Cell sorting system and methods of use

Abstract

The presently disclosed subject matter provides methods and systems for isolating cells expressing a particular construct. In certain non-limiting embodiments, the system comprises a membrane-bound polypeptide and a soluble polypeptide capable of dimerizing with the membrane-bound polypeptide.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 765,129, filed August 16, 2018, and U.S. Provisional Application No. 62 / 798,206, filed January 29, 2019, the contents of each of which are incorporated by reference in their entirety and priority to each is claimed.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on September 16, 2019, is named 072734_0919_SL.txt and is 233,171 bytes in size.

[0005] SUMMARY

[0006] The presently disclosed subject matter provides methods and compositions for isolating cells expressing particular constructs. It relates to systems comprising a membrane-bound polypeptide and a soluble polypeptide and methods of using the same. BACKGROUND

[0007] Stable integration of large amounts of genetic information into primary T cells represents a current limitation of cell engineering. Both retroviral and lentiviral vectors show a significant reduction in viral titer when the size of the viral vector insert exceeds the packaging limit of the virus (approximately 6-8 kb for retroviruses and approximately 10-12 kb for lentiviruses). Low viral titer results in low transduction efficiency and low copy number integration per cell, leading to poor expression of genetic constructs. Thus, there remains a need for gene expression systems comprising multiple vectors and for isolating cells comprising such systems. SUMMARY

[0008] The presently disclosed subject matter provides membrane-bound polypeptides. The membrane-bound polypeptides can be used to sort cells.

[0009] In certain embodiments, the membrane-bound polypeptide comprises: a) a transmembrane domain, and b) an extracellular domain comprising a first dimerization domain and a second dimerization domain capable of dimerizing with the first dimerization domain at the cell surface. In certain embodiments, each of the first and second dimerization domains comprises a leucine zipper domain. In certain embodiments, the first dimerization domain comprises an amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106, and the second dimerization domain comprises an amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106. In certain embodiments, the extracellular domain further comprises a linker between the first dimerization domain and the second dimerization domain. In certain embodiments, the linker comprises an amino acid sequence set forth as SEQ ID NO: 3.

[0010] In certain embodiments, the extracellular domain further comprises a spacer / hinge domain between the first dimerization domain and the transmembrane domain. In certain embodiments, the spacer / hinge domain comprises an epitope recognized by an antibody, wherein binding of the antibody to the epitope mediates depletion of cells expressing the membrane-bound polypeptide. In certain embodiments, the spacer / hinge domain comprises a Thyl.1 molecule or a truncated EGFR molecule (EGFRt). In certain embodiments, the Thyl.1 molecule comprises or has an amino acid sequence set forth as SEQ ID NO: 68. In certain embodiments, the EGFRt comprises or has an amino acid sequence set forth as SEQ ID NO: 70.

[0011] In certain embodiments, the extracellular domain further comprises a costimulatory ligand. In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, CD30L, and combinations thereof. In certain embodiments, the costimulatory ligand is 4-1BBL.

[0012] In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the costimulatory ligand is CD80.

[0013] In certain embodiments, the extracellular domain further comprises a dominant negative of a molecule. In certain embodiments, the molecule is selected from the group consisting of an inhibitor of an immune checkpoint molecule, a tumor necrosis factor receptor superfamily (TNFRSF) member, and a transforming growth factor beta (TGF ) receptor. In certain embodiments, the immune checkpoint molecule is selected from the group consisting of PD-1, CTLA-4, B7-H3 (also known as "CD276"), B7-H4, BTLA, TIM-3, LAG-3, TIGIT, LAIR1, CD200, CD200R, HVEM, 2B4, CD160, Galectin 9, and combinations thereof. In certain embodiments, the immune checkpoint molecule is PD-1. In certain embodiments, the TNFRSF member is selected from the group consisting of Fas, tumor necrosis factor receptor, OX40, CD40, CD27, CD30, 4-1BB (also known as "CD137"), and combinations thereof. In certain embodiments, the dominant negative receptor comprises an extracellular domain of TGFRII or a fragment thereof.

[0014] In certain embodiments, the membrane-bound polypeptide further comprises an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3-zeta domain, a costimulatory domain, a suicide gene, or a combination thereof.

[0015] In certain embodiments, the membrane-bound polypeptide is expressed from a vector.

[0016] The presently disclosed subject matter also provides a system for isolating a cell comprising at least two expression vectors.

[0017] In certain embodiments, the at least two expression vectors comprise: a) a membrane-bound polypeptide disclosed herein encoded by a first expression vector, and b) a soluble polypeptide encoded by a second expression vector, which comprises a tag and a third dimerization domain capable of dimerizing with the first dimerization domain. In certain embodiments, the third dimerization domain dimerizes with the first dimerization domain prior to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain dimerizes with the first dimerization domain in the endoplasmic reticulum. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from the same cell, the two are capable of forming a dimer. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from the same cell, the two are capable of forming a dimer in the endoplasmic reticulum. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from different cells, the two are not capable of forming a dimer due to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106.

[0018] In certain embodiments, the tag comprises an epitope tag recognized by a first antibody. In certain embodiments, the epitope tag is selected from the group consisting of a Myc tag, a HA tag, a Flag tag, a V5 tag, a T7 tag, a CD34 tag, and combinations thereof. In certain embodiments, the tag comprises an affinity tag that binds to a substrate. In certain embodiments, the affinity tag is selected from the group consisting of a His tag, a Strep tag, an E tag, a streptavidin binding protein tag (SBP tag), and combinations thereof.

[0019] In certain embodiments, the tag further comprises a mimotope recognized by a second antibody. In certain embodiments, binding of the second antibody to the mimotope mediates depletion of cells comprising the membrane-bound polypeptide. In certain embodiments, the mimotope is a CD20 mimotope and the second antibody is an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is rituximab.

[0020] In certain embodiments, the soluble polypeptide further comprises an antigen binding domain. In certain embodiments, the antigen binding domain comprises a single chain variable fragment (scFv), a soluble ligand, a cytokine, a non-scFv based antigen recognition motif, or a combination thereof.

[0021] In certain embodiments, the soluble polypeptide further comprises a cytokine or a chemokine. In certain embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-36, and combinations thereof. In certain embodiments, the chemokine is selected from the group consisting of CCL1, CCL8, CCL16, CCL17, CCL18, CCL22, and combinations thereof. In certain embodiments, the membrane-bound polypeptide is expressed from a first vector. In certain embodiments, the soluble polypeptide is expressed from a second vector. The first vector can be the same as the second vector. In certain embodiments, the first vector is the same as the second vector, e.g., the vector backbones of the first and second vectors can be the same, while the polypeptides or proteins encoded / expressed by the first and second vectors can be different.

[0022] In certain embodiments, the at least two expression vectors comprise: a) a membrane-bound polypeptide encoded by a first expression vector, comprising a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerization domain and a blocking spacer; and b) a soluble polypeptide encoded by a second expression vector, comprising a tag and a second dimerization domain, wherein the first and second dimerization domains each comprise a leucine zipper domain, and wherein the blocking spacer prevents dimerization of the membrane-bound polypeptide and the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell.

[0023] In certain embodiments, the first dimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106, and the second dimerization domain comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106.

[0024] In certain embodiments, the blocking spacer is no more than about 25 amino acid residues. In certain embodiments, the blocking spacer is between about 5 amino acid residues to about 25 amino acid residues. In certain embodiments, the blocking spacer is a truncated CD28 spacer or an IgGl hinge.

[0025] Further, the presently disclosed subject matter provides a method of isolating a cell comprising at least two expression vectors, and a method of sorting a plurality of cells comprising at least two expression vectors.

[0026] In certain embodiments, the method of isolating a cell comprising at least two expression vectors comprises: a) expressing in the cell i) a membrane-bound polypeptide disclosed herein encoded by a first expression vector, and ii) a soluble polypeptide disclosed herein encoded by a second expression vector, which comprises a tag and a third dimerization domain capable of dimerizing with the first dimerization domain, b) contacting the cell with a substrate that binds to the tag, and isolating the cell bound to the substrate.

[0027] In certain embodiments, the method of sorting a plurality of cells comprising at least two expression vectors comprises: a) transfecting a plurality of cells with i) and ii) i) a first expression vector encoding a membrane-bound polypeptide disclosed herein, and ii) a second vector encoding a soluble polypeptide disclosed herein, e.g., the soluble polypeptide comprises a tag and a third dimerization domain capable of dimerizing with the first dimerization domain, b) contacting the cells with a substrate that binds to the tag, and c) isolating one or more cells bound to the substrate.

[0028] In certain embodiments, the third dimerization domain is capable of dimerizing with the first dimerization domain prior to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain is capable of dimerizing with the first dimerization domain in the endoplasmic reticulum. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from the same cell, both are capable of forming a dimer. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from the same cell, both are capable of forming a dimer in the endoplasmic reticulum. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from different cells, both are not capable of forming a dimer due to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, step c) is preceded by a step of washing the substrate to remove cells that do not bind to the substrate.

[0029] In certain embodiments, a method of isolating a cell comprising at least two expression vectors comprises: a) expressing in the cell i) a membrane-bound polypeptide disclosed herein, e.g., the membrane-bound polypeptide comprises a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerization domain and a blocking spacer, and ii) a soluble polypeptide disclosed herein, e.g., the soluble polypeptide comprises a tag and a second dimerization domain, wherein the first and second dimerization domains each comprise a leucine zipper domain, and wherein the blocking spacer prevents dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell, b) contacting the cell with a substrate that binds to the tag, and c) isolating the cell bound to the substrate.

[0030] In certain embodiments, a method of isolating a cell comprising at least two expression vectors comprises: a) expressing in the cell i) a membrane-bound polypeptide disclosed herein, e.g., the membrane-bound polypeptide comprises a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerization domain and a blocking spacer, and ii) a soluble polypeptide disclosed herein, e.g., the soluble polypeptide comprises a tag and a second dimerization domain, wherein the first and second dimerization domains each comprise a leucine zipper domain, and wherein the blocking spacer prevents dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell, b) contacting the cell with a substrate that binds to the tag, and c) isolating the cell bound to the substrate.

[0031] In certain embodiments, the cell is selected from a T cell, a natural killer (NK) cell, a stem cell from which a lymphoid cell can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is selected from a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell. In certain embodiments, the cell is autologous.

[0032] In certain embodiments, the leucine zippers are orthogonal zippers.

[0033] The presently disclosed subject matter also provides nucleic acid molecules encoding the membrane-bound polypeptides disclosed herein, including vectors comprising such nucleic acid molecules. The presently disclosed subject matter also provides host cells comprising the nucleic acid molecules and vectors disclosed herein. In certain embodiments, the host cell is a T cell. In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a retroviral vector, e.g., a lentiviral vector. In certain embodiments, the vector is a transposon-based vector. BRIEF DESCRIPTION OF DRAWINGS

[0034] The following detailed description can be understood when read in conjunction with the drawings, which are included to provide a thorough understanding of the disclosure. The drawings disclosed herein are not exhaustive or limiting of the scope of the disclosure, but rather, the purpose of the drawings is to illustrate key inventive aspects of the disclosure.

[0035] Figure 1A A cell sorting system according to certain embodiments of the disclosed subject matter is depicted. Figure 1B A cell sorting system according to certain embodiments of the disclosed subject matter is depicted. Figure 1A Co-transduction with the two vectors shown in the middle allows magnetic beads to sort cells with integration of both viral vectors.

[0036] Figure 2 In certain cases, secreted affinity tagged leucine zippers can pair with membrane bound leucine zippers extracellularly.

[0037] Figure 3 Generation of "blocked" membrane bound leucine zippers results in intracellular pairing over extracellular pairing.

[0038] Figure 4 Purification of double transduced cells intentionally contaminated with non-double transduced cells is depicted.

[0039] Figure 5 Purification and testing of cells double transduced with iCaspase 9 and CD20-CAR is depicted. T cells containing RR12EE345L-FLAG iCaspase 9 (vector 1) and RR12EE345L-linker-EE12RR345L-Thyl.l CD20-CAR (vector 2) were incubated with EL4-CD19 and EL4-CD20 targets in the presence or absence of dimerization chemical inducer of dimerization (CID).

[0040] Figure 6 depicts the use of the sorting system of the disclosure for purifying multifunctional CAR T cells (A) Leucine zipper sorting system for purifying cells to greater than 95% with the cells comprising two vectors expressing CD19-CAR, CD20-CAR, and iCaspase9, and optionally IL-18 (top left panel). These cells are capable of killing CD19 or CD20+ targets (right panel), and incubation with iCaspase9 dimer can result in about 90% cell death (bottom left panel). (B) Multifunctional CAR T cells specific for CD19 and CD20 and engineered to secrete IL-18. Mouse T cells were co-transduced with vectors encoding (1) FLAG-RR12EE345L leucine zipper, iCaspase9, and CD19-CAR and (2) linker-blocked RR12EE345L / EE12RR345L Thy1.1 leucine zipper (sorting-depletion construct), CD20-CAR + / - IL-18 with either intact pro-peptide (pro-IL-18) or mouse IL-2 signal peptide (sIL-18). T cells were single-step MACS sorted to >90% purity with anti-FLAG beads and tested for target lysis against C1498 CD19, C1498 CD20, and CD1498. IL-18 secretion was assessed by ELISA. Interferon gamma secretion was assessed by cytometric bead array.

[0041] Figures 7A-7C Depiction of a linker-blocked truncated CD28 membrane proximal hinge-spacer transmembrane leucine zipper facilitating MACS sorting of dual transduced cell populations. Figure 7A Depiction of C1498 cell line double transduced with vector 1 (FLAG-RR12EE345L 2A CBR-2A-GFP) and vector 2 (EE12RR345L Myc CD28EC-9C CD2TM 2A Thy1.1). Figure 7B Depiction of FLAG staining limited to GFP+BFP+ dual transduced population due to truncated nine amino acid hinge-spacer. Figure 7C Depiction of purified GFP+BFP+ dual transduced population resulting from single-step anti-FLAG magnetic bead MACS sorting.

[0042] Figures 8A-8C Depiction of a linker-blocked truncated CD28 membrane proximal hinge-spacer transmembrane leucine zipper facilitating MACS sorting of dual transduced cell populations. Figure 8A Depiction of C1498 cell line double transduced with vector 1 (FLAG-RR12EE345L 2A CBR-2A-GFP) and vector 2 (EE12RR345L Myc CD28EC-9C CD2TM 2A Thy1.1). Figure 8BDisplay of high purity MACS sorting of dual transduced T cells (left panel). Myc tag staining of the tandem CD20-CD19 CAR is weaker than the CD19 single CAR (right panel, contrast Figure 8C Display of purified GFP+BFP+dual transduced population generated by single step anti-FLAG magnetic bead MACS sorting.

[0043] Figure 9 A dual tandem CAR construct is described that binds to the iCaspase9 and blocked Thy1.1 leucine zipper suicide construct. Two retroviral vectors encoding the leucine zipper sorting system construct and the tandem CAR are used to transduce T cells. Vector 1 encodes a tagged secreted leucine zipper, iCaspase9, and the following tandem CAR that contains a CD38 scFv linked to an IL-3 cytokine (interchain linker), a CD8 hinge, a CD8 TM, and a CD28 zeta signaling motif. Vector 2 encodes a blocked Thy1.1 leucine zipper and the following tandem CAR that contains a CD20 scFv linked to a CD19 scFv (interchain linker), a CD8 hinge, a CD8 TM, and a CD28 zeta signaling motif.

[0044] Figure 10A and 10B Depiction of the leucine zipper sorting system enabling single step MACS sorting of T cells expressing a dual tandem CAR. Figure 10A Display of high purity MACS sorting of dual transduced T cells (left panel). Myc tag staining of the tandem CD20-CD19 CAR is weaker than the CD19 single CAR (right panel, contrast Figure 5 ). However, Thy1.1 is co-expressed on the CD20-CD19 CAR vector and shows high co-purification with the CD38-IL-3 tandem CAR (middle panel). Figure 10B Display of individual T cell line lysis of targets each expressing one of 4 separate antigens. Target lysis was determined by detecting residual luciferase activity in firefly luciferase transduced C1498 target cells at 24 hours after initiation of culture.

[0045] Figure 11A and 11B Depiction of the leucine zipper sorting system enabling deletion of sorted T cells using two suicide genes. T cells were transduced with two retroviral vectors encoding a tagged secreted leucine zipper sorting construct + tandem CD38-IL-3 CAR + iCaspase9 (vector 1) and a tandem CD20-CD19 CAR + blocked Thy1.1 leucine zipper sorting suicide construct (vector 2). After single step simultaneous MACS sorting of cells transduced with both vectors with >90% purity, T cells were tested for suicide gene activity. Figure 11ASorted or mock transduced T cells were shown to be incubated with anti-Thy1.1 and 10% rabbit complement for 40 minutes. Control cells were incubated with media only. Figure 11B Sorted or mock transduced T cells were shown to be incubated in 100 nM homodimeric AP20187 or media for 24 hours. In panels A and B, relative survival was calculated as the percentage of surviving cells in treated cells versus control cells. Surviving T cells were quantified by flow cytometry using CountBright beads and DAPI.

[0046] Figures 12A-12C Truncated EGFR spacer (EGFRt) fused to a linker-blocked leucine zipper was shown to facilitate cell sorting and antibody-dependent cell-mediated cytotoxicity (ADCC). Figure 12A BM185 cell line co-transduced with FLAG-RR12EE345L 2AiCaspase9 and RR12EE345L / EE12RR345L-EGFRt BFP vectors and MACS sorted with anti-FLAG microbeads were shown. Figure 12B Sorted BM185 FLAG-RR iC9|RR / EE-EGFRt or control BM185 (co-expressing firefly luciferase) were shown to be incubated in media or in 10 nM AP20187 dimer overnight to activate iCaspase9. Figure 12C : Sorted BM185 FLAG-RR iC9|RR / EE-EGFRt or control BM185 cells were incubated overnight with various ratios of effector NK cell line NK92-MI + / - 10 pg / mL cetuximab. In Figure 12B and 12C In panels A and B, relative survival was calculated as the percentage of surviving cells in treated cells versus control cells. Surviving T cells were quantified by flow cytometry using CountBright beads and DAPI.

[0047] Figure 13 Leucine-zipper “zip-factors” tagged with cytokines were shown to be engineered to facilitate secretion and trans-presentation of cytokines while retaining the sorting function of affinity-tag secreted leucine zippers. Cytokines (e.g., IL-7, IL-15, and IL-21) can be fused to affinity tags and heterodimeric leucine zippers. Zip-factors can be secreted to interact with cytokine receptors on T cells or co-expressed with intrinsically blocked transmembrane leucine zippers to facilitate sorting of dual vector co-transduced cells and trans-presentation of cytokines.

[0048] Figures 14A-14C Zip-factors were shown to retain the functional sorting characteristics of leucine zipper sorting systems and facilitate T cell proliferation. Figure 14AC1498 cell line co-transduced with retroviral vectors displaying coding (Cytokine- RR12EE345L-FLAG 2A BFP) and intrinsic blocking transmembrane leucine zippers (EE12RR345L-Myc-CD28EC-9C CD28TM CD3zδ2A Thy1.1). IL-7, IL-15 and IL-21 zippers were detected on the cell surface presented in trans (FLAG staining) and cells were sorted with anti-FLAG beads to obtain highly purified co-transduced cells. Figure 14B Primary T cells transduced with IL-15-RR12EE345L-FLAG 2A BFP and EE12RR345L- Myc-CD28EC-9C CD28TM CD3zδ2A Thy1.1 and sorted with anti-FLAG beads. FLAG staining (top) and BFP (bottom) confirm vector 1 expression, while Thy1.1 proves vector 2 expression. Figure 14C Primary T cells incubated with irradiated splenocytes, 0.5ug / mL anti-CD3 and irradiated sorted C1498 cells presenting IL-7, IL-15 and IL-21 zippers in trans or with control C1498 cells transduced with transmembrane leucine zippers only. T cells were counted by flow cytometry 72 hours later.

[0049] Figure 15 A sorting system according to certain embodiments of the presently disclosed subject matter is shown.

[0050] Figure 16 Efficient CD34 and CD20 staining by capture of leucine zippers using IgGl hinge CD28TM CD3zΔ and CD28-9C CD28TM CD3zΔ is depicted.

[0051] Figure 17 Uniform surface display of round CD20 mimotopes in cells sorted by anti-CD34 magnetic beads is depicted.

[0052] Figure 18 Selective depletion of double transduced cells using anti-CD20 antibodies is depicted.

[0053] Figure 19 Selective magnetic sorting and antibody-mediated depletion by separate CD20 and CD34 binding domains is depicted.

[0054] Figure 20 Effect of mutant blocking leucine zippers on capture and presentation of secreted leucine zippers is depicted.

[0055] Figure 21A and 21BCD80 (B7-1) molecules functionalized to present a blocking, capture leucine zipper are depicted, allowing magnetic sorting with FLAG-RR12EE345L leucine zippers. Figure 21A Sorted cells show high purity for CD19 and CD20 CAR (Myc, Streptag, respectively) and CD80 functionalized leucine zippers. Figure 21B T cells expressing RR12EE345L linker EE12RR345L CD80 are shown to form conjugates in culture and bind to soluble CD28-Fc. DETAILED DESCRIPTION

[0056] The presently disclosed subject matter provides a membrane-bound polypeptide comprising a transmembrane domain and an extracellular domain, the extracellular domain comprising a first dimerization domain and a second dimerization domain capable of dimerizing with the first dimerization domain at the cell surface, wherein the first and second dimerization domains each comprise a leucine zipper. The membrane-bound polypeptides disclosed herein can be used to sort cells comprising such membrane-bound polypeptides. Furthermore, the presently disclosed subject matter provides a system for isolating cells expressing a particular construct, e.g., a membrane-bound polypeptide disclosed herein. In certain embodiments, the system comprises a membrane-bound polypeptide and a soluble polypeptide, wherein the soluble polypeptide is capable of dimerizing with the membrane-bound polypeptide. In certain embodiments, when the soluble polypeptide and the membrane-bound polypeptide are expressed from different cells, the two are unable to form a dimer, which enables sorting of cells expressing a particular combination of constructs.

[0057] 1. Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this application: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0059] As used herein, the term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, "about" can mean ranges approximately 20%, preferably about 10%, more preferably about 5%, and more preferably about 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold and preferably within 2-fold, of a value.

[0060] As used herein, the term "antibody" refers not only to whole antibody molecules but also to fragments of antibody molecules that retain immunogen binding ability. Such fragments are also well known in the art and are frequently used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well known active fragments F(ab')2and Fab. F(ab')2and Fab fragments lack the Fc fragment of intact antibodies and are cleared more rapidly from the circulation and can have less non-specific tissue binding of intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies of the present application include intact native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (herein referred to as V H ) and a heavy chain constant (C H ) region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (herein referred to as V L ) and a light chain constant C L region. The light chain constant region is comprised of one domain, C L . The V H region and the V L region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0061] As used herein, the term "single chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (V H ) and light (V L ) chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a V H ::V L heterodimer. The heavy (V H ) and light (V L ) chains are either directly linked or linked through a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids) that links the VH The N-terminus and V L C-end or V H C-end and V L The N end is connected.

[0062] As used herein, a "connector" refers to a functional group (e.g., chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids together to link them to each other. In some embodiments, the connector includes a function for coupling two proteins together (e.g., coupling V). H and V L A linker is one or more amino acids (or two dimerized domains coupled together). Linkers are typically glycine-rich for flexibility and serine or threonine-rich for solubility.

[0063] As used herein, the term "vector" refers to any genetic element that, when associated with appropriate control elements, is capable of replication and can transfer a gene sequence into a cell, such as plasmids, bacteriophages, transposons, kinases, chromosomes, viruses, viral particles, etc. Therefore, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.

[0064] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, such as a recombinant DNA molecule, containing the desired coding sequence operatively linked to an appropriate nucleic acid sequence necessary for the expression of the coding sequence in a specific host organism. The nucleic acid sequence necessary for expression in prokaryotes typically includes a promoter, an operon (optional), and a ribosome-binding site, usually along with other sequences. The nucleic acid sequence necessary for expression in eukaryotic cells may include, but is not limited to, promoters, enhancers, and termination and polyadenylation signals.

[0065] In some embodiments, nucleic acid molecules that can be used for the purposes of this disclosure include nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. Such nucleic acid molecules do not need to be 100% identical to endogenous nucleic acid sequences, but will generally exhibit substantial identity. Polynucleotides having “substantialgesic homology” or “substantialgesic identity” with endogenous sequences are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

[0066] As used herein, the term "disease" refers to any condition or symptom that impairs or interferes with the normal function of cells, tissues, or organs. Examples of diseases include tumor formation or pathogen infection of cells, tissues, or organs.

[0067] An“effective amount” (or“therapeutically effective amount”) is an amount that is sufficient to produce a beneficial or desired clinical result after treatment. An effective amount can be administered to a subject in one or more doses. For purposes of treatment, an effective amount is an amount that is sufficient to reduce, ameliorate, stabilize, reverse or slow the progression of a disease (e.g., a neoplasia), or reduce the pathological consequences of a disease (e.g., a neoplasia). The dosage containing an effective amount is typically determined by a physician, based on the particular circumstances of the case, and such determination is within the ability of one of ordinary skill in the art. When determining the appropriate dosage to achieve an effective amount, several factors are typically taken into account. These factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells (e.g., engineered immune cells) being administered.

[0068] As used herein, the term“neoplasm” refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. The growth of a neoplasia is typically uncontrolled and progressive and occurs under conditions that do not cause normal cells to proliferate or cause normal cell proliferation to cease. A neoplasia can affect a variety of cell types, tissues or organs, including but not limited to an organ selected from the group consisting of skin, bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, neural tissue, ovary, pleura, pancreas, prostate, skeletal muscle, spinal cord, spleen, stomach, testicle, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus and vagina, or a tissue or cell type thereof. A neoplasia includes a cancer, such as a melanoma, sarcoma, carcinoma or plasmacytoma (a malignant tumor of plasma cells).

[0069] As used herein, the term“immunoresponsive cell” refers to a cell that plays a role in an immune response, and includes progenitors of the cell and descendants of the cell.

[0070] As used herein, the term“isolated cell” refers to a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0071] As used herein, the term "isolated," "purified," or "biologically pure," means a substance that is not contained in its natural state. "Isolated" indicates a degree of separation from the original source or environment. "Purified" indicates a higher degree of separation than isolated. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, if a nucleic acid or peptide of the presently disclosed subject matter is produced by recombinant DNA technology with essentially no contamination from cellular material, viral material, or culture medium, or is produced by chemical synthesis with essentially no chemical precursors or other chemicals, then it is purified. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially one band on an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated proteins, which can be purified separately.

[0072] As used herein, the term "secreted" refers to the release of a polypeptide from a cell through the secretory pathway via the endoplasmic reticulum, Golgi apparatus, and as vesicles that transiently fuse on the plasma membrane to release the protein outside the cell.

[0073] As used herein, the term "treating" or "treatment" refers to clinical intervention with the intent to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviating symptoms, reducing any pathological consequences of the disease, either directly or indirectly, preventing metastasis, decreasing the rate of disease progression, ameliorating or palliating a disease state, and remission or amelioration of the prognosis. Treatment can prevent worsening due to the condition not only in the affected or diagnosed subject, but also in a subject at risk for, or suspected of having, the condition, by preventing progression of the disease or condition.

[0074] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like (e.g., who is the recipient of a particular treatment).

[0075] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell and a transmembrane domain. In certain embodiments, the extracellular antigen binding domain of the CAR comprises a scFv. The scFv can be derived from fusing the variable heavy and light regions of an antibody. Alternatively or additionally, the scFv can be derived from Fab's (rather than antibodies, e.g., obtained from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain, which is then fused to an intracellular signaling domain. In certain embodiments, the CAR has a high binding affinity or avidity for an antigen.

[0076] In certain non-limiting embodiments, the intracellular signaling domain of the CAR or ZipR-CAR comprises a CD3 zeta polypeptide, which can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T cell). CD3 zeta comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) and transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T cell) following antigen binding. The intracellular signaling domain of the CD3 zeta chain is the primary transmitter of signals from the endogenous TCR.

[0077] In certain non-limiting embodiments, the CAR or ZipR-CAR can further comprise a spacer / hinge region linking the extracellular antigen binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The spacer region can be a hinge region from IgGl, or a fragment of the CH2CH3 region of an immunoglobulin and a fragment of CD3, a fragment of a CD28 polypeptide, a fragment of a CD8 polypeptide, a variant thereof, or a synthetic spacer sequence.

[0078] As used herein, a "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand required for a lymphocyte response to an antigen. The at least one costimulatory signaling region can include a CD28 polypeptide (e.g., an intracellular domain of CD28 or a fragment thereof), a 4-1BB polypeptide (e.g., an intracellular domain of 4-1BB or a fragment thereof), an OX40 polypeptide (e.g., an intracellular domain of OX40 or a fragment thereof), an ICOS polypeptide (e.g., an intracellular domain of ICOS or a fragment thereof), a DAP-10 polypeptide (e.g., an intracellular domain of DAP-10 or a fragment thereof), or a combination thereof. The costimulatory molecule can bind a costimulatory ligand. As used herein, the term "costimulatory ligand" refers to a protein expressed on the cell surface that, upon binding to its receptor, produces a costimulatory response, i.e., an intracellular response that affects the stimulation provided by an activation signaling domain (e.g., a CD3 zeta signaling domain). Non-limiting examples of costimulatory ligands include a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, or a combination thereof. The costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof. Non-limiting examples of TNF family members include 4-1BBL, OX40L, CD70, GITRL, CD40L, and CD30L. Non-limiting examples of Ig superfamily members include CD80, CD86, and ICOSLG. For example, 4-1BBL can bind 4-1BB to provide an intracellular signal that, together with the CAR signal, induces CAR + effector functions of T cells. CARs comprising intracellular signaling domains comprising costimulatory signaling regions comprising 4-1BB, ICOS, or DAP-10 costimulatory signaling domains are disclosed in U.S. 7,446,190, the entirety of which is incorporated herein by reference.

[0079] As used herein, the term "multimerization" refers to the formation of multimers, including dimers. Multimerization includes dimerization.

[0080] As used herein, the term "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of a polypeptide of the present disclosure comprising an amino acid sequence (e.g., an extracellular antigen binding domain of a polypeptide). Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the human scFv of a polypeptide of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be divided into groups based on their physicochemical properties (e.g., charge and polarity). Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function (i.e., the functions listed in (c) through (1) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or CDR region are altered.

[0081] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), where the number of positions is determined by the alignment of the two sequences and the introduction of gaps, if any, to achieve the maximum percent homology. The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.

[0082] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program (version 10) using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length penalty of 1, 2, 3, 4, 5, or 6.

[0083] Additionally or alternatively, the amino acid sequences of the presently disclosed subject matter can be further used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. ((1990) J. Mol. Biol. 215:403-10). BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900) disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0084] 2. Membrane-bound polypeptide and soluble polypeptide

[0085] The presently disclosed subject matter provides a system comprising a membrane-bound polypeptide and a soluble polypeptide, wherein the soluble polypeptide is capable of dimerizing with the membrane-bound polypeptide.

[0086] 2.1 Membrane-bound polypeptides

[0087] In certain embodiments, the membrane-bound polypeptide comprises a transmembrane domain and an extracellular domain. In certain embodiments, the membrane-bound polypeptide further comprises an intracellular domain.

[0088] 2.1.1 Extracellular domain

[0089] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises a dimerization domain comprising a leucine zipper domain. In certain embodiments, the dimerization domain is capable of dimerizing with one or more dimerization domains comprised in the membrane-bound polypeptide. In certain embodiments, the dimerization domain is capable of dimerizing with one or more dimerization domains within a soluble polypeptide disclosed herein.

[0090] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises a first dimerization domain and a second dimerization domain capable of dimerizing with the first dimerization domain at the cell surface. In certain embodiments, the first and second dimerization domains each comprise a leucine zipper domain. In certain embodiments, the first dimerization domain comprises a first leucine zipper domain. In certain embodiments, the second dimerization domain comprises a second leucine zipper domain.

[0091] In certain embodiments, the leucine zipper domain comprises a dimerization domain of the basic region leucine zipper (bZIP) class of eukaryotic transcription factors. In certain embodiments, the leucine zipper domain comprises a particular alpha-helix monomer that can dimerize with another alpha-helix monomer. In certain embodiments, the leucine zipper domain comprises an EE domain comprising one or more acidic amino acids, such as glutamic acid (E). In certain embodiments, the leucine zipper domain comprises an RR domain comprising one or more basic amino acids, such as arginine (R). In certain embodiments, the first leucine zipper domain comprises an RR domain and the second leucine zipper domain comprises an EE domain.

[0092] In certain embodiments, the RR domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth as SEQ ID NO: 1, or a fragment thereof. In certain embodiments, the RR domain comprises a modification of SEQ ID NO: 1, or a fragment thereof. In certain embodiments, the modification comprises at most one, at most two, or at most three amino acid substitutions. SEQ ID NO: 1 is provided below.

[0093] LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPL GGGK [SEQ ID NO: 1]

[0094] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is set forth as SEQ ID NO: 97, which is provided below.

[0095]

[0096] In certain embodiments, the RR domain comprises a modification of SEQ ID NO: 1, wherein the modification consists of or has one amino acid substitution. In certain embodiments, the RR domain comprises an amino acid sequence as set forth in SEQ ID NO: 98 or SEQ ID NO: 99. SEQ ID NO: 98 and SEQ ID NO: 99 are provided below.

[0097]

[0098] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 98 is set forth in SEQ ID NO: 100, which is provided below.

[0099]

[0100] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 99 is set forth in SEQ ID NO: 101, which is provided below.

[0101]

[0102] In certain embodiments, the RR domain comprises a modification of SEQ ID NO: 1, wherein the modification consists of or has two amino acid substitutions. In certain embodiments, the RR domain comprises an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 103. SEQ ID NO: 102 and SEQ ID NO: 103 are provided below.

[0103]

[0104] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 102 is set forth in SEQ ID NO: 104, which is provided below.

[0105]

[0106] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 103 is set forth in SEQ ID NO: 105, which is provided below.

[0107]

[0108] In certain embodiments, the RR domain comprises a modification of SEQ ID NO: 1, wherein the modification consists of or has three amino acid substitutions. In certain embodiments, the RR domain comprises an amino acid sequence as set forth in SEQ ID NO: 106 or SEQ ID NO: 107. SEQ ID NO: 106 and SEQ ID NO: 107 are provided below.

[0109]

[0110] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 106 is set forth in SEQ ID NO: 108, which is provided below.

[0111]

[0112] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 107 is set forth in SEQ ID NO: 109, which is provided below.

[0113]

[0114] In certain embodiments, the modification is at the "g" residue of the RR domain of the leucine zipper. In certain embodiments, the modification reduces the heterodimerization affinity between the membrane-bound polypeptide and the linked soluble polypeptide.

[0115] In certain embodiments, the EE domain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof. In certain embodiments, the EE domain comprises a modification of SEQ ID NO: 2 or a fragment thereof. In certain embodiments, the modification comprises at most one, at most two, or at most three amino acid substitutions. SEQ ID NO: 2 is provided below.

[0116] LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPL GGGK [SEQ ID NO: 2]

[0117] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 is set forth in SEQ ID NO: 110, which is provided below.

[0118]

[0119] In certain embodiments, the extracellular domain further comprises a linker between the first dimerization domain and the second dimerization domain. In certain embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 20. SEQ ID NO: 3 and SEQ ID NO: 20 are provided below.

[0120]

[0121] In certain embodiments, the dimerization domain comprises an orthogonal zipper. Orthogonal zippers are coiled helical domains that form heterodimers with their specific partners only, and not with other zipper domains. In certain embodiments, orthogonality refers to a set of molecules (e.g., leucine zippers) that are not cross-reactive (i.e., “orthogonal”) with other sets of molecules. For example, A+B=AB, C+D=CD, but neither A nor B bind to C or D, and vice versa.

[0122] In certain embodiments, the first and second leucine zipper domains of the membrane-bound polypeptide are a pair of orthogonal zippers, i.e., the first and second leucine zipper domains are specific partners that form heterodimers with each other. Orthogonal zippers include, but are not limited to, RR / EE zippers, Fos / Jun zippers, and Fos / synZip zippers. Fos / Jun zippers have been previously disclosed in Ransone et al., Genes Dev. 1989 Jun; 3(6): 770-81; Kohler et al., Biochemistry. (2001 Jan); 9; 40(1): 130-42, which are incorporated herein by reference. Fos / synZip zippers have been previously disclosed in Grigoryan et al., Nature. (2009); 458, 859-864; Reinke et al., J Am Chem Soc. (2010); 132, 6025-6031, which are incorporated herein by reference.

[0123] In certain embodiments, the orthogonal zipper comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a RR / EE zipper, a Fos / Jun zipper, or a Fos / synZip zipper, or a fragment thereof, and / or can comprise at most one, at most two, or at most three amino acid substitutions.

[0124] Examples of synZip-9, Fos, and Jun zippers are set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0125]

[0126] In certain embodiments, the extracellular domain of the membrane-bound polypeptide further comprises a spacer / hinge domain between the dimerization domain and the transmembrane domain.

[0127] In certain embodiments, the spacer / hinge domain can be sufficiently flexible to allow the dimerization domain to orient in different directions to facilitate antigen recognition following dimerization with the soluble polypeptides disclosed herein. The spacer can be a hinge region from IgGl, or a fragment of the CH2CH3 region of an immunoglobulin and CD3, a fragment of a CD28 polypeptide, a fragment of a CD8 polypeptide, a variant of any of the foregoing (at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical thereto), or a synthetic spacer sequence.

[0128] In certain embodiments, the spacer / hinge domain comprises an epitope recognized by an antibody. In certain embodiments, binding of the antibody to the epitope mediates depletion of cells comprising the membrane-bound polypeptide. In certain embodiments, the spacer / hinge domain comprises a Thy1.1 molecule, a truncated EGFR molecule (EGFRt), a CD22 immunoglobulin-like domain epitope, an IgG / Fc domain (which can be Fc from any IgG), CD2, CD20 cyclic mimotope, CD30, CD52, or HER2.

[0129] In certain embodiments, the membrane-bound polypeptide further comprises a blocking spacer, wherein the blocking spacer is capable of preventing dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell. In certain embodiments, the blocking spacer comprises a minimal spacer of no more than about 20 to about 30 amino acid residues. In certain embodiments, the blocking spacer comprises no more than about 25 amino acid residues. In certain embodiments, the blocking spacer comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid residues. In certain embodiments, the blocking spacer comprises about 5 amino acid residues to about 25 amino acid residues, about 5 amino acid residues to about 20 amino acid residues, about 10 amino acid residues to about 25 amino acid residues, or about 10 amino acid residues to about 20 amino acid residues.

[0130] In certain embodiments, the blocking spacer comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a truncated CD28 spacer set forth in SEQ ID NO: 7 or SEQ ID NO: 21, or a fragment thereof. In certain embodiments, the blocking spacer comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous to an IgGl hinge set forth in SEQ ID NO: 8 or 22, or a fragment thereof. In certain embodiments, the blocking spacer comprises a modification of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 21, SEQ ID NO: 22, wherein the modification is at most one, at most two, or at most three amino acid substitutions.

[0131]

[0132] In certain embodiments, the blocking spacer is no more than about 25 amino acids in length. In certain embodiments, the blocking spacer is from about 5 amino acids to about 25 amino acids in length. In certain embodiments, the blocking spacer is a truncated CD28 spacer or an IgGl hinge.

[0133] In certain non-limiting embodiments, the extracellular domain of the membrane-bound polypeptide comprises at least one costimulatory ligand or fragment thereof.

[0134] In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof. In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and CD30L.

[0135] In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, and ICOSLG.

[0136] In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the CD80 is a mouse CD80. In certain embodiments, the CD80 comprises an amino acid sequence as set forth in SEQ ID NO: 111. In certain embodiments, the CD80 is a human CD80. In certain embodiments, the CD80 comprises an amino acid sequence as set forth in SEQ ID NO: 112. SEQ ID NO: 111 and SEQ ID NO: 112 are provided below.

[0137]

[0138] In certain embodiments, the costimulatory ligand is 4-1BBL. In certain embodiments, the 4-1BBL is a mouse 4-1BBL. In certain embodiments, the 4-1BBL comprises an amino acid sequence as set forth in SEQ ID NO: 113. In certain embodiments, the 4-1BBL is a human 4-1BBL. In certain embodiments, the 4-1BBL comprises an amino acid sequence as set forth in SEQ ID NO: 114. SEQ ID NO: 113 and 114 are provided below.

[0139]

[0140] In certain non-limiting embodiments, the extracellular domain of the membrane-bound polypeptide further comprises a dominant negative molecule or a fragment thereof. In certain embodiments, the dominant negative molecule is selected from an inhibitor of an immune checkpoint molecule, a tumor necrosis factor receptor superfamily (TNFRSF) member, and a TGF receptor. In certain embodiments, the immune checkpoint molecule is selected from PD-1, CTLA-4, B7-H3, B7-H4, BTLA, TIM-3, LAG-3, TIGIT, LAIR1, CD200, CD200R, HVEM, 2B4, CD160, galectin 9, and combinations thereof. In certain embodiments, the immune checkpoint molecule is PD-1. In certain embodiments, the TNFRSF member is selected from Fas, tumor necrosis factor receptor, OX40, CD40, CD27, CD30, 4-1BB, and combinations thereof. In certain embodiments, the dominant negative receptor comprises an extracellular domain of TGFpRII or a fragment thereof.

[0141] In certain non-limiting embodiments, the dominant negative molecule is an inhibitor of an immune checkpoint molecule. Details of dominant negative (DN) versions of inhibitors of immune checkpoint molecules are disclosed in WO2017 / 040945 and WO2017 / 100428, the contents of each of which are incorporated herein in their entirety by reference. In certain embodiments, the extracellular domain of the membrane-bound polypeptide further comprises a dominant negative version of an inhibitor of an immune checkpoint disclosed in WO2017 / 040945. In certain embodiments, the extracellular domain of the membrane-bound polypeptide further comprises a dominant negative version of an inhibitor of an immune checkpoint disclosed in WO2017 / 100428.

[0142] In certain embodiments, the dominant negative molecule is a PD-1 dominant negative (i.e., PD-1DN) molecule. In certain embodiments, the PD-1DN comprises (a) at least one fragment of the extracellular domain of PD-1 comprising a ligand binding region and (b) a transmembrane domain.

[0143] In certain embodiments, the PD-1DN is a mouse PD-1DN. In certain embodiments, the PD-1DN comprises or has an amino acid sequence as set forth in SEQ ID NO: 115, which is provided below. In certain embodiments, the PD-1DN is a human PD-1DN.

[0144]

[0145] In certain embodiments, the extracellular domain of the membrane-bound polypeptide further comprises a tag. In certain embodiments, the tag comprises an epitope tag recognized by a first antibody. Non-limiting examples of epitope tags include a Myc tag, a HA tag, a Flag tag, a V5 tag, a T7 tag, and a CD34 tag. In certain embodiments, the epitope tag is a CD34 tag.

[0146] In certain embodiments, the tag comprises an affinity tag that binds to a substrate. Non-limiting examples of affinity tags include a His tag, a Strep tag, an E tag, and a streptavidin binding protein tag (SBP tag).

[0147] In addition, the extracellular domain of the membrane-bound polypeptide can further comprise a mimotope recognized by a second antibody. Binding of the second antibody to the mimotope can mediate depletion of cells comprising the membrane-bound polypeptide. In certain embodiments, the mimotope is a CD20 mimotope recognized by an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is rituximab.

[0148] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises an epitope tag recognized by a first antibody and a mimotope recognized by a second antibody. In certain embodiments, the epitope tag is a CD34 epitope tag, the first antibody is an anti-CD34 antibody, the mimotope is a CD20 mimotope, and the second antibody is an anti-CD20 antibody. In certain embodiments, the anti-CD34 antibody is QBEND10. In certain embodiments, the anti-CD20 antibody is rituximab. In certain embodiments, the CD20 mimotope is a cyclic CD20 mimotope.

[0149] In certain embodiments, the CD20 mimotope comprises or has an amino acid sequence as set forth in SEQ ID NO: 116, which is provided below.

[0150] CPYSNPSLC [SEQ ID NO: 116]

[0151] In certain embodiments, the CD34 epitope tag comprises or has an amino acid sequence as set forth in SEQ ID NO: 117, which is provided below.

[0152] ELPTQGTFSNVSTNVs [SEQ ID NO: 117]

[0153] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises two CD34 epitope tags, e.g., each CD34 epitope tag comprises or has an amino acid sequence as set forth in SEQ ID NO: 117. In certain embodiments, the two CD34 epitope tags are connected by a linker. In certain embodiments, the linker comprises or has an amino acid sequence as set forth in SEQ ID NO: 118, which is provided below.

[0154] GGGGSGGGS [SEQ ID NO: 118]

[0155] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 119, which is provided below. SEQ ID NO: 119 has two CD34 epitope tags connected by a linker having an amino acid sequence as set forth in SEQ ID NO: 118, and SEQ ID NO: 119 is referred to as “Q2” or “Q2 sequence”.

[0156] ELPTQGTFSNVSTNVSGGGGSGGGSELPTQGTFSNVSTNVS [SEQ ID NO: 119]

[0157] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises two CD20 mimotope epitopes, e.g., each CD20 mimotope epitope comprises or has an amino acid sequence as set forth in SEQ ID NO: 116. In certain embodiments, the two CD20 mimotope epitopes are connected by a linker. In certain embodiments, the linker comprises or has an amino acid sequence as set forth in SEQ ID NO: 120, which is provided below.

[0158] SGGGGSSGGGGSD [SEQ ID NO: 120]

[0159] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 121, which is provided below. SEQ ID NO: 121 has two CD20 mimotope epitopes connected by a linker having an amino acid sequence as set forth in SEQ ID NO: 120, and SEQ ID NO: 121 is referred to as “R2” or “R2 sequence.”

[0160] CPYSNPSLCSGGGGSSGGGGSDCPYSNPSLC [SEQ ID NO: 121]

[0161] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises two CD20 mimotope epitopes and one CD34 epitope tag, e.g., each CD20 mimotope epitope comprises or has an amino acid sequence as set forth in SEQ ID NO: 116, and the CD34 epitope tag comprises or has an amino acid sequence as set forth in SEQ ID NO: 117. In certain embodiments, the CD34 epitope tag is connected to each CD20 mimotope epitope by a linker. In certain embodiments, the linker is a human CD8 polypeptide, e.g., a human CD8 polypeptide comprising or having an amino acid sequence as set forth in SEQ ID NO: 122, which is provided below.

[0162] PAKPTTT [SEQ ID NO: 122]

[0163] In certain embodiments, the linker comprises or has an amino acid sequence as set forth in SEQ ID NO: 123, which is provided below.

[0164] SGGGGS [SEQ ID NO: 123]

[0165] In certain embodiments, the extracellular domain of the membrane-bound polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 124, which is provided below. SEQ ID NO: 124 has two CD20 mimotope and one CD34 epitope tag, wherein the CD34 epitope tag is connected to one CD20 mimotope by a linker having an amino acid sequence as set forth in SEQ ID NO: 122, and connected to another CD20 mimotope by a linker having an amino acid sequence as set forth in SEQ ID NO: 123, SEQ ID NO: 124 is referred to as “RQR” or “RQR sequence”.

[0166] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLC [SEQ ID NO: 124]

[0167] 2.1.2 Transmembrane domain

[0168] Different transmembrane domains can result in different receptor stability. According to the presently disclosed subject matter, the transmembrane domain can comprise: a CD8 polypeptide (e.g., a transmembrane domain of CD8 or a fragment thereof), a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a fragment thereof), a CD3 zeta polypeptide (e.g., a transmembrane domain of CD3 zeta or a fragment thereof), a CD4 polypeptide (e.g., a transmembrane domain of CD4 or a fragment thereof), a 4-1BB polypeptide (e.g., a transmembrane domain of 4-1BB or a fragment thereof), an OX40 polypeptide (e.g., a transmembrane domain of OX40 or a fragment thereof), an ICOS polypeptide (e.g., a transmembrane domain of ICOS or a fragment thereof), a CD2 polypeptide (e.g., a transmembrane domain of CD2 or a fragment thereof), a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0169] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a fragment thereof). In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_001139345.1 (SEQ ID NO: 9) or a fragment thereof, and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 9 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 235 amino acids in length. Alternatively or additionally, in various embodiments without limitation, the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 183 to 203, or 200 to 235 of SEQ ID NO: 9. In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD8 polypeptide that comprises or has an amino acid sequence of amino acids 183 to 203 of SEQ ID NO: 9.

[0170] SEQ ID NO: 9 is provided below.

[0171]

[0172] In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number AAA92533.1 (SEQ ID NO: 10) or a fragment thereof, and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 10 that is at least 20, or at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 100, or at least 200 amino acids in length and up to 247 amino acids in length. Alternatively or additionally, in various embodiments without limitation, the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 247 of SEQ ID NO: 10. SEQ ID NO: 10 is provided below.

[0173]

[0174] In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence as set forth in SEQ ID NO: 11, which is provided below:

[0175] IYIWAPLAGICVALLLSLIITLICY [SEQ ID NO: 11]

[0176] In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence as set forth in SEQ ID NO: 12, which is provided below:

[0177] IYIWAPLAGTCGVLLLSLVIT [SEQ ID NO: 12]

[0178] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a fragment thereof). The CD28 polypeptide can have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number P10747 or NP_006130 (SEQ ID NO: 14) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 14 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 220 amino acids in length. Alternatively or additionally, in various embodiments without limitation, the CD28 polypeptide comprises or has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 14. In certain embodiments, the transmembrane domain of the membrane-bound polypeptide disclosed herein comprises a CD28 polypeptide comprising or having an amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 14. SEQ ID NO: 14 is provided below:

[0179]

[0180] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD28 polypeptide comprising or having an amino acid sequence as set forth in SEQ ID NO: 152, which is provided below.

[0181] FWVLVVVGGVLACYSLLVTVAFIIFWV [SEQ ID NO: 152]

[0182] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD28 polypeptide comprising or having an amino acid sequence set forth in SEQ ID NO: 23 as provided below.

[0183] FWALVVVAGVLFCYGLLVTVALCVIWT [SEQ ID NO: 23]

[0184] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD4 polypeptide (e.g., a transmembrane domain of CD4 or a fragment thereof). The CD4 polypeptide can have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_038516.1 (SEQ ID NO: 125) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD4 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 125 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 457 amino acids. Alternatively or additionally, in various embodiments without limitation, the CD4 polypeptide comprises or has an amino acid sequence of amino acids 1 to 457, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 395 to 417, or 400 to 457 of SEQ ID NO: 125. In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD4 polypeptide comprising or having amino acids 395 to 417 of SEQ ID NO: 125. SEQ ID NO: 125 is provided below:

[0185]

[0186] In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD4 polypeptide (e.g., the transmembrane domain of CD4 or a fragment thereof). The CD4 polypeptide can have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_000607.1 (SEQ ID NO: 126) or a fragment thereof, and / or can optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD4 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 126 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 458 amino acids. Alternatively or additionally, in various non-limiting embodiments, the CD4 polypeptide comprises or has the amino acid sequence of amino acids 1 to 457, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 397 to 418, or 400 to 457 of SEQ ID NO: 126. In certain embodiments, the transmembrane domain of the membrane-bound polypeptide comprises a CD4 polypeptide comprising or having the amino acid sequence of amino acids 397 to 418 of SEQ ID NO: 126. SEQ ID NO: 126 is provided below:

[0187]

[0188] 2.1.3 Intracellular Domain

[0189] In certain non-limiting embodiments, the membrane-bound polypeptide further comprises an intracellular domain. In certain non-limiting embodiments, the intracellular domain provides an activation signal to a cell (e.g., a cell of the lymphoid lineage, e.g., a T cell). In certain embodiments, the intracellular domain of the membrane-bound polypeptide comprises an immune activation molecule. In certain embodiments, the immune activation molecule is a CD3 zeta polypeptide.

[0190] In certain non-limiting embodiments, the intracellular domain of the membrane-bound polypeptide comprises a CD3 zeta polypeptide or fragment thereof. The CD3 zeta polypeptide can activate or stimulate a cell. CD3 zeta comprises 3 ITAMs and transmits an activation signal to a cell (e.g., a cell of lymphoid lineage, e.g., a T cell) following antigen binding. The intracellular signaling domain of the CD3 zeta-chain is the primary transmitter of signals from the endogenous TCR. In certain embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_932170 (SEQ ID NO: 15) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 15 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 164 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3 zeta polypeptide comprises or has the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 15. In certain embodiments, the CD3 zeta polypeptide comprises or has the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 15. SEQ ID NO: 15 is provided below:

[0191]

[0192] In certain embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_001106864.2 (SEQ ID NO: 13), or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 13 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100 and up to 188 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 142, 100 to 150, or 150 to 188 of SEQ ID NO: 13. SEQ ID NO: 13 is provided below:

[0193]

[0194] In certain embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence as set forth in SEQ ID NO: 17, which is provided below:

[0195]

[0196] In certain embodiments, the intracellular domain of the membrane-bound polypeptide comprises a murine CD3 zeta polypeptide.

[0197] In certain embodiments, the intracellular domain of the membrane-bound polypeptide comprises a human CD3 zeta polypeptide.

[0198] In certain non-limiting embodiments, the intracellular domain of the membrane-bound polypeptide provides an activation signal and a stimulatory signal to the cell. In certain embodiments, the intracellular domain of the membrane-bound polypeptide comprises at least one costimulatory molecule or fragment thereof.

[0199] In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide (e.g., an intracellular domain of CD28 or a fragment thereof), a 4-1BB polypeptide (e.g., an intracellular domain of 4-1BB or a fragment thereof), an OX40 polypeptide (e.g., an intracellular domain of OX40 or a fragment thereof), an ICOS polypeptide (e.g., an intracellular domain of ICOS or a fragment thereof), a DAP-10 polypeptide (e.g., an intracellular domain of DAP-10 or a fragment thereof), or a fragment or combination thereof. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide. In certain embodiments, the at least one costimulatory signaling region comprises an intracellular domain of CD28 or a fragment thereof.

[0200] In certain embodiments, the costimulatory molecule is a CD28 polypeptide. The CD28 polypeptide can comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 100% homologous or identical to the sequence of NCBI Reference Number P10747 or NP_006130 (SEQ ID NO: 14) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 14 that is at least 20, or at least 30, or at least 40, or at least 50 amino acids in length and up to 220 amino acids in length. Alternatively or additionally, in various embodiments without limitation, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 220 of SEQ ID NO: 14. In certain embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 181 to 220 of SEQ ID NO: 14.

[0201] In certain embodiments, the CD28 polypeptide can comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_031668.3 (SEQ ID NO: 16) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a contiguous fragment of SEQ ID NO: 16 that is at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to 218 amino acids in length. Alternatively or additionally, in various embodiments without limitation, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 218 of SEQ ID NO: 16. SEQ ID NO: 16 is provided below:

[0202]

[0203] In certain embodiments, the costimulatory molecule is a mouse CD28 polypeptide. In certain embodiments, the costimulatory molecule is a human CD28 polypeptide.

[0204] In certain embodiments, the intracellular domain of the membrane-bound polypeptide comprises two costimulatory molecules, e.g., CD28 and 4-1BB or CD28 and OX40.

[0205] In certain embodiments, the at least one costimulatory signaling region comprises a 4-1BB polypeptide. In certain embodiments, the at least one costimulatory signaling region comprises an intracellular domain of 4-1BB or a fragment thereof.

[0206] In certain embodiments, the costimulatory molecule is a 4-1BB polypeptide (e.g., an intracellular domain of 4-1BB or a fragment thereof). 4-1BB can act as a tumor necrosis factor (TNF) ligand and has stimulatory activity. The 4-1BB polypeptide can comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number P41273 or NP_001552 (SEQ ID NO: 151) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 151 is provided below:

[0207]

[0208] According to the presently disclosed subject matter, a“4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-1BB polypeptide.

[0209] In certain embodiments, the at least one costimulatory signaling region comprises an OX40 polypeptide. In certain embodiments, the at least one costimulatory signaling region comprises an intracellular domain of OX40 or a fragment thereof.

[0210] In certain embodiments, the costimulatory molecule is an OX40 polypeptide (e.g., an intracellular domain of OX40 or a fragment thereof). An OX40 polypeptide can comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number P43489 or NP_003318 (SEQ ID NO: 18) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 18 is provided below:

[0211]

[0212] According to the presently disclosed subject matter, an“OX40 nucleic acid molecule” refers to a polynucleotide encoding an OX40 polypeptide.

[0213] In certain embodiments, the at least one costimulatory signaling region comprises an ICOS polypeptide. In certain embodiments, the at least one costimulatory signaling region comprises an intracellular domain of ICOS or a fragment thereof.

[0214] In certain embodiments, the costimulatory molecule is an ICOS polypeptide. An ICOS polypeptide can comprise or have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference Number NP_036224 (SEQ ID NO: 19) or a fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 19 is provided below:

[0215]

[0216] According to the presently disclosed subject matter, an“ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide.

[0217] In certain embodiments, the at least one costimulatory signaling region comprises two costimulatory molecules or fragments thereof. In certain embodiments, the at least one costimulatory signaling region comprises a CD28 polypeptide (e.g., an intracellular domain of CD28 or a fragment thereof) and a 4-1BB polypeptide (e.g., an intracellular domain of 4-1BB or a fragment thereof).

[0218] In certain non-limiting embodiments, the intracellular domain of the membrane-bound polypeptide alone does not provide an activation signal to the cell. In certain embodiments, the intracellular domain of the membrane-bound polypeptide does not comprise a costimulatory molecule. In certain embodiments, the intracellular domain of the membrane-bound polypeptide does not comprise a CD3 zeta polypeptide.

[0219] In certain embodiments, the intracellular domain of the membrane-bound polypeptide further comprises a suicide gene. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk) and inducible caspase-9 suicide gene (iCasp-9). In certain embodiments, the intracellular domain of the membrane-bound polypeptide further comprises a truncated human epidermal growth factor receptor (EGFRt) polypeptide. The truncated EGFRt polypeptide can be eliminated from the T cell by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab).

[0220] In certain embodiments, the membrane-bound polypeptide comprises a synNotch module. SynNotch modules are disclosed in U.S. Patent Application No. 9,670,281 and Morsut et al., Cell, 164, 780-791, 2016, each of which is incorporated by reference herein in its entirety.

[0221] 2.2 Soluble polypeptides

[0222] The systems and methods for isolating a cell comprising at least two expression vectors of the present disclosure comprise a membrane-bound polypeptide encoded by a first expression vector and a soluble polypeptide encoded by a second expression vector. In certain embodiments, the membrane-bound polypeptide is a membrane-bound polypeptide disclosed herein, e.g., in Section 2.1.

[0223] In certain embodiments, the soluble polypeptide comprises a dimerization domain capable of dimerizing with a dimerization domain comprised in a membrane-bound polypeptide disclosed herein. In certain embodiments, the dimerization domain comprises a leucine zipper domain. The dimerization domain can be any dimerization domain disclosed in Section 2.1.1.

[0224] In certain embodiments, the soluble polypeptide comprises a dimerization domain and an antigen binding domain capable of binding an antigen.

[0225] In certain embodiments, the soluble polypeptide comprises a dimerization domain and a cytokine or chemokine. In certain embodiments, the soluble polypeptide further comprises a tag.

[0226] In certain embodiments, the leucine zipper domain of the membrane-bound polypeptide and the leucine zipper domain of the soluble polypeptide are a pair of orthogonal zippers, i.e., they are specific partners that form heterodimers with each other.

[0227] 2.2.1 Cytokine / chemokine

[0228] In certain embodiments, the soluble polypeptide further comprises a cytokine or chemokine. In certain embodiments, the cytokine / chemokine is capable of enhancing an immune response of an immune response cell and / or causing cell death of a malignant or infected cell. In certain embodiments, the cytokine / chemokine is an anti-tumor cytokine / chemokine. In certain embodiments, the cytokine or chemokine comprises or has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to a native cytokine / chemokine or fragment thereof, and / or can optionally comprise up to one or up to two or up to three conservative amino acid substitutions. Non-limiting examples of cytokines include IL-1, IL-2, IL-3, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-36, granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-gamma, CXCL1, IL-23, and CXCL10. Non-limiting examples of chemokines include CCL1, CCL8, CCL16, CCL17, CCL18, CCL22, or a combination thereof.

[0229] In certain embodiments, the chemokine is CCL1. In certain embodiments, the CCL1 is mouse CCL1. In certain embodiments, the CCL1 comprises the amino acid sequence set forth in SEQ ID NO: 127. In certain embodiments, the CCL1 is human CCL1. In certain embodiments, the CCL1 comprises the amino acid sequence set forth in SEQ ID NO: 128. SEQ ID NO: 127 and SEQ ID NO: 128 are provided below.

[0230]

[0231] In certain embodiments, the chemokine is CCL17. In certain embodiments, the CCL17 is mouse CCL17. In certain embodiments, the CCL17 comprises the amino acid sequence set forth in SEQ ID NO: 129. In certain embodiments, the CCL17 is human CCL17. In certain embodiments, the CCL17 comprises the amino acid sequence set forth in SEQ ID NO: 130. SEQ ID NO: 129 and SEQ ID NO: 130 are provided below.

[0232]

[0233] In certain embodiments, the chemokine is CCL18. In certain embodiments, the CCL18 is human CCL18. In certain embodiments, the CCL18 comprises the amino acid sequence set forth in SEQ ID NO: 131. SEQ ID NO: 131 is provided below.

[0234]

[0235] In certain embodiments, the chemokine is CCL22. In certain embodiments, the CCL22 is mouse CCL22. In certain embodiments, the CCL22 chemokine comprises the amino acid sequence set forth in SEQ ID NO: 132. In certain embodiments, the CCL22 is human CCL22. In certain embodiments, the CCL22 comprises the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NO: 132 and SEQ ID NO: 133 are provided below.

[0236]

[0237] 2.2.2 Antigen binding domain

[0238] In certain embodiments, the antigen binding domain of the soluble polypeptide comprises a single-chain variable fragment (scFv), a soluble ligand, a cytokine, or a non-scFv based antigen recognition motif, or a combination thereof.

[0239] In certain non-limiting embodiments, the antigen binding domain of the soluble polypeptide (specifically embodied, for example, as an scFv or an analog thereof) binds to an antigen with a dissociation constant (K d ) of about 2 x 10 -7 M or less. In certain embodiments, the K d is about 2 x 10 -7 M or less, about 1 x 10 -7 M or less, about 9 x 10 -8 M or less, about 1 x 10 -8 M or less, about 9 x 10-9 M or less, about 5 x 10 -9 M or less, about 4 x 10 -9 M or less, about 3 x 10 -9 or less, about 2 x 10 -9 M or less, or about 1 x 10 -9 M or less. In certain non-limiting embodiments, K d is about 3 x 10 -9 M or less. In certain non-limiting embodiments, K d is about 1 x 10 -9 M to about 3 x 10 -7 M. In certain non-limiting embodiments, K d is about 1.5 x 10 -9 M to about 3 x 10 -7 M.

[0240] Binding of an antigen binding domain (e.g., in a scFv or analog thereof) can be confirmed by, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular target protein-antibody complex by employing a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, a scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). The radioisotope can be detected by methods such as the use of a gamma counter or a scintillation counter, or by autoradiography. In certain embodiments, an extracellular antigen binding domain is labeled with a fluorescent label. Non-limiting examples of fluorescent labels include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

[0241] In certain embodiments, the antigen binding domain of the soluble polypeptide specifically binds an antigen. In certain embodiments, the antigen binding domain is a scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a murine scFv. In certain embodiments, the antigen binding domain is a Fab, which is optionally cross-linked. In certain embodiments, the antigen binding domain is a F(ab)2. In certain embodiments, any of the foregoing molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen binding domain. In certain embodiments, the scFv is identified by screening a scFv phage library with an antigen-Fc fusion protein. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen.

[0242] 2.2.3 Antigen

[0243] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to a tumor antigen. Any tumor antigen can be used in the tumor-associated embodiments described herein. The antigen can be expressed as a peptide or an intact protein or fragment thereof. The intact protein or fragment thereof can be native or mutagenized. Non-limiting examples of tumor antigens include CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, VpPreB, CD30, CD33, CD38, CD40, CD44v6, CD70, CD79a, CD70b, CLL-1 / CLEC12A, CD123, IL-3R complex, TIM-3, BCMA, CD244, E-cadherin, B7-H3, B7-H4, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD10, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2, 3, 4 (erb-B2, 3, 4), folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor alpha, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin 13 receptor subunit alpha-2 (IL-13Ra2), kappa light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), NKCS1, EGFlR, EGFR-VIII, CD99, ADGRE2, CCR1, LILRB2, PRAME, and ERBB.

[0244] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to a human CD19 polypeptide. In certain embodiments, the antigen binding domain of the soluble polypeptide binds to the extracellular domain of a human CD19 protein.

[0245] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to an immune checkpoint molecule. Non-limiting examples of immune checkpoint molecules include PD-L1, CD200, B7-H3, B7-H4, HVEM, Galectin 9, PD-1, CTLA-4, CD200R, TIM-3, Lag-3, and TIGIT.

[0246] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to an activating receptor, wherein binding of the antigen binding domain to the activating receptor is capable of activating an antigen presenting cell (APC). Non-limiting examples of immune checkpoint molecules include CD40, Toll-like receptors (TLRs), FLT3, RANK, and GM-CSF receptor.

[0247] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to a biomarker of hematopoietic lineage cells. Non-limiting examples of immune checkpoint molecules include CD3, CD16, CD33, c-Kit, CD161, CD19, CD20, vPreB (preB cell receptor), luteinizing hormone-releasing receptor (LHCGR), CD123, IL-3R complex, CLEC12A / CLL-1.

[0248] In certain embodiments, the antigen binding domain of the soluble polypeptide binds to a pathogen antigen, e.g., for the treatment and / or prevention of a pathogen infection or other infectious disease, e.g., in an immunocompromised subject. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that are capable of causing disease.

[0249] Non-limiting examples of viruses include, Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 (also called HTLV-III, LAVE, or HTLV-III / LAV or HIV-III; as well as other isolates, such as HIV-LP); Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis in humans); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue fever viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps viruses, Newcastle disease viruses, respiratory syncytial viruses); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hanta viruses, bunga viruses, phleboviruses, and nairoviruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses, and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (e.g., herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes B virus); Poxviridae (e.g., variola virus, vaccinia virus, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the originator of hepatitis delta virus (thought to be a defective satellite of hepatitis B virus), the originator of non-A, non-B hepatitis (class 1 = parenteral; class 2 = enterically transmitted (i.e., hepatitis C); Norwalk and related viruses; and the astroviruses).

[0250] Non-limiting examples of bacteria and / or fungi include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of bacteria that cause infection include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp.), Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Aspergillus species, and Actinomyces israelli.

[0251] 2.2.4 Tags

[0252] In certain embodiments, the soluble polypeptide comprises a tag. In certain embodiments, the tag comprises an epitope tag comprising an epitope recognized by a first antibody. In certain embodiments, the epitope tag is selected from the group consisting of a Myc tag, a HA tag, a Flag tag, a V5 tag, a T7 tag, a CD34 tag, and combinations thereof. In certain embodiments, the tag comprises an affinity tag that binds to a substrate. In certain embodiments, the affinity tag is selected from the group consisting of a His tag, a Strep tag, an E tag, a streptavidin binding protein tag (SBP tag), and combinations thereof.

[0253] 2.2.5. Mimic Epitopes

[0254] In certain embodiments, the soluble polypeptide further comprises a mimic epitope recognized by a second antibody. Binding of the second antibody to the mimic epitope can mediate depletion of cells comprising the soluble polypeptide.

[0255] In certain embodiments, the soluble polypeptide comprises an epitope tag recognized by a first antibody and a mimic epitope recognized by a second antibody. In certain embodiments, the epitope tag is a CD34 epitope tag, the first antibody is an anti-CD34 antibody, the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody. In certain embodiments, the anti-CD34 antibody is QBEND10. In certain embodiments, the anti-CD20 antibody is rituximab. In certain embodiments, the CD20 mimic epitope is a circular CD20 mimic epitope.

[0256] In certain embodiments, the CD20 mimotope comprises or has the amino acid sequence set forth in SEQ ID NO: 116, which is provided below.

[0257] CPYSNPSLC [SEQ ID NO: 116]

[0258] In certain embodiments, the CD34 epitope tag comprises or has the amino acid sequence set forth in SEQ ID NO: 117, which is provided below.

[0259] ELPTQGTFSNVSTNVS [SEQ ID NO: 117]

[0260] In certain embodiments, the soluble polypeptide comprises two CD34 epitope tags, e.g., each CD34 epitope tag comprises or has the amino acid sequence set forth in SEQ ID NO: 117. In certain embodiments, the two CD34 epitope tags are connected by a linker. In certain embodiments, the linker comprises or has the amino acid sequence set forth in SEQ ID NO: 118, which is provided below.

[0261] GGGGSGGGS [SEQ ID NO: 118]

[0262] In certain embodiments, the soluble polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 119, which is provided below. SEQ ID NO: 119 has two CD34 epitope tags connected by a linker having the amino acid sequence set forth in SEQ ID NO: 118, and SEQ ID NO: 119 is referred to as “Q2”.

[0263] ELPTQGTFSNVSTNVSGGGGSGGGSELPTQGTFSNVSTNVS [SEQ ID NO: 119]

[0264] In certain embodiments, the soluble polypeptide comprises two CD20 mimotopes, e.g., each CD20 mimotope comprises or has the amino acid sequence set forth in SEQ ID NO: 116. In certain embodiments, the two CD20 mimotopes are connected by a linker. In certain embodiments, the linker comprises or has the amino acid sequence set forth in SEQ ID NO: 120, which is provided below.

[0265] sGGGGSSGGGGSD [SEQ ID NO: 120]

[0266] In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 121, which is provided below. SEQ ID NO: 121 has two CD20 mimotope epitopes connected by a linker set forth in an amino acid sequence as set forth in SEQ ID NO: 120, and SEQ ID NO: 121 is referred to as "R2."

[0267] CPYSNPSLCSGGGGSSGGGGSDCPYSNPSLC [SEQ ID NO: 121]

[0268] In certain embodiments, the soluble polypeptide comprises two CD20 mimotope epitopes and a CD34 epitope tag, e.g., each CD20 mimotope epitope comprises or has an amino acid sequence set forth in SEQ ID NO: 116, and the CD34 epitope tag comprises or has an amino acid sequence set forth in SEQ ID NO: 117. In certain embodiments, the CD34 epitope tag is connected to each CD20 mimotope epitope by a linker. In certain embodiments, the linker is a human CD8 polypeptide, e.g., a human CD8 polypeptide comprising or having an amino acid sequence set forth in SEQ ID NO: 122, which is provided below.

[0269] PAKPTTT [SEQ ID NO: 122]

[0270] In certain embodiments, the linker comprises or has an amino acid sequence set forth in SEQ ID NO: 123, which is provided below.

[0271] SGGGGS [SEQ ID NO: 123]

[0272] In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 124, which is provided below. SEQ ID NO: 124 has two CD20 mimotope epitopes and a CD34 epitope tag, wherein the CD34 epitope tag is connected to one CD20 mimotope epitope by a linker set forth in an amino acid sequence as set forth in SEQ ID NO: 124, and the CD34 epitope tag is connected to the other CD20 mimotope epitope by a linker set forth in an amino acid sequence as set forth in SEQ ID NO: 124, and SEQ ID NO: 124 is referred to as "RQR."

[0273] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLC [SEQ ID NO: 124]

[0274] The presently disclosed subject matter also provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide of the present disclosure (e.g., a membrane-bound polypeptide or a soluble polypeptide). In certain embodiments, the nucleic acid further comprises a promoter for expressing the nucleic acid sequence in a human cell. The promoter for expressing the polypeptide can be a constitutive promoter (e.g., ubiquitin C (UbiC) promoter, MSCV, SFFV, EF1a, RSV, PGK, and MMLV LTR) or an inducible promoter (e.g., NFAT transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, IL-6 response element, sis-inducible element (SIE), interferon gamma response element, GAS / IRES element, NFkB response element, Gal response element, and tetracycline response element).

[0275] Also provided herein are expression vectors comprising a nucleic acid molecule encoding a membrane-bound polypeptide as disclosed herein or a soluble polypeptide as disclosed herein. The expression vector can be a viral vector or a transposon-based vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the retroviral vector is a lentiviral vector. The presently disclosed subject matter also provides a host cell comprising a nucleic acid molecule of the present disclosure. In certain embodiments, the host cell is a T cell.

[0276] 3. System

[0277] The presently disclosed subject matter provides a system for isolating a cell (e.g., for isolating a cell comprising at least two expression vectors) and / or for immunotherapy. In certain embodiments, the system comprises a membrane-bound polypeptide of the present disclosure encoded by a first expression vector and a soluble polypeptide of the present disclosure encoded by a second expression vector.

[0278] 3.1. Cell sorting system comprising a membrane-bound polypeptide with a self- blocking feature

[0279] The presently disclosed subject matter provides a system for isolating a cell comprising at least two expression vectors. In certain embodiments, the system comprises: a) a membrane-bound polypeptide of the present disclosure encoded by a first expression vector, and b) a soluble polypeptide of the present disclosure encoded by a second expression vector. In certain embodiments, the soluble polypeptide comprises a tag and a third dimerization domain capable of dimerizing with the first dimerization domain. In certain embodiments, the third dimerization domain forms a dimer with the first dimerization domain prior to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain forms a dimer with the first dimerization domain in the endoplasmic reticulum. In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are capable of forming a dimer when expressed from the same cell. In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are capable of forming a dimer in the endoplasmic reticulum when expressed from the same cell. In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are not capable of forming a dimer when expressed from different cells due to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106.

[0280] 3.2. Cell sorting system comprising a membrane-bound polypeptide with a blocking spacer

[0281] The presently disclosed subject matter provides a system for isolating a cell comprising at least two expression vectors. In certain embodiments, the system comprises: a) a membrane-bound polypeptide encoded by a first expression vector, wherein the membrane-bound polypeptide comprises a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerization domain and a blocking spacer, and b) a soluble polypeptide encoded by a second expression vector, wherein the soluble polypeptide comprises a tag and a second dimerization domain. In certain embodiments, the first and second dimerization domains each comprise a leucine zipper domain, and wherein the blocking spacer prevents dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell. In certain embodiments, the first dimerization domain comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106. In certain embodiments, the second dimerization domain comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 106. In certain embodiments, the blocking spacer is no more than about 25 amino acid residues. In certain embodiments, the blocking spacer is between about 5 amino acid residues to about 25 amino acid residues. In certain embodiments, the blocking spacer is a truncated CD28 spacer or an IgGl hinge.

[0282] 3.3. Common features of the systems disclosed herein

[0283] Any features of the membrane-bound polypeptides or soluble polypeptides disclosed herein (e.g., as disclosed in Section 2) can be applied to the systems disclosed herein.

[0284] In certain embodiments, the tag comprises an epitope tag recognized by the first antibody. In certain embodiments, the epitope tag is selected from the group consisting of a Myc tag, an HA tag, a Flag tag, a V5 tag, a T7 tag, a CD34 tag, and combinations thereof. In certain embodiments, the tag comprises an affinity tag that binds to a substrate. In certain embodiments, the affinity tag is selected from the group consisting of a His tag, a Strep tag, an E tag, a streptavidin binding protein tag (SBP tag), and combinations thereof.

[0285] In certain embodiments, the soluble polypeptide further comprises an antigen binding domain. In certain embodiments, the antigen binding domain comprises a single chain variable fragment (scFv), a soluble ligand, a cytokine, a chemokine, a non-scFv based antigen recognition motif, or combinations thereof. In certain embodiments, the soluble polypeptide further comprises a cytokine or a chemokine.

[0286] In certain embodiments, the membrane-bound polypeptide is expressed from a first vector. In certain embodiments, the soluble polypeptide is expressed from a second vector. The first vector can be the same as, or different from, the second vector. In certain embodiments, the first vector is the same as the second vector, e.g., the vector backbones of the first and second vectors can be the same, while the polypeptides or proteins encoded / expressed by the first and second vectors can be different.

[0287] 3.4. Exemplary membrane-bound polypeptides, soluble polypeptides, and systems

[0288] In certain embodiments, the membrane-bound polypeptide comprises a V5 tag, an EE12RR345L leucine zipper, a CD28 EC-9C hinge, a CD2 transmembrane (TM) domain, and a truncated cytoplasmic domain, an E2A peptide, and a Thy 1.1 peptide. (V5 tag staining identifies surface expression of the membrane-bound polypeptide). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0289]

[0290] In certain embodiments, the membrane-bound polypeptide comprises a V5 tag, an EE12RR345L leucine zipper, a CD28 EC-9C hinge, a CD28 TM domain, a CD3 zeta domain, an E2A peptide, and a Thyl.l peptide. (V5 tag staining identifies surface expression of the membrane-bound polypeptide). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25.

[0291]

[0292] In certain embodiments, the membrane-bound polypeptide comprises an EE12RR345L leucine zipper, a myc tag, a CD28 EC-9C hinge, a CD2 TM domain, and a truncated cytoplasmic domain, an E2A peptide, and a Thyl.l peptide. (No myc staining). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0293]

[0294] In certain embodiments, the membrane-bound polypeptide comprises an EE12RR345L leucine zipper, a myc tag, a CD28 EC-9C hinge, a CD28 TM domain, a CD3 zeta domain, an E2A peptide, and a Thyl.l peptide. (No myc staining). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0295]

[0296] In certain embodiments, the membrane-bound polypeptide comprises a V5 tag, an EE12RR345L leucine zipper, an IgGl hinge, a CD2 TM domain, and a truncated cytoplasmic domain, an E2A peptide, and a Thyl.l peptide. (V5 tag staining identifies surface expression of the membrane-bound polypeptide). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0297]

[0298] In certain embodiments, the membrane-bound polypeptide comprises a V5 tag, an EE12RR345L leucine zipper, an IgGl hinge, a CD28 TM domain, a CD3 zeta domain, an E2A peptide, and a Thyl.l peptide. (V5 tag staining identifies surface expression of the membrane-bound polypeptide). In certain embodiments, the membrane-bound polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0299]

[0300] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), an EGFRt polypeptide, a P2A peptide, and a blue fluorescent protein (BFP). In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 30.

[0301]

[0302] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), a Thy1.1 polypeptide, a P2A peptide, and a blue fluorescent protein (BFP). In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 31.

[0303]

[0304] In certain embodiments, the membrane-bound polypeptide comprises an EE12RR345L leucine zipper, a myc tag, a MHC-I hinge / TM domain, a P2A peptide, and a blue fluorescent protein (BFP). (No myc staining). In certain embodiments, the membrane-bound polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 32.

[0305]

[0306] In certain embodiments, the soluble polypeptide comprises a FLAG-tagged RR12EE345L leucine zipper, a P2A peptide, an iCaspase9 polypeptide, and a F2A peptide. In certain embodiments, the soluble polypeptide construct comprises an amino acid sequence set forth in SEQ ID NO: 33.

[0307]

[0308] In certain embodiments, the soluble polypeptide comprises a FLAG-tagged RR12EE345L leucine zipper, a P2A peptide, an iCaspase9 polypeptide, a F2A peptide, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3z polypeptide. In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 34 and SEQ ID NO: 35.

[0309]

[0310] In certain embodiments, the soluble polypeptide comprises a RR12EE345L leucine zipper with a FLAG tag, a P2A peptide, an iCaspase9 polypeptide, a F2A peptide, an IL-3 polypeptide, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3 zeta polypeptide. In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 36.

[0311]

[0312] In certain embodiments, the soluble polypeptide comprises a RR12EE345L leucine zipper with a FLAG tag, a P2A peptide, an iCaspase9 polypeptide, a F2A peptide, a CD38 scFv, an interchain linker, an IL-3 polypeptide, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3 zeta polypeptide. In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 37 and SEQ ID NO: 38.

[0313]

[0314] In certain embodiments, the system comprises a soluble polypeptide (e.g., a RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., a EE12RR345L leucine zipper), a Thyl.l polypeptide, a P2A peptide, a CD20 scFv, a twin streptag-II, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, and an E2A polypeptide. In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 39 and SEQ ID NO: 40.

[0315]

[0316] In certain embodiments, the system comprises a soluble polypeptide (e.g., a RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., a EE12RR345L leucine zipper), a Thyl.l polypeptide, a P2A peptide, a CD20 scFv, a twin streptag-II, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, an E2A polypeptide, and a pro-IL-18 polypeptide (comprising an IL-18 pro-peptide sequence). In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 41 and SEQ ID NO: 42.

[0317]

[0318] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), a Thyl.1 polypeptide, a P2A peptide, a CD20 scFv, a twin streptag-II, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, an E2A polypeptide, and a sIL-18 polypeptide (comprising a mouse IL-2 signal peptide sequence). In certain embodiments, the system comprises the amino acid sequences set forth in SEQ ID NO: 43 and SEQ ID NO: 44.

[0319]

[0320] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), a Thyl.1 polypeptide, a P2A peptide, a CD20 scFv, an interchain linker, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, and an E2A polypeptide. In certain embodiments, the system comprises the amino acid sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

[0321]

[0322] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), a Thyl.1 polypeptide, a P2A peptide, a CD20 scFv, an interchain linker, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, and an E2A polypeptide. In certain embodiments, the system comprises the amino acid sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

[0323]

[0324] In certain embodiments, the system comprises a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide (e.g., EE12RR345L leucine zipper), a Thyl.1 polypeptide, a P2A peptide, an IL-3 polypeptide, an interchain linker, a CD20 scFv, a twin streptag-II, a CD8EC hinge / TM domain, a CD28 costimulatory domain, a CD3 zeta polypeptide, and an E2A polypeptide. In certain embodiments, the system comprises the amino acid sequences set forth in SEQ ID NO: 50 and SEQ ID NO: 51.

[0325]

[0326] In certain embodiments, the soluble polypeptide comprises a mouse IL-7 polypeptide fused to a FLAG-tagged RR12EE345L leucine zipper, a P2A peptide, an iCaspase9 polypeptide, an F2A peptide, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3 zeta polypeptide. In certain embodiments, the soluble polypeptide comprises the amino acid sequences set forth in SEQ ID NO: 52 and SEQ ID NO: 53.

[0327]

[0328] In certain embodiments, the soluble polypeptide comprises a mouse IL-15 polypeptide fused to a FLAG-tagged RR12EE345L leucine zipper, a P2A peptide, an iCaspase9 polypeptide, an F2A peptide, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3 zeta polypeptide. In certain embodiments, the soluble polypeptide comprises the amino acid sequences set forth in SEQ ID NO: 54 and SEQ ID NO: 55.

[0329]

[0330] In certain embodiments, the soluble polypeptide comprises a mouse IL-21 polypeptide fused to a FLAG-tagged RR12EE345L leucine zipper, a P2A peptide, an iCaspase9 polypeptide, an F2A peptide, a CD19 scFv, a myc tag, a CD8EC hinge / TM domain, a CD28 costimulatory domain, and a CD3 zeta polypeptide. In certain embodiments, the soluble polypeptide comprises the amino acid sequences set forth in SEQ ID NO: 56 and SEQ ID NO: 57.

[0331]

[0332] In certain embodiments, the soluble polypeptide comprises a RQR sequence (with two CD20 mimotopes and one CD34 epitope), a linker, and a RR12EE345L leucine zipper. In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 134, which is provided below.

[0333]

[0334] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 134 is set forth in SEQ ID NO: 135, which is provided below.

[0335]

[0336] In certain embodiments, the soluble polypeptide comprises a Q2 sequence (with two CD34 epitopes), a linker, and a RR12EE345L leucine zipper. In certain embodiments, the soluble polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 136, which is provided below.

[0337]

[0338] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 136 is set forth in SEQ ID NO: 137, which is provided below.

[0339]

[0340] In certain embodiments, the membrane-bound polypeptide comprises a R2 sequence (with two CD20 mimotopes), a linker, a EE12RR345L leucine zipper, a CD28-9C hinge, a CD28 transmembrane domain, and a truncated CD3 zeta (delta). In certain embodiments, the membrane-bound polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 138, which is provided below.

[0341]

[0342] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 is set forth in SEQ ID NO: 139, which is provided below.

[0343]

[0344] In certain embodiments, the system comprises a soluble polypeptide comprising a R2 sequence and a RR12EE345L leucine zipper, a linker, a membrane-bound polypeptide comprising an EE12RR345L leucine zipper, a PD1 dominant negative molecule, a CD4 TM domain, and a truncated CD3z (delta). In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 140, which is provided below.

[0345]

[0346] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 is set forth in SEQ ID NO: 141, which is provided below.

[0347]

[0348] In certain embodiments, the system comprises a kappa signal peptide, a soluble polypeptide (e.g., RR12EE345L leucine zipper), a linker, a membrane-bound polypeptide having an EE12RR345L leucine zipper and a CD80 polypeptide. In certain embodiments, the system comprises an amino acid sequence set forth in SEQ ID NO: 142, which is provided below.

[0349]

[0350] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 142 is set forth in SEQ ID NO: 143, which is provided below.

[0351]

[0352]

[0353] In certain embodiments, the membrane-bound polypeptide comprises an EE12RR345L leucine zipper, a linker, and a 4-1BBL polypeptide. In certain embodiments, the membrane-bound polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 144, which is provided below.

[0354]

[0355] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 144 is set forth in SEQ ID NO: 145, which is provided below.

[0356]

[0357] Exemplary sequences of the elements included in the leucine zipper constructs are as follows.

[0358] Interchain linker: GGGGSGGGGSGGGGSGGGGSGGGS [SEQ ID NO: 59]

[0359] Alternative interchain linker 1: GGGGSGGGGSGGGGSGGGGSGGGS [SEQ ID NO: 59]

[0360] Alternative interchain linker 2: GGGGSSGGGGSD [SEQ ID NO: 146]

[0361] Alternative interchain linker 3: GGGGSGGGS [SEQ ID NO: 118]

[0362] Alternative interchain linker 4: GSTSGSGKPGSGEGSTKG [SEQ ID NO: 147]

[0363] Alternative interchain linker 5: EFTGSTSGSGKPGSGEGSTKG [SEQ ID NO: 148]

[0364] Alternative interchain linker 6: GGGGSGGGSALG [SEQ ID NO: 149]

[0365] Mouse IL-3 sequence used in the cytokine-based receptor binding region:

[0366]

[0367] Human IL-3 sequence to be used in a similar IL-3-based CAR:

[0368]

[0369] Mouse CD8 spacer for CAR, also a non-blocking spacer for ZipR-CAR:

[0370] STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACD [SEQ ID NO: 62]

[0371] Human CD8 spacer equivalent sequence:

[0372] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD [SEQ ID NO: 63]

[0373] Mouse CD28 long spacer for CAR:

[0374] IEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKL [SEQ ID NO: 64]

[0375] Human CD28 long spacer region equivalent sequence:

[0376] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP [SEQ ID NO: 65]

[0377] Mouse Thy1.1 (CD90) spacer region (non-blocking):

[0378]

[0379] Mouse Thyl.1 (CD90) propeptide (used as part of the Thyl.1 / CD90 spacer region to direct the construct to the cell membrane, immediately following the Thyl.1 spacer sequence):

[0380] GGIsLLVQNTSWMLLLLLSLsLLQALDFISL [SEQ ID NO: 67]

[0381] Human CD90 spacer region - deduced to be non-blocking based on homology to mouse (Thyl.1 / Thyl.2 allelic variants are restricted to mouse):

[0382]

[0383] Human CD90 propeptide (used as part of the CD90 spacer region to direct the construct to the cell membrane, immediately following the CD90 spacer sequence):

[0384] EGISLLAQNTSWLLLLLLSLSLLQATDFMSL [SEQ ID NO: 69]

[0385] Human EGFRt spacer region (non-blocking):

[0386]

[0387] Human EGFRt transmembrane domain: IATGMVGALLLLLVVALGIGLFM [SEQ ID NO: 71]

[0388] Mouse CD2 transmembrane domain and short intracellular region:

[0389] FYVTVGVGAGGLLLVLLVALFIFCIC KRRK (underlined is the transmembrane sequence) [SEQ ID NO: 72]

[0390] This element can promote weaker construct expression compared to CD28 transmembrane + CD3 zeta and can be used to control cell surface leucine zipper density expression. KRKK (SEQ ID NO: 150) can act as an endoplasmic reticulum retention signal.

[0391] Human CD2 transmembrane domain and short intracellular region:

[0392] IYLIIGICGGGSLLMVFVALLVFYIT KRKK (underlined is the transmembrane sequence) [SEQ ID NO: 73]

[0393] Mouse MHC class I transmembrane domain (H2-Kd) and short cytoplasmic linker:

[0394] VIIAVLVVLGAAIVTGAVVAFVMK GSG [SEQ ID NO: 74]

[0395] Mouse IL-7 sequence including signal peptide:

[0396]

[0397] Mouse IL-15 sequence + mouse IL-2 signal peptide:

[0398]

[0399] Mouse IL-21 sequence + mouse IL-2 signal peptide:

[0400]

[0401] Leucine zipper linker + FLAG tag + RR12EE345L (sequence after the cytokine sequence listed above):

[0402]

[0403] 2A peptide sequence:

[0404] E2A QCTNYALLKLAGDVESNPGP [SEQ ID NO: 79] F2A VKQTLNFDLLKLAGDVESNPGP [SEQ ID NO: 80] P2A ATNFSLLKQAGDVEENPGP [SEQ ID NO: 81] T2A EGRGSLLTCGDVEENPGP [SEQ ID NO: 82]

[0405] Tag sequence:

[0406]

[0407] Signal peptide sequence:

[0408] Mouse kappa leader METDTLLLWVLLLWVPGSTG [SEQ ID NO: 87] Mouse CD8a MASPLTRFLSLNLLLLGESIILGSGEA [SEQ ID NO: 88] Mouse IL-2 MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 89] Mouse IL-3 MVLASSTTSIHTMLLLLLMLFHLGLQ [SEQ ID NO: 90] Mouse IL-7 MFHVSFRYIFGIPPLILVLLPVTSS [SEQ ID NO: 91] Mouse IL-21 MERTLVCLVVIFLGTVA [SEQ ID NO: 92]

[0409] Other elements:

[0410]

[0411] 4. Methods of use

[0412] The presently disclosed subject matter provides a method of isolating a cell comprising at least two expression vectors. In certain embodiments, the method comprises:

[0413] a) expressing in the cell i) a membrane-bound polypeptide of the disclosure encoded by a first expression vector, and ii) a soluble polypeptide of the disclosure encoded by a second expression vector,

[0414] b) contacting the cell with a substrate that binds to the tag, and

[0415] c) isolating the cell bound to the substrate.

[0416] In certain embodiments, the method of isolating a cell comprising at least two expression vectors comprises:

[0417] a) expressing in the cell i) a membrane-bound polypeptide encoded by a first expression vector, comprising a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerization domain and a blocking spacer, and ii) a soluble polypeptide encoded by a second expression vector, comprising a tag and a second dimerization domain, wherein the first and second dimerization domains each comprise a leucine zipper domain, and wherein the blocking spacer prevents dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell,

[0418] b) contacting the cell with a substrate that binds to the tag, and

[0419] c) isolating the cell bound to the substrate.

[0420] Further, the presently disclosed subject matter provides a method of sorting a plurality of cells comprising at least two vectors. In certain embodiments, the method comprises:

[0421] a) transfecting a plurality of cells with i) a first expression vector encoding a membrane-bound polypeptide disclosed herein, and ii) a second expression vector encoding a soluble polypeptide disclosed herein,

[0422] b) contacting the cell with a substrate that binds to the tag, and

[0423] c) isolating one or more cells bound to the substrate.

[0424] In certain embodiments, step c), e.g., the step of isolating one or more cells bound to the substrate, is preceded by step d), e.g., washing the substrate to remove cells that are not bound to the substrate.

[0425] In certain embodiments, the method of sorting a plurality of cells comprising at least two expression vectors comprises:

[0426] a) transfecting a plurality of cells with i) a first expression vector encoding a membrane-bound polypeptide comprising a transmembrane domain and an extracellular domain comprising a first dimerization domain, and ii) a second expression vector encoding a soluble polypeptide comprising a tag and a second dimerization domain capable of dimerizing with the first dimerization domain, wherein the first and second dimerization domains each comprise a leucine zipper domain, and wherein the membrane-bound polypeptide does not dimerize with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell,

[0427] b) contacting the cells with a substrate that binds to the tag, and

[0428] c) isolating one or more cells bound to the substrate.

[0429] In certain embodiments, the soluble polypeptide comprises a tag and a third dimerization domain capable of dimerizing with the first dimerization domain comprised in the membrane-bound polypeptide. In certain embodiments, the third dimerization domain is capable of dimerizing with the first dimerization domain prior to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, the third dimerization domain is capable of dimerizing with the first dimerization domain in the endoplasmic reticulum.

[0430] In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are capable of forming a dimer when expressed from the same cell. In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are capable of forming a dimer in the endoplasmic reticulum when expressed from the same cell. In certain embodiments, the soluble polypeptide and the membrane-bound polypeptide are not capable of forming a dimer when expressed from different cells due to dimerization between the first dimerization domain and the second dimerization domain. In certain embodiments, step c), e.g., the step of isolating one or more cells bound to the substrate, is preceded by step d), washing the substrate to remove cells that are not bound to the substrate.

[0431] In certain embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the cell is autologous. In certain embodiments, the leucine zipper is an orthogonal zipper. In certain embodiments, the orthogonal zipper is an RR / EE zipper, a Fos / Jun zipper, or a Fos / synZip zipper. Examples of synZip-9, Fos, and Jun zippers are set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0432] 5. The cell of claim 1, wherein the first dimerization domain and the second dimerization domain each comprise a leucine zipper domain.

[0433] The presently disclosed subject matter provides cells comprising the membrane-bound polypeptides, soluble polypeptides, and / or systems disclosed herein. In certain embodiments, the polypeptides and / or systems are capable of activating or inhibiting an immune responsive cell. In certain embodiments, the polypeptides and / or systems are capable of promoting an anti-tumor effect of an immune responsive cell. The cells can be transduced with the polypeptides and / or systems such that the cells co-express the polypeptides and / or systems. In certain embodiments, the cells are immune responsive cells. The cells can be of the lymphoid lineage or of the myeloid lineage.

[0434] Cells of the lymphoid lineage can produce antibodies, regulate the cellular immune system, detect foreign substances in the blood, and detect foreign cells in the host, among other things. Non-limiting examples of cells of the lymphoid lineage include B cells, T cells, natural killer (NK) cells, dendritic cells, stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0435] In certain embodiments, the cells are T cells. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cell, including but not limited to: helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., T EM cells and T EMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that are capable of inducing death of infected somatic cells or tumor cells. By introducing any of the polypeptides or systems disclosed herein, a patient’s own T cells can be genetically modified to target specific antigens. The T cells can be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cells are CD4 + T cells. In certain embodiments, the T cells are CD8 + T cells.

[0436] In certain embodiments, the cells are natural killer (NK) cells. Natural killer (NK) cells can be lymphocytes that are part of the cell-mediated immunity and play a role in the innate immune response. NK cells do not require prior activation to perform cytotoxic effects on target cells.

[0437] In certain embodiments, the cell is a human lymphocyte. In certain embodiments, the human lymphocyte includes, but is not limited to, peripheral donor lymphocytes, such as those disclosed in Sadelain, M. et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, R.A. et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptors complexes comprising alpha and beta heterodimers), Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G.A., et al. 2003 Blood 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells).

[0438] The cell (e.g., T cell) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0439] In certain embodiments, the cell of the presently disclosed subject matter is a cell of the myeloid lineage. In certain embodiments, the cell of the myeloid lineage includes, but is not limited to, monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, and platelet cells.

[0440] The cells of the present disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment that can suppress the host immune response by any of a number of mechanisms, thereby protecting themselves from immune surveillance, recognition, and elimination. Immunosuppressive factors include, but are not limited to, infiltrating regulatory CD4 +T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands targeting immunosuppressive receptors (CTLA-4 and PD-1) expressed by activated T cells. These immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses, but within the tumor microenvironment, these mechanisms prevent an effective anti-tumor immune response. These immunosuppressive factors can collectively induce profound anergy or apoptosis of adoptively transferred modified T cells (e.g., CAR T cells) upon encountering target tumor cells.

[0441] In certain embodiments, the cells of the present disclosure have enhanced cell persistence. In certain embodiments, the cells of the present disclosure have reduced apoptosis and / or anergy.

[0442] The unpurified source of CTLs can be any source known in the art, such as bone marrow, fetal, neonatal, or adult or other hematopoietic cell sources, such as fetal liver, peripheral blood, or umbilical cord blood. Various techniques can be employed to isolate the cells. For example, negative selection methods can initially remove non-CTLs. Monoclonal antibodies (mAbs) are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages for positive and negative selection.

[0443] A substantial portion of terminally differentiated cells can be removed initially by a relatively crude separation. For example, a large number of irrelevant cells can be removed initially using magnetic bead separation. In certain embodiments, at least about 80%, and typically at least about 70% of total hematopoietic cells will be removed prior to isolating the cells.

[0444] Isolation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents linked to or used in conjunction with mAbs, including but not limited to complement and cytotoxins; and panning with antibodies attached to a solid substrate (e.g., plates, chips, panning) or any other convenient technique.

[0445] Techniques for isolation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scatter detection channels, impedance channels.

[0446] Cells can be distinguished from dead cells by using a dye associated with dead cells, such as propidium iodide (PI). In certain embodiments, the cells are collected in culture medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable medium, such as sterile isotonic medium.

[0447] 6. Vectors

[0448] Genetic modification of an immunoresponsive cell (e.g., a T cell) can be achieved by transducing a substantially homogeneous cell component with a recombinant DNA construct. In certain embodiments, a retroviral vector is used to introduce the DNA construct into the cell. For example, a polynucleotide encoding any of the polypeptides or systems disclosed herein can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for the target cell type of interest. In certain embodiments, the retroviral vector is a gamma retroviral vector. In certain embodiments, the retroviral vector is a lentiviral vector. Non-viral vectors can also be used.

[0449] For initial genetic modification of an immunoresponsive cell to include a polypeptide and / or system disclosed herein, a retroviral vector is typically employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. The polypeptide and / or system can be constructed in a single multi-cistronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors. Examples of elements that produce a multi-cistronic expression cassette include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κΒ IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A, and F2A peptides). Combinations of retroviral vectors and suitable packaging lines are also suitable, where the capsid proteins will have the function of infecting human cells. Various amphotropic cell lines are known that produce viral particles, including but not limited to PA12 (Miller et al., (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al., (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos et al., (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, such as pseudotyped particles with VSVG, RD114, or GALV envelopes and any other particles known in the art.

[0450] Possible transduction methods also include direct co-culture of cells with producer cells, for example by the method of Bregni et al. (1992) Blood 80:1418-1422, or separate incubation with viral supernatant or concentrated vector stock with or without appropriate growth factors and poly-cations, for example by the methods of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0451] Other transducing viral vectors can be used to modify immune response cells. In certain embodiments, the selected vector exhibits high infectivity efficiency, stable integration into the host cell genome, and persistent expression of the recombinant gene product (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes virus, such as Epstein-Barr virus (see also, e.g., vectors in Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Egli tis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors have developed particularly well and have been used in the clinic (Rosenberg et al., N. Engl. J. Med. 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0452] Non-viral methods can also be used for genetic modification of the immunoresponsive cells. For example, nucleic acid molecules can be introduced into the immunoresponsive cells by administration of the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), desialylated serum-mucin-polylysine conjugates (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Other non-viral methods of gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be beneficial for delivering DNA to cells. Transplantation of normal genes into affected tissues of a subject can also be accomplished by transferring normal nucleic acids into a cell type that can be cultured ex vivo (e.g., autologous or heterologous primary cells or progeny thereof), after which the cells (or progeny thereof) are injected into the target tissue or systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALEN nucleases, CRISPR). Transient expression can be obtained by RNA electroporation. In certain embodiments, the recombinant receptor can be introduced by a transposition-based vector. In certain embodiments, the transposition-based vector comprises a transposon (also known as a transposable element). In certain embodiments, the transposon can be recognized by a transposase. In certain embodiments, the transposase is a Sleeping Beauty transposase.

[0453] The resulting cells can be grown under conditions similar to unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

[0454] 7. Polypeptides and analogs

[0455] Also included in the presently disclosed subject matter are CD28, CD8, CD80, 4-1BBL, PD-1, and CD3 zeta polypeptides, the membrane-bound polypeptides disclosed herein, and the soluble polypeptides disclosed herein, or fragments thereof modified in a manner that enhances their therapeutic efficacy when expressed in an immune response cell. The presently disclosed subject matter provides methods of optimizing an amino acid sequence or a nucleic acid sequence by making sequence alterations. Such alterations can include certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter also includes analogs of any of the naturally occurring polypeptides disclosed herein (including, but not limited to, CD8, CD28, CD80, 4-1BBL, PD-1, and CD3 zeta). The analogs can differ from the naturally occurring polypeptides disclosed herein by amino acid sequence differences, by post-translational modifications, or by both. The analogs can exhibit at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology to all or a portion of the naturally occurring amino acid sequences of the presently disclosed subject matter. The length of the sequence comparison is at least 5, 10, 15, or 20 amino acid residues, such as at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Also, in exemplary methods of determining the degree of identity, the BLAST program can be used, with probability scores indicating closely related sequences between e -3 and e -100 Modifications include in vivo and in vitro chemical derivatization of the polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications can occur during polypeptide synthesis or processing or after treatment with isolated modifying enzymes. The analogs can also differ from the naturally occurring polypeptides by alterations in the primary sequence. These include natural and induced genetic variations (e.g., as described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra, due to random mutagenesis by irradiation or exposure to ethane methyl sulfonate or by site-specific mutagenesis). Also included are cyclized peptides, molecules, and analogs that contain residues other than L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids, such as beta or gamma amino acids.

[0456] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any of the polypeptides or peptide domains disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60 to 80, 100, 200, 300, or more contiguous amino acids. Fragments can be produced by methods known to those of skill in the art, or can be produced by normal protein processing (e.g., removal of amino acids not needed for biological activity from nascent polypeptides, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0457] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins / peptides disclosed herein. Such analogs can exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be performed according to such methods by modifying the chemical structure to result in an analog that, when expressed in an immune responsive cell, increases the anti-tumor activity of the original polypeptide. These chemical modifications include, but are not limited to, substitution of alternative R groups and alteration of the saturation of the reference polypeptide at particular carbon atoms. In certain embodiments, the protein analogs are relatively resistant to degradation in vivo, resulting in a longer lasting therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0458] Examples

[0459] Unless otherwise stated, the practice of this disclosure employs conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. These techniques are well explained in the following literature: for example, "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology"; "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" These techniques are well explained in *In Immunology* (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of this invention, and therefore can be considered in the preparation and implementation of this invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0460] The following examples are provided to provide a complete disclosure and description to those skilled in the art regarding how to prepare and use the compositions of the present invention and the methods for determining, screening and treating diseases, and are not intended to limit the scope of the inventors’ claims.

[0461] Example 1 - Leucine Zipper Cell Sorting Affinity Tag

[0462] A method was developed that allows for the selective sorting of cells that are co-transduced with two viral vectors to allow for the transfer of larger amounts of genetic information without exceeding the viral packaging limit. To achieve this goal, a leucine zipper based bi-affinity tag method was designed to allow for the magnetic selection of cells co-transduced with two viral vectors, where each vector expresses half of a leucine zipper based affinity tag system. In this system, one viral vector encodes a leucine zipper with an attached affinity tag (e.g. FLAG, streptag, myc, etc.) (Figure 1). The second viral vector encodes a membrane bound leucine zipper (e.g. EE12RR345L) that has a high preference for heterodimerization with the RR12EE345L-affinity tag zipper. When co-transduced into the same target cell, surface capture of the membrane bound EE12RR345L leucine zipper to the RR12EE345L-affinity tag occurs, allowing for surface detection of the affinity tag by flow cytometry and immunomagnetic selection using magnetic beads conjugated to antibodies specific for the affinity tag.

[0463] In some cases, secreted RR12EE345L-affinity tag leucine zippers are able to pair extracellularly with cells expressing only the membrane bound EE12RR345L leucine zipper Figure 2 ). To prevent this pairing, the membrane bound EE12RR345L leucine zipper is modified to have a "self-blocking" function, such that pairing of the RR12EE345L-affinity tag leucine zipper to the membrane bound EE12RR345L leucine zipper only occurs within cells transduced with both retroviral vectors, while extracellular pairing pathways are inhibited Figure 3 and Figure 4 ). As shown in Figure 4 , sorting of the FLAG tagged leucine zipper sorting construct with anti-FLAG magnetic beads resulted in a purified population of double transduced primary mouse T cells. The "self-blocking" feature was achieved by generating a membrane bound EE12RR345L leucine zipper with the attached RR12EE345L chain, but lacking the affinity tag. Possible genes for co-expression with the leucine zipper sorting system include, but are not limited to: chimeric antigen receptors (CARs), costimulatory chimeric receptors (CCRs), cytokines and chemokines, suicide genes, synNotch receptors and corresponding transactivator gene constructs, and costimulatory ligands.

[0464] Initial studies with the iCaspase 9 suicide gene on one vector and the CD20-CAR on another vector demonstrated that CD20-CAR T cells have high cytolytic activity against target cells and that activation of iCaspase 9 by the chemical inducer of dimerization induces more than 90% T cell apoptosis Figure 5

[0465] In addition, Figure 6 shows that the leucine zipper sorting system is used to purify cells to more than 95% that contain two vectors expressing CD19-CAR, CD20-CAR and iCaspase9 and optionally IL-18. These cells are able to kill CD19 or CD20+ targets and have enhanced cytokine expression and incubation with iCaspase9 dimer can lead to about 90% cell death.

[0466] In addition, monoclonal antibodies can be targeted to the spacer molecule contained within the membrane-bound leucine zippers to allow for in vivo depletion of cells expressing the construct Figure 3 For mouse and human T cells, in addition to the specific cell sorting method that integrates the two vectors together, the Thy1.1 and truncated EGFR (EGFRt) molecules can also be used as spacers to enable antibody-mediated depletion, respectively. For example, Figures 12A-12C It was demonstrated that a truncated EGFR spacer (EGFRt) fusion to a linker-blocked leucine zipper promotes cell sorting and antibody-dependent cell-mediated cytotoxicity (ADCC).

[0467] Figures 8A-8C It was further demonstrated that a linker-blocked truncated EGFR-spacer transmembrane zipper promotes MACS sorting of dual transduced cell populations by blocking pairing between membrane-bound leucine zippers expressed by different cells and soluble leucine zippers.

[0468] As another example of a binary system, Figure 9 A dual-tandem CAR configuration in conjunction with an iCaspase9 and blocked Thy1.1 leucine zipper sorting suicide construct is depicted. Two retroviral vectors encoding the leucine zipper sorting system construct and the tandem CAR are used to transduce T cells. The use of the same spacer / hinge (such as CD8) in the two independent CARs expressed on the same cell can promote heterodimerization. Different spacer combinations (such as CD8 spacer / CD28 spacer) can be used to avoid heterodimerization. Figures 10A-10B It was demonstrated that the leucine zipper sorting system is able to perform a single step MACS sorting of T cells expressing a dual-tandem CAR. Figures 11A-11B It was further demonstrated that the leucine zipper sorting system is able to deplete sorted T cells with two suicide genes. ​

[0469] Example 2 - Leucine Zipper Cell Sorting System comprising a short spacer / hinge region in the membrane-bound polypeptide

[0470] A new design of membrane-bound leucine zippers was developed that can inhibit the binding of other cell-secreted soluble tagged leucine zippers, but still allow the binding of internally produced tagged leucine zippers without the self- blocking feature described in Example 1. This membrane-bound polypeptide comprises a very small extracellular domain that does not contain an antibody epitope such as Thy1.1 or EGFRt. For example, a membrane-bound leucine zipper polypeptide comprising a CD8 spacer demonstrated the binding of soluble scFv leucine zippers expressed from the same cell expressing the membrane-bound polypeptide and other cells. However, membrane-bound leucine zipper polypeptides comprising a truncated CD28 nine amino acid spacer or IgGl hinge only bound soluble scFv leucine zippers expressed in the same cell as the membrane-bound polypeptide.

[0471] As shown in Figures 7A-7C , the truncated CD28 membrane proximal hinge-spacer transmembrane leucine zipper facilitates MACS sorting of double transduced cell populations by blocking pairing between membrane-bound leucine zippers and soluble leucine zippers expressed from different cells.

[0472] Example 3 - Leucine Zipper Cell Sorting System comprising a translocated cytokine in the soluble polypeptide

[0473] As shown in Figure 13 , a cytokine-tagged zipper - "ZipCyt" - was engineered to facilitate the secretion and trans-presentation of cytokines while retaining the sorting function of the affinity-tagged secreted leucine zippers. Cytokines (e.g., IL-7, IL-15, and IL-21) can be fused to the affinity tag and heterodimeric leucine zippers. Secreted ZipCyt interacts with cytokine receptors on T cells or is co-expressed with inherently blocked transmembrane leucine zippers to facilitate the sorting of double-carrier co-transduced cells and the trans-presentation of cytokines. Figures 14A-14C It was shown that ZipCyt retains the functional sorting characteristics of the leucine zipper sorting system and facilitates the proliferation of T cells.

[0474] Example 4 - Leucine Zipper Cell Sorting System comprising an epitope tag and a mimotope

[0475] As shown in Figure 15 , a sorting system comprising two CD20 mimotopes in tandem and a CD34 epitope tag (RQR-RR12EE345L) was generated. The ability of the sorting system was evaluated by using beads comprising an anti-CD34 antibody. As shown in Figure 16As shown, the optimal CD20 mimotopic CD34 leucine zipper tag capture and presentation required high expression of the truncated capture leucine zipper. Efficient CD34 and CD20 staining was observed when using the IgGl-hinge CD28TM CD3zA and CD28-9C CD28TM CD3zA capture leucine zippers. Cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently magnetically sorted using anti-CD34 magnetic beads. As shown, cells were sorted by anti-CD34 magnetic beads. Figure 17 C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. Figure 17 C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab.

[0476] C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. Figure 18 C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. Figure 18 C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab.

[0477] C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. Figure 19 C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab. C1498 cells were double transduced with the capture leucine zippers and a second vector encoding the CD20 mimotopic CD34 leucine zipper tag (RQR-RR12EE345L) as shown. Cells were subsequently incubated with the anti-CD20 antibody rituximab or the irrelevant antibody cetuximab in the presence of complement. As shown, only the cells transduced with the RQR tagged leucine zippers and capture leucine zipper vectors were depleted by the anti-CD20 antibody. Thus, selective elimination of the double transduced cells was achieved by using the anti-CD20 antibody rituximab.

[0478] Example 5 - Leucine zipper cell sorting system comprising a mutant membrane-bound polypeptide

[0479] Mutants blocking leucine zippers increase capture and presentation of secreted leucine zippers, but can show increased extracellular pairing patterns ("Surface Painting"). A series of mutations were made in the "g" residues of the blocking leucine zippers to reduce the heterodimerization affinity between the captured leucine zippers and the attached blocking leucine zippers. The following six mutants were generated: 1N mutant: E1R2EE345L (amino acid sequence set forth in SEQ ID NO: 98), 1M mutant: RR123E45L (amino acid sequence set forth in SEQ ID NO: 99), 2N mutant: EE12345L (amino acid sequence set forth in SEQ ID NO: 102), 2M mutant: RR1234E5L (amino acid sequence set forth in SEQ ID NO: 103), 3N mutant: EE12R3E45L (amino acid sequence set forth in SEQ ID NO: 106), and 3C mutant: RR12345L (amino acid sequence set forth in SEQ ID NO: 107).

[0480] C1498 cells were co-transduced with the FLAG-RR12EE345L GFP vector and one of the six mutants as part of the RR12EE345L linker EE12RR345L GFP vector construct. As shown in Figure 20 the 3N mutant. The 3N mutant showed increased FLAG binding not only in double transduced cells (intracellular pairing), but also in single transduced cells that only captured leucine zippers (extracellular pairing). The symmetric use of EE12RR345L for both capture and blocking leucine zippers resulted in strong capture of the FLAG zippers in both single and double transduced cells. Thus, mutating the RR12EE345L leucine zippers at the "g" position residues to include repulsive amino acid interactions can reduce the degree of blocking by the mutant leucine zippers based on the attached RR12EE345L.

[0481] Example 6 - Leucine Zipper Cell Sorting System Including Functionalized Membrane Bound Polypeptides

[0482] A CD80 (B7-1) molecule was functionalized to present a blocking captured leucine zipper, allowing magnetic sorting with a FLAG-RR12EE345L leucine zipper. T cells were transduced with vectors encoding (a) FLAG-RR12EE345L iCaspase9 CD19-myc-CAR and (b) RR12EE345L linker EE12RR345L CD80 CD20-streptag-CAR. Next, the cells were magnetically sorted with anti-FLAG magnetic beads. As shown in Figure 21ASorted cells showed high purity for CD19 and CD20 CAR (Myc, Streptag, respectively) and CD80 functionalized leucine zippers, as shown. Figure 21B T cells expressing RR12EE345L linker EE12RR345L CD80 formed conjugates in culture and bound to soluble CD28-Fc, as shown.

[0483] Embodiments of the presently disclosed subject matter

[0484] From the foregoing description, it will be apparent that variations and modifications can be made to the presently disclosed subject matter to apply to a variety of uses and conditions. Such embodiments are also within the scope of the following claims.

[0485] Any list of elements in the definition of a variable herein recited includes each individual element of the list or combinations (or subcombinations) of elements recited. Embodiments recited herein include the embodiment as any single embodiment or in combination with any other embodiment or portions thereof.

[0486] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A system for isolating cells containing at least two expression vectors, comprising: A) A membrane-binding polypeptide encoded by a first expression vector, said membrane-binding polypeptide comprising: i) Transmembrane structural domains, and ii) An extracellular domain comprising a first dimerizing domain and a second dimerizing domain capable of dimerizing with the first dimerizing domain on the cell surface. The first dimerization domain and the second dimerization domain each contain a leucine zipper domain, and B) A soluble polypeptide encoded by a second expression vector, comprising a tag and a third dimerizing domain capable of dimerizing with the first dimerizing domain. The third dimerizing domain forms a dimer with the first dimerizing domain prior to dimerization between the first dimerizing domain and the second dimerizing domain; wherein: (a) The amino acid sequence of the first dimerizing domain is as shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:107; the amino acid sequence of the second dimerizing domain is as shown in SEQ ID NO:2; and the amino acid sequence of the third dimerizing domain is as shown in SEQ ID NO:2; or (b) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:2, the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:107, and the amino acid sequence of the third dimerizing domain is shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:

107.

2. The system according to claim 1, wherein the third dimerizing domain forms a dimer with the first dimerizing domain in the endoplasmic reticulum.

3. The system of claim 1, wherein when the soluble polypeptide and the membrane-bound polypeptide are expressed from the same cell, they are able to form a dimer.

4. The system according to claim 1, wherein the soluble polypeptide and the membrane-bound polypeptide, when expressed from different cells, cannot form dimers due to dimerization between the first dimerizing domain and the second dimerizing domain.

5. The system according to claim 1, wherein: (i) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO:1, the amino acid sequence of the second dimerization domain is shown in SEQ ID NO:2, and the amino acid sequence of the third dimerization domain is shown in SEQ ID NO:

2. (ii) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO:2, the amino acid sequence of the second dimerization domain is shown in SEQ ID NO:1, and the amino acid sequence of the third dimerization domain is shown in SEQ ID NO:

1. (iii) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:106, the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:2, and the amino acid sequence of the third dimerizing domain is shown in SEQ ID NO:2; or (iv) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO:2, the amino acid sequence of the second dimerization domain is shown in SEQ ID NO:106, and the amino acid sequence of the third dimerization domain is shown in SEQ ID NO:

106.

6. The system of claim 1, wherein the extracellular domain further comprises a connector between the first dimerizing domain and the second dimerizing domain.

7. The system according to claim 6, wherein the amino acid sequence of the linker is as shown in SEQ ID NO:

3.

8. The system of claim 1, wherein the extracellular domain further comprises a spacer / hinge domain between the first dimerizing domain and the transmembrane domain.

9. The system of claim 8, wherein the spacer / hinge domain comprises an antibody-recognized epitope, wherein the binding of the antibody to the epitope mediates the depletion of cells expressing the membrane-binding polypeptide.

10. The system according to claim 8 or 9, wherein the spacer / hinge domain comprises a Thy1.1 molecule or a truncated EGFR molecule (EGFRt), wherein the amino acid sequence of the EGFRt molecule is as shown in SEQ ID NO:

70.

11. The system of claim 1, further comprising an intracellular domain.

12. The system of claim 11, wherein the intracellular domain comprises a CD3ζ domain, a co-stimulatory domain, or a suicide gene or a combination thereof.

13. The system of claim 1, wherein the membrane-binding polypeptide is expressed from a carrier.

14. The system of claim 1, wherein the extracellular domain further comprises a co-stimulatory ligand.

15. The system of claim 14, wherein the co-stimulatory ligand is selected from members of the tumor necrosis factor (TNF) family, members of the immunoglobulin (Ig) superfamily, and combinations thereof.

16. The system of claim 15, wherein the TNF family member is selected from 4-1BBL, OX40L, CD70, GITRL, CD40L, CD30L, and combinations thereof.

17. The system of claim 16, wherein the co-stimulatory ligand is 4-1BBL.

18. The system of claim 15, wherein the Ig superfamily member is selected from CD80, CD86, ICOSLG, and combinations thereof.

19. The system of claim 18, wherein the co-stimulatory ligand is CD80.

20. The system of claim 1, wherein the extracellular domain further comprises the dominant-negative form of the molecule.

21. The system of claim 20, wherein the molecule is selected from inhibitors of immune checkpoint molecules, members of the tumor necrosis factor receptor superfamily (TNFRSF), transforming growth factor β (TGFβ) receptor, and combinations thereof.

22. The system of claim 21, wherein the immune checkpoint molecule is selected from PD-1, CTLA-4, B7-H3, B7-H4, BTLA, TIM-3, LAG-3, TIGIT, LAIR1, CD200, CD200R, HVEM, 2B4, CD160, galactagogue 9, and combinations thereof.

23. The system of claim 21, wherein the TNFRSF member is selected from Fas, tumor necrosis factor receptor, OX40, CD40, CD27, CD30, 4-1BB, and combinations thereof.

24. The system of claim 1, wherein the tag comprises an epitope tag recognized by a first antibody.

25. The system of claim 24, wherein the epitope tag is selected from Myc tags, HA tags, Flag tags, V5 tags, T7 tags, CD34 tags, and combinations thereof.

26. The system of claim 24, wherein the soluble polypeptide further comprises a mimic epitope recognized by a second antibody, wherein the binding of the second antibody to the mimic epitope mediates the depletion of the cell.

27. The system of claim 26, wherein the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody.

28. The system of claim 27, wherein the anti-CD20 antibody is rituximab.

29. The system of claim 1, wherein the tag comprises an affinity tag that binds to the substrate.

30. The system of claim 29, wherein the affinity tag is selected from His tag, Strep tag, E tag, streptavidin-binding protein tag (SBP tag), and combinations thereof.

31. The system of claim 1, wherein the soluble polypeptide further comprises an antigen-binding domain.

32. The system of claim 31, wherein the antigen-binding domain comprises a single-chain variable fragment (scFv), a soluble ligand, a cytokine, or a non-scFv-based antigen recognition motif, or a combination thereof.

33. The system of claim 1, wherein the soluble polypeptide further comprises cytokines or chemokines.

34. The system of claim 1, wherein the first expression vector and the second expression vector have the same vector backbone, but the polypeptides or proteins encoded by the first expression vector and the second expression vector are different.

35. A system for isolating cells containing at least two expression vectors, comprising: A) A membrane-binding polypeptide encoded by a first expression vector, the membrane-binding polypeptide comprising a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerizing domain and a blocking spacer region, and B) A soluble polypeptide encoded by a second expression vector, the soluble polypeptide comprising a tag and a second dimerizing domain. The first dimerizing domain and the second dimerizing domain each contain a leucine zipper domain, and the blocking spacer region prevents the dimerization of the membrane-bound polypeptide and the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell; wherein: (a) The amino acid sequence of the first dimerizing domain is as shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:107, and the amino acid sequence of the second dimerizing domain is as shown in SEQ ID NO:2; or (b) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:2, and the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106, or SEQ ID NO:107; and The amino acid sequence of the blocking spacer region is shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:21 or SEQ ID NO:

22.

36. The system according to claim 35, wherein: (a) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO: 1, and the amino acid sequence of the second dimerization domain is shown in SEQ ID NO: 2; (b) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO: 2, and the amino acid sequence of the second dimerization domain is shown in SEQ ID NO: 1; (c) The amino acid sequence of the first dimerizing domain is as shown in SEQ ID NO: 106, and the amino acid sequence of the second dimerizing domain is as shown in SEQ ID NO: 2; or (d) The amino acid sequence of the first dimerization domain is shown in SEQ ID NO: 2, and the amino acid sequence of the second dimerization domain is shown in SEQ ID NO:

106.

37. The system of claim 35, wherein the tag comprises an epitope tag recognized by a first antibody.

38. The system of claim 37, wherein the epitope tag is selected from Myc tags, HA tags, Flag tags, V5 tags, T7 tags, CD34 tags, and combinations thereof.

39. The system of claim 38, wherein the epitope tag is a CD34 tag.

40. The system of claim 37, wherein the soluble polypeptide further comprises a mimic epitope recognized by a second antibody, wherein the binding of the second antibody to the mimic epitope mediates the depletion of the cell.

41. The system of claim 40, wherein the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody.

42. The system of claim 41, wherein the anti-CD20 antibody is rituximab.

43. The system of claim 35, wherein the tag comprises an affinity tag that binds to the substrate.

44. The system of claim 43, wherein the affinity tag is selected from His tag, Strep tag, E tag, streptavidin-binding protein tag (SBP tag), and combinations thereof.

45. The system of claim 35, wherein the soluble polypeptide further comprises an antigen-binding domain.

46. ​​The system of claim 45, wherein the antigen-binding domain is a single-stranded variable fragment (scFv).

47. The system of claim 35, wherein the soluble polypeptide further comprises cytokines or chemokines.

48. The system of claim 35, wherein the membrane-bound polypeptide and the soluble polypeptide are expressed from two carriers.

49. The system of claim 37, wherein the membrane-binding polypeptide further comprises a mimic epitope recognized by a second antibody, wherein the binding of the second antibody to the mimic epitope mediates the depletion of the cell.

50. The system of claim 49, wherein the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody.

51. The system of claim 50, wherein the anti-CD20 antibody is rituximab.

52. A method for isolating cells containing at least two expression vectors, comprising: a) Expression in cells i) The system according to claims 1-34; or ii) The system according to claims 35-51, b) Contact the cells with the substrate bound to the tag, and c) Isolate the cells that have bound the substrate.

53. A method for sorting multiple cells comprising at least two expression vectors, comprising: a) Transfect multiple cells using either i) or ii) below i) A first expression vector encoding the membrane-bound polypeptide of any one of claims 1-34 and a second expression vector encoding the soluble polypeptide of any one of claims 1-34; ii) A first expression vector encoding the membrane-bound polypeptide of any one of claims 35-51 and a second expression vector encoding the soluble polypeptide of any one of claims 35-51, b) Contact the cells with the substrate bound to the tag, and c) Isolate one or more cells that have bound the substrate.

54. The method of claim 52, wherein c) is preceded by d) washing the substrate to remove cells that do not bind to the substrate.

55. A method for isolating cells containing at least two expression vectors, comprising: A) Expressed in cells i) A membrane-binding polypeptide encoded by a first expression vector, comprising a transmembrane domain and an extracellular domain, wherein the extracellular domain comprises a first dimerizing domain and a blocking spacer region, and ii) A soluble polypeptide encoded by a second expression vector, comprising a tag and a second dimerizing domain, wherein both the first and second dimerizing domains comprise a leucine zipper domain, and wherein the blocking spacer region prevents dimerization of the membrane-bound polypeptide with the soluble polypeptide when the membrane-bound polypeptide and the soluble polypeptide are not expressed from the same cell. B) Contact the cells with the substrate bound to the tag, and C) Isolate cells that have bound the substrate; in: a) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:107, and the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:2, or b) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:2, and the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:1 and SEQ ID NO:

2.

98. SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: As shown in SEQ ID NO:106 or SEQ ID NO:107, and The amino acid sequence of the blocking spacer region is shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:21 or SEQ ID NO:

22.

56. A method for sorting multiple cells comprising at least two expression vectors, comprising: A) Transfect multiple cells using the following i) and ii) i) A first expression vector encoding a membrane-binding polypeptide, the membrane-binding polypeptide comprising a transmembrane domain and an extracellular domain, the extracellular domain comprising a first dimerizing domain and a second dimerizing domain capable of dimerizing with the first dimerizing domain on the cell surface, and ii) A second expression vector encoding a soluble polypeptide comprising a tag and a third dimerizing domain capable of dimerizing with the first dimerizing domain, wherein the first dimerizing domain and the second dimerizing domain each comprise a leucine zipper domain, and wherein the third dimerizing domain dimerizes with the first dimerizing domain prior to dimerization between the first dimerizing domain and the second dimerizing domain. B) Contact the cells with the substrate bound to the tag, and C) Isolate one or more cells that have bound the substrate; in: a) The amino acid sequence of the first dimerization domain is as shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:107, the amino acid sequence of the second dimerization domain is as shown in SEQ ID NO:2, and the amino acid sequence of the third dimerization domain is as shown in SEQ ID NO:2, or b) The amino acid sequence of the first dimerizing domain is shown in SEQ ID NO:2, and the amino acid sequence of the second dimerizing domain is shown in SEQ ID NO:1 and SEQ ID NO:

2.

98. SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: As shown in SEQ ID NO:106 or SEQ ID NO:107, and the amino acid sequence of the third dimerizing domain is as shown in SEQ ID NO:1, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: As shown in SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:106 or SEQ ID NO:

107.

57. The method of claim 55, wherein c) comprises d) washing the substrate to remove cells that do not bind to the substrate.

58. The method of claim 55, wherein the tag comprises an epitope tag recognized by the first antibody.

59. The method of claim 58, wherein the epitope tag is selected from Myc tags, HA tags, Flag tags, V5 tags, T7 tags, CD34 tags, and combinations thereof.

60. The method of claim 59, wherein the epitope tag is a CD34 tag.

61. The method of claim 58, wherein the soluble polypeptide further comprises a mimic epitope recognized by a second antibody, wherein the binding of the second antibody to the mimic epitope mediates the depletion of the cell.

62. The method of claim 61, wherein the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody.

63. The method of claim 62, wherein the anti-CD20 antibody is rituximab.

64. The method of claim 55, wherein the label comprises an affinity label that binds to the substrate.

65. The method of claim 64, wherein the affinity tag is selected from His tag, Strep tag, E tag, streptavidin-binding protein tag (SBP tag), and combinations thereof.

66. The method of claim 58, wherein the membrane-binding polypeptide further comprises a mimic epitope recognized by a second antibody, wherein the binding of the second antibody to the mimic epitope mediates the depletion of the cell.

67. The method of claim 66, wherein the mimic epitope is a CD20 mimic epitope, and the second antibody is an anti-CD20 antibody.

68. The method of claim 67, wherein the anti-CD20 antibody is rituximab.

69. The system of claim 1 or 35 or the method of claim 55 or 56, wherein the cell is selected from natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, human embryonic stem cells and pluripotent stem cells from which lymphoid cells can differentiate.

70. The system of claim 1 or 35, or the method of claim 55 or 56, wherein the cell is a T cell.

71. The system or method according to claim 70, wherein the T cell is selected from cytotoxic T lymphocytes (CTL), regulatory T cells, and natural killer T (NKT) cells.

72. The system of claim 1 or 35 or the method of claim 55, wherein the cell is autologous.

73. The system of claim 1 or 35 or the method of claim 55, wherein the leucine zipper is an orthogonal zipper.

74. A nucleic acid molecule encoding a membrane-bound polypeptide and a soluble polypeptide as defined in claim 1 or a membrane-bound polypeptide and a soluble polypeptide as defined in claim 35.

75. An expression vector comprising the nucleic acid molecule of claim 74.

76. The expression vector according to claim 75, wherein the vector is a viral vector.

77. The expression vector according to claim 76, wherein the viral vector is a retroviral vector.

78. The expression vector according to claim 77, wherein the retroviral vector is a lentiviral vector.

79. The expression vector according to claim 75, wherein the vector is a transposon-based vector.

80. A host cell comprising the nucleic acid molecule of claim 74.

81. The host cell of claim 80, wherein the host cell is a T cell.

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