Cell selection methods and related compositions

Abstract

Methods are provided for selecting cells that contain two or more separate expression constructs. In certain embodiments, the methods include contacting a cell population with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the cell population. The two or more separate expression constructs include a first expression construct that encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The second expression construct encodes a protein required for cell surface expression of the selection marker. Such methods further include selecting for cells that exhibit cell surface expression of the selection marker. Related cells, compositions, kits, and methods of treatment are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 171,841, filed April 7, 2021, which is incorporated by reference herein in its entirety.

[0002] Incorporation by reference of sequence listing provided as a text file A sequence listing is provided herewith as a text file (STAN-1819WO_SEQ_LIST_ST25) having a file size of 420,000 bytes, created on April 5, 2022. The contents of the text file are incorporated herein by reference in their entirety.

[0003] Introduction Currently, there is a lack of techniques that can be used to purify cells engineered with multiple genetic modifications. Current limitations in payload capacity necessitate the use of multiple expression constructs to deliver transgenes. Sequential sorting against multiple surface markers is expensive, time-consuming, and results in large amounts of cell loss. These purification issues impose limitations on the ability to engineer cells, for example, for cell-based therapies. Thus, improved approaches to purify cells engineered with multiple genetic modifications are needed. Summary of the Invention

[0004] Methods are provided for selecting cells that contain two or more separate expression constructs. In certain embodiments, the methods include contacting a cell population with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the cell population. The two or more separate expression constructs include a first expression construct that encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The second expression construct encodes a protein required for cell surface expression of the selection marker. Such methods further include selecting for cells that exhibit cell surface expression of the selection marker. Related cells, compositions, kits, and methods of treatment are also provided. [Brief description of the drawings]

[0005] [Figure 1] Figure 1A: Schematic of a bilateral cell selection system according to some embodiments of the present disclosure. The selection system of the present disclosure may be referred to herein as a "STASH selection" system because the selection marker is "stashed" in the cell if the expression constructs present in the cell are not the desired combination. Figure 1B: Schematic of two separate expression constructs (or "expression constructs" as used interchangeably herein) encoding the components of the selection system shown in Figure 1A, which are required for cell surface expression of the selection marker. [Diagram 2] Figure 2A: Schematic of AND gate logic that can be implemented using the selection system of the present disclosure. Cells that meet the two input requirements (expression of construct A and expression of construct B) result in output surface expression of a selection marker. Figure 2B: Schematic of four possible outcomes of cells exposed to construct A and construct B. Cells that express only construct A have the selection marker retained within the cell. Cells that express only construct B have the protease retained within the cell. Cells that express both construct A and construct B have the selection marker expressed on the surface of the cell that can be used for enrichment and detection. [Diagram 3]Figure 3A: Schematic of separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figure 3B: Flow cytometry data demonstrating high cell surface expression of the selection marker only in the presence of both expression constructs. [Figure 4] Figure 4A: Schematic of separate expression constructs of a cell selection system according to some embodiments of the present disclosure. Figure 4B: Flow cytometry data demonstrating high cell surface expression of the selection marker only in the presence of both expression constructs. [Diagram 5] Figure 5A: Schematic of a tripartite cell selection system, according to some embodiments of the present disclosure. Figure 5B: Schematic of three separate expression constructs encoding components of the selection system shown in Figure 5A, which are required for cell surface expression of a selection marker. [Figure 6] Figure 6A: Schematic of three separate expression constructs of a tripartite cell selection system according to some embodiments of the present disclosure. Figure 6B: Flow cytometry data demonstrating high cell surface expression of the selection marker only in the presence of all three expression constructs. [Figure 7] Figure 7A: Schematic of a penta-way cell selection system according to some embodiments of the present disclosure. Figure 7B: Schematic of five separate expression constructs encoding components of the selection system shown in Figure 7A, which are required for cell surface expression of a selection marker. [Figure 8] Figure 8A: Schematic of five separate expression constructs of a penta-way cell selection system according to some embodiments of the present disclosure. Figure 8B: Flow cytometry data demonstrating high cell surface expression of the selection marker in cells positive for all five expression constructs. [Figure 9]FIG. 9A: Schematic of a bilateral cell selection system, according to some embodiments of the present disclosure. In this example, a truncated receptor (here, truncated EGFR or "EGFRt") serves as the selection marker. FIG. 9B (adapted from Labaniieh et al. (2018) Nature Biomedical Engineering vol. 2:377-391): Schematic of a truncated receptor serving as a suicide switch. In this example, cells containing both expression constructs express the truncated receptor on their surface. Cells administered to an individual for therapeutic purposes can then be removed, if desired, by administration of an antibody specific for the truncated receptor. In the particular example shown in FIG. 9, the suicide switch is truncated EGFR (EGFRt) and the cells can be removed by administration of an anti-EGFR antibody, such as cetuximab. [Figure 10] Figure 10A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figures 10B-10C: Flow cytometry data assessing surface expression of a selection marker (here, EGFRt) when using a specific ER localization tag. [Figure 11] Figure 11A: Schematic of a bilateral cell selection system according to some embodiments of the present disclosure. In this example, a truncated receptor (here, truncated EGFR or "EGFRt") serves as a selection marker. Figure 11B: Sequences of fusion proteins including a hinge domain, a transmembrane domain, various ER localization tags, and a protease cleavage site located between the transmembrane domain and the particular ER localization tag. [Figure 12] Figure 12A: Schematic of a bilateral cell selection system according to some embodiments of the present disclosure. In this example, a truncated receptor (here, truncated EGFR or "EGFRt") serves as a selection marker. Figure 12B: Sequences of fusion proteins including transmembrane domains, intracellular domains (ICDs) of various ER resident membrane proteins, and a protease cleavage site located between the transmembrane domain and the particular intracellular domain. In Figure 12B, each ER resident protein is a human ER resident protein, except for those in constructs 506 and 507. [Figure 13]Figure 13A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figures 13B-E: Flow cytometry data showing the identification of constructs that showed high performance among the various ER localization tags used. [Figure 14] FIG. 14A: An example workflow for selecting (or "purifying") cells exhibiting cell surface expression of a selection marker, according to some embodiments of the present disclosure. In this example, selection based on magnetic activated cell sorting (MACS) is used. In this particular example, the selection marker is EGFRt. FIG. 14B: A plot of the percentage of double positive (BFP+GFP+) cells from the purified cell fraction after MACS-based selection. This data demonstrates that multiple EGFRt-STASH ER localization tag variants can be used to isolate a highly pure double positive population using a single selection marker. [Figure 15] Flow cytometry data demonstrating highly pure double positive cell populations for multiple ER localization tag variants after EGFR MACS-based selection. [Figure 16] Figure 16A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figure 16B: Flow cytometry data showing a highly pure double positive cell population for a specific ER localization tag variant after EGFR MACS-based selection. [Figure 17] Figure 17A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figure 17B: Flow cytometry data showing a highly pure double positive cell population for a specific ER localization tag variant after EGFR MACS-based selection. [Figure 18] Figure 18A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figure 18B: Flow cytometry data showing a highly pure double positive cell population for a specific ER localization tag variant after EGFR MACS-based selection. [Figure 19] Figure 19A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figure 19B: Flow cytometry data showing a highly pure double positive cell population for a specific ER localization tag variant after EGFR MACS-based selection. [Figure 20] Figure 20A: Schematic of a tripartite cell selection system, according to some embodiments of the present disclosure. Figure 20B: Schematic of three separate expression constructs encoding the components of the selection system shown in Figure 20A, which are required for cell surface expression of a selection marker. Figures 20C-D: Flow cytometry data demonstrating a highly pure population of trispecific cells (here trispecific CAR-T cells) transduced with three separate expression constructs. [Figure 21] 21A: Schematic of a bilateral cell selection system, according to some embodiments of the present disclosure. FIG. 21B: Schematic of a bilateral cell selection system, according to some embodiments of the present disclosure. [Figure 22] Figure 22A: Schematic of two separate expression constructs of a bilateral cell selection system according to some embodiments of the present disclosure. Figures 22B-C: Flow cytometry data demonstrating selection of double positive cells using selection markers. [Diagram 23] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Figure 24] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 25] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Figure 26] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Figure 27] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Figure 28] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Figure 29] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 30] 23-30 are schematic diagrams of 3-way, 4-way, 5-way, 6-way, 7-way, 8-way, 9-way, and 10-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 31] 31-33 are schematic diagrams of 5-way, 9-way, and 13-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 32] 31-33 are schematic diagrams of 5-way, 9-way, and 13-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 33] 31-33 are schematic diagrams of 5-way, 9-way, and 13-way cell selection systems, respectively, according to some embodiments of the present disclosure. [Diagram 34] Flow cytometry histograms of surface CD34 staining using QBEnd / 10 antibody on primary human T cells. As can be seen in the data, only double positive cells show high surface expression of the CD34 epitope. [Diagram 35] Flow cytometry histograms showing surface EGFR expression of primary human T cells transduced with EGFRt-STASH variants and TEV protease carrying the CISD2 ER retention tag. [Diagram 36]Flow cytometry histograms showing surface EGFRt expression on primary human T cells transduced in a three-way STASH selection system with EGFRt-STASH variants carrying the CD8α or CD28 Tm domain and the CISD2 ER retention signal. [Figure 37] Flow cytometry histograms showing surface EGFR expression on primary human T cells transduced in a three-way STASH selection system using EGFRt-STASH variants carrying the CD8α or CD28 Tm domain and the IBV S protein retention signal. [Figure 38] Flow cytometry histograms showing surface EGFR expression in primary human T cells transduced in a three-way STASH selection system using EGFRt-STASH variants carrying a degron fused to the CD8α or CD28 Tm domain and the adenoviral E3-19K retention signal. [Figure 39] Figure 39A: Schematic of three separate expression constructs of a tripartite cell selection system, according to some embodiments of the present disclosure. Figure 39B: Flow plot histograms showing surface expression of EGFR, cJun, CD19.BBz, and HER2.BBz CARs. [Diagram 40] Figure 40A: A series of flow plots of primary human T cells demonstrating BFP and GFP expression after staining with anti-EGFR-biotin at the dilutions indicated on the flow plot and MACS selection diagram. In this example, the variant 497ER retained tag was used. Figure 40B: Bar graph showing the yield of double positive cells after MACS selection of the samples shown in Figure 40A. [Diagram 41] Figure 41A: A series of flow plots of primary human T cells demonstrating BFP and GFP expression after staining with anti-EGFR-biotin at the dilution factors indicated on the flow plot and MACS selection figures. In this example, the variant 501ER retaining tag was used. Figure 41B: Bar graph showing the yield of double positive cells after MACS selection of the samples shown in Figure 41A. [Diagram 42]A series of flow plots demonstrating BFP and GFP expression in primary human T cells. [Diagram 43] A series of flow plots demonstrating surface EGFR expression on primary human T cells. [Diagram 44] A series of flow plots demonstrating surface EGFR expression in primary human T cells transduced with EGFR STASH variants and minimized TEV protease constructs as indicated on each flow plot. [Diagram 45] Data demonstrating the identification of human proteases that find use in the STASH selection system. Figure 45D shows the amino acid sequences of the protease cleavage sites used (#834-#837, SEQ ID NOs: 132-135, respectively; #839-#840, SEQ ID NOs: 136-137, respectively). [Figure 46] Schematic and data demonstrating two-way STASH selection using a combination of CRISPR knock-in and retroviral gene delivery methods. [Figure 47] A series of flow plots demonstrating surface EGFR expression on primary human T cells in a bilateral STASH selection system with various truncated EGFRs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Before describing the methods and compositions of the present disclosure in more detail, it should be understood that the methods and compositions are not limited to the specific embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the methods and compositions is limited only by the scope of the appended claims.

[0007] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed in the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

[0008] Certain ranges are presented herein with numerical values ​​preceded by the term "about." The term "about," when it precedes it, is used to provide literal support for the number itself, as well as a number that is close to or approximately the number. In determining whether a number is close to or approximately a specifically stated number, the close or approximate, but unstated number may be a number that provides a substantial equivalent to the specifically stated number in the context in which it is presented.

[0009] 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 the method and composition belongs. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative exemplary methods and compositions are described herein.

[0010] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the materials and / or methods for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present methods and composition kits are not entitled to antedate such publications, since the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0011] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0012] It is understood that, for clarity, certain features of the methods and compositions described in the context of another embodiment may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the methods and compositions described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically embraced by the present disclosure and, to the extent that such combinations encompass processes and / or compositions capable of being performed, are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations recited in the embodiments describing such variations are also specifically embraced by the present methods and compositions and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0013] As will be apparent to one of ordinary skill in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0014] Cell Selection Method Aspects of the present disclosure include a method for selecting cells containing two or more distinct expression constructs. The method is used in a variety of applications, including both research and clinical applications. By way of example, the method is used in any application in which it is desirable to efficiently manipulate and select cells containing multiple genetic modifications (e.g., transgenic modifications and / or gene knockouts, etc.), where such genetic modifications are difficult or not feasible using a single expression construct, for example due to limitations in expression construct payload capacity. The method of the present disclosure allows for the selection of cells containing multiple desired genetic modifications using a single selection marker. That is, cell surface expression of a single selection marker is a readout for cells containing each of the multiple desired genetic modifications, thereby avoiding the need for sequential sorting with multiple surface markers to obtain cells containing multiple modifications. Cells containing multiple desired genetic modifications can be easily selected (sometimes referred to herein as "purified" or "enriched") based on a single selection marker using existing reagents and systems for magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), and the like.

[0015] According to some embodiments, multiple genetic modifications find use in the context of cell-based therapy, such that the methods of the present disclosure find use in the production and selection of cells for such therapy. Non-limiting examples of genetic modifications used in cell-based therapy include transgenic modifications that express recombinant receptors (e.g., chimeric antigen receptors (CARs) and T cell receptors (TCRs) and the like) for targeting undesirable cells (e.g., cancer cells) when administered to an individual, transgenic and / or knockout modifications that reduce the immunogenicity of the engineered cells when administered to an individual, transgenic and / or knockout modifications that render the cells resistant to cell exhaustion when administered to an individual, and / or transgenic and / or knockout modifications that enhance the efficacy of the cells in the tumor microenvironment (TME) for the treatment of solid tumors. Any desired combination of such modifications can be made and selected according to the methods of the present disclosure.

[0016] The selection approach of the present disclosure may be referred to herein as a "STASH selection system," "STASH selection," or the like, because in the absence of the desired combination of expression constructs present in the cell, the selection marker is "stashed" in the cell. According to the selection system, one of the expression constructs encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. In the absence of one or more additional expression constructs that provide a protease capable of cleaving the protease cleavage site, the selection marker remains localized (i.e., retained or stored) in a subcellular location (e.g., an organelle) determined by the particular protein localization tag used. When one or more additional expression constructs are present in the cell, thereby providing a protease capable of cleaving the protease cleavage site, the selection marker is cleaved from the protein localization tag and transported to the surface of the cell, whereby cells containing the desired multiple genetic modifications exhibit cell surface expression of the selection marker. The selection system of the present disclosure is modular and includes configurations where delivery of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more separate expression constructs (each of which may provide the desired genetic modification, e.g., a transgene, a targeted gene knockout, etc.) to a cell is required to provide the protease activity necessary for cell surface expression of a selection marker.

[0017] FIG. 1A shows an example of a cell selection system in which delivery of two expression constructs to a cell is required for cell surface expression of a selection marker (a "two-way" system). In this particular example, an epitope-based selection marker (such as EGFRt, CD34, Myc tag, etc.) is fused to a protease cleavage site and a subcellular localization (or "retention") tag, such as an endoplasmic reticulum (ER) localization tag. A co-expressed protease localized within the cell recognizes the protease cleavage site and cleaves the fusion protein at the protease cleavage site. This cleavage event liberates the selection marker from the retention tag, allowing it to move to the surface of the cell. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing both the selection marker and the protease. Two expression constructs encoding the components of the two-way selection system shown in FIG. 1A are shown diagrammatically in FIG. 1B. In this example, each of the two expression constructs encodes a protein of interest (protein A and protein B from construct A and construct B, respectively), such that cell surface expression of the selection marker is a marker for cells expressing proteins of interest A and B, and such cells can be selected (enriched, purified) using a single selection marker. As shown in FIG. 1B, a ribosome skipping site (in this example, P2A from porcine teschovirus) can be provided to allow bicistronic expression of the protein of interest and the selection system components. That is, the ribosome skipping site allows the protein of interest and the selection system components to be expressed as separate proteins.

[0018] Figure 2A shows a schematic of the AND gate logic that can be implemented using the STASH selection system. Cells that meet the two input requirements (expression of construct A and expression of construct B) result in an output surface expression of a selection marker. Figure 2B shows a schematic of four possible outcomes of cells exposed to construct A and construct B. Cells that express only construct A have the selection marker retained within the cell. Cells that express only construct B have the protease retained within the cell. Cells that express both construct A and construct B have the selection marker expressed on the surface of the cell that can be used for detection and enrichment.

[0019] In certain embodiments, the disclosed method includes contacting a cell population with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the cell population. The two or more separate expression constructs include a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The two or more separate expression constructs further include a second expression construct encoding a protein required for cell surface expression of the selection marker. The method further includes selecting for cells exhibiting cell surface expression of the selection marker.

[0020] The contacting step may include contacting the cell population with the two or more separate expression constructs simultaneously, for example, by combining the cells with each of the two or more separate expression constructs in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of each of the two or more separate expression constructs to the cells of the cell population. The contacting step may include contacting the cell population with the two or more separate expression constructs sequentially, for example, where the cell population is first combined with less than each of the two or more separate expression constructs under expression construct delivery conditions, followed by combining the cell population with the remaining two or more separate expression constructs in one or more further combination steps under suitable conditions.

[0021] Various suitable approaches and conditions for delivering expression constructs to cells are known. According to some embodiments, two or more separate expression constructs are delivered to cells of the cell population by microinjection, transfection, lipofection, heat shock, electroporation, transduction, and / or gene gun, DEAE-dextran mediated transfer, etc. In certain embodiments, two or more separate expression constructs are introduced into cells of the cell population by AAV transduction. The AAV vector may include ITRs from AAV2 and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. According to some embodiments, the AAV vector includes ITRs from AAV2 and a serotype from AAV6. In certain embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is introduced into cells (e.g., T cells) by lentiviral or retroviral transduction. Lentiviral vector backbones can be derived from HIV-1, HIV-2, Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), or Simian Immunodeficiency Virus (SIV). Lentiviral vectors can be integration-competent or integrase-deficient lentiviral vectors (TDLV). In one embodiment, an IDLV vector containing an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used. Non-limiting examples of approaches for the preparation of retroviral expression constructs and the transduction of cells with such constructs are provided in the experimental section below.

[0022] As used herein, an "expression construct" is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) that includes a region encoding a component of a cell selection system (e.g., a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site; and / or a protein required for cell surface expression of the selection marker) operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the cell selection system component is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoters, enhancers, etc., present in the expression construct and operably linked to the region encoding the cell selection system component prior to contacting the cell population. In some embodiments, expression of the cell selection system component may be controlled by one or more endogenous regulatory elements, e.g., promoters, enhancers, etc., at or near the genomic locus where the expression construct is inserted.

[0023] One or more of the two or more separate expression constructs may further comprise one or more regions encoding one or more proteins of interest (e.g., any of the proteins of interest described elsewhere herein), each operably linked to a suitable promoter, where the promoter may be a single shared promoter between each of the protein coding regions of the expression constructs (including the cell selection system components), or at least one of the protein coding regions may be operably linked to a promoter that is not shared with any other protein coding region of the expression construct. In certain embodiments, when an expression construct comprises one or more protein coding regions in addition to a region encoding a component of the cell selection system, the expression construct may be configured to allow polycistronic expression of two or more (e.g., each) protein coding regions. That is, two or more (e.g., each) of the proteins encoded by the expression construct may be expressed as separate proteins from the same promoter. In certain embodiments, the expression construct comprises a ribosome skip site to allow polycistronic expression of two or more (e.g., each) protein coding regions. A non-limiting example of a suitable ribosomal skipping site that can be incorporated into the expression construct is the P2A ribosomal skipping site from the porcine teschovirus.

[0024] An expression construct (e.g., a vector) may be suitable for replication and integration in prokaryotes, eukaryotes, or both. An expression construct may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of nucleic acids encoding components of a cell selection system. An expression construct optionally contains a generic expression cassette that contains at least one independent terminator sequence, a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, such as those found in shuttle vectors, and a selection marker for both prokaryotic and eukaryotic systems.

[0025] To obtain high levels of expression of a cloned nucleic acid, it is common to construct an expression construct that typically contains a strong promoter to direct transcription, a ribosome binding site for translation initiation, and a transcription / translation terminator, each in a functional orientation relative to each other and to the protein coding sequence. Examples of regulatory regions suitable for this purpose in E. coli include the promoter and operator regions of the E. coli tryptophan biosynthetic pathway, phage lambda (P L ), the left promoter of ribosomal RNA, and the L-arabinose (araBAD) operon. It is also useful to include a selection marker in the DNA vector transformed in E. coli. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the selection system components are available, for example, with E. coli, Bacillus, and Salmonella. E. coli systems can also be used. Nucleic acid encoding the selection system components. Transducing a cell with a nucleic acid can include, for example, incubating lipid microparticles containing the nucleic acid with the cell, or incubating a viral vector containing the nucleic acid with a cell within the host range of the vector.

[0026] The two or more expression constructs are "distinct," meaning that none of the two or more expression constructs are part of the same polynucleotide molecule.

[0027] In certain embodiments, when two or more separate expression constructs are delivered to cells of a cell population, one or more of the expression constructs is episomal (e.g., extrachromosomal), where "episome" or "episomal" refers to a polynucleotide that replicates independently of the chromosomal DNA of the cell. A non-limiting example of an episome that can be used in the present methods is a plasmid.

[0028] According to some embodiments, when delivering two or more separate expression constructs to cells of a cell population, one or more expression constructs are integrated into the genome of the cell. In certain embodiments, one or more expression constructs are adapted for site-specific integration into the genome. For example, the expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a target gene in the genome of the cell. As an example, the site-specific integration may knock out a target gene by knocking in the expression construct. Any suitable approach for site-specific gene editing and functional integration may be used. The functional integration of the expression construct may be achieved by various means, including by using an integration vector, including viral vectors and non-viral vectors. In some examples, a retroviral vector, such as a lentiviral vector, may be used. In some examples, a non-retroviral integration vector may be used. The integration vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells to promote functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0029] Strategies for site-specific integration that find use in the methods of the present disclosure include those using homologous recombination and / or nonhomologous end-joining (NHEJ), etc. Such strategies may use non-naturally occurring or engineered nucleases, including but not limited to zinc-ringer nucleases (ZNFs), meganucleases, transcription activator-like effector nucleases (TALENs), or CRISPR-Cas systems. Eukaryotic cells utilize two different DNA repair mechanisms in response to DNA double strand breaks (DSBs): homologous recombination (HR) and nonhomologous end joining (NHEJ). Mechanistically, HR is an error-free DNA repair mechanism since it requires a homologous template to repair the damaged DNA strand. Due to its homology-based mechanism, HR has been used as a tool to site-specifically engineer genomes. Gene targeting by HR requires the use of two homologous arms flanking the transgene / target site of interest. HR efficiency can be increased by introducing DSBs at the target site using specific rare-cutting endonucleases. The discovery of this phenomenon prompted the development of methods to create site-specific DSBs in the genomes of different species. Over the past decade, various chimeric enzymes have been designed for this purpose, namely ZFNs, meganucleases, and TALENs. ZFNs are modular chimeric proteins that contain a ZF-based DNA binding domain (DBD) and a Fokl nuclease domain. The DBD is usually composed of three ZF domains, each with three base pair specificity; the Fokl nuclease domain provides the DNA nicking activity that is targeted by two adjacent ZFNs. Due to the modularity of the DBD, any site in the genome could be targeted.TALENs are similar to ZFNs, except that the DBD is derived from a TAL effector nuclease (TALE). The TALE DBD is modular and composed of 34-residue repeats, whose DNA specificity is determined by the number and order of the repeats. Each repeat binds to a single nucleotide in the target sequence via only two residues.

[0030] The methods of the present disclosure may be performed on any cell population of interest. In certain embodiments, the cell population is a prokaryotic cell population (e.g., bacteria), a yeast cell population, an insect (e.g., Drosophila) cell population, an amphibian (e.g., frog, e.g., Xenopus) cell population, a plant cell population, and the like. According to some embodiments, the cell population is a mammalian cell population. Mammalian cells of interest include human cells, rodent cells, and the like. According to some embodiments, the cell population is a population of peripheral blood mononuclear cells (PBMCs). In certain embodiments, the cell population is an immune cell population. The immune cell population may include one or any combination of T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. When the immune cells include T cells, the T cells may be naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), gamma delta T cells (T γδ). According to some embodiments, the cell population is a cell population that includes stem cells, such as mammalian (e.g., human) stem cells. For example, the stem cells of the cell population may include embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), or any combination thereof.

[0031] Protein localization tags As used herein, the term "protein localization tag" refers to an amino acid sequence that directs the cellular localization of a fusion protein comprising a selection marker (and optionally any other cell selection system components expressed by two or more separate expression constructs) to a specific cellular compartment. In certain embodiments, the protein localization tag is selected from an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosomal localization tag, a plasma membrane localization tag, a mitochondrial localization tag, a peroxisomal localization tag, a cytoplasmic localization tag, and a nuclear localization tag.

[0032] The fusion protein comprising the selection marker (and optionally any other cell selection system component(s) expressed by the two or more separate expression constructs) may comprise any suitable protein localization tag to direct localization to a desired cellular compartment. In some embodiments, when two or more cell selection system components comprise protein localization tags, the protein localization tag of each component may direct each component to the same cellular compartment (e.g., organelle). For example, in certain embodiments, when two or more cell selection system components comprise protein localization tags, the protein localization tags are identical or substantially identical to one another.

[0033] Suitable protein localization tags are known. In certain embodiments, the cell selection system components include protein localization tags in LocSigDB (a database of protein localization signals / tags available at genome.unmc.edu / LocSigDB / and described in Negi et al. (2015) Database vol. 2015: pp. 1-7); DBSubLoc (a database of protein subcellular localization available at bioinfo.tsinghua.edu.cn / dbsubloc.html); LOCATE (a mammalian protein subcellular localization database available at locate.imb.uq.edu.au); LocDB (a protein localization database available at rostlab.org / services / locDB); eSLDB (a eukaryotic subcellular localization database available at gpcr.biocomp.unibo.it / esldb); and / or any other convenient database of protein localization tags. In some embodiments, the protein localization tag is located at the N-terminus of the cell selection system component, for example, type II membrane proteins (see, e.g., Schutz et al., 1994, EMBO J. 13(7):1696-1705) and other proteins have naturally occurring N-terminal protein localization tags.

[0034] According to some embodiments, the protein localization tag is an ER localization tag. In certain embodiments, the ER localization tag comprises the amino acid sequence KKMP. Non-limiting examples of ER localization tags that may be included in the cell selection system components disclosed herein are: LYKYKSRRSFIDEKKMP (SEQ ID NO: 1); AEKDEL (SEQ ID NO: 2); EQKLISEEDLKDEL (SEQ ID NO: 3); GGGGSGGGGSKDEL (SEQ ID NO: 4); GGGGSGGGGSGGGGGSGGGGSKDEL (SEQ ID NO: 5); GGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 6); KYKSRRSFIEEKKMP (SEQ ID NO: 7); LKYKSRRSFIEEKKMP (SEQ ID NO: 8); LYKYKSRRSFIEEKKMP (SEQ ID NO: 9); LYCKYKSRRSFIEEKKMP (SEQ ID NO: 10); LYCNKYKSRRSFIEEKKMP (SEQ ID NO: 11); LYCNKYKSRRSFIDEKKMP (SEQ ID NO: 12). ;LYEQKLISEEDLKYKSRRSFIEEKKMP (SEQ ID NO:13);LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO:14);LYKKLETFKKTN (SEQ ID NO:15);LYEQKLISEEDLKKLETFKKTN (SEQ ID NO:16);LYYQRL (SEQ ID NO:17);LYEQKLISEEDLYQRL (SEQ ID NO:18);LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO:19);LYRNIKCD (SEQ ID NO:20); and LYEQKLISEEDLRNIKCD (SEQ ID NO:21). Another example of an ER localization tag that may be included in a cell selection system component of the present disclosure is the following: PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCCCGCFGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO:31); NRSPRNRKPRRE (SEQ ID NO:32); LYKYKSRRSFIEEKKMP (SEQ ID NO:9); TKVLKGKKLSLPA (SEQ ID NO:33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and The ER localization tag comprises an amino acid sequence selected from, consisting of, or present within an ER localization tag having 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within an amino acid sequence selected from: KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35).

[0035] In certain embodiments, the protein localization tag is a Golgi localization tag. A non-limiting example of a Golgi localization tag that can be included in the cell selection system components of the present disclosure is a Golgi localization tag that comprises the amino acid sequence YQRL (SEQ ID NO: 36).

[0036] According to some embodiments, the protein localization tag is a lysosomal localization tag. A non-limiting example of a lysosomal localization tag that can be included in the cell selection system components of the present disclosure is the lysosomal localization tag comprising the amino acid sequence KFERQ (SEQ ID NO: 37).

[0037] Protease cleavage sites and proteases As described above, the first expression construct encodes a fusion protein that includes a protease cleavage site disposed between the selection marker and the protein localization tag. The term "cleavage site" refers to a bond (e.g., a cleavable bond) that is cleaved by an agent, such as a protease. A protease cleavage site includes a specific amino acid sequence that is recognized by a protease during proteolytic cleavage and may include surrounding amino acids (e.g., 1-6 amino acids) on either side of the cleavable bond that bind to the active site of the protease and are necessary for recognition as a substrate. In some embodiments, the cleavage site is provided as a cleavable linker, and "cleavable linker" refers to a linker that includes a protease cleavage site. A cleavable linker is typically cleavable under physiological conditions.

[0038] According to some embodiments, the protease cleavage site is a viral protease cleavage site. Non-limiting examples of viral protease cleavage sites include cleavage sites of Potyviridae proteases. Potyviridae proteases of interest include tobacco etch virus (TEV) protease, plum pox virus protease (PPVp), soybean mosaic virus protease (SbMVp), sunflower mild mosaic virus protease (SuMMVp), tobacco vein mottle virus protease (TVMVp), and West Nile virus protease (WNVp). In certain embodiments, the viral protease cleavage site is a TEV protease cleavage site. The amino acid sequence of an exemplary TEV protease cleavage site is ENLYFQS. The amino acid sequence of an exemplary TEV protease is as follows: GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN (SEQ ID NO: 38) In some embodiments, the protease is a TEV protease comprising an amino acid sequence as set forth above, or a functional (proteolytic) variant thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to such a sequence, and / or a functional (proteolytic) fragment thereof, such as a fragment having a length of 100-125, 125-150, 150-175, 175-200, 200-225, or 225-235 amino acids. Such a protease may be provided by two or more (e.g., two) complementary fragments of the protease, where the two or more (e.g., two) complementary fragments form an active protease complex.

[0039] According to some embodiments, the viral protease cleavage site is for HCV protease. In certain embodiments, the viral protease cleavage site is for viral protease derived from HCV nonstructural protein 3 (NS3). NS3 is composed of an N-terminal serine protease domain and a C-terminal helicase domain. "Derived from HCV NS3" means that the protease is the serine protease domain of HCV NS3 that can cleave the cleavage site of the serine protease domain of HCV NS3, or a proteolytically active variant thereof. The protease domain of NS3 forms a heterodimer with HCV nonstructural protein 4A (NS4A) and activates the proteolytic activity. The protease derived from HCV NS3 may include the entire NS3 protein or a proteolytically active fragment thereof, and may further include a cofactor polypeptide, such as a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A), for example, an activated NS4A region. NS3 protease is highly selective and can be inhibited by many non-toxic cell-permeable drugs that are currently available for humans.The NS3 protease inhibitors that can be used include, but are not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, and any combination thereof.The non-limiting examples of proteases derived from HCV NS3 are provided below.

[0040] Examples of proteases derived from HCV NS3 APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 39) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 40) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 41) In some embodiments, the protease comprises one of the sequences set forth above or is a functional (proteolytic) variant thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to one of such sequences, and / or a functional (proteolytic) fragment thereof, such as a fragment having a length of 100-185, 120-185, 140-185, 160-185, 170-185, 180-185, 182-185, or 184-185 amino acids.

[0041] In some embodiments, the protease cleavage site is a viral protease cleavage site. For example, when a protease derived from HCV NS3 is used, the cleavage site should include the NS3 protease cleavage site. The NS3 protease cleavage site may include the four junctions between the nonstructural (NS) proteins of the HCV polyprotein that are normally cleaved by the NS3 protease during HCV infection, including the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B junction cleavage sites. For a description of the NS3 protease and the representative sequences of its cleavage sites for various HCV strains, see, for example, Hepatitis C Viruses: Genomes and Molecular Biology (SLTan, ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; the disclosure of which is incorporated herein by reference in its entirety.

[0042] In some embodiments, the protease is derived from HCV NS3 and engineered to include one or more amino acid substitutions relative to the HCV NS3 protease amino acid sequence described above. For example, the protease can include a substitution at a position corresponding to position 54 of the amino acid sequence APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 39). In some embodiments, such a substitution is a threonine to alanine substitution.

[0043] The NS3 nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. Numerous NS3 nucleic acid and protein sequences are known and are described, for example, in USSN 15 / 737,712, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Additional representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. For example, NCBI entries: accession numbers YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99 041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, and JX171063, all of which sequences are incorporated herein by reference. Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof can be used.

[0044] The NS4A nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., the seven genotypes 1-7) or subtype. Numerous NS4A nucleic acid and protein sequences are known. Representative NS4A sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199. All of the sequences (registered as of the filing date of this application) are incorporated herein by reference. Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, can be used.

[0045] HCV polyprotein nucleic acid and protein sequences can be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. Numerous HCV polyprotein nucleic acid and protein sequences are known. Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. YP_001469631, NP_671491, YP_001469633, YP_001469630, YP_001469634, YP_001469632, NC_009824, NC_004102, NC_009825, NC_009827, NC_009823, NC_009826, and EF108306. All of the sequences (registered as of the filing date of this application) are incorporated herein by reference. Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, can be used.

[0046] In some embodiments, the protease is derived from HCV NS3 and the cleavage site comprises an NS3 protease cleavage site. The NS3 protease cleavage site may comprise the HCV polyprotein NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B junction cleavage sites. Exemplary HCV NS4A / 4B protease cleavage sites include DEMEECSQH and DEMEECSQH. Exemplary HCV NS5A / 5B protease cleavage sites include EDVVPCSMG and EDVVPCSMGS. Exemplary NS4B / 5A protease cleavage sites are ECTTPCSGSWL. Additional NS3 protease cleavage sites that may be included in the recombinant polypeptides of the present disclosure include those described in Shiryaev et al. (2012) PLoS One 7(4):e35759.

[0047] In certain embodiments, the protease cleavage site is a human protease cleavage site, non-limiting examples of which include cleavage sites for human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloproteases (MMPs), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, or human cathepsins. According to some embodiments, the protease cleavage site is a cleavage site for human kallikrein (KLK) proteases, non-limiting examples of which include human KLK3 (UniProtKB-Q546G3), human KLK4 (UniProtKB-Q9Y5K2), human KLK6 (UniProtKB-Q92876), human KLK8 (UniProtKB-O60259), human KLK11 (UniProtKB-Q9UBX7), human KLK13 (UniProtKB-Q9UKR3), human KLK14 (UniProtKB-Q9P0G3), and human KLK15 (UniProtKB-Q9H2R5). Data demonstrating the utility of human KLK proteases and corresponding cleavage sites in the STASH selection system are provided in the experimental section herein. Any of these sequences, or functional variants thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof can be used.

[0048] In certain embodiments, the protease cleavage site is selected from the group consisting of acrosin (ACR), AGBL carboxypeptidase 1 (AGBL1), AGBL carboxypeptidase 2 (AGBL2), AGBL carboxypeptidase 3 (AGBL3), AGBL carboxypeptidase 4 (AGBL4), AGBL carboxypeptidase 5 (AGBL5), ATP / GTP-binding carboxypeptidase 1 (AGTPBP1), asparaginase and isoaspartyl peptidase 1 (ASRGL1), astacin-like metalloendopeptidase (ASTL), ATP23 metallopeptidase and ATP synthase assembly factor homolog (ATP23), ataxin 3 (ATXN3), ataxin 3-like (ATXN3L), azurocidin 1 (AZU1), beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), bone morphogenetic protein 1 (BMP1), BRCA1 / BRCA2-containing complex subunit 3 (BRCC3), calpain 14 (CAPN14), calpain 3 (CAPN3), caspase recruitment domain family member 8 (CARD8), caspase 4 (CASP4), chymotrypsin-like elastase 1 (CELA1), chymotrypsin-like elastase 2A (CELA2A), chymotrypsin-like elastase 2B (CELA2B), chymotrypsin-like elastase 3A (CELA3A), chymotrypsin-like elastase 3B (CELA3B), CUGBP Elav-like family member 3 (CELF3), CUGBP Elav-like family member 4 (CELF4), CUGBP Elav-like family member 5 (CELF5), CUGBP Elav-like family member 6 (CELF6), cell growth regulator with EF-hand domain 1 (CGREF1), charged multivesicular body protein 3 (CHMP3), CLN5 intracellular transport protein (CLN5), chymase 1 (CMA1), collectin subfamily member 11 (COLEC11), COP9 signalosome subunit 5 (COPS5), choline, serine peptidase (CORIN), carboxypeptidase A4 (CPA4), carboxypeptidase cytopregenesis-like (CPVL), cystatin SN (CST1), cystatin 11 (CST11), cystatin C (CST3), cystatin S (CST4), cystatin D (CST5),Cystatin E / M (CST6), Cystatin 8 (CST8), Cystatin 9 (CST9), Cystatin-like 1 (CSTL1), Chymotrypsinogen B2 (CTRB2), Chymotrypsin-like (CTRL), Cathepsin L (CTSL), DNA damage-inducible 1 homolog 2 (DDI2), DAP3-binding cell death promoter 1 (DELE1), Adipsin (DF), Dikopf WNT signaling pathway inhibitor 2 (DKK2), Dikopf WNT signaling pathway inhibitor 4 (DKK4), Dipeptidase 1 (DPEP1), Dipeptidyl peptidase 3 (DPP3), Dipeptidyl peptidase 9 (DPP9), FAM111 trypsin-like peptidase A (FAM111A), Ficolin 1 (FCN1), Ficolin 2 (FCN2), Ficolin 3 (FCN3), G3BP stress granule assembly factor 1 (G3BP1), hepsin (HPN), HtrA serine peptidase 1 (HTRA1), insulin degrading enzyme (IDE), mitochondrial inner membrane peptidase subunit 2 (IMMP2L), janthin domain containing 7 (JMJD7), josephin domain containing 2 (JOSD2), kallikrein 1 (KLK1), kallikrein-related peptidase 10 (KLK10), kallikrein-related peptidase 11 (KLK11), kallikrein-related peptidase 12 (KLK12), kallikrein-related peptidase 13 (KLK13), kallikrein-related peptidase 14 (KLK14), kallikrein-related peptidase 15 (KLK15), kallikrein-related peptidase 2 (KLK2), kallikrein-related peptidase 3 (KLK3), kallikrein-related peptidase 4 (KLK4), kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 6 (KLK6 ...5 (KLK15), kallikrein-related peptidase 2 (KLK2), kallikrein-related peptidase 3 (KLK3), kallikrein-related peptidase 4 (KLK4), kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 6 (KLK6), kallikrein-related p lein-related peptidase 7 (KLK7), kallikrein-related peptidase 8 (KLK8), kallikrein-related peptidase 9 (KLK9), kallikrein pseudogene 1 (KLKP1), lipocalin 2 (LCN2), legumain (LGMN), leishmanolysin-like peptidase (LMLN), MAS1 proto-oncogene-like, G protein-coupled receptor (MAS1L), MBL-associated serine protease 1 (MASP1), MBL-associated serine protease 2 (MASP2), mannose-binding lectin 2 (MBL2), matrix metallopeptidase 10 (MMP10),Matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 13 (MMP13), matrix metallopeptidase 16 (MMP16), matrix metallopeptidase 2 (MMP2), napsin A aspartic peptidase (NAPSA), neuroligin (NLN), NLR family CARD domain containing 4 (NLRC4), NLR family pyrin domain containing 1 (NLRP1), aminopeptidase puromycin sensitive (NPEPPS), opyruphin prepropeptide (OPRPN), OTU deubiquitinase, ubiquitin aldehyde-binding 2 (OTUB2), poly(ADP-ribose) polymerase family member 9 (PARP9), proprotein convertase subtilisin / kexin type 1 (PCSK1), proprotein convertase Subtilisin / kexin type 1 inhibitor (PCSK1N), proprotein convertase subtilisin / kexin type 2 (PCSK2), proprotein convertase subtilisin / kexin type 4 (PCSK4), proprotein convertase subtilisin / kexin type 5 (PCSK5), proprotein convertase subtilisin / kexin type 6 (PCSK6), proprotein convertase subtilisin / kexin type 7 (PCSK7), proprotein convertase subtilisin / kexin type 9 (PCSK9), platelet-derived growth factor C (PDGFC), pepsinogen A3 (PGA3), pepsinogen A4 (PGA4), pepsinogen A5 (PGA5), pyroglutamyl peptidase I-like (PGPEP1L), PTEN-induced kinase 1 (PINK1), prolyl endopeptidase-like (PREPL), parkin RBR E3 ubiquitin protein ligase (PRKN), serine protease gene cluster (PRSS), serine protease 2 (PRSS2), serine protease 21 (PRSS21), serine protease 22 (PRSS22), serine protease 23 (PRSS23), serine protease 27 (PRSS27), serine protease 33 (PRSS33), serine protease 46, pseudogene (PRSS46P), serine protease 55 (PRSS55), serine protease 8 (PRSS8), proteinase 3 (PRTN3), presenilin 2 (PSEN2), PYD and CARD domain containing (PYCARD),Retinoic acid receptor responder 1 (RARRES1), RING finger and FYVE-like domain-containing E3 ubiquitin protein ligase (RFFL), rhomboid-like 2 (RHBDL2), SEC11 homolog A, signal peptidase complex subunit (SEC11A), SEC11 homolog B, signal peptidase complex subunit (SEC11B), SEC11 homolog C, signal peptidase complex subunit (SEC11BC), NEDD8-specific SUMO peptidase family member (SENP8), SET nuclear protoma Oncogenic factor (SET), synaptosomal associated protein 25 (SNAP25), secreted phosphoprotein 2 (SPP2), small proline-rich protein 3 (SPRR3), spleen-associated tyrosine kinase (SYK), transcription factor EB (TFEB), transglutaminase 2 (TGM2), toll-like receptor adaptor molecule 1 (TICAM1), tubulointerstitial nephritis antigen-like 1 (TINAGL1), transmembrane serine protease 11D (TMPRSS11D), transmembrane serine protease 11E (TMPRSS11E), transmembrane serine protease 4 (TMPRSS 4), transmembrane serine protease 5 (TMPRSS5), transmembrane serine protease 6 (TMPRSS6), transmembrane serine protease 7 (TMPRSS7), TNF receptor superfamily member 10a (TNFRSF10A), tryptase alpha / beta 1 (TPSAB1), tryptase beta 2 (TPSB2), tryptase delta 1 (TPSD1), tryptase gamma 1 (TPSG1), tryptase pseudogene 2 (TPSP2), tyrosylprotein sulfotransferase 1 (TPST1), tyrosylprotein sulfotransferase 2 (TPST2), protease cleavage sites for human proteases selected from tyrosine sulfotransferase 2 (TPST2), tyrosylprotein sulfotransferase 2 pseudogene 1 (TPST2P1), thyrotropin-releasing hormone degrading enzyme (TRHDE), thyroid hormone receptor interactor 4 (TRIP4), ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin-specific peptidase 27X-conjugating (USP27X), vasohibin 2 (VASH2), valosin-containing protein (VCP), and WAP4-disulfide core domain 1 (WFDC1).

[0049] In some embodiments, proteases are highly selective for their cleavage site. Moreover, protease activity may be amenable to inhibition by known small molecule inhibitors that are cell permeable and non-toxic to the cell or individual being studied or treated. For a discussion of proteases, see, e.g., VYH Hook, "Proteolytic and cellular mechanisms in prohormone and proprotein processing", RG Landes Company, Austin, Tex., USA (1998); NM Hooper et al., Biochem. J. 321:265-279 (1997); Z. Werb, Cell 91:439-442 (1997); TG Wolfsberg et al., J. Cell Biol. 131:275-278 (1995); T. Berg et al., Biochem. J. 307:313-326 (1995); MJ Myth and JA Trapani, Immunology Today 16:202-206 (1995); RV Talanian et al., J. Biol. Chem. 272:9677-9682 (1997); and NA Thornberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein by reference in their entireties for all purposes. In some embodiments, the protease used is a sequence-specific non-human protease for which FDA-approved pharmacological inhibitors are available.

[0050] Any of the proteases used in accordance with the methods of the disclosure, including any of the proteases described above, may be provided by two or more (e.g., two) complementary fragments of the protease, where the two or more (e.g., two) complementary fragments form an active protease complex. The protease may be provided by two or more (e.g., two) complementary fragments of the protease, for example, to increase the number of separate expression constructs required for cell surface expression of a selectable marker.

[0051] Membrane Association Domain, Hinge Domain, and Dimerization Domain Any of the cell selection system components of the present disclosure may include a membrane-associated domain. Non-limiting examples of membrane-associated domains include transmembrane domains. Transmembrane (Tm) domains may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the Tm domain is derived from (e.g., including at least the transmembrane region(s) or functional portion thereof) CD35, CD3zeta, CD3gamma, CD3delta, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or the alpha or beta chain of PD-1. In certain embodiments, the transmembrane domain is a CD8alpha transmembrane domain. According to some embodiments, the transmembrane domain is a CD28 transmembrane domain. Non-limiting examples of transmembrane domains that may be included in one or more (e.g., each) of the cell selection system components include the following: WLRLPFLGVLALLGYLAVRPFL (SEQ ID NO: 42); VLWWSIAQTVILILTGIW (SEQ ID NO: 43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO: 44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO: 45); IAFLLACVATMIFMITKCCLF (SEQ ID NO: 46); VIGFLLAVVLTVAFITF (SEQ ID NO: 47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO: 48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO: 49); TFCSTALLITALALVCTLLYL (SEQ ID NO: 50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO: 51); WLWVVYILT A transmembrane domain that comprises, consists of, or has 80% or more, 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to a transmembrane domain that comprises, consists of, or is present within an amino acid sequence selected from VALPVFLVILFC (SEQ ID NO: 52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 53); and FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 54).

[0052] Any of the cell selection system components of the present disclosure may include a hinge domain, such as a CD8α hinge domain, a CD28 hinge domain, and the like.

[0053] Provided herein are examples of amino acid sequences of transmembrane and hinge domains that may be included in one or more (eg, each) of the cell selection system components.

[0054] Non-limiting examples of membrane-associated domains also include post-translational modifications that anchor the cell selection system component to a membrane. That is, the cell selection system component may include a membrane-tethering domain that is added post-translationally. "Membrane-tethering domain" refers to a domain (e.g., a portion) that can stably associate with a membrane of a cell (e.g., an ER membrane). Suitable membrane-tethering domains include, but are not limited to, post-translational modifications such as palmitoylation, myristoylation, prenylation, and glycosylphosphatidylinositol (GPI) anchors.

[0055] In some embodiments, when two or more cell selection system components comprise a membrane-associated domain (eg, a transmembrane domain), the membrane-associated domain of each component can be identical or substantially identical to one another.

[0056] In some embodiments, to increase the number of separate expression constructs required for cell surface expression of the selection marker, two or more cell selection system components each contain a dimerization domain, where dimerization of the cell selection system components is required for cell surface expression of the selection marker. Examples of cell selection system configurations using one or more pairs of dimerization domains are described elsewhere. Non-limiting examples of dimerization domains that can be used include domains that contain coiled coil structures. When the dimerization domain contains a coiled coil structure, in some embodiments, the dimerization domain contains a leucine zipper domain.

[0057] Genetic modification – target protein and gene inactivation The two or more separate expression constructs may each provide a genetic modification to the cell to which the two or more separate expression constructs are delivered. Non-limiting examples of genetic modifications include providing a region of the expression construct that encodes a protein of interest. Non-limiting examples of proteins of interest include receptors, ligands, transcription factors, antibodies, bispecific T-cell engagers (BiTEs), enzymes, cytokines, chemokines, toxins, proteins that confer resistance to cell exhaustion, and suicide switch proteins.

[0058] In some embodiments, the protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is a receptor. For example, one or more expression constructs of the two or more separate expression constructs is a chimeric antigen receptor (CAR), a T cell receptor (TCR), e.g., a recombinant TCR, a chimeric cytokine receptor (CCR), a chimeric chemokine receptor, a synthetic Notch receptor (synNotch), a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a systemic extracellular molecular sensor (GEMS) receptor, a growth factor receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrate molecule, a fusion protein ... gliomas, inhibitory receptors, stimulatory receptors, immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors, hormone receptors, receptor tyrosine kinases, immunoreceptors, e.g., CD28, CD80, ICOS, CTLA4, PD1, PD-L1, BTLA, HVEM, CD27, 4-1BB, 4-1BBL, OX40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226, CD16 type I cytokine receptors, such as interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-9 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-16 receptor, interleukin-17 receptor, interleukin-18 receptor, interleukin-19 receptor, interleukin-20 receptor, interleukin-21 receptor, interleukin-22 receptor, interleukin-23 receptor, interleukin-24 receptor, interleukin-25 receptor, interleukin-26 receptor, interleukin-27 receptor, interleukin-28 receptor, interleukin-29 receptor, interleukin-30 receptor, interleukin-31 receptor, interleukin-32 receptor, interleukin-33 receptor, interleukin-34 receptor, interleukin-35 receptor, interleukin-36 receptor, interleukin-37 receptor, interleukin-38 receptor, interleukin-39 receptor, interleukin-40 receptor, interleukin-41 receptor, interleukin-42 receptor, interleukin-43 receptor, interleukin-44 receptor, interleukin-45 receptor, interleukin-46 receptor, interleukin-47 receptor, interleukin-48 receptor, interleukin-49 receptor, interleukin-50 receptor, interleukin-51 receptor, interleukin-52 receptor, interleukin-53 receptor, interleukin-54 receptor, interleukin-55 receptor, interleukin-56 receptor, interleukin-57 receptor, interleukin-58 receptor, interleukin-59 receptor, interleukin-60 18 receptor, interleukin-21 receptor, interleukin-23 receptor, interleukin-27 receptor, erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, leptin receptor, oncostatin M receptor, leukemia inhibitory factor, type II cytokine receptors such as interferon-alpha / beta receptor, interferon-gamma receptor, interferon type III receptor, interleukin-10 receptor, interleukin-20 receptor,Interleukin-22 receptor, Interleukin-28 receptor, receptors of the tumor necrosis factor receptor superfamily, e.g. tumor necrosis factor receptor 2(1B), tumor necrosis factor receptor 1, lymphotoxin beta receptor, OX40, CD40, Fas receptor, decoy receptor 3, CD27, CD30, 4-1BB, decoy receptor 2, decoy receptor 1, cell death receptor 5, cell death receptor 4, RANK, osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, herpes virus entry mediator, nerve growth factor receptor, B cell maturation antigen, glucocorticoid-inducible TNFR-related, TROY, cell death receptor 6, cell death receptor 3, ectodysplasin A2 receptor, chemokine receptors, e.g. CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2 , CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, ACKR1, ACKR2, ACKR3, ACKR4, CCRL2, receptors of the epidermal growth factor receptor (EGFR) family, receptors of the fibroblast growth factor receptor (FGFR) family, receptors of the vascular endothelial growth factor receptor (VEGFR) family, receptors of the receptor rearranged during transfection (RET) receptor family, receptors of the Eph receptor family, receptors capable of inducing cell differentiation (e.g., Notch receptors), adhesion receptors such as cell adhesion molecules (CAMs), integrin receptors, cadherins, selectins, and receptors of the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1, and transforming growth factor beta receptor 2. In some embodiments, such receptors are immune cell receptors selected from T cell receptors, B cell receptors, natural killer (NK) cell receptors, macrophage receptors, monocyte receptors, neutrophil receptors, dendritic cell receptors, mast cell receptors, basophil receptors, and eosinophil receptors.

[0059] According to some embodiments, one or more of the two or more separate expression constructs may encode a CAR. When the two or more separate expression constructs encode a CAR, the CAR may be the same CAR, or the two or more separate expression constructs may encode two or more different CARs. In certain embodiments, when the protein of interest is a CAR, the extracellular binding domain of the CAR comprises a single chain antibody. The single chain antibody may be a monoclonal single chain antibody, a chimeric single chain antibody, a humanized single chain antibody, a fully human single chain antibody, and the like. In one non-limiting example, the single chain antibody is a single chain variable fragment (scFv). Suitable CAR extracellular binding domains include those described in Labaniieh et al. (2018) Nature Biomedical Engineering 2:377-391. In some embodiments, the extracellular binding domain of the CAR is a single chain version (e.g., an scFv version) of an antibody approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use therapeutically, e.g., as an antibody to induce antibody-dependent cellular cytotoxicity (ADCC) of specific disease-associated cells in a patient. When the protein of interest is a CAR, non-limiting examples of single chain antibodies that can be used include adecatumumab, ascrimbacumab, cixutumumab, conatumumab, daratumumab, drozitumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumumab, fizitumumab, ganitumab, glembatumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narunatumab, necitumumab, nesbacumab, ofatumumab, olaratumumab, panitumumab, patitutumumab, and the like. Mab, Pritumumab, Ladoltumab, Ramucirumab, Rilotumumab, Lobatumumab, Seribantumab, Talextumab, Teprotumumab, Tobetumab, Vantictumab, Besencumab, Votumumab, Zalutumumab, Franvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minletumomab, Mitsumomab, Moxetumomab, Naputumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab,Tremelimumab, abagovomab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab, dellotuximab, dinutuximab, ensituximab, futuximab, girentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ubrituximab, Tuximab, ecromeximab, avituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontixtuzumab, cantuzumab, cantuzumab, sitatuzumab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblitzumab, etaracizumab, farletuzumab, ficratuzumab zumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, rifastuzumab, lintuzumab, lorvotuzumab, lumuletuzumab, matuzumab, milatuzumab, nimotuzumab, obinutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, opoltuzumab, palsatuzumab, pertuzumab, pinatuzumab, polatuzumab, sibrotuzumab, simutuzumab In some embodiments, the protein of interest is a CAR, and the extracellular binding domain of the CAR specifically binds to an antigen expressed on the surface of a cancer cell. For example, the extracellular binding domain may bind to a surface antigen of a cancer cell selected from B7-H3 (CD276), CD19, GD2, CD22, and HER2.

[0060] According to some embodiments, one or more of the two or more separate expression constructs may encode an antibody. When the two or more separate expression constructs encode an antibody, the antibody may be the same antibody, or the two or more separate expression constructs may encode two or more different antibodies. The term "antibody" (also used interchangeably with "immunoglobulin") encompasses antibodies of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer, which in turn is composed of two dimers of a heavy chain polypeptide and a light chain polypeptide), single chain antibodies (e.g., scFv), single chain Fv (scFv), Fab, (Fab')2, (scFv')2, and diabodies, fragments of antibodies (e.g., whole antibodies or single chain antibody fragments) that retain specific binding to an antigen, including, but not limited to, chimeric antibodies, monoclonal antibodies, humanized antibodies, human antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0061] Immunoglobulin polypeptides include kappa and lambda light chains and alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains, or equivalents in other species. A full-length immunoglobulin "light chain" (usually of about 25 kDa or about 214 amino acids) contains a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (of about 150 kDa or about 446 amino acids) similarly contains a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0062] Immunoglobulin light or heavy chain variable regions (V L and V H) is composed of a "framework" region (FR) separated by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The extent of the framework regions and CDRs have been defined (E. Kabat et al., "Sequences of proteins of immunological interest", 4th ed., USDept. Health and Human Services, Public Health Services, Bethesda, MD (1987); Lefranc et al., IMGT, the international ImMunoGeneTics information system®. Nucl. Acids Res., 2005, 33, D593-D597). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and frameworks provided by this disclosure are defined according to Kabat above, unless otherwise indicated.

[0063] Thus, "antibody" encompasses a protein having one or more polypeptides genetically encodable, for example, by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, the antibodies of the present disclosure are IgG antibodies, e.g., IgG1 antibodies, such as human IgG1 antibodies. In some embodiments, the antibodies of the present disclosure comprise a human Fc domain.

[0064] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The variable light chain (VLC) is L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0065] Antibodies encompass whole immunoglobulins and a number of well-characterized fragments that may be genetically encoded or produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies under the disulfide bond in the hinge region to produce F(ab)'2 (a dimer of Fab, itself a light chain bound to VH-CHI by a disulfide bond). F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see "Fundamental Immunology, WE", Paul, ed., Raven Press, NY (1993)). While various antibody fragments have been defined with respect to digestion of whole antibodies, one skilled in the art will understand that such Fab' fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments, either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. In certain embodiments, the antibody of the present disclosure is selected from an IgG, an Fv, a single chain antibody, a scFv, a Fab, a F(ab')2, and a Fab'.

[0066] One or more of the two or more separate expression constructs may encode a protein of interest for use in the context of cell therapy (e.g., cell-based cancer therapy), non-limiting examples of which include therapies involving administration of therapeutic immune cells, such as T cells (e.g., CART cells, T cells expressing an engineered T cell receptor (TCR), etc.), NK cells (e.g., CAR NK cells), macrophages (e.g., CAR macrophages), etc. Examples of such proteins that may be expressed by one or more of the two or more separate expression constructs include those described in Rodriguez-Garcia et al. (2020) Front Immunol. 11:1109; Martinez and Moon (2019) Front. Immunol. 10:128; Knochelmann et al. (2018) Front Immunol. 9:1740, etc., the disclosures of which are incorporated herein in their entireties for all purposes.

[0067] In some embodiments, one or more of the two or more separate expression constructs express a protein independently selected from a protein that reduces the immunogenicity of an engineered cell upon administration to an individual, a protein that confers resistance to cell exhaustion to a cell upon administration to an individual (e.g., cJun, etc.), a protein that enhances the effectiveness of a cell in the tumor microenvironment (TME) for the treatment of a solid tumor (e.g., a switch receptor, a dominant negative receptor), an HLA-E protein, a CD47 protein, a homing protein (e.g., a chemokine receptor), a persistence-promoting protein (e.g., a cytokine receptor), an autonomous control unit protein (e.g., a gene circuit protein, an oscillator protein, etc.), a protein that rewires the metabolism of a cell, a logic gate protein (e.g., SynNotch, iCAR), a suicide switch protein (e.g., EGFRt, iCASP9, etc.), and any other protein useful in the context of cell therapy.

[0068] Non-limiting examples of genetic modification also include inactivating (e.g., knocking out) one or more genes in the genome of a cell.Thus, in some embodiments, one or more of the two or more separate expression constructs are configured to be site-specifically integrated into the genome of a cell in a manner that inactivates one or more target genes.

[0069] In some embodiments, one or more of the two or more separate expression constructs are configured to site-specifically integrate into the genome of a cell in a manner that inactivates one or more target genes, where such gene inactivation is used in the context of cell therapy (e.g., cell-based cancer therapy), non-limiting examples of which include therapies that include administration of therapeutic immune cells, such as T cells (e.g., CART cells, T cells expressing an engineered T cell receptor (TCR), etc.), NK cells (e.g., CAR NK cells), macrophages (e.g., CAR macrophages), etc. Examples of such gene inactivation include those described in Rodriguez-Garcia et al. (2020) Front Immunol. 11:1109; Martinez and Moon (2019) Front. Immunol. 10:128; Knochelmann et al. (2018) Front Immunol. 9:1740, the disclosures of which are incorporated herein in their entireties for all purposes.

[0070] In some embodiments, one or more of the two or more separate expression constructs are configured to be site-specifically integrated into the genome of a cell in a manner that inactivates one or more target genes, where such gene inactivation reduces immunogenicity of the engineered cells upon administration to an individual (e.g., knockout of one or more T cell receptor genes, e.g., TRAC knockout), renders the cells resistant to cellular exhaustion upon administration to an individual, enhances the efficacy of the cells in the tumor microenvironment (TME) for the treatment of solid tumors, promotes persistence of the cells, and any other gene inactivation useful in the context of cell therapy.

[0071] Selection markers and cell selection The first expression construct may encode a fusion protein containing any convenient selection marker that allows for the selection of cells containing two or more separate expression constructs. In some embodiments, the selection marker is one that is already used for cell selection purposes, for which existing reagents (e.g., antibodies, etc.) and devices exist for selecting cells that exhibit cell surface expression of the selection marker. For example, the selection marker may be one that is currently used in magnetic activated cell sorting (MACS) workflows, flow cytometry workflows (e.g., fluorescence activated cell sorting (FACS) workflows), etc.

[0072] The selection marker can be a protein tag. For example, the selection marker can be a Myc-tag, His-tag, HA-tag, FLAG-tag, Strep-tag, NE-tag, Xpress tag, Avi-tag, polyglutamic acid tag, polyarginine tag, etc.

[0073] In certain embodiments, the selection marker comprises a cluster of differentiation (CD) protein. A non-limiting example of a CD protein that finds use as a selection marker is CD34.

[0074] According to some embodiments, the selection marker comprises a truncated receptor comprising the extracellular domain of the receptor. Examples of truncated receptors used as selection markers include truncated epidermal growth factor receptor (EGFRt), truncated nerve growth factor receptor (NGFRt), truncated CD19 (CD19t), and truncated CD20 (CD20t).

[0075] As will be appreciated by the benefit of the present disclosure, the selection marker can be selected such that in addition to facilitating the selection of cells containing each of the two or more expression constructs, the selection marker provides a functionality. For example, the selection marker can further serve a useful function in the context of cell therapy, for example, during the cell manufacturing process or after administration of the cells to an individual in need thereof. In one non-limiting example, the selection marker can further serve as a suicide switch that allows for removal of cells if an individual experiences excessive adverse side effects from cell therapy. The use of the selection marker as a suicide switch is shown diagrammatically in FIG. 9B. The suicide switch in that particular example is a truncated EGFR (EGFRt), which allows for targeting of cells with an anti-EGFR antibody such as cetuximab.

[0076] Any convenient approach can be used to select for cells exhibiting cell surface expression of the selection marker. In certain embodiments, a magnetic-based cell selection approach is used. As an example, cells exhibiting cell surface expression of the selection marker can be selected (purified, enriched) by magnetic activated cell sorting (MACS). MACS involves labeling cells exhibiting cell surface expression of the selection marker with magnetic beads, for example, by combining a cell population with magnetic beads coated with a moiety (antibody, lectin, enzyme, etc.) that binds to the selection marker on the cell surface. The labeled cells can then be transferred to a column, where a magnetic field is applied to magnetically attract the labeled cells to the wall of the column, while unlabeled cells flow through the column. The magnetic field can then be removed and the labeled cells (i.e., those exhibiting cell surface expression of the selection marker) can be collected from the column.

[0077] By way of example, cells exhibiting cell surface expression of a selection marker can also be selected (purified, enriched) by flow cytometry, e.g., fluorescence-activated cell sorting (FACS). FACS involves labeling cells exhibiting cell surface expression of a selection marker with a fluorophore, e.g., by combining a cell population with a fluorophore-labeled antibody that binds to the selection marker on the cell surface. The fluorescently labeled cells may then be separated from unlabeled cells using a fluorescence-activated cell sorter according to the manufacturer's instructions.

[0078] Further non-limiting examples of cell selection systems according to embodiments of the present disclosure are then described.

[0079] An example of a tripartite cell selection system is shown diagrammatically in FIG. 5A. In this example, an epitope-based selection marker (such as EGFRt, CD34, Myc tag) is fused to a protease cleavage site and an intracellular retention tag (e.g., an endoplasmic reticulum retention tag). Co-expression of a split protease (a protease comprising a first complementary fragment and a second complementary fragment that form an active protease complex) results in the reconstitution of an active protease complex, whereby the N-terminal domain of the protease is tethered to one transmembrane protein and the C-terminal domain is tethered to another transmembrane protein. The active protease complex cleaves the selection marker at the protease cleavage site, which releases the selection marker from the protein localization tag (here, the ER retention tag) and translocates the selection marker to the surface of the cell. The surface expressed selection tag can then be used as a selection handle to isolate cells expressing both the selection marker and the two protease domains (the N-terminal protease and the C-terminal protease).

[0080] Figure 5B is a schematic diagram showing separate expression constructs encoding the three proteins of interest (Protein A, Protein B, and Protein C) and the components of the selection system (Stashed selection marker, N-terminal protease, and C-terminal protease) shown in Figure 5A. A ribosomal skip site (here, P2A from a porcine teschovirus) allows for bicistronic expression of the proteins of interest and the STASH selection system components.

[0081] An example of a penta-way cell selection system is shown diagrammatically in Figure 7A. This system consists of: (1) an epitope-based selection marker (such as EGFRt, CD34, Myc tag, etc.) fused to a protease cleavage site and an intracellular retention tag (e.g., endoplasmic reticulum retention tag); (2) a transmembrane domain fused to a leucine zipper (Zip2) and an ER retention tag; (3) another transmembrane domain fused to an orthogonal leucine zipper (Zip3) and an ER retention tag; (4) an N-terminal protease domain fused to a leucine zipper (Zip4) that binds Zip2; (5) a C-terminal protease domain fused to a leucine zipper (Zip5) that binds Zip3. Binding events between Zip2+Zip4, Zip3+Zip5, and the two transmembrane domains result in the reconstitution of an active protease complex. The active protease complex cleaves the selection marker at the protease cleavage site, which releases the selection marker from the ER retention tag and translocates the selection marker to the surface of the cell. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing the selection marker, the two protease domains (the N-terminal protease and the C-terminal protease), and the two transmembrane domains.

[0082] Figure 7B is a schematic diagram showing separate expression constructs encoding five proteins of interest (Protein A, Protein B, Protein C, Protein D, and Protein E) and the components of the selection system shown in Figure 7A (stashed selection marker, transmembrane Zip2, transmembrane Zip3, N-terminal protease Zip4, and C-terminal protease Zip5). A ribosomal skip site (here, P2A from porcine teschovirus) allows for bicistronic expression of the proteins of interest and the STASH selection system components.

[0083] An example of a cell selection system using a truncated epidermal growth factor receptor (EGFRt) as a selectable surface marker and suicide switch is shown diagrammatically in Figure 9 A. Figure 9B is a schematic of the EGFRt suicide switch, whereby cells expressing EGFRt can be eliminated by administration of an anti-EGFR antibody, such as cetuximab.

[0084] An example of a tripartite cell selection system is shown diagrammatically in FIG. 23. The first component of the system (1) is an epitope-based selection marker fused to a cleavage site A (Cut A) and a protein localization tag, here an ER retention tag ("ER tag"). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (Cut B), a degron that induces protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to protease B (Prot B) and an ER retention tag. Cut A and Cut B are the cleavage sites for Prot A and Prot B, respectively. When all three components are present in the same cell, they associate at the ER membrane. Prot B of component 3 cleaves component 2 at Cut B, removing the degron from component 2, resulting in high levels of component 2 expression. Component 2 then cleaves cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all three constructs.

[0085] As used herein, a "degron" is a sequence of amino acids that provides a degradation signal that directs a polypeptide into an intracellular pathway for protein degradation. A degron can promote degradation of an associated polypeptide via either the proteasome pathway or the autophagy-lysosomal pathway. In some embodiments, a degron induces rapid degradation of a polypeptide. For a discussion of degrons and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425; Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221; Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822; Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690; Tasaki et al. (2007) Trends Biochem Sci. 32(Issue ll):520-528; Meinnel et al. (2006) Biol. Chem. 387(Issue 7):839-851; Kim et al. (2013) Autophagy 9(Issue 7):1100-1103; Varshavsky (2012) Methods Mol. Biol. 832:1-11; and Fayadat et al. (2003) Mol Biol Cell. 14(Issue 3):1268-1278; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0086] According to some embodiments, the degron is a degron found in p53, HIF1 alpha, ubiquitin, or a functional variant thereof. In certain embodiments, the degron comprises a portion of the HCV nonstructural proteins NS3 and NS4A. According to some embodiments, the degron comprises or consists of the amino acid sequence PITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLST (amino acid sequence of a degron from HCV genotype 1a; SEQ ID NO: 55), or a functional variant thereof having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more amino acid sequence identity to such an amino acid sequence, or a fragment thereof, such as a fragment having a length of 30-41 amino acids, 32-41 amino acids, 34-41 amino acids, 36-41 amino acids, or 38-41 amino acids, etc., wherein the functional variant of the degron is capable of promoting degradation of the polypeptide.

[0087] An example of a four-way cell selection system is shown diagrammatically in FIG. 24. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (Cut A) and an ER retention tag (ER Tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER Tag), a transmembrane domain, protease A (Prot A), cleavage site B (Cut B), a degron that induces protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) is a transmembrane domain fused to a c-terminal fragment of protease B (cB) and an ER retention tag. Cut A and Cut B are the cleavage sites of Prot A and Prot B, respectively. When all four components are present in the same cell, they associate at the ER membrane. Protease B is reconstituted into an active form by association of the two protease fragments on components 3 and 4. Reconstituted protease B cleaves component 2 at cut B, removing the degron from component 2, resulting in high levels of expression of component 2. Component 2 then cleaves cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all four constructs.

[0088] An example of a tripartite cell selection system is shown diagrammatically in FIG. 25. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and an ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a c-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and an ER retention tag. The fifth component (5) is a transmembrane domain fused to protease C and an ER retention tag.

[0089] Cut A, Cut B, and Cut C are the cleavage sites for Prot A, Prot B, and Prot C, respectively. When all five components are present in the same cell, they associate at the ER membrane. Prot C on component 5 cleaves at cut C, removing the degron from component 4 and causing it to be expressed at high levels. Protease B is reconstituted by the association of components 3 and 4, cleaving at cut B, removing the degron from component 2 and causing component 2 to be expressed at high levels. Prot A on component 2 then cleaves at cut A, removing the ER tag from the epitope marker and moving it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all five constructs.

[0090] An example of a six-way cell selection system is shown diagrammatically in FIG. 26. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a c-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and the ER retention tag.

[0091] The fifth component (5) is a transmembrane domain fused to the N-terminal fragment (nC) of protease C and an ER retention tag. The sixth component (6) is a transmembrane domain fused to the C-terminal fragment (cC) of protease C and an ER retention tag. Cut A, Cut B, and Cut C are the cleavage sites of Prot A, Prot B, and Prot C, respectively. When all six components are present in the same cell, they associate at the ER membrane. Prot C is reconstituted by the association of components 5 and 6, and the reconstituted Prot C cleaves component 4 at cut C, removes the degron, and expresses it at a high level. Protease B, which is reconstituted by the association of components 3 and 4, cleaves component 2 at cut B, removes the degron from component 2, and expresses component 2 at a high level. Next, Prot A on component 2 cleaves the epitope marker at cut A, removing the ER tag from the epitope marker and moving it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all six constructs.

[0092] An example of a seven-way cell selection system is shown diagrammatically in FIG. 27. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a C-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and the ER retention tag.

[0093] The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of protease C (nC) and an ER retention tag. The sixth component (6) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease C (cC), cleavage site D (Cut D), a degron that induces protein degradation, and an ER retention tag. The seventh component (7) consists of a transmembrane domain fused to protease D (Prot D) and an ER retention tag. Cut A, Cut B, Cut C, and Cut D are the cleavage sites of Prot A, Prot B, Prot C, and Prot D, respectively. When all seven components are present in the same cell, they associate at the ER membrane. Prot D on component 7 cleaves component 6 at Cut D, removing the degron from component 6 and allowing component 6 to be expressed at high levels. The association of components 5 and 6 reconstitutes Prot C, which cleaves component 4 at cut C, removing the degron from component 4 and expressing it at a high level. Protease B, reconstituted by the association of components 3 and 4, cleaves component 2 at cut B, removing the degron from component 2 and expressing it at a high level. Next, Prot A on component 2 cleaves the epitope marker at cut A, removing the ER tag from the epitope marker and moving it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all seven constructs.

[0094] An example of an eight-way cell selection system is shown diagrammatically in FIG. 28. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a C-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and the ER retention tag.

[0095] The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of protease C (nC) and an ER retention tag. The sixth component (6) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease C (cC), cleavage site D (Cut D), a degron that induces protein degradation, and an ER retention tag.

[0096] The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of protease D (nD) and an ER retention tag. The eighth component (8) is a transmembrane domain fused to the C-terminal fragment of protease D (cD) and an ER retention tag. Cut A, Cut B, Cut C, and Cut D are the cleavage sites of Prot A, Prot B, Prot C, and Prot D, respectively. When all eight components are present in the same cell, they associate at the ER membrane. Prot D, which is reconstituted by the association of components 7 and 8, cleaves component 6 at cut D, removing the degron from component 6 and allowing component 6 to be expressed at a high level. Prot C, which is reconstituted by the association of components 5 and 6, cleaves component 4 at cut C, removing the degron from component 4 and allowing it to be expressed at a high level. Protease B, reconstituted by the association of components 3 and 4, cleaves component 2 at cut B, removing the degron from component 2 and allowing high levels of expression of component 2. Prot A on component 2 then cleaves the epitope marker at cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all eight constructs.

[0097] An example of a nine-way cell selection system is shown diagrammatically in FIG. 29. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a C-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and the ER retention tag.

[0098] The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of protease C (nC) and an ER retention tag. The sixth component (6) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease C (cC), a cleavage site D (Cut D), a degron that induces proteolysis, and an ER retention tag. The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of protease D (nD) and an ER retention tag. The eighth component (8) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease D (cD), a cleavage site E (Cut E), a degron that induces proteolysis, and an ER retention tag. The ninth component (9) consists of a transmembrane domain fused to protease E (Prot E) and an ER retention tag.

[0099] Cut A, Cut B, Cut C, Cut D, and Cut E are the cleavage sites for Prot A, Prot B, Prot C, Prot D, and Prot E, respectively. When all nine components are present in the same cell, they associate at the ER membrane. Protease E on component 9 cleaves component 8 at cut E, removing the degron from component 8, causing component 8 to be expressed at a high level. Prot D, reconstituted by the association of components 7 and 8, cleaves component 6 at cut D, removing the degron from component 6, causing component 6 to be expressed at a high level. Prot C, reconstituted by the association of components 5 and 6, cleaves component 4 at cut C, removing the degron from component 4, causing it to be expressed at a high level. Protease B, reconstituted by the association of components 3 and 4, cleaves component 2 at cut B, removing the degron from component 2, causing component 2 to be expressed at a high level. Prot A on component 2 then removes the ER tag from the epitope marker and translocates it to the cell surface by cleaving the epitope marker with Cut A. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all nine constructs.

[0100] An example of a ten-way cell selection system is shown diagrammatically in FIG. 30. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of an N-terminal ER retention tag (N-terminal ER tag), a transmembrane domain, protease A (Prot A), cleavage site B (CutB), a degron inducing protein degradation, and the ER retention tag. The third component (3) is a transmembrane domain fused to an n-terminal fragment of protease B (nB) and an ER retention tag. The fourth component (4) consists of an N-terminal ER retention tag, a transmembrane domain fused to a C-terminal fragment of protease B (cB), cleavage site C (CutC), a degron inducing protein degradation, and the ER retention tag.

[0101] The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of protease C (nC) and an ER retention tag. The sixth component (6) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease C (cC), cleavage site D (Cut D), a degron that induces proteolysis, and an ER retention tag. The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of protease D (nD) and an ER retention tag. The eighth component (8) consists of an N-terminal ER retention tag, a transmembrane domain fused to the C-terminal fragment of protease D (cD), cleavage site E (Cut E), a degron that induces proteolysis, and an ER retention tag.

[0102] The ninth component (9) is a transmembrane domain and an ER retention tag fused to the N-terminal fragment (nE) of Protease E. The tenth component (10) is a transmembrane domain and an ER retention tag fused to the C-terminal fragment (cE) of Protease E.

[0103] Cut A, Cut B, Cut C, Cut D, and Cut E are cleavage sites for Prot A, Prot B, Prot C, Prot D, and Prot E, respectively. When all 10 components are present in the same cell, they associate at the ER membrane. Protease E reconstituted by the association of components 9 and 10 cleaves component 8 at cut E, removing the degron from component 8, and component 8 is expressed at a high level. Prot D reconstituted by the association of components 7 and 8 cleaves component 6 at cut D, removing the degron from component 6, and component 6 is expressed at a high level. Prot C reconstituted by the association of components 5 and 6, and the reconstituted Prot C cleaves component 4 at cut C, removing the degron from component 4, and expressing it at a high level. Protease B reconstituted by the association of components 3 and 4 cleaves component 2 at cut B, removing the degron from component 2, and expressing component 2 at a high level. Prot A on component 2 then removes the ER tag from the epitope marker and translocates it to the cell surface by cleaving the epitope marker with Cut A. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all 10 constructs.

[0104] An example of a penta-way cell selection system is shown diagrammatically in FIG. 31. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ER tag). The second component (2) consists of a transmembrane domain, a leucine zipper (Zip2), and an ER retention tag. The third component (3) consists of a transmembrane domain, a leucine zipper (Zip3), and an ER retention tag. The fourth component (4) consists of a leucine zipper (Zip4), which is the leucine zipper homologous to Zip2, and an N-terminal fragment of protease A (nA). The fifth component (5) consists of a leucine zipper (Zip5), which is the leucine zipper homologous to Zip3, and a C-terminal fragment of protease A (cA).

[0105] Binding events between Zip2+Zip4, Zip3+Zip5 and the transmembrane domains result in the reconstitution of proteolytic complex A and its localization in the ER in close proximity to component 1. Proteolytic complex A cleaves the epitope marker with cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all five constructs.

[0106] An example of a nine-way cell selection system is shown diagrammatically in FIG. 32. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ERTag). The second component (2) consists of a transmembrane domain, a leucine zipper (Zip2), and an ER retention tag. The third component (3) consists of a transmembrane domain, a leucine zipper (Zip3), and an ER retention tag. The fourth component (4) consists of a leucine zipper (Zip4), which is the leucine zipper homologous to Zip2, and an N-terminal fragment of protease A (nA). The fifth component (5) consists of a leucine zipper (Zip5), which is the leucine zipper homologous to Zip3, a C-terminal fragment of protease A (cA), a cleavage site B (CutB), and a degron that induces protein degradation. The sixth component (6) consists of a transmembrane domain, a leucine zipper (Zip6), and an ER retention tag. The seventh component (7) consists of a transmembrane domain, a leucine zipper (Zip7), and an ER retention tag. The eighth component (8) consists of a leucine zipper homologous to Zip6 (Zip8), and an N-terminal fragment of protease B (nB). The ninth component (9) consists of a leucine zipper homologous to Zip7 (Zip9), and a C-terminal fragment of protease B (cB).

[0107] Binding events between Zip6+Zip8, Zip7+Zip9 and the transmembrane domains result in the reconstitution of proteolytic complex B and its localization in the ER in close proximity to component proteolytic complex A. Protease complex B removes the degron from component 5 by cleaving component 5 at cut B, resulting in high levels of component 5 expression.

[0108] Binding between Zip2+Zip4, Zip3+Zip5 and the transmembrane domains results in the reconstitution of proteolytic complex A and its localization in the ER adjacent to component 1. Proteolytic complex A cleaves the epitope marker with cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all nine constructs.

[0109] An example of a tripartite cell selection system is shown diagrammatically in FIG. 33. The first component of the system (1) is an epitope-based selection marker fused to cleavage site A (CutA) and an ER retention tag (ERTag). The second component (2) consists of a transmembrane domain, a leucine zipper (Zip2), and an ER retention tag. The third component (3) consists of a transmembrane domain, a leucine zipper (Zip3), and an ER retention tag. The fourth component (4) consists of a leucine zipper (Zip4), which is the leucine zipper homologous to Zip2, and an N-terminal fragment of protease A (nA). The fifth component (5) consists of a leucine zipper (Zip5), which is the leucine zipper homologous to Zip3, a C-terminal fragment of protease A (cA), a cleavage site B (CutB), and a degron that induces protein degradation. The sixth component (6) consists of a transmembrane domain, a leucine zipper (Zip6), and an ER retention tag. The seventh component (7) consists of a transmembrane domain, a leucine zipper (Zip7), and an ER retention tag. The eighth component (8) consists of a leucine zipper (Zip8), which is a homologous leucine zipper to Zip6, and an N-terminal fragment of protease B (nB). The ninth component (9) consists of a leucine zipper (Zip9), which is a homologous leucine zipper to Zip7, a C-terminal fragment of protease B (cB), a cleavage site C (CutC), and a degron that induces protein degradation. The tenth component (10) consists of a transmembrane domain, a leucine zipper (Zip10), and an ER retention tag. The eleventh component (11) consists of a transmembrane domain, a leucine zipper (Zip11), and an ER retention tag. The twelfth component (12) consists of a leucine zipper (Zip12), which is a leucine zipper homologous to Zip10, and the N-terminal fragment (nC) of protease C. The thirteenth component (13) consists of a leucine zipper (Zip13), which is a leucine zipper homologous to Zip11, and the C-terminal fragment (cC) of protease C.

[0110] Binding events between Zip10+Zip12, Zip11+Zip13 and the transmembrane domains result in the reconstitution of proteolytic complex C and its localization in the ER in close proximity to proteolytic complex B. Protease complex C removes the degron from component 9 by cleaving component 9 with cut C, resulting in high levels of component 9 expression.

[0111] Binding between Zip6+Zip8, Zip7+Zip9 and the transmembrane domains leads to the reconstitution of proteolytic complex B and its localization in the ER adjacent to proteolytic complex A. Protease complex B removes the degron from component 5 by cleaving component 5 at cut B, resulting in high-level expression of component 5.

[0112] Binding events between Zip2+Zip4, Zip3+Zip5 and the transmembrane domains result in the reconstitution of proteolytic complex A and its localization in the ER in close proximity to component 1. Proteolytic complex A cleaves the epitope marker with cut A, removing the ER tag from the epitope marker and translocating it to the cell surface. The surface expression selection tag can then be used as a selection handle to isolate cells expressing all 13 constructs.

[0113] Fusion proteins Fusion proteins are also provided by the present disclosure. In some embodiments, any fusion protein used in the above cell selection method is provided, for example, any of the fusion proteins encoded by the first, second, etc. expression constructs described elsewhere herein, including any of the fusion proteins or their equivalents whose amino acid sequences are provided herein, for example, in the sequence listing(s) herein. Nucleic acids encoding such fusion proteins, and expression vectors comprising such nucleic acids are also provided. Cells comprising such fusion proteins, nucleic acids, and / or expression vectors are also provided.

[0114] In certain embodiments, a fusion protein is provided comprising a protein fused to an ER localization tag, where the ER localization tag is one of the following: PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCC The ER localization tag comprises, consists of, or is present within an amino acid sequence selected from: CGCFGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 31); NRSPRNRKPRRE (SEQ ID NO: 32); LYKYKSRRSFIEEKKMP (SEQ ID NO: 9); TKVLKGKKLSLPA (SEQ ID NO: 33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35).

[0115] According to some embodiments, a fusion protein is provided comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises a transmembrane (Tm) domain, an intracellular domain (ICD), or both, of an ER localization tag of a polypeptide listed in Table 1, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER. By "variant" Tm and / or ICD is meant a variant comprising an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to a parent / reference sequence, or a fragment thereof, wherein the variant retains the ability of the ER localization tag to localize the polypeptide to the ER.

[0116] In certain embodiments, a fusion protein is provided that includes a protein fused to an ER localization tag, wherein the ER localization tag comprises a Tm domain, an ICD, or both, of an ER localization tag of a human ER resident protein, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER. For example, as demonstrated in the experimental section herein, various aspects of the present disclosure include novel human ER localization tags that find use in localizing proteins to the ER. According to some embodiments, the human ER resident protein is CDGSH iron-sulfur domain 2 (CISD2). In certain embodiments, such an ER localization tag comprises a Tm domain, an ICD, or both, of a polypeptide set forth in SEQ ID NO:91, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER. According to some embodiments, the human ER resident protein is UDP glucuronosyltransferase family 2 member B17 (UGT2B17). In certain embodiments, such an ER localization tag comprises the Tm domain, the ICD, or both, of a polypeptide set forth in SEQ ID NO: 95, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0117] The fusion protein may be directly fused to the ER localization tag or may be indirectly fused via one or more domains, such as other protein coding domain(s) and / or linker(s). The fusion protein may further comprise a protease cleavage site, for example, located between the protein and the ER localization tag. The fusion protein may further comprise a membrane association domain, such as any of the transmembrane domains described elsewhere herein. The fusion protein may further comprise a protein localization tag, such as any of the protein localization tags described elsewhere herein. Nucleic acids encoding such fusion proteins, and expression vectors comprising such nucleic acids are also provided. Cells comprising such fusion proteins, nucleic acids, and / or expression vectors are also provided. Methods of producing such fusion proteins are also provided. In some embodiments, such methods comprise culturing a cell comprising an expression vector encoding the fusion protein under conditions suitable for the cell to express the fusion protein, where the fusion protein is produced.

[0118] Also provided is a fusion protein comprising a protein fused to a transmembrane domain, wherein the transmembrane domain is one of the following: WRLLPFLGVLALLGYLAVRPFL (SEQ ID NO: 42); VLWWSIAQTVILILTGIW (SEQ ID NO: 43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO: 44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO: 45); IAFLLACVATMIFMITKCCLF (SEQ ID NO: 46); VIGFLLAVVLTVAFITF (SEQ ID NO: 47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO: 48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO: 49); TFCSTALLITALALVCTLLYL (SEQ ID NO: 50); The amino acid sequence comprises, consists of, or is present within an amino acid sequence selected from: WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO: 51); WLWVVYILT VALPVFLVILFC (SEQ ID NO: 52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 53); and FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 54).

[0119] The fusion protein may be directly fused to the transmembrane domain or indirectly fused via one or more domains, such as other protein coding domain(s) and / or linker(s). The fusion protein may further comprise a protease cleavage site. The fusion protein may further comprise a membrane association domain, such as any of the transmembrane domains described elsewhere herein. The fusion protein may further comprise a protein localization tag, such as any of the protein localization tags described elsewhere herein. Nucleic acids encoding such fusion proteins, and expression vectors comprising such nucleic acids, are also provided. Cells comprising such fusion proteins, nucleic acids, and / or expression vectors are also provided. Methods of producing such fusion proteins are also provided. In some embodiments, such methods comprise culturing a cell comprising an expression vector encoding the fusion protein under conditions suitable for the cell to express the fusion protein, where the fusion protein is produced.

[0120] Examples of amino acid sequences of cell selection system components are provided in Table 1 below. For each sequence, the domains ordered from N-terminus to C-terminus are listed in the left column. The sequences in the right column indicate the domains by intermittent underlining. The present disclosure provides each of the proteins provided in Table 1, and each individual domain therein, as well as nucleic acids encoding such proteins and individual domains. Cells containing such proteins and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the proteins and individual domains therein, and in some examples, the variant protein or domain thereof comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to a parent / reference sequence, or a functional fragment thereof, wherein the variant retains the functionality (e.g., protease activity, cleavability by a protease, localization / retention (e.g., in the ER), and / or selectability by a cellular selection system, etc.) of the parent / reference sequence.

[0121] [Table 1-1]

[0122] [Table 1-2]

[0123] [Table 1-3]

[0124] [Table 1-4] TIFF2024513235000006.tif82170

[0125] [Table 1-5]

[0126]

Table 1-6

[0127]

Table 1-7

[0128]

Table 1-8

[0129]

Table 1-9

[0130]

Table 1-10

[0131]

Table 1-11

[0132]

Table 1-12

[0133]

Table 1-13

[0134]

Table 1-14

[0135]

Table 1-15

[0136]

Table 1-16

[0137]

Table 1-17

[0138]

Table 1-18

[0139]

Table 1-19

[0140]

Table 1-20

[0141]

Table 1-21

[0142]

Table 1-22

[0143]

Table 1-23

[0144]

Table 1-24

[0145]

Table 1-25

[0146] Cells, Compositions, and Methods of Use cell Cells are also provided by the present disclosure. According to some embodiments, cells are provided that include two or more separate expression constructs, where the two or more separate expression constructs include a first expression construct that encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The two or more separate expression constructs further include a second expression construct that encodes a protein required for cell surface expression of the selection marker. The first expression construct and / or the second expression construct may further encode a protein of interest, such as any of the proteins of interest described elsewhere herein. In certain embodiments, the first expression construct and / or the second expression construct are site-specifically integrated into the genome of the cell. The site-specific integration may result in the inactivation of one or more target genes in the genome of the cell.

[0147] The present disclosure also provides a cell, or a progeny thereof, selected according to the cell selection method of the present disclosure.

[0148] Cells of the present disclosure can be autologous / autogeneic ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autologous" refers to cells derived from the same individual to which they are subsequently administered. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the comparative cell. As used herein, "syngeneic" refers to cells of a different individual that are genetically identical to the comparative cell.

[0149] In some embodiments, the cell is a T cell obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0150] T cells can be obtained from many sources, including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of known techniques, such as sedimentation, e.g., FICOLL™ separation.

[0151] In some embodiments, an isolated or purified population of T cells is used. CTL and T H The lymphocytes are purified from PBMCs. CTL and T H Lymphocytes may be naive (T N ), Memory (T MEM ), stem cell memory (T SCM ), Central Memory (T CM ), Effector Memory (T EM ), and effector (T EFF ) T cell subpopulations. Suitable approaches for such selection are known in the art, for example, TN is selected from the CD45RA + CD62L + CD95 - TSCM is a method for detecting CD45RA + CD62L + CD95 + TCM is CD45RO + CD62L + CD95 + and TEM is CD45RO + CD62L - CD95 + An example of such a selection approach is described in Wang et al. (2016) Blood 127(24):2980-90.

[0152] Specific subpopulations of T cells expressing one or more of the following markers can be further isolated by positive or negative selection techniques. In some embodiments, specific subpopulations of T cells expressing one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38 are further isolated by positive or negative selection techniques. In some embodiments, the produced T cell compositions do not express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the produced T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0153] To achieve a therapeutically effective dose of the T cell composition, the T cells may be subjected to one or more rounds of stimulation, activation and / or expansion. T cells can generally be activated and expanded using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated by reference in its entirety for all purposes. In some embodiments, the T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, the T cells are activated and expanded for about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 3 days, about 2 to about 4 days, about 3 to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introducing a nucleic acid (e.g., an expression vector) encoding a polypeptide into the T cells.

[0154] In some embodiments, the T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours, or about 24 hours prior to introducing a nucleic acid (e.g., an expression vector) encoding a cell surface receptor into the T cells. In some embodiments, the T cells are activated simultaneously with the introduction of a nucleic acid (e.g., an expression vector) encoding a cell surface receptor into the T cells.

[0155] In some embodiments, suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) and one or more factors necessary for proliferation and survival, including, but not limited to, serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives suitable for growing cells known to one of skill in the art. Further representative examples of cell culture media include, but are not limited to, RPMI 1640, Click, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate and vitamins and are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in amounts sufficient for the growth and proliferation of T cells.

[0156] composition Compositions are also provided by the present disclosure. According to some embodiments, compositions are provided that include any of the cells of the present disclosure or their progeny, such as cells selected according to the methods of the present disclosure.

[0157] Such compositions may include cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffer, etc. One or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), lysis agents, detergents (e.g., non-ionic detergents such as Tween-20), nuclease inhibitors, glycerol, chelating agents, etc., may be present in such compositions. In certain embodiments, the liquid medium is a cell culture medium. Non-limiting examples of cell culture media include Minimal Essential Media, DMEM, a-MEM, RPMI Media, Clicks, F-12, X-Vivo 15, X-Vivo 20, Optimizer, and the like.

[0158] In certain embodiments, a pharmaceutical composition is provided that includes cells or progeny thereof selected according to the disclosed methods. The pharmaceutical composition may include such cells and a pharma- ceutically acceptable carrier. The pharmaceutical composition generally includes a therapeutically effective amount of cells. By "therapeutically effective amount" is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to produce a beneficial or desired therapeutic result (including a prophylactic result), such as the alleviation of symptoms of a disease (e.g., cancer) or disorder associated with a target cell or population thereof (e.g., cancer cells) compared to a control. An effective amount can be administered in one or more administrations. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount effective to "treat" an individual, e.g., a patient. When a therapeutic amount is indicated, the exact amount of the composition contemplated in a particular embodiment to be administered can be determined by the physician, taking into account the specifications and individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, the pharmaceutical composition of the present disclosure is administered in an amount of 1×10 6 ~5×10 10 The present disclosure includes the cells of the present disclosure.

[0159] The cells of the present disclosure can be incorporated into various formulations for therapeutic administration. More specifically, the cells of the present disclosure can be formulated into a pharmaceutical composition by combining with a suitable pharma- ceutically acceptable excipient or diluent.

[0160] Preparations of cells suitable for administration to a patient (e.g., suitable for human administration) will generally be sterile and further will be free of detectable pyrogens or other contaminants that would contraindicate administration to a patient according to a selected route of administration.

[0161] The cells may be formulated for parenteral (e.g., intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0162] Pharmaceutical compositions comprising the cells of the present disclosure can be prepared by mixing the cells having the desired purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, glycine, glycerol ... amino acids such as sucrose, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid, and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0163] Aqueous formulations of cells can be prepared in pH buffers, for example, at a pH in the range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or about 5.5. Examples of buffers suitable for a pH in this range include phosphate buffers, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and other organic acid buffers. The buffer concentration can be, for example, about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0164] To adjust the tonicity of the formulation, an isotonicity agent may be included in the formulation. Examples of isotonicity agents include sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids, sugars, and combinations thereof. In some embodiments, the aqueous formulation is an isotonic solution, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological saline or serum. The isotonicity agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 100 mM to 350 mM.

[0165] Surfactants may also be added to the formulation to reduce aggregation in the formulation and / or minimize the formation of fine particles and / or reduce adsorption. Examples of surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of the surfactant can range from about 0.001% to about 1% (w / v).

[0166] In some embodiments, the pharmaceutical composition comprises the cells of the present disclosure and one or more of the agents set forth above (e.g., surfactants, buffers, stabilizers, isotonicity agents), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, for example, at a concentration ranging from about 0.001 to about 2% (w / v).

[0167] How to use The present disclosure also provides methods of using the cells and compositions of the present disclosure, in certain embodiments, the methods comprise administering to an individual in need thereof a therapeutically effective amount of any of the pharmaceutical compositions of the present disclosure.

[0168] In some embodiments, the individual in need thereof has cancer, and one or more of the two or more distinct expression constructs encodes a receptor (such as, for example, a CAR and / or a TCR) that binds to a molecule on the surface of a cancer cell. The pharmaceutical composition typically includes a therapeutically effective amount of such cells as described above. The cell can be any cell capable of performing the desired treatment. In some embodiments, the cell is an immune cell. Non-limiting examples of immune cells that may be administered include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cell is a T cell. According to some embodiments, the cell is a T cell, and the protein of interest expressed by one or more of the two or more expression constructs is a CAR, such that the cell is a CAR T cell. In certain embodiments, the cell is a stem cell, such as an embryonic stem cell or an adult stem cell. In some embodiments, the pharmaceutical composition is an autologous composition produced by a method comprising removing cells from an individual, introducing two or more desired expression constructs into the removed cells or their progeny, and subsequently selecting such cells based on cell surface expression of a selection marker.

[0169] In certain embodiments, the individual in need thereof has a cell proliferative disorder. By "cell proliferative disorder" is meant a disorder in which unwanted cell proliferation of one or more subsets of cells of a multicellular organism occurs, resulting in harm to the organism (e.g., pain or reduced life expectancy). Cell proliferative disorders include, but are not limited to, cancer, precancer, benign tumors, vascular proliferative disorders (e.g., arthritis, restenosis, etc.), fibrotic disorders (e.g., hepatic cirrhosis, atherosclerosis, etc.), psoriasis, epidermal and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteogenic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.

[0170] In some embodiments, the individual has cancer. The subject method can be used to treat a wide variety of cancers. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, or thyroid cancer, liver cancer, various types of head and neck cancer, and the like. In certain embodiments, the individual has a cancer selected from a solid tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube carcinoma, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In certain aspects, the individual has a cancer selected from melanoma, Hodgkin's lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0171] kit Kits are also provided by the present disclosure. In certain embodiments, kits are provided that include any reagents used in carrying out the methods of the present disclosure. By way of example, in certain embodiments, kits are provided that include a first expression construct that encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site located between the selection marker and the protein localization tag, and a second expression construct that encodes a protein required for cell surface expression of the selection marker. The first expression construct and / or the second expression construct may further include a cloning site for a nucleic acid encoding a protein of interest. In certain embodiments, the first expression construct and / or the second expression construct further encodes one or more proteins of interest, such as any of the proteins of interest described elsewhere herein.

[0172] The kits of the present disclosure may further include any other reagents useful for practicing the methods of the present disclosure, such as transfection / transduction reagents useful for introducing the expression construct into a cell of interest, such as an immune cell (e.g., a T cell) or other cell of interest.

[0173] The components of the kit may be in separate containers, or multiple components may be in a single container. For example, a first expression construct and a second expression construct may be provided in separate containers or in the same container. Suitable containers include single tubes (e.g., vials), plates of one or more wells (e.g., 96-well plates, 384-well plates, etc.), and the like.

[0174] The kits of the present disclosure may further comprise instructions for contacting a cell population with two or more expression constructs under conditions whereby the two or more expression constructs are delivered to cells of the cell population.The kits of the present disclosure may further comprise instructions for selecting cells that exhibit cell surface expression of a selection marker.

[0175] The instructions for the kit can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Thus, the instructions can be present in the kit as a package insert, in the label of the container of the kit or a component thereof (i.e., associated with the packaging or subpackaging), and the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, such as a portable flash drive, DVD, CD-ROM, diskette, and the like. In still other embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0176] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following embodiments: 1. A method for selecting cells containing two or more distinct expression constructs, comprising: contacting a cell population with two or more separate expression constructs under conditions such that the two or more expression constructs are delivered to cells of the cell population, the two or more separate expression constructs comprising: a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag; and a second expression construct encoding a protein required for cell surface expression of the selectable marker; and selecting cells exhibiting cell surface expression of the selection marker; A method comprising:

[0177] 2. The method of embodiment 1, wherein said first expression construct further encodes a protein of interest.

[0178] 3. The method of embodiment 1 or embodiment 2, wherein the first expression construct is site-specifically integrated into the genome of the cell.

[0179] 4. The method of embodiment 3, wherein site-specific integration of said first expression construct into said genome of said cell inactivates a target gene within said genome of said cell.

[0180] 5. The method of any one of embodiments 1 to 4, wherein the second expression construct further encodes a protein of interest.

[0181] 6. The method of any one of embodiments 1 to 5, wherein the second expression construct integrates site-specifically into the genome of the cell.

[0182] 7. The method of embodiment 6, wherein site-specific integration of said second expression construct into said genome of said cell inactivates a target gene within said genome of said cell.

[0183] 8. The method of any one of embodiments 1 to 7, wherein the protein localization tag is selected from the group consisting of an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosomal localization tag, a plasma membrane localization tag, a mitochondrial localization tag, a peroxisomal localization tag, a cytoplasmic localization tag, and a nuclear localization tag.

[0184] 9. The method of any one of embodiments 1 to 7, wherein the protein localization tag is an ER localization tag.

[0185] 10. The method of embodiment 9, wherein said ER localization tag comprises the amino acid sequence KKMP.

[0186] 11. The ER localization tag is selected from the group consisting of: LYKYKSRRSFIDEKKMP (SEQ ID NO: 1); AEKDEL (SEQ ID NO: 2); EQKLISEEDLKDEL (SEQ ID NO: 3); GGGGSGGGGSKDEL (SEQ ID NO: 4); GGGGSGGGGSGGGGGSGGGGSKDEL (SEQ ID NO: 5); GGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO: 6); KYKSRRSFIEEKKMP (SEQ ID NO: 7); LKYKSRRSFIEEKKMP (SEQ ID NO: 8); LYKYKSRRSFIEEKKMP (SEQ ID NO: 9); LYCKYKSRRSFIEEKKMP (SEQ ID NO: 10); LYCNKYKSRRSFIEEKKMP (SEQ ID NO: 11); LYCNKYKSRRSFIDEKKMP (SEQ ID NO: 12); LYEQKLISEEDLKY The method of embodiment 9, comprising 85% or more, 90% or more, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within an amino acid sequence selected from the group consisting of KSRRSFIEEKKMP (SEQ ID NO: 13); LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO: 14); LYKKLETFKKTN (SEQ ID NO: 15); LYEQKLISEEDLKKLETFKKTN (SEQ ID NO: 16); LYYQRL (SEQ ID NO: 17); LYEQKLISEEDLYQRL (SEQ ID NO: 18); LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO: 19); LYRNIKCD (SEQ ID NO: 20); and LYEQKLISEEDLRNIKCD (SEQ ID NO: 21).

[0187] 12. The ER localization tag is selected from the group consisting of: PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCCCGCFGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO:31); NRSPRNRKPRRE (SEQ ID NO:32); LYKYKSRRSFIEEKKMP (SEQ ID NO:9); TKVLKGKKLSLPA (SEQ ID NO:33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and 10. The method of embodiment 9, comprising 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within an amino acid sequence selected from the group consisting of: KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35).

[0188] 13. The method of embodiment 11 or embodiment 12, wherein the C-terminus of the ER localization tag comprises the four C-terminal residues of one of the sequences listed in embodiment 11 or embodiment 12.

[0189] 14. The method of embodiment 9, wherein the ER localization tag comprises the transmembrane (Tm) domain, the intracellular domain (ICD), or both, of an ER localization tag of a polypeptide listed in Table 1, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0190] 15. The method of embodiment 9, wherein the ER localization tag comprises the Tm domain, ICD, or both, of an ER localization tag of a human ER resident protein, or a variant Tm and / or ICD thereof that retains the ability to localize a polypeptide to the ER.

[0191] 16. The method of embodiment 15, wherein the human ER resident protein is CDGSH iron-sulfur domain 2 (CISD2).

[0192] 17. The method of embodiment 16, wherein the ER localization tag comprises the Tm domain of the polypeptide set forth in SEQ ID NO:91, the ICD, or both, or a variant Tm and / or ICD thereof that retains the ability to localize a polypeptide to the ER.

[0193] 18. The method of embodiment 15, wherein the human ER resident protein is UDP glucuronosyltransferase family 2 member B17 (UGT2B17).

[0194] 19. The method of embodiment 18, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO: 95, or a variant Tm and / or ICD thereof that retains the ability to localize a polypeptide to the ER.

[0195] 20. The method of any one of embodiments 1 to 7, wherein the protein localization tag is a Golgi localization tag.

[0196] 21. The method of embodiment 20, wherein the Golgi localization tag comprises the amino acid sequence YQRL (SEQ ID NO:36).

[0197] 22. The method of any one of embodiments 1 to 7, wherein the protein localization tag is a lysosomal localization tag.

[0198] 23. The method of embodiment 22, wherein the lysosomal localization tag comprises the amino acid sequence KFERQ (sequence number 37).

[0199] 24. The method of any one of embodiments 1 to 23, wherein the protease cleavage site is a viral protease cleavage site.

[0200] 25. The method of embodiment 24, wherein the viral protease cleavage site is a cleavage site for a Potyviridae protease.

[0201] 26. The method of embodiment 25, wherein the Potyviridae protease is Tobacco etch virus (TEV) protease, Plum pox virus protease (PPVp), Soybean mosaic virus protease (SbMVp), Sunflower mild mosaic virus protease (SuMMVp), Tobacco vein mottle virus protease (TVMVp), or West Nile virus protease (WNVp).

[0202] 27. The method of embodiment 25, wherein the viral protease cleavage site is a TEV protease cleavage site.

[0203] 28. The method of embodiment 24, wherein the viral protease cleavage site is for a viral protease derived from Hepatitis C virus (HCV) nonstructural protein 3 (NS3).

[0204] 29. The method of embodiment 28, wherein the viral protease cleavage site is for a viral protease that further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A).

[0205] 30. The method of embodiment 28 or embodiment 29, wherein the viral protease cleavage site is selected from the group consisting of an NS4A / 4B junction cleavage site, an NS3 / NS4A junction cleavage site, an NS4A / NS4B junction cleavage site, an NS4B / NS5A junction cleavage site, an NS5A / NS5B junction cleavage site, and variants thereof cleavable by the viral protease.

[0206] 31. The method of any one of embodiments 1 to 23, wherein the protease cleavage site is a human protease cleavage site.

[0207] 32. The method of embodiment 31, wherein the human protease cleavage site is a cleavage site for a human protease selected from the group consisting of human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloprotease (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, and human cathepsin.

[0208] 33. The method of embodiment 32, wherein the human kallikrein protease is selected from the group consisting of human KLK3, human KLK4, human KLK6, human KLK8, human KLK11, human KLK13, human KLK14, and human KLK15.

[0209] 34. The method of any one of embodiments 1 to 33, wherein said protein required for cell surface expression of said selectable marker is a protease and said protease cleavage site is a cleavage site for said protease.

[0210] 35. The method of embodiment 34, wherein the protease is fused to a protein localization tag that localizes the protease in the same cellular compartment as the fusion protein containing the selectable marker.

[0211] 36. The method of embodiment 35, wherein the protease is fused to a protein localization tag having the same amino acid sequence as the protein localization tag of the fusion protein comprising the selection marker.

[0212] 37. The method of embodiment 35 or embodiment 36, wherein the protease is fused to a membrane association domain.

[0213] 38. The method of embodiment 37, wherein the membrane-associated domain is a transmembrane domain.

[0214] 39. The method of embodiment 38, wherein the transmembrane domain is the CD8α transmembrane domain.

[0215] 40. The method of embodiment 38, wherein the transmembrane domain is the CD28 transmembrane domain.

[0216] 41. The transmembrane domain is selected from the group consisting of: WLRLLPFLGVLALLGYLAVRPFL (SEQ ID NO:42); VLWWSIAQTVILILTGIW (SEQ ID NO:43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO:44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO:45); IAFLLACVATMIFMITKCCLF (SEQ ID NO:46); VIGFLLAVVLTVAFITF (SEQ ID NO:47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO:48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO:49); TFCSTALLITALALVCTLLYL (SEQ ID NO:50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO:51); WLWVVYILT VALPVFLVILFC (SEQ ID NO:52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:53); and 39. The method of embodiment 38, comprising 80% or more, 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to a transmembrane domain comprising, consisting of, or present within an amino acid sequence selected from the group consisting of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:54).

[0217] 42. The method of embodiment 38 or embodiment 41, wherein the protease is fused to a hinge domain.

[0218] 43. The method of embodiment 42, wherein the hinge domain is a CD8α hinge domain.

[0219] 44. The method of embodiment 34, wherein the protease is fused to a dimerization domain.

[0220] 45. The method of embodiment 44, wherein the method comprises contacting the cell population with a third expression construct encoding a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the protease, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker.

[0221] 46. ​​The method of embodiment 45, wherein the third expression construct further encodes a protein of interest.

[0222] 47. The method of embodiment 45 or embodiment 46, wherein the first expression construct is site-specifically integrated into the genome of the cell.

[0223] 48. The method of embodiment 47, wherein site-specific integration of the first expression construct into the genome of the cell inactivates a target gene within the genome of the cell.

[0224] 49. The method of any one of embodiments 1 to 33, wherein the protein required for cell surface expression of the selection marker is a first complementary fragment of a protease and the protease cleavage site is a cleavage site for the protease.

[0225] 50. The method of embodiment 49, wherein the two or more expression constructs include a third expression construct encoding a second complementary fragment of the protease, and the first complementary fragment and the second complementary fragment form an active protease complex.

[0226] 51. The method of embodiment 50, wherein the third expression construct further encodes a protein of interest.

[0227] 52. The method of embodiment 50 or embodiment 51, wherein the first expression construct is site-specifically integrated into the genome of the cell.

[0228] 53. The method of embodiment 52, wherein site-specific integration of the first expression construct into the genome of the cell inactivates a target gene in the genome of the cell.

[0229] 54. The method of any one of embodiments 50 to 53, wherein the first complementary fragment and the second complementary fragment are each fused to a protein localization tag that localizes the first complementary fragment and the second complementary fragment in the same cellular compartment as the fusion protein containing the selection marker.

[0230] 55. The method of embodiment 54, wherein each of the first complementary fragment and the second complementary fragment is fused to a protein localization tag having the same amino acid sequence as the protein localization tag of the fusion protein comprising the selection marker.

[0231] 56. The method of any one of embodiments 50 to 55, wherein each of the first and second complementary fragments is fused to a membrane-associated domain.

[0232] 57. The method of embodiment 56, wherein the membrane-associated domain is a transmembrane domain.

[0233] 58. The method of embodiment 57, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.

[0234] 59. The method of embodiment 50, wherein each of the first complementary fragment and the second complementary fragment is fused to a dimerization domain.

[0235] 60. The two or more expression constructs comprise: a fourth expression construct encoding a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the first complementary fragment, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker; a fifth expression construct encoding a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the second complementary fragment, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker; 60. The method of embodiment 59, comprising:

[0236] 61. The method of embodiment 60, wherein the fourth expression construct further encodes a protein of interest.

[0237] 62. The method of embodiment 60 or embodiment 61, wherein the fourth expression construct is site-specifically integrated into the genome of the cell.

[0238] 63. The method of embodiment 62, wherein site-specific integration of the fourth expression construct into the genome of the cell inactivates a target gene in the genome of the cell.

[0239] 64. The method of any one of embodiments 60 to 63, wherein the fifth expression construct further encodes a protein of interest.

[0240] 65. The method of any one of embodiments 60 to 64, wherein the fifth expression construct integrates site-specifically into the genome of the cell.

[0241] 66. The method of embodiment 65, wherein site-specific integration of the fourth expression construct into the genome of the cell inactivates a target gene in the genome of the cell.

[0242] 67. The method of any one of embodiments 60-66, wherein the membrane-associated domain of the fusion protein encoded by each of the fourth and fifth expression constructs is, independently, a transmembrane domain as defined in any one of embodiments 39-41.

[0243] 68. The method of any one of embodiments 44, 45, or 59-67, wherein the dimerization domain comprises a coiled-coil structure.

[0244] 69. The method of embodiment 68, wherein the dimerization domain comprises a leucine zipper domain.

[0245] 70. The method of any one of embodiments 2-69, wherein the protein of interest further encoded by one or more of the two or more separate expression constructs is independently selected from the group consisting of a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein that confers resistance to cellular exhaustion, and a suicide switch protein.

[0246] 71. The method of embodiment 70, wherein the protein of interest further encoded by one or more of the two or more separate expression constructs is a receptor.

[0247] 72. The method of embodiment 71, wherein the receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a systemic extracellular molecular sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor.

[0248] 73. The method of embodiment 72, wherein the receptor is CAR.

[0249] 74. The method of any one of embodiments 1 to 73, wherein the selection marker comprises a protein tag.

[0250] 75. The method of embodiment 74, wherein the protein tag is selected from the group consisting of Myc-tag, His-tag, HA-tag, FLAG-tag, Strep-tag, NE-tag, Xpress-tag, Avi-tag, polyglutamic acid tag, and polyarginine tag.

[0251] 76. The method of any one of embodiments 1 to 75, wherein the selection marker comprises a cluster of differentiation (CD) protein.

[0252] 77. The method of embodiment 76, wherein the CD protein is CD34.

[0253] 78. The method of any one of embodiments 1-75, wherein the selection marker comprises a truncated receptor comprising the extracellular domain of the receptor.

[0254] 79. The method of embodiment 78, wherein the truncated receptor is a truncated epidermal growth factor receptor (EGFRt), a truncated nerve growth factor receptor (NGFRt), a truncated CD19 (CD19t), or a truncated CD20 (CD20t).

[0255] 80. The method of any one of embodiments 1 to 79, wherein the selection marker is fused to a membrane-associated domain.

[0256] 81. The method of embodiment 80, wherein the membrane-associated domain is a transmembrane domain as defined in any one of embodiments 39 to 41.

[0257] 82. The method of any one of embodiments 1 to 81, wherein the fusion protein encoded by the first expression construct further comprises a degron, and the protease cleavage site is located between the selection marker and the degron.

[0258] 83. The method of any one of embodiments 1-82, wherein the fusion protein encoded by the first expression construct further comprises a domain that confers antibiotic resistance.

[0259] 84. The method of embodiment 83, wherein the domain that confers antibiotic resistance is located between the selection marker and the protease cleavage site.

[0260] 85. The method of embodiment 83 or embodiment 84, wherein the domain that confers antibiotic resistance confers puromycin resistance.

[0261] 86. The method of embodiment 85, wherein the domain that confers puromycin resistance comprises puromycin-N-acetyltransferase (PuroR).

[0262] 87. The method of any one of embodiments 1 to 86, wherein the selecting comprises magnetic activated cell sorting (MACS).

[0263] 88. The method of any one of embodiments 1 to 86, wherein the selecting comprises flow cytometry.

[0264] 89. The method of embodiment 88, wherein said flow cytometry comprises fluorescence-activated cell sorting (FACS).

[0265] 90. The method of any one of embodiments 1-89, wherein the cell population is a mammalian cell population.

[0266] 91. The method of embodiment 90, wherein the mammalian cells comprise immune cells.

[0267] 92. The method of embodiment 91, wherein the immune cells include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, eosinophils, and any combination thereof.

[0268] 93. The method of embodiment 91, wherein the immune cells comprise T cells.

[0269] 94. The T cells are naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), gamma delta T cells (T γδ 94. The method of embodiment 93, comprising:

[0270] 95. The method of embodiment 90, wherein the mammalian cells comprise stem cells.

[0271] 6. The method of embodiment 95, wherein the stem cells comprise embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), or any combination thereof.

[0272] 97. A cell comprising two or more separate expression constructs, the two or more separate expression constructs comprising: a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site located between the selection marker and the protein localization tag; a second expression construct encoding proteins required for cell surface expression of the selectable marker; , comprising a cell.

[0273] 98. The cell of embodiment 97, wherein the first expression construct further encodes a protein of interest.

[0274] 99. The cell of embodiment 97 or embodiment 98, wherein the first expression construct is site-specifically integrated into the genome of the cell.

[0275] 100. The cell of embodiment 99, wherein a target gene in the genome of the cell is inactivated as a result of the site-specific integration of the first expression construct.

[0276] 101. The cell of any one of embodiments 97 to 100, wherein the second expression construct further encodes a protein of interest.

[0277] 102. The cell of any one of embodiments 97 to 101, wherein the second expression construct is site-specifically integrated into the genome of the cell.

[0278] 103. The cell of embodiment 102, wherein a target gene in the genome of the cell is inactivated as a result of the site-specific integration of the second expression construct.

[0279] 104. The cell according to any one of embodiments 97 to 103, wherein the cell is a mammalian cell.

[0280] 105. The cell of embodiment 104, wherein the mammalian cell is a human cell.

[0281] 106. The cell described in embodiment 104 or embodiment 105, wherein the cell is an immune cell.

[0282] 107. The cell of embodiment 106, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, or an eosinophil.

[0283] 108. The cell described in embodiment 106, wherein the immune cell is a T cell.

[0284] 109. The T cells are naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REGs ), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), or gamma delta T cells (T γδ 109. The cell of embodiment 108, wherein said cell is

[0285] 110. The cell described in embodiment 104 or embodiment 105, wherein the cell is a stem cell.

[0286] 111. The cell described in embodiment 110, wherein the stem cell is an embryonic stem (ES) cell, an adult stem cell, a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), or a neural stem cell (NSC).

[0287] 112. A kit comprising two or more separate expression constructs, the two or more separate expression constructs comprising: a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site located between the selection marker and the protein localization tag; a second expression construct encoding proteins required for cell surface expression of the selectable marker; Including the kit.

[0288] 113. The kit of embodiment 112, wherein the first expression construct further encodes a protein of interest.

[0289] 114. The kit of embodiment 112, wherein the first expression construct comprises a cloning site for a nucleic acid encoding a protein of interest.

[0290] 115. A kit according to any one of embodiments 112 to 114, wherein the second expression construct further encodes a protein of interest.

[0291] 116. A kit according to any one of embodiments 112 to 114, wherein the second expression construct comprises a cloning site for a nucleic acid encoding a protein of interest.

[0292] 117. The kit of any one of embodiments 112-116, further comprising instructions for contacting the cell population with the two or more expression constructs under conditions such that the two or more expression constructs are delivered to cells of the cell population.

[0293] 118. The kit of any one of embodiments 112-117, further comprising instructions for selecting cells exhibiting cell surface expression of said selection marker.

[0294] 119. The cell according to any one of embodiments 97 to 118, wherein the protein localization tag is as defined in any one of embodiments 8 to 23.

[0295] 120. The cell or kit according to any one of embodiments 97 to 119, wherein the protease cleavage site tag is as defined in any one of embodiments 24 to 33.

[0296] 121. The cell or kit according to any one of embodiments 97 to 120, wherein the protein required for cell surface expression of the selection marker is a protease and the protease cleavage site is a cleavage site for the protease.

[0297] 122. The cell or kit according to embodiment 121, wherein the protease is fused to a protein localization tag that localizes the protease in the same cellular compartment as the fusion protein containing the selection marker.

[0298] 123. The cell or kit according to embodiment 122, wherein the protease is fused to a protein localization tag having the same amino acid sequence as the protein localization tag of the fusion protein comprising the selection marker.

[0299] 124. The cell or kit according to embodiment 122 or embodiment 123, wherein the protease is fused to a membrane-associated domain.

[0300] 125. The cell or kit according to embodiment 124, wherein the membrane-associated domain is a transmembrane domain as defined in any one of embodiments 39 to 41.

[0301] 126. The cell or kit according to embodiment 121, wherein the protease is fused to a dimerization domain.

[0302] 127. The cell or kit of embodiment 126, comprising a third expression construct encoding a fusion protein comprising a transmembrane domain, a dimerization domain that dimerizes with the dimerization domain fused to the protease, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker.

[0303] 128. The cell or kit according to any one of embodiments 97 to 120, wherein the protein required for cell surface expression of the selection marker is a first complementary fragment of a protease and the protease cleavage site is a cleavage site for the protease.

[0304] 129. The cell or kit of embodiment 128, comprising a third expression construct encoding a second complementary fragment of the protease, wherein the first complementary fragment and the second complementary fragment form an active protease complex.

[0305] 130. The cell or kit of embodiment 129, wherein the third expression construct further encodes a protein of interest.

[0306] 131. The cell or kit of embodiment 129 or embodiment 130, wherein each of the first complementary fragment and the second complementary fragment is fused to a protein localization tag that localizes the protease in the same cellular compartment as the fusion protein containing the selection marker.

[0307] 132. The cell or kit of embodiment 131, wherein each of the first complementary fragment and the second complementary fragment is fused to a protein localization tag having the same amino acid sequence as the protein localization tag of the fusion protein comprising the selection marker.

[0308] 133. The cell or kit according to any one of embodiments 129 to 132, wherein each of the first and second complementary fragments is fused to a membrane-associated domain.

[0309] 134. The cell or kit according to embodiment 133, wherein the membrane-associated domain is a transmembrane domain.

[0310] 135. The cell or kit according to embodiment 134, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.

[0311] 136. The cell or kit according to any one of embodiments 129 to 132, wherein each of the first and second complementary fragments is fused to a dimerization domain.

[0312] 137. Below: a fourth expression construct encoding a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the first complementary fragment, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker; a fifth expression construct encoding a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the second complementary fragment, and a protein localization tag that localizes the dimerization domain in the same cellular compartment as the fusion protein containing the selection marker; 137. The cell or kit of embodiment 136, comprising:

[0313] 138. The cell or kit of embodiment 137, wherein the fourth expression construct further encodes a protein of interest.

[0314] 139. The cell or kit according to embodiment 137 or embodiment 138, wherein the fifth expression construct further encodes a protein of interest.

[0315] 140. The cell or kit of any one of embodiments 137 to 139, wherein the membrane-associated domain of the fusion protein encoded by each of the fourth and fifth expression constructs is, independently, a transmembrane domain as defined in any one of embodiments 39 to 41.

[0316] 141. The cell or kit according to any one of embodiments 126, 127, or 136 to 140, wherein the dimerization domain comprises a coiled-coil structure.

[0317] 142. The cell or kit of embodiment 141, wherein the dimerization domain comprises a leucine zipper domain.

[0318] 143. The cell or kit of any one of embodiments 97 to 142, wherein the protein of interest further encoded by one or more of the two or more separate expression constructs is independently selected from the group consisting of a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein that confers resistance to cell exhaustion, and a suicide switch protein.

[0319] 144. The cell or kit according to embodiment 143, wherein the protein of interest further encoded by one or more of the two or more separate expression constructs is a receptor.

[0320] 145. The cell or kit of embodiment 144, wherein the receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a systemic extracellular molecular sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor.

[0321] 146. The cell or kit according to embodiment 144, wherein the receptor is a CAR.

[0322] 147. The cell or kit according to any one of embodiments 97 to 146, wherein the selection marker is as defined in any one of embodiments 74 to 81.

[0323] 148. The cell or kit according to any one of embodiments 97 to 147, wherein the fusion protein encoded by the first expression construct further comprises a degron, and the protease cleavage site is located between the selection marker and the degron.

[0324] 149. The cell or kit according to any one of embodiments 97 to 148, wherein the fusion protein encoded by the first expression construct further comprises a domain that confers antibiotic resistance.

[0325] 150. The cell or kit according to embodiment 149, wherein the domain conferring antibiotic resistance is located between the selection marker and the protease cleavage site.

[0326] 151. The cell or kit according to embodiment 149 or embodiment 150, wherein the domain that confers antibiotic resistance confers puromycin resistance.

[0327] 152. The cell or kit according to embodiment 151, wherein the domain that confers puromycin resistance comprises puromycin-N-acetyltransferase (PuroR).

[0328] 153. A composition comprising a cell or a progeny thereof selected according to the method of any one of embodiments 1 to 96, present in a liquid medium.

[0329] 154. A composition comprising a cell according to any one of embodiments 97 to 111 or embodiments 119 to 152, which is present in a liquid medium.

[0330] 155. The composition according to embodiment 153 or embodiment 154, wherein the liquid medium is a cell culture medium.

[0331] 156. The composition according to embodiment 153 or embodiment 154, wherein the liquid medium is suitable for administration of the composition to an individual in need thereof.

[0332] 157. The composition of embodiment 156, formulated for parenteral administration to the individual.

[0333] 158. A method comprising administering a therapeutically effective amount of the composition of embodiment 156 or embodiment 157 to an individual in need thereof.

[0334] 159. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag is one of the following: PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCCCGCFGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO:31); NRSPRNRKPRRE (SEQ ID NO:32); LYKYKSRRSFIEEKKMP (SEQ ID NO:9); TKVLKGKKLSLPA (SEQ ID NO:33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and A fusion protein comprising an amino acid sequence selected from the group consisting of: KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35), consisting of, or comprising 80% or more, 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to an ER localization tag present within the same.

[0335] 160. The fusion protein of embodiment 159, wherein the C-terminus of the ER localization tag comprises the four C-terminal residues of one of the sequences listed in embodiment 159.

[0336] 161. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises the Tm domain, ICD, or both, of an ER localization tag of a polypeptide listed in Table 1, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0337] 162. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises the Tm domain, ICD, or both, of an ER localization tag of a human ER resident protein, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0338] 163. The fusion protein of embodiment 162, wherein the human ER resident protein is CISD2.

[0339] 164. The fusion protein of embodiment 163, wherein the ER localization tag comprises the Tm domain of the polypeptide set forth in SEQ ID NO: 91, the ICD, or both, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0340] 165. The fusion protein of embodiment 162, wherein the human ER resident protein is UGT2B17.

[0341] 166. The fusion protein of embodiment 165, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO: 95, or a variant Tm and / or ICD thereof that retains the ability to localize the polypeptide to the ER.

[0342] 167. The fusion protein of embodiment 159, wherein the protein is fused directly to the ER localization tag.

[0343] 168. The fusion protein of embodiment 159, wherein the protein is indirectly fused to the ER localization tag.

[0344] 169. A fusion protein according to any one of embodiments 159 to 168, further comprising a protease cleavage site.

[0345] 170. The fusion protein of embodiment 169, wherein the protease cleavage site is located between the protein and the ER localization tag.

[0346] 171. The fusion protein according to embodiment 169 or embodiment 170, wherein the protease cleavage site is as defined in any one of embodiments 24 to 33.

[0347] 172. A fusion protein according to any one of embodiments 159 to 171, further comprising a transmembrane domain.

[0348] 173. The fusion protein according to embodiment 172, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.

[0349] 174. A fusion protein comprising a protein fused to a transmembrane domain, the transmembrane domain being: WLRLLPFLGVLALLGYLAVRPFL (SEQ ID NO:42); VLWWSIAQTVILILTGIW (SEQ ID NO:43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO:44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO:45); IAFLLACVATMIFMITKCCLF (SEQ ID NO:46); VIGFLLAVVLTVAFITF (SEQ ID NO:47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO:48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO:49); TFCSTALLITALALVCTLLYL (SEQ ID NO:50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO:51); WLWVVYILT VALPVFLVILFC (SEQ ID NO:52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:53); and A fusion protein comprising an amino acid sequence selected from the group consisting of: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:54), or comprising 80% or more, 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to a transmembrane domain present therein.

[0350] 175. The fusion protein of embodiment 174, wherein the protein is fused directly to the transmembrane domain.

[0351] 176. The fusion protein of embodiment 174, wherein the protein is indirectly fused to the transmembrane domain.

[0352] 177. The fusion protein according to any one of embodiments 174 to 176, further comprising a protease cleavage site.

[0353] 178. The fusion protein according to embodiment 177, wherein the protease cleavage site is as defined in any one of embodiments 24 to 33.

[0354] 179. The fusion protein of any one of embodiments 174 to 177, further comprising a protein localization tag.

[0355] 180. The fusion protein according to embodiment 179, wherein the protein localization tag is as defined in any one of embodiments 8 to 23.

[0356] 181. The fusion protein of any one of embodiments 159 to 180, wherein the protein is a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein that confers resistance to cell exhaustion, and a suicide switch protein.

[0357] 182. The fusion protein of any one of embodiments 159 to 180, wherein the protein is a receptor selected from the group consisting of chimeric antigen receptors (CARs), T cell receptors (TCRs), synthetic Notch (SynNotch) receptors, modular extracellular sensor architecture (MESA) receptors, Tango receptors, ChaCha receptors, systemic extracellular molecular sensor (GEMS) receptors, cytokine receptors, chemokine receptors, switch receptors, adhesion molecules, integrins, inhibitory receptors, stimulatory receptors, immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, and immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors.

[0358] 183. The fusion protein of embodiment 182, wherein the receptor is a CAR.

[0359] 184. The fusion protein according to any one of embodiments 159 to 176, wherein the protein is a selection marker.

[0360] 185. A nucleic acid encoding a fusion protein according to any one of embodiments 159 to 184.

[0361] 186. An expression construct comprising a nucleic acid according to embodiment 185.

[0362] 187. A cell comprising a nucleic acid according to embodiment 185 or an expression construct according to embodiment 186.

[0363] 188. A method for producing a fusion protein according to any one of embodiments 159 to 184, comprising culturing a cell according to embodiment 186 or embodiment 187 under conditions suitable for the cell to express the fusion protein, thereby producing the fusion protein.

[0364] The following examples are offered by way of illustration and not by way of limitation.

[0365] experiment Selection of cells carrying multiple genetic modifications using a single selectable marker Described herein is a cell selection system according to embodiments of the present disclosure. This system may be referred to herein as a "STASH selection system," "STASH selection," or the like, because in the absence of a desired combination of expression constructs present in a cell, a selection marker is "stashed" in the cell. According to the selection system, one of the expression constructs encodes a fusion protein that includes a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. In the absence of one or more additional expression constructs that provide a protease capable of cleaving the protease cleavage site, the selection marker remains localized (i.e., retained or stored) in a subcellular location (e.g., an organelle) determined by the particular protein localization tag used. When one or more additional expression constructs are present in the cell, thereby providing a protease capable of cleaving the protease cleavage site, the selection marker is cleaved from the protein localization tag and transported to the surface of the cell, such that cells containing the desired multiple genetic modifications exhibit cell surface expression of the selection marker. Non-limiting examples and data are then described that provide proof of concept of a cell selection system according to embodiments of the present disclosure.

[0366] High surface expression of Myc-tagged selectable marker in cells double positive for separate expression constructs A and B (NS3 protease) A schematic of an exemplary embodiment of the STASH selection system is shown in Figure 3A. Expression construct A encodes a Myc-tag fused to the N-terminus of a fusion protein containing a CD8a hinge, a CD8 transmembrane, a GFP, an HCV NS3 cleavage site, and an ER retention tag. Expression construct B consists of a BFP-P2A-membrane-tethered HCV NS3 protease fused to an ER retention tag.

[0367] FIG. 3B is a series of flow cytometry histograms of surface Myc staining in primary human T cells retrovirally transduced with expression construct A and expression construct B (from FIG. 3A). As can be seen in the data, only cells engineered with expression construct A and expression construct B have high surface expression of the Myc tag. Cells exposed to expression construct A and expression construct B result in four populations of cells: non-transduced (BFP-GFP-), single transduced expression of construct A (BFP-GFP+), single transduced expression of construct B (BFP+GFP-), and double transduced (BFP+GFP+). Only the double transduced cell population has high surface expression of the Myc selection marker, whereas the single transduced expression of construct A (BFP-GFP+) population has negligible levels of Myc staining. Addition of the NS3 protease inhibitor grazoprevir resulted in a dramatic decrease in surface expression of the Myc tag, demonstrating that surface expression of the Myc tag is dependent on the proteolytic activity of the NS3 protease.

[0368] High surface expression of Myc-tagged selectable marker in cells double positive for separate expression construct A and expression construct B (TEV protease variant) Figure 4A is a schematic diagram of an example embodiment of the STASH selection system. Expression construct A encodes a Myc-tag fused to the N-terminus of a fusion protein containing a CD8a hinge, CD8 transmembrane, GFP, a TEV cleavage site, and an ER retention tag. Expression construct B consists of a BFP-P2A-membrane-tethered TEV protease fused to an ER retention tag.

[0369] Figure 4B is a series of flow cytometry histograms of surface Myc staining in primary human T cells retrovirally transduced with expression construct A and expression construct B (from Figure 4A). As can be seen in the data, only cells engineered with expression construct A and expression construct B have high surface expression of the Myc tag.

[0370] High surface expression of Myc-tag selectable marker in cells triple positive for separate expression constructs A, B, and C. 6A is a schematic diagram of an exemplary embodiment of the three-way STASH selection system. Expression construct A encodes a Myc-tag fused to the N-terminus of a fusion protein comprising CD8a hinge, CD8a transmembrane, GFP, a TEV cleavage site, and an ER retention tag. Expression construct B consists of BFP-P2A-CD8a hinge-CD8a N-terminal TEV protease domain fused to an ER retention tag. Expression construct C consists of RFP-P2A-CD8a hinge-CD8a N-terminal TEV protease domain fused to an ER retention tag.

[0371] FIG. 6B is a series of flow cytometry histograms of surface Myc staining in primary human T cells retrovirally transduced with expression construct A, expression construct B, and expression construct C (from FIG. 6A). As can be seen in the data, only cells engineered with expression construct A, expression construct B, and expression construct C have high surface expression of the Myc tag. Cells that are only positive for expression construct A and expression construct B or expression construct A and expression construct C have minimal surface expression of the Myc tag selection marker. These data demonstrate that the three-way STASH selection system can be used to identify and enrich cells that are triple positive from untransduced, single positive, or double positive cells.

[0372] High surface expression of Myc-tagged selectable marker in cells that are quintuple positive for separate expression construct A, expression construct B, expression construct C, expression construct D, and expression construct E. 8A is a schematic diagram of an exemplary embodiment of the penta-way STASH selection system. Expression construct A encodes a Myc-tag fused to the N-terminus of a fusion protein comprising a CD8a hinge, a CD8 transmembrane, a GFP, a TEV cleavage site, and an ER retention tag. Expression construct B consists of a FLAG tag tethered to a CD8a hinge, a CD8a transmembrane domain, a leucine zipper domain (Zip2), and an ER retention tag. Expression construct C consists of a HA tag tethered to a CD8a hinge, a CD8a transmembrane domain, a leucine zipper domain (Zip3), and an ER retention tag. Expression construct D consists of a BFP-P2A-cytosolic N-terminal TEV protease domain fused to a leucine zipper (Zip4). Expression construct E consists of a RFP-P2A-cytosolic C-terminal TEV protease domain fused to a leucine zipper (Zip5).

[0373] FIG. 8B is a series of flow cytometry dot plots of surface Myc staining in primary human T cells retrovirally transduced with expression construct A, expression construct B, expression construct C, expression construct D, and expression construct E (from FIG. 8A). As can be seen in the data, cells engineered with expression construct A, expression construct B, expression construct C, expression construct D, and expression construct E have a quintuple positive cell population with high surface expression of the Myc tag. Cells that are only positive for expression construct A do not contain this population of high Myc tag surface expression. These data demonstrate that the five-way STASH selection system can be used to identify and enrich for cells that are quintuple positive.

[0374] Evaluation and development of various ER localization tags 10A is a schematic diagram of a bilateral STASH selection system with EGFRt as the STASHed surface marker. The first expression construct encodes GFP, P2A ribosomal skipping sequence, EGFRt, a TEV cleavage site, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1). Expression construct 2 encodes BFP, P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0375] Figure 10B is a series of flow cytometry histograms of surface EGFR in primary human T cells retrovirally transduced with expression construct 1 and expression construct 2. As can be seen in the data, cells positive for GFP but not BFP (single+) have high residual surface EGFR expression, suggesting that the E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1) is a STASH tag that results in suboptimal intracellular retention.

[0376] 10C is a series of flow cytometry histograms of surface EGFR on primary human T cells retrovirally transduced with expression construct 2 and modified expression construct 1, whereby the EGFRt extracellular domain (ECD) was fused to the CD8α hinge and transmembrane domain (CD8αH / Tm), a TEV cleavage site, and the E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1). As can be seen in the data, cells positive for GFP but not BFP (single+) had high residual surface EGFR expression, suggesting that the E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1) is a STASH tag that results in suboptimal intracellular retention even when EGFRt ECD is fused to CD8αH / Tm.

[0377] EGFRt fusion proteins with various ER tags (Set 1) were then generated. Figure 11A is a schematic of the bilateral STASH selection system with EGFRt as the STASHed surface marker, in which the extracellular domain (ECD) of EGFRt is fused to the CD8α hinge and transmembrane domain (CD8αH / Tm), a TEV cleavage site, and an ER retention tag.

[0378] FIG. 11B is a table of EGFRt-STASH variants comprising the EGFRt ECD fused to CD8αH / Tm, a TEV cleavage site, and the indicated ER retention tag variant.

[0379] Additional EGFRt fusion proteins with various ER tags (Set 2) were then produced. Figure 12A is a schematic of a bilateral STASH selection system with EGFRt as the STASHed surface marker, in which the extracellular domain (ECD) of EGFRt is fused to the transmembrane domain (Tm) and intracellular domain (ICD) of an ER-resident membrane protein, separated by a linker containing a TEV cleavage site.

[0380] FIG. 12B is a table of EGFRt-STASH variants comprising the EGFRt ECD fused to the Tm and ICD of the indicated ER-resident membrane proteins separated by a linker containing a TEV cleavage site.

[0381] Developing high performance Stash constructs 13A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant as a STASHed surface marker. The first expression construct encodes GFP, P2A ribosomal skipping sequence, EGFRt STASH variant. Expression construct 2 encodes BFP, P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0382] FIG. 13B is a series of flow cytometry histograms of surface EGFRt in primary human T cells retrovirally transduced with expression construct 1 and expression construct 2. The numbers above each flow plot indicate the ER tag variant used. Mock negative control cells, cells that are solely positive for GFP, and cells that are double positive for GFP and BFP are shown. As seen in the data, some ER tag variants show different surface EGFRt expression levels between single and double positive populations, which allows selective purification of the double positive population by surface expressed EGFRt. Some of the high-performing ER tag variants are novel sequences derived from human proteins, which are preferred for clinical applications in humans due to the low risk of immunogenicity.

[0383] MACS-based enrichment of EGFRt-STASH-selected variants Figure 14A is a schematic diagram of the workflow for EGFR-based purification using magnetic activated cell sorting (MACS). Cells are labeled with anti-EGFR-biotin, which only binds to surface-expressed EGFR. Cells are washed and then labeled with anti-biotin microbeads. Cells are applied to a magnetic separation column, the column is washed to remove the negative cell fraction, and then the purified cell fraction is eluted from the column.

[0384] Figure 14B is a plot of the percentage of double positives (BFP+GFP+) from purified cell fractions after EGFR MACS selection (enrichment) of the samples shown in Figure 13. These data demonstrate that several EGFRt-STASH variants can be used to isolate highly pure double positive populations using a single selection marker.

[0385] Bilateral STASH selection using EGFRt with a novel ER tag Figure 15 is a series of flow plots showing BFP, GFP, and surface EGFR expression on primary human T cells for five EGFRt STASH variants before and after enrichment by EGFR MACS selection. For each variant (variants indicated by numbers above the histogram plots), histograms of surface EGFR expression are shown for the single+ and double+ populations along with dot plots showing BFP and GFP expression before and after enrichment. Mock negative control cells, cells that are solely positive for GFP, and cells that are double positive for GFP and BFP are shown. As can be seen in the data, variants 493, 497, 501, and 503 have highly differential surface EGFR expression between the single and double positive populations, which results in a highly pure double population after EGFR MACS selection, whereas construct 487, which has less distinct surface EGFR expression, results in an impure population after MACS selection as indicated by the relatively large single+ fraction (GFP+BFP-) in the enrichment sample.

[0386] Bilateral STASH selection using EGFRt-STASH variant 497 in a low percentage of early double-positive cells 16A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant 497 as a STASHed surface marker. The first expression construct encodes GFP, a P2A ribosomal skipping sequence, EGFRt STASH variant 497. Expression construct 2 encodes BFP, a P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0387] Figure 16B is a series of flow plots showing BFP, GFP, and surface EGFR expression for EGFRt STASH variant 497 before and after enrichment by EGFR MACS selection. As can be seen in the data, variant 497 yields a highly pure doublet population (96.3%) after EGFR MACS selection, even when starting from a low percentage initial double positive population (16.4%).

[0388] Bilateral STASH selection using EGFRt-STASH variant 493 in a low percentage of early double-positive cells 17A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant 493 as a STASHed surface marker. The first expression construct encodes GFP, a P2A ribosomal skipping sequence, EGFRt STASH variant 493. Expression construct 2 encodes BFP, a P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0389] Figure 17B is a series of flow plots showing BFP, GFP, and surface EGFR expression for EGFRt STASH variant 493 before and after enrichment by EGFR MACS selection. As can be seen in the data, variant 493 yields a highly pure doublet population (91.3%) after EGFR MACS selection, even when starting from a low percentage initial double positive population (17.0%).

[0390] Bilateral STASH selection using EGFRt-STASH variant 491 in a low percentage of early double-positive cells 18A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant 491 as a STASHed surface marker. The first expression construct encodes GFP, a P2A ribosomal skipping sequence, EGFRt STASH variant 491. Expression construct 2 encodes BFP, a P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0391] Figure 18B is a series of flow plots showing BFP, GFP, and surface EGFR expression for EGFRt STASH variant 491 before and after enrichment by EGFR MACS selection. As can be seen in the data, variant 491 yields a highly pure doublet population (87.1%) after EGFR MACS selection, even when starting from a low percentage initial double positive population (13.1%).

[0392] Bilateral STASH selection using EGFRt-STASH variant 501 in a low percentage of early double-positive cells 19A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant 501 as a STASHed surface marker. The first expression construct encodes GFP, a P2A ribosomal skipping sequence, EGFRt STASH variant 501. Expression construct 2 encodes BFP, a P2A ribosomal skipping sequence, CD8 H / Tm, TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0393] Figure 19B is a series of flow plots showing BFP, GFP, and surface EGFR expression for EGFRt STASH variant 501 before and after enrichment by EGFR MACS selection. As can be seen in the data, variant 501 yields a highly pure doublet population (96.3%) after EGFR MACS selection, even when starting from a low percentage initial double positive population (12.3%).

[0394] Three-way STASH selection of CD22, CD19, and HER2 CAR-T cells with EGFRt-STASH variant 497 Figure 20A is a schematic diagram of the tripartite STASH selection system. An epitope-based selection marker (EGFRt) is fused to a protease cleavage site and an intracellular retention tag (e.g., an endoplasmic reticulum retention tag). Co-expression of the split protease results in the reconstitution of an active protease complex, whereby the N-terminal domain of the protease is tethered to one transmembrane protein and the C-terminal domain is tethered to another transmembrane protein. The active protease complex cleaves the selection marker at the protease cleavage site, which releases the selection marker from the ER retention tag and translocates the selection marker to the surface of the cell. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing both the selection marker and the two protease domains (the N-terminal protease and the C-terminal protease).

[0395] Figure 20B is a schematic diagram showing expression constructs encoding three proteins of interest (CD22, CD19, and HER2.BBz CAR) and components of the STASH selection system (EGFRt-STASH variant 497, N-terminal protease, and C-terminal protease). A ribosomal skip site (P2A from porcine teschovirus) allows for bicistronic expression of the proteins of interest and the STASH selection system components.

[0396] FIG. 20C is a series of flow plot histograms showing surface expression of EGFR, CD22, CD19, and HER2.BBz CAR-T cells. Primary human T cells were activated with CD3 / CD28 activation beads on day 0. On day 2, they were exposed to viral expression construct 1 for 24 hours. On day 3, T cells were exposed to a 1:1 mixture of viral expression construct 2 and viral expression construct 3. On day 7, T cells were harvested, purified by EGFR MACS, stained for the indicated surface markers, and analyzed by flow cytometry. As can be seen in the data, three-way STASH selection allows for the isolation of a highly pure population of trispecific CAR-T cells transduced with three separate viral expression constructs. Isolation was achieved using a single EGFR MACS selection.

[0397] STASH-selectable variant 493 containing a degron domain allows antibiotic selection FIG. 21A is a schematic diagram of a bilateral STASH selection system using EGFRt-STASH variant 493, which consists of the extracellular domain (ECD) of EGFRt fused to the CD8α hinge and transmembrane domains, a TEV cleavage site, a degron, and an ER retention tag. The ER retention tag and degron reduce surface expression of EGFRt-STASH in the absence of proteases by retaining EGFRt inside the cell and labeling the protein for degradation. Co-expression of TEV protease tethered to the CD8a transmembrane protein results in cleavage of the selection marker at the TEV cleavage site, which liberates the selection marker from the ER retention tag and degron and allows it to move to the surface of the cell at high expression levels. Cells expressing both the STASHed selection marker and the protease component can then be isolated using the surface expression selection tag as a selection handle.

[0398] FIG. 21B is a schematic diagram of a bilateral STASH selection system using EGFRt-STASH variant 493, which consists of the extracellular domain (ECD) of EGFRt fused to the CD8α hinge and transmembrane domains, a puromycin resistance gene (PuroR, puromycin-N-acetyltransferase), a TEV cleavage site, a degron, and an ER retention tag. The ER retention tag and degron reduce expression of EGFRt-STASH in the absence of proteases by retaining EGFRt inside the cell and labeling the protein for degradation. Co-expression of TEV protease tethered to the CD8a transmembrane protein results in cleavage of the selection marker at the TEV cleavage site, which releases the selection marker from the ER retention tag and degron and allows the selection marker to move to the surface of the cell at high expression levels. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing both the STASHed selection marker and the protease component by puromycin antibiotic selection or by MACS using surface-expressed EGFRt.

[0399] Chemical-based selection of double-positive cells using a single antibiotic selection marker 22A is a schematic diagram of a bilateral STASH selection system with EGFRt-STASH variant 493 with an integrated puromycin resistance gene as a STASHed selection marker. The first expression construct encodes GFP, a P2A ribosomal skip sequence, the extracellular domain (ECD) of EGFRt fused to the CD8α hinge and transmembrane domains, a puromycin resistance gene (PuroR, puromycin-N-acetyltransferase), a TEV cleavage site, a degron, and an ER retention tag. Expression construct 2 encodes BFP, a P2A ribosomal skip sequence, CD8 H / Tm, a TEV protease, and an E3-19K protein ER retention tag (LYKYKSRRSFIDEKKMP; SEQ ID NO:1).

[0400] Figure 22B is a series of flow plots of primary human T cells transduced with a mixture of expression construct 1 and expression construct 2 shown in Figure 22A, demonstrating BFP and GFP expression after 96 hours of puromycin exposure at the indicated puromycin concentrations. As can be seen in the data, cells that are double positive for expression construct 1 and expression construct 2 (BFP+ and GFP+) are progressively enriched with increasing concentrations of puromycin. These data demonstrate that this variant of STASH selection allows for the purification of double positive cells using a single chemical selection marker.

[0401] Bilateral STASH selection using CD34 as an epitope marker Figure 34 is a series of flow cytometry histograms of surface CD34 staining with QBEnd / 10 antibody on primary human T cells. As can be seen in the data, only double positive cells show high surface expression of the CD34 epitope.

[0402] Bilateral STASH selection using TEV protease carrying the CISD2 ER retention tag Figure 35 is a series of flow cytometry histograms showing surface EGFR expression on primary human T cells transduced with EGFRt-STASH variants and TEV proteases carrying the CISD2 ER retention tag. The specific EGFRt-STASH variants are indicated above each plot. These data demonstrate that TEV proteases with the CISD2 ER retention tag are functional in the ER STASH selection system and are compatible with several EGFRt-STASH variants.

[0403] Three-way STASH selection using various Tm combinations for EGFRt-STASH variants with CISD2 retention signals Figure 36 is a series of flow cytometry histograms showing surface EGFR expression on primary human T cells transduced in a three-way STASH selection system using EGFRt-STASH variants carrying CD8α or CD28 Tm domains and a CISD2 ER retention signal. Cells were co-transduced with split TEV variants fused to CD8α or CD28 transmembrane (Tm) domains. These data demonstrate that both CD8α and CD28 Tm domains are compatible in three-way STASH selection using split TEV protease and EGFRt-STASH with a CISD2 ER retention signal.

[0404] Three-way STASH selection using various Tm combinations for EGFRt-STASH variants with IBV S protein ER tags Figure 37 is a series of flow cytometry histograms showing surface EGFR expression on primary human T cells transduced in a three-way STASH selection system using EGFRt-STASH variants carrying CD8α or CD28 Tm domains and an IBV S protein retention signal. Cells were co-transduced with split TEV variants fused to CD8α or CD28 transmembrane (Tm) domains. These data demonstrate that both CD8α and CD28 Tm domains are compatible in three-way STASH selection using EGFRt-STASH with split TEV protease and IBV S protein ER retention signal.

[0405] Three-way STASH selection with various Tm combinations for EGFRt-STASH variants with degron-fused ER tags Figure 38 is a series of flow cytometry histograms showing surface EGFR expression on primary human T cells transduced in a three-way STASH selection system using EGFRt-STASH variants carrying a degron fused to the CD8α or CD28 Tm domain and the adenoviral E3-19K retention signal. Cells were co-transduced with a split TEV variant fused to the CD8α or CD28 transmembrane (Tm) domain. These data demonstrate that both CD8α and CD28 Tm domains are compatible in three-way STASH selection using split TEV protease and EGFRt-STASH with a degron fused to the adenoviral E3-19K retention signal.

[0406] Three-way STASH selection of cJun, CD19, and HER2 CAR-T cells with EGFRt-STASH variant 507 Figure 39A is a schematic diagram of the tripartite STASH selection expression constructs used in this experiment. Expression construct A encodes the transcription factor cJun, which renders T cells resistant to exhaustion, and the bicistronic expression of EGFRt-STASH variant 507. Expression construct 2 encodes the CD19.BBz CAR and the bicistronic expression of an N-terminal fragment of a split TEV fused to the CD28 hinge Tm domain. Expression construct 3 encodes the HER2.BBz CAR and the bicistronic expression of a C-terminal fragment of a split TEV fused to the CD28 hinge Tm domain.

[0407] Figure 39B is a series of flow plot histograms showing surface expression of EGFR, cJun, CD19.BBz, and HER2.BBz CARs. Primary human T cells were activated with CD3 / CD28 activation beads on day 0. On day 2, they were exposed to viral expression construct 1 for 24 hours. On day 3, T cells were exposed to a 1:1 mixture of viral expression construct 2 and viral expression construct 3. On day 7, T cells were harvested, purified by EGFR MACS, stained for the indicated surface markers, and analyzed by flow cytometry. As can be seen in the data, three-way STASH selection allows for the isolation of a highly pure population of bispecific CAR-T cells (CD19 and HER2CAR+) expressing the transcription factor cJun. Isolation was achieved using a single single EGFR MACS selection. These data demonstrate that the STASH selection system can be used to purify cells expressing three proteins of interest, one of which is a transcript and two of which are receptors, from three expression constructs.

[0408] Anti-EGFR-biotin antibody titration for MACS using EGFR STASH 497 Figure 40A is a series of flow plots of primary human T cells transduced with a mixture of Vector 1 and Vector 2 shown in Figure 16A, demonstrating BFP and GFP expression after staining with anti-EGFR-biotin at the dilutions shown above the flow plots and MACS selection. As can be seen in the data, the concentration of anti-EGFR-biotin antibody during the MACS procedure affects the purity of the selected product.

[0409] FIG. 40B is a bar graph showing the yield of double positive cells after MACS selection of the samples shown in FIG. 40A.

[0410] Anti-EGFR-biotin antibody titration for MACS using EGFR STASH 501 Figure 41A is a series of flow plots of primary human T cells transduced with a mixture of Vector 1 and Vector 2 shown in Figure 19A, demonstrating BFP and GFP expression after staining with anti-EGFR-biotin at the dilutions shown above the flow plots and MACS selection. As can be seen in the data, the concentration of anti-EGFR-biotin antibody during the MACS procedure affects the purity of the selected product.

[0411] FIG. 41B is a bar graph showing the yield of double positive cells after MACS selection of the samples shown in FIG. 41A.

[0412] Viral Supernatant Dilutions of EGFR STASH 501 and TEV Protease 413: BFP and GFP Expression Figure 42 is a series of flow plots demonstrating EGFR and GFP expression in primary human T cells transduced with a mixture of Vector 1 and Vector 2 shown in Figure 19A. Viral supernatant dilutions for each vector are shown above and to the side of the flow plot. Color scale intensity indicates EGFR expression.

[0413] Viral Supernatant Dilutions of EGFR STASH 501 and TEV Protease 413: Surface EGFR Expression Figure 43 is a series of flow plots demonstrating surface EGFR expression in primary human T cells transduced with a mixture of Vector 1 and Vector 2 shown in Figure 19A. Viral supernatant dilutions for each vector are shown above and to the side of the flow plot. Mock untransduced T cells serve as negative controls. As can be seen in the data, all combinations of viral supernatant dilutions achieved a high degree of differential surface EGFR expression between the single and double positive populations.

[0414] Surface EGFR expression by Protease 797, a minimized protease construct with an ER retention tag Figure 44 is a series of flow plots demonstrating surface EGFR expression in primary human T cells transduced with the EGFR STASH variants shown above each flow plot, and minimized TEV protease construct 797 consisting of BFP-P2A-UGT2B17 membrane-tethered TEV protease fused to the variant 501 ER retention tag. As can be seen in the data, widely differing surface EGFR expression between single and double positive populations was achieved using TEV protease construct 797.

[0415] Identification of human proteases for use in STASH selection Figure 45A is a series of flow plots demonstrating surface EGFR expression in primary human T cells co-transduced with two vectors. The first vector is a modified version of EGFR STASH 501 that contains a human protease cleavage site instead of the TEV protease cleavage site. The second vector contains a human protease cognate to the human cleavage site. As can be seen in the data, some human protease and protease cleavage site combinations result in significantly different surface EGFR expression between single and double positive populations.

[0416] Figure 45B is a table showing the constructs used to transduce each sample number. Figure 45C is a table showing the identity of the human protease used in each protease construct. Figure 45D is a table showing the amino acid sequence of the protease cleavage site used.

[0417] Bilateral STASH selection using a combination of CRISPR knock-in and retroviral gene delivery methods Figure 46A is a schematic of the AND gate logic that can be implemented using the STASH selection system. Cells that meet two input requirements (expression of vector A delivered by CRISPR knock-in and expression of vector B delivered by a retroviral vector) result in output surface expression of the selection marker.

[0418] Figure 46B is a schematic diagram of the bilateral STASH selection vectors used in this experiment. The first vector is an AAV6 vector containing construct 776, a nucleotide sequence with a left homology arm to the TRAC locus, an EGFR-STASH501 domain, and a right homology arm to the TRAC locus. The second vector is a retroviral expression vector 413 consisting of a BFP-P2A membrane-tethered TEV protease fused to an ER retention tag.

[0419] Figure 46C is a series of flow plots demonstrating surface EGFR expression in primary human T cells. Cells were electroporated with Cas9 ribonucleoprotein with guide specific to the TRAC locus, and then exposed to the AAV6 vector shown in Figure 46B, either alone or in combination with a retroviral vector. Non-electroporated cells serve as negative controls. As can be seen in the data, only cells that are electroporated with Cas9 ribonucleoprotein with guide specific to the TRAC locus, exposed to AAV6 vector, and express BFP from a retroviral expression vector have high levels of surface EGFR. These data indicate that STASH selection components can be delivered by a combination of retroviral expression vectors and site-specific CRISPR-based genome engineering.

[0420] STASH selection between two parties with various EGFR truncations Figure 47 is a series of flow plots demonstrating surface EGFR expression in primary human T cells transduced with truncated EGFR STASH variant 501 and TEV protease construct #413. Specific truncations were made in domain IV of the EGFR extracellular domain and these are indicated above each flow plot. The anti-EGFR antibodies used for staining are indicated next to the flow plots. As can be seen in the data, vastly different surface EGFR expression between single and double positive populations was achieved by various truncations of EGFR.

[0421] Materials and Methods A. Construction of Protease Expression Constructs HCV NS3 protease expression construct for bilateral STASH selection The protease modules were expressed as bicistronic fusion constructs, from N- to C-terminus, containing the following: payload protein (BFP, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (TCR-β leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm), linker, NS4A cofactor domain, linker, HCV NS3 protease, NS3 helicase fragment, linker, and ER retention tag (adenovirus E3-19K tag).

[0422] TEV protease expression construct for bilateral STASH selection Protease modules were expressed as bicistronic fusion constructs, from N- to C-terminus: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (TCR-β leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, TEV protease, linker, and ER retention tag (adenovirus E3-19K tag or CISD2 intracellular domain).

[0423] Human protease expression constructs for bilateral STASH selection Protease modules were expressed as bicistronic fusion constructs, from N- to C-terminus: payload protein (such as BFP, tdTomato, CAR, cJun), ribosomal skipping sequence (P2A), leader sequence (TCR-β leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, human protease (such as kallikrein-15 or enterokinase light chain), linker, and ER retention tag (adenovirus E3-19K tag or CISD2 intracellular domain).

[0424] nTEV protease expression construct for three-way STASH selection Protease modules were expressed as bicistronic fusion constructs, from N- to C-terminus: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (TCR-β leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, nTEV protease (the N-terminal domain of split TEV protease containing the 118 N-terminal amino acids of the protease), linker, and ER retention tag (adenovirus E3-19K tag).

[0425] cTEV protease expression construct for three-way STASH selection Protease modules were expressed as bicistronic fusion constructs, from N- to C-terminus, of the following: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (TCR-β leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, cTEV protease (the C-terminal domain of split TEV protease containing the 118 C-terminal amino acids of the protease), linker, and ER retention tag (adenovirus E3-19K tag).

[0426] nTEV protease expression construct for five-way STASH selection Protease modules were expressed as bicistronic fusion constructs from N- to C-terminus of the following: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and nTEV protease (the N-terminal domain of split TEV protease containing the 118 N-terminal amino acids of the protease).

[0427] cTEV protease expression construct for five-way STASH selection Protease modules were expressed as bicistronic fusion constructs from N- to C-terminus of the following: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and cTEV protease (the C-terminal domain of split TEV protease containing the 118 C-terminal amino acids of the protease).

[0428] B. Construction of protease-recruiting transmembrane protein expression constructs Protease-recruiting transmembrane protein 1 expression constructs for five-way STASH selection Protease recruitment transmembrane protein modules were expressed as bicistronic fusion constructs, from N- to C-terminus, containing the following: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (GM-CSFR leader), detection tag (FLAG tag or Myc tag), transmembrane domain (CD8α hinge and Tm or CD28 hinge and Tm), linker, leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and ER retention tag (adenovirus E3-19K tag).

[0429] Protease-recruiting transmembrane protein 2 expression constructs for five-way STASH selection Protease recruitment transmembrane protein modules were expressed as bicistronic fusion constructs, from N- to C-terminus, containing the following: payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosomal skipping sequence (P2A), leader sequence (GM-CSFR leader), detection tag (HA tag), transmembrane domain (CD8α hinge and Tm or CD28 hinge and Tm), linker, leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and ER retention tag (adenovirus E3-19K tag).

[0430] C. Construction of STASH selectable marker expression constructs.

[0431] STASH selection marker expression construct STASH selection modules were expressed as bicistronic fusion constructs from N- to C-terminus: payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosomal skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt, CD34, Myc tag, NGFRt), linker, transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, CISD2 Tm, TMED4 Tm, Sel1L Tm, DDOST Tm, UGT2B17 Tm, UGT1A1 Tm, TAPBP Tm, TMED4 Tm, TRIQK Tm, mastadenovirus C E3 19K Tm, IBV STm, or calnexin Tm), linker, protease cleavage site (TEV cleavage site, HCV NS3 cleavage site, or human enterokinase light chain cleavage site), a linker, and an ER retention tag (adenovirus E3-19K tag, adenovirus E3-19K variant 1 tag, adenovirus E3-19K variant 2 tag, adenovirus E3-19K variant 3 tag, KDELR2 ICD, carboxypeptidase D ICD, coronavirus infectious bronchitis virus (IBV) S protein ER retention motif, HCV NS3 helix, HCV helicase domain, CISD2 ICD, TMED4 ICD, Sel1L ICD, DDOST ICD, UGT2B17 ICD, UGT1A1 ICD, TAPBP ICD, TRIQK ICD, mast adenovirus C E3 19K ICD, IBV S ICD, or calnexin ICD).

[0432] STASH selection marker with degron domain expression construct STASH selection modules were expressed as bicistronic fusion constructs, from N- to C-terminus of the following: payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosomal skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt, CD34, Myc tag, NGFRt), linker, transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm), linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention tag (adenovirus E3-19K tag).

[0433] STASH selection marker with puromycin resistance and degron domain expression construct STASH selection modules were expressed as bicistronic fusion constructs, from N- to C-terminus, containing the following: payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosomal skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt), linker, transmembrane domain (CD8α hinge and Tm), linker, puromycin-N-acetyltransferase (PuroR) linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention tag (adenovirus E3-19K tag).

[0434] STASH selection marker with puromycin resistance, degron domain, and dual cleavage site expression construct STASH selection modules were expressed as bicistronic fusion constructs, from N- to C-terminus, containing the following: payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosomal skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt), linker, transmembrane domain (CD8α hinge and Tm), linker, protease cleavage site (TEV cleavage site), puromycin-N-acetyltransferase (PuroR) linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention tag (adenovirus E3-19K tag).

[0435] D. Isolation of Primary Human T Cells from Blood Donors Primary human T cells were extracted from buffy coats by negative selection using the RosetteSep Human T Cell Enrichment Kit (Stem Cell Technologies) and SepMate-50 tubes. T cells were cryopreserved in CryoStor CS10 cryopreservation medium (Stem Cell Technologies) until use.

[0436] E. Construction of Retroviral Plasmid Expression Constructs DNA sequences were synthesized as gBlocks or oligonucleotides (Integrated DNA Technologies) and cloned into MSGV1 retroviral expression constructs by In-Fusion cloning. In-Fusion reaction products were transformed into chemically competent cells (Stellar Cell, Takara Bio) by heat shock method. Transformants were sequence verified by Sanger sequencing. Bacterial cultures from sequence verified clones were grown for 16 h at 37°C with shaking. Bacterial cells were then harvested and DNA was extracted using a miniprep kit (QIAprep Spin Miniprep Kit, Qiagen).

[0437] F. Virus Production Retroviral supernatants were prepared using 293GP cells and RD114 envelope plasmid. Briefly, 22 μg of the corresponding MSGV1 transfer plasmid and 11 μg of RD114 were delivered to 293GP cells grown on poly-D-lysine dishes (Corning) to approximately 80% confluency by transient transfection using Lipofectamine 2000 reagent (Thermo Fisher). 293GP cells were cultured in medium (DMEM, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, Gibco) at 37°C in a 5% CO2 environment. The medium was changed every 24 hours. Retroviral supernatants were harvested 48 and 72 hours after transfection, centrifuged to remove dead cells and debris, and stored at -80°C until further use.

[0438] G. Adeno-associated Virus (AAV) Production The EGFR-STASH TRAC knock-in template was cloned into the AAV plasmid backbone with the following configuration: ITR, TRAC left homology arm, EF1a promoter, EGFR STASH variant 501, bGH poly(A) signal, TRAC right homology arm, and ITR. AAV was produced by transfecting five 150 mm plates of 293T cells with 30 μg of template plasmid and 110 μg of AAV6 helper plasmid (pDGM6). 293T cells were cultured in medium (DMEM, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin, Gibco) at 37°C in a 5% CO2 environment. The medium was changed every 24 hours. After 72 hours, AAV6 particles were extracted using the AAVpro® Purification Kit Maxi kit (Takara, Cat. No. 6666) according to the manufacturer's instructions. For related methods, see Wiebking et al., Nat. Biotechnology (2020).

[0439] HT cell retroviral transduction Primary human T cells were thawed on day 0 and activated with anti-CD3 / CD28 Human T-Expander Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 3:1. On day 2, virus-coated culture plates were prepared by incubating 0.1-1 mL of each retroviral component diluted in DMEM on non-TC-treated 12-well or 24-well plates pre-coated with RetroNectin (Takara Bio) according to the manufacturer's instructions, followed by centrifugation at 3200 RPM and 32°C for approximately 2 hours. The supernatant was then aspirated from the wells and 0.25-0.5 × 10 6 T cells were added to 1 mL of T cell media consisting of AIM V (Thermo Fisher), 5% fetal bovine serum (FBS), 100 U / mL penicillin (Gibco), 2 mM L-glutamine (Gibco), 100 mg / mL streptomycin (Gibco), 10 mM HEPES (Gibco), and 100 U / mL rhIL-2 (Peprotech). After addition of T cells, plates were gently spun down at 1200 RPM for 2 minutes and then incubated at 37 °C with 5% CO2 for 24 hours. This transduction process was repeated on day 3. In some experiments with more than three expression constructs, this process was repeated on day 4. Dynabeads were removed on day 4 by magnetic separation. Cells were transduced at 0.4–2 × 10 6 cells / mL and expanded from days 7 to 21. T cells were transduced with one to five different viruses on each transduction day.

[0440] I. CRISPR and AAV6-based site-specific knock-in CRISPR guides were synthesized by Synthego and resuspended according to the manufacturer's instructions. Alt-R® SpCas9 Nuclease V3 was purchased from IDT. To generate Cas9 ribonucleoprotein, 0.5uL of sgRNA was added to 0.4uL of Cas9 to allow complexation at room temperature and then placed on ice until electroporation. Primary human T cells were thawed on day 0 and activated with anti-CD3 / CD28 Human T-Expander Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 3:1. On day 2, beads were removed from the T cells by magnetic separation. 1x106 T cells were resuspended in 20uL of P3 buffer (Lonza), added to the cas9 ribonucleoprotein complex, transferred to electroporation strips, and then electroporated using a Lonza Nucleofector 4D system with program EH-115. Immediately after electroporation, the cells were transferred to a 96-well plate containing T cell culture medium and AAV6 viral particles.

[0441] J. Antibodies and Flow Cytometry Recombinant CD19 idiotype antibodies, HER2-Fc, and CD22-Fc fluorescently labeled with DyLight650 Microscale Antibody Labeling Kit (Thermo Fisher) were used for CAR detection. The following antibodies were used for staining: anti-human EGFR antibody (clone AY13, BioLegend), Myc-tag (71D10) rabbit mAb (Cell Signaling Techology), anti-human CD271 (NGFR) antibody (clone ME20.4, BioLegend), CD34 monoclonal antibody (QBEND / 10, Thermo Fisher Scientific), DYKDDDDK (SEQ ID NO: 131) tag (9A3) mouse mAb (Cell Signaling Technology), anti-HA.11 epitope tag antibody (BioLegend), EGF receptor antibody anti-human biotin (Miltenyi Biotec), and c-myc antibody biotin (Miltenyi Biotec). Flow cytometry was performed on a BD Fortessa instrument and analyzed by FlowJo software (Tree Star).

[0442] K. Magnetic Activated Cell Sorting (MACS) Cells were stained with the indicated biotinylated antibodies according to the manufacturer's instructions. Cells were then labeled with magnetic microbeads (Streptavidin MicroBeads or Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) according to the manufacturer's instructions. Cells were loaded onto an LS column, washed with MACS buffer, and magnetically separated using a QuadroMACS separator (Miltenyi Biotec) according to the manufacturer's instructions.

[0443] L. Puromycin selection Primary human T cells transduced with the indicated expression constructs were expanded as above until days 7–10 post-activation and then cultured for 48–96 h in T cell medium containing the indicated concentrations or puromycin dihydrochloride (Thermo Fisher Scientific).

[0444] Thus, the above merely illustrates the principles of the disclosure. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the present invention and are within the spirit and scope thereof, although not explicitly described or illustrated herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts that the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not limited to the exemplary embodiments shown and described herein.

Claims

1. 1. A method for selecting cells containing two or more distinct expression constructs, the method comprising: contacting a cell population with two or more separate expression constructs under conditions such that said two or more expression constructs are delivered to cells in said cell population, said two or more separate expression constructs comprising: a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site located between the selection marker and the protein localization tag; and a second expression construct encoding a protein required for cell surface expression of the selectable marker; and selecting cells exhibiting cell surface expression of said selection marker; A method comprising:

2. The method of claim 1 , wherein the second expression construct further encodes a protein of interest.

3. 2. The method of claim 1, wherein the protein localization tag is selected from the group consisting of an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosomal localization tag, a plasma membrane localization tag, a mitochondrial localization tag, a peroxisomal localization tag, a cytoplasmic localization tag, and a nuclear localization tag.

4. wherein the protein localization tag is an ER localization tag, and optionally the ER localization tag comprises the amino acid sequence KKMP; or The ER localization tag is LYKYKSRRSFIDEKKMP (SEQ ID NO: 1); AEKDEL (SEQ ID NO:2); EQKLISEEDLKDEL (SEQ ID NO:3); GGGGSGGGGSKDEL (SEQ ID NO:4); GGGGSGGGGSGGGGSGGGGSGGGGSKDEL (SEQ ID NO:5); GGGGSGGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO:6); KYKSRRSFIEEKKMP (SEQ ID NO:7); LKYKSRRSFIEKKMP (SEQ ID NO:8); LYKYKSRRSFIEKKMP (SEQ ID NO:9); LYCKYKSRRSFIEKKMP (SEQ ID NO: 10); LYCNKYKSRRSFIEKKMP (SEQ ID NO:11); LYCNKYKSRRSFIDEKKMP (SEQ ID NO: 12); LYEQKLISEEDLKYKSRRSFIEKKMP (SEQ ID NO: 13); LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO: 14); LYKKLETFKKTN (SEQ ID NO: 15); LYEQKLISEEDLKKLETFKKTN (SEQ ID NO: 16); LYYQRL (SEQ ID NO: 17); LYEQKLISEEDLYQRL (SEQ ID NO: 18); LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO: 19); LYRNIKCD (SEQ ID NO:20); and LYEQKLISEEDLRNIKCD (SEQ ID NO:21) or The ER localization tag is PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCCCGC FGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 31); NRSPRNRKPRRE (SEQ ID NO:32); LYKYKSRRSFIEKKMP (SEQ ID NO:9); TKVLKGKKLSLPA (SEQ ID NO:33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35) or comprising 85% or more, 90% or more, 95% or more, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within an amino acid sequence selected from the group consisting of: the ER localization tag comprises the Tm domain, ICD, or both of an ER localization tag of a human ER resident protein, or the Tm and / or ICD of a variant thereof that retains the ability to localize a polypeptide to the ER, and optionally the human ER resident protein is CDGSH iron-sulfur domain 2 (CISD2) or UDP glucuronosyltransferase family 2 member B17 (UGT2B17); The method according to any one of claims 1 to 3.

5. wherein the protease cleavage site is a human protease cleavage site, and optionally the human protease cleavage site is a cleavage site for a human protease selected from the group consisting of human kallikrein (KLK) protease, human enterokinase protease, human thrombin, human matrix metalloproteases (MMPs), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, and human cathepsins, and optionally the human kallikrein protease is selected from the group consisting of human KLK3, human KLK4, human KLK6, human KLK8, human KLK11, human KLK13, human KLK14, and human KLK15; The method according to any one of claims 1 to 3.

6. The method of any one of claims 1 to 3, wherein the protein required for cell surface expression of the selection marker is a protease and the protease cleavage site is a cleavage site for the protease.

7. 7. The method of claim 6, wherein the protease is fused to a protein localization tag that localizes the protease in the same cellular compartment as the fusion protein containing the selection marker, and optionally, the protease is fused to a transmembrane domain.

8. wherein the transmembrane domain is a CD8α transmembrane domain or a CD28 transmembrane domain, and optionally the transmembrane domain is WLRLLPFLGVLALLGYLAVRPFL (SEQ ID NO:42); VLWWSIAQTVILILTGIW (SEQ ID NO:43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO:44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO:45); IAFLLACVATMIFMITKCCLF (SEQ ID NO:46); VIGFLLAVVLTVAFITF (SEQ ID NO:47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO:48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO:49); TFCSTALLITALALVCTLLYL (SEQ ID NO:50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO:51); WLWVVYILT VALPVFLVILFC (SEQ ID NO:52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:53); and FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 54) comprising an amino acid sequence selected from the group consisting of: The method according to claim 7.

9. said protein required for cell surface expression of said selectable marker is a first complementary fragment of a protease, said protease cleavage site is a cleavage site for said protease, and optionally 4. The method of any one of claims 1 to 3, wherein the two or more expression constructs further comprise a third expression construct encoding a second complementary fragment of the protease, wherein the first complementary fragment and the second complementary fragment form an active protease complex.

10. the protein of interest further encoded by one or more of the two or more separate expression constructs is independently selected from the group consisting of a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein that confers resistance to cellular exhaustion, and a suicide switch protein; and optionally the receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a modular extracellular sensor architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a systemic extracellular molecular sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor; The method of claim 2.

11. 4. The method of any one of claims 1 to 3, wherein the selection marker comprises a protein tag, optionally wherein the protein tag is selected from the group consisting of Myc-tag, His-tag, HA-tag, FLAG-tag, Strep-tag, NE-tag, Xpress tag, Avi-tag, polyglutamic acid tag, and polyarginine tag.

12. 4. The method of any one of claims 1 to 3, wherein the selection marker comprises a cluster of differentiation (CD) protein or truncated receptor, optionally wherein the CD protein or truncated receptor is CD34, a truncated epidermal growth factor receptor (EGFRt), a truncated nerve growth factor receptor (NGFRt), a truncated CD19 (CD19t), or a truncated CD20 (CD20t).

13. the cell population is a mammalian cell population, optionally wherein the mammalian cells comprise immune cells, and optionally the immune cells include T cells or natural killer (NK) cells, and optionally the T cells are CD4+ T cells, CD8+ T cells, regulatory T cells (TREG), or central memory T cells (TCM); The method according to any one of claims 1 to 3.

14. 1. A cell comprising two or more separate expression constructs, said two or more separate expression constructs comprising: a first expression construct encoding a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site located between the selection marker and the protein localization tag; a second expression construct encoding a protein required for cell surface expression of the selectable marker; and optionally, the cell is an immune cell, optionally, the immune cell is a T cell or a natural killer (NK) cell, and optionally, the T cell is a CD4+ T cell, a CD8+ T cell, a regulatory T cell (TREG), or a central memory T cell (TCM). cell.

15. 4. A composition comprising a cell or a progeny thereof selected according to the method of any one of claims 1 to 3, present in a liquid medium, optionally said liquid medium being suitable for administration of said composition to an individual in need thereof.

16. A fusion protein comprising a protein fused to an ER localization tag, a transmembrane domain, or both, When the fusion protein comprises an ER localization tag, the ER localization tag is PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKEV (SEQ ID NO:22); QMRHLKSFFEAKKLV (SEQ ID NO:23); AYRQRQHQDMPAPRPPGPRPAPPQQEGPPEQQPPQ (SEQ ID NO:24); HMKEKEKSD (SEQ ID NO:25); CFRKLAKTGKKKKRD (SEQ ID NO:26); KCCAYGYRKCLGKKGRVKKAHKSKTH (SEQ ID NO:27); YLSTCKDSKKKAE (SEQ ID NO:28); RLTTDVDPDLDQDED (SEQ ID NO:29); KYKSRRSFIDEKKMP (SEQ ID NO:30); MTGCCGCCCGC FGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 31); NRSPRNRKPRRE (SEQ ID NO:32); LYKYKSRRSFIEKKMP (SEQ ID NO:9); TKVLKGKKLSLPA (SEQ ID NO:33); KSNRHKDGFHRLRGHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEETLYSSKH (SEQ ID NO: 34); and KCGKKSSYYTTFDNDVVIEQYRPKKSV (SEQ ID NO: 35) comprising an amino acid sequence selected from the group consisting of: When the fusion protein comprises a transmembrane domain, the transmembrane domain is WLRLLPFLGVLALLGYLAVRPFL (SEQ ID NO:42); VLWWSIAQTVILILTGIW (SEQ ID NO:43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO:44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO:45); IAFLLACVATMIFMITKCCLF (SEQ ID NO:46); VIGFLLAVVLTVAFITF (SEQ ID NO:47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO:48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO:49); TFCSTALLITALALVCTLLYL (SEQ ID NO:50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO:51); WLWVVYILT VALPVFLVILFC (SEQ ID NO:52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:53); and FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 54) comprising an amino acid sequence selected from the group consisting of: Fusion proteins.

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