoma, rhabdomyomas, fibroma and lipoma), mediastinum and pleura and other intrathoracic organs, vascular tumors and tumor-related vascular tissue; respiratory tract, such as nasal cavity and middle ear, paranasal sinuses, larynx, trachea, bronchi, and lungs , such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchial proto-carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchial) carcinoma, bronchial adenoma, sarcoma , lymphoma, chondrohamartoma, mesothelioma; gastrointestinal system such as esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (cancer, lymphoma, leiomyosarcoma), stomach, pancreas Dirty (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, pancreatic tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma), smooth Fibroids, hemangiomas, lipomas, neurofibromas, fibroids), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors appearing anywhere and Neuroendocrine tumors; genitourinary tract such as kidney (adenocarcinoma, Wilm's tumor [Wilm's tumor], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma , adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, mesenchymal cell carcinoma, fibroma, fibroadenoma, adenoid tumor , lipoma); liver, e.g., hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (e.g., pheochromocytoma, insulinoma, Vasoactive intestinal peptide tumors, islet cell tumors, and glucagonomas); bones, such as osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma tumor (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondral exostoses), benign chondroma, chondroblastoma, chondromyxoid fibroma, bone Osteoid tumors and giant cell tumors; nervous system, e.g., central nervous system (CNS) neoplasms, primary CNS lymphomas, skull cancers (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges Carcinoma (meningioma, meningiosarcoma, excess glia), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pineal tumor], pleomorphism Glioblastoma, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas); reproductive system e.g. gynecology, uterus (uterus endometrial cancer), cervix (uterus) cervical cancer, preneoplastic cervical dysplasia), ovary (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], mesothelial cell tumor, Sertoli-Reading cell tumor (Sertoli- Leydig cell tumor), dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, grape-shaped sarcoma (embryonic rhabdomyosarcoma), fallopian tubes (cancer), and other parts of the female reproductive organs; penis, prostate, testis, and other parts of the male reproductive organs; hematological systems, such as blood (myeloid leukemia [ acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma Tumors [Malignant Lymphomas]; Oral cavity such as lips, tongue, gums, floor of mouth, palate and other parts of oral cavity, parotid and other parts of salivary glands, tonsils, oropharynx, nasopharynx, pyriform recesses, hypopharynx and lips, oral cavity and other parts of the pharynx; skin, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, dermatofibroma and keloids; adrenal glands: neuroblastoma; and other tissues, including connective and soft tissues, retroperitoneal cavity and peritoneum, eye, intraocular melanoma and appendages, breast, head or/and neck, anal area, thyroid, Parathyroid, adrenal and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites.

Embodiment 80. The method of any one of embodiments 74-79, wherein the cancer cell line is a solid tumor cell.

Embodiment 81. The method of any one of embodiments 74-79, wherein the cancer cells are not cells of a solid tumor, optionally wherein the cancer cells are leukemia cancer cells.

Embodiment 82. The method of any one of embodiments 74 to 81, wherein the cancer cells are primary cancer cells or metastatic cancer cells.

Embodiment 83. The method of any one of embodiments 74 to 82, wherein the cancer cell line is from a carcinoma, sarcoma, myeloma, leukemia or lymphoma.

Embodiment 84. The method of any one of embodiments 75 and 79-83, wherein the contacting is performed in vivo or in vitro.

Embodiment 85. The method of any one of Embodiments 74-84, further comprising contacting the cell with or administering a monotherapy.

Embodiment 86. The method of embodiment 85, wherein the monotherapy comprises surgical resection, chemotherapy, radiation therapy, immunotherapy, and targeted therapy.

Embodiment 87. The method of embodiment 85 or 86, wherein the monotherapy is first-line therapy, second-line therapy, third-line therapy or fourth-line therapy.

Embodiment 88. An NK cell, or a population thereof, produced or prepared by the method of any one of embodiments 7-74.

Embodiment 89. A composition comprising the cells of Embodiment 88 or a population thereof and a carrier, optionally a pharmaceutically acceptable carrier.

Embodiment 90. The cell or population thereof of embodiment 88 or the composition of embodiment 89, comprising less than 0.5% aAPC.

Embodiment 91. The cell or population thereof of embodiment 88 or 90 or the composition of embodiment 89 or 90, comprising less than 0.1% or less than 0.2% or less than 0.3%, less than 0.4% or less than 0.5% aAPC.

Embodiment 92. The cell or population thereof of any one of embodiments 88 and 90 to 91 or the composition of any one of embodiments 89 to 91, which has one or more of the following properties: (A) Not less than 70% survival rate; (B) Sterile; (C) Contains less than 5 EU Kg ⁻¹ h ⁻¹ endotoxin; (D) Negative for one or more of the following: Mycoplasma, foreign virus or HHV (HHV6 or HHV7); (E) include greater than ¹ x 106 cells/kg body weight of the subject to be treated; (F) include greater than 40% of total cells expressing CAR; (G) include greater than 95% of cells expressing CD56; (H) including less than 0.3% of total cells expressing CD3; (I) including less than 4 polynucleotide copies/cell; (J) in the absence of IL-2 or IL No proliferation at -21; (K) has efficacy, eg, greater than 10-fold increase in IFNy secretion or CD107 assays; and (L) expresses one or more antigens (eg, CD19, NKp46, or NKG2D).

Embodiment 93. The cell or population thereof of embodiment 92 or the composition of embodiment 92, having one or both of the following properties: (E') comprising greater than ¹ x 107 cells/kg of the individual to be treated body weight; or (F') including greater than 50% of CAR-expressing cells in total cells.

Example 94. An engineered aAPC expressing an antigen and one or more of the following cell surface markers: 4-1BBL, membrane bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86 , OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, fusion protein with IL-15RαSushi-Fc Complex mb IL-15-N72D superagonist (IL-15SA/IL-15RαSu-Fc; ALT-803) or cell surface markers that mediate CD122/CD132 signaling.

Embodiment 95. The aAPCs of embodiment 94, wherein the aAPCs are engineered K562 cells.

Embodiment 96. The aAPCs of embodiment 94 or 95, wherein the aAPCs are irradiated thereby lacking cell proliferation or long-term survival or both.

Embodiment 97. The aAPCs of Embodiment 96, wherein the aAPCs are irradiated at 50 Gy, 100 Gy, 150 Gy, or 200 Gy.

Embodiment 98. The aAPCs of any one of embodiments 94-97, wherein the aAPCs do not substantially survive for more than 14 days.

Embodiment 99. A kit comprising one or more agents suitable for use in the method of any one of Embodiments 7-87 and optional instructions.

Embodiment 100. The kit of embodiment 99, wherein the agents are selected from one or more of the following: a polynucleotide encoding a CAR or another therapeutic protein, a vector comprising the polynucleotide, a Antibodies for detecting cell phenotype, antibodies for isolating or enriching or purifying immune cells, primers for detecting polynucleotides, interleukins and aAPCs.

Example 101. A method of making a population of γδ T cells, comprising culturing a population of cells comprising one or more of the following with an immune cell activator (eg, a γδ T cell activator): γδ T cells, Progenitor cells capable of deriving γδ T cells or stem cells capable of deriving γδ T cells, wherein the cell population is depleted in the cell population of cells expressing one or more of the following: T cell receptor (TCR) alpha chain, TCR beta chain or alpha beta TCR, and wherein the activator is selected from one or more of the following (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of γδ T cells, progenitor cells or stem cells, thereby activating or proliferating γδ T cells, (iii) one or more cytokines, thereby activating or proliferating γδ T cells, or (iv) one or more chemical moieties, thereby activating or proliferating γδ T cells, which chemical moieties are optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs).

Embodiment 102. The method of Embodiment 101, further comprising introducing a polynucleotide encoding a CAR or another therapeutic protein optionally selected from an antibody or fragment thereof, enzyme, ligand or receptor into the cultured cell population For expression, wherein the CAR specifically recognizes and binds to the tumor-associated antigen (TAA) or viral antigen.

Embodiment 103. The method of embodiment 102, further comprising culturing the cell population with an immune cell activator (eg, a gamma delta T cell activator) after the introducing step of embodiment 102, wherein the activator is selected from the group consisting of One or more of (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of γδ T cells, progenitor cells or stem cells, thereby activating or proliferating γδ T cells, (iii) one or more cytokines, thereby activating or proliferating γδ T cells, or (iv) one or more chemical moieties, thereby activating or proliferating γδ T cells, optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs); Where appropriate, the incubation step is repeated one, two, three or more times with the same or different activators.

Embodiment 104. The method of any one of embodiments 101 to 103, wherein the cell population comprises one or more of the following: γδ T cells or a substantially purified composition thereof.

Embodiment 105. The method of any one of embodiments 101 to 104, wherein the depleted cell population comprises one or more of the following: γδ T cells, HSCs, iPSCs, or substantially purified of each of them group.

Embodiment 106. The method of embodiment 105, wherein one or more of the progenitor cells, HSCs or iPSCs are capable of deriving γδ T cells.

Embodiment 107. The method of embodiment 101 or 106, wherein the γδ T cells comprise cells derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, Embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or immortalized cells.

Embodiment 108. The method of any one of embodiments 101 to 107, wherein the cell population is substantially free of cells expressing TCR alpha chains or TCR beta chains.

Example 109. A viral packaging system for the production of pseudotyped gamma retroviral particles, comprising: (a) a plastid expressing a vector gene body; (b) a packaging plastid; and (c) one or more expressing RD114TR And the membranous plastid of BaEVTR.

Embodiment 110. The viral packaging system of embodiment 109, wherein the vector genome comprises one or more of the following flanked by two long terminal repeats (LTRs): (A) a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein, optionally selected from antibodies or fragments thereof, enzymes, ligands or receptors, (B) the reverse complement of (A), or (C) A polynucleotide comprising one or more recognition sites recognized by restriction enzymes suitable for insertion of a CAR coding sequence or its reverse complement into the polynucleotide.

Embodiment 111. The viral packaging system of any one of embodiments 109 to 110, wherein the vector genome further comprises one or more of the following: 5' LTR, 5' cap, 3' poly-A tail and 3' LTR.

Embodiment 112. The viral packaging system of any one of embodiments 109 to 111, wherein the pseudotyped gamma retroviral particle is selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried murine embryonic stem cell virus (FMEV), heterophilic MuLB-related virus, feline sarcoid virus, heterophilic murine leukemia virus-related virus (XMRV), and feline leukemia virus.

Embodiment 113. The viral packaging system of any one of Embodiments 109-112, further comprising a packaging cell line.

Embodiment 114. The viral packaging system of embodiment 113, wherein the packaging cell line is the 293T cell line.

Embodiment 115. A method of producing a pseudotyped gamma retroviral particle comprising transducing packaging using the system of any one of embodiments 109 to 112 under conditions suitable for packaging the pseudotyped gamma retroviral particle cell line.

Embodiment 116. The method of embodiment 115, wherein the packaging cell line is the 293T cell line.

Embodiment 117. The method of embodiment 115 or 116, wherein the cell line is transduced with the plastids of (a), (b) and (c) at a ratio of 1.5:1.5:1.

Embodiment 118. The method of any one of embodiments 115-116, wherein the packaging system comprises at least two mantle plastids, one expressing RD114TR and the other expressing BaEVTR.

Embodiment 119. The method of embodiment 118, wherein the cell line uses (a) plastids, (b) plastids, RD114TR-expressing plastids, and BaEVTR-expressing plastids at a ratio of 1.5:1.5:1:1 to transduce.

Figure 1 provides an illustration of gamma reverse transcription vector (PCIR)-based therapeutic gene delivery to human primary NK cells.

Figures 2A - 2B provide growth curves and determination of the time points at which primary NK cells entered a state of significant proliferation for gene delivery. The time point of retroviral transduction of expanded primary NK cells was optimized. Growth curves and time points at which primary NK cells entered a state of significant proliferation for gene delivery were determined. Primary human NK cell lines used in the present invention were derived from peripheral blood (PB) and isolated using the MACSxpress Human Whole Blood NK Cell Isolation Kit (Miltenyi Biotec, 130-098-185). Purity was determined by staining cells with CD56 and CD3 antibodies, and NK cells were defined as the CD56+/CD3- population ( Figure 2A ). NK cells were cultured with NK MACS medium (130-114-429) in the presence of 50 IU/ml human IL-2 and irradiated K562-mb21-41BBL feeder cells (1:1 ratio). It was evident that after isolation and culture for 5 days, the primary NK cells entered a state of significant proliferation, whereby the activated NK cells on days 6-10 were used for gene delivery ( Fig. 2B ).

Figures 3A - 3D show the efficacy of RD114TR and BaEVTR pseudotyped gamma retroviral particles (PCIRs) using RetroNectin, which allows greater gene transfer in NK cells with high viability and high yield. The RetroNectin binding virus (RBV) infection strategy was used in the present invention and the details are illustrated in Figure 3A . There were NK cells with significantly higher viability (between 73.51% - 82.81%) after infection (approximately viable cells can be obtained by excluding low forward scatter (FSC) and high side scatter (SSC) events gate) ( Fig. 3B ), the transduction efficiency of the EGFR CAR gene (Fab-AF647 positive) in the three donors was between 65.11% - 72.75% ( Fig. 3C ), and the three donors at day 8 post-infection The high proliferative state yields 25, 34 and 45 times the initial infection NK numbers ( Fig. 3D ).

Figure 4 provides the determination of retroviral titration based on direct infection of activated NK cells of the same proliferative state using the same delivery strategy. In the present invention, for the development of gamma reverse transcription vector (PCIR)-based therapeutic gene delivery to human primary NK cells, the titration assay method is based on the exact same infection strategy for activated NK cells in the same proliferative state on day 7 . In this PCIR-EGFR-CAR-NK delivery assay shown in Figures 3A to 3D , the empty vector system PCIR-EV-EGFP was used. Serial dilutions of pooled primary viral supernatants harvested at 48- and 72 hours post-transfection were added to RetroNectin-coated plates and the procedure was the same as in Figures 1 and 3A . ALEXA ^FLUOR® 647 AffiniPure Goat Anti-Mouse IgG (F(ab') ₂ Fragment Specific, Jackson ImmunoResearch_115-605-006) was used to detect the mouse single chain variable fragment (ScFv) of the CAR. Transduction units/mL were calculated using the following formula: TU/mL = (number of transduced cells x percent fluorescence x dilution factor)/(transduction volume (in mL)). To obtain a more accurate titer, obtain the average of multiple dilutions. MOI 3 was used in this study (see Figures 3A to 3D ).

Figures 5A to 5C show that CAR-NKs engineered with gamma reverse transcription vectors outperformed CAR-NKs engineered with lentiviral vectors with highly stable expression of the transduced gene in long-term culture. Lentivirus-engineered PCIL-EGFR-CAR-NK and EV-GFP controls were enriched based on sorting GFP-positive populations, and retrovirus-engineered PCIR-EGFR-CAR-NK was based on goat anti-mouse Fab-AF647 Sorting of positive cells. PCIL-EGFR-CAR-NK transduction rates and performance were assessed using flow cytometry and fluorescence microscopy imaging at day 10 post-sort, and in PCIL-EGFR-CAR-NK and PCIL-GFP empty vector controls The number of GFP-positive cells dropped significantly from about 100% to 39% ( FIGS. 5A and 5C ). However, flow analysis confirmed that PCIR-EGFR-CAR-NK had a highly stable expression of the transduced gene at days 10, 14 and 25 after sorting ( Figures 5B and 5C ).

Figures 6A and 6B show the high transduction efficiency and cytotoxicity of EGFR-CAR-NK cells engineered with the gamma reverse transcription vector PCIR/BaEVTR obtained using real-time cell analysis (RTCA). NovoCyte 3005 flow cytometry analysis confirmed the high transduction efficiency of EGFR-CAR-NK engineered with the gamma reverse transcription vector PCIR/BaEVTR from 4 donors. The transduction rates of the empty vector carrying truncated CD19 (EV-Tcd19) and the EGFR transgene averaged 85% (positive for CD19-PE) and 79.6% (positive for goat anti-mouse Fab-AF647), respectively ( Fig. 6A ). Immune cell killing assays were performed using the xCELLigence RTCA MP Bundle (ACEA Biosciences, catalog number: 00380601040). The plots show impedance data for experiments with two donors using LN229 at 5,000 cells/well and adding 500 cells/well of effector (1:10 E:T). The engineered EGFR-CAR-NK had dynamic real-time killing compared to the empty vector mock-transduced group and the non-transduced NK group with the lowest viable cell index ( FIG. 6B ).

Figure 7 shows NK cell expansion from 17-24 days.

Figures 8A - 8C provide expression of retroviral entry receptors by 293Vec-GalV and 293Vec-BaEV packaging cells and the two cell lines Jurkat T and HT1080 used for titration. As shown in Figure 8A , among retroviruses, baboon endogenous virus (BaEV) and feline endogenous retrovirus (RD114) use a common cell surface receptor ASCT2 (sodium-dependent neutral amino acid transporter) protein) into the cell. In addition to ASCT2, baboon endogenous virus (BaEV) also uses ASCT1 as a cell entry receptor. Gibbon leukemia virus (GALV) uses the sodium-dependent phosphate transporter (Pit1) as its entry receptor. Figure 8B provides immunostaining for ASCT1, ASCT2 and Pit1 on two packaging cell lines 293Vec-GalV and 293Vec-BaEV and titer cell lines Jurkat T and HT1080. Histograms represent the mean fluorescence intensity (MFI) of a rabbit IgG isotype control (Invitrogen, catalog number: 02-6102) and a goat anti-rabbit IgG conjugated to Alexa Fluor 647 (A-21245) with the corresponding secondary antibody ; Invitrogen) stained cells, cells stained with anti-ASCT1 (LifeSpan BioSciences, LS-C179222) and corresponding secondary antibodies, cells stained with anti-ASCT2 (Cell Signaling Technology, 8057S) and corresponding secondary antibodies and anti-Pit1 ( ThermoFisher, PA5-98650) stained cells. Figure 8C shows that the three retroviral envelope proteins (BaEV, RD114 and GALV) can be titrated using two cell lines (Jurkat T and HT1080), due to the ubiquity of the corresponding entry receptors for these retroviral envelope proteins Performance. For the 293-based packaging cell lines 293Vec GALV and 293Vec BaEV, only the GALV pseudotyped retrovirus can infect them using the entry receptor Pit1. For each cell type, three bars are plotted in Figure 8C : the left bar represents the expression of ASCT1, the middle bar represents the expression of ASCT2, and the right bar represents the expression of Pit1.

Figure 9 illustrates the workflow of the transduction strategy used to generate the stable retroviral producer-293Vec-BaEV. A Moloney murine leukemia virus (Mo-MuLV) based retroviral vector (PCIR) was used as the target transgene delivery vehicle. The BaEV-pseudotyped 293Vec-BaEV packaging cell line was used to generate vehicle to infect cord blood-derived NK cells. This production was performed using the following two packaging cell lines: 293Vec-GALV (to generate transient Gibbon Leukemia Virus (GALV) pseudotyped supernatant) and 293Vec-BaEV (to generate final vector). Both cell lines were supplied by BioVec Pharma. This supernatant was then used to transduce the BaEV pseudotyped packaging cell line 293Vec-BaEV to generate bulk producers. For high titer clone generation, this 293Vec-BaEV bulk producer can be further sorted or single cell cloned by limiting dilution and using the high titer clone selected by titration from each clone generation supernatant . The supernatant can then be continuously produced from the high titer stable retrovirus producer-293Vec-BaEV.

Figures 10A and 10B show improved titers of the primary supernatant from the stable retroviral producer - 293Vec-BaEV, which produces a vector with a large-sized transgene. "Multiple targets in one" is a unique therapeutic strategy developed by the applicant. The BaEV pseudotyped 293Vec-BaEV packaging cell line was used to generate vehicle. Figure 10A shows that representative retroviruses with large transgenic inserts (>11 kb) can be transiently and stably produced by 293Vec-BaEV packaging cells. A total of 3.5 million 293Vec-BaEV packaging cells were seeded in 100 mm tissue culture dishes and incubated at 37 °C for 48-72 h. When cells reached 90-100% confluence, the original supernatant was collected and spun at 1500 g for 5 min. The above supernatant was titered on Jurkat T cells. A series of viral supernatants were loaded onto RetroNectin precoated non-tissue culture treated 24-well plates and made up to 500 μl using complete DMEM medium including 10% heat-inactivated fetal bovine serum. The plate was spun at 32°C, 2000 g for 2 hours, and 0.1 million Jurkat T cells were added and spun at 1000 g for 5 min. Plates were incubated at 37°C for 48h, then flow analysis was performed to determine titers (transduction units/ml) according to the following formula: Transduction units (TU)/mL = [(Number of target cells) × (% positive cells)] . In this example, the expression of the reporter gene RQR8 was detected by a flow cytometric antibody against human CD34 antibody (QBEnd/10) (Allophycocyanin) (Novus Biologicals, LLC, #FAB7227A). As shown in Figure 10B , the original virus from the stable retroviral producer-293Vec-BaEV (TU=7.2E5) for the vector with the large size transgene was compared to the transiently produced virus (TU=2.2E5). Serum has improved potency.

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<![CDATA[<210 > 58]]> <![CDATA[<400> 58]]> 000 <![CDATA[<210> 59]]> <![CDATA[<400> 59]]> 000 <![CDATA[<210 > 60]]> <![CDATA[<400> 60]]> 000 <![CDATA[<210> 61]]> <![CDATA[<400> 61]]> 000 <![CDATA[<210 > 62]]> <![CDATA[<400> 62]]> 000 <![CDATA[<210> 63]]> <![CDATA[<400> 63]]> 000 <![CDATA[<210 > 64]]> <![CDATA[<400> 64]]> 000 <![CDATA[<210> 65]]> <![CDATA[<400> 65]]> 000 <![CDATA[<210 > 66]]> <![CDATA[<400> 66]]> 000 <![CDATA[<210> 67]]> <![CDATA[<400> 67]]> 000 <![CDATA[<210 > 68]]> <![CDATA[<400> 68]]> 000 <![CDATA[<210> 69]]> <![CDATA[<400> 69]]> 000 <![CDATA[<210 > 70]]> <![CDATA[<400> 70]]> 000 <![CDATA[<210> 71]]> <![CDATA[<400> 71]]> 000 <![CDATA[<210 > 72]]> <![CDATA[<400> 72]]> 000 <![CDATA[<210> 73]]> <![CDATA[<400> 73]]> 000 <![CDATA[<210 > 74]]> <![CDATA[<400> 74]]> 000 <![CDATA[<210> 75]]> <![CDATA[<400> 75]]> 000 <![CDATA[<210 > 76]]> <![CDATA[<400> 76]]> 000 <![CDATA[<210> 77]]> <![CDATA[<400> 77]]> 000 < ![CDATA[<210> 78]]> <![CDATA[<400> 78]]> 000 <![CDATA[<210> 79]]> <![CDATA[<400> 79]]> 000 < ![CDATA[<210> 80]]> <![CDATA[<400> 80]]> 000 <![CDATA[<210> 81]]> <![CDATA[<211> 229]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 81]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <![CDATA[<210> 82]]> <![CDATA[<211> 229]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence] ]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 82]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His As n His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <![CDATA[<210> 83]]> <![CDATA[<211> 20]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 83]]> Pro Ser Gly Gln Ala Gly Ala Ala Ala Ser Glu Ser Leu Phe Val Ser 1 5 10 15 Asn His Ala Tyr 20 <![CDATA[<210> 84]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 84]]> Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <![CDATA[<210> 85]]> <![CDATA[<211> 23]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 85]]> His Val Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro 20 <![CDATA[<210> 86]]> <![CDATA[<211> 54]]> <![ CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthesis of Oligonucleotides acid]]> <![CDATA[<400> 86]]> atggggtggt caagcattat tctgtttctg gtcgctaccg ctacaggcgt ccat 54 <![CDATA[<210> 87]]> <![CDATA[<211> 60]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> Description of Artificial Sequences: Synthetic Oligonucleotides]]> <![CDATA[<400> 87]]> atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60 <![CDATA[<210> 88]]> <![CDATA[<211> 45]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> Description of Artificial Sequences: Synthetic Oligonucleotides]]> <![CDATA[<400> 88]]> ggtgggggcg gctctggtgg cggtggcagc ggcggaggtg gcagt 45 <![CDATA[<210> 89]]> < ![CDATA[<211> 204]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Unknown]]> <![CDATA[<220>]]> <![CDATA [<223> 未知之說明：跨膜結構域及細胞質結構域]]> <![CDATA[<400> 89]]> ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120 aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180 cgcgacttcg cagcctatcg ctcc 204 <![CDATA[<210> 90]]> <![CDATA[<211> 336]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Unknown]]> <![CDATA[<220>]]> <![CDATA[<223> Unknown Description: Signaling Domain]]> <![CDATA[<400> 90]]> agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <![CDATA[<210> 91]]> <![CDATA[<211> 69]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Unknown]]> <![CDATA[<220>]]> <![ CDATA[<223> Unknown specification: cleavable sequence]]> <![CDATA[<400> 91]]> cacgtgggtt ctggagaagg acgcggttcc ttgttgacgt gtggcgatgt agaggaaaat 60 ccgggtcca 69 <![CDATA[<210> 92]]> <! [CDATA[<211> 60]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA [<223> Description of artificial sequences: synthetic oligonucleotides]]> <![CDATA[<400> 92]]> ccgagcggcc aggcgggcgc ggcggcatcg gagtccctgt ttgtgtcaaa tcacgcctac 60 <![CDATA[<210> 93]]> <! [CDATA[<400> 93]]> 000 <![CDATA[<210> 94]]> <![CDATA[<400> 94]]> 000 <![CDATA[<210> 95]]> <! [CDATA[<400> 95]]> 000 <![CDATA[<210> 96]]> <![CDA TA[<400> 96]]> 000 <![CDATA[<210> 97]]> <![CDATA[<400> 97]]> 000 <![CDATA[<210> 98]]> <![ CDATA[<400> 98]]> 000 <![CDATA[<210> 99]]> <![CDATA[<400> 99]]> 000 <![CDATA[<210> 100]]> <![ CDATA[<400> 100]]> 000 <![CDATA[<210> 101]]> <![CDATA[<400> 101]]> 000 <![CDATA[<210> 102]]> <![ CDATA[<400> 102]]> 000 <![CDATA[<210> 103]]> <![CDATA[<400> 103]]> 000 <![CDATA[<210> 104]]> <![ CDATA[<400> 104]]> 000 <![CDATA[<210> 105]]> <![CDATA[<400> 105]]> 000 <![CDATA[<210> 106]]> <![ CDATA[<400> 106]]> 000 <![CDATA[<210> 107]]> <![CDATA[<400> 107]]> 000 <![CDATA[<210> 108]]> <![ CDATA[<400> 108]]> 000 <![CDATA[<210> 109]]> <![CDATA[<211> 63]]> <![CDATA[<212> DNA]]> <![CDATA [<213> Unknown]]> <![CDATA[<220>]]> <![CDATA[<223> Unknown description: hinge domain sequence]]> <![CDATA[<400> 109]]> ctcgagccca aatcttgtga caaaactcac acatgcccac cgtgcccgga tcccaaaggt 60 acc 63 <![CDATA[<210> 110]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<220>]]> <! [CDATA[<221> SITE]]> <![CDATA[<222> (1)..(12) ]]> <![CDATA[<223> This sequence can cover 1-6 repeating units of "Gly Ser"]]> <![CDATA[< 400> 110]]> Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <![CDATA[<210> 111]]> <![CDATA[<211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthesize 6xHis Tag]]> <![CDATA[<400> 111]]> His His His His His His 1 5 <![CDATA[<210> 112]]> <![ CDATA[<211> 48]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Description of Artificial Sequences: Synthetic Oligonucleotides]]> <![CDATA[<400> 112]]> ctcgagccca aatcttgtga caaaactcac acatgcccac cgtgcccg 48 <![CDATA[<210> 113]]> <![CDATA [<211> 81]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Description of artificial sequences: synthetic oligonucleotides]]> <![CDATA[<400> 113]]> ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt g 81 <![CDATA[<210> 114]]> <![CDATA[<211> 126]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> Description of Artificial Sequences: Synthetic Polynucleotides]]> <![CDATA[<400> 114]]> aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <![CDATA [<210> 115]]> <![CDATA[<211> 123]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Polynucleotides]]> <![CDATA[<400> 115]]> aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccac cc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <![CDATA[<210> 116]]> <![CDATA[<211> 339]]> <![CDATA[<212> DNA]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polynucleotide]]> <![CDATA[<400> 116]] > agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <![CDATA[<210> 117 ]]> <![CDATA[<211> 51]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 117] ]> Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Pro Thr Pro Ala 1 5 10 15 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 20 25 30 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 35 40 45 Asp Ile Tyr 50 <![CDATA[<210> 118]]> <![CDAT A[<211> 49]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mice]]> <![CDATA[<400> 118]]> Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro 1 5 10 15 Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg 20 25 30 Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile 35 40 45 Tyr <![CDATA[<210> 119]]> <![CDATA[<211> 51]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Feline]]> <![CDATA[<400> 119]]> Pro Val Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Gln Ala 1 5 10 15 Pro Ile Thr Thr Ser Gln Arg Val Ser Leu Arg Pro Gly Thr Cys Gln 20 25 30 Pro Ser Ala Gly Ser Thr Val Glu Ala Ser Gly Leu Asp Leu Ser Cys 35 40 45 Asp Ile Tyr 50 <![CDATA[<210> 120]]> <![CDATA[<211> 21 ]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 120]]> Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <![CDATA[<210> 121]]> <![CDATA[<211> 21]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Mice]]> <![CDATA[<400> 121]]> Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu 1 5 10 15 Ile Ile Thr Leu Ile 20 <![CDATA[<210> 122]]> <![CDATA[<211> 21]]> <![CDATA[<212> PRT] ]> <![CDATA[<213> Rat]]> <![CDATA[<400> 122]]> Ile Trp Ala Pro Leu Ala Gly Ile Cys Ala Val Leu Leu Leu Ser Leu 1 5 10 15 Val Ile Thr Leu Ile 20 <![CDATA[<210> 123]]> <![CDATA[<211> 42]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 123]]> Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <![CDATA[ <210> 124]]> <![CDATA[<211> 220]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[< 220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 124]]> Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205 Tyr Al a Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220 <![CDATA[<210> 125]]> <![CDATA[<211> 105]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polynucleotide]]> <![CDATA[< 400> 125]]> acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60 gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105 <![CDATA[<1210> 126]]> <![CDATA[<211> 108]]> <![CDATA[<2 > DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polynucleotide]]> < ![CDATA[<400> 126]]> agggaccaga ggctgcccccc cgatgcccac aagccccctg ggggaggcag tttccggacc 60 cccatccaag aggagcaggc cgacgcccac tccaccctgg ccaagatc 108 <![CDATA[<210> 127]]> <![CDATA[<400> 127] ![CDATA[<210> 128]]> <![CDATA[<400> 128]]> 000 <![CDATA[<210> 129]]> <![CDATA[<400> 129]]> 000 < ![CDATA[<210> 130]]> <![CDATA[<400> 130]]> 000 <![CDATA[<210> 131]]> <![CDATA[<400> 131]]> 000 < ![CDATA[<210> 132]]> <![CDATA[<400> 132]]> 000 <![CDATA[<210> 133]]> <![CDATA[<400> 133]]> 000 < ![CD ATA[<210> 134]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 134]]> Gly Gly Gly Gly Ser 1 5 <![CDATA[< 210> 135]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 135]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5 10 <![ CDATA[<210> 136]]> <![CDATA[<211> 20]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 136]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <![CDATA[<210> 137]]> <![CDATA[<211> 25]]> <![CDATA[<212> PRT]]> <! [CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptides]]> <![CDATA[<400> 137] ]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 20 25 <![CDATA[<210> 138]]> <![CDATA[ <211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDA TA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 138]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <![CDATA[<210> 139]]> <![CDATA[<211> 35]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide ]]> <![CDATA[<400> 139]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser 35 <![CDATA[<210> 140]]> <![CDATA[<211> 40]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 140]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Gly Ser 35 40 <![ CDATA[<210> 141]]> <![CDATA[<211> 45]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthesis Peptide]]> <![CDATA[<400> 141]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 35 40 45 <![CDATA[<210> 142]]> <![CDATA[<211> 50]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> < ![CDATA[<400> 142]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50 <![CDATA[<210> 143]]> <![CDATA[<211> 55]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 143]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 4 0 45 Gly Ser Gly Gly Gly Gly Ser 50 55 <![CDATA[<210> 144]]> <![CDATA[<211> 60]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 144]] > Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 <![CDATA[<210> 145]]> <![CDATA[<211> 65]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide] ]> <![CDATA[<400> 145]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly 50 55 60 Ser 65 <![CDATA[<210 > 146]]> <![CDATA[<211> 70]]> <![CDATA[<212> PRT]]> < ![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 146 ]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 50 55 60 Ser Gly Gly Gly Gly Gly Ser 65 70 <![CDATA[<210> 147]]> <![CDATA[<211> 75]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 147]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 50 55 60 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 65 70 75 <![CDATA[<210> 148]]> <![CDATA[<211> 546]]> <![CDATA[<212 > DNA]]> <![CDATA[<213 > 智人]]> <![CDATA[<400> 148]]> aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60 cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240 acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300 ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360 tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420 cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480 actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540 gcctac 546 <![CDATA[<210> 149]]> <![CDATA[ <400> 149]]> 000 <![CDATA[<210> 150]]> <![CDATA[<400> 150]]> 000 <![CDATA[<210> 151]]> <![CDATA[ <211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223 > Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 151]]> Ala Val Gly Thr Ile Val Asp Gln Ser Ala Lys 1 5 10 <![CDATA[<210> 152]]> < ![ CDATA[<400> 152]]> 000 <![CDATA[<210> 153]]> <![CDATA[<400> 153]]> 000 <![CDATA[<210> 154]]> <![ CDATA[<400> 154]]> 000 <![CDATA[<210> 155]]> <![CDATA[<400> 155]]> 000 <![CDATA[<210> 156]]> <![ CDATA[<400> 156]]> 000 <![CDATA[<210> 157]]> <![CDATA[<400> 157]]> 000 <![CDATA[<210> 158]]> <![ CDATA[<400> 158]]> 000 <![CDATA[<210> 159]]> <![CDATA[<400> 159]]> 000 <![CDATA[<210> 160]]> <![ CDATA[<400> 160]]> 000 <![CDATA[<210> 161]]> <![CDATA[<400> 161]]> 000 <![CDATA[<210> 162]]> <![ CDATA[<400> 162]]> 000 <![CDATA[<210> 163]]> <![CDATA[<400> 163]]> 000 <![CDATA[<210> 164]]> <![ CDATA[<400> 164]]> 000 <![CDATA[<210> 165]]> <![CDATA[<400> 165]]> 000 <![CDATA[<210> 166]]> <![ CDATA[<400> 166]]> 000 <![CDATA[<210> 167]]> <![CDATA[<400> 167]]> 000 <![CDATA[<210> 168]]> <![ CDATA[<211> 57]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Description of artificial sequences: synthetic oligonucleotides]]> <![CDATA[<400> 168]]> atggaatttg ggctgcgctg ggttttcctt gttgctattt taaaagatgt ccagtgt 57 <![CDATA[<210> 169]]> <![ CDATA [<211> 19]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Unknown]]> <![CDATA[<220>]]> <![CDATA[<223 > Unknown description: Peptide sequence]]> <![CDATA[<400> 169]]> Met Glu Phe Gly Leu Arg Trp Val Phe Leu Val Ala Ile Leu Lys Asp 1 5 10 15 Val Gln Cys <![CDATA[ <210> 170]]> <![CDATA[<211> 903]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[< 220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Polynucleotides]]> <![CDATA[<400> 170]]> gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctccc 120 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660 tacacacaga agagcctctc cctgtctccg gagctgcaac tggaggagag ctgtgcggag 720 gcgcaggacg gggagctgga cgggctgtgg acgaccatca ccatcttcat cacactcttc 780 ctgttaagcg tgtgctacag tgccaccgtc accttcttca aggtgaagtg gatcttctcc 840 tcggtggtgg acctgaagca gaccatcatc cccgactaca ggaacatgat cggacagggg 900 gcc 903 <![CDATA[< 210> 171]]> <![CDATA[<211> 301]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![ CDATA[<400> 171]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu 225 230 235 240 Ala Gln Asp Gly Glu Leu Asp Gly Leu Trp Thr Thr Ile Thr Ile Phe 245 250 255 Ile Thr Leu Phe Leu Leu Ser Val Cys Tyr Ser Ala Thr Val Thr Phe 260 265 270 Phe Lys Val Lys Trp Ile Phe Ser Ser Val Val Asp Leu Lys Gln Thr 275 280 285 Ile Ile Pro Asp Tyr Arg Asn Met Ile Gly Gln Gly Ala 290 295 300 <![CDATA[<210> 172]]> <![CDATA[<211> 563]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 172]]> Met Lys Leu Pro Thr Gly Met Val Ile Leu Cys Ser Leu Ile Ile Val 1 5 10 15 Arg Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Ala Leu Val Gln Lys 20 25 30 Gln His Gly Lys Pro Cys Glu Cys Ser Gly Gly Gln Val Ser Glu Ala 35 40 45 Pro Pro Asn Ser Ile Gln Gln Val Thr Cys Pro Gly Lys Thr Ala Tyr 50 55 60 Leu Met Thr Asn Gln Lys Trp Lys Cys Arg Val Thr Pro Lys Ile Ser 65 70 75 80 Pro Ser Gly Gly Glu Leu Gln Asn Cys Pro Cys Asn Thr Phe Gln Asp 85 90 95 Ser Met His Ser Ser Cys Tyr Thr Glu Tyr Arg Gln Cys Arg Arg Ile 100 105 110 Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Ile Arg Ser Gly Ser 115 120 125 Leu Asn Glu Val Gln Ile Leu Gln Asn Pro Asn Gln Leu Leu Gln Ser 130 135 140 Pro Cys Arg Gly Ser Ile Asn Gln Pro Val Cys Trp Ser Ala Thr Ala 145 150 155 160 Pro Ile His Ile Ser Asp Gly Gly Gly Pro Leu Asp Thr L ys Arg Val 165 170 175 Trp Thr Val Gln Lys Arg Leu Glu Gln Ile His Lys Ala Met Thr Pro 180 185 190 Glu Leu Gln Tyr His Pro Leu Ala Leu Pro Lys Val Arg Asp Asp Leu 195 200 205 Ser Leu Asp Ala Arg Thr Phe Asp Ile Leu Asn Thr Thr Phe Arg Leu 210 215 220 Leu Gln Met Ser Asn Phe Ser Leu Ala Gln Asp Cys Trp Leu Cys Leu 225 230 235 240 Lys Leu Gly Thr Pro Thr Pro Leu Ala Ile Pro Thr Pro Ser Leu Thr 245 250 255 Tyr Ser Leu Ala Asp Ser Leu Ala Asn Ala Ser Cys Gln Ile Ile Pro 260 265 270 Pro Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Ser 275 280 285 Ser Pro Phe Ile Asn Asp Thr Glu Gln Ile Asp Leu Gly Ala Val Thr 290 295 300 Phe Thr Asn Cys Thr Ser Val Ala Asn Val Ser Ser Pro Leu Cys A la 305 310 315 320 Leu Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr 325 330 335 Leu Pro Gln Asn Trp Thr Arg Leu Cys Val Gln Ala Ser Leu Leu Pro 340 345 350 Asp Ile Asp Ile Asn Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile 355 360 365 Asp His Tyr Ile His Arg Pro Lys Arg Ala Val Gln Phe Ile Pro Leu 370 375 380 Leu Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly 385 390 395 400 Leu Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser His Gln Leu Ile 405 410 415 Ser Asp Val Gln Val Leu Ser Gly Thr Ile Gln Asp Leu Gln Asp Gln 420 425 430 Val Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp 435 440 445 Leu Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala L eu Gln Glu Lys 450 455 460 Cys Cys Phe Tyr Ala Asn Lys Ser Gly Ile Val Arg Asn Lys Ile Arg 465 470 475 480 Thr Leu Gln Glu Glu Leu Gln Lys Arg Arg Glu Ser Leu Ala Thr Asn 485 490 495 Pro Leu Trp Thr Gly Leu Gln Gly Phe Leu Pro Tyr Leu Leu Pro Leu 500 505 510 Leu Gly Pro Leu Leu Thr Leu Leu Leu Ile Leu Thr Ile Gly Pro Cys 515 520 525 Val Phe Asn Arg Leu Val Gln Phe Val Lys Asp Arg Ile Ser Val Val 530 535 540 Gln Ala Leu Val Leu Thr Gln Gln Tyr His Gln Leu Lys Pro Leu Glu 545 550 555 560 Tyr Glu Pro <![CDATA[<210> 173]]> <![CDATA[<211> 562]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthesis Peptide]]> <![CDATA[<400> 173]]> Met Gly Phe Thr Thr Lys Ile Ile Phe Leu Tyr Asn Leu Val Leu Val 1 5 10 15 Tyr Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Glu Leu Val Gln Lys 20 25 30 Arg Tyr Gly Arg Pro Cys Asp Cys Ser Gly Gly Gln Val Ser Glu Pro 35 40 45 Pro Ser Asp Arg Val Ser Gln Val Thr Cys Ser Gly Lys Thr Ala Tyr 50 55 60 Leu Met Pro Asp Gln Arg Trp Lys Cys Lys Ser Ile Pro Lys Asp Thr 65 70 75 80 Ser Pro Ser Gly Pro Leu Gln Glu Cys Pro Cys Asn Ser Tyr Gln Ser 85 90 95 Ser Val His Ser Ser Cys Tyr Thr Ser Tyr Gln Gln Cys Arg Ser Gly 100 105 110 Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Thr Gln Thr Gly Gly 115 120 125 Thr Ser Asp Val Gln Val Leu Gly Ser Thr Asn Lys Leu Ile Gln Ser 130 135 140 Pro Cys Asn Gly Ile Lys Gly Gln Ser Ile Cys Trp Ser Thr Thr Ala 145 150 155 160 Pro Ile His Val Ser Asp Gly Gly Gly Pro Leu Asp Thr Thr Arg Ile 165 170 175 Lys Ser Val Gln Arg Lys Leu Glu Glu Ile His Lys Ala Leu Tyr Pro 180 185 190 Glu Leu Gln Tyr His Pro Leu Ala Ile Pro Lys Val Arg Asp Asn Leu 195 200 205 Met Val Asp Ala Gln Thr Leu Asn Ile Leu Asn Ala Thr Tyr Asn Leu 210 215 220 Leu Leu Met Ser Asn Thr Ser Leu Val Asp Asp Cys Trp Leu Cys Leu 225 230 235 240 Lys Leu Gly Pro Pro Thr Pro Leu Ala Ile Pro Asn Phe Leu Leu Ser 245 250 255 Tyr Val Thr Arg Ser Ser Asp Asn Ile Ser Cys Leu Ile Ile Pro Pro Pro 260 265 270 Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Phe Ser 275 280 285 Pro Ser Tyr Asn Ser Thr Glu Glu Ile Asp Leu Gly His Val Ala Phe 290 295 300 Ser Asn Cys Thr Ser Ile Thr Asn Val Thr Gly Pro Ile Cys Ala Val 305 310 315 320 Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr Leu 325 330 335 Pro Thr Asn Trp Thr Gly Leu Cys Val Leu Ala Thr Leu Leu Pro Asp 340 345 350 Ile Asp Ile Ile Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile Asp 355 360 365 His Phe Ile Tyr Arg Pro Lys Arg Ala Ile Gln Phe Ile Pro Leu Leu 370 375 380 Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly Leu 385 390 395 400 Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser Asn Gln Leu Ile Ser 405 410 415 Asp Val Gln Ile Leu Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val 420 425 430 Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu 435 440 445 Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys Cys 450 455 460 Cys Phe Tyr Val Asn Lys Ser Gly Ile Val Arg Asp Lys Ile Lys Thr 465 470 475 480 Leu Gln Glu Glu Leu Glu Arg Arg Arg Lys Asp Leu Ala Ser Asn Pro 485 490 495 Leu Trp Thr Gly Leu Gln Gly Leu Leu Pro Tyr Leu Leu Pro Phe Leu 500 505 510 Gly Pro Leu Leu Thr Leu Leu Leu Leu Leu Thr Ile Gly Pro Cys Ile 515 520 525 Phe Asn Arg Leu Val Gln Phe Val Lys Asp Arg Ile Ser Val Val Gln 530 535 540 Ala Leu Val Leu Thr Gln Gln Tyr His Gln Leu Lys Pro Leu Glu Tyr 545 550 555 560 Glu Pro <![CDATA[<210> 174]]> <![CDATA[<211> 655]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Murine Leukemia Virus] ]> <![CDATA[<400> 174]]> Met Ala Ala Arg Ser Thr Leu Ser Lys Pro Pro Gln Asp Lys Ile Asn 1 5 10 15 Pro Trp Lys Pro Leu Ile Val Met Gly Val Leu Leu Gly Val Gly Met 20 25 30 Ala Glu Ser Pro His Gln Val Phe Asn Val Thr Trp Arg Val Thr Asn 35 40 45 Leu Met Thr Gly Arg Thr Ala Asn Ala Thr Ser L eu Leu Gly Thr Val 50 55 60 Gln Asp Ala Phe Pro Lys Leu Tyr Phe Asp Leu Cys Asp Leu Val Gly 65 70 75 80 Glu Glu Trp Asp Pro Ser Asp Gln Glu Pro Tyr Val Gly Tyr Gly Cys 85 90 95 Lys Tyr Pro Ala Gly Arg Gln Arg Thr Arg Thr Phe Asp Phe Tyr Val 100 105 110 Cys Pro Gly His Thr Val Lys Ser Gly Cys Gly Gly Pro Gly Glu Gly 115 120 125 Tyr Cys Gly Lys Trp Gly Cys Glu Thr Thr Gly Gln Ala Tyr Trp Lys 130 135 140 Pro Thr Ser Ser Trp Asp Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro 145 150 155 160 Trp Asp Thr Gly Cys Ser Lys Val Ala Cys Gly Pro Cys Tyr Asp Leu 165 170 175 Ser Lys Val Ser Asn Ser Phe Gln Gly Ala Thr Arg Gly Gly Arg Cys 180 185 190 Asn Pro Leu Val Leu Glu Phe Thr Asp Ala Gly Lys Lys Ala Asn Trp 195 200 205 Asp Gly Pro Lys Ser Trp Gly Leu Arg Leu Tyr Arg Thr Gly Thr Asp 210 215 220 Pro Ile Thr Met Phe Ser Leu Thr Arg Gln Val Leu Asn Val Gly Pro 225 230 235 240 Arg Val Pro Ile Gly Pro Asn Pro Val Leu Pro Asp Gln Arg Leu Pro 245 250 255 Ser Ser Pro Ile Glu Ile Val Pro Ala Pro Gln Pro Pro Ser Pro Leu 260 265 270 Asn Thr Ser Tyr Pro Pro Ser Thr Thr Ser Thr Pro Ser Thr Ser Pro 275 280 285 Thr Ser Pro Ser Val Pro Gln Pro Pro Pro Gly Thr Gly Asp Arg Leu 290 295 300 Leu Ala Leu Val Lys Gly Ala Tyr Gln Ala Leu Asn Leu Thr Asn Pro 305 310 315 320 Asp Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr 325 330 335 Tyr Glu Gly Val Ala Val Val Gly Thr Tyr Thr Asn His Ser Thr Ala 340 345 350 Pro Ala Asn Cys Thr Ala Thr Ser Gln His Lys Leu Thr Leu Ser Glu 355 360 365 Val Thr Gly Gln Gly Leu Cys Met Gly Ala Val Pro Lys Thr His Gln 370 375 380 Ala Leu Cys Asn Thr Thr Gln Ser Ala Gly Ser Gly Ser Tyr Tyr Leu 385 390 395 400 Ala Ala Pro Ala Gly Thr Met Trp Ala Cys Ser Thr Gly Leu Thr Pro 405 410 415 Cys Leu Ser Thr Thr Val Leu Asn Leu Thr Thr Asp Tyr Cys Val Leu 420 425 430 Val Glu Leu Trp Pro Arg Val Ile Tyr His Ser Pro Asp Tyr Met Tyr 435 440 445 Gly Gln Leu Glu Gln Arg Thr Lys Tyr Lys Arg Glu Pro Val Ser Leu 450 455 460 Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly Gly Ile Ala Ala 465 470 475 480 Gly Ile Gly Thr Gly Thr Thr Ala Leu Ile Lys Thr Gln Gln Phe Glu 485 490 495 Gln Leu His Ala Ala Ile Gln Thr Asp Leu Asn Glu Val Glu Lys Ser 500 505 510 Ile Thr Asn Leu Glu Lys Ser Leu Thr Ser Leu Ser Glu Val Val Leu 515 520 525 Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu 530 535 540 Cys Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp His Thr Gly 545 550 555 560 Leu Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Asn Gln Arg 565 570 575 Gln Lys Leu Phe Glu Thr Gly Gln Gly Trp Phe Glu Gly Leu Phe Asn 580 585 590 Arg Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met Gly Pro Leu 595 600 605 Ile Val Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg 610 615 620 Leu Val Gln Phe Val Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val 625 630 635 640 Leu Thr Gln Gln Tyr His Gln Leu Lys Pro Leu Glu Tyr Glu Pro 645 650 655 <![CDATA[<210> 175]]> <![CDATA[<211> 563]]> <![CDATA[<212> PRT]] > <![CDATA[<213> Baboon Endogenous Virus]]> <![ CDATA[<400> 175]]> Met Gly Phe Thr Thr Lys Ile Ile Phe Leu Tyr Asn Leu Val Leu Val 1 5 10 15 Tyr Ala Gly Phe Asp Asp Pro Arg Lys Ala Ile Glu Leu Val Gln Lys 20 25 30 Arg Tyr Gly Arg Pro Cys Asp Cys Ser Gly Gly Gln Val Ser Glu Pro 35 40 45 Pro Ser Asp Arg Val Ser Gln Val Thr Cys Ser Gly Lys Thr Ala Tyr 50 55 60 Leu Met Pro Asp Gln Arg Trp Lys Cys Lys Ser Ile Pro Lys Asp Thr 65 70 75 80 Ser Pro Ser Gly Pro Leu Gln Glu Cys Pro Cys Asn Ser Tyr Gln Ser 85 90 95 Ser Val His Ser Ser Cys Tyr Thr Ser Tyr Gln Gln Cys Arg Ser Gly 100 105 110 Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Thr Gln Thr Gly Gly 115 120 125 Thr Ser Asp Val Gln Val Leu Gly Ser Thr Asn Lys Leu Ile Gln Ser 130 135 140 Pro Cys Asn Gly Ile Lys Gly Gln Ser Ile Cys Trp Ser Thr Thr Ala 145 150 155 160 Pro Ile His Val Ser Asp Gly Gly Gly Pro Leu Asp Thr Thr Arg Ile 165 170 175 Lys Ser Val Gln Arg Lys Leu Glu Glu Ile His Lys Ala Leu Tyr Pro 180 185 190 Glu Leu Gln Tyr His Pro Leu Ala Ile Pro Lys Val Arg Asp Asn Leu 195 200 205 Met Val Asp Ala Gln Thr Leu Asn Ile Leu Asn Ala Thr Tyr Asn Leu 210 215 220 Leu Leu Met Ser Asn Thr Ser Leu Val Asp Asp Cys Trp Leu Cys Leu 225 230 235 240 Lys Leu Gly Pro Pro Thr Pro Leu Ala Ile Pro Asn Phe Leu Leu Ser 245 250 255 Tyr Val Thr Arg Ser Ser Asp Asn Ile Ser Cys Leu Ile Ile Pro Pro Pro 260 265 270 Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Phe Ser 275 280 285 Pro Ser Tyr Asn Ser Thr Glu Glu Ile Asp Leu Gly His Val Ala Phe 290 295 300 Ser Asn Cys Thr Ser Ile Thr Asn Val Thr Gly Pro Ile Cys Ala Val 305 310 315 320 Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr Leu 325 330 335 Pro Thr Asn Trp Thr Gly Leu Cys Val Leu Ala Thr Leu Leu Pro Asp 340 345 350 Ile Asp Ile Ile Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile Asp 355 360 365 His Phe Ile Tyr Arg Pro Lys Arg Ala Ile Gln Phe Ile Pro Leu Leu 370 375 380 Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly Leu 385 390 395 400 Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser Asn Gln Leu Ile Ser 405 410 415 Asp Val Gln Ile Leu Ser Ser Thr Ile Gln Asp Leu Gln Asp Gln Val 420 425 430 Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu 435 440 445 Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys Cys 450 455 460 Cys Phe Tyr Val Asn Lys Ser Gly Ile Val Arg Asp Lys Ile Lys Thr 465 470 475 480 Leu Gln Glu Glu Leu Glu Arg Arg Arg Lys Asp Leu Ala Ser Asn Pro 485 490 495 Leu Trp Thr Gly Leu Gln Gly Leu Leu Pro Tyr Leu Leu Pro Phe Leu 500 505 510 Gly Pro Leu Leu Thr Leu Leu Leu Leu Leu Leu Thr Ile Gly Pro Cys Ile 515 520 525 Phe Asn Arg Leu Thr Ala Phe Ile Asn Asp Lys Leu Asn Ile Ile His 530 535 540 Ala Met Val Leu Thr Gln Gln Tyr Gln Val Leu Arg Thr Asp Glu Glu 545 550 555 560 Ala Gln Asp <![CDATA[<210> 176]]> <![CDATA[<211> 564]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Unknown]]> <![CDATA[<220>]]> <![CDATA[<223> Unknown Description: RD114 Retrovirus Sequence]]> <![CDATA[<400> 176]]> Met Lys Leu Pro Thr Gly Met Val Ile Leu Cys Ser Leu Ile Ile Val 1 5 10 15 Arg Ala Gly Phe Asp Asp Pro Arg Lys A la Ile Ala Leu Val Gln Lys 20 25 30 Gln His Gly Lys Pro Cys Glu Cys Ser Gly Gly Gln Val Ser Glu Ala 35 40 45 Pro Pro Asn Ser Ile Gln Gln Val Thr Cys Pro Gly Lys Thr Ala Tyr 50 55 60 Leu Met Thr Asn Gln Lys Trp Lys Cys Arg Val Thr Pro Lys Ile Ser 65 70 75 80 Pro Ser Gly Gly Glu Leu Gln Asn Cys Pro Cys Asn Thr Phe Gln Asp 85 90 95 Ser Met His Ser Ser Cys Tyr Thr Glu Tyr Arg Gln Cys Arg Arg Ile 100 105 110 Asn Lys Thr Tyr Tyr Thr Ala Thr Leu Leu Lys Ile Arg Ser Gly Ser 115 120 125 Leu Asn Glu Val Gln Ile Leu Gln Asn Pro Asn Gln Leu Leu Gln Ser 130 135 140 Pro Cys Arg Gly Ser Ile Asn Gln Pro Val Cys Trp Ser Ala Thr Ala 145 150 155 160 Pro Ile His Ile Ser Asp Gly Gly Gly Pro Leu Asp Thr Lys Arg Val 165 170 175 Trp Thr Val Gln Lys Arg Leu Glu Gln Ile His Lys Ala Met Thr Pro 180 185 190G lu Leu Gln Tyr His Pro Leu Ala Leu Pro Lys Val Arg Asp Asp Leu 195 200 205 Ser Leu Asp Ala Arg Thr Phe Asp Ile Leu Asn Thr Thr Phe Arg Leu 210 215 220 Leu Gln Met Ser Asn Phe Ser Leu Ala Gln Asp Cys Trp Leu Cys Leu 225 230 235 240 Lys Leu Gly Thr Pro Thr Pro Leu Ala Ile Pro Thr Pro Ser Leu Thr 245 250 255 Tyr Ser Leu Ala Asp Ser Leu Ala Asn Ala Ser Cys Gln Ile Ile Pro 260 265 270 Pro Leu Leu Val Gln Pro Met Gln Phe Ser Asn Ser Ser Cys Leu Ser 275 280 285 Ser Pro Phe Ile Asn Asp Thr Glu Gln Ile Asp Leu Gly Ala Val Thr 290 295 300 Phe Thr Asn Cys Thr Ser Val Ala Asn Val Ser Ser Pro Leu Cys Ala 305 310 315 320 Leu Asn Gly Ser Val Phe Leu Cys Gly Asn Asn Met Ala Tyr Thr Tyr 325 330 335 Leu Pro Gln Asn Trp Thr Arg Leu Cys Val Gln Ala Ser Leu Leu Pro 340 345 350 Asp Ile Asp Ile Asn Pro Gly Asp Glu Pro Val Pro Ile Pro Ala Ile 355 360 365 Asp His Tyr Ile His Arg Pro Lys Arg Ala Val Gln Phe Ile Pro Leu 370 375 380 Leu Ala Gly Leu Gly Ile Thr Ala Ala Phe Thr Thr Gly Ala Thr Gly 385 390 395 400 Leu Gly Val Ser Val Thr Gln Tyr Thr Lys Leu Ser His Gln Leu Ile 405 410 415 Ser Asp Val Gln Val Leu Ser Gly Thr Ile Gln Asp Leu Gln Asp Gln 420 425 430 Val Asp Ser Leu Ala Glu Val Val Leu Gln Asn Arg Arg Gly Leu Asp 435 440 445 Leu Leu Thr Ala Glu Gln Gly Gly Ile Cys Leu Ala Leu Gln Glu Lys 450 455 460 Cys Cys Phe Tyr Ala Asn Lys Ser Gly Ile Val Arg Asn Lys Ile Arg 465 470 4 75 480 Thr Leu Gln Glu Glu Leu Gln Lys Arg Arg Glu Ser Leu Ala Thr Asn 485 490 495 Pro Leu Trp Thr Gly Leu Gln Gly Phe Leu Pro Tyr Leu Leu Pro Leu 500 505 510 Leu Gly Pro Leu Leu Thr Leu Leu Leu Ile Leu Thr Ile Gly Pro Cys 515 520 525 Val Phe Ser Arg Leu Met Ala Phe Ile Asn Asp Arg Leu Asn Val Val 530 535 540 His Ala Met Val Leu Ala Gln Gln Tyr Gln Ala Leu Lys Ala Glu Glu 545 550 555 560 Glu Ala Gln Asp <![CDATA[<210> 177]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]] > <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<220>]]> <![CDATA[<221> MOD_RES ]]> <![CDATA[<222> (2)..(2)]]> <![CDATA[<223> Ile or Val]]> <![CDATA[<220>]]> <![ CDATA[<221> MOD_RES]]> <![CDATA[<222> (4)..(4)]]> <![CDATA[<223> Any amino acid that is generally considered self-cleaving]]> < ![CDATA[<400> 177]]> Asp Xaa Glu Xaa Asn Pro Gly Pro 1 5 <![CDATA [<210> 178]]> <![CDATA[<211> 153]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[ <400> 178]]> Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <![CDATA[<210> 179 ]]> <![CDATA[<211> 132]]> <![CDATA[<212> PRT]]> <![CDAT A[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 179]] > Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu 1 5 10 15 Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 20 25 30 Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys 35 40 45 Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro 50 55 60 Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg 65 70 75 80 Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys 85 90 95 Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr 100 105 110 Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile 115 120 125 Ser Thr Leu Thr 130 <![CDATA[<210> 180]]> <![CDATA[<211> 134]]> <![CDATA[<212> PRT]]> <![CDATA [<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polypeptide]]> <![CDATA[<400> 180]]> Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 1 5 10 15 H is Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 20 25 30 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro 35 40 45 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 50 55 60 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His 65 70 75 80 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 85 90 95 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser 115 120 125 Ile Ile Ser Thr Leu Thr 130 <![CDATA[<210> 181 ]]> <![CDATA[<211> 162]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 181] ]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser <![CDATA[<210> 182]]> <![CDATA[<211> 135]]> <![CDATA [<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 182]]> Met Val Leu Gly Thr Ile Asp Leu Cys Ser Cys Phe Ser Ala Gly Leu 1 5 10 15 Pro Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys 20 25 30 Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr 35 40 45 Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala M et Lys Cys Phe 50 55 60 Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile 65 70 75 80 His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser 85 90 95 Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu 100 105 110 Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val 115 120 125 Gln Met Phe Ile Asn Thr Ser 130 135 <![CDATA[<210 > 183]]> <![CDATA[<211> 685]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Gibbon Leukemia Virus]]> <![ CDATA[<400> 183]]> Met Val Leu Leu Pro Gly Ser Met Leu Leu Thr Ser Asn Leu His His 1 5 10 15 Leu Arg His Gln Met Ser Pro Gly Ser Trp Lys Arg Leu Ile Ile Leu 20 25 30 Leu Ser Cys Val Phe Gly Gly Gly Gly Thr Ser Leu Gln Asn Lys Asn 35 40 45 Pro His Gln Pro Met Thr Leu Thr Trp Gln Val Leu Ser Gln Thr Gly 50 55 60 Asp Val Val Trp Asp Thr Lys Ala Val Gln Pro Pro Trp Thr Trp Trp 65 70 75 80 Pro Thr Leu Lys Pro Asp Val Cys Ala Leu Ala Ala Ser Leu Glu Ser 85 90 95 Trp Asp Ile Pro Gly Thr Asp Val Ser Ser Ser Lys Arg Val Arg Pro 100 105 110 Pro Asp Ser Asp Tyr Thr Ala Ala Tyr Lys Gln Ile Thr Trp Gly Ala 115 120 125 Ile Gly Cys Ser Tyr Pro Arg Ala Arg Thr Arg Met Ala Ser Ser Thr 130 135 140 Phe Tyr Val Cys Pro Arg Asp Gly Arg Thr Leu Ser Glu Ala Arg Arg 145 150 155 160 Cys Gly Gly Leu Glu Ser Leu Tyr Cys Lys Glu Trp Asp Cys Glu Thr 165 170 175 Thr Gly Thr Gly Tyr Trp Leu Ser Lys Ser Ser Lys Asp Leu Ile Thr 180 185 190 Val Lys Trp Asp Gln Asn Ser Glu Trp Thr Gln Lys Phe Gln Gln Cys 195 200 205 His Gln Thr Gly Trp Cys Asn Pro Leu Lys Ile Asp Phe Thr Asp Lys 210 215 220 Gly Lys Leu Ser Lys Asp Trp Ile Thr Gly Lys Thr Trp Gly Leu Arg 225 230 235 240 Phe Tyr Val Ser Gly His Pro Gly Val Gln Phe Thr Ile Arg Leu Lys 245 250 255 Ile Thr Asn Met Pro Ala Val Ala Val Gly Pro Asp Leu Val Leu Val 260 265 270 Glu Gln Gly Pro Pro Arg Thr Ser Leu Ala Leu Pro Pro Pro Leu Pro 275 280 285 Pro Arg Glu Ala Pro Pro Pro Ser Leu Pro Asp Ser Asn Ser Thr Ala 290 295 300 Leu Ala Thr Ser Ala Gln Thr Pro Thr Val Arg Lys Thr Ile Val Thr 305 310 315 320 Leu Asn Thr Pro Pro Pro Thr Thr Gly Asp Arg Leu Phe Asp Leu Val 325 330 335 Gln Gly Ala Phe Leu Thr Leu Asn Ala Thr Asn Pro Gly Ala Thr Glu 340 345 350 Ser Cys Trp Leu Cys Leu Ala Met Gly Pro Pro Tyr Tyr Glu Ala Ile 355 360 365 Ala Ser Ser Gly Glu Val Ala Tyr Ser Thr Asp Leu Asp Arg Cys Arg 370 375 380 Trp Gly Thr Gln Gly Lys Leu Thr Leu Thr Glu Val Ser Gly His Gly 385 390 395 400 Leu Cys Ile Gly Lys Val Pro Phe Thr His Gln His Leu Cys Asn Gln 405 410 415 Thr Leu Ser Ile Asn Ser Ser Gly Asp His Gln Tyr Leu Leu Pro Ser 420 425 430 Asn His Ser Trp Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Leu Ser 435 440 445 Thr Ser Val Phe Asn Gln Thr Arg Asp Phe Cys Ile Gln Val Gln Leu 450 455 460 Ile Pro Arg Ile Tyr Tyr Tyr Pro Glu Glu Val Leu Leu Gln Ala Tyr 465 470 475 480 Asp Asn Ser His Pro Arg Thr Lys Arg Glu Ala Val Ser Leu Thr Leu 485 490 495 Ala Val Leu Leu Gly Leu Gly Ile Thr Ala Gly Ile Gly Thr Gly Ser 500 505 510 Thr Ala Leu Ile Lys Gly Pro Ile Asp Leu Gln Gln Gly Leu Thr Ser 515 520 525 Leu Gln Ile Ala Ile Asp Ala Asp Leu Arg Ala Leu Gln Asp Ser Val 530 535 540 Ser Lys Leu Glu Asp Ser Leu Thr Ser Leu Ser Glu Val Val Leu Gln 545 550 555 560 Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys 565 570 575 Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ile Asp His Ser Gly Ala 580 585 590 Val Arg Asp Ser Met Lys Lys Leu Lys Glu Lys Leu Asp Lys Arg Gln 595 600 605 Leu Glu Arg Gln Lys Ser Gln Asn Trp Tyr Glu Gly Trp Phe Asn Asn 610 615 620 Ser Pro Trp Phe Thr Thr Leu Leu Ser Thr Ile Ala Gly Pro Leu Leu 625 630 635 640 Leu Leu Leu Leu Leu Leu Ile Leu Gly Pro Cys Ile Ile Asn Lys Leu 645 650 655 Val Gln Phe Ile Asn Asp Arg Ile Ser Ala Val Lys Ile Leu Val Leu 660 665 670 Arg Gln Lys Tyr Gln Ala Leu Glu Asn Glu Gly Asn Leu 675 680 685
        

Claims (33)

A pseudotyped gamma retroviral particle comprising modified RD114 feline endogenous retrovirus envelope glycoprotein (RD114TR) and modified baboon envelope glycoprotein (BaEVTR), wherein: a. the RD114TR glycoprotein includes the extracellular and transmembrane domains of the RD114 glycoprotein and the cytoplasmic domain of the amphiphilic murine leukemia virus (MLV-A) glycoprotein; and b. wherein the BaEVTR glycoprotein comprises the extracellular domain and the transmembrane domain of the baboon envelope glycoprotein (BaEV) and the cytoplasmic domain of the MLV-A glycoprotein, optionally wherein the RD114TR and the BaEVTR are incorporated into the envelope of the gamma retroviral particle as membrane proteins; and Also optionally wherein the pseudotyped gamma retroviral particle further comprises a vector gene body, an optional reverse transcriptase and an optional integrase encapsulated in the envelope, wherein the vector gene body comprises the following flanked by two long One or more of terminal repeats (LTR): (A) a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein or polypeptide optionally selected from antibodies or fragments thereof, enzymes, ligands or receptors, (B) the reverse complement of (A), or (C) A polynucleotide comprising one or more recognition sites that are optionally recognized and cleaved by restriction enzymes suitable for insertion of a CAR coding sequence or its reverse complement into the polynucleotide. The pseudotyped gamma retroviral particle of claim 1, wherein the vector genome further comprises one or more of the following: a 5' LTR, a 5' cap, a 3' poly-A tail, and a 3' LTR. The pseudotyped gamma retroviral particle of claim 1 or 2, which is selected from any one of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV) , Fried murine embryonic stem cell virus (friend murine embryonic stem cell virus, FMEV), heterophilic MuLB-related virus, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus. A method of preparing a population of natural killer (NK) cells, comprising culturing a population of cells comprising one or more of the following with an immune cell activator (eg, an NK cell activator): NK cells, capable of deriving NK Progenitor cells of cells or stem cells capable of deriving NK cells, wherein the cell population is depleted in the cell population of cells expressing one or more of the following: CD3, CD4, CD8, T cell receptor (TCR) alpha chain , TCR beta chain or alpha beta TCR, and wherein the immune cell activator is selected from one or more of the following (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of NK cells, progenitor cells or stem cells, thereby activating or proliferating NK cells, as appropriate wherein the The antibody system is selected from one or more of the following: anti-CD2 antibody, anti-CD16 antibody, anti-NKG2D antibody, anti-DNAM-1 antibody, anti-2B4 antibody, anti-NTB-A antibody or anti-NKp46 (natural cytotoxicity receptor 1 ( NCR1)) antibody, (iii) one or more cytokines thereby activating or proliferating NK cells, or (iv) one or more chemical moieties thereby activating or proliferating NK cells, optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs). The method of claim 4, further comprising one or both of the following steps: introducing a polynucleotide encoding a CAR or another therapeutic protein or polypeptide optionally selected from an antibody or fragment thereof, enzyme, ligand or receptor for expression into the cultured cell population, wherein the CAR specifically recognizes and Binds to the tumor-associated antigen (TAA) or viral antigen; or Such as after the introducing step of claim 4, the cell population is cultured with an immune cell activator, wherein the immune cell activator is selected from one or more of the following (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies or antigen-binding fragments thereof that specifically recognize and bind to stimulatory receptors on one or more of NK cells, progenitor cells or stem cells, thereby activating or proliferating NK cells, (iii) one or more cytokines thereby activating or proliferating NK cells, or (iv) one or more chemical moieties thereby activating or proliferating NK cells, optionally selected from mTOR inhibitors, PI3K inhibitors or STING activating cyclic dinucleotides (CDNs), Where appropriate, the culturing step is repeated one, two, three or more times with the same or different immune cell activating agents or a combination thereof. The method of claim 4 or 5, wherein the aAPCs further express one or more of the following: 4-1BBL, membrane bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86 , OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, fusion protein with IL-15RαSushi-Fc Complexed mb IL-15-N72D superagonists (IL-15SA/IL-15RαSu-Fc; ALT-803) or cell surface markers that mediate CD122/CD132 signaling, as appropriate where the aAPCs further express mb IL -21 and 4-1BBL, optionally wherein the aAPCs are engineered K562 cells, optionally wherein the aAPCs are irradiated, thereby lacking cell proliferation or lacking long-term survival, and optionally wherein at 50 Gy, 100 Gy, 150 Gy The aAPCs are irradiated at Gy or 200 Gy, and where appropriate at about 10:1, about 5:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3 , a cell number ratio of about 1:5 or about 1:10 to culture the aAPC with the cell population. The method of claim 4 or 5, wherein the cytokines are selected from the group consisting of B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, low Toxic IL-2, IL-2 mutant lacking CD25 binding, IL-7, IL-15-N72D superagonist complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT- 803 soluble), IL-15, IL-18, IL-21, LEC, OX40L, ICOSL (B7H2, B7RP1), or MICA, as appropriate where the cell population was mixed with 100-500 IU/ml IL-2, 20 ng/ Either, or both, or all three of ml IL-15 or 25 ng/ml IL-21, as appropriate, were incubated with the cell population with 50 IU/ml IL-2 and 0.5 ng/ml IL- Either or both of 15 were cultured together, as appropriate where the cell population was cultured with 50 IU/ml IL-2. The method of claim 5, wherein the activators cultured with the cell population before and after the introducing step are the same, or wherein the activators cultured with the cell population before and after the introducing step are different from each other . The method of claim 5, comprising introducing a pseudotyped gamma retroviral particle into the cultured cell population, thereby introducing the CAR-encoding polynucleotide into the cultured cell, wherein the particle comprises flanking two The CAR-encoding polynucleotide of long terminal repeat (LTR) or its reverse complement, RD114TR and BaEVTR, optionally wherein the pseudotyped gamma retroviral particle comprises a vector genome comprising 5' LTR, 5' A cap, the CAR-encoding polynucleotide or its reverse complement, a 3' poly-A tail, and a 3' LTR, optionally wherein the pseudotyped gamma retroviral particle further comprises any one of reverse transcriptase and integrase, or Both, where the pseudotyped gamma retroviral particle is selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried Murine Embryonic Stem Cell Virus (FMEV) ), heterophilic MuLB-related virus, feline sarcoid virus, heterophilic murine leukemia virus-related virus (XMRV), and feline leukemia virus, as the case may be, wherein about 0.1, about 0.2, about 0.5, about 1, about 2, about 3. A multiplicity of infection (MOI) of about 4, about 5, about 6, about 7, about 8, about 9, or about 10 to introduce the pseudotyped gamma retroviral particle into the cultured cell population. The method of claim 5, wherein the cell population is cultured prior to the introducing step for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days or at least about 10 days, optionally wherein the cell population is cultured prior to the introducing step for no more than 7 days, no more than 8 days, no more than 9 days, no more than 10 days, no more than 11 days, no more than 12 days , no more than 13 days, no more than 14 days, no more than 15 days, no more than 3 weeks, or no more than 1 month, as the case may be, wherein the cell population is cultured for about 5 days, about 6 days, about 7 days prior to the introducing step days, about 8 days, about 9 days, or about 10 days. The method of claim 5, wherein the CAR-encoding polynucleotide is introduced into the population of cells via transduction of a viral vector comprising the CAR-encoding polynucleotide or its reverse complement in the presence of RetroNectin, optionally wherein the RetroNectin Coated on the inner surface of the container in which the cell population is transduced, and optionally wherein the cell population expresses either or both of integrin α4β1 (VLA-4) and integrin α5β1 (VLA-5) . The method of claim 4 or 5, further comprising enriching the cell population for cells expressing any one or more of CD56, CD25, CD122, CD212, CD215, CD218, CD360, TGF-βR or IL-10R , and optionally enriched for either or both of CD56 ^dim cells and CD56 ^bright cells. The method of claim 4 or 5, wherein any cell population comprises natural killer (NK) cells or a substantially purified composition thereof, optionally wherein the depleted cell population comprises one or more of the following: NK Substantially purified populations of cells, progenitor cells, HSCs, iPSCs, or each thereof, where the NK cells include cells derived from one or more of the following: progenitor cells, embryonic stem cells, embryonic stem cell-derived cells, embryonic germ cells, embryonic germ cell-derived cells, stem cells, stem cell-derived cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or immortalized cells, as the case may be wherein such progenitors One or more of the cells, HSCs or iPSCs are capable of deriving NK cells, optionally any of the cell populations are substantially free of T cells, optionally any of the cell populations are substantially free of T regulatory cells, optionally any of the cell populations Isolated, enriched or purified. The method of claim 4 or 5, further comprising either or both of the following steps: formulating the CAR-expressing population into a composition; or cryopreserving the CAR-expressing population. A use of a population of CAR-expressing cells prepared by the method of any one of claims 5 to 14 for the manufacture of a medicament for inhibiting the growth of cancer cells, wherein the antigen recognized by the CAR is derived from the cancer Cell-expressed TAA. An in vitro method of inhibiting the growth of cancer cells, comprising contacting a population of CAR-expressing cells prepared by the method of any one of claims 5 to 14 with the cancer cells, wherein the antigen recognized by the CAR is produced by the CAR TAA expressed by cancer cells. A use of a population of CAR-expressing immune cells prepared by the method of any one of claims 5 to 14 for the manufacture of a medicament for the treatment of cancer, wherein the antigen recognized by the CAR is derived from the cancer TAA expressed by the cells. A NK cell, or a population thereof, produced or prepared by the method of any one of claims 4 to 14. The cell or population of claim 18, comprising less than 0.5% aAPC, less than 0.1% or less than 0.2% or less than 0.3%, less than 0.4% or less than 0.5% aAPC as appropriate. A composition comprising the cells of claim 18 or 19 or a population thereof and a carrier, optionally a pharmaceutically acceptable carrier. The composition of claim 20, comprising less than 0.5% aAPC, less than 0.1% or less than 0.2% or less than 0.3%, less than 0.4% or less than 0.5% aAPC as appropriate. An engineered aAPC expressing an antigen and one or more of the following cell surface markers: 4-1BBL, membrane bound (mb) IL-15, mb IL-21, CD64, CD80, CD83, CD86, OX40L, ICOSL (B7H2, B7RP1), MICA, CD 40L, CD137L, mb IL-2, mb IL-18, mbIL-12, mb IL-2 mutant lacking CD25 binding, mb IL complexed with IL-15RαSushi-Fc fusion protein - 15-N72D superagonists (IL-15SA/IL-15RαSu-Fc; ALT-803) or cell surface markers that mediate CD122/CD132 signaling, as appropriate wherein the aAPCs are engineered K562 cells, as appropriate wherein the aAPCs are irradiated, thereby lacking cell proliferation or long-term survival, or both, and wherein the aAPCs are irradiated at 50 Gy, 100 Gy, 150 Gy, or 200 Gy, as the case may be, wherein the aAPCs are substantially will not survive for more than 14 days. A kit comprising one or more agents suitable for use in the method of any one of claims 4 to 14 and optional instructions, optionally wherein the agents are selected from one or more of the following: Polynucleotides encoding CAR or another therapeutic protein, vectors including polynucleotides, antibodies for detecting cell phenotypes, antibodies for isolating or enriching or purifying immune cells, primers for detecting polynucleotides , interleukin and aAPC. A method of preparing a population of γδ T cells, comprising culturing a population of cells comprising one or more of the following with an immune cell activator (eg, a γδ T cell activator): γδ T cells, capable of deriving γδ T cells Progenitor cells of cells or stem cells capable of deriving γδ T cells, wherein the cell population is depleted in the cell population of cells expressing one or more of the following: T cell receptor (TCR) alpha chain, TCR beta chain or αβTCR, and wherein the activator is selected from one or more of the following (i) to (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of γδ T cells, progenitor cells or stem cells, thereby activating or proliferating γδ T cells, (iii) one or more cytokines, thereby activating or proliferating γδ T cells, or One or more chemical moieties, optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs), thereby activate or proliferate γδ T cells. The method of claim 24, further comprising introducing a polynucleotide encoding a CAR or another therapeutic protein optionally selected from an antibody or fragment thereof, enzyme, ligand or receptor into the cultured cell population for expression , wherein the CAR specifically recognizes and binds to the tumor-associated antigen (TAA) or viral antigen. The method of claim 25, further comprising culturing the cell population with an immune cell activator (eg, a γδ T cell activator) after the introducing step of claim 25, wherein the activator is selected from the following (i) To one or more of (iv): (i) artificial antigen presenting cells (aAPCs) that express tumor-associated antigens (TAAs) or viral antigens and as appropriate activate and/or stimulate immune cell growth, (ii) one or more antibodies, or antigen-binding fragments thereof, that specifically recognize and bind to stimulatory receptors on one or more of γδ T cells, progenitor cells or stem cells, thereby activating or proliferating γδ T cells, (iii) one or more cytokines, thereby activating or proliferating γδ T cells, or (iv) one or more chemical moieties, thereby activating or proliferating γδ T cells, optionally selected from mTOR inhibitors, PI3K inhibitors, or STING activating cyclic dinucleotides (CDNs); Where appropriate, the incubation step is repeated one, two, three or more times with the same or different activating agents. A viral packaging system for the production of pseudotyped gamma retroviral particles, comprising: (a) a plastid expressing a vector gene body; (b) a packaging plastid; and (c) one or more sets expressing RD114TR and BaEVTR Membranoplasts, optionally wherein the vector genome comprises one or more of the following flanked by two long terminal repeats (LTRs): (A) a polynucleotide encoding a chimeric antigen receptor (CAR) or another therapeutic protein, optionally selected from antibodies or fragments thereof, enzymes, ligands or receptors, (B) the reverse complement of (A), or (C) a polynucleotide comprising one or more recognition sites recognized by restriction enzymes suitable for insertion of a CAR coding sequence or its reverse complement into the polynucleotide, Optionally wherein the vector genome further comprises one or more of the following: 5' LTR, 5' cap, 3' poly-A tail and 3' LTR, Optionally wherein the pseudotyped gamma retroparticle is selected from any of the following species: Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), Fried Murine Embryonic Stem Cell Virus (FMEV), Heterophile MuLB-related virus, feline sarcoma virus, heterophilic murine leukemia virus-related virus (XMRV) and feline leukemia virus. The viral packaging system of claim 27, further comprising a packaging cell line, optionally wherein the packaging cell line is the 293T cell line. A method of producing pseudotyped gamma retroviral particles, comprising using the system of claim 27 or 28 to transduce a packaging cell line under conditions suitable for packaging the pseudotyped gamma retroviral particles, optionally wherein the packaging cells Line 293T cell line, optionally wherein the cell line is transduced with plastids of (a), (b) and (c) at a ratio of 1.5:1.5:1, optionally wherein the packaging system includes at least two Mantle plastids, one expressing RD114TR and the other expressing BaEVTR, additionally where the cell line uses (a) plastids, (b) plastids, RD114TR expressing plastids at a ratio of 1.5:1.5:1:1 and BaEVTR expressing plastids for transduction. A method of producing retroviral particles comprising (i) introducing a vector expressing the vector genome, optionally a plasmid, into a first packaging cell line suitable for packaging the vector genome into a first retroviral particle, (ii) transducing the first retroviral particle into a second packaging cell line suitable for replicating the first retroviral particle; and (iii) isolating the replicated retroviral particle. The method of claim 30, wherein the second packaging cell line includes a retroviral envelope protein in the cell membrane but does not include an entry receptor for the retroviral envelope protein in the cell membrane, optionally wherein the retroviral envelope protein The viral envelope protein is baboon envelope glycoprotein (BaEV) and its entry receptor system alanine/serine/cysteine/threonine transporter 1 (ASCT1) or sodium-dependent neutral amino acid transporter Type 2 (ASCT2), optionally wherein the retrovirus envelope protein is the envelope glycoprotein of the RD114 retrovirus (RD114), and it enters the acceptor system ASCT2, optionally wherein the retrovirus envelope protein is gibbon leukemia The viral envelope glycoprotein (GALV) and its entry into the receptor is the sodium-dependent phosphate transporter (Pit1). A retroviral particle produced by the method of claim 30 or 31. A method of producing engineered immune cells comprising transducing a retroviral particle as claimed in claim 32 into immune cells or precursor cells thereof.

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