Expression vectors for chimeric phagocytosis receptors, genetically modified host cells, and uses thereof

Abstract

The present disclosure relates to tandem expression cassettes encoding chimeric phagocytosis receptor molecules and chimeric antigen receptor / or T cell receptor binding proteins, host cells modified to comprise the tandem expression cassettes, and methods of making and using such receptor molecules and modified cells.

Description

[0001] Declaration of sequence list

[0002] A sequence list relating to this application is provided in the form of a text file in lieu of a paper copy, and is incorporated herein by reference. The text file containing the sequence list is named 200265_406WO_SEQUENCE_LISTING.txt. This text file is 539KB in size, was created on March 26, 2019, and submitted electronically via EFS-Web. Background Technology

[0003] The use of genetically engineered receptor-modified T cells targeting cancer antigens in hematologic malignancies has shown clinical success (e.g., CD19-specific chimeric antigen receptor therapy in leukemia). To name just a few, numerous clinical trials are underway for adoptive cell immunotherapy of solid tumors using engineered receptors targeting CEA, GD2, mesothelin, IL13Rα, HER2, FAP, and L1CAM. Engineered receptors include chimeric antigen receptors (CARs) and affinity-enhanced T-cell receptors (TCRs). However, treating solid tumors presents unique challenges, including: transport to the tumor site, physical barriers in the tumor microenvironment, stress-induced metabolic conditions, and immunosuppressive mechanisms (e.g., expression of immune checkpoint molecules and production of inhibitory cytokines). Efforts to increase T-cell persistence and activity in adoptive immunotherapy continue. Attached Figure Description

[0004] Figure 1A -G shows a vector diagram of an exemplary tandem expression cassette. This tandem expression cassette contains a human papillomavirus 16 (HPV16) E7 protein-specific TCR to induce a tumor-specific cytolysis response (e.g., cervix) and a phosphatidylserine-specific chimeric phagocytic receptor (CER) to stimulate tumor-specific phagocytic activity following cytolysis-induced phosphatidylserine exposure. Figure 1A An exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a chimeric phagocytic receptor 5 (CER5) construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER5 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER5 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER5 includes a Tim4 binding domain, a Tim4 transmembrane domain, and a TLR4 phagocytic signaling domain. Figure 1BAn exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER19 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER19 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER19 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER19 comprises a Tim4 binding domain, a Tim4 transmembrane domain, and a TLR5 signaling domain. Figure 1C An exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER21 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER21 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER21 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER21 comprises a Tim4 binding domain, a Tim4 transmembrane domain, and a TLR8 signaling domain. Figure 1D An exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER25 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER25 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER25 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER25 contains a Tim4 binding domain, a Tim4 transmembrane domain, and an NFAM1 signaling domain. Figure 1E An exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER27 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER27 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER27 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER27 comprises a Tim4 binding domain, a Tim4 transmembrane domain, and a TLR2 signaling domain. Figure 1F An exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER29 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER29 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER29 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER29 comprises a Tim4 binding domain, a Tim4 transmembrane domain, and a Traf6 signaling domain. Figure 1GAn exemplary tandem expression cassette is shown, comprising a polynucleotide encoding a CER31 construct and a polynucleotide encoding an HPV16 E7-specific TCR. CER31 is located upstream of the HPV16 E7-specific TCR. The sequences encoding CER31 and the HPV16 E7 TCR are operatively linked to the EF-1α promoter and separated by a T2A peptide. CER31 contains a Tim4 binding domain, a Tim4 transmembrane domain, and a Traf3 signaling domain.

[0005] Figure 2 The cytotoxicity of human primary CD8+ T cells modified with a tandem expression construct containing the CER21-HPV16 E7 TCR is shown. Images show caspase 3 / 7 fluorescent indicator dye emitted from HPV16 E7+ head and neck squamous cell carcinoma (SCC152) cells at different time points (2 h, 4 h, and 6 h). In timed co-culture experiments using SCC152 cells, CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette (top row) were compared with CD8+ T cells transduced with HPV16 E7 TCR alone (bottom row). The tandem expression construct endowed CD8+ T cells with cytolytic and phagocytic activity and enhanced cytotoxicity. Effector CD8+ T cells:target SCC152 cells were incubated at a 1:1 ratio.

[0006] Figure 3 This is a line graph showing the induction of caspase 3 / 7 over time in HPV16 E7+ SCC152 cells after co-culturing with human primary CD8+ T cells transduced with a lentiviral vector containing a tandem cassette of HPV16 E7 TCR and chimeric phagocytic receptor 21 (CER21) separated by a T2A sequence. The tandem expression construct encoding HPV16 E7 TCR and CER21 conferred greater target cell killing ability on host CD8+ T cells compared to host CD8+ T cells containing only HPV16 E7 TCR. Effector CD8+ T cells were co-incubated with target SCC152 cells at a 1:1 ratio. Total caspase 3 / 7 fluorescence was quantified over time.

[0007] Figure 4 This is a bar graph showing the induction of Caspase 3 / 7 in HPV16 E7+ SCC152 cells after co-culturing with CD8+ T cells transduced with a lentiviral vector containing a tandem cassette of HPV16 E7 TCRs and CERs separated by a T2A sequence. Simulated transduced cells were used as a negative control. SCC152 cells were labeled with caspase 3 / 7 red apoptosis reagent, which can detect cells undergoing apoptosis (red fluorescence). Measurements were taken over time from co-culture experiments, comparing CD8+ T cells transduced using the tandem CER-HPV16 E7 TCR cassette with CD8+ T cells transduced using the HPV16 E7 TCR control.

[0008] Figure 5 Images of caspase 3 / 7 fluorescent indicator dye emitted from HPV+ head and neck squamous cell carcinoma (SCC152) cells 6 hours after the start of co-culture are shown. Co-culture of CD8+ T cells transduced using the CER-HPV16 E7 TCR tandem expression cassette is compared to co-culture of CD8+ T cells transduced using only HPV16 E7 TCR (second from the top). CD8+ T cells transduced using the CER-HPV16 E7 TCR tandem expression cassette showed higher caspase induction compared to control CD8+ T cells.

[0009] Figure 6 This is a bar graph showing the quantification of phagocytosis in CD8+ T cells transduced using HPV16 E7 TCR, CER21-HPV16 E7 TCR tandem expression cassette, CER29-HPV16 E7 TCR tandem expression cassette, or CER31-HPV16 E7 TCR tandem expression cassette after 6 hours of co-culture. Phagocytosis was quantified using hybrid capture software on the Keyence BZ-X710 imaging system, where phagocytosis was determined by identifying the number of red fluorescent targets (SCC152 cells) in blue-stained effector cells (purple-labeled CD8+ T cells transduced using the CER-HPV16 E7 TCR tandem expression cassette - internalized red # / blue #) x 100.

[0010] Figure 7 These are fluorescence micrographs showing CER21-HPV16 E7 TCR tandem expression cassette-transduced CD8+ T cells (purple) engulfing SCC152 target cells (pHrodo Red labeled). The CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette were co-cultured with a 1:1 mixture of SCC152 target cells for 6 hours before imaging. The arrows indicate representative images of SCC152 cells (red) internalized in the endosomal compartments of the purple CD8+ T cells (left image = bright field overlap, right image = fluorescence overlap). The tandem cassette-engineered CD8+ T cells clearly engulfed the SCC152 tumor cell line.

[0011] Figure 8This study demonstrates that CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette phagocytose target cells in a Rac1-dependent manner. Activation and membrane recruitment of the small GTPase Rac1 triggered phagocytosis. The left image shows the phagocytosis of pH rodo-labeled target cells by blue-labeled CD8+ cells transduced with the CER21-HPV16 E7 TCR. The Rac1 inhibitor NSC23766 (50 μM) was added to the co-culture experiment (right image), and in vitro phagocytosis / phagocytosis was quantified. Fluorescence micrographs show that the inhibition of Rac1 by the small molecule eliminated in vitro phagocytosis by CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette (right).

[0012] Figure 9 This image shows CD8+ T cells transduced with the CER29-HPV16 E7 TCR tandem expression cassette phagocytizing target cells in a Rac1-dependent manner. Activation and membrane recruitment of the small GTPase Rac1 triggered phagocytosis. The left image shows blue-labeled CD8+ cells transduced with the CER29-HPV16 E7 TCR phagocytizing pH rodo-labeled target cells. The Rac1 inhibitor NSC23766 (50 μM) was added to the co-culture experiment (right image), and in vitro phagocytosis / phagocytosis was quantified. Fluorescence micrographs show that the inhibition of Rac1 by the small molecule eliminated in vitro phagocytosis by CER29-HPV16 E7 TCR tandem expression cassette transduced CD8+ T cells (right).

[0013] Figure 10 This image shows CD8+ T cells transduced with the CER31-HPV16 E7 TCR tandem expression cassette phagocytizing target cells in a Rac1-dependent manner. Activation and membrane recruitment of the small GTPase Rac1 triggered phagocytosis. The left image shows blue-labeled CD8+ cells transduced with the CER31-HPV16 E7 TCR phagocytizing pH rodo-labeled target cells. The Rac1 inhibitor NSC23766 (50 μM) was added to the co-culture experiment (right image), and in vitro phagocytosis / phagocytosis was quantified. Fluorescence micrographs show that the inhibition of Rac1 by the small molecule eliminated in vitro phagocytosis by CER31-HPV16 E7 TCR tandem expression cassette transduced CD8+ T cells (right).

[0014] Figure 11This study demonstrates that CD8+ T cells transduced using a tandem expression cassette containing the CER21-HPV16 E7 TCR possess cytolysis and phagocytic functions. CER21-HPV16 E7 TCR-transduced CD8+ T cells exhibit the ability to phagocytose and take up phosphatidylserine-coated beads in vitro. Streptavidin-coated latex beads were conjugated with biotin-phosphatidylserine and used in a phagocytosis assay. After 30 minutes of incubation, CER21-HPV16 E7 TCR-transduced CD8+ T cells showed up to take up phosphatidylserine-coated beads (white arrows indicate representative images of the phagocytic event).

[0015] Figure 12 High-magnification images of CD8+ T cells transduced by CER21-HPV16 E7 TCR are shown, demonstrating their in vitro phagocytic uptake of phosphatidylserine-coated beads.

[0016] Figure 13 Optical micrographs are shown of HPV16 E7 TCR-transduced CD8+ T cells co-cultured with phosphatidylserine-coated latex beads at 30 minutes post-incubation. HPV16 E7 TCR-transduced CD8+ T cells alone did not show uptake of the phosphatidylserine-coated latex beads.

[0017] Figure 14 This is a 3D bar chart showing the cytokine secretion pattern of CD8+ T cells transduced with the CER21-HPV16E7 TCR tandem expression cassette or solely with HPV16E7TCR and co-cultured with SCC152 target cells. To determine the cytokine secretion pattern, CER21-HPV16E7 TCR-modified CD8+ T cells were co-cultured with SCC152 target cells. The secretion of antigen-specific cytokines was determined by measuring cytokine concentrations in the cell supernatant of each co-culture using a medium-sized multiarray cytokine plate. The following cytokines were measured in the assay: IFNγ, IL-2, TNFα, IL-4, IL-6, IL-12b, IL-13, IL-1b, and IL-10. As indicated by cytokine secretion (e.g., IFNγ), CD8+ T cells transduced with the CER21-HPV16E7 TCR tandem expression cassette exhibited antigen-specific effector function.

[0018] Figures 15A-15B This shows that the phagocytic activity of T cells is specifically induced by CER. Figure 15AFACS analysis of phagocytosis assays is shown. CellTrace-purple labeled simulated transduced T cells, HPV E7-TCR-transduced T cells, and HPV E7-TCR / CER29 tandem expression cassette-transduced T cells were co-cultured with pHrodo red labeled head and neck cancer cells. Figure 15B Quantitative FACS data were presented, showing no difference in phagocytosis between simulated transduced T cells and E7 TCR transduced T cells. T cells co-expressing CER29 and E7 TCR exhibited phagocytic activity. Detailed Implementation

[0019] In one aspect, this disclosure provides a tandem expression cassette for co-expressing a first transgene encoding a first adoptive immunotherapy molecule and a second transgene encoding a second adoptive immunotherapy molecule in the same host cell. Embodiments of the tandem expression cassette described herein include a polynucleotide encoding a chimeric phagocytic receptor (CER); and a polynucleotide encoding a chimeric antigen receptor (CAR) or recombinant T-cell receptor (TCR). The tandem expression cassette of this disclosure can be used to confer tandem cell lysis and phagocytosis phenotypes in the same host cell. In some embodiments, cytotoxic activity of a CAR or TCR specific to a first target antigen induces apoptosis in target cells expressing the first target antigen, thereby exposing the second target antigen; and co-expression of a CER specific to the second target antigen induces phagocytosis of target cells or particles expressing the second target antigen. This interaction may be separated from the same cells that produce environmental / phosphatidylserine expression and have different interactions with specific tumors or tumor cells, wherein the environmental / phosphatidylserine expression is associated with phagocytosis of target cells.

[0020] Additionally, cells modified to express the contents of this disclosure in a tandem expression cassette are provided, as well as methods and compositions for delivering such modified cells to objects in need of them.

[0021] Before elaborating on this disclosure in more detail, providing definitions for certain terms used herein may help in understanding it.

[0022] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, and where appropriate, to include fractions (e.g., one-tenth and one-hundredth of an integer). Furthermore, unless otherwise stated, any numerical range relating to any physical characteristic (such as polymer subunits, size, or thickness) described herein should be understood to include any integer within the range. As used herein, unless otherwise stated, the term “about” means ±20% of the range, value, or structure shown. It should be understood that, as used herein, the term “a / some” refers to “one / some or more / some” of the listed components. The use of alternative words (e.g., “or”) should be understood to mean one, both, or any combination thereof. The terms “comprising,” “having,” and “including” as used herein are used synonymously, and these terms and variations thereof are intended to be construed as non-limiting.

[0023] Unless otherwise explicitly defined herein, those skilled in the art should understand the terminology as it is given in the art. The term "antibody" is used in the broadest sense and includes both polyclonal and monoclonal antibodies. "Antibody" can refer to a complete antibody comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds, and an antigen-binding moiety (or antigen-binding domain) of the complete antibody having or retaining the ability to bind to a target molecule. Antibodies can be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, such as intracellular antibodies, peptide bodies, nanobodies, single-domain antibodies, SMIPs, and multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem bi-scFVs, tandem tri-scFVs, ADAPTIR). Monoclonal antibodies or their antigen-binding moiety can be non-human, chimeric, humanized, or human, preferably humanized or human. The structure and function of immunoglobulins are reviewed in, for example, Chapter 14 of *Antibodies: A Laboratory Manual*, edited by Harlow et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The term "antigen-binding portion" or "antigen-binding domain" of a complete antibody is intended to include "antibody fragments" that represent a portion of the complete antibody and refer to the antigen-determining variable region or complementarity-determining region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, Fab'-SH, F(ab')2, bifunctional antibodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. A "Fab" (antigen-binding fragment) is a portion of an antibody that binds to an antigen and contains a variable region of the heavy chain and a CH1 region linked to the light chain via interchain disulfide bonds. Antibodies can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0024] The term "variable region" or "variable domain" in antibody refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, each containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated by screening libraries of complementary VL or VH domains, using the VH or VL domains of antibodies that bind to that antigen, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0025] As is known in the art, the terms “complementarity-determining region” and “CDR”, synonymous with “hypervariate region” or “HVR,” refer to discontinuous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0026] As used herein, the terms “binding domain,” “binding region,” and “binding moiety” refer to molecules (such as peptides, oligopeptides, polypeptides, or proteins) that have the ability to specifically and non-covalently bind, associate, unite, recognize, or combine with target molecules (e.g., tumor antigens). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate against a target biomolecule or other target. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or its functional binding domain or antigen-binding moiety. Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), receptor extracellular domains (e.g., TNF-α), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific binding ability to biomolecules.

[0027] A T-cell receptor (TCR) is a molecule present on the surface of T cells (also called T lymphocytes) and typically responsible for recognizing antigens that bind to the major histocompatibility complex (MHC) molecule. In most T cells, the TCR is usually composed of a heterodimer of highly variable α and β chains linked by disulfide bonds (also called TCRα and TCRβ, respectively). In a small subset of T cells, the TCR is composed of a heterodimer of γ and δ chains (also called TCRγ and TCRδ, respectively). Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus (see Janeway et al., Immunobiology: The Immune System in Health and Disease, 3). rd Ed., Current Biology Publications, p. 4:33, 1997. TCRs in this disclosure can be derived from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs can be cell-bound (i.e., having transmembrane regions or domains) or soluble. TCRs include recombinant-produced, genetically engineered, fusion, or modified forms of TCRs, including, for example, scTCRs, soluble TCRs, and TCR fusion constructs (TRuCs). TM (See U.S. Patent Publication No. 2017 / 0166622).

[0028] The "variable regions" or "variable domains" (Vα and Vβ) of the TCR α-chain and β-chain, or Vγ and Vδ of the TCR, are involved in the binding of the TCR to the antigen. The Vα and Vβ domains of the natural TCR... α and V β They typically have similar structures, with each variable structural domain containing four conservative FRs and three CDRs. α The domain is encoded by two separate DNA segments: the variable gene segment (V gene) and the linker gene segment (J gene); V β The domain is encoded by three separate DNA segments: the variable gene segment (V gene), the diverse gene segment (D gene), and the connecting gene segment (J gene). A single V gene... α or V β The domain may be sufficient to confer antigen binding specificity.

[0029] Major histocompatibility complex (MHC) molecules are glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a transmembrane α chain (with three α domains) and a non-covalently bound β2-microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which are transmembrane. Each chain has two domains. MHC class I molecules deliver peptides originating from the cytoplasm to the cell surface, where the peptide:MHC complex is activated by CD8+. + T cell recognition. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, where they are recognized by CD4+. + T-cell recognition. MHC molecules can originate from various animal species, including humans, mice, rats, or other mammals.

[0030] A chimeric antigen receptor (CAR) is a chimeric protein containing two or more distinct domains that can act as a receptor when expressed on the cell surface. A CAR typically consists of an extracellular domain (containing a binding domain that binds to the target antigen), optionally an extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., containing a T-cell activation motif based on an immune receptor tyrosine activation motif (ITAM), and optionally an intracellular co-stimulatory domain). In some embodiments, the intracellular signaling domain of the CAR has a T-cell activation domain containing an ITAM (e.g., CD3ζ) and an intracellular co-stimulatory domain (e.g., CD28). In some embodiments, the CAR is synthesized as a single polypeptide chain or encoded by a nucleic acid molecule as a single-chain polypeptide.

[0031] Various assays are known to be used to identify the binding domains of this disclosure that specifically bind to a particular target, and to determine the affinity of the binding domains, such as Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. Analysis and MHC tetramer analysis (see also, for example, Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; Altman et al., Science 274:94-96 ,1996; and U.S. Patent Nos. 5,283,173, 5,468,614 or equivalents thereof. As used herein, “specific binding” means binding a domain or its fusion protein at a ratio of 10 or greater. 5 M -1 Affinity or K a (i.e., the equilibrium association constant of a specific binding interaction, in units of 1 / M) associates or binds with the target molecule, but does not significantly associate or bind with any other molecule or component in the sample.

[0032] The terms “antigen” and “Ag” refer to molecules capable of inducing an immune response. The induced immune response may involve antibody production, activation of specific immune cells, or both. Macromolecules (including proteins, glycoproteins, and glycolipids) can serve as antigens. Antigens can be derived from recombinant or genomic DNA. As considered herein, an antigen (i) does not need to be encoded solely by the full-length nucleotide sequence of a gene or (ii) does not need to be encoded by a “gene” at all. Antigens can be produced or synthesized, or they can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0033] The term "epitaph" or "antigenic epitope" refers to any molecule, structure, amino acid sequence, or protein determinant within an antigen that is specifically bound by an associated immune-binding molecule, such as an antibody or fragment thereof (e.g., scFv), T-cell receptor (TCR), CAR, chimeric phagocytic receptor, or other binding molecules, domains, or proteins. Epitope determinants typically contain chemically active surface groups of the molecule, such as amino acid or sugar side chains, and may possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be linear or conformational.

[0034] As used herein, an "effect domain" is the intracellular portion of a fusion protein or chimeric receptor that, upon receiving an appropriate signal, can directly or indirectly promote a biological or physiological response in cells expressing the effector domain. In some embodiments, the effector domain is part of a protein or protein complex that receives a signal upon binding. In other embodiments, the effector domain is part of a protein or protein complex that directly binds to a target molecule, which triggers a signal from the effector domain. For example, in response to the binding of a CER to a target molecule, the effector domain can transduce signals into the host cell to stimulate effector functions, such as phagocytosis, phagolysosomal maturation, or secretion of anti-inflammatory and / or immunosuppressive cytokines. When the effector domain contains one or more signal transduction domains or motifs, it can directly promote a cellular response. In other embodiments, the effector domain will indirectly promote a cellular response by binding to one or more other proteins that directly promote a cellular response.

[0035] A "phagocytic signaling domain" refers to an intracellular effector domain that, upon binding to a target molecule (e.g., phosphatidylserine) targeted by the extracellular domain of the CER expressed by the host cell, activates one or more signaling pathways in the host cell, thereby leading to phagocytosis (including, in certain embodiments, cytoskeleton rearrangement of the host cell and internalization of target cells or particles associated with the target antigen). In some embodiments, the phagocytic signaling domain activates one or more signaling pathways to result in the phagocytosis of target cells or particles. In further embodiments, the phagocytic signaling domain includes a primary phagocytic signaling domain and a secondary phagocytic signaling domain.

[0036] "Linking amino acid" or "linking amino acid residue" refers to one or more (e.g., about 2-20) amino acid residues located between two adjacent motifs, regions, or domains of a polypeptide. Linking amino acids can be generated by the design of chimeric protein constructs (e.g., amino acid residues generated using restriction enzyme sites during the construction of nucleic acid molecules encoding fusion proteins).

[0037] "Disease" is a state of health in which the object cannot maintain homeostasis, and where, if the disease is not improved, the object's health will continue to deteriorate. In contrast, an object's "disorder" or "adverse condition" refers to a state of health in which the object can maintain homeostasis, but in which case the object's health is worse than it would be without disorder or adverse condition. Without treatment, disorder or adverse condition does not necessarily lead to a further decline in the object's health.

[0038] "Nucleic acid molecules" and "polynucleotides" can be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. Nucleic acid molecules can consist of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), naturally occurring nucleotide analogs (e.g., α-enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications or substitutions of a sugar moiety, or a pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester bonds include thiophosphates, dithiophosphates, selenite phosphates, diselenophosphates, thiophosphate sulfates, aniline phosphates, aminophosphates, etc. Nucleic acid molecules can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding strand (antisense strand). Coding molecules can have the same coding sequence as those known in the art, or can have different coding sequences that encode the same polypeptide due to redundancy or degeneracy of the genetic code, or through splicing.

[0039] "Encoding" refers to the inherent properties of a specific polynucleotide sequence (such as DNA, cDNA, and mRNA sequences) that serve as a template for the synthesis of other polymers and macromolecules during bioprocessing, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to that polynucleotide produces a protein in a cell or other biological system, then that polynucleotide encodes a protein. Both coding and non-coding strands can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

[0040] As used herein, the terms “endogenous” or “natural” refer to a gene, protein, compound, molecule, or activity that is normally present in the host or host cells, including naturally occurring variants of said gene, protein, compound, molecule, or activity.

[0041] As used herein, the term "homologous" or "homogeneous" refers to a molecule or activity derived from a host cell that is related to a second gene or activity through ancestry, for example, from the same host cell, from different host cells, from different organisms, from different strains, or from different species. For example, a heterologous molecule or a heterologous gene encoding said heterologous molecule may be homologous to the natural host cell molecule or the gene encoding said molecule, but it may have altered structure, sequence, expression level, or a combination thereof.

[0042] As used herein, a "heterologous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule or part of a nucleic acid molecule that is not naturally present in the host cell, but may be homologous to a nucleic acid molecule or part of a nucleic acid molecule derived from the host cell. The source of a heterologous nucleic acid molecule, construct, or sequence may be from a different genus or species. In some embodiments, the heterologous nucleic acid molecule is not naturally present. In some embodiments, a heterologous nucleic acid molecule (i.e., not endogenous or natural) is added to a host cell or host genome by means of, for example, conjugation, transformation, transfection, transduction, electroporation, etc., wherein the added molecule may be integrated into the host cell genome or exist in the form of extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may exist in multiple copies. Furthermore, "heterologous" refers to a non-natural enzyme, protein, or other activity encoded by a non-endogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.

[0043] As used herein, the terms “engineered,” “recombinant,” “modified,” or “non-natural” refer to organisms, microorganisms, cells, nucleic acid molecules, or vectors that have been modified by introducing exogenous nucleic acid molecules, or to cells or microorganisms that have been genetically engineered through human intervention, i.e., modified by introducing heterologous nucleic acid molecules, or to cells or microorganisms that have been altered to control, dysregulate, or constitutively modify the expression of endogenous nucleic acid molecules or genes, wherein such alterations or modifications may be introduced through genetic engineering. Human-derived genetic alterations may include, for example, modifications such as the introduction of nucleic acid molecules encoding one or more proteins, chimeric receptors, or enzymes (which may include expression control elements such as promoters), or the addition, deletion, substitution, or functional disruption or addition of other cellular genetic material. Exemplary modifications include modifications in the coding region or functional fragment of a heterologous or homologous polypeptide from a reference or parent molecule. Other exemplary modifications include, for example, modifications in non-coding regulatory regions, wherein said modifications alter the expression of a gene or operon.

[0044] As used herein, the term "transgenic" refers to a gene or polynucleotide encoding a target protein (e.g., CER, CAR, TCR) that is expected to be expressed in a host cell and has been transferred into the cell using genetic engineering techniques. Transgenics can encode proteins of therapeutic significance, or they can be reporter proteins, tags, biomarkers, suicide proteins, etc. Transgenics can be molecules derived from natural sources, modified or recombined from natural genes, or synthetic molecules. In some embodiments, the transgenic is a component of a vector.

[0045] The term "overexpressed" or "overexpressed" antigen refers to an abnormally high level of antigen expression in cells. Overexpressed antigens or antigen overexpression are often associated with disease states, such as in hematologic malignancies and in cells that form solid tumors within specific tissues or organs of a subject. Solid tumors or hematologic malignancies characterized by tumor antigen overexpression can be identified by standard assays known in the art.

[0046] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains and longer chains; short chains are generally referred to in the art, for example, as peptides, oligopeptides, and oligomers, while longer chains are generally referred to in the art, and there are many types of proteins. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0047] As used in this application, the terms "mature polypeptide" or "mature protein" refer to a protein or polypeptide that is secreted or located within the cell membrane or certain organelles (e.g., endoplasmic reticulum, Golgi apparatus, or introns) and does not contain an N-terminal signal peptide.

[0048] A signal peptide, also known as a signal sequence, leader sequence, or localization signal, is a short peptide (typically 15-30 amino acids long) located at the N-terminus of a newly synthesized protein and transported to the secretory pathway. A signal peptide typically consists of a short, positively charged hydrophilic amino acid at the N-terminus, a hydrophobic domain of 5-15 residues in the middle, and a C-terminal region containing a signal peptidase cleavage site. In eukaryotes, the signal peptide prompts the translocation of the newly synthesized protein to the endoplasmic reticulum (ER), where it is cleaved by a signal peptidase to produce the mature protein, which then proceeds to its appropriate destination.

[0049] The "percentage of identity" between two or more nucleic acid or amino acid sequences is a function of the number of common positions shared by the sequences (i.e., identity % = number of common positions / total number of positions x 100), taking into account the number of gaps that need to be introduced to optimize the alignment of the two or more sequences and the length of each gap. Mathematical algorithms, such as the BLAST and Gapped BLAST programs, can be used with their default parameters (e.g., Altschul et al., J.Mol.Biol.215:403,1990; see also BLASTN at www.ncbi.nlm.nih.gov / BLAST) to perform sequence alignment and determine the percentage of identity between two or more sequences.

[0050] In the art, a “conservative substitution” is considered to be the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO 97 / 09433, p. 10, publication date: March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).

[0051] The term "chimera" refers to any non-endogenous nucleic acid molecule or protein that contains sequences that are bound or linked together (sequences that would not normally be bound or linked together in nature). For example, a chimeric nucleic acid molecule may include nucleic acids encoding multiple domains from multiple different genes. In another instance, a chimeric nucleic acid molecule may include regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from that found in nature.

[0052] As used herein, the term “promoter” is defined as a DNA sequence that is required to initiate specific transcription of a polynucleotide sequence and is recognized by the cell’s synthetic mechanism or an introduced synthetic mechanism.

[0053] As used herein, the term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product operatively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence; in others, it may contain enhancer sequences and other regulatory elements required for the expression of the gene product. A promoter / regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

[0054] A "constitutive" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide sequence encoding or defining a gene product, results in the production of that gene product in the cell under most or all physiological conditions.

[0055] An "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide sequence encoding or defining a gene product, results in the adequate production of the gene product in a cell only when an inducer corresponding to the promoter is present in the cell.

[0056] A tissue-specific promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or defined by a gene, results in adequate production of the gene product in the cell only if the cell is a cell in a tissue type corresponding to the promoter.

[0057] As used herein, the phrase “under transcriptional control” or “operably linked” refers to the promoter being in the correct position and orientation relative to the polynucleotide to control the transcriptional initiation of RNA polymerase and the expression of that polynucleotide.

[0058] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, granules, viruses, or bacteriophages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells. An "expression vector" is a vector that, when present in a suitable environment, can direct the expression of a protein encoded by one or more genes carried by the vector.

[0059] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentiviral vectors. A "retrovirus" is a virus with an RNA genome. A "gamma retrovirus" refers to a genus within the family Retroviridae. Examples of gamma retroviruses include mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendothelial cell proliferation virus. A "lentivirus" refers to a genus of retroviruses capable of infecting both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0060] In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, closed linear double-stranded (CELiD) DNA, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty). When using a non-viral delivery system, the delivery carrier can be a liposome. Lipid formulations can be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. Nucleic acids can be encapsulated within liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes by linker molecules that bind nucleic acids to liposomes, contained within or complexed with micelles, or otherwise bound to lipids.

[0061] As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid containing at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell. A tandem expression cassette refers to a vector nucleic acid component containing at least two transgenes under the control of the same set of regulatory sequences for tandem expression of the transgenes. In some embodiments, the tandem expression cassette contains at least two transgenes under the control of the same promoter. In some embodiments, the first and second transgenes are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to enable co-expression of two proteins from a single mRNA.

[0062] "Particle" refers to cell debris or small objects with a diameter of at least 10 nm and at most 50 μm. Particles can originate from living cells or organisms, the environment, or be synthetic. Particles can be viral particles, prions, protein particles, synthetic particles, small mineral particles, or cell debris.

[0063] As used herein, the term “phagocytosis” refers to a receptor-mediated process in which endogenous or exogenous cells or particles larger than 10 nm in diameter are internalized by the phagocytes or host cells of this disclosure. Phagocytosis typically consists of several steps: (1) binding of the target cell or particle to a prophagocytic marker or antigen marker directly or indirectly (via a bridging molecule) to the phagocytic receptor; and (2) internalization or phagocytosis of the entire target cell or particle or a portion thereof. In some embodiments, internalization may occur by rearranging the cytoskeleton of the phagocyte or host cell to form a phagosome (containing a membrane-bound chamber containing the internalized target). Phagocytosis may also include the maturation of the phagosome, in which the phagosome becomes more acidic and fuses with a lysosome (to form a phagolysosome), after which the phagosome is degraded (e.g., “phagocytosis”). Alternatively, phagosome-lysosome fusion may not be observed in phagocytosis. In yet another embodiment, the phagosome may refluxing or expelling its contents into the extracellular environment before complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, phagocytes that engulf host cells comprising the present disclosure bind target cells or particles but do not internalize them. In some embodiments, phagocytes that engulf host cells comprising the present disclosure bind target cells or particles and partially internalize target cells or particles.

[0064] As used herein, the term "phagocytosis" refers to the process of phagocytosis of cells or large particles (>0.5 μm), wherein the binding of a target cell or particle, phagocytosis of the target cell or particle, and degradation of the internalized target cell or particle occur. In some embodiments, phagocytosis includes the formation of a phagosome that surrounds the internalized target cell or particle, and the fusion of the phagosome with a lysosome to form a phagolysosome, wherein the contents are degraded. In some embodiments, during phagocytosis, a phagocytic synapse is formed after the CER expressed on the host cell of the present disclosure binds to the target antigen expressed by the target cell or particle; an actin-rich phagocytic cup is generated at the phagocytic synapse; phagocytic arms extend around the target cell or particle via cytoskeleton rearrangement; and finally, the target cell or particle is pulled into the phagocytic cell or host cell by forces generated by motor proteins. As used herein, "phagocytosis" includes the process of "cell burial," which specifically refers to the phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

[0065] The term "immune system cells" or "immune cells" refers to any cell in the immune system that originates from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells produce two main lineages: myeloid progenitor cells (myeloid cells that produce monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (lymphoid cells that produce T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 cells. + T cells, CD8 + T cells, CD4 - CD8 - Double-negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells, also known as "antigen-presenting cells" or "APCs," are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of APCs, which are complexed with peptides, interacts with the TCR on the surface of T cells.

[0066] The term "T cell" refers to cells of the T cell lineage. "Cells of the T cell lineage" are cells exhibiting at least one phenotypic characteristic of a T cell or its precursor or progenitor cell, distinct from other lymphocytes and erythrocyte or myeloid cell lineage cells. Such phenotypic characteristics may include the expression of one or more T cell-specific proteins (e.g., CD3). + CD4 + CD8 + ( ), or T cell-specific physiological, morphological, functional, or immunological characteristics. For example, cells in a T cell lineage can be progenitor cells or precursor cells directed towards a T cell lineage; CD25 +Immature and unactivated T cells; cells that have undergone CD4 or CD8 lineage orientation; CD4+CD8+ double-positive thymocyte progenitor cells; single-positive CD4+ T cells. + or CD8 + TCRαβ or TCRγδ; or mature and functional or activated T cells. The term "T cell" includes naïve T cells (CD45RA). + CCR7 + CD62L + CD27 + CD45RO - ), central memory T cells (CD45RO) + CD62L + CD8 + ), effector memory T cells (CD45RA) + CD45RO - CCR7 - CD62L - CD27 - Mucosa-associated inertial T cells (MAIT), regulatory T cells (Tregs), natural killer T cells, and tissue-resident T cells.

[0067] The term "B cell" refers to cells of the B cell lineage. "Cells of the B cell lineage" are cells exhibiting at least one phenotypic characteristic of B cells or their precursors or progenitors, distinguishing them from other lymphocytes and erythrocyte or myeloid lineage cells. Such phenotypic characteristics may include the expression of one or more B cell-specific proteins (e.g., CD19). + CD72 + CD24 + CD20 + B cells can be defined by their specific physiological, morphological, functional, or immunological characteristics. For example, cells in a B cell lineage can be progenitor or precursor cells directed towards that lineage (e.g., pre-progenitor B cells, progenitor B cells, and pre-B cells); immature and unactivated B cells; or mature and functional or activated B cells. Therefore, “B cells” include naïve B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic-like cells, plasmablasts, and memory B cells (e.g., CD27 cells). + IgD - ).

[0068] For cells (e.g., T cells) that express immune receptors (e.g., TCRs) on their cell surfaces, the term "cytotoxic activity," also known as "cytolytic activity," refers to the induction of apoptosis in target cells following antigen-specific signaling (e.g., via TCRs). In some embodiments, cytotoxic cells can induce apoptosis in target cells by releasing cytotoxins (such as perforin, granzymes, and granzymes) from granules. Perforin inserts into the target cell membrane and forms pores to allow rapid entry of water and salts into the target cell. Granzymes are serine proteases that induce apoptosis in target cells. Granzymes are also capable of forming pores on the target cell membrane and are pro-inflammatory molecules. In some embodiments, cytotoxic cells can induce apoptosis in target cells through the interaction of Fas ligands, which are upregulated on T cells following antigen-specific signaling as Fas molecules are expressed on target cells. Fas are apoptosis signaling receptor molecules on a variety of different cell surfaces.

[0069] "Disease" is a state of health in which the object cannot maintain homeostasis, and where, if the disease is not improved, the object's health will continue to deteriorate. In contrast, an object's "disorder" or "adverse condition" refers to a state of health in which the object can maintain homeostasis, but in which case the object's health is worse than it would be without disorder or adverse condition. Without treatment, disorder or adverse condition does not necessarily lead to a further decline in the object's health.

[0070] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. These abnormal cells can form solid tumors or constitute malignant hematologic disorders. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

[0071] The terms “object,” “patient,” and “individual” are used interchangeably in this document and are intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of objects include humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0072] "Adoptive cell immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cell immunotherapy can be autologous (immune cells come from the recipient), allogeneic (immune cells come from a donor of the same species), or genetically identical (immune cells come from a donor with the same genes as the recipient).

[0073] "Autologous" refers to a graft (e.g., an organ, tissue, cell) that originates from the same object from which it will subsequently be reintroduced.

[0074] "Also-heterogeneous" refers to grafts from different objects of the same species.

[0075] The term "therapeutic effective amount" or "effective amount" for chimeric proteins or cells expressing chimeric proteins (e.g., tandem expression cassettes or cells expressing tandem expression cassettes) in this disclosure refers to the amount of protein or cells sufficient to improve one or more symptoms of the treated disease, disorder, or adverse condition. When referring to the administration of a single active ingredient alone or cells expressing a single active ingredient, the therapeutic effective amount refers to the effective amount of the ingredient or cells expressing the single ingredient. When referring to combination therapy, the therapeutic effective amount refers to the amount of the active ingredient that produces the therapeutic effect, or the amount of an adjuvant active ingredient combined with cells expressing the active ingredient, whether administered sequentially or simultaneously.

[0076] "Treatment" or "improvement" refers to the medical management of a disease, disorder, or adverse condition. Typically, it involves administering an appropriate dose or treatment regimen of host cells expressing the chimeric protein of this disclosure in an amount sufficient to elicit a therapeutic or preventative benefit. The therapeutic or preventative / avoidant benefits include improved clinical outcomes; reduction or alleviation of symptoms associated with the disease, disorder, or adverse condition; reduction of symptom occurrence; improved quality of life; prolongation of disease-free status; reduction of the severity of the disease, disorder, or adverse condition; stabilization of disease status; delay of disease progression; remission; survival; extended survival; or any combination thereof.

[0077] The term "anti-tumor effect" refers to biological effects that can manifest as shrinking tumor volume, reducing the number of tumor cells, reducing the number of metastases, prolonging life expectancy, or improving various physiological symptoms associated with cancer. The "anti-tumor effect" can also manifest as preventing the formation of malignant hematological diseases or tumors.

[0078] Other definitions are provided throughout this disclosure.

[0079] genetically modified

[0080] The tandem expression cassettes of this disclosure comprise at least two transgenes encoding a chimeric phagocytic receptor (CER) and a chimeric antigen receptor (CAR) / or a T-cell receptor (TCR). Certain embodiments of the tandem expression cassettes provided herein include: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a binding domain for binding to a target antigen; a phagocytic signaling domain; and a transmembrane domain located between and connecting the extracellular and phagocytic signaling domains; and (b) a polynucleotide encoding a CAR comprising: an extracellular domain including a binding domain for binding to a target antigen; an intracellular signaling domain; and a transmembrane domain located between and connecting the extracellular and intracellular signaling domains. Other embodiments of the tandem expression cassette provided herein include: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a binding domain for binding to a target antigen; a phagocytic signaling domain; and a transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; and (b) a polynucleotide encoding a recombinant TCR-binding protein. In some embodiments, the polynucleotide encoding the CER is located at the 5' end of the polynucleotide encoding a CAR or TCR. In other embodiments, the polynucleotide encoding the CER is located at the 3' end of the polynucleotide encoding a CAR or TCR.

[0081] The following provides other aspects of genetically modified organisms and the cellular immunotherapy molecules they encode.

[0082] I. Chimeric phagocytic receptor (CER)

[0083] The tandem expression cassette of this disclosure contains at least one polynucleotide encoding a CER. Chimeric phagocytic receptors typically comprise: (a) an extracellular domain containing a binding domain that binds to a target antigen; (b) a phagocytic signaling domain; and (c) a transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain. In some embodiments, the extracellular domain of the chimeric phagocytic receptor described herein optionally includes an extracellular spacer domain located between and connecting the binding domain and the transmembrane domain.

[0084] The chimeric phagocytic receptors described herein can confer a phagocytic phenotype specific to a target antigen onto host cells modified to express the chimeric phagocytic receptor. In some embodiments, expression of the CER as described herein confers a phagocytic phenotype onto host cells that do not naturally possess such a phagocytic phenotype. In some embodiments, phagocytic activity is phagocytic cell activity. The CER of this disclosure can be used to redirect phagocytic specificity to target cells expressing a target antigen.

[0085] extracellular domain

[0086] As described herein, the CER contains an extracellular domain specifically targeting a target antigen. In some embodiments, the extracellular domain contains a binding domain that specifically binds to the target antigen (e.g., phosphatidylserine). Binding of a target molecule through the binding domain can block the interaction between the target molecule (e.g., a receptor or ligand) and another molecule, and, for example, interfere with, attenuate, or eliminate certain functions of the target molecule (e.g., signal transduction). In some embodiments, binding of the target molecule can induce certain biological pathways or identify the target molecule or cells expressing the target molecule for elimination.

[0087] The binding domain suitable for use in the CER of this disclosure can be any polypeptide or peptide that specifically binds to a target molecule (e.g., phosphatidylserine). Sources of the binding domain include extracellular domains of receptors, ligands of cell surface receptors or molecules, and antibody or antigen-binding moieties, such as antibody variable regions from various species. For example, the binding domain can contain sFv, scFv, Fab, scFv-based grababody, VH domain, VL domain, single-domain camel antibody (VHH), or domain antibody. The binding domain can be derived from humans, primates, rodents, birds, or sheep. Other sources of the binding domain include antibody variable regions from other species, such as camelids (from camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett. 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature). 363:446,1993 and Nguyen et al., J.Mol.Biol.275:413,1998), nurse shark (Roux et al., Proc.Nat′l.Acad.Sci.(USA)95:11804,1998), spotted chinchilla (Nguyen et al., Immunogen.54:39,2002) or lamprey (Herrin et al., Proc.Nat′l.Acad.Sci.(USA)105:2040,2008 and Alder et al., Nat.Immunol.9:319,2008). These antibodies use only the variable region of the heavy chain to form the antigen-binding region; that is, these functional antibodies are simply homodimers of the heavy chain (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008). In some embodiments, the binding domain is mouse, chimeric, human, or humanized.

[0088] In some embodiments, the CER-binding domain comprises an antibody or an antigen-binding fragment thereof, such as a single-chain Fv fragment (scFv), which includes VH and VL regions specific to the target disease antigen. In some embodiments, the antibody or antigen-binding fragment is chimeric, human, or humanized. In a further embodiment, V H and V L The area is human or humanized.

[0089] Target molecules that specifically bind to the extracellular domain of the CER of this disclosure may be present on or bound to target cells (“target cells”). Exemplary target cells include cancer cells, cells associated with autoimmune diseases or disorders or inflammatory diseases or disorders, and infectious microorganisms (e.g., bacteria, viruses, or fungi) or infected cells (e.g., virus-infected cells). Cells of infectious organisms (such as mammalian parasites) are also considered target cells.

[0090] In some embodiments, the extracellular domain binds a prophagocytic marker. As used herein, a prophagocytic marker is a portion (e.g., a protein, lipid, or polysaccharide) displayed on the surface of apoptotic, necrotic, pyroptotic, or infected cells that distinguishes them from non-apoptotic, non-necrotic, non-pyroptotic, oncolytic, or non-infected cells. A prophagocytic marker can be an intracellular portion exposed on the surface of apoptotic or necrotic cells, a portion with altered glycosylation or altered surface charge on apoptotic or necrotic cells, or a serum portion bound to apoptotic, necrotic, pyroptotic, or oncolytic cells. Examples of prophagocytic markers targeting apoptotic cells include phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, and platelet-reactive protein. Necrotic, oncolytic, and pyroptotic cells also expose the PtdSer prophagocytic marker on their cell surface. Phagocytic receptors can utilize soluble bridging molecules as intermediates for binding pro-phagocytic markers to directly or indirectly detect (or bind) pro-phagocytic markers on target cells (e.g., damaged, infected, apoptotic, necrotic, pyroptotic, or oncolytic cells). In some such embodiments, the extracellular domain-targeted pro-phagocytic markers are phosphatidylserine (PtdSer), ICAM-3, oxidized low-density lipoprotein, calreticulin, annexin I, complement C1q, or platelet-reactive protein. An exemplary Tim4 binding domain for phosphatidylserine includes an amino acid sequence comprising amino acids 23-279 of SEQ ID NO:91, SEQ ID NO:96, SEQ ID NO:91, or amino acids 25-314 of SEQ ID NO:96.

[0091] In some implementations, the extracellular domain binds to tumor antigens. Exemplary tumor antigens include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, liver glycoside A2, liver glycoside B2, Lewis A antigen, Lewis... Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucose GM1, GM3, o-acetyl GD2 and GD2.

[0092] In some implementations, the extracellular domain binds to viral antigens, bacterial antigens, fungal antigens, protozoan antigens, or parasitic antigens.

[0093] In some embodiments, the extracellular domain optionally includes an extracellular, non-signal transduction spacer region or a linker domain. When included, such spacers or linker domains can position the binding domains away from the host cell surface to further facilitate suitable cell-to-cell contact, binding, and activation. The extracellular spacer region typically lies between the extracellular binding domain and the transmembrane domain of the CER. The length of the extracellular spacer region can be varied depending on the selected target molecule, the selected binding epitope, the size of the binding domain, and the affinity, to optimize target molecule binding (see, for example, Guest et al., J. Immunother. 28:203-11, 2005; PCT Publication No. WO2014 / 031687). In some embodiments, the extracellular spacer region is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The immunoglobulin hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is as described in PCT Publication No. WO2014 / 031687, the entire contents of which are incorporated herein by reference. In one particular embodiment, the extracellular spacer domain comprises the modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 63).

[0094] Other examples of hinge regions that can be used in the CER described herein include hinge regions present in the extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD7), which may be wild-type or variants thereof. In a further embodiment, the extracellular spacer region domain comprises all or part of the immunoglobulin Fc domain selected from the group consisting of: CH1 domain, CH2 domain, CH3 domain, or combinations thereof (see, for example, PCT Publication WO2014 / 031687, the entire contents of which are spacers are incorporated herein by reference). In yet another embodiment, the extracellular spacer region domain may comprise the stem region of type II C-lectins (the extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In yet another embodiment, the extracellular spacer region domain may be derived from the juxtamembrane domain of a toll-like receptor (TLR). The TLR juxtamembrane domain comprises an acidic amino acid located between a leucine-rich repeat sequence (LRR) and the transmembrane domain of the TLR. In some embodiments, the TLR juxtamembrane domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain. An exemplary TLR juxtamembrane domain is a TLR4 juxtamembrane domain comprising the amino acid sequence shown in SEQ ID NO:1.

[0095] The extracellular domain can be derived from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species. In some embodiments, the extracellular domain is mouse, chimeric, human, or humanized.

[0096] Swallowing signal transduction domain

[0097] The phagocytic signaling domain of the cytokinetic echogenic molecule (CER) is an intracellular effector domain and is capable of transmitting functional signals to the cell in response to the binding of the CER's extracellular domain to target molecules. The phagocytic signaling domain can be any portion of a phagocytic signaling molecule that retains sufficient signaling activity. In some embodiments, the full-length or full-length intracellular portion of the phagocytic signaling molecule is used. In some embodiments, a truncated portion of the phagocytic signaling molecule or its intracellular portion is used, provided that the truncated portion retains sufficient signaling activity. In a further embodiment, the phagocytic signaling domain is a variant of the complete or truncated portion of the phagocytic signaling molecule, provided that the variant retains sufficient signaling activity (i.e., a functional variant).

[0098] Exemplary signal transduction domains that can be used in CER include the MRC1 signal transduction domain, MERTK signal transduction domain, Tyro3 signal transduction domain, Axl signal transduction domain, ELMO signal transduction domain, Traf6 signal transduction domain, Syk signal transduction domain, MyD88 signal transduction domain, PI3K signal transduction domain, and FcR signal transduction domain (e.g., FcγR1, FcγR2A, FcγR2C, FcγR2B2, FcγR3A, FcγR2C, FcγR3A, FcεR1, or FcαR1 signal transduction domains). The signal transduction domains include: B cell activating factor receptor (BAFF-R) signal transduction domain, DAP12 (also known as TYRO protein tyrosine kinase binding protein (TYROBP)) signal transduction domain, NFAT activating protein with ITAM motif 1 (NFAM1) signal transduction domain, CD79b signal transduction domain, TLR signal transduction domains (e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 signal transduction domains), Traf2 signal transduction domain, or Traf3 signal transduction domain.

[0099] In some embodiments, the phagocytic signal transduction domain comprises a sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the following sequences: an MRC1 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:2; a MERTK signal transduction domain containing the amino acid sequence shown in SEQ ID NO:3; a Tyro3 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:5; an Axl signal transduction domain containing the amino acid sequence shown in SEQ ID NO:6; an ELMO signal transduction domain containing the amino acid sequence shown in SEQ ID NO:7; a Traf6 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:8; a Syk signal transduction domain containing the amino acid sequence shown in SEQ ID NO:9; a MyD88 signal transduction domain containing the amino acid sequence shown in SEQ ID NO:10; and a sequence containing SEQ ID NO:2. The amino acid sequence shown in NO:11 contains the PI3K signal transduction domain, the amino acid sequence shown in SEQ ID NO:12 contains the FcγR1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:13 contains the FcγR2A signal transduction domain, the amino acid sequence shown in SEQ ID NO:14 contains the FcγR2C signal transduction domain, the amino acid sequence shown in SEQ ID NO:15 contains the FcγR3A signal transduction domain, the amino acid sequence shown in SEQ ID NO:16 contains the BAFF-R signal transduction domain, the amino acid sequence shown in SEQ ID NO:17 contains the DAP12 signal transduction domain, the amino acid sequence shown in SEQ ID NO:18 contains the NFAM1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:19 contains the CD79b signal transduction domain, the amino acid sequence shown in SEQ ID NO:21 contains the TLR1 signal transduction domain, the amino acid sequence shown in SEQ ID NO:22 contains the TLR2 signal transduction domain, and the amino acid sequence shown in SEQ ID NO:23 contains the SEQ ID The amino acid sequence shown in NO:24 contains the TLR3 signal transduction domain, the amino acid sequence shown in SEQ ID NO:25 contains the TLR4 signal transduction domain, the amino acid sequence shown in SEQ ID NO:26 contains the TLR5 signal transduction domain, the amino acid sequence shown in SEQ ID NO:27 contains the TLR6 signal transduction domain, and the amino acid sequence shown in SEQ ID NO:28 contains the TLR7 signal transduction domain.The signal transduction domain contains the TLR8 domain of the amino acid sequence shown in SEQ ID NO:29, the TLR9 domain of the amino acid sequence shown in SEQ ID NO:30, the Traf2 domain of the amino acid sequence shown in SEQ ID NO:31, or the Traf3 domain of the amino acid sequence shown in SEQ ID NO:32.

[0100] In some embodiments, the phagocytic signal transduction domain is an MRC1 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:2, a MERTK signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:3, a Tyro3 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:5, an Axl signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:6, an ELMO signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:7, a Traf6 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:8, a Syk signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:9, a MyD88 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:10, a PI3K signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:11, an FcεRIγ signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:12, an FcγR1 signal transduction domain comprising or composed of the amino acid sequence shown in SEQ ID NO:13, or a domain comprising or composed of the amino acid sequence shown in SEQ ID NO:2. The FcγR2A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:14, the FcγR2C signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:15, the FcγR3A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:16, the BAFF-R signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:17, the DAP-12 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:18, the NFAM1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:19, the CD79b signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:21, the TLR1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:22, the TLR2 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:23, the TLR3 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:24, the TLR4 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:25, and the FcγR2A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:14, the FcγR2C signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:15, the FcγR3A signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:16, the BAFF-R signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:17, the DAP-12 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:18, the NFAM1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:19, the CD79b signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:21, the TLR1 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:22, the TLR2 signal transduction domain composed of the amino acid sequence shown in SEQ ID NO:23, The TLR5 signal transduction domain, composed of the amino acid sequence shown in SEQ ID NO:26, contains or is composed of the TLR6 signal transduction domain, composed of the amino acid sequence shown in SEQ ID NO:27.The signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:28, the TLR7 signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:29, the TLR9 signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:30, the Traf2 signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:31, or the Traf3 signal transduction domain comprises or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0101] A truncated swallowing signal transduction domain can be truncated at its N-terminus, its C-terminus, or both its N-terminus and C-terminus. In some embodiments, the MRC1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:2; the MERTK phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:3; the Tyro3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:5; the Axl phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:6; the ELMO phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:7; the Traf6 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:8; and the Syk phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:8. The N-terminus of the amino acid sequence shown in NO:9 is truncated by 1, 2, 3, 4, 5 or more amino acids; the MyD88 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:10; the PI3K phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:11; the FcεRIγ phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:12; the FcγR1 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:13; the FcγR2A phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:14; the FcγR2C phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:14; The N-terminus of the amino acid sequence shown in NO:15 is truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:16; the BAFF-R phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:17.The DAP-12 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 18; the NFAM1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 19; the CD79b phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 21; the TLR1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 22; the TLR2 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 23; the TLR3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 24; and the TLR4 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 24. The N-terminus of the amino acid sequence shown in NO:25 is truncated by 1, 2, 3, 4, 5 or more amino acids; the phagocytic signal transduction domain of TLR5 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:26; the phagocytic signal transduction domain of TLR6 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:27; the phagocytic signal transduction domain of TLR7 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:28; the phagocytic signal transduction domain of TLR8 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:29; the phagocytic signal transduction domain of TLR9 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:30; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:29; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid sequence corresponding to SEQ ID NO:30; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the N-terminus of the amino acid The N-terminus of the amino acid sequence shown in NO:31 is truncated by 1, 2, 3, 4, 5, or more amino acids; or the Traf3 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5, or more amino acids at the N-terminus corresponding to the amino acid sequence shown in SEQ ID NO:32.

[0102] In some embodiments, the MRC1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:2; the MERTK phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:3; the Tyro3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:5; the Axl phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:6; the ELMO phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:7; the Traf6 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:8; and the Syk phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:8. The C-terminus of the amino acid sequence shown in NO:9 is truncated by 1, 2, 3, 4, 5 or more amino acids; the MyD88 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:10; the PI3K phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:11; the FcεRIγ phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:12; the FcγR1 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:13; the FcγR2A phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:14; the FcγR2C phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:14; The C-terminus of the amino acid sequence shown in NO:15 is truncated by 1, 2, 3, 4, 5 or more amino acids; the FcγR3A phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:16; the BAFF-R phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:17.The DAP-12 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:18; the NFAM1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:19; the CD79b phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:21; the TLR1 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:22; the TLR2 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:23; the TLR3 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:24; and the TLR4 phagocytic signal transduction domain is formed by truncating 1, 2, 3, 4, 5, or more amino acids at the C-terminus of the amino acid sequence corresponding to SEQ ID NO:24. The C-terminus of the amino acid sequence shown in NO:25 is truncated by 1, 2, 3, 4, 5 or more amino acids; the phagocytic signal transduction domain of TLR5 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:26; the phagocytic signal transduction domain of TLR6 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:27; the phagocytic signal transduction domain of TLR7 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:28; the phagocytic signal transduction domain of TLR8 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:29; the phagocytic signal transduction domain of TLR9 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:30; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:25; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:29; the phagocytic signal transduction domain of Traf2 is truncated by 1, 2, 3, 4, 5 or more amino acids at the C- The C-terminus of the amino acid sequence shown in NO:31 is truncated by 1, 2, 3, 4, 5, or more amino acids; or the Traf3 phagocytic signal transduction domain is truncated by 1, 2, 3, 4, 5, or more amino acids at the C-terminus corresponding to the amino acid sequence shown in SEQ ID NO:32.

[0103] In some embodiments, the truncated MyD88 phagocytic signaling domain includes a death domain but lacks the Toll / interleukin 1 receptor (TIR) ​​homologous domain. Examples of such truncated MyD88 phagocytic signaling domains include the amino acid sequence shown in SEQ ID NO:33. In some embodiments, the truncated MyD88 phagocytic signaling domain includes a TIR domain. Examples of truncated MyD88 phagocytic signaling domains include a TIR domain containing the amino acid sequence shown in SEQ ID NO:106. An exemplary truncated Traf6 signaling domain contains the amino acid sequence shown in SEQ ID NO:34. An exemplary truncated NFAM1 signaling domain contains the amino acid sequence shown in SEQ ID NO:35. An exemplary truncated CD79b signaling domain contains the amino acid sequence shown in SEQ ID NO:20.

[0104] In some embodiments, the CER includes a first phagocytic signaling domain and a second phagocytic signaling domain. In some embodiments, the CER includes a first phagocytic signaling domain and a second phagocytic signaling domain from the same molecule. In other embodiments, the first phagocytic signaling domain and the second phagocytic signaling domain originate from different molecules.

[0105] Phagocytic signal transduction domains can originate from mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0106] Transmembrane domain

[0107] The CER of this disclosure includes a transmembrane domain that connects and lies between the extracellular domain and the phagocytic signaling domain. The transmembrane domain is a hydrophobic α-helix that traverses the host cell membrane and anchors the CER within it. The transmembrane domain may be directly fused to a binding domain or an extracellular spacer domain (if present). In some embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptors, differentiation cluster (CD) molecules, enzymes, transport proteins, cell adhesion molecules, etc.). The transmembrane domain may be selected from the same molecule as the extracellular domain or the phagocytic signaling domain (e.g., the CER contains a TLR4 phagocytic signaling domain and a TLR4 transmembrane domain, or the CER contains a Tim4 binding domain and a Tim4 transmembrane domain). In some embodiments, the transmembrane domain and the extracellular domain are selected from different molecules. In other embodiments, the transmembrane domain and the phagocytic signaling domain are selected from different molecules. In still other embodiments, the transmembrane domain, the extracellular domain, and the phagocytic signaling domain are selected from different molecules.

[0108] In some embodiments, the transmembrane domains include Tim1, Tim4, Tim3, FcR (e.g., FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, FcεR1 or FcαR1), CD8a, CD28, MERTK, Axl, Tyro3, ​​CD4, DAP12, MRC1, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9 transmembrane domains.

[0109] In some embodiments, the transmembrane domain comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99% of the following sequences.Sequences with 5% or 100% identity: The Tim1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:36; the Tim4 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:37 or 38; the Tim3 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:39; the FcγR1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:40; the FcγR2A transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:41; the FcγR2B2 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:42; the FcγR2C transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:43; the FcγR3A transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:44; the FcεR1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:45; the FcαR1 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:46; the CD8a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:47; and the sequence comprising SEQ ID NO:36. The amino acid sequence shown in NO:107 contains the CD28 transmembrane domain, the amino acid sequence shown in SEQ ID NO:48 contains the MERTK transmembrane domain, the amino acid sequence shown in SEQ ID NO:49 contains the Axl transmembrane domain, the amino acid sequence shown in SEQ ID NO:50 contains the Tyro3 transmembrane domain, the amino acid sequence shown in SEQ ID NO:51 contains the CD4 transmembrane domain, the amino acid sequence shown in SEQ ID NO:52 contains the DAP12 transmembrane domain, the amino acid sequence shown in SEQ ID NO:53 contains the MRC1 transmembrane domain, the amino acid sequence shown in SEQ ID NO:54 contains the TLR1 transmembrane domain, the amino acid sequence shown in SEQ ID NO:55 contains the TLR2 transmembrane domain, the amino acid sequence shown in SEQ ID NO:56 contains the TLR3 transmembrane domain, the amino acid sequence shown in SEQ ID NO:57 contains the TLR4 transmembrane domain, the amino acid sequence shown in SEQ ID NO:58 contains the TLR5 transmembrane domain, and the amino acid sequence shown in SEQ ID NO:58 contains the CD28 transmembrane domain, the amino acid sequence shown in SEQ ID NO:57 contains the TLR4 transmembrane domain, and the amino acid sequence shown in SEQ ID NO:58 contains the CD28 transmembrane domain, the amino acid sequence shown in SEQ ID NO:57 contains the CD28 transmembrane domain, ... The TLR6 transmembrane domain of the amino acid sequence shown in NO:59 includes the TLR7 transmembrane domain of the amino acid sequence shown in SEQ ID NO:60, the TLR8 transmembrane domain of the amino acid sequence shown in SEQ ID NO:61, or the TLR9 transmembrane domain of the amino acid sequence shown in SEQ ID NO:62.

[0110] In some embodiments, the transmembrane domain is a Tim1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:36, a Tim4 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:37 or 38, a Tim3 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:39, an FcγR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:40, an FcγR2A transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:41, an FcγR2B2 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:42, an FcγR2C transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:43, an FcγR3A transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:44, an FcεR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:45, an FcαR1 transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:46, and a transmembrane domain comprising or composed of the amino acid sequence shown in SEQ ID NO:46. The following domains are included: the CD8a transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:47; the CD28 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:107; the MERTK transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:48; the Axl transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:49; the Tyro3 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:50; the CD4 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:51; the DAP12 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:52; the MRC1 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:53; the TLR1 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:54; the TLR2 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:55; the TLR3 transmembrane domain formed by the amino acid sequence shown in SEQ ID NO:56; and the domains containing or formed by the amino acid sequence shown in SEQ ID NO:47. The TLR4 transmembrane domain, composed of the amino acid sequence shown in SEQ ID NO:57, includes or is composed of the TLR5 transmembrane domain, the TLR6 transmembrane domain, the TLR7 transmembrane domain, the TLR8 transmembrane domain, or the TLR8 transmembrane domain, which includes or is composed of the amino acid sequence shown in SEQ ID NO:58, the TLR7 transmembrane domain, the TLR8 transmembrane domain, or the TLR8 transmembrane domain, which includes or is composed of the amino acid sequence shown in SEQ ID NO:61.It may contain or consist of a TLR9 transmembrane domain composed of the amino acid sequence shown in SEQ ID NO:62.

[0111] Transmembrane domains can originate from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0112] In some embodiments, the chimeric phagocytic receptor comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species, or any combination thereof. In some embodiments, the chimeric phagocytic receptor is mouse, chimeric, human, or humanized.

[0113] It should be understood that the direct fusion of one domain of a CER to another described herein does not preclude the presence of connecting amino acids in between. Connecting amino acids can be natural or non-natural (e.g., the result of chimeric protein construct design).

[0114] Implementations of the CER used in the tandem expression box of this disclosure are provided in Table 1 and are also described in PCT applications PCT / 2017 / 053553 and PCT / US2018 / 52297, the entire contents of which are incorporated herein by reference.

[0115] Table 1: Exemplary chimeric phagocytic receptors

[0116]

[0117]

[0118]

[0119] II. Chimeric antigen receptor

[0120] In some embodiments, the tandem expression cassette of this disclosure includes a transgene encoding a chimeric antigen receptor (CAR). A chimeric antigen receptor is a recombinant receptor that typically includes: an extracellular domain containing a binding domain for binding to a target antigen; an intracellular signaling domain; and a transmembrane domain located between and connecting the extracellular and intracellular signaling domains.

[0121] The binding domain of the CAR applicable to this disclosure includes any antigen-binding polypeptide. The binding domain may comprise an antibody or an antigen-binding fragment thereof, including, for example, a full-length heavy chain, a Fab fragment, Fab', F(ab')2, sFv, a VH domain, a VL domain, dAb, VHH, CDR, and scFv. In some embodiments, the CAR binding domain is mouse, chimeric, human, or humanized.

[0122] In some embodiments, the extracellular domain of the CAR provided in this disclosure optionally includes an extracellular, non-signal transduction spacer region or a linker domain. When included, such spacer regions or linker domains can position the binding domain away from the host cell surface to further facilitate suitable cell-to-cell contact, binding, and activation. The extracellular spacer region typically lies between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer region can be varied depending on the selected target molecule, the selected binding epitope, the size of the binding domain, and the affinity, to optimize target molecule binding (see, for example, Guest et al., J. Immunother. 28:203-11, 2005; PCT Publication No. WO2014 / 031687). In some embodiments, the extracellular spacer region is an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD). The hinge region can be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. The modified IgG4 hinge region is as described in PCT Publication No. WO2014 / 031687, the entire contents of which are incorporated herein by reference. In one particular embodiment, the extracellular spacer domain comprises the modified IgG4 hinge region having the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 63).

[0123] Other examples of hinge regions that can be used in the CAR described herein include hinge regions of extracellular regions of type 1 membrane proteins (such as CD8a, CD4, CD28, and CD7), which may be wild-type or variants thereof. In a further embodiment, the extracellular spacer region domain comprises all or part of the immunoglobulin Fc domains selected from the group consisting of: CH1 domain, CH2 domain, CH3 domain, or combinations thereof (see, for example, PCT Publication WO2014 / 031687, the entire contents of which are spacers are incorporated herein by reference). In yet another further embodiment, the extracellular spacer region domain may comprise the stem region of a type II C-lectin (the extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.

[0124] The CAR disclosed herein includes a transmembrane domain that connects and lies between the extracellular and intracellular signaling domains. The transmembrane domain is a hydrophobic α-helix that traverses the host cell membrane and anchors the CAR within it. The transmembrane domain may be directly fused to a binding domain or an extracellular spacer domain (if present). In some embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptors, differentiation cluster (CD) molecules, enzymes, transport proteins, cell adhesion molecules, etc.). The transmembrane domain may be selected from the same molecule as the extracellular or phagocytic signaling domain (e.g., the CAR includes a CD28 co-stimulatory signaling domain and a CD28 transmembrane domain). In some embodiments, the transmembrane domain and the extracellular domain are selected from different molecules. In other embodiments, the transmembrane domain and the intracellular signaling domain are selected from different molecules. In still other embodiments, the transmembrane domain, the extracellular domain, and the phagocytic signaling domain are selected from different molecules.

[0125] Exemplary transmembrane domains of the CAR used in this disclosure include CD28, CD2, CD3ε, CD3δ, CD3ζ, CD25, CD27, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), and CD273 (PD-L2). CD274 (PD-L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70. An exemplary CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:107.

[0126] The intracellular signaling domain of a CAR is an intracellular effector domain and is capable of delivering functional signals to the cell in response to the binding of the CAR's extracellular domain to a target molecule. The intracellular signaling domain can be any portion of an intracellular signaling molecule that retains sufficient signal transduction activity. In some embodiments, the full-length or full-length intracellular component of the intracellular signaling molecule is used. In some embodiments, a truncated portion or an intracellular component of the intracellular signaling molecule is used, provided that the truncated portion retains sufficient signal transduction activity. In a further embodiment, the intracellular signaling domain is a variant of the complete or truncated portion of the intracellular signaling molecule, provided that the variant retains sufficient signal transduction activity (i.e., is a functional variant).

[0127] In some embodiments, the intracellular signal transduction domain of the CAR includes an immune receptor tyrosine activation motif (ITAM) containing the signal transduction domain. The ITAM containing the signal transduction domain typically comprises at least one (one, two, three, four, or more) ITAMs, designated YXXL / IX. 6-8 The conserved motif of -YXXL / I. ITAMs containing signal transduction domains can initiate T cell activation signaling upon antigen or ligand binding. ITAM signal transduction domains include, for example, intracellular signal transduction domains of CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, and CD66d. An exemplary CD3ζ signal transduction domain that can be used in a CAR according to this disclosure contains the amino acid sequence shown in SEQ ID NO: 166 or 167.

[0128] The intracellular signal transduction domain of the CAR optionally includes a co-stimulatory signal transduction domain that, when activated by binding to a major or classical (e.g., ITAM-driven) activation signal, promotes or enhances T cell responses such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. The co-stimulatory signal transduction domains used in CAR include, for example, CD27, CD28, CD40L, GITR, NKG2C, CARD1, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX-40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD226, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, LFA-1, LIGHT, NKG2C, SLP76, TRIM, ZAP70, or any combination thereof. In one particular embodiment, the co-stimulatory signal transduction domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137) signal transduction domain. An exemplary CD28 co-stimulatory signal transduction domain that can be used in a CAR of this disclosure comprises the amino acid sequence shown in SEQ ID NO: 169 or 170. An exemplary 4-1BB co-stimulatory signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 168. In some embodiments, the CAR comprises one, two, or more co-stimulatory signal transduction domains.

[0129] In some embodiments, the chimeric antigen receptor comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In some embodiments, the chimeric antigen receptor is mouse, chimeric, human, or humanized.

[0130] In some implementations, the CAR is a first-generation CAR, a second-generation CAR, or a third-generation CAR. First-generation CARs typically have an intracellular signaling domain containing an activation domain of CD3ζ, FcγRI, or other ITAM-containing domains to provide T cell activation signals. Second-generation CARs also contain a co-stimulatory signaling domain (e.g., a co-stimulatory signaling domain derived from an endogenous T cell co-stimulatory receptor, such as CD28, 4-1BB, or ICOS). Third-generation CARs contain an activation domain containing an ITAM, a first co-stimulatory signaling domain, and a second co-stimulatory signaling domain.

[0131] In some implementations, the CAR is a chimeric antigen receptor based on the T-cell receptor (TCR-CAR). A TCR-CAR is a heterodimeric fusion protein that typically comprises a soluble TCR (a polypeptide chain containing Vα and Cα domains and a polypeptide chain containing Vβ and Cβ domains), wherein the VβCβ polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an activation domain containing ITAM and optionally a co-stimulatory signaling domain) (see, for example, Walseng et al., 2017 Scientific Reports 7:10713).

[0132] The CAR disclosed herein can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasitic antigens, tumor antigens, and autoimmune disease antigens. Exemplary tumor antigens that CARs can target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79b, mesothelin, PSMA, BCMA, ROR1, MUC-16, L1CAM, CD22, CD19, CD20, CD23, CD24, CD37, CD30, CA125, CD56, c-Met, EGFR, GD-3, HPV E6, HPV E7, MUC-1, HER2, folate receptor α, CD97, CD171, CD179a, CD44v6, WT1, VEGF-α, VEGFR1, IL-13Rα1, IL-13Rα2, IL-11Rα, PSA, FcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, liver glycoside A2, liver glycoside B2, Lewis... A antigen, Lewis Y antigen, MAGE, MAGE-A1, RAGE-1, folate receptor β, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucose GM1, GM3, o-acetyl GD2 and GD2.

[0133] III. T cell receptor-binding protein

[0134] In some embodiments, the tandem expression cassette of this disclosure includes a transgene encoding a recombinant TCR-binding protein. The recombinant TCR-binding protein includes a “conventional” TCR composed of a heterodimer of an α-chain polypeptide and a β-chain polypeptide or a heterodimer of a γ-chain polypeptide and a δ-chain polypeptide, its binding fragment, and a fusion protein, including, for example: single-chain TCRs, single-domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (TRuC). TMIn some embodiments, the tandem expression cassette comprises a polynucleotide encoding a recombinant TCRβ chain and a polynucleotide encoding a recombinant TCRα chain, wherein the recombinant TCRβ chain includes a TCRβ variable region and a TCRβ constant region, and the recombinant TCRα chain includes a TCRα variable region and a TCRα constant region. In some embodiments, the recombinant TCR is an affinity-enhanced TCR.

[0135] In some embodiments, the recombinant TCR-binding protein is a single-chain TCR (scTCR) containing Vα linked to Vβ via a flexible linker. In some embodiments, the scTCR contains a Vα-linker-Vβ polypeptide. In other embodiments, the scTCR contains a Vβ-linker-Vα polypeptide.

[0136] In some implementations, the recombinant TCR-binding protein is a single-domain TCR (e.g., Vβ).

[0137] In some embodiments, the recombinant TCR-binding protein is a single-chain TCR (scTCR) fusion protein. The scTCR fusion protein comprises a binding domain containing the scTCR (TCR Vα domain linked to the TCR Vβ domain), optionally an extracellular spacer region, a transmembrane domain, and an intracellular signal transduction domain containing a CD3ζITAM activation domain and optionally a co-stimulatory signal transduction domain (see Aggen et al., 2012, Gene Ther. 19:365-374; Stone et al., Cancer Immunol. Immunother. 2014, 63:1163-76).

[0138] In some implementations, the recombinant TCR-binding protein is a TCR fusion construct (TRuC). TM (See, U.S. Patent Publication No. 2017 / 0166622). TRuC TM The construct contains an antigen-specific binding domain (e.g., scFv) that fuses with at least one component of the TCR complex (CD3γ, CD3ε, or CD3δ) to form a TCR complex component fusion protein. The human TCR complex contains CD3ε polypeptide, CD3γ polypeptide, CD3δ polypeptide, CD3ζ polypeptide, TCRα chain polypeptide, and TCRβ chain polypeptide. The TCR complex component fusion protein can bind to other components of the TCR complex to form a functional, complete TCR fusion complex. Unlike TCR, TruC... TM The construct is able to bind to the target antigen in a manner independent of MHC.

[0139] In some embodiments, the TCR-binding protein comprises a multinucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, their transgenic species, or any combination thereof. In some embodiments, the TCR-binding protein is mouse-derived, chimeric, human-derived, or humanized.

[0140] The TCR-binding protein of this disclosure can bind to a variety of antigens, including tumor antigens, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, and autoimmune disease antigens. Exemplary tumor antigens that the recombinant TCR-binding protein can target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, alpha-fetoprotein, and tumor-specific neoantigens. An exemplary HPV16 E7-specific TCR that can be used in the tandem expression cassette of this disclosure is provided in PCT Publication No. WO2015 / 184228 (incorporated in its entirety by reference). In some embodiments, the HPV16 E7-specific TCR comprises the amino acid sequence shown in SEQ ID NO:90. The amino acid sequence shown in SEQ ID NO:90 contains a P2A self-cleaving peptide between the TCRβ chain sequence and the TCRα chain sequence, which will cleave in the host cell to form two polypeptide chains. Therefore, in some embodiments, the TCR represented by SEQ ID NO:90 comprises separate TCRβ and TCRα polypeptide chains capable of dimerizing to form an αβTCR. In some embodiments, the HPV16 E7-specific TCR comprises Vβ, which contains the amino acid sequence shown in SEQ ID NO:92. In some embodiments, the HPV16 E7-specific TCR comprises Vα, which contains the amino acid sequence shown in SEQ ID NO:94. In a further embodiment, the HPV16-specific E7 TCR comprises Vβ containing the amino acid sequence shown in SEQ ID NO:92 and Vα containing the amino acid sequence shown in SEQ ID NO:94.

[0141] In some embodiments, the Cα domain, Cβ domain, or both of the TCR contain cysteine ​​substitutions to form interchain disulfide bonds between cysteine ​​residues in the two constant domains, which are absent in the unmodified TCR. Such modified TCRs can form more stable heterodimers. In a particular embodiment, the Cα domain contains a Thr→Cys substitution at position 48 of the wild-type protein sequence, and the Cβ domain contains a Ser→Cys substitution at position 56 of the wild-type protein sequence (see PCT publication WO2015 / 184228). An exemplary cysteine-modified TCR Cβ constant region contains the amino acid sequence shown in SEQ ID NO:93.

[0142] In some embodiments, the TCR includes replacing one, two, or three amino acids in the transmembrane domain of one or both of the α and β chains with hydrophobic amino acids to increase the hydrophobicity of the transmembrane domain. In some embodiments, one, two, or three residues of the TCR α chain selected from Ser112, Met114, and Gly115 are substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. An exemplary cysteine-modified, “LVL”-substituted TCR Cα region comprises the amino acid sequence shown in SEQ ID NO: 95.

[0143] In some embodiments, the CER and CAR / or TCR-binding proteins encoded by the tandem expression cassette target the same antigen. In other embodiments, the CER and CAR / or TCR-binding proteins encoded by the tandem expression cassette target different antigens.

[0144] Polynucleotides, expression cassettes, vectors, and modified host cells

[0145] In some aspects, this disclosure provides nucleic acid molecules encoding any one or more receptors described herein (e.g., CER, CAR, and TCR-binding proteins). Nucleic acids can refer to single-stranded or double-stranded DNA, cDNA, or RNA, and can include positive and negative strands of nucleic acids complementary to each other, including antisense DNA, cDNA, and RNA. Nucleic acids can be naturally occurring or synthetically produced DNA or RNA. The nucleic acid sequence encoding the desired receptor can be obtained or generated using standard techniques and recombination methods known in the art, such as by screening a library from cells expressing the desired sequence or a portion thereof, by obtaining the sequence from a vector known to contain the same sequence, or by directly isolating the sequence or a portion thereof from cells or tissues containing the same sequence, as described, for example, in Sambrook et al. (1989 and 2001; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). Alternatively, the target sequence can be produced synthetically rather than cloned.

[0146] The polynucleotide encoding the receptor composition provided herein can be derived from any animal, such as human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, pig, or a combination thereof. In some embodiments, the polynucleotide encoding at least one or two receptors contained in the tandem expression cassette is derived from the same animal species as the host cell into which the polynucleotide is inserted.

[0147] In some embodiments, the polynucleotide encoding the receptor includes a 5' end sequence encoding a signal peptide (also referred to as a leader peptide or signal sequence) for targeting the precursor protein to the secretion pathway. Optionally, the signal peptide is cleaved from the N-terminus of the extracellular domain during cellular processing and receptor localization to the host cell membrane. A polypeptide with the signal peptide sequence cleaved or removed may also be referred to as a mature polypeptide. Examples of signal peptides that may be used in the receptors of this disclosure include signal peptides derived from endogenous secretory proteins, including, for example, GM-CSF (amino acid sequence shown in SEQ ID NO: 64) or Tim4 (amino acid sequence shown in SEQ ID NO: 65). As used herein, the receptor polynucleotide or polypeptide sequences (e.g., CER, CAR, or TCR-binding proteins) provided herein may or may not include a signal sequence. Those skilled in the art will understand that for sequences including a signal peptide sequence disclosed herein, another signal peptide capable of transporting the encoded protein to the extracellular membrane may be used instead of that signal peptide sequence.

[0148] In some embodiments, the polynucleotide encoding the receptor in this disclosure is codon-optimized for efficient expression in target host cells containing the polynucleotide (see, for example, Scholten et al., Clin. Immunol. 119:135-145 (2006)). As used herein, a “codon-optimized” polynucleotide comprises a heteropolynucleotide having a codon modified by a silent mutation corresponding to the abundance of tRNA in the target host cell.

[0149] The polynucleotides encoding at least two transgenes (e.g., CER and CAR, CER and TCR binding proteins) provided in this disclosure can be used to form tandem expression cassettes. A tandem expression cassette refers to a component of a vector nucleic acid containing at least two transgenes, under the control of or effectively linked to the same set of regulatory sequences for the tandem or co-expression of at least two transgenes. Regulatory sequences that can be used in the tandem expression cassettes of this disclosure include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; sequences enhancing protein secretion; or any combination thereof.

[0150] In some embodiments, tandem expression cassettes can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette may include promoter elements to optimize spatial and temporal control. In some embodiments, the tandem expression cassette includes a tissue-specific promoter or enhancer capable of specifically delivering the tandem expression cassette to an organ, cell type (e.g., immune cells), or pathological microenvironment, such as tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In some embodiments, the tandem expression cassette includes a constitutive promoter. An exemplary constitutive promoter used in the tandem expression cassettes used in this disclosure is the EF-1α promoter. In some embodiments, the tandem expression cassette includes an inducible promoter. In some embodiments, the tandem expression cassette includes a tissue-specific promoter.

[0151] The at least two transgenes contained in a tandem expression cassette can be in any order. For example, a tandem expression cassette containing a polynucleotide encoding CER and a polynucleotide encoding CAR can be arranged from 5' to 3' as CER-CAR or CAR-CER. In another example, a tandem expression cassette containing a polynucleotide encoding CER and a polynucleotide encoding TCR can be arranged from 5' to 3' as CER-TCR or TCR-CER.

[0152] In some embodiments, a receptor comprising two or more polypeptide chains that bind to form a multimer or complex may be encoded by two or more polynucleotide molecules in a tandem expression construct. Exemplary multimeric receptors contemplated for expression in the tandem expression constructs of this disclosure include multi-chain CARs, TCRs, TCR-CARs, and TruCs. TM Constructs. Therefore, embodiments of exemplary tandem expression cassettes encoding CER and TCR may include polynucleotides encoding CER, polynucleotides encoding the TCRα chain polypeptide, and polynucleotides encoding the TCRβ chain polypeptide.

[0153] In some embodiments, the tandem expression cassette of this disclosure may include an internal ribosome entry site (IRES) or a peptide cleavage site, such as a furin cleavage site or a viral 2A peptide, located between each polynucleotide contained within the tandem expression cassette, to enable the co-expression of multiple proteins from a single mRNA. For example, an IRES, furin cleavage site, or viral 2A peptide may be located between a polynucleotide encoding CER and a polynucleotide encoding CAR within the tandem expression cassette. In another example, an IRES, furin cleavage site, or viral 2A peptide may be located between each polynucleotide encoding CER, a polynucleotide encoding a TCRα chain polypeptide, and a polynucleotide encoding a TCRβ chain polypeptide. In some embodiments, the viral 2A peptide is porcine swine cisvirus-1 (P2A), Thoseea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. An exemplary T2A peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 67, 68, 69, and 75. The exemplary P2A peptide sequence comprises the amino acid sequence shown in SEQ ID NO:70 or 71. The exemplary E2A peptide sequence comprises the amino acid sequence shown in SEQ ID NO:72. The exemplary F2A peptide sequence comprises the amino acid sequence shown in SEQ ID NO:73.

[0154] Some embodiments of the tandem expression cassettes disclosed herein comprise polynucleotides encoding CARs and / or TCRs specifically targeting a target antigen (e.g., a tumor antigen) and polynucleotides encoding CERs that bind to prophagocytic markers (e.g., apoptosis markers such as phosphatidylserine). After target cells expressing the target antigen via CAR / or TCR binding, cells modified to express this tandem expression cassette induce apoptosis in the target cells. Apoptosis induces exposure of the prophagocytic marker (such as phosphatidylserine) on the target cells, which can then be targeted to damaged or apoptotic cells for phagocytosis by the CER.

[0155] An exemplary tandem expression cassette of this disclosure comprises: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds to phosphatidylserine; a TLR4 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1AIn some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER5-HPV16 E7 TCR”) comprises CER5 containing the amino acid sequence shown in SEQ ID NO:97 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0156] Another exemplary tandem expression cassette of this disclosure includes: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds phosphatidylserine; a TLR5 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1B In some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER19-HPV16 E7 TCR”) comprises CER19 containing the amino acid sequence shown in SEQ ID NO:98 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0157] Another exemplary tandem expression cassette of this disclosure includes: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds to phosphatidylserine; a TLR8 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1C In some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER21-HPV16 E7 TCR”) comprises CER21 containing the amino acid sequence shown in SEQ ID NO:99 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0158] Another exemplary tandem expression cassette of this disclosure includes: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds phosphatidylserine; an NFAM1 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1DIn some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER25-HPV16 E7 TCR”) comprises CER25 containing the amino acid sequence shown in SEQ ID NO:100 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0159] Another exemplary tandem expression cassette of this disclosure includes: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds to phosphatidylserine; a TLR2 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1E In some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER27-HPV16 E7 TCR”) comprises CER27 containing the amino acid sequence shown in SEQ ID NO:101 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0160] Another exemplary tandem expression cassette of this disclosure includes: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds to phosphatidylserine; a Traf6 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1F In some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER29-HPV16 E7 TCR”) comprises CER29 containing the amino acid sequence shown in SEQ ID NO:102 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0161] Another exemplary tandem expression cassette of this disclosure comprises: (a) a polynucleotide encoding a CER comprising: an extracellular domain including a Tim4 binding domain that binds to phosphatidylserine; a Traf3 phagocytic signaling domain; and a Tim4 transmembrane domain located between and connecting the extracellular domain and the phagocytic signaling domain; (b) a polynucleotide encoding an HPV-E7-specific recombinant T-cell receptor (TCR) β chain including a TCRβ variable region and a TCRβ constant region; and (c) a polynucleotide encoding an HPV-E7-specific recombinant TCRα chain including a TCRα variable region and a TCRα constant region (see [link to documentation]). Figure 1GIn some embodiments, the tandem expression cassette comprises a 2A peptide sequence dispersed between a polynucleotide encoding the CER, a polynucleotide encoding the TCRβ chain, and a polynucleotide encoding the TCRα chain. In some embodiments, the tandem expression cassette comprises an EF-1α promoter operatively linked to the CER and TCR polynucleotides. In some embodiments, such an exemplary tandem expression cassette (“CER31-HPV16 E7 TCR”) comprises CER31 containing the amino acid sequence shown in SEQ ID NO:103 and HPV16 E7 TCR containing the amino acid sequence shown in SEQ ID NO:90.

[0162] The polynucleotide encoding the desired tandem expression cassette can be inserted into a suitable vector, such as a viral vector, a non-viral plasmid vector, or a non-viral vector like a lipid-based DNA vector, modified mRNA (modRNA), self-amplified mRNA, CELID, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), to introduce it into a target host cell (e.g., immune cells). The polynucleotide encoding the tandem expression cassette disclosed herein can be cloned into any suitable vector, such as an expression vector, replication vector, probe generation vector, or sequencing vector. In some embodiments, the polynucleotide encoding CER and the polynucleotide encoding a CAR or TCR-binding protein are linked together as a single polynucleotide and then inserted into the vector. In other embodiments, the polynucleotide encoding CER and the polynucleotide encoding a CAR or TCR-binding protein can be inserted into the vector separately, such that the expressed amino acid sequence produces a functional CER and CAR / or TCR. The vector encoding the tandem expression cassette is referred to herein as a "tandem expression vector".

[0163] In some embodiments, the vector comprises a polynucleotide encoding a tandem expression vector. In some embodiments, the vector comprises a tandem expression cassette encoding a CER and CAR / or TCR-binding protein.

[0164] In some implementations, vectors that allow tandem expression cassettes to integrate long-term and proliferate into daughter cells are used. Examples include viral vectors such as adenovirus, adeno-associated virus, vaccinia virus, herpesvirus, cytomegalovirus, poxvirus, or retroviral vectors such as lentiviral vectors. Lentiviral-derived vectors can be used to achieve long-term gene transfer and offer several advantages over traditional vectors, including the ability to transduce non-proliferating cells (such as hepatocytes) and low immunogenicity.

[0165] In some embodiments, a free, non-integrating vector is used in the tandem expression cassette of this disclosure. Examples of non-integrating viral vectors include adenovirus vectors and integrated viral vectors that have been mutated to be non-integrating, such as non-integrating lentiviral vectors and non-integrating foamy virus vectors.

[0166] In this document, the vector encoding the core virus is referred to as a "viral vector." A wide variety of viral vectors are suitable for the compositions of this disclosure, including those identified for human gene therapy applications (see, Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as vectors derived from retroviruses, for example, vectors derived from Moloney mouse leukemia virus (MLV), and more complex vectors derived from retroviruses, such as vectors derived from lentiviruses. Vectors derived from HIV-1 fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods for transducing viral particles containing chimeric receptor transgenes into mammalian host cells using retroviral and lentiviral vectors and packaging cells are well known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0167] In some embodiments, the viral vector is used to introduce a non-endogenous polynucleotide sequence encoding a tandem expression cassette into host cells. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also contain a nucleic acid sequence encoding a marker for transduction. Transduction markers used with the viral vector are known in the art and include selectable markers that may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In certain embodiments, the viral vector also contains a genetic marker for transduction, which includes a fluorescent protein (e.g., green, yellow), the extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see Wang et al., Blood 118:1255, 2011). An exemplary tEGFR sequence contains the amino acid sequence shown in SEQ ID NO:82.

[0168] Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0169] Other viral vectors recently developed for gene therapy applications can also be used in conjunction with the compositions and methods disclosed herein. Such vectors include those derived from baculoviruses and alpha viruses (Jolly, DJ. 1999. Emerging Viral Vectors. pp. 209-40, Friedmann T. ed., The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0170] Where time control is required, tandem expression vectors can contain elements capable of inducing depletion of transduced cells. For example, such vectors can contain inducible suicide genes. Suicide genes can be apoptosis genes or genes that confer sensitivity to reagents (e.g., drugs). Exemplary suicide genes include chemically inducible caspase 9 (iCASP9) (US Patent Publication No. 2013 / 0071414), chemically inducible Fas or herpes simplex virus thymidine kinase (HSV-TK) (conferring sensitivity to ganciclovir). In a further embodiment, tandem expression vectors can be designed to express known cell surface antigens that, upon infusion of an associated antibody, deplete transduced cells. Examples of cell surface antigens and associated antibodies that can be used to deplete transduced cells include CD20 and rituximab, RQR8 (a combination of CD34 and CD20 epitopes capable of CD34 selection and anti-CD20 deletion) and rituximab, and EGFR and cetuximab.

[0171] Inducible vector systems can also be used for the induction of tandem expression cassettes, such as the tetracycline-On vector system, which uses doxycycline to activate transgene expression (Heinz et al., Hum. Gene Ther. 2011, 22:166-76). Small molecule reactive transcription factors can also be used to regulate expression. The induction of tandem expression cassettes can also be accomplished using a selective hook (RUSH) system, which is based on hooking streptavidin anchored to the endoplasmic reticulum membrane and introducing streptavidin-binding proteins into CER and CAR / or TCR structures. In this system, the addition of biotin leads to the release of CER and CAR / or TCR from the endoplasmic reticulum (Agaugue et al., 2015, Mol. Ther. 23(Suppl. 1):S88).

[0172] In some implementations, host cells modified with tandem expression cassettes can also be modified to co-express one or more small GTPases. Rho GTPases are a small (~21 kDa) family of signaling G proteins and a subfamily of the Ras superfamily. They regulate actin cytoskeleton organization in various cell types during phagocytosis and promote pseudopodia expansion and phagosome closure (see, for example, Castellano et al., 2000, J. Cell Sci. 113:2955-2961). Phagocytosis requires the recruitment of F-actin to tethered cells or granules, and F-actin rearrangement to allow membrane extension leading to cell or granule internalization. Rho GTPases include RhoA, Rac1, Rac2, RhoG, and CDC42. Other small GTPases (such as Rap1) are involved in the regulation of complement-mediated phagocytosis. Co-expression of small GTPases with tandem expression cassettes encoding CER can promote or enhance the internalization of target cells or granules and / or phagosome formation by host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on a separate vector compared to a vector containing a tandem expression cassette. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase and the tandem expression cassette are encoded on the same vector. The GTPase and the tandem expression cassette can be expressed on the same vector under the regulation of different promoters (e.g., at different multiple cloning sites). Alternatively, the tandem expression cassette and the GTPase can be expressed under the regulation of one promoter in a polycistronic vector.

[0173] Examples of GTPases that can be co-expressed with a tandem expression cassette include Rac1, Rac2, Rab5 (also known as Rab5a), Rab7, Rap1, RhoA, RhoG, CDC42, or any combination thereof. In a particular embodiment, the GTPase comprises or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence of Rac1 shown in SEQ ID NO:83, the amino acid sequence of Rab5 shown in SEQ ID NO:84, the amino acid sequence of Rab7 shown in SEQ ID NO:85, the amino acid sequence of Rap1 shown in SEQ ID NO:86, the amino acid sequence of RhoA shown in SEQ ID NO:87, the amino acid sequence of CDC42 shown in SEQ ID NO:108, or any combination thereof. In some embodiments, GTPase expression is induced or modulated in host cells such that GTPase expression is activated only after a sufficiently long period of time has elapsed for the CER-encoded tandem expression cassette to bind to its target antigen. In a further embodiment, GTPase expression can be deactivated after a sufficient period of time for the CER-mediated phagocytosis of the target antigen-expressing cell.

[0174] In some embodiments, cells obtained from a subject (such as immune cells) can be genetically modified to form non-natural or recombinant cells (e.g., non-natural or recombinant immune cells) by introducing a tandem expression cassette as described herein, thereby enabling the cells to express CERs and CARs / / or TCRs located on their cell surfaces. In some embodiments, the host cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell. Exemplary immune cells that can be modified to include a tandem expression cassette or a carrier containing a tandem expression cassette include T cells, natural killer cells, B cells, lymphoid progenitor cells, antigen-presenting cells, dendritic cells, Langerhans cells, myeloid progenitor cells, mature myeloid cells, monocytes, or macrophages.

[0175] In some embodiments, B cells are genetically modified to express a tandem expression cassette of the present disclosure. B cells possess certain properties that may be advantageous as host cells, including: transport to sites of inflammation, ability to internalize and present antigens, ability to co-stimulate T cells, high proliferation and self-renewal (persistent survival). In some embodiments, tandem expression cassette-modified B cells are capable of digesting phagocytosed target cells or phagocytosed target particles into smaller peptides and presenting them to T cells via MHC molecules. Antigen presentation by tandem expression cassette-modified B cells may facilitate antigen diffusion in immune responses against untargeted antigens. B cells include progenitor cells or precursor cells associated with B cell lineages (e.g., pre-progenitor B cells, progenitor B cells, and pre-B cells); immature and unactivated B cells; or mature and functional or activated B cells. In some embodiments, B cells may be naïve B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytic-like cells, plasmablasts, memory B cells, or any combination thereof. Memory B cells can be distinguished from naive B cells based on the lack of CD27 expression on naive B cells. In some embodiments, B cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. B cell lines are well known to those skilled in the art. If obtained from mammals, B cells can be obtained from many sources, including blood, bone marrow, spleen, lymph nodes, or other tissues or body fluids. B cell components may be enriched or purified.

[0176] In some embodiments, T cells are genetically modified to express a tandem expression cassette of the present disclosure. Exemplary T cells include CD4+. + Helper cells, CD8 + Effector (cytotoxic) cells, naïve (CD45 RA+, CCR7+, CD62L+, CD27+, CD45RO-) cells, central memory (CD45RO-) cells + CD62L + CD8 +The T cells may include effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-) cells, T memory stem cells, regulatory cells, mucosa-associated invariance (MAIT) cells, γδ (gd) cells, tissue-resident T cells, natural killer T cells, or any combination thereof. In some embodiments, the T cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. If derived from mammals, the T cells may be obtained from a variety of sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell composition may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In some embodiments, the T cells lack the TCRα gene, the TCRβ gene, or endogenous expression of both. These T cells may naturally lack endogenous expression of TCRα and β chains, or may have been modified to prevent expression (e.g., T cells from transgenic mice that do not express TCRα and β chains or cells that have been manipulated to suppress TCRα and β chain expression) or have the TCRα chain, TCRβ chain, or both genes knocked out.

[0177] In some embodiments, the host cell expressing the tandem expression cassette of this disclosure is not a T cell or a cell of the T cell lineage, but a progenitor cell, stem cell, or a cell modified to express cell surface anti-CD3.

[0178] In some embodiments, gene editing methods are used to modify the host cell genome to include a tandem expression cassette encoding the disclosed herein. Gene editing, or genome editing, is a genetic engineering method in which genetically engineered endonucleases are used to insert, replace, or delete DNA from the host cell's genome. The nucleases form specific double-strand breaks at target loci in the genome. The host cell's endogenous DNA repair pathways then repair one or more induced breaks, for example, through non-homologous end joining (NHEJ) and homologous recombination. Exemplary endonucleases used in gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, clustered regularly spaced short palindromic repeat (CRISPR) / Cas nuclease systems (e.g., CRISPR-Cas9), large-scale nucleases, or combinations thereof. Methods for disrupting or knocking out genes or gene expression in immune cells, including B cells and T cells, using gene-editing endonucleases are known in the art and are described, for example, in PCT Publications WO 2015 / 066262; WO 2013 / 074916; WO 2014 / 059173; Cheong et al., Nat. Comm. 2016 7:10934; Chu et al., Proc. Natl. Acad. Sci. USA 2016 113:12514-12519; the entire contents of the methods from each of these publications are incorporated herein by reference.

[0179] In some implementations, the expression of endogenous genes in host cells is suppressed, knocked down, or eliminated. Examples of endogenous genes that can be suppressed, knocked down, or eliminated in B cells include IGH, IGκ, IGλ, or any combination thereof. Examples of endogenous genes that can be suppressed, knocked down, or eliminated in T cells include TCR genes (TRA or TRB), HLA genes (class I or class II HLA genes), immune checkpoint molecules (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, or PVRIG / CD112R) or any combination thereof. Endogenous gene expression can be suppressed, knocked down, or eliminated at the gene level, transcription level, translation level, or a combination thereof. This suppression, knockdown, or elimination of endogenous genes can be achieved through, for example, RNA interference agents (e.g., siRNA, shRNA, miRNA, etc.) or engineered endonucleases (e.g., CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), large-scale nucleases) or any combination thereof. In some embodiments, endogenous B-cell genes (e.g., IGH, IGκ, or IGλ) are knocked out by inserting the tandem expression cassette of this disclosure into the locus of an endogenous B-cell gene, such as using an engineered endonuclease. In some embodiments, endogenous T-cell genes (e.g., TCR genes, HLA genes, or immune checkpoint genes) are knocked out by inserting a polynucleotide encoding the tandem expression cassette of this disclosure into the locus of an endogenous T-cell gene, such as using an engineered endonuclease.

[0180] This disclosure also provides compositions comprising host cell populations modified with tandem expression cassettes. In some embodiments, the host cell population modified with tandem expression cassettes may be a population of B cells, T cells, natural killer cells, lymphocyte precursor cells, antigen-presenting cells, dendritic cells, Langerhans cells, bone marrow precursor cells, mature myeloid cells, or any combination thereof. Furthermore, the host cell population modified with tandem expression cassettes for a specific cell type may consist of one or more subtypes. For example, the B cell population may consist of tandemly expressed B cells, plasma cells, regulatory B cells, marginal zone B cells, lymphoplasmacytic-like cells, plasmablasts, memory B cells, or any combination thereof. In another example, the T cell population may consist of CD4+ modified with tandem expression cassettes. + Helper T cells, CD8 +Effector (cytotoxic) T cells, naive (CD45RA+, CCR7+, CD62L+, CD27+, CD45RO-) T cells, central memory (CD45RO-) T cells + CD62L + CD8 + T cells, effector memory (CD45RA+, CD45RO-, CCR7-, CD62L-, CD27-) T cells, T memory stem cells, regulatory T cells, mucosa-associated invariant T cells (MAIT), γδ (gd), tissue-resident T cells, natural killer T cells, or any combination thereof.

[0181] In some embodiments, the host cell population consists of cells that each express the same tandem expression cassette. In other embodiments, the host cell population consists of a mixture of two or more host cell subpopulations, each expressing a different tandem expression cassette.

[0182] In some embodiments, when preparing host cells modified with tandem expression cassettes (e.g., B cells or T cells), one or more growth factor cytokines that promote the proliferation of host cells (e.g., B cells or T cells) may be added to the cell culture medium. The cytokines may be human or non-human. Exemplary growth factor cytokines that can be used to promote T cell proliferation include IL-2, IL-15, etc. Exemplary growth factor cytokines that can be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, etc.

[0183] Before genetically modifying host cells using a tandem expression cassette vector, the host cells (e.g., T cells, B cells, natural killer cells, etc.) are derived from an object (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue), from which host cells are isolated using methods known in the art. Specific subpopulations of host cells can be collected according to known techniques and enriched or depleted according to known techniques, such as antibody affinity binding, flow cytometry, and / or immunomagnetic selection. After the enrichment and / or depletion steps and the introduction of the tandem expression cassette, the desired modified host cells can be expanded in vitro according to known techniques or variations of such techniques that will be obvious to those skilled in the art.

[0184] The expression of receptors encoded by tandem expression cassettes on host cells can be functionally characterized using many methods recognized in the art for assessing host cell (e.g., T cell) activity, including determining T cell binding, activation, or induction, as well as determining antigen-specific T cell responses. Examples include determining T cell proliferation, T cytokine release, antigen-specific T cell stimulation, and CTL activity (e.g., by detecting preloaded target cells). 51 The release of Cr or europium, the induction of caspase activity in target cells, the extracellular release of lactate dehydrogenase from target cells, changes in the expression of T cell phenotypic markers, and other indicators of T cell function. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii, eds., Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and the references cited therein. Cytokine levels can be determined using methods known in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., intracellular cytokine staining and flow cytometry). The proliferation and clonal expansion of immune cells caused by antigen-specific initiation or stimulation of an immune response can be determined by isolating lymphocytes (such as peripheral blood cells or circulating lymphocytes from cell samples from lymph nodes), stimulating the cells with antigens, and measuring cytokine production, cell proliferation, and / or cell viability (such as by incorporation of tritium-substituted thymine or by non-radioactive assays such as MTT assays).

[0185] In some implementations, host cells modified with tandem expression cassettes containing CERs exhibit a phagocytic index of approximately 20 to approximately 1,500 for target cells. The “phagocytic index” is a measure of the phagocytic activity of transduced host cells, determined by counting the number of target cells or particles ingested by each tandem expression cassette-modified host cell in a suspension of target cells or particles and tandem expression cassette-modified host cells incubated in a culture medium over a set time period. It can be expressed as a multiplier [total number of phagocytosed target cells / total number of counted tandem expression cassette-modified cells (e.g., phagocytic frequency)] x [per tandem expression cassette]. + [Average area of ​​host cell or particle staining x 100 (e.g., hybridization capture)] or [Total number of phagocytosed particles / Total number of tandem expression cassette modified host cells] x [Number of tandem expression cassette modified host cells containing phagocytosed particles / Number of tandem expression cassette cells] +The phagocytic index is calculated by multiplying the total number of cells by 100. In some embodiments, the tandem expression cassette modified cells have approximately 30 to approximately 1,500; approximately 40 to approximately 1,500; approximately 50 to approximately 1,500; approximately 75 to approximately 1,500; approximately 100 to approximately 1,500; approximately 200 to approximately 1,500; approximately 300 to approximately 1,500; approximately 400 to approximately 1,500; approximately 500 to approximately 1,500; approximately 20 to approximately 1,400; approximately 30 to approximately 1,400; approximately 40 to approximately 1,400; approximately 50 to approximately 1,400; approximately 100 to approximately 1,400; approximately 200 to approximately 1,400; approximately 300 to approximately 1, 400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 3 00 to about 1,200; about 400 to about 1,200; about 500 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about Phagocytic index of 1,000; about 300 to about 1,000; about 400 to about 1,000; or about 500 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750; about 300 to about 750; about 400 to about 750; or about 500 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or about 300 to about 500. In a further embodiment, the incubation time is about 2 hours to about 4 hours, for example about 2 hours, about 3 hours, or about 4 hours. In yet another embodiment, cells modified with tandem expression cassettes showed a statistically significantly higher phagocytic index than control cells transduced using truncated EGFR.The phagocytic index can be calculated using methods known in the art and further described in the examples and PCT application number PCT / US2017 / 053553 (which are incorporated herein by reference in their entirety), including quantification by flow cytometry or fluorescence microscopy.

[0186] The host cells can be derived from animals such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, or combinations thereof. In a preferred embodiment, the animal is a human. The host cells can be derived from healthy subjects or subjects suffering from diseases associated with the expression or overexpression of antigens.

[0187] How to use

[0188] In one aspect, this disclosure provides methods for conferring antigen-specific cytolytic and phagocytic activity to a cell, comprising introducing a tandem expression cassette according to any embodiment herein into a host cell; and expressing CER and CAR / or TCR-binding proteins in the host cell. In some embodiments, the CER and CAR / or TCR-binding proteins bind to the same target antigen. In some embodiments, the CER and CAR / or TCR bind to different target antigens. In some embodiments, host cells modified with the tandem expression cassette of this disclosure are capable of phagocytosing target cells or a portion of target cells. Therefore, cells modified with the tandem expression cassette of this disclosure can possess targeted cell-killing capabilities in a variety of ways: cell lysis of target cells, phagocytosis of whole target cells, phagocytosis of a portion of target cells, or any combination thereof.

[0189] In another aspect, this disclosure provides a method for enhancing cell antigen-specific cytotoxic activity, comprising introducing a tandem expression cassette according to any embodiment herein into a host cell; and expressing a CER and a CAR / or TCR-binding protein in the host cell, wherein expression of the tandem expression cassette enhances the cytotoxic activity of the host cell compared to host cells expressing only the CAR / or TCR-binding protein. In some embodiments, the cytotoxic activity of the host cell is increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more compared to host cells expressing only CAR / or TCR. In a further embodiment, the tandem expression cassette confers a synergistic cytotoxic response. In some implementations, the host cells are T cells or NK cells. Methods for measuring the cytotoxic activity of host cells (particularly immune cells such as T cells and NK cells) include chromium (…). 51Cr) release assays, β-gal or firefly luciferase release assays, and flow cytometry methods for measuring target cell death and effector cell activity (see, for example, Expert Rev. Vaccines, 2010, 9:601-616). In some embodiments, host cell cytotoxic activity, such as caspase 3 / 7 activity and lactate dehydrogenase release, can be measured by monitoring apoptosis of target cells after exposure to host cells.

[0190] In another aspect, the tandem expression cassettes described according to any of the embodiments provided herein can be used in methods for treating subjects suffering from diseases, disorders, or adverse conditions. Embodiments of these methods include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising one or more tandem expression cassettes as described herein, a polynucleotide encoding one or more tandem expression cassettes, a vector containing one or more tandem expression cassettes, or a host cell population genetically modified to express one or more tandem expression cassettes.

[0191] Diseases for which cell therapy expressing the expression tandem expression cassettes described in this disclosure can be used include cancer, autoimmune diseases, and infectious diseases (viral, bacterial, fungal, protozoan infections). Adoptive immunotherapy and gene therapy are promising therapies for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2011).

[0192] A variety of cancers, including solid tumors and leukemias, are suitable for treatment using the tandem expression cassette compositions provided herein. Exemplary cancers that can be treated using the receptors, modified host cells, and compositions described herein include breast, prostate, and colonic adenocarcinoma; all forms of bronchogenic carcinoma; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papilloma; amine precursor uptake and decarboxylation cell tumors; vacuole tumors; branchial protozoa; malignant carcinoid syndromes; carcinoid heart disease; and carcinomas (e.g., Walker carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scleroderma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Other cancer types that can be treated using the receptors, modified host cells, and compositions described herein include histiocytosis; malignant histiocytosis; leukemia; Hodgkin's disease; immunoproliferative enteropathy; non-Hodgkin's lymphoma; plasmacytoma; multiple myeloma; plasmacytoma; reticuloendothelial proliferation; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; stromal tumor; mesonephroma; sarcoma; ameloblastoma; cementum tumor; odontoma; teratoma; thymoma; and trophoblastic tumor. Furthermore, it is also considered that the receptors, modified host cells, and compositions described herein may be used to treat the following types of cancer: adenoma; cholangioma; cholesteatoma; cylindrica; cystadenocarcinoma; cystadenoma; granulosa cell tumor; germ cell tumor; hepatocellular carcinoma; hidradenoma; islet cell tumor; Ledich's cell tumor; papilloma; Sertoli cell tumor; theca cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; sarcoma; rhabdomyosarcoma; rhabdomyosarcoma; ependymoma; ganglionoma; glioma; medulloblastoma; meningioma; schwannoma; neuroblastoma; neuroepithelial tumor; neurofibroma; neuroma; paraganglioma; paraganglioma non-pheochromocytoma. Treatable cancer types also include angiokeratoma; angiolymphoid hyperplasia with eosinophilia; hemangioma sclerosis; hemangiomatosis; glomus tumor; hemangioendothelioma; hemangioma; hemangiopericytoma; angiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pineal tumor; carcinosarcoma; chondrosarcoma; phyllodes sarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphangiosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; growths; neurofibromatosis; and cervical dysplasia.

[0193] Exemplary hyperproliferative disorders suitable for use with the tandem expression cassette composition therapy described herein are B-cell cancers, including B-cell lymphomas (such as various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL), or central nervous system lymphoma), leukemias (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, B-blast transformation of chronic myeloid leukemia), and myelomas (such as multiple myeloma). Other B-cell cancers that can be treated with the receptors, modified host cells, and compositions described herein include small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-joint marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), lymph node marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferation with uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders.

[0194] Infectious diseases include those associated with infectious agents and include any number of bacteria (e.g., pathogenic E. coli, S. typhimurium, P. aeruginosa, B. anthracis, C. botulinum, C. difficile, C. perfringens, H. pylori, V. cholerae, Listeria spp., Rickettsia spp., Chlamydiaspp., etc.), mycobacteria, and parasites (including any known parasitic member of protozoa). Infectious viruses include eukaryotic viruses such as adenoviruses, Bunyaviruses, herpesviruses, polymorphonuclear papillomaviruses, papillomaviruses (e.g., HPV), paramyxoviruses, picornaviruses, rhabdoviruses (e.g., rabies virus), orthomyxoviruses (e.g., influenza virus), poxviruses (e.g., vaccinia virus), reoviruses, retroviruses, lentiviruses (e.g., HIV), flaviviruses (e.g., HCV, HBV), etc. In some embodiments, a composition comprising a tandem expression cassette according to the present disclosure is used to treat infections caused by microorganisms that establish persistent infection in a subject.

[0195] The treatment method involves administering an effective amount of cells modified with a tandem expression cassette (i.e., recombinant cells expressing a tandem expression cassette). The cells modified with the tandem expression cassette can be allogeneic, syngeneic, allogeneic, or autologous to the subject.

[0196] Pharmaceutical compositions comprising cells modified with a tandem expression cassette can be administered in a manner suitable for the disease or condition to be treated (or prevented) by a person skilled in the medical field. The appropriate dose, duration, and frequency of administration of the composition will be determined by factors such as the patient's condition, physique, weight, body surface area, age, sex, type and severity of the disease, the specific therapy administered, the specific form of the active ingredient, the time and method of administration, and any other medications administered concurrently. This disclosure provides pharmaceutical compositions comprising cells modified with a tandem expression cassette and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextran, glycerol, and combinations thereof. Other suitable infusion media may be any isotonic formulation, including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% aqueous dextran solution, or Ringer's lactate.

[0197] The therapeutically effective amount of cells in the pharmaceutical composition is at least one cell (e.g., one tandem expression cassette-modified T cell) or more generally more than 10. 2 Cells, for example, up to 10 6 Up to 10 7 Up to 10 8 Cells, up to 10 9 Cells, up to 10 10 One cell or up to 10 11 One or more cells. In some embodiments, cells are arranged from about 10 6 To about 10 10 cells / m 2 Application within a range, preferably about 10 7 To about 10 9 The range is cells / m². In a particular embodiment, it is at least about 1 x 10⁻⁶. 6 1 cell, 2x10 6 1 cell, 3x10 6 1 cell, 4x10 6 1 cell, 5x10 6 1 cell, 6x10 6 1 cell, 7x10 6 1 cell, 8x10 6 1 cell, 9x10 6 1 cell, 1x10 7 1 cell, 2x10 7 1 cell, 3x10 7 1 cell, 4x10 7 1 cell, 5x10 7 1 cell, 6x10 7 1 cell, 7x10 71 cell, 8x10 7 1 cell, 9x10 7 1 cell, 1x10 8 1 cell, 2x10 8 1 cell, 3x10 8 1 cell, 4x10 8 1 cell, 5x10 8 1 cell, 6x10 8 1 cell, 7x10 8 1 cell, 8x10 8 1 cell, 9x10 8 1 cell, 1x10 9 1 cell, 2x10 9 1 cell, 3x10 9 1 cell, 4x10 9 1 cell, 5x10 9 1 cell, 6x10 9 1 cell, 7x10 9 1 cell, 8x10 9 1 cell, 9x10 9 1 cell, 1x10 10 1 cell, 2x10 10 1 cell, 3x10 10 1 cell, 4x10 10 1 cell, 5x10 10 1 cell, 6x10 10 1 cell, 7x10 10 1 cell, 8x10 10 1 cell, 9x10 10 1 cell, 1x10 11 1 cell, 2x10 11 1 cell, 3x10 11 1 cell, 4x10 11 1 cell, 5x10 11 1 cell, 6x10 11 1 cell, 7x10 11 1 cell, 8x10 11 1 cell or 9x10 11The amount of cells applied is the amount of cells modified with the tandem expression cassette. The number of cells will depend on the intended end use of the composition and the cell types contained therein. For example, a composition containing cells modified to contain the tandem expression cassette will contain a cell population containing about 5% to about 95% or more of such cells. In some embodiments, the composition containing the tandem expression cassette-modified cells contains a cell population containing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the purposes provided herein, the cells are typically in volumes of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the desired cell density is typically greater than 10. 4 cells / ml, and generally greater than 10 7 10 cells / ml, typically 10 8 Cells / ml or greater. Cells can be administered as a single infusion or multiple infusions over a period of time. If there is a relapse of disease or disease activity, repeated infusions of tandem expression cassette-modified cells can be administered at intervals of days, weeks, months, or even years. Clinically relevant numbers of immune cells can be allocated to multiple infusions, accumulating to 10⁻⁶ cells / ml or more. 6 10 7 10 8 10 9 10 10 Or 10 11 One cell. The preferred dose for administering host cells containing the recombinant expression vector as described herein is about 10. 7 cells / m 2 Approximately 5x10 7 cells / m 2 Approximately 10 8 cells / m 2 Approximately 5x10 8 cells / m 2 Approximately 10 9 cells / m 2 Approximately 5x10 9 cells / m 2 Approximately 10 10 cells / m 2 Approximately 5x10 10 cells / m 2 Or about 10 11 cells / m 2 .

[0198] Compositions containing tandem expression cassettes, vectors containing tandem expression cassettes, or cells modified with tandem expression cassettes as described herein can be administered intravenously, intraperitoneally, intranasally, intratumorally, intramedullary, intramedullary, intralymphatic, and / or intracerebrospinal fluid.

[0199] The tandem expression cassette composition can be administered to a subject in combination with one or more other therapeutic agents. Examples of therapeutic agents that can be administered in combination with the tandem expression cassette composition according to this specification include radiotherapy, genetically engineered cell immunotherapy (e.g., T-cell, dendritic cell, natural killer cell, macrophage, chimeric antigen receptor therapy), antibody therapy, immune checkpoint molecule inhibitor therapy, UV phototherapy, electroporation therapy, high-intensity focused ultrasound therapy, oncolytic virus therapy, or drug therapy such as chemotherapeutic agents, therapeutic peptides, hormones, aptamers, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, and small molecule therapies.

[0200] Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity modulated radiation therapy, volume modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, whole-body radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

[0201] Exemplary antibodies used in conjunction with the tandem expression cassette compositions described herein include rituximab, pertuzumab, trastuzumab, alemtuzumab, teimomab, bentuximab, cetuximab, bevacizumab, abciximab, adalimumab, afasicept, basilizumab, belimumab, bezoolomab, canakizumab, cetuzumab, dacrolimus, denosumab, efazolinumab, golimumab, olaratumab, palizumab, panitumumab, and tocilizumab.

[0202] Inhibitors of exemplary immune checkpoint molecules that can be used in conjunction with the tandem expression cassette compositions described herein include checkpoint inhibitors targeting PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244 / 2B4, CD160, TIGIT, LAIR-1, PVRIG / CD112R, or any combination thereof. In some embodiments, the immune checkpoint inhibitor may be an antibody, peptide, RNAi agent, or small molecule. An antibody specific to CTLA-4 may be ipilimumab or trimemumab. An antibody specific to PD-1 may be pidilizumab, nivolumab, or pembrolizumab. Antibodies that specifically target PD-L1 can be durvalumab, atezolizumab, or avelumab.

[0203] Chemotherapy agents include nonspecific cytotoxic agents that inhibit mitosis or cell division, and molecularly targeted therapies that stop cancer cell growth and spread by targeting specific molecules (e.g., oncogenes) associated with tumor growth, progression, and metastasis. Exemplary nonspecific chemotherapeutic agents used in conjunction with the tandem expression cassette compositions described herein include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs, and glycomodification analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors.

[0204] Examples of chemotherapeutic agents considered for use in the combination therapies discussed herein include vemurafenib, dabrafenib, trametinib, cobimetinib, and anastrozole. Bicalutamide Bleomycin sulfate Bai Xiaoan Busulfan Injection Capecitabine N4-pentoxycarbonyl-5-deoxy-5-fluorocytosine nucleoside, carboplatin Camustin Chlorinated nitrogen mustard Cisplatin Kratsubin Cyclophosphamide ( or ), cytarabine, cytarabine Cytarabine liposome injection Dacarbazine Actinomycin (Actinomycin D, Cosmegan), Daunorubicin Hydrochloride Daunorubicin Citrate Liposome Injection Dexamethasone, Docetaxel Doxorubicin Hydrochloride Etoposide Fludarabine phosphate 5-Fluorouracil Flutamide Tezatabin, gemcitabine (difluorodeoxycytidine), hydroxyurea Idabi Star Ifosfamide Irinotecan L-asparaginase Calcium formyltetrahydrofolate, melphalan 6-Mercaptopurine Methotrexate Mitothrone Gemtuzumab, Paclitaxel Phoenix (Yttrium 90 / MX-DTPA), Pentostatin, and polyphenylene 20 containing carmustine implants Tamoxifen Citrate teniposide 6-Thioguanine, thiotepa, telazamine Topotecan Hydrochloride for Injection Vincristine Changchun New Alkali Ibrutinib, Venetocin, Crizotinib, Alectinib, Brigatinib, Ceritinib, and Vinorelbine

[0205] Exemplary alkylating agents used in the combination therapies considered herein include nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazines, uramustine (Aminouracil) Uracilnitrogen nitrogen mustard Cyclophosphamide ( Revimmune TM ), ifosfamide Meifalun Chlorinated nitrogen mustard piperobromine Triethylene melamine Triethylenethiophosphoramide, temozolomide Thiotepa Bai Xiaoan Camustin Lomustine streptozotocin and dacarbazine Other exemplary alkylating agents used in the combination therapies considered in this article include, but are not limited to, oxaliplatin. Temozolomide ( and Actinomycin (also known as actinomycin D), ); Mefarin (also known as L-PAM, L-oncolytic toxin, and phenylalanine mustard) ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), ); Camustin Bendamustine Bai Xiaoan ( and Carboplatin Lomustine (also known as CCNU, ); Cisplatin (also known as CDDP, and ); Chlorobutyric acid mustard Cyclophosphamide ( and ); Dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, ); Hexamethylmelamine (also known as hexamethylmelamine (HMM), Ifosfamide Prednumustine; Methylbenzylhydrazine Dichloromethyldiethylamine (also known as nitrogen mustard, nitrogen mustard, and methylchloroethylamine hydrochloride) Streptozotocin Thiotepa (also known as thiophosphoramide, TESPA, and TSPA) ); Cyclophosphamide and bendamustine hydrochloride

[0206] Exemplary platinum-based agents used for the combination therapies considered herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, pyridine, saxaplatin, phenanthreneplatin, and triplatintetranitrate.

[0207] Exemplary molecularly targeted inhibitors that can be used in conjunction with the tandem expression cassette compositions described herein include small molecules that target molecules associated with cancer cell growth and survival, including, for example, hormone antagonists, signal transduction inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF / EGFR pathway inhibitors), growth factor inhibitors, GTPase inhibitors, serine / threonine kinase inhibitors, transcription factor inhibitors, inhibitors of cancer-associated driver mutations, B-Raf inhibitors, MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, BCR-ABL inhibitors, HER2 inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, ALK inhibitors, ROS1 inhibitors, and BTK inhibitors. In some embodiments, the use of molecularly targeted therapy comprises administering a molecularly targeted therapy specific to the molecular target to a subject identified as having a tumor having a molecular target (e.g., a driver oncogene). In some embodiments, the molecular target has an activating mutation. In some embodiments, combining tandem expression cassette-modified cells with a molecularly targeted inhibitor can increase the intensity, durability, or both of the antitumor response. In some embodiments, molecularly targeted therapy is combined with tandem expression cassette-modified cells at a lower dose than typically used.

[0208] Exemplary angiogenesis inhibitors that can be used in conjunction with the tandem expression cassette compositions described herein include, but are not limited to, A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (Atrasentan) (Abbott Laboratories / Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Promasitol) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP O2) (Alnylam Pharmaceuticals), and AMG. 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Desfotiam / MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis / Antisoma), Atemod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bevacizumab) (Genentech), AZD2171 (Sildenafil / Recentin) (AstraZeneca), BAY 57-9352 (Tiratitinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim) Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (brivanib) (Bristol-Myers Squibb),BMS-690514 (Bristol-Myers Squibb), Calcitriol, CCI-779 (Wyeth), CDP-791 (ImClone Systems), High-Crude Torreya (HHT) (ChemGenex Therapeutics), Celebrex (Celexicob) (Pfizer), CEP-7055 (Cephalon / Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex Pharmaceuticals), Comparepine (Oxigene), CP-751, 871 (Figitumumab) (Pfizer), CP-547, 632 (Pfizer), CS-7017 (Daiichi Sankyo) Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fanamin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Silengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone / Bristol-Myers Squibb), EZN-2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals) Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (Ramucumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Iressa (ZD1839 / Gefitinib), LBH589 (Faridak / Panobinostst) (Novartis), Lucentis (Ramucumab) (Genentech / Novartis),LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (Pegatani) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (Tandutinib) (Millennium), Neovastat (AE941 / Benifen) (Aeterna Zentaris), Nexavar (Bayer / Onyx), NM-3 (Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals, Inc.), Panzem Capsules (2ME2) ​​(EntreMed), Panzem NCD (2ME2) ​​(EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen) Industries / Medigen Biotechnology), PKC412 (Novartis), Tea Polyphenol E (Green Tea Extract) (Polypheno E International, Inc.), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Valtarani) (Novartis), PXD101 (Belisitar) (CuraGen Corporation), RAD001 (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Anacostat (Alcon) Research), SN38 (liposomes) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (paretide) (Novartis), squalamine (Genaera), suramin, sunitin (Pfizer), taraxer (Genentech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis),TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK474.

[0209] Exemplary vascular endothelial growth factor (VEGF) receptor inhibitors that can be used in combination with the tandem expression cassette compositions described herein include, but are not limited to, bevacizumab. Axitinib Alanine brinib (BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-yloxy)propyl-2-yl)2-aminopropionate); sorafenib Pazopanib Sunitinib malate Sildenafil (AZD2171, CAS 288383-20-1); Nintedanib (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Tilatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib Panatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brinib (BMS-540215, CAS 649735-46-6); Vandetanib ( Or AZD6474); Moteseni diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridylmethyl)amino]-3-pyridinecarboxamide, as described in PCT Publication No. WO 02 / 066470); Dovirtinib dilactate (TKI258, CAS852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lettatinib (CAS 111358-88-4); N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidine-3-ol (BMS690514); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopentan[c]pyrrolo-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-methyl-3-[[1-methyl-6-(3-pyridyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and aflibercept

[0210] Exemplary EGF pathway inhibitors that can be used in conjunction with the tandem expression cassette compositions described herein include, but are not limited to, tyrosine phosphorylation inhibitors 46, EKB-569, and erlotinib. Gefitinib Cerbitux, Nimotuzumab, Lapatinib Cetuximab (anti-EGFR mAb) 188 Re-labeled nimotuzumab (anti-EGFR mAb), and those compounds generally and specifically disclosed in WO 97 / 02266, EP 0 564 409, WO 99 / 03854, EP 0 520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Patent Nos. 5,747,498, WO 98 / 10767, WO97 / 30034, WO 97 / 49688, WO 97 / 38983, and WO 96 / 33980. Exemplary EGFR antibodies include, but are not limited to, cetuximab. Panitumumab Martuzumab (EMD-72000); Trastuzumab Nimotuzumab (hR3); Zatumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, osimertinib. Erlotinib Hydrochloride Brigatinib N-[4-[(3-chloro-4-fluorobenzene)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, ); Van der Tani Lapatinib (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); cannabinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS497839-62-0); linotinib (TAK165); pelitinib (EKB569) Afatinib (BIBW2992); Lenatinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]carbamic acid, (3S)-3-morpholinomethyl ester (BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopentan[c]pyrrolo-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI166, CAS 187724-61-4).

[0211] Exemplary mTOR inhibitors that can be used in combination with the tandem expression cassette compositions described herein include, but are not limited to, rapamycin. and its analogues and derivatives; SDZ-RAD; tesimolimus ( Also known as CCI-779); deferolimus (formerly known as deferolimus, dimethylphosphonic acid (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentoxy-11,36-dioxa-4-azatricyclo[30.3.1.0]). 4,9 [36-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl ester, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383); everolimus ( Or RAD001); Rapamycin (AY22989, ); Simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N 2 -[1,4-dioxo-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholin-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-asparticyl-L-serine-inner salt (SF1126, CAS 936487-67-1).

[0212] Exemplary phosphoinositol 3-kinase (PI3K) inhibitors that can be used in conjunction with the tandem expression cassette compositions described herein include, but are not limited to, 4-[2-(1H-indazol-4-yl)-6-[[4-(methanesulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941 and described in PCT publications WO 09 / 036082 and WO 09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication WO 09 / 036082 and WO 09 / 055730). 06 / 122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridine-2-amine (also known as BKM120 or NVP-BKM120, and described in PCT Publication No. WO2007 / 084786); Tóuzhärtyl (VX680 or MK-0457, CAS) 639089-54-6); (5Z)-5-[[4-(4-pyridyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(acetoxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopentan[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).Exemplary protein kinase B (PKB) or AKT inhibitors include, but are not limited to, 8-[4-(1-aminocyclobutyl)benzene]-9-phenyl-1,2,4-thiazo[3,4-f][1,6]naphthyl-3(2H)-one (MK-2206, CAS 1032349-93-1); perifoxine (KRX0401); 4-dodecyl-N-1,3,4-thiadiazol-2-ylbenzenesulfonamide (PHT-427, CAS1191951-57-1); 4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-[(3S)-3-piperidinylmethoxy]-1H-imidazo[4,5-c]pyridin-4-yl]-2-methyl-3-butyn-2-ol (GSK690693, CAS 937174-76-0); 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]-6H-dibenzo[b,d]pyran-6-one (palomid 529, P529 or SG-00529); Tricirbine (6-amino-4-methyl-8-(β-D-furanribosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimidino[4,5-c]pyridazine); (αS)-α-[[[5-(3-methyl-1H-indazol-5-yl)-3-pyridyl]oxy]methyl]phenethylamine (A674563, CAS) 552325-73-2); 4-[(4-chlorophenyl)methyl]-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidineamine (CCT128930, CAS 885499-61-6); 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]-piperidine (AT7867, CAS 857531-00-1); and Archexin (RX-0201, CAS663232-27-7).

[0213] In some embodiments, the tyrosine kinase inhibitor used in combination with CER-modified cells is an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, alectinib, brigatinib, dalantercept, entrectinib, and lorlatinib.

[0214] In some embodiments where tandem expression cassette-modified cells are administered in combination with one or more other therapies, the one or more other therapies may be administered at a dose that would otherwise be considered subtherapeutic. In such embodiments, the tandem expression cassette can provide an additive or synergistic effect, allowing one or more other therapies to be administered at a lower dose. Combination therapies include administering the tandem expression cassette composition described herein before (e.g., 1 to 30 days or longer before other therapies), concurrently with (on the same day) with other therapies, or after (e.g., 1 to 30 days or longer after other therapies). In some embodiments, the tandem expression cassette-modified cells are administered after one or more other therapies. In further embodiments, the tandem expression vector-modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after one or more other therapies. In a further embodiment, the tandem expression cassette-modified cells are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after administration of one or more other therapies. When one or more other therapies involve multiple doses, the tandem expression cassette-modified cells may be administered after the initial dose of one or more other therapies, after the final dose of one or more other therapies, or between multiple doses of one or more other therapies.

[0215] In some embodiments, the methods of this disclosure include an exhaustion step. An exhaustion step can be performed after a sufficiently long period of therapeutic benefit to remove tandem expression cassette-modified cells from the subject, thereby reducing toxicity to the subject. In such embodiments, the vector containing the tandem expression cassette may contain an inducible suicide gene, such as iCASP9, inducible Fas, or HSV-TK. Similarly, the vector can be engineered to express known cell surface antigens, such as CD20 or a truncated EGFR (SEQ ID NO:82), which promotes exhaustion of transduced cells by infusion of an associated monoclonal antibody (mAb). For example, rituximab targeting CD20 or cetuximab targeting EGFR. Alemtuzumab, which targets CD52 present on the surface of mature lymphocytes, can also be used to exhaust transduced B cells, T cells, or natural killer cells.

[0216] Subjects that can be treated with the compositions and methods disclosed herein include animals such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. Subjects can be male or female, and can be of any suitable age, including infants, adolescents, teenagers, adults, and the elderly.

[0217] Example

[0218] Example 1: Construction of Tandem Expression Boxes

[0219] A polynucleotide comprising the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of TLR4 to form a chimeric phagocytic receptor “CER5” encoding the amino acid sequence shown in SEQ ID NO:97. A polynucleotide comprising the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of TLR5 to form a chimeric phagocytic receptor “CER19” encoding the amino acid sequence shown in SEQ ID NO:98. A polynucleotide comprising the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of TLR8 to form a chimeric phagocytic receptor “CER21” encoding the amino acid sequence shown in SEQ ID NO:99. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of NFAM1 to form the chimeric phagocytic receptor “CER25” encoding the amino acid sequence shown in SEQ ID NO:100. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of TLR2 to form the chimeric phagocytic receptor “CER27” encoding the amino acid sequence shown in SEQ ID NO:101. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signaling domain of Traf6 to form the chimeric phagocytic receptor “CER29” encoding the amino acid sequence shown in SEQ ID NO:102. A polynucleotide containing the extracellular domain and transmembrane domain of phosphatidylserine-binding protein Tim4 was fused with the intracellular signal transduction domain of Traf3 to form a chimeric phagocytic receptor “CER31” encoding the amino acid sequence shown in SEQ ID NO:103.

[0220] Using the sequence of the P2A self-cleaving peptide, a polynucleotide encoding the TCRβ chain and a polynucleotide encoding the TCRα chain of the HPV16 E7-specific TCR (see PCT Publication No. WO2015 / 184228) were fused. The TCR Vα domain contains the amino acid sequence shown in SEQ ID NO:94, and the TCR Vβ region contains the amino acid sequence shown in SEQ ID NO:92. The Cα domain contains a cysteine ​​substitution and LVL substitutions at positions 12, 14, and 15, and contains the amino acid sequence shown in SEQ ID NO:95. The Cβ domain also contains a cysteine ​​substitution and contains the amino acid sequence shown in SEQ ID NO:93. The encoded HPV16 E7-specific TCR contains the amino acid sequence shown in SEQ ID NO:90. The amino acid sequences of the tandem expression constructs described in this embodiment are provided in Table 2.

[0221] Table 2: Exemplary Tandem Expression Boxes

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230] The selected CER polynucleotide and HPV16 E7 TCR polynucleotide were inserted into the same pLenti lentiviral vector, with a T2A sequence (encoding the amino acid shown in SEQ ID NO:68) between them. (See also...) Figure 1A-1GPeripheral blood was collected from human donors via venipuncture, and human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using lymphocyte separation medium. CD8+ T cells were enriched from PBMCs using a commercially available isolation kit and activated in complete cell growth medium with anti-CD3 and anti-CD28 agents. 50 μl of a viral vector expressing the CER-HPV16 E7 TCR conjugate was diluted in 0.5 ml of complete cell growth medium and added to the CD8+ T cells. The transduced CD8+ T cells were then centrifuged at 270 x g rpm for 1 h in a preheated centrifuge at 32 °C. The CD8+ T cells were incubated at 37 °C for 24 h. The T cells were then further expanded in complete cell growth medium for 72 h, the beads were removed, and the cells were allowed to expand for 5 days before being used for functional assays.

[0231] Example 2: CD8 T cells transduced using CER-TCR tandem expression cassettes exhibit antigen-specific cell lysis. and phagocytic activity

[0232] Using caspase 3 / 7 apoptosis reagent The cytotoxic activity of CD8+ T cells transduced with the tandem expression cassette was assessed by conjugating an activated caspase 3 / 7 recognition motif to a red reagent that fluoresces upon lysis. Fluorescence signals were measured using fluorescence microscopy. Transduced CD8+ T cells were co-cultured with HPV16 E7+ head and neck squamous cell carcinoma cells (SCC152) at a 1:1 ratio, and the caspase 3 / 7 apoptosis reagent was added to the co-culture. CD8+ T cells containing the CER21-HPV16 E7 TCR tandem expression cassette exhibited cytotoxic activity against SCC152 cells (see [link to article]). Figure 2 At 6 hours, the cytotoxic response of CD8+ T cells transduced with the CER21-HPV16 E7 TCR tandem expression cassette appeared to be exponentially higher than that of CD8+ T cells containing only the HPV16 E7 TCR (see [link to relevant documentation]). Figure 3 CD8+ T cells transduced with CER21-HPV16 E7 TCR tandem expression cassette, CER29-HPV16 E7 TCR tandem expression cassette, or CER31-HPV16 E7 TCR tandem expression cassette were co-cultured with SCC152 cells at a target cell to effector cell ratio of 1:1. Caspase 3 / 7 apoptosis reagent was added to the co-culture, and cytotoxic activity was measured by changes in fluorescence over time (see [link to relevant documentation]). Figure 4 and Figure 5 The control samples were CD8+ T cells transduced with HPV16 E7 TCR alone or simulated transduced T cells.

[0233] tandem expression cassette-transduced CD8+ T cells were co-cultured with SCC152 cells at a 1:1 ratio for 6 hours to detect the phagocytic activity of tandem expression cassette-transduced CD8+ T cells. Phagocytic events were visualized and quantified using a KEYENCE BZ-X710 fluorescence microscope with a 20X objective and hybrid capture software. CD8+ T cells transduced with CER21-HPV16 E7 TCR, CER29-HPV16 E7 TCR, or CER31-HPV16 E7 TCR tandem cassettes were able to phagocytose SCC152 cells (see [link to documentation]). Figure 6 and 7 The Rac1 inhibitor NSC23766 (50 μM) was also added to the co-culture experiment, and in vitro phagocytosis was measured. After treatment with the Rac1 inhibitor, it was found that T cells transduced by CER21-HPV16 E7 TCR, CER29-HPV16 E7 TCR, or CER31-HPV16 E7 TCR phagocytosed SCC152 cells in a Rac1-dependent manner (see [link to Rac1 inhibitor]). Figure 8-10 ). Figure 11 and Figure 12 The results showed that CD8+ T cells transduced with the CER21-HPV16 E7TCR tandem expression cassette engulfed streptavidin-coated latex beads, which were coated with biotin-conjugated phosphatidylserine. After incubation for approximately 30 minutes, phosphatidylserine-coated beads were visible inside the CER21-HPV16 E7 TCR+ T cells.

[0234] During co-culture experiments with SCC152 cells, cytokine responses in CD8+ T cells transduced using the CER21-HPV16 E7 TCR tandem expression cassette were measured by sampling of cell supernatant, demonstrating that CER21-HPV16 E7 TCR+ T cells exhibit antigen-specific effector function, as measured by IFNγ response (see [link to study]). Figure 14 ).

[0235] Example 3: Phagocytic activity of T cells induced specifically by CER

[0236] The ability of CER-modified T cells to phagocytose target cells and generalize phagocytic signaling events was evaluated in a co-culture assay. T cells were transduced with nucleic acid encoding an HPV E7-specific TCR (containing the polypeptide sequence of SEQ ID NO:158) or a tandem expression cassette encoding an HPV E7-specific TCR+CER29 (containing the polypeptide sequence of SEQ ID NO:169). pHrodo-labeled HPV+SCC152 head and neck cancer cells were co-cultured with simulated transduced T cells, HPV E7 TCR-transduced T cells, or HPV E7 TCR / CER29-transduced T cells. Phagocytosis was analyzed by FACS, and the results showed a large number of trace amounts of purple-labeled E7 TCR / CER29-T cells with pHrodo-positive double staining (see [link to FACS]). Figure 15A Quantitative analysis of FACS data showed no difference between simulated transduced T cells and E7 TCR transduced T cells (see [link to FACS data]). Figure 15B ).

[0237] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the application data sheets, including but not limited to U.S. Provisional Patent Application No. 62 / 649,499, filed March 28, 2018, are incorporated herein by reference in their entirety. Various aspects of the embodiments may be modified if it is necessary to employ concepts from various patents, applications, and publications to provide other embodiments.

[0238] These and other changes can be made to the embodiments based on the detailed description above. Generally, the terminology used in the claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of equivalents enjoyed by these claims. Therefore, the claims are not limited by the disclosure. sequence list <110> Shinro Therapeutic Company D.M. Corey I. Ibarra <120> Chimeric phagocytic receptor expression vectors, genetically modified host cells, and their applications <130> 200265.406WO <140> PCT <141> 2019-03-27 <150> US 62 / 649,499 <151> 2018-03-28 <160> 195 <170> FastSEQ, version 4.0 for Windows <210> 1 <211> 13 <212> PRT <213> Homo sapiens <220> <223> TLR4 near-membrane domain <400> 1 Pro Val Leu Ser Leu Asn Ile Thr Cys Gln Met Asn Lys 1 5 10 <210> 2 <211> 46 <212> PRT <213> Homo sapiens <220> <223> MRC1 signal conduction domain <400> 2 Tyr Lys Lys Arg Arg Val His Leu Pro Gln Glu Gly Ala Phe Glu Asn 1 5 10 15 Thr Leu Tyr Phe Asn Ser Gln Ser Ser Pro Gly Thr Ser Asp Met Lys 20 25 30 Asp Leu Val Gly Asn Ile Glu Gln Asn Glu His Ser Val Ile 35 40 45 <210> 3 <211> 473 <212> PRT <213> Homo sapiens <220> <223> MERTK signal conduction domain <400> 3 Lys Arg Val Gln Glu Thr Lys Phe Gly Asn Ala Phe Thr Glu Glu Asp 1 5 10 15 Ser Glu Leu Val Val Asn Tyr Ile Ala Lys Lys Ser Phe Cys Arg Arg 20 25 30 Ala Ile Glu Leu Thr Leu His Ser Leu Gly Val Ser Glu Glu Leu Gln 35 40 45 Asn Lys Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu Ile Leu Gly 50 55 60 Lys Ile Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu Gly Asn Leu 65 70 75 80 Lys Gln Glu Asp Gly Thr Ser Leu Lys Val Ala Val Lys Thr Met Lys 85 90 95 Leu Asp Asn Ser Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala 100 105 110 Ala Cys Met Lys Asp Phe Ser His Pro Asn Val Ile Arg Leu Leu Gly 115 120 125 Val Cys Ile Glu Met Ser Ser Gln Gly Ile Pro Lys Pro Met Val Ile 130 135 140 Leu Pro Phe Met Lys Tyr Gly Asp Leu His Thr Tyr Leu Leu Tyr Ser 145 150 155 160 Arg Leu Glu Thr Gly Pro Lys His Ile Pro Leu Gln Thr Leu Leu Lys 165 170 175 Phe Met Val Asp Ile Ala Leu Gly Met Glu Tyr Leu Ser Asn Arg Asn 180 185 190 Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Arg Asp Asp 195 200 205 Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Ser 210 215 220 Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp 225 230 235 240 Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp 245 250 255 Val Trp Ala Phe Gly Val Thr Met Trp Glu Ile Ala Thr Arg Gly Met 260 265 270 Thr Pro Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp Tyr Leu Leu 275 280 285 His Gly His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp Glu Leu Tyr 290 295 300 Glu Ile Met Tyr Ser Cys Trp Arg Thr Asp Pro Leu Asp Arg Pro Thr 305 310 315 320 Phe Ser Val Leu Arg Leu Gln Leu Glu Lys Leu Leu Glu Ser Leu Pro 325 330 335 Asp Val Arg Asn Gln Ala Asp Val Ile Tyr Val Asn Thr Gln Leu Leu 340 345 350 Glu Ser Ser Glu Gly Leu Ala Gln Gly Ser Thr Leu Ala Pro Leu Asp 355 360 365 Leu Asn Ile Asp Pro Asp Ser Ile Ile Ala Ser Cys Thr Pro Arg Ala 370 375 380 Ala Ile Ser Val Val Thr Ala Glu Val His Asp Ser Lys Pro His Glu 385 390 395 400 Gly Arg Tyr Ile Leu Asn Gly Gly Ser Glu Glu Trp Glu Asp Leu Thr 405 410 415 Ser Ala Pro Ser Ala Ala Val Thr Ala Glu Lys Asn Ser Val Leu Pro 420 425 430 Gly Glu Arg Leu Val Arg Asn Gly Val Ser Trp Ser His Ser Ser Met 435 440 445 Leu Pro Leu Gly Ser Ser Leu Pro Asp Glu Leu Leu Phe Ala Asp Asp 450 455 460 Ser Ser Glu Gly Ser Glu Val Leu Met 465 470 <210> 4 <211> 476 <212> PRT <213> Mus musculus <220> <223> MERTK signaling domain <400> 4 Ala Leu Arg Arg Arg Val Gln Glu Thr Lys Phe Gly Gly Ala Phe Ser 1 5 10 15 Glu Glu Asp Ser Gln Leu Val Val Asn Tyr Arg Ala Lys Lys Ser Phe 20 25 30 Cys Arg Arg Ala Ile Glu Leu Thr Leu Gln Ser Leu Gly Val Ser Glu 35 40 45 Glu Leu Gln Asn Lys Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu 50 55 60 Val Leu Gly Lys Val Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu 65 70 75 80 Gly Asn Leu Lys Gln Glu Asp Gly Thr Ser Gln Lys Val Ala Val Lys 85 90 95 Thr Met Lys Leu Asp Asn Phe Ser Gln Arg Glu Ile Glu Glu Phe Leu 100 105 110 Ser Glu Ala Ala Cys Met Lys Asp Phe Asn His Pro Asn Val Ile Arg 115 120 125 Leu Leu Gly Val Cys Ile Glu Leu Ser Ser Gln Gly Ile Pro Lys Pro 130 135 140 Met Val Ile Leu Pro Phe Met Lys Tyr Gly Asp Leu His Thr Phe Leu 145 150 155 160 Leu Tyr Ser Arg Leu Asn Thr Gly Pro Lys Tyr Ile His Leu Gln Thr 165 170 175 Leu Leu Lys Phe Met Met Asp Ile Ala Gln Gly Met Glu Tyr Leu Ser 180 185 190 Asn Arg Asn Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu 195 200 205 Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys 210 215 220 Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro 225 230 235 240 Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser 245 250 255 Lys Ser Asp Val Trp Ala Phe Gly Val Thr Met Trp Glu Ile Thr Thr 260 265 270 Arg Gly Met Thr Pro Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp 275 280 285 Tyr Leu Leu His Gly His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp 290 295 300 Glu Leu Tyr Asp Ile Met Tyr Ser Cys Trp Ser Ala Asp Pro Leu Asp 305 310 315 320 Arg Pro Thr Phe Ser Val Leu Arg Leu Gln Leu Glu Lys Leu Ser Glu 325 330 335 Ser Leu Pro Asp Ala Gln Asp Lys Glu Ser Ile Ile Tyr Ile Asn Thr 340 345 350 Gln Leu Leu Glu Ser Cys Glu Gly Ile Ala Asn Gly Pro Ser Leu Thr 355 360 365 Gly Leu Asp Met Asn Ile Asp Pro Asp Ser Ile Ile Ala Ser Cys Thr 370 375 380 Pro Gly Ala Ala Val Ser Val Val Thr Ala Glu Val His Glu Asn Asn 385 390 395 400 Leu Arg Glu Glu Arg Tyr Ile Leu Asn Gly Gly Asn Glu Glu Trp Glu 405 410 415 Asp Val Ser Ser Thr Pro Phe Ala Ala Val Thr Pro Glu Lys Asp Gly 420 425 430 Val Leu Pro Glu Asp Arg Leu Thr Lys Asn Gly Val Ser Trp Ser His 435 440 445 His Ser Thr Leu Pro Leu Gly Ser Pro Ser Pro Asp Glu Leu Leu Phe 450 455 460 Val Asp Asp Ser Leu Glu Asp Ser Glu Val Leu Met 465 470 475 <210> 5 <211> 440 <212> PRT <213> Homo sapiens <220> <223> Tyro3 signaling domain <400> 5 Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe Asp Ser 1 5 10 15 Val Met Ala Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala Arg Ser 20 25 30 Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu Asp Ser Leu 35 40 45 Gly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu Ile Pro 50 55 60 Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu Phe Gly 65 70 75 80 Ser Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe Val Lys 85 90 95 Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser Asp Ile 100 105 110 Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp His Pro 115 120 125 His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala Lys Gly 130 135 140 Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His Gly Asp 145 150 155 160 Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu Asn Pro Phe Asn 165 170 175 Leu Pro Leu Gln Thr Leu Ile Arg Phe Met Val Asp Ile Ala Cys Gly 180 185 190 Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile His Arg Asp Leu Ala Ala 195 200 205 Arg Asn Cys Met Leu Ala Glu Asp Met Thr Val Cys Val Ala Asp Phe 210 215 220 Gly Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Cys 225 230 235 240 Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu Ala Asp 245 250 255 Asn Leu Tyr Thr Val Gln Ser Asp Val Trp Ala Phe Gly Val Thr Met 260 265 270 Trp Glu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly Ile Glu Asn 275 280 285 Ala Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys Gln Pro 290 295 300 Pro Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gln Cys Trp Ser 305 310 315 320 Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met Glu Leu 325 330 335 Glu Asn Ile Leu Gly Gln Leu Ser Val Leu Ser Ala Ser Gln Asp Pro 340 345 350 Leu Tyr Ile Asn Ile Glu Arg Ala Glu Glu Pro Thr Ala Gly Gly Ser 355 360 365 Leu Glu Leu Pro Gly Arg Asp Gln Pro Tyr Ser Gly Ala Gly Asp Gly 370 375 380 Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp Cys Arg Tyr Ile 385 390 395 400 Leu Thr Pro Gly Gly Leu Ala Glu Gln Pro Gly Gln Ala Glu His Gln 405 410 415 Pro Glu Ser Pro Leu Asn Glu Thr Gln Arg Leu Leu Leu Leu Gln Gln 420 425 430 Gly Leu Leu Pro His Ser Ser Cys 435 440 <210> 6 <211> 422 <212> PRT <213> Homo sapiens <220> <223> Axl signaling domain <400> 6 His Arg Arg Lys Lys Glu Thr Arg Tyr Gly Glu Val Phe Glu Pro Thr 1 5 10 15 Val Glu Arg Gly Glu Leu Val Val Arg Tyr Arg Val Arg Lys Ser Tyr 20 25 30 Ser Arg Arg Thr Thr Glu Ala Thr Leu Asn Ser Leu Gly Ile Ser Glu 35 40 45 Glu Leu Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His Lys Val 50 55 60 Ala Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe Gly Ala Val Met Glu 65 70 75 80 Gly Gln Leu Asn Gln Asp Asp Ser Ile Leu Lys Val Ala Val Lys Thr 85 90 95 Met Lys Ile Ala Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe Leu Ser 100 105 110 Glu Ala Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met Arg Leu 115 120 125 Ile Gly Val Cys Phe Gln Gly Ser Glu Arg Glu Ser Phe Pro Ala Pro 130 135 140 Val Val Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe Leu 145 150 155 160 Leu Tyr Ser Arg Leu Gly Asp Gln Pro Val Tyr Leu Pro Thr Gln Met 165 170 175 Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr Leu Ser 180 185 190 Thr Lys Arg Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu 195 200 205 Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys 210 215 220 Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro 225 230 235 240 Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser 245 250 255 Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met Trp Glu Ile Ala Thr 260 265 270 Arg Gly Gln Thr Pro Tyr Pro Gly Val Glu Asn Ser Glu Ile Tyr Asp 275 280 285 Tyr Leu Arg Gln Gly Asn Arg Leu Lys Gln Pro Ala Asp Cys Leu Asp 290 295 300 Gly Leu Tyr Ala Leu Met Ser Arg Cys Trp Glu Leu Asn Pro Gln Asp 305 310 315 320 Arg Pro Ser Phe Thr Glu Leu Arg Glu Asp Leu Glu Asn Thr Leu Lys 325 330 335 Ala Leu Pro Pro Ala Gln Glu Pro Asp Glu Ile Leu Tyr Val Asn Met 340 345 350 Asp Glu Gly Gly Gly Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly Ala 355 360 365 Asp Pro Pro Thr Gln Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu Thr 370 375 380 Ala Ala Glu Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser Thr 385 390 395 400 Thr Pro Ser Pro Ala Gln Pro Ala Asp Arg Gly Ser Pro Ala Ala Pro 405 410 415 Gly Gln Glu Asp Gly Ala 420 <210> 7 <211> 727 <212> PRT <213> Homo sapiens <220> <223> ELMO signal transmission structure <400> 7 Met Pro Pro Pro Ala Asp Ile Val Lys Val Ala Ile Glu Trp Pro Gly 1 5 10 15 Ala Tyr Pro Lys Leu Met Glu Ile Asp Gln Lys Lys Pro Leu Ser Ala 20 25 30 Ile Ile Lys Glu Val Cys Asp Gly Trp Ser Leu Ala Asn His Glu Tyr 35 40 45 Phe Ala Leu Gln His Ala Asp Ser Ser Asn Phe Tyr Ile Thr Glu Lys 50 55 60 Asn Arg Asn Glu Ile Lys Asn Gly Thr Ile Leu Arg Leu Thr Thr Ser 65 70 75 80 Pro Ala Gln Asn Ala Gln Gln Leu His Glu Arg Ile Gln Ser Ser Ser 85 90 95 Met Asp Ala Lys Leu Glu Ala Leu Lys Asp Leu Ala Ser Leu Ser Arg 100 105 110 Asp Val Thr Phe Ala Gln Glu Phe Ile Asn Leu Asp Gly Ile Ser Leu 115 120 125 Leu Thr Gln Met Val Glu Ser Gly Thr Glu Arg Tyr Gln Lys Leu Gln 130 135 140 Lys Ile Met Lys Pro Cys Phe Gly Asp Met Leu Ser Phe Thr Leu Thr 145 150 155 160 Ala Phe Val Glu Leu Met Asp His Gly Ile Val Ser Trp Asp Thr Phe 165 170 175 Ser Val Ala Phe Ile Lys Lys Ile Ala Ser Phe Val Asn Lys Ser Ala 180 185 190 Ile Asp Ile Ser Ile Leu Gln Arg Ser Leu Ala Ile Leu Glu Ser Met 195 200 205 Val Leu Asn Ser His Asp Leu Tyr Gln Lys Val Ala Gln Glu Ile Thr 210 215 220 Ile Gly Gln Leu Ile Pro His Leu Gln Gly Ser Asp Gln Glu Ile Gln 225 230 235 240 Thr Tyr Thr Ile Ala Val Ile Asn Ala Leu Phe Leu Lys Ala Pro Asp 245 250 255 Glu Arg Arg Gln Glu Met Ala Asn Ile Leu Ala Gln Lys Gln Leu Arg 260 265 270 Ser Ile Ile Leu Thr His Val Ile Arg Ala Gln Arg Ala Ile Asn Asn 275 280 285 Glu Met Ala His Gln Leu Tyr Val Leu Gln Val Leu Thr Phe Asn Leu 290 295 300 Leu Glu Asp Arg Met Met Thr Lys Met Asp Pro Gln Asp Gln Ala Gln 305 310 315 320 Arg Asp Ile Ile Phe Glu Leu Arg Arg Ile Ala Phe Asp Ala Glu Ser 325 330 335 Glu Pro Asn Asn Ser Ser Gly Ser Met Glu Lys Arg Lys Ser Met Tyr 340 345 350 Thr Arg Asp Tyr Lys Lys Leu Gly Phe Ile Asn His Val Asn Pro Ala 355 360 365 Met Asp Phe Thr Gln Thr Pro Pro Gly Met Leu Ala Leu Asp Asn Met 370 375 380 Leu Tyr Phe Ala Lys His His Gln Asp Ala Tyr Ile Arg Ile Val Leu 385 390 395 400 Glu Asn Ser Ser Arg Glu Asp Lys His Glu Cys Pro Phe Gly Arg Ser 405 410 415 Ser Ile Glu Leu Thr Lys Met Leu Cys Glu Ile Leu Lys Val Gly Glu 420 425 430 Leu Pro Ser Glu Thr Cys Asn Asp Phe His Pro Met Phe Phe Thr His 435 440 445 Asp Arg Ser Phe Glu Glu Phe Phe Cys Ile Cys Ile Gln Leu Leu Asn 450 455 460 Lys Thr Trp Lys Glu Met Arg Ala Thr Ser Glu Asp Phe Asn Lys Val 465 470 475 480 Met Gln Val Val Lys Glu Gln Val Met Arg Ala Leu Thr Thr Lys Pro 485 490 495 Ser Ser Leu Asp Gln Phe Lys Ser Lys Leu Gln Asn Leu Ser Tyr Thr 500 505 510 Glu Ile Leu Lys Ile Arg Gln Ser Glu Arg Met Asn Gln Glu Asp Phe 515 520 525 Gln Ser Arg Pro Ile Leu Glu Leu Lys Glu Lys Ile Gln Pro Glu Ile 530 535 540 Leu Glu Leu Ile Lys Gln Gln Arg Leu Asn Arg Leu Val Glu Gly Thr 545 550 555 560 Cys Phe Arg Lys Leu Asn Ala Arg Arg Arg Gln Asp Lys Phe Trp Tyr 565 570 575 Cys Arg Leu Ser Pro Asn His Lys Val Leu His Tyr Gly Asp Leu Glu 580 585 590 Glu Ser Pro Gln Gly Glu Val Pro His Asp Ser Leu Gln Asp Lys Leu 595 600 605 Pro Val Ala Asp Ile Lys Ala Val Val Thr Gly Lys Asp Cys Pro His 610 615 620 Met Lys Glu Lys Gly Ala Leu Lys Gln Asn Lys Glu Val Leu Glu Leu 625 630 635 640 Ala Phe Ser Ile Leu Tyr Asp Ser Asn Cys Gln Leu Asn Phe Ile Ala 645 650 655 Pro Asp Lys His Glu Tyr Cys Ile Trp Thr Asp Gly Leu Asn Ala Leu 660 665 670 Leu Gly Lys Asp Met Met Ser Asp Leu Thr Arg Asn Asp Leu Asp Thr 675 680 685 Leu Leu Ser Met Glu Ile Lys Leu Arg Leu Leu Asp Leu Glu Asn Ile 690 695 700 Gln Ile Pro Asp Ala Pro Pro Pro Ile Pro Lys Glu Pro Ser Asn Tyr 705 710 715 720 Asp Phe Val Tyr Asp Cys Asn 725 <210> 8 <211> 522 <212> PRT <213> Homo sapiens <220> <223> Traf6 signaling domain - full length <400> 8 Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu 1 5 10 15 Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys 20 25 30 Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser 35 40 45 Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu 50 55 60 Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala 65 70 75 80 Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys 85 90 95 Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu 100 105 110 Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu 115 120 125 Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu 130 135 140 Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met 145 150 155 160 Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile 165 170 175 His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys 180 185 190 Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys 195 200 205 Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg 210 215 220 Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile 225 230 235 240 Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn 245 250 255 His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met 260 265 270 Leu Ala Gln Ala Val His Ser Leu Ser Val Ile Pro Asp Ser Gly Tyr 275 280 285 Ile Ser Glu Val Arg Asn Phe Gln Glu Thr Ile His Gln Leu Glu Gly 290 295 300 Arg Leu Val Arg Gln Asp His Gln Ile Arg Glu Leu Thr Ala Lys Met 305 310 315 320 Glu Thr Gln Ser Met Tyr Val Ser Glu Leu Lys Arg Thr Ile Arg Thr 325 330 335 Leu Glu Asp Lys Val Ala Glu Ile Glu Ala Gln Gln Cys Asn Gly Ile 340 345 350 Tyr Ile Trp Lys Ile Gly Asn Phe Gly Met His Leu Lys Cys Gln Glu 355 360 365 Glu Glu Lys Pro Val Val Ile His Ser Pro Gly Phe Tyr Thr Gly Lys 370 375 380 Pro Gly Tyr Lys Leu Cys Met Arg Leu His Leu Gln Leu Pro Thr Ala 385 390 395 400 Gln Arg Cys Ala Asn Tyr Ile Ser Leu Phe Val His Thr Met Gln Gly 405 410 415 Glu Tyr Asp Ser His Leu Pro Trp Pro Phe Gln Gly Thr Ile Arg Leu 420 425 430 Thr Ile Leu Asp Gln Ser Glu Ala Pro Val Arg Gln Asn His Glu Glu 435 440 445 Ile Met Asp Ala Lys Pro Glu Leu Leu Ala Phe Gln Arg Pro Thr Ile 450 455 460 Pro Arg Asn Pro Lys Gly Phe Gly Tyr Val Thr Phe Met His Leu Glu 465 470 475 480 Ala Leu Arg Gln Arg Thr Phe Ile Lys Asp Asp Thr Leu Leu Val Arg 485 490 495 Cys Glu Val Ser Thr Arg Phe Asp Met Gly Ser Leu Arg Arg Glu Gly 500 505 510 Phe Gln Pro Arg Ser Thr Asp Ala Gly Val 515 520 <210> 9 <211> 261 <212> PRT <213> Homo sapiens <220> <223> Syk signaling domain <400> 9 Thr Leu Glu Asp Lys Glu Leu Gly Ser Gly Asn Phe Gly Thr Val Lys 1 5 10 15 Lys Gly Tyr Tyr Gln Met Lys Lys Val Val Lys Thr Val Ala Val Lys 20 25 30 Ile Leu Lys Asn Glu Ala Asn Asp Pro Ala Leu Lys Asp Glu Leu Leu 35 40 45 Ala Glu Ala Asn Val Met Gln Gln Leu Asp Asn Pro Tyr Ile Val Arg 50 55 60 Met Ile Gly Ile Cys Glu Ala Glu Ser Trp Met Leu Val Met Glu Met 65 70 75 80 Ala Glu Leu Gly Pro Leu Asn Lys Tyr Leu Gln Gln Asn Arg His Val 85 90 95 Lys Asp Lys Asn Ile Ile Glu Leu Val His Gln Val Ser Met Gly Met 100 105 110 Lys Tyr Leu Glu Glu Ser Asn Phe Val His Arg Asp Leu Ala Ala Arg 115 120 125 Asn Val Leu Leu Val Thr Gln His Tyr Ala Lys Ile Ser Asp Phe Gly 130 135 140 Leu Ser Lys Ala Leu Arg Ala Asp Glu Asn Tyr Tyr Lys Ala Gln Thr 145 150 155 160 His Gly Lys Trp Pro Val Lys Trp Tyr Ala Pro Glu Cys Ile Asn Tyr 165 170 175 Tyr Lys Phe Ser Ser Lys Ser Asp Val Trp Ser Phe Gly Val Leu Met 180 185 190 Trp Glu Ala Phe Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met Lys Gly 195 200 205 Ser Glu Val Thr Ala Met Leu Glu Lys Gly Glu Arg Met Gly Cys Pro 210 215 220 Ala Gly Cys Pro Arg Glu Met Tyr Asp Leu Met Asn Leu Cys Trp Thr 225 230 235 240 Tyr Asp Val Glu Asn Arg Pro Gly Phe Ala Ala Val Glu Leu Arg Leu 245 250 255 Arg Asn Tyr Tyr Tyr 260 <210> 10 <211> 296 <212> PRT <213> Homo sapiens <220> <223> MyD88 signaling domain <400> 10 Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser 1 5 10 15 Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu 50 55 60 Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly 65 70 75 80 Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu 85 90 95 Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp 100 105 110 Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro 115 120 125 Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg 145 150 155 160 Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln 165 170 175 Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys 180 185 190 Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala 195 200 205 Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser 210 215 220 Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala 225 230 235 240 Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys 245 250 255 Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr 260 265 270 Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg 275 280 285 Leu Ala Lys Ala Leu Ser Leu Pro 290 295 <210> 11 <400> 11 000 <210> 12 <211> 42 <212> PRT <213> Homo sapiens <220> <223> FcεR1γ signal conduction domain <400> 12 Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys 1 5 10 15 Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr 20 25 30 Glu Thr Leu Lys His Glu Lys Pro Pro Gln 35 40 <210> 13 <211> 61 <212> PRT <213> Homo sapiens <220> <223> FcγR1 signal conduction domain <400> 13 Arg Lys Glu Leu Lys Arg Lys Lys Lys Trp Asp Leu Glu Ile Ser Leu 1 5 10 15 Asp Ser Gly His Glu Lys Lys Val Ile Ser Ser Leu Gln Glu Asp Arg 20 25 30 His Leu Glu Glu Glu Leu Lys Cys Gln Glu Gln Lys Glu Glu Gln Leu 35 40 45 Gln Glu Gly Val His Arg Lys Glu Pro Gln Gly Ala Thr 50 55 60 <210> 14 <211> 77 <212> PRT <213> Homo sapiens <220> <223> FcγR2A signal conduction domain <400> 14 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 1 5 10 15 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 20 25 30 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 35 40 45 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 50 55 60 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 65 70 75 <210> 15 <211> 77 <212> PRT <213> Homo sapiens <220> <223> FcγR2c signal conduction domain <400> 15 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 1 5 10 15 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 20 25 30 Gln Pro Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 35 40 45 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 50 55 60 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 65 70 75 <210> 16 <211> 25 <212> PRT <213> Homo sapiens <220> <223> FcγR3A signal conduction domain <400> 16 Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe 1 5 10 15 Lys Trp Arg Lys Asp Pro Gln Asp Lys 20 25 <210> 17 <211> 85 <212> PRT <213> Homo sapiens <220> <223> BAFF-R signal conduction domain <400> 17 Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser Ser Ala Glu 1 5 10 15 Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp Lys Val Ile 20 25 30 Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala Trp Pro Pro 35 40 45 Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His Ser Val Pro Val 50 55 60 Pro Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr Lys Thr Ala 65 70 75 80 Gly Pro Glu Gln Gln 85 <210> 18 <211> 52 <212> PRT <213> Homo sapiens <220> <223> DAP12 signal conduction domain <400> 18 Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala 1 5 10 15 Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu 20 25 30 Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg 35 40 45 Pro Tyr Tyr Lys 50 <210> 19 <211> 86 <212> PRT <213> Homo sapiens <220> <223> NFAM1 signal transduction domain <400> 19 Leu Trp Asn Lys Lys Arg Met Arg Gly Pro Gly Lys Asp Pro Thr Arg 1 5 10 15 Lys Cys Pro Asp Pro Arg Ser Ala Ser Ser Pro Lys Gln His Pro Ser 20 25 30 Glu Ser Val Tyr Thr Ala Leu Gln Arg Arg Glu Thr Glu Val Tyr Ala 35 40 45 Cys Ile Glu Asn Glu Asp Gly Ser Ser Pro Thr Ala Lys Gln Ser Pro 50 55 60 Leu Ser Gln Glu Arg Pro His Arg Phe Glu Asp Asp Gly Glu Leu Asn 65 70 75 80 Leu Val Tyr Glu Asn Leu 85 <210> 20 <211> 29 <212> PRT <213> Homo sapiens <220> <223> CD79b signal transduction domain (amino acids 185-229) <400> 20 Asp Ser Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp 1 5 10 15 Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile Val Thr Leu 20 25 <210> twenty one <211> 45 <212> PRT <213> Homo sapiens <220> <223> CD79b signal transduction domain (amino acids 185-213) <400> twenty one Asp Ser Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp 1 5 10 15 Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile Val Thr Leu Arg Thr Gly 20 25 30 Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln Glu 35 40 45 <210> twenty two <211> 185 <212> PRT <213> Homo sapiens <220> <223> TLR1 signal conduction domain <400> twenty two Ser Tyr Leu Asp Leu Pro Trp Tyr Leu Arg Met Val Cys Gln Trp Thr 1 5 10 15 Gln Thr Arg Arg Arg Ala Arg Asn Ile Pro Leu Glu Glu Leu Gln Arg 20 25 30 Asn Leu Gln Phe His Ala Phe Ile Ser Tyr Ser Gly His Asp Ser Phe 35 40 45 Trp Val Lys Asn Glu Leu Leu Pro Asn Leu Glu Lys Glu Gly Met Gln 50 55 60 Ile Cys Leu His Glu Arg Asn Phe Val Pro Gly Lys Ser Ile Val Glu 65 70 75 80 Asn Ile Ile Thr Cys Ile Glu Lys Ser Tyr Lys Ser Ile Phe Val Leu 85 90 95 Ser Pro Asn Phe Val Gln Ser Glu Trp Cys His Tyr Glu Leu Tyr Phe 100 105 110 Ala His His Asn Leu Phe His Glu Gly Ser Asn Ser Leu Ile Leu Ile 115 120 125 Leu Leu Glu Pro Ile Pro Gln Tyr Ser Ile Pro Ser Ser Tyr His Lys 130 135 140 Leu Lys Ser Leu Met Ala Arg Arg Thr Tyr Leu Glu Trp Pro Lys Glu 145 150 155 160 Lys Ser Lys Arg Gly Leu Phe Trp Ala Asn Leu Arg Ala Ala Ile Asn 165 170 175 Ile Lys Leu Thr Glu Gln Ala Lys Lys 180 185 <210> 23 <211> 175 <212> PRT <213> Homo sapiens <220> <223> TLR2 signaling domain <400> 23 His Arg Phe His Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu 1 5 10 15 Gln Ala Lys Arg Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr 20 25 30 Asp Ala Phe Val Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn 35 40 45 Leu Met Val Gln Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys 50 55 60 Leu His Lys Arg Asp Phe Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile 65 70 75 80 Ile Asp Ser Ile Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu 85 90 95 Asn Phe Val Lys Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His 100 105 110 Phe Arg Leu Phe Asp Glu Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu 115 120 125 Glu Pro Ile Glu Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg 130 135 140 Lys Ile Met Asn Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala 145 150 155 160 Gln Arg Glu Gly Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser 165 170 175 <210> 24 <211> 179 <212> PRT <213> Homo sapiens <220> <223> TLR3 doesn't work. <400> 24 Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val Ser Val His Arg Val 1 5 10 15 Leu Gly Phe Lys Glu Ile Asp Arg Gln Thr Glu Gln Phe Glu Tyr Ala 20 25 30 Only Tyr Ile Ile His Only Tyr Lys Asp Lys Asp Trp Val Trp Glu His 35 40 45 Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu Lys Phe Cys Leu Glu 50 55 60 Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu Glu Ala Ile Val Asn 65 70 75 80 Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val Ile Thr His His Leu 85 90 95 Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val His Ala Val Gln 100 105 110 Gln Ala With Glu Gln Asn With Asp Ser With With Val Phe With Glu 115 120 125 Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Cys Leu Arg Arg Gly 130 135 140 Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro Val Gln Lys Glu Arg 145 150 155 160 Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala Leu Gly Ser Lys Asn 165 170 175 Ser Val His <210> 25 <211> 187 <212> PRT <213> Homo sapiens <220> <223> TLR4 signaling domain[[ID=二十一]] [[ID=二十二]]<400> 25[[ID=二十三]] [[ID=二十四]]Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly[[ID=二十五]] [[ID=二十六]]1 5 10 15[[ID=二十七]] [[ID=二十八]]Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp[[ID=二十九]] [[ID=三十]]20 25 30[[ID=三十一]] [[ID=三十二]]Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val[[ID=三十三]] [[ID=三十四]]35 40 45[[ID=三十五]] [[ID=三十六]]Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val[[ID=三十七]] [[ID=三十八]]50 55 60[[ID=三十九]] [[ID=四十]]Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys[[ID=四十一]] [[ID=四十二]]65 70 75 80[[ID=四十三]] [[ID=四十四]]Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile[[ID=四十五]] [[ID=四十六]]85 90 95[[ID=四十七]] It should be noted that there is an error in the translation of "<213> 智人 ", which should be "Homo sapiens" instead of "智人". And in the translation process, the numbers and tags are kept as they are according to the requirements. Also, the text "[[ID=二十一]] " etc. seems to have some incorrect numbering in the original, but the translation follows the rules of keeping the tags and text as is.Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala 100 105 110 Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg 115 120 125 Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu 130 135 140 Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg 145 150 155 160 Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly 165 170 175 Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile 180 185 <210> 26 <211> 198 <212> PRT <213> Homo sapiens <220> <223> TLR5 signaling domain <400> 26 Thr Lys Phe Arg Gly Phe Cys Phe Ile Cys Tyr Lys Thr Ala Gln Arg 1 5 10 15 Leu Val Phe Lys Asp His Pro Gln Gly Thr Glu Pro Asp Met Tyr Lys 20 25 30 Tyr Asp Ala Tyr Leu Cys Phe Ser Ser Lys Asp Phe Thr Trp Val Gln 35 40 45 Asn Ala Leu Leu Lys His Leu Asp Thr Gln Tyr Ser Asp Gln Asn Arg 50 55 60 Phe Asn Leu Cys Phe Glu Glu Arg Asp Phe Val Pro Gly Glu Asn Arg 65 70 75 80 Ile Ala Asn Ile Gln Asp Ala Ile Trp Asn Ser Arg Lys Ile Val Cys 85 90 95 Leu Val Ser Arg His Phe Leu Arg Asp Gly Trp Cys Leu Glu Ala Phe 100 105 110 Ser Tyr Ala Gln Gly Arg Cys Leu Ser Asp Leu Asn Ser Ala Leu Ile 115 120 125 Met Val Val Val Gly Ser Leu Ser Gln Tyr Gln Leu Met Lys His Gln 130 135 140 Ser Ile Arg Gly Phe Val Gln Lys Gln Gln Tyr Leu Arg Trp Pro Glu 145 150 155 160 Asp Phe Gln Asp Val Gly Trp Phe Leu His Lys Leu Ser Gln Gln Ile 165 170 175 Leu Lys Lys Glu Lys Glu Lys Lys Lys Asp Asn Asn Ile Pro Leu Gln 180 185 190 Thr Val Ala Thr Ile Ser 195 <210> 27 <211> 189 <212> PRT <213> Homo sapiens <220> <223> TLR6 signaling domain <400> 27 Tyr Leu Asp Leu Pro Trp Tyr Leu Arg Met Val Cys Gln Trp Thr Gln 1 5 10 15 Thr Arg Arg Arg Ala Arg Asn Ile Pro Leu Glu Glu Leu Gln Arg Asn 20 25 30 Leu Gln Phe His Ala Phe Ile Ser Tyr Ser Glu His Asp Ser Ala Trp 35 40 45 Val Lys Ser Glu Leu Val Pro Tyr Leu Glu Lys Glu Asp Ile Gln Ile 50 55 60 Cys Leu His Glu Arg Asn Phe Val Pro Gly Lys Ser Ile Val Glu Asn 65 70 75 80 Ile Ile Asn Cys Ile Glu Lys Ser Tyr Lys Ser Ile Phe Val Leu Ser 85 90 95 Pro Asn Phe Val Gln Ser Glu Trp Cys His Tyr Glu Leu Tyr Phe Ala 100 105 110 His His Asn Leu Phe His Glu Gly Ser Asn Asn Leu Ile Leu Ile Leu 115 120 125 Leu Glu Pro Ile Pro Gln Asn Ser Ile Pro Asn Lys Tyr His Lys Leu 130 135 140 Lys Ala Leu Met Thr Gln Arg Thr Tyr Leu Gln Trp Pro Lys Glu Lys 145 150 155 160 Ser Lys Arg Gly Leu Phe Trp Ala Asn Ile Arg Ala Ala Phe Asn Met 165 170 175 Lys Leu Thr Leu Val Thr Glu Asn Asn Asp Val Lys Ser 180 185 <210> 28 <211> 189 <212> PRT <213> Homo sapiens <220> <223> TLR7 signaling domain <400> 28 His Leu Tyr Phe Trp Asp Val Trp Tyr Ile Tyr His Phe Cys Lys Ala 1 5 10 15 Lys Ile Lys Gly Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp 20 25 30 Ala Phe Ile Val Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val 35 40 45 Leu Ala Glu Leu Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe 50 55 60 Asn Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu 65 70 75 80 Glu Asn Leu Ser Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val 85 90 95 Met Thr Asp Lys Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr 100 105 110 Leu Ser His Gln Arg Leu Met Asp Glu Lys Val Asp Val Ile Ile Leu 115 120 125 Ile Phe Leu Glu Lys Pro Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg 130 135 140 Lys Arg Leu Cys Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln 145 150 155 160 Ala His Pro Tyr Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp 165 170 175 Asn His Val Ala Tyr Ser Gln Val Phe Lys Glu Thr Val 180 185 <210> 29 <211> 193 <212> PRT <213> Homo sapiens <220> <223> TLR8 signaling domain <400> 29 His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu 1 5 10 15 Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr 20 25 30 Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp 35 40 45 Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn 50 55 60 Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile 65 70 75 80 Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe 85 90 95 Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe 100 105 110 Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile 115 120 125 Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu 130 135 140 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro 145 150 155 160 Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr 165 170 175 Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln 180 185 190 Tyr <210> 30 <211> 193 <212> PRT <213> Homo sapiens <220> <223> TLR9 signaling domain <400> 30 Gly Trp Asp Leu Trp Tyr Cys Phe His Leu Cys Leu Ala Trp Leu Pro 1 5 10 15 Trp Arg Gly Arg Gln Ser Gly Arg Asp Glu Asp Ala Leu Pro Tyr Asp 20 25 30 Ala Phe Val Val Phe Asp Lys Thr Gln Ser Ala Val Ala Asp Trp Val 35 40 45 Tyr Asn Glu Leu Arg Gly Gln Leu Glu Glu Cys Arg Gly Arg Trp Ala 50 55 60 Leu Arg Leu Cys Leu Glu Glu Arg Asp Trp Leu Pro Gly Lys Thr Leu 65 70 75 80 Phe Glu Asn Leu Trp Ala Ser Val Tyr Gly Ser Arg Lys Thr Leu Phe 85 90 95 Val Leu Ala His Thr Asp Arg Val Ser Gly Leu Leu Arg Ala Ser Phe 100 105 110 Leu Leu Ala Gln Gln Arg Leu Leu Glu Asp Arg Lys Asp Val Val Val 115 120 125 Leu Val Ile Leu Ser Pro Asp Gly Arg Arg Ser Arg Tyr Val Arg Leu 130 135 140 Arg Gln Arg Leu Cys Arg Gln Ser Val Leu Leu Trp Pro His Gln Pro 145 150 155 160 Ser Gly Gln Arg Ser Phe Trp Ala Gln Leu Gly Met Ala Leu Thr Arg 165 170 175 Asp Asn His His Phe Tyr Asn Arg Asn Phe Cys Gln Gly Pro Thr Ala 180 185 190 Glu <210> 31 <211> 303 <212> PRT <213> Homo sapiens <220> <223> TRAF2 signaling domain <400> 31 Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln 1 5 10 15 Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr 20 25 30 Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln 35 40 45 Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser 50 55 60 Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu 65 70 75 80 Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala 85 90 95 Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys 100 105 110 Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg 115 120 125 Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg 130 135 140 Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser 145 150 155 160 Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys 165 170 175 Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys 180 185 190 Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr 195 200 205 Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu 210 215 220 Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu 225 230 235 240 Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro 245 250 255 Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys 260 265 270 Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val 275 280 285 Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys 290 295 300 <210> 32 <211> 274 <212> PRT <213> Homo sapiens <220> <223> TRAF3 signaling domain <400> 32 Met Glu Ser Ser Lys Lys Met Asp Ser Pro Gly Ala Leu Gln Thr Asn 1 5 10 15 Pro Pro Leu Lys Leu His Thr Asp Arg Ser Ala Gly Thr Pro Val Phe 20 25 30 Val Pro Glu Gln Gly Gly Tyr Lys Glu Lys Phe Val Lys Thr Val Glu 35 40 45 Asp Lys Tyr Lys Glu Lys Cys His Leu Val Leu Cys Ser Pro Lys 50 55 60 Gln Thr Glu Cys Gly His Arg Phe Cys Glu Ser Cys Met Ala Ala Leu 65 70 75 80 Leu Ser Ser Ser Ser Pro Lys Cys Thr Ala Cys Gln Glu Ser Ile Val 85 90 95 Lys Asp Lys Val Phe Lys Asp Asn Cys Cys Lys Arg Glu Ile Leu Ala 100 105 110 Leu Gln With Tyr Cys Arg Asn Glu Ser Arg Gly Cys Ala Glu Gln Leu 115 120 125 Met Leu Gly His Leu Leu Val His Leu Lys Asn Asp Cys His Phe Glu 130 135 140 Glu Leu Pro Cys Val Arg Pro Asp Cys Lys Glu Lys Val Leu Arg Lys 145 150 155 160 Asp Leu Arg Asp His Val Glu Lys Ala Cys Lys Tyr Arg Glu Ala Thr 165 170 175 Cys Ser His Cys Lys Ser Gln Val Pro Met Ile Ala Leu Gln Lys His 180 185 190 Glu Asp Thr Asp Cys Pro Cys Val Val Val Ser Cys Pro His Lys Cys 195 200 205 Ser Val Gln Thr Leu Leu Arg Ser Glu Leu Ser Ala His Leu Ser Glu 210 215 220 Cys Val Asn Ala Pro Ser Thr Cys Ser Phe Lys Arg Tyr Gly Cys Val 225 230 235 240 Phe Gln Gly Thr Asn Gln Gln Ile Lys Ala His Glu Ala Ser Ser Ala 245 250 255 Val Gln His Val Asn Leu Leu Lys Glu Trp Ser Asn Ser Leu Glu Lys 260 265 270 Lys Val <210> 33 <211> 155 <212> PRT <213> Homo sapiens <220> <223> Truncated MyD88 signal conduction domain, without TIR domain <400> 33 Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser 1 5 10 15 Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu 50 55 60 Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly 65 70 75 80 Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu 85 90 95 Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp 100 105 110 Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro 115 120 125 Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly 145 150 155 <210> 34 <211> 274 <212> PRT <213> Homo sapiens <220> <223> Truncated TRAF6 signaling domain <400> 34 Met Ser Leu Leu Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu 1 5 10 15 Ser Asp Cys Cys Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys 20 25 30 Asp Asp Ser Val Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser 35 40 45 Phe Met Glu Glu Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu 50 55 60 Glu Ser Lys Tyr Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala 65 70 75 80 Val Gln Thr Pro Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys 85 90 95 Ser Ile Arg Asp Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu 100 105 110 Leu Glu Asn Gln Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu 115 120 125 Ser Leu Met Val Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu 130 135 140 Leu Arg His Leu Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met 145 150 155 160 Asp Cys Pro Gln Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile 165 170 175 His Ile Leu Lys Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys 180 185 190 Ala Ala Ser Met Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys 195 200 205 Pro Leu Ala Asn Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg 210 215 220 Glu Gln Met Pro Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile 225 230 235 240 Pro Cys Thr Phe Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn 245 250 255 His Leu Ala Arg His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met 260 265 270 Leu Ala <210> 35 <211> 29 <212> PRT <213> Homo sapiens <220> <223> Truncated NFAM1 signaling domain <400> 35 Ser Ser Pro Lys Gln His Pro Ser Glu Ser Val Tyr Thr Ala Leu Gln 1 5 10 15 Arg Arg Glu Thr Glu Val Tyr Ala Cys Ile Glu Asn Glu 20 25 <210> 36 <211> 21 <212> PRT <213> Homo sapiens <220> <223> Tim1 transmembrane domain <400> 36 Ile Tyr Ala Gly Val Cys Ile Ser Val Leu Val Leu Leu Ala Leu Leu 1 5 10 15 Gly Val Ile Ile Ala 20 <210> 37 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> Tim4 transmembrane domain <400> 37 Leu Leu Met Ile Ile Ala Pro Ser Leu Gly Phe Val Leu Phe Ala Leu 1 5 10 15 Phe Val Ala Phe Leu 20 <210> 38 <211> twenty one <212> PRT <213> House mouse <220> <223> Tim4 transmembrane domain <400> 38 Ile Leu Ile Ile Ala Cys Cys Val Gly Phe Val Leu Met Val Leu Leu 1 5 10 15 Phe Leu Ala Phe Leu 20 <210> 39 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> Tim3 transmembrane domain <400> 39 Ile Tyr Ile Gly Ala Gly Ile Cys Ala Gly Leu Ala Leu Ala Leu Ile 1 5 10 15 Phe Gly Ala Leu Ile 20 <210> 40 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> FcγR1 transmembrane domain <400> 40 Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu Val Asn Thr Val 1 5 10 15 Leu Trp Val Thr Ile 20 <210> 41 <211> twenty three <212> PRT <213> Homo sapiens <220> <223> FcγR2A transmembrane domain <400> 41 Ile Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile Val Ala 1 5 10 15 Ala Val Val Ala Leu Ile Tyr 20 <210> 42 <211> twenty three <212> PRT <213> Homo sapiens <220> <223> FcγR2B2 transmembrane domain <400> 42 Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Thr Gly Ile Ala 1 5 10 15 Val Ala Ala Ile Val Ala Ala 20 <210> 43 <211> twenty three <212> PRT <213> Homo sapiens <220> <223> FcγR2C transmembrane domain <400> 43 Ile Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala 1 5 10 15 Ala Val Val Ala Leu Ile Tyr 20 <210> 44 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> FcγR3A transmembrane domain <400> 44 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 1 5 10 15 Leu Tyr Phe Ser Val 20 <210> 45 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> FcεR1 transmembrane domain <400> 45 Leu Cys Tyr Ile Leu Asp Ala Ile Leu Phe Leu Tyr Gly Ile Val Leu 1 5 10 15 Thr Leu Leu Tyr Cys 20 <210> 46 <211> 19 <212> PRT <213> Homo sapiens <220> <223> FcαR1 transmembrane domain <400> 46 Leu Ile Arg Met Ala Val Ala Gly Leu Val Leu Val Ala Leu Leu Ala 1 5 10 15 Ile Leu Val <210> 47 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> CD8 transmembrane domain <400> 47 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 48 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> MERTK transmembrane domain <400> 48 Phe Gly Cys Phe Cys Gly Phe Ile Leu Ile Gly Leu Ile Leu Tyr Ile 1 5 10 15 Ser Leu Ala Ile Arg 20 <210> 49 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> Axl transmembrane domain <400> 49 Tyr Val Leu Leu Gly Ala Val Val Ala Ala Ala Cys Val Leu Ile Leu 1 5 10 15 Ala Leu Phe Leu Val 20 <210> 50 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> Tyro3 transmembrane domain <400> 50 Val Pro Val Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala Ala Ala 1 5 10 15 Leu Ala Leu Ile Leu 20 <210> 51 <211> twenty two <212> PRT <213> Homo sapiens <220> <223> CD4 transmembrane domain <400> 51 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile 1 5 10 15 Gly Leu Gly Ile Phe Phe 20 <210> 52 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> DAP12 transmembrane domain <400> 52 Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu 1 5 10 15 Ile Ala Leu Ala Val 20 <210> 53 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> MRC1 transmembrane domain <400> 53 Gly Val Val Ile Ile Val Ile Leu Leu Ile Leu Thr Gly Ala Gly Leu 1 5 10 15 Ala Ala Tyr Phe Phe 20 <210> 54 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR1 transmembrane domain <400> 54 Leu Leu Ile Val Thr Ile Val Ala Thr Met Leu Val Leu Ala Val Thr 1 5 10 15 Val Thr Ser Leu Cys 20 <210> 55 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR2 transmembrane domain <400> 55 Ala Leu Val Ser Gly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu 1 5 10 15 Thr Gly Val Leu Cys 20 <210> 56 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR3 transmembrane domain <400> 56 Phe Phe Met Ile Asn Thr Ser Ile Leu Leu Ile Phe Ile Phe Ile Val 1 5 10 15 Leu Leu Ile His Phe 20 <210> 57 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR4 transmembrane domain <400> 57 Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val 1 5 10 15 Ala Val Leu Val Tyr 20 <210> 58 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR5 transmembrane domain <400> 58 Phe Ser Leu Phe Ile Val Cys Thr Val Thr Leu Thr Leu Phe Leu Met 1 5 10 15 Thr Ile Leu Thr Val 20 <210> 59 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR6 transmembrane domain <400> 59 Ala Leu Val Ser Gly Met Cys Cys Ala Leu Phe Leu Leu Ile Leu Leu 1 5 10 15 Thr Gly Val Leu Cys 20 <210> 60 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR7 transmembrane domain <400> 60 Leu Ile Leu Phe Ser Leu Ser Ile Ser Val Ser Leu Phe Leu Met Val 1 5 10 15 Met Met Thr Ala Ser 20 <210> 61 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR8 transmembrane domain <400> 61 Ala Val Ile Leu Phe Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met 1 5 10 15 Leu Ala Ala Leu Ala 20 <210> 62 <211> twenty one <212> PRT <213> Homo sapiens <220> <223> TLR9 transmembrane domain <400> 62 Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val Pro Met 1 5 10 15 Leu His His Leu Cys 20 <210> 63 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Modified IgG4 hinge <400> 63 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 64 <211> twenty two <212> PRT <213> Homo sapiens <220> <223> GM-CSF signal peptide <400> 64 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 65 <211> twenty four <212> PRT <213> Homo sapiens <220> <223> Tim4 signal peptide <400> 65 Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Val Thr Ser 20 <210> 66 <211> twenty two <212> PRT <213> House mouse <220> <223> Tim4 signal peptide <400> 66 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala 20 <210> 67 <211> 18 <212> PRT <213> Artificial sequence <220> <223> T2A autolytic peptide <400> 67 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 68 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> T2A self-cleaving peptide variant <400> 68 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro 20 <210> 69 <211> 19 <212> PRT <213> Artificial sequence <220> <223> T2A self-cleaving peptide variant <400> 69 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro Arg <210> 70 <211> 19 <212> PRT <213> Artificial sequence <220> <223> P2A self-cleaving peptide <400> 70 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 71 <211> 27 <212> PRT <213> Artificial sequence <220> <223> P2A self-cleaving peptide variant <400> 71 Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 72 <211> 20 <212> PRT <213> Artificial sequence <220> <223> E2A self-cleaving peptide <400> 72 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 73 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> F2A self-cleaving peptide <400> 73 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 74 <400> 74 000 <210> 75 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> T2A self-cleaving peptide variant <400> 75 Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro Arg 20 <210> 76 <400> 76 000 <210> 77 <400> 77 000 <210> 78 <400> 78 000 <210> 79 <400> 79 000 <210> 80 <400> 80 000 <210> 81 <400> 81 000 <210> 82 <211> 285 <212> PRT <213> Homo sapiens <220> <223> Truncated EGFR <400> 82 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 275 280 285 <210> 83 <211> 192 <212> PRT <213> Homo sapiens <220> <223> Rac1 <400> 83 Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys 1 5 10 15 Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr 20 25 30 Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly 35 40 45 Lys Pro Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr 50 55 60 Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile 65 70 75 80 Cys Phe Ser Leu Val Ser Pro Ala Ser Phe Glu Asn Val Arg Ala Lys 85 90 95 Trp Tyr Pro Glu Val Arg His His Cys Pro Asn Thr Pro Ile Ile Leu 100 105 110 Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Asp Thr Ile Glu Lys 115 120 125 Leu Lys Glu Lys Lys Leu Thr Pro Ile Thr Tyr Pro Gln Gly Leu Ala 130 135 140 Met Ala Lys Glu Ile Gly Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu 145 150 155 160 Thr Gln Arg Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val 165 170 175 Leu Cys Pro Pro Pro Val Lys Lys Arg Lys Arg Lys Cys Leu Leu Leu 180 185 190 <210> 84 <211> 215 <212> PRT <213> Homo sapiens <220> <223> Rab5 <400> 84 Met Ala Ser Arg Gly Ala Thr Arg Pro Asn Gly Pro Asn Thr Gly Asn 1 5 10 15 Lys Ile Cys Gln Phe Lys Leu Val Leu Leu Gly Glu Ser Ala Val Gly 20 25 30 Lys Ser Ser Leu Val Leu Arg Phe Val Lys Gly Gln Phe His Glu Phe 35 40 45 Gln Glu Ser Thr Ile Gly Ala Ala Phe Leu Thr Gln Thr Val Cys Leu 50 55 60 Asp Asp Thr Thr Val Lys Phe Glu Ile Trp Asp Thr Ala Gly Gln Glu 65 70 75 80 Arg Tyr His Ser Leu Ala Pro Met Tyr Tyr Arg Gly Ala Gln Ala Ala 85 90 95 Ile Val Val Tyr Asp Ile Thr Asn Glu Glu Ser Phe Ala Arg Ala Lys 100 105 110 Asn Trp Val Lys Glu Leu Gln Arg Gln Ala Ser Pro Asn Ile Val Ile 115 120 125 Ala Leu Ser Gly Asn Lys Ala Asp Leu Ala Asn Lys Arg Ala Val Asp 130 135 140 Phe Gln Glu Ala Gln Ser Tyr Ala Asp Asp Asn Ser Leu Leu Phe Met 145 150 155 160 Glu Thr Ser Ala Lys Thr Ser Met Asn Val Asn Glu Ile Phe Met Ala 165 170 175 Ile Ala Lys Lys Leu Pro Lys Asn Glu Pro Gln Asn Pro Gly Ala Asn 180 185 190 Ser Ala Arg Gly Arg Gly Val Asp Leu Thr Glu Pro Thr Gln Pro Thr 195 200 205 Arg Asn Gln Cys Cys Ser Asn 210 215 <210> 85 <211> 207 <212> PRT <213> Homo sapiens <220> <223> Rab7 <400> 85 Met Thr Ser Arg Lys Lys Val Leu Leu Lys Val Ile Ile Leu Gly Asp 1 5 10 15 Ser Gly Val Gly Lys Thr Ser Leu Met Asn Gln Tyr Val Asn Lys Lys 20 25 30 Phe Ser Asn Gln Tyr Lys Ala Thr Ile Gly Ala Asp Phe Leu Thr Lys 35 40 45 Glu Val Met Val Asp Asp Arg Leu Val Thr Met Gln Ile Trp Asp Thr 50 55 60 Ala Gly Gln Glu Arg Phe Gln Ser Leu Gly Val Ala Phe Tyr Arg Gly 65 70 75 80 Ala Asp Cys Cys Val Leu Val Phe Asp Val Thr Ala Pro Asn Thr Phe 85 90 95 Lys Thr Leu Asp Ser Trp Arg Asp Glu Phe Leu Ile Gln Ala Ser Pro 100 105 110 Arg Asp Pro Glu Asn Phe Pro Phe Val Val Leu Gly Asn Lys Ile Asp 115 120 125 Leu Glu Asn Arg Gln Val Ala Thr Lys Arg Ala Gln Ala Trp Cys Tyr 130 135 140 Ser Lys Asn Asn Ile Pro Tyr Phe Glu Thr Ser Ala Lys Glu Ala Ile 145 150 155 160 Asn Val Glu Gln Ala Phe Gln Thr Ile Ala Arg Asn Ala Leu Lys Gln 165 170 175 Glu Thr Glu Val Glu Leu Tyr Asn Glu Phe Pro Glu Pro Ile Lys Leu 180 185 190 Asp Lys Asn Asp Arg Ala Lys Ala Ser Ala Glu Ser Cys Ser Cys 195 200 205 <210> 86 <211> 184 <212> PRT <213> Homo sapiens <220> <223> Rap1 <400> 86 Met Arg Glu Tyr Lys Leu Val Val Leu Gly Ser Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Cys 35 40 45 Gln Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe 50 55 60 Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Ala Leu 65 70 75 80 Val Tyr Ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu 85 90 95 Arg Glu Gln Ile Leu Arg Val Lys Asp Thr Glu Asp Val Pro Met Ile 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Glu Asp Glu Arg Val Val Gly Lys 115 120 125 Glu Gln Gly Gln Asn Leu Ala Arg Gln Trp Cys Asn Cys Ala Phe Leu 130 135 140 Glu Ser Ser Ala Lys Ser Lys Ile Asn Val Asn Glu Ile Phe Tyr Asp 145 150 155 160 Leu Val Arg Gln Ile Asn Arg Lys Thr Pro Val Glu Lys Lys Lys Pro 165 170 175 Lys Lys Lys Ser Cys Leu Leu Leu 180 <210> 87 <211> 193 <212> PRT <213> Homo sapiens <220> <223> RhoA <400> 87 Met Ala Ala Ile Arg Lys Lys Leu Val Ile Val Gly Asp Gly Ala Cys 1 5 10 15 Gly Lys Thr Cys Leu Leu Ile Val Phe Ser Lys Asp Gln Phe Pro Glu 20 25 30 Val Tyr Val Pro Thr Val Phe Glu Asn Tyr Val Ala Asp Ile Glu Val 35 40 45 Asp Gly Lys Gln Val Glu Leu Ala Leu Trp Asp Thr Ala Gly Gln Glu 50 55 60 Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr Pro Asp Thr Asp Val Ile 65 70 75 80 Leu Met Cys Phe Ser Ile Asp Ser Pro Asp Ser Leu Glu Asn Ile Pro 85 90 95 Glu Lys Trp Thr Pro Glu Val Lys His Phe Cys Pro Asn Val Pro Ile 100 105 110 Ile Leu Val Gly Asn Lys Lys Asp Leu Arg Asn Asp Glu His Thr Arg 115 120 125 Arg Glu Leu Ala Lys Met Lys Gln Glu Pro Val Lys Pro Glu Glu Gly 130 135 140 Arg Asp Met Ala Asn Arg Ile Gly Ala Phe Gly Tyr Met Glu Cys Ser 145 150 155 160 Ala Lys Thr Lys Asp Gly Val Arg Glu Val Phe Glu Met Ala Thr Arg 165 170 175 Ala Ala Leu Gln Ala Arg Arg Gly Lys Lys Lys Ser Gly Cys Leu Val 180 185 190 Leu <210> 88 <400> 88 000 <210> 89 <400> 89 000 <210> 90 <211> 598 <212> PRT <213> Artificial sequence <220> <223> HPV16 E7 TCRβ chain - P2A - TCRα chain <400> 90 Met Ala Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His 35 40 45 Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe 50 55 60 Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro 65 70 75 80 Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn 85 90 95 Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Gly Trp Arg Gly Gly Arg Tyr Asn Glu Gln Phe Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro 130 135 140 Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys 180 185 190 Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp 225 230 235 240 Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys 290 295 300 Arg Lys Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe 305 310 315 320 Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met 325 330 335 Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Thr 340 345 350 Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly Ala 355 360 365 Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly Leu 370 375 380 Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser Tyr 385 390 395 400 Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe Leu 405 410 415 Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln Met 420 425 430 Lys Asp Ser Ala Ser Tyr Leu Cys Ala Ser Val Asp Gly Asn Asn Arg 435 440 445 Leu Ala Phe Gly Lys Gly Asn Gln Val Val Val Ile Pro Asn Ile Gln 450 455 460 Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp 465 470 475 480 Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro 485 490 495 Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Cys Val Leu Asp 500 505 510 Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn 515 520 525 Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr 530 535 540 Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser 545 550 555 560 Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Leu Val Ile Val 565 570 575 Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr 580 585 590 Leu Arg Leu Trp Ser Ser 595 <210> 91 <211> 279 <212> PRT <213> House mouse <220> <223> Tim4 binding domain, amino acids 1-22 are signal peptides <400> 91 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr 275 <210> 92 <211> 135 <212> PRT <213> Artificial sequence <220> <223> TCR Vβ region <400> 92 Met Ala Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His 35 40 45 Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe 50 55 60 Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro 65 70 75 80 Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn 85 90 95 Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Gly Trp Arg Gly Gly Arg Tyr Asn Glu Gln Phe Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu 130 135 <210> 93 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> TCR Cβ region, Cys-substituted <400> 93 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 94 <211> 126 <212> PRT <213> artificial sequence <220> <223> TCR Vα district <400> 94 Met Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr 1 5 10 15 Thr Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly 20 25 30 Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly 35 40 45 Leu Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser 50 55 60 Tyr Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe 65 70 75 80 Leu Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln 85 90 95 Met Lys Asp Ser Ala Ser Tyr Leu Cys Ala Ser Val Asp Gly Asn Asn 100 105 110 Arg Leu Ala Phe Gly Lys Gly Asn Gln Val Val Val Ile Pro 115 120 125 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr 85 90 95 Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Leu 100 105 110 Val Ile Val Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu 115 120 125 Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 <210> 96 <211> 314 <212> PRT <213> Homo sapiens <220> <223> Tim4 binding domain, amino acid signal peptide 1-24 <400> 96 Met Ser Lys Glu Pro Leu Ile Leu Trp Leu Met Ile Glu Phe Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Val Thr Ser Glu Thr Val Val Thr Glu Val Leu 20 25 30 Gly His Arg Val Thr Leu Pro Cys Leu Tyr Ser Ser Trp Ser His Asn 35 40 45 Ser Asn Ser Met Cys Trp Gly Lys Asp Gln Cys Pro Tyr Ser Gly Cys 50 55 60 Lys Glu Ala Leu Ile Arg Thr Asp Gly Met Arg Val Thr Ser Arg Lys 65 70 75 80 Ser Ala Lys Tyr Arg Leu Gln Gly Thr Ile Pro Arg Gly Asp Val Ser 85 90 95 Leu Thr Ile Leu Asn Pro Ser Glu Ser Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Ile Asn Val Arg 115 120 125 Leu Asn Leu Gln Arg Ala Ser Thr Thr Thr His Arg Thr Ala Thr Thr 130 135 140 Thr Thr Arg Arg Thr Thr Thr Thr Ser Pro Thr Thr Thr Arg Gln Met 145 150 155 160 Thr Thr Thr Pro Ala Ala Leu Pro Thr Thr Val Val Thr Thr Pro Asp 165 170 175 Leu Thr Thr Gly Thr Pro Leu Gln Met Thr Thr Ile Ala Val Phe Thr 180 185 190 Thr Ala Asn Thr Cys Leu Ser Leu Thr Pro Ser Thr Leu Pro Glu Glu 195 200 205 Ala Thr Gly Leu Leu Thr Pro Glu Pro Ser Lys Glu Gly Pro Ile Leu 210 215 220 Thr Ala Glu Ser Glu Thr Val Leu Pro Ser Asp Ser Trp Ser Ser Val 225 230 235 240 Glu Ser Thr Ser Ala Asp Thr Val Leu Leu Thr Ser Lys Glu Ser Lys 245 250 255 Val Trp Asp Leu Pro Ser Thr Ser His Val Ser Met Trp Lys Thr Ser 260 265 270 Asp Ser Val Ser Ser Pro Gln Pro Gly Ala Ser Asp Thr Ala Val Pro 275 280 285 Glu Gln Asn Lys Thr Thr Lys Thr Gly Gln Met Asp Gly Ile Pro Met 290 295 300 Ser Met Lys Asn Glu Met Pro Ile Ser Gln 305 310 <210> 97 <211> 487 <212> PRT <213> Artificial sequence <220> <223> CER5, amino acids 1-22 are signal peptides. <400> 97 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Lys Phe Tyr Phe 290 295 300 His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 305 310 315 320 Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 325 330 335 Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 340 345 350 Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala 355 360 365 Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 370 375 380 Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 385 390 395 400 Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 405 410 415 Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 420 425 430 Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser 435 440 445 Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 450 455 460 Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 465 470 475 480 Trp Gln Glu Ala Thr Ser Ile 485 <210> 98 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> CER19, Amino acids 1 - 22 are the signal peptide <400> 98 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Thr Lys Phe Arg 290 295 300 Gly Phe Cys Phe Ile Cys Tyr Lys Thr Ala Gln Arg Leu Val Phe Lys 305 310 315 320 Asp His Pro Gln Gly Thr Glu Pro Asp Met Tyr Lys Tyr Asp Ala Tyr 325 330 335 Leu Cys Phe Ser Ser Lys Asp Phe Thr Trp Val Gln Asn Ala Leu Leu 340 345 350 Lys His Leu Asp Thr Gln Tyr Ser Asp Gln Asn Arg Phe Asn Leu Cys 355 360 365 Phe Glu Glu Arg Asp Phe Val Pro Gly Glu Asn Arg Ile Ala Asn Ile 370 375 380 Gln Asp Ala Ile Trp Asn Ser Arg Lys Ile Val Cys Leu Val Ser Arg 385 390 395 400 His Phe Leu Arg Asp Gly Trp Cys Leu Glu Ala Phe Ser Tyr Ala Gln 405 410 415 Gly Arg Cys Leu Ser Asp Leu Asn Ser Ala Leu Ile Met Val Val Val 420 425 430 Gly Ser Leu Ser Gln Tyr Gln Leu Met Lys His Gln Ser Ile Arg Gly 435 440 445 Phe Val Gln Lys Gln Gln Tyr Leu Arg Trp Pro Glu Asp Phe Gln Asp 450 455 460 Val Gly Trp Phe Leu His Lys Leu Ser Gln Gln Ile Leu Lys Lys Glu 465 470 475 480 Lys Glu Lys Lys Lys Asp Asn Asn Ile Pro Leu Gln Thr Val Ala Thr 485 490 495 Ile Ser <210> 99 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> CER21, amino acids 1-22 are the signal peptide <400> 99 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu His His Leu Phe 290 295 300 Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu Ala Lys Val Lys 305 310 315 320 Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr Asp Ala Tyr Ile 325 330 335 Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp Val Ile Asn Glu 340 345 350 Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn Val Leu Leu Cys 355 360 365 Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile Ile Asp Asn Leu 370 375 380 Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe Val Leu Thr Lys 385 390 395 400 Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe Tyr Leu Ala Leu 405 410 415 Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile Phe Ile Leu Leu 420 425 430 Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu Arg Gln Arg Ile 435 440 445 Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro Lys Ala Glu Gly 450 455 460 Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr Glu Asn Asp Ser 465 470 475 480 Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln Tyr 485 490 <210> 100 <211> 386 <212> PRT <213> Artificial sequence <220> <223> CER25; Amino acids 1-22 are signal peptides. <400> 100 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Leu Trp Asn Lys 290 295 300 Lys Arg Met Arg Gly Pro Gly Lys Asp Pro Thr Arg Lys Cys Pro Asp 305 310 315 320 Pro Arg Ser Ala Ser Ser Pro Lys Gln His Pro Ser Glu Ser Val Tyr 325 330 335 Thr Ala Leu Gln Arg Arg Glu Thr Glu Val Tyr Ala Cys Ile Glu Asn 340 345 350 Glu Asp Gly Ser Ser Pro Thr Ala Lys Gln Ser Pro Leu Ser Gln Glu 355 360 365 Arg Pro His Arg Phe Glu Asp Asp Gly Glu Leu Asn Leu Val Tyr Glu 370 375 380 Asn Leu 385 <210> 101 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> CER27, Amino acids 1 - 22 are the signal peptide <400> 101 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu His Arg Phe His 290,295,300 Gly Leu Trp Tyr Met Lys Met Met Trp Ala Trp Leu Gln Ala Lys Arg 305 310 315 320 Lys Pro Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val 325 330 335 Ser Tyr Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln 340 345 350 Glu Leu Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg 355 360 365 Asp Phe Isolated Pro Gly Lys Trp Isolated Asp Asn Isolated Asp Ser 370 375 380 Glu Lys Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys 385 390 395 400 Ser Glu Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe 405 410 415 Asp Glu Asn Asn Asp Ala Ala Glu Pro Glu 420 425 430 Lys Lys Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn 435 440 445 Thr Lys Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly 450 455 460 Phe Trp Val Asn Leu Arg Ala Ala Ile Lys Ser 465 470 475 <210> 102 <211> 574 <212> PRT <213> artificial sequence <220> <223> CER29, amino acids 1‑22 is a signal peptide <400> 102 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Met Ser Leu Leu 290 295 300 Asn Cys Glu Asn Ser Cys Gly Ser Ser Gln Ser Glu Ser Asp Cys Cys 305 310 315 320 Val Ala Met Ala Ser Ser Cys Ser Ala Val Thr Lys Asp Asp Ser Val 325 330 335 Gly Gly Thr Ala Ser Thr Gly Asn Leu Ser Ser Ser Phe Met Glu Glu 340 345 350 Ile Gln Gly Tyr Asp Val Glu Phe Asp Pro Pro Leu Glu Ser Lys Tyr 355 360 365 Glu Cys Pro Ile Cys Leu Met Ala Leu Arg Glu Ala Val Gln Thr Pro 370 375 380 Cys Gly His Arg Phe Cys Lys Ala Cys Ile Ile Lys Ser Ile Arg Asp 385 390 395 400 Ala Gly His Lys Cys Pro Val Asp Asn Glu Ile Leu Leu Glu Asn Gln 405 410 415 Leu Phe Pro Asp Asn Phe Ala Lys Arg Glu Ile Leu Ser Leu Met Val 420 425 430 Lys Cys Pro Asn Glu Gly Cys Leu His Lys Met Glu Leu Arg His Leu 435 440 445 Glu Asp His Gln Ala His Cys Glu Phe Ala Leu Met Asp Cys Pro Gln 450 455 460 Cys Gln Arg Pro Phe Gln Lys Phe His Ile Asn Ile His Ile Leu Lys 465 470 475 480 Asp Cys Pro Arg Arg Gln Val Ser Cys Asp Asn Cys Ala Ala Ser Met 485 490 495 Ala Phe Glu Asp Lys Glu Ile His Asp Gln Asn Cys Pro Leu Ala Asn 500 505 510 Val Ile Cys Glu Tyr Cys Asn Thr Ile Leu Ile Arg Glu Gln Met Pro 515 520 525 Asn His Tyr Asp Leu Asp Cys Pro Thr Ala Pro Ile Pro Cys Thr Phe 530 535 540 Ser Thr Phe Gly Cys His Glu Lys Met Gln Arg Asn His Leu Ala Arg 545 550 555 560 His Leu Gln Glu Asn Thr Gln Ser His Met Arg Met Leu Ala 565 570 <210> 103 <211> 574 <212> PRT <213> Artificial sequence <220> <223> CER31, amino acids 1-22 are the signal peptide <400> 103 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Met Glu Ser Ser 290 295 300 Lys Lys Met Asp Ser Pro Gly Ala Leu Gln Thr Asn Pro Pro Leu Lys 305 310 315 320 Leu His Thr Asp Arg Ser Ala Gly Thr Pro Val Phe Val Pro Glu Gln 325 330 335 Gly Gly Tyr Lys Glu Lys Phe Val Lys Thr Val Glu Asp Lys Tyr Lys 340 345 350 Cys Glu Lys Cys His Leu Val Leu Cys Ser Pro Lys Gln Thr Glu Cys 355 360 365 Gly His Arg Phe Cys Glu Ser Cys Met Ala Ala Leu Leu Ser Ser Ser 370 375 380 Ser Pro Lys Cys Thr Ala Cys Gln Glu Ser Ile Val Lys Asp Lys Val 385 390 395 400 Phe Lys Asp Asn Cys Cys Lys Arg Glu Ile Leu Ala Leu Gln Ile Tyr 405 410 415 Cys Arg Asn Glu Ser Arg Gly Cys Ala Glu Gln Leu Met Leu Gly His 420 425 430 Leu Leu Val His Leu Lys Asn Asp Cys His Phe Glu Glu Leu Pro Cys 435 440 445 Val Arg Pro Asp Cys Lys Glu Lys Val Leu Arg Lys Asp Leu Arg Asp 450 455 460 His Val Glu Lys Ala Cys Lys Tyr Arg Glu Ala Thr Cys Ser His Cys 465 470 475 480 Lys Ser Gln Val Pro Met Ile Ala Leu Gln Lys His Glu Asp Thr Asp 485 490 495 Cys Pro Cys Val Val Val Ser Cys Pro His Lys Cys Ser Val Gln Thr 500 505 510 Leu Leu Arg Ser Glu Leu Ser Ala His Leu Ser Glu Cys Val Asn Ala 515 520 525 Pro Ser Thr Cys Ser Phe Lys Arg Tyr Gly Cys Val Phe Gln Gly Thr 530 535 540 Asn Gln Gln Ile Lys Ala His Glu Ala Ser Ser Ala Val Gln His Val 545 550 555 560 Asn Leu Leu Lys Glu Trp Ser Asn Ser Leu Glu Lys Lys Val 565 570 <210> 104 <211> 70 <212> PRT <213> Homo sapiens <220> <223> FcγR2B2 signaling domain <400> 104 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Leu Pro 1 5 10 15 Gly Tyr Pro Glu Cys Arg Glu Met Gly Glu Thr Leu Pro Glu Lys Pro 20 25 30 Ala Asn Pro Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn 35 40 45 Thr Ile Thr Tyr Ser Leu Leu Met His Pro Asp Ala Leu Glu Glu Pro 50 55 60 Asp Asp Gln Asn Arg Ile 65 70 <210> 105 <211> 41 <212> PRT <213> Homo sapiens <220> <223> FcαR1 signal conduction domain <400> 105 Glu Asn Trp His Ser His Thr Ala Leu Asn Lys Glu Ala Ser Ala Asp 1 5 10 15 Val Ala Glu Pro Ser Trp Ser Gln Gln Met Cys Gln Pro Gly Leu Thr 20 25 30 Phe Ala Arg Thr Pro Ser Val Cys Lys 35 40 <210> 106 <211> 141 <212> PRT <213> Homo sapiens <220> <223> MyD88 TIR domain <400> 106 His Met Pro Glu Arg Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp 1 5 10 15 Ile Gln Phe Val Gln Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr 20 25 30 Arg Leu Lys Leu Cys Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys 35 40 45 Val Trp Ser Ile Ala Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met 50 55 60 Val Val Val Val Ser Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe 65 70 75 80 Gln Thr Lys Phe Ala Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg 85 90 95 Leu Ile Pro Ile Lys Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile 100 105 110 Leu Arg Phe Ile Thr Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser 115 120 125 Trp Phe Trp Thr Arg Leu Ala Lys Ala Leu Ser Leu Pro 130 135 140 <210> 107 <211> 27 <212> PRT <213> Homo sapiens <220> <223> CD28 transmembrane domain <400> 107 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 108 <211> 191 <212> PRT <213> Homo sapiens <220> <223> CDC42 <400> 108 Met Gln Thr Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys 1 5 10 15 Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Lys Phe Pro Ser Glu Tyr 20 25 30 Val Pro Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met Ile Gly Gly 35 40 45 Glu Pro Tyr Thr Leu Gly Leu Phe Asp Thr Ala Gly Gln Glu Asp Tyr 50 55 60 Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val 65 70 75 80 Cys Phe Ser Val Val Ser Pro Ser Ser Phe Glu Asn Val Lys Glu Lys 85 90 95 Trp Val Pro Glu Ile Thr His His Cys Pro Lys Thr Pro Phe Leu Leu 100 105 110 Val Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Ser Thr Ile Glu Lys 115 120 125 Leu Ala Lys Asn Lys Gln Lys Pro Ile Thr Pro Glu Thr Ala Glu Lys 130 135 140 Leu Ala Arg Asp Leu Lys Ala Val Lys Tyr Val Glu Cys Ser Ala Leu 145 150 155 160 Thr Gln Lys Gly Leu Lys Asn Val Phe Asp Glu Ala Ile Leu Ala Ala 165 170 175 Leu Glu Pro Pro Glu Pro Lys Lys Ser Arg Arg Cys Val Leu Leu 180 185 190 <210> 109 <211> 776 <212> PRT <213> artificial sequence <220> <223> CER1, amino acid 1‑22 is the signal peptide <400> 109 Met Ser Lys Gly Leu Leu Leu Leu Trp Leu Val Thr Glu Leu Trp Trp 1 5 10 15 Leu Tyr Leu Thr Pro Ala Ala Ser Glu Asp Thr Ile Ile Gly Phe Leu 20 25 30 Gly Gln Pro Val Thr Leu Pro Cys His Tyr Leu Ser Trp Ser Gln Ser 35 40 45 Arg Asn Ser Met Cys Trp Gly Lys Gly Ser Cys Pro Asn Ser Lys Cys 50 55 60 Asn Ala Glu Leu Leu Arg Thr Asp Gly Thr Arg Ile Ile Ser Arg Lys 65 70 75 80 Ser Thr Lys Tyr Thr Leu Leu Gly Lys Val Gln Phe Gly Glu Val Ser 85 90 95 Leu Thr Ile Ser Asn Thr Asn Arg Gly Asp Ser Gly Val Tyr Cys Cys 100 105 110 Arg Ile Glu Val Pro Gly Trp Phe Asn Asp Val Lys Lys Asn Val Arg 115 120 125 Leu Glu Leu Arg Arg Ala Thr Thr Thr Lys Lys Pro Thr Thr Thr Thr 130 135 140 Arg Pro Thr Thr Thr Pro Tyr Val Thr Thr Thr Thr Pro Glu Leu Leu 145 150 155 160 Pro Thr Thr Val Met Thr Thr Ser Val Leu Pro Thr Thr Thr Pro Pro 165 170 175 Gln Thr Leu Ala Thr Thr Ala Phe Ser Thr Ala Val Thr Thr Cys Pro 180 185 190 Ser Thr Thr Pro Gly Ser Phe Ser Gln Glu Thr Thr Lys Gly Ser Ala 195 200 205 Phe Thr Thr Glu Ser Glu Thr Leu Pro Ala Ser Asn His Ser Gln Arg 210 215 220 Ser Met Met Thr Ile Ser Thr Asp Ile Ala Val Leu Arg Pro Thr Gly 225 230 235 240 Ser Asn Pro Gly Ile Leu Pro Ser Thr Ser Gln Leu Thr Thr Gln Lys 245 250 255 Thr Thr Leu Thr Thr Ser Glu Ser Leu Gln Lys Thr Thr Lys Ser His 260 265 270 Gln Ile Asn Ser Arg Gln Thr Ile Leu Ile Ile Ala Cys Cys Val Gly 275 280 285 Phe Val Leu Met Val Leu Leu Phe Leu Ala Phe Leu Ala Leu Arg Arg 290 295 300 Arg Val Gln Glu Thr Lys Phe Gly Gly Ala Phe Ser Glu Glu Asp Ser 305 310 315 320 Gln Leu Val Val Asn Tyr Arg Ala Lys Lys Ser Phe Cys Arg Arg Ala 325 330 335 Ile Glu Leu Thr Leu Gln Ser Leu Gly Val Ser Glu Glu Leu Gln Asn 340 345 350 Lys Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu Val Leu Gly Lys 355 360 365 Val Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu Gly Asn Leu Lys 370 375 380 Gln Glu Asp Gly Thr Ser Gln Lys Val Ala Val Lys Thr Met Lys Leu 385 390 395 400 Asp Asn Phe Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala Ala 405 410 415 Cys Met Lys Asp Phe Asn His...

Claims

1. An expression cassette comprising: (a) a polynucleotide encoding a chimeric engulfment receptor (CER) comprising: an extracellular domain comprising a Tim4 binding domain that binds a first target antigen, wherein the first target antigen is phosphatidylserine; an engulfment signaling domain comprising a TLR1 signaling domain, a TLR2 signaling domain, a TLR3 signaling domain, a TLR4 signaling domain, a TLR5 signaling domain, a TLR6 signaling domain, a TLR7 signaling domain, a TLR8 signaling domain, a TLR9 signaling domain, a Traf2 signaling domain, a Traf3 signaling domain, a Traf6 signaling domain, a DAP-12 signaling domain, a NFAM1 signaling domain, a BAFF-R signaling domain, a CD79b signaling domain, a MERTK signaling domain, a Tyro3 signaling domain, an Axl signaling domain, or a MyD88 signaling domain; a transmembrane domain between and connecting the extracellular domain and the engulfment signaling domain; (b) a polynucleotide encoding a recombinant T cell receptor (TCR) binding protein beta chain comprising a TCR beta variable region and a TCR beta constant region; and (c) a polynucleotide encoding a recombinant TCR a chain comprising a TCR a variable region TCR a constant region, wherein, the TCR binding protein binds to a second target antigen.

2. The expression cassette of claim 1, wherein the CER extracellular domain further comprises a spacer domain between the binding domain and the transmembrane domain.

3. The expression cassette of claim 2, wherein the spacer domain of the CER is selected from an immunoglobulin hinge region, a type 1 membrane protein hinge region, a stem region of a type II C-lectin, an immunoglobulin constant domain, or a TLR juxtamembrane domain.

4. The expression cassette of claim 3, wherein the immunoglobulin hinge region is selected from an IgGl, IgG2, IgG3, IgG4, IgA, or IgD hinge region.

5. The expression cassette of claim 4, wherein the amino acid sequence of the IgG4 hinge region is set forth in SEQ ID NO:

63.

6. The expression cassette of claim 1, wherein the CER transmembrane domain comprises a Timl, Tim4, Tim3, CD8, CD28, MERTK, Axl, Tyro3, BAI1, CD4, DAP-12, MRC1, FcR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

7. The expression cassette of claim 6, wherein the CER transmembrane domain comprises: a Timl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 36, a Tim4 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 37 or 38, a Tim3 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 39, a FcyRl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 40, a FcyR2A transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 41, a FcyR2B2 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 42, a FcyR2C transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 43, a FcyR3A transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 44, a FcƐRl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 45, a FcaRl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 46, a CD8a transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 47, a CD28 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 107, a MERTK transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 48, an Axl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 49, a Tyro3 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 50, a CD4 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 51, a DAP12 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 52, a MRC1 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 53, a TLRl transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 54, a TLR2 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 55, a TLR3 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 56, a TLR4 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 57, a TLR5 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 58, a TLR6 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 59, a TLR7 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 60, a TLR8 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO: 61, or a TLR9 transmembrane domain as set forth in the amino acid sequence of SEQ ID NO:

62.

8. The expression cassette of claim 1, wherein the CER engulfment signaling domain comprises: a MERTK signaling domain as set forth in the amino acid sequence of SEQ ID NO: 3, a Tyro3 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 5, an Axl signaling domain as set forth in the amino acid sequence of SEQ ID NO: 6, a Traf6 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 34, a MyD88 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 106, or SEQ ID NO: 33, a BAFF-R signaling domain as set forth in the amino acid sequence of SEQ ID NO: 17, a DAP- 12 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 18, a NFAM1 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 35, a CD79b signaling domain as set forth in the amino acid sequence of SEQ ID NO: 21, a TLR1 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 22, a TLR2 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 23, a TLR3 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 24, a TLR4 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 25, a TLR5 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 26, a TLR6 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 27, a TLR7 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 28, a TLR8 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 29, a TLR9 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 30, a Traf2 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 31, or a Traf3 signaling domain as set forth in the amino acid sequence of SEQ ID NO:

32.

9. The expression cassette of any one of claims 1-8, wherein the CER phagocytic signaling domain comprises a primary phagocytic signaling domain and a secondary phagocytic signaling domain, wherein the primary phagocytic signaling domain is a TLR1 signaling domain, a TLR2 signaling domain, a TLR3 signaling domain, a TLR4 signaling domain, a TLR5 signaling domain, a TLR6 signaling domain, a TLR7 signaling domain, a TLR8 signaling domain, a TLR9 signaling domain, a Traf2 signaling domain, a Traf3 signaling domain, a Traf6 signaling domain, a DAP-12 signaling domain, a NFAM1 signaling domain, a BAFF-R signaling domain, a CD79b signaling domain, a MERTK signaling domain, a Tyro3 signaling domain, an Axl signaling domain, or a MyD88 signaling domain.

10. The expression cassette of claim 9, wherein the primary and secondary phagocytic signaling domains of the CER are different.

11. The expression cassette of claim 9, wherein the primary and secondary phagocytic signaling domains of the CER are each independently selected from the group consisting of MERTK, Tyro3, Axl, MyD88, BAFF-R, DAP-12, NFAM1, CD79b, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, Traf6, Traf2, and Traf3 signaling domains.

12. The expression cassette of claim 11, wherein the primary phagocytic signaling domain and the secondary phagocytic signaling domain of the CER are each independently selected from a phagocytic signaling domain comprising a MERTK signaling domain as set forth in the amino acid sequence of SEQ ID NO:3, a Tyro3 signaling domain as set forth in the amino acid sequence of SEQ ID NO:5, an Axl signaling domain as set forth in the amino acid sequence of SEQ ID NO:6, a Traf6 signaling domain as set forth in the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:34, a MyD88 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 106, or SEQ ID NO:33, a BAFF-R signaling domain as set forth in the amino acid sequence of SEQ ID NO: 17, a DAP- 12 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 18, a NFAM1 signaling domain as set forth in the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO:35, a CD79b signaling domain as set forth in the amino acid sequence of SEQ ID NO:21, a TLR1 signaling domain as set forth in the amino acid sequence of SEQ ID NO:22, a TLR2 signaling domain as set forth in the amino acid sequence of SEQ ID NO:23, a TLR3 signaling domain as set forth in the amino acid sequence of SEQ ID NO:24, a TLR4 signaling domain as set forth in the amino acid sequence of SEQ ID NO:25, a TLR5 signaling domain as set forth in the amino acid sequence of SEQ ID NO:26, a TLR6 signaling domain as set forth in the amino acid sequence of SEQ ID NO:27, a TLR7 signaling domain as set forth in the amino acid sequence of SEQ ID NO:28, a TLR8 signaling domain as set forth in the amino acid sequence of SEQ ID NO:29, a TLR9 signaling domain as set forth in the amino acid sequence of SEQ ID NO:30, a Traf2 signaling domain as set forth in the amino acid sequence of SEQ ID NO:31, or a Traf3 signaling domain as set forth in the amino acid sequence of SEQ ID NO:

32.

13. The expression cassette of claim 1, wherein the amino acid sequence of the Tim4 binding domain is set forth in SEQ ID NO:96 or amino acids 25-314 of SEQ ID NO:

96.

14. The expression cassette of claim 9, wherein the amino acid sequence of the Tim4 binding domain is set forth in SEQ ID NO:96 or amino acids 25-314 of SEQ ID NO:

96.

15. The expression cassette of claim 1, wherein the second target antigen of the recombinant TCR is WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, or alpha fetoprotein.

16. The expression cassette of claim 1, wherein the polynucleotide encoding the CER is upstream of the polynucleotide encoding the TCR.

17. The expression cassette of claim 1, wherein the polynucleotide encoding the CER, the polynucleotide encoding the TCR alpha chain, and the polynucleotide encoding the TCR beta chain are separated from each other by a first polynucleotide encoding an IRES element or a 2A peptide and a second polynucleotide encoding an IRES element or a 2A peptide.

18. The expression cassette of claim 17, wherein the 2A peptide comprises a T2A, P2A, E2A, or F2A peptide.

19. The expression cassette of claim 18, wherein each 2A peptide comprises: (a) a T2A peptide as set forth in the amino acid sequence of any one of SEQ ID NOs: 67, 68, 69, and 75; (b) a P2A peptide as set forth in the amino acid sequence of SEQ ID NO: 70 or 71; (c) an E2A peptide as set forth in the amino acid sequence of SEQ ID NO: 72; or (d) an F2A peptide as set forth in the amino acid sequence of SEQ ID NO:

73.

20. The expression cassette of claim 1, wherein: (a) the amino acid sequence of the CER is set forth in any one of SEQ ID NOs: 97-103, 109-115, 118-134, 136-165, 171-175, and 183-195; and (b) the amino acid sequence of the TCR is set forth in SEQ ID NO:

90.

21. The expression cassette of claim 1, further comprising a promoter operably linked to the expression cassette.

22. The expression cassette of claim 21, wherein the promoter is an EF-1 alpha promoter.

23. The expression cassette of claim 1, further comprising a polynucleic acid sequence encoding a transduction marker protein.

24. The expression cassette of claim 23, wherein the transduction marker protein comprises a fluorescent protein, an extracellular domain of CD2, or a truncated EGFR.

25. The expression cassette of claim 24, wherein the amino acid sequence of the truncated EGFR is set forth in SEQ ID NO:

82.

26. A vector comprising the expression cassette of any one of claims 1-25.

27. The vector of claim 26, wherein the vector is a viral vector.

28. The vector of claim 27, wherein the viral vector is a retroviral vector or a lentiviral vector.

29. A T cell comprising the vector of claim 26.

30. The T cell of claim 29, wherein the T cell is a CD4 T cell, a CD8 T cell, or both. ​ ​ 31. The T cell of claim 30, wherein the T cell is a naive T cell, a central memory T cell, an effector T cell, or any combination thereof.

32. The T cell of claim 29, wherein the T cell is from a human.

33. The T cell of claim 29, wherein the T cell exhibits cytolytic activity against cells expressing an antigen targeted by the TCR and exhibits phagocytic activity against cells expressing phosphatidylserine on the cell surface.

34. Use of the T cell of any one of claims 29-33 in the manufacture of a medicament for treating squamous cell carcinoma of the head and neck.

35. The use of claim 34, wherein the T cell is autologous or allogeneic to the subject.

36. The use of claim 34, wherein the medicament further comprises an additional therapeutic agent.

37. The use of claim 36, wherein the additional therapeutic agent is an antibody, a chemotherapeutic agent, an oncolytic virus, an antibiotic, an antifungal agent, or an antiviral agent.

38. The use of claim 36, wherein the additional therapeutic agent is at a subtherapeutic dose.

39. The use of claim 37, wherein the additional therapeutic agent is at a subtherapeutic dose.

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