HK40013683A

HK40013683A - Chimeric polypeptides and methods of altering their localisation in the cell membrane

Info

Publication number: HK40013683A
Application number: HK62020003641.1A
Authority: HK
Inventors: Stephen Santoro; Scott COYLE; Levi RUPP; Peter Emtage
Original assignee: 细胞设计实验室股份有限公司
Priority date: 2017-03-28
Filing date: 2018-03-28
Publication date: 2020-08-07

Description

Chimeric polypeptides and methods for altering their localization in cell membranes

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application serial No. 62/477,733 filed on 28.3.2017 and U.S. provisional patent application serial No. 62/587,262 filed on 16.11.2017; the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the field of biotechnology, and more specifically, to modulation of cellular localization of chimeric polypeptides.

Background

Chimeric Antigen Receptors (CARs) are a subset of single-chain and multi-chain polypeptides. Chimeric antigen receptors generally include, for example, an antigen binding domain, a transmembrane domain, a costimulatory domain, and a CD3 ζ or other signaling domain. CARs have been investigated for use in clinical treatment of various cancers in mammals, such as hematological cancers, with considerable success. However, the limiting factor has been the uncontrollable toxicity associated with CAR anti-cancer activity.

Nuclear hormone receptors are a class of polypeptides that include several domains, including DNA binding motifs and ligand binding domains. In the absence of ligands, naturally occurring nuclear hormone receptors associate with various chaperones and other interacting proteins, sequestering the nuclear hormone receptors outside the nucleus. Ligand binding induces changes in nuclear hormone receptor conformation and interaction partners and allows nuclear translocation and transcriptional activation or repression of nuclear hormone target genes. While the classical view of steroid signaling suggests that these hormones bind to intracellular receptors that act as ligand-activated transcription factors, it has been reported that steroids may also act through non-genomic mechanisms at the cell surface.

Summary of The Invention

The present disclosure is based, at least in part, on the following findings: the reversible outer cell membrane of the chimeric polypeptide including the transmembrane domain can be localized for use in medical applications where it is desired to reversibly express a protein or peptide of interest on the outer cell membrane. An exemplary feature of the disclosure is that toxicity associated with CAR T cells can be modulated by reversibly expressing the antigen binding domain of the CAR on its surface.

The present disclosure is further based, at least in part, on the following findings: the plasma membrane localization or extracellular membrane localization of a chimeric polypeptide comprising a transmembrane domain and a hormone receptor ligand binding domain in a mammalian cell can be modulated by contacting the mammalian cell with a hormone or hormone analog, wherein the transmembrane domain and the hormone receptor ligand binding domain are directly adjacent to each other or separated by 1 to about 700 amino acids (e.g., 1 to about 500 amino acids), and both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single chain polypeptide (e.g., single chain chimeric antigen receptor) in the mammal. In view of this finding, provided herein are: single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric antigen receptors; a nucleic acid encoding the same; a vector comprising any of these nucleic acids; mammalian cells comprising any of these vectors; and methods of reversibly inducing membrane localization of the outer membrane of cells and methods of altering the membrane localization of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric polypeptides.

In some embodiments, reversibly inducing plasma membrane localization or extracellular membrane localization of a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, and/or a multi-chain chimeric antigen receptor) using one or more compositions or methods disclosed herein is useful in the field of cell-based adoptive immunotherapy, in which immune cells isolated from a subject may be modified to express synthetic proteins that enable the cells to perform new therapeutic functions after being subsequently transferred back into the subject. Non-limiting examples of such synthetic proteins are Chimeric Antigen Receptors (CARs) and engineered T Cell Receptors (TCRs).

In some embodiments, reversibly inducing plasma membrane localization or extracellular membrane localization of a polypeptide (e.g., a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, and/or a multi-chain chimeric antigen receptor) using one or more compositions or methods disclosed herein is useful in controlling the toxic effects of chimeric antigen T cells (CAR-T cells) that participate in the elimination of cancer cells by modulating the presence or absence of an antigen-binding moiety on the plasma membrane or extracellular membrane of the cell.

Provided herein are single chain chimeric polypeptides comprising: a transmembrane domain; and a hormone receptor ligand binding domain, wherein: the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 500 amino acids; and both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single chain polypeptide in the mammal. In some embodiments of any of the single chain chimeric polypeptides described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the single chain chimeric polypeptides described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the single chain chimeric polypeptides described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

In some embodiments of any of the single chain chimeric polypeptides described herein, the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other. In some embodiments of any of the single chain chimeric polypeptides described herein, the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids or about 1 to 50 amino acids).

Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein is a vector comprising any nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein are mammalian cells comprising any of the vectors described herein.

Also provided herein is a method of inducing membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, comprising: contacting a mammalian cell expressing any of the single chain chimeric polypeptides described herein with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell.

Also provided is a method of reversibly altering the membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single chain chimeric polypeptides described herein with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with a reduced amount of a hormone or hormone analog that results in a reduced level of the single chain chimeric polypeptide outside of the cell of the plasma membrane of the mammalian cell. Some embodiments of any of the methods described herein further comprise: (c) contacting the mammalian cell with an increased amount of a hormone or hormone analog which results in an increased level of the single chain chimeric polypeptide outside the cell of the plasma membrane of the cell as compared to the level in step (b).

Some embodiments of any of the methods described herein further comprise: introducing a nucleic acid encoding a single-chain chimeric polypeptide into a mammalian cell to produce a mammalian cell expressing the single-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the mammalian cell is a T cell (e.g., a T cell selected from the group consisting of CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells).

In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from the subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. Some embodiments of any of the methods described herein further comprise: mammalian cells are obtained from a subject. In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments of any of the methods described herein, the contacting step is performed in a mammal.

Also provided herein are single-chain chimeric antigen receptors comprising: an extracellular antigen-binding domain; a transmembrane domain; a hormone receptor ligand binding domain; a co-stimulatory domain; and an immunoreceptor tyrosine-based activation motif (ITAM); wherein: the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 500 amino acids; and both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single chain polypeptide in the mammal. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to a single antigen. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single antigen is a tumor antigen. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD 19. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to two different antigens.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C and B7-H3. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the co-stimulatory domain is 4-1BB or CD 28. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the ITAM comprises a cytoplasmic signaling sequence from CD3 ζ.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids or 1 to 50 amino acids).

Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided herein are vectors comprising a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided herein are mammalian cells comprising any of the vectors described herein.

Also provided is a method of inducing membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, comprising: contacting a mammalian cell expressing any of the single-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce the single-chain chimeric antigen-binding receptor to localize to the extracellular side of the plasma membrane of the cell.

Also provided herein is a method of altering the membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with an amount of a hormone or hormone analog that results in a decrease in the level of the single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of these methods further comprise: (c) contacting a mammalian cell with an amount of a hormone or hormone analog which results in an increase in the level of single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell as compared to the level in (b).

Some embodiments of any of the methods described herein further comprise: introducing a nucleic acid encoding a single-chain chimeric antigen receptor into a mammalian cell to produce a mammalian cell expressing the single-chain chimeric antigen receptor. In some embodiments of any of the methods described herein, the mammalian cell is a T cell. In some embodiments of any of the methods described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from the subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. Some embodiments of any of the methods described herein further comprise: mammalian cells are obtained from a subject. In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments of any of the methods described herein, the contacting step is performed in a mammal. In some embodiments of any of the methods described herein, the mammal has cancer. In some embodiments of any of the methods described herein, the contacting step results in the treatment of cancer in the mammal.

Also provided herein are multi-chain chimeric antigen receptors comprising at least one first polypeptide comprising: an extracellular antigen-binding domain; a transmembrane domain; and a hormone receptor ligand binding domain; wherein: the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 500 amino acids; and both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single chain polypeptide in the mammal. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to a single antigen. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the single antigen is a tumor antigen. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD 19. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to two different antigens.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids (e.g., 1 to 250 amino acids or 1 to 50 amino acids).

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one polypeptide further comprises a co-stimulatory domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C or B7-H3. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the co-stimulatory domain is 4-1BB or CD 28.

Also provided herein is a method of inducing membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, comprising: contacting a mammalian cell expressing any of the multi-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce the multi-chain chimeric antigen-binding receptor to localize to the extracellular side of the plasma membrane of the cell.

Also provided herein are methods of altering the membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the multi-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog(ii) an amount of the hormone or hormone analog sufficient to induce localization of the multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with an amount of a hormone or hormone analog that results in a decrease in the level of the multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of any of the methods described herein further comprise: (c) contacting a mammalian cell with an amount of a hormone or hormone analog that results in an increase in the level of a multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of any of the methods described herein further comprise: introducing a nucleic acid encoding a single-chain chimeric antigen receptor into a mammalian cell to produce a mammalian cell expressing the single-chain chimeric antigen receptor. In some embodiments of any of the methods described herein, the mammalian cell is a T cell. In some embodiments of any of the methods described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

Also provided herein are methods of treating cancer in a subject (e.g., a human), comprising: (a) administering to the subject a mammalian cell expressing any of the single chain chimeric antigen receptors described herein or any of the multi-chain chimeric antigen receptors described herein, wherein the antigen binding domain specifically binds to an antigen expressed by a cancer in the subject; (b) administering to the subject an amount of a hormone or hormone sufficient to induce single-chain or multi-chain chimeric antigen receptor localization to the extracellular side of the plasma membrane of the cell; (c) determining the presence or severity of one or more symptoms associated with toxicity, the level of one or more cytokines associated with a cytokine storm, or both in the subject following (b); (d) comparing the determined presence or severity of one or more symptoms associated with toxicity, the determined level of one or more cytokines associated with the cytokine storm, or both, to a control; and (e) administering to the subject an amount of a hormone or hormone analog sufficient to reduce the level of single-chain or multi-chain chimeric antigen receptors on the extracellular side of the plasma membrane of the cell, such that the presence or severity of one or more symptoms associated with toxicity, the level of one or more cytokines associated with a cytokine storm, or both, is reduced in the subject following (e).

In some embodiments of any of the methods described herein, the amount of hormone or hormone analogue administered to the subject in step (e) is reduced by at least 25% compared to the amount of hormone or hormone analogue administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analogue administered to the subject in step (e) is reduced by at least 50% compared to the amount of hormone or hormone analogue administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analogue administered to the subject in step (e) is reduced by at least 75% compared to the level of hormone or hormone analogue administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analogue administered to the subject in step (e) is reduced by 100% compared to the level of hormone or hormone analogue administered to the subject in step (b).

In some embodiments of any of the methods described herein, the one or more symptoms associated with toxicity are selected from the group consisting of: high fever, swelling, chills, hypotension, tachycardia, weakness, headache, rash, itching throat, dyspnea, redness, extreme fatigue, nausea and encephaledema. In some embodiments of these methods, the one or more cytokines associated with the cytokine storm are selected from the group consisting of: including tumor necrosis factor alpha, IFN gamma, IL-10, IL-1 beta, IL-2, IL-6, IL-8 and IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5.

Also provided herein is a single chain chimeric polypeptide comprising: an extracellular hormone receptor ligand binding domain; and a transmembrane domain, wherein: the hormone receptor ligand binding domain and the extracellular transmembrane domain are directly contiguous with each other or are separated by 1 to 700 amino acids; and both the transmembrane domain and the extracellular hormone receptor ligand binding domain are absent from a single endogenous single chain polypeptide in a mammal. In some embodiments of any of the single chain chimeric polypeptides described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the single chain chimeric polypeptides described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the single chain chimeric polypeptides described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor. In some embodiments of any of the single chain chimeric polypeptides described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor. In some embodiments of any of the single chain chimeric polypeptides described herein, the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it comprises one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

In some embodiments of any of the single chain chimeric polypeptides described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other. In some embodiments of any of the single chain chimeric polypeptides described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids (e.g., 1 to 500 amino acids, 1 to 250 amino acids, 1 to 100 amino acids, 1 to 50 amino acids, or 1 to 25 amino acids).

In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction.

Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein are vectors comprising a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein are mammalian cells comprising any of the vectors described herein. In some embodiments of any of the mammalian cells described herein, the mammalian cell is a T cell. In some embodiments of any of the mammalian cells described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

Also provided herein is a method of inducing plasma membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single chain chimeric polypeptides described herein with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell.

Also provided herein is a method of reversibly altering plasma membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single chain chimeric polypeptides described herein with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with a reduced amount of a hormone or hormone analog that results in a reduced level of the single chain chimeric polypeptide outside of the cell of the plasma membrane of the mammalian cell. Some embodiments of any of the methods described herein further comprise: (c) contacting the mammalian cell with an increased amount of a hormone or hormone analog which results in an increased level of the single chain chimeric polypeptide outside the cell of the plasma membrane of the cell as compared to the level in step (b). Some embodiments of any of the methods described herein further comprise, prior to step (a), introducing a nucleic acid encoding a single-chain chimeric polypeptide into a mammalian cell to generate a mammalian cell expressing the single-chain chimeric polypeptide.

In some embodiments of any of the methods described herein, the mammalian cell is a T cell. In some embodiments of any of the methods described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells. In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from the subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. Some embodiments of any of the methods described herein further comprise: mammalian cells are obtained from a subject.

In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments of any of the methods described herein, the contacting step is performed in a mammal.

Also provided herein are single-chain chimeric antigen receptors comprising: an extracellular antigen-binding domain; an extracellular hormone receptor ligand binding domain; a transmembrane domain; an intracellular co-stimulatory domain; and an intracellular immunoreceptor tyrosine-based activation motif (ITAM); wherein: the transmembrane domain and the extracellular hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 700 amino acids; and both the transmembrane domain and the extracellular hormone receptor ligand binding domain are absent from a single endogenous single chain polypeptide in a mammal. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to a single antigen. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single antigen is a tumor antigen. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD 19. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to two different antigens. In some embodiments of any of the single-chain chimeric antigen receptors described herein, at least one of the two different antigens is a tumor antigen. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD 19.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it comprises one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C or B7-H3. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from 4-1BB or CD 28. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the intracellular ITAM comprises a cytoplasmic signaling sequence from CD3 ζ.

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids (e.g., 1 to 500 amino acids, 1 to 250 amino acids, 1 to 100 amino acids, 1 to 50 amino acids, or 1 to 25 amino acids).

In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the N-terminal to C-terminal direction. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the C-terminal to N-terminal direction. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the N-terminal to C-terminal direction. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric polypeptide comprises, when moved in the C-terminal to N-terminal direction, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs.

Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided herein are vectors comprising a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided herein are mammalian cells comprising any of the vectors described herein. In some embodiments of any of the mammalian cells described herein, the mammalian cell is a T cell. In some embodiments of any of the mammalian cells described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T is thinCells, memory T cells, Treg cells, and natural killer T cells.

Also provided herein is a method of inducing plasma membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce the single-chain chimeric antigen-binding receptor to localize to the extracellular side of the plasma membrane of the cell.

Also provided herein are methods of altering plasma membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with an amount of a hormone or hormone analog that results in a decrease in the level of the single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of any of the methods described herein further comprise: (c) contacting a mammalian cell with an amount of a hormone or hormone analog which results in an increase in the level of single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell as compared to the level in (b). Some embodiments of any of the methods described herein further comprise, prior to step (a): introducing a nucleic acid encoding a single-chain chimeric antigen receptor into a mammalian cell to produce a mammalian cell expressing the single-chain chimeric antigen receptor.

In some embodiments of any of the methods described herein, the mammalian cell is a T cell. In some embodiments of any of the methods described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells. In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from the subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as havingThere is cancer. Some embodiments of any of the methods described herein further comprise: mammalian cells are obtained from a subject. In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments of any of the methods described herein, the contacting step is performed in a mammal. In some embodiments of any of the methods described herein, the mammal has cancer. In some embodiments of any of the methods described herein, the contacting step results in the treatment of cancer in the mammal.

Also provided herein are multi-chain chimeric antigen receptors comprising at least one first polypeptide comprising: an extracellular antigen-binding domain; an extracellular hormone receptor ligand binding domain; and a transmembrane domain; wherein: the transmembrane domain and the extracellular hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 700 amino acids; and both the transmembrane domain and the extracellular hormone receptor ligand binding domain are absent from a single endogenous single chain polypeptide in a mammal. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to a single antigen. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the single antigen is a tumor antigen. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD 19. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds to two different antigens. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one of the two different antigens is a tumor antigen. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD 19.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it includes one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids (e.g., 1 to 500 amino acids, 1 to 250 amino acids, 1 to 100 amino acids, 1 to 50 amino acids, or 1 to 25 amino acids).

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one polypeptide further comprises an intracellular co-stimulatory domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C or B7-H3. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from 4-1BB or CD 28.

In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction.

Also provided herein are nucleic acids encoding any of the multi-chain chimeric antigen receptors described herein. Also provided herein are sets of nucleic acids that together encode any of the multi-chain chimeric antigen receptors described herein. Also provided herein are mammalian cells comprising a nucleic acid encoding any of the multi-chain chimeric antigen receptors described herein. Also provided herein are chimeric constructs comprising a plurality of strands encoding together any of the polypeptides described hereinA mammalian cell of the group of nucleic acids of antigen receptors. In some embodiments of any of the mammalian cells described herein, the mammalian cell is a T cell. In some embodiments of any of the mammalian cells described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

Also provided herein are methods of inducing plasma membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the multi-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce the multi-chain chimeric antigen-binding receptor to localize to the extracellular side of the plasma membrane of the cell.

Also provided herein are methods of altering plasma membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the multi-chain chimeric antigen receptors described herein with an amount of a hormone or hormone analog sufficient to induce the multi-chain chimeric antigen-binding receptor to localize to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with an amount of a hormone or hormone analog that results in a decrease in the level of the multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of any of the methods described herein further comprise: (c) contacting a mammalian cell with an amount of a hormone or hormone analog that results in an increase in the level of a multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of the cell. Some embodiments of any of the methods described herein further comprise, prior to step (a): introducing a nucleic acid encoding a multi-chain chimeric antigen receptor, or a set of nucleic acids together encoding a multi-chain chimeric receptor, into a mammalian cell to generate a mammalian cell expressing a multi-chain chimeric antigen receptor. In some embodiments of any of the methods described herein, the mammalian cell is a T cell. In some embodiments of any of the methods described herein, the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells. In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from the subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. Some embodiments of any of the methods described herein further comprise obtaining the mammalian cell from a subject. In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments of any of the methods described herein, the contacting step is performed in a mammal. In some embodiments of any of the methods described herein, the mammal has cancer. In some embodiments of any of the methods described herein, the contacting step results in the treatment of cancer in the mammal.

Also provided herein are methods of treating cancer in a subject, comprising: (a) administering to the subject a mammalian cell expressing any of the single chain chimeric antigen receptors described herein or any of the multi-chain chimeric antigen receptors described herein, wherein the antigen binding domain specifically binds to an antigen expressed by a cancer in the subject; (b) administering to the subject an amount of a hormone or hormone sufficient to induce single-chain or multi-chain chimeric antigen receptor localization to the extracellular side of the plasma membrane of the cell; (c) determining the presence or severity of one or more symptoms associated with toxicity or the level of one or more cytokines associated with a cytokine storm, or both, in the subject following (b); (d) comparing the determined presence or severity of one or more symptoms associated with toxicity or the determined level of one or more cytokines associated with a cytokine storm, or both, to a control; and (e) administering to the subject an amount of a hormone or hormone analog sufficient to reduce the level of single-chain or multi-chain chimeric antigen receptors on the extracellular side of the plasma membrane of the cell such that the presence or severity of one or more symptoms associated with toxicity or the level of one or more cytokines associated with a cytokine storm, or both, is reduced in the subject following (e).

In some embodiments of any of the methods described herein, the one or more symptoms associated with toxicity are selected from the group consisting of: high fever, swelling, chills, hypotension, tachycardia, weakness, headache, rash, itching throat, dyspnea, redness, extreme fatigue, nausea and encephaledema. In some embodiments of any of the methods described herein, the one or more cytokines associated with the cytokine storm are selected from the group consisting of: including tumor necrosis factor alpha, IFN gamma, IL-10, IL-1 beta, IL-2, IL-6, IL-8 and IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5.

The use of the term "a" or "an" preceding a noun means "one or more" of that particular noun. For example, the phrase "a mammalian cell" means "one or more mammalian cells.

The term "single chain chimeric polypeptide" means a single chain polypeptide comprising a first contiguous amino acid sequence (e.g., a first domain) and a second contiguous amino acid sequence (e.g., a second domain), wherein the first contiguous amino acid sequence and the second contiguous amino acid sequence are not present in the same endogenous single chain polypeptide in a mammal. In some examples, the first contiguous amino acid sequence and the second contiguous amino acid sequence are directly adjacent to each other in the single-chain chimeric polypeptide. In some examples, the first contiguous amino acid sequence and the second contiguous amino acid sequence are separated by one or more intervening amino acids (e.g., one or more domains). The term "single chain chimeric polypeptide" is not intended to limit in any way the relative positions of a first contiguous amino acid sequence and a second contiguous amino acid sequence in a single chain polypeptide. Non-limiting examples of single-chain chimeric polypeptides (e.g., single-chain chimeric antigen receptors) are described herein. Additional examples of single-chain chimeric polypeptides (e.g., single-chain chimeric antigen receptors) are known in the art.

The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein and refer to an artificial, multi-modular molecule capable of triggering or inhibiting immune cell activation, typically, but not exclusively, comprising an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. CAR molecules and derivatives thereof (e.g., CAR variants) are described in PCT application nos. US 2014/016527; fedorov et al.Sci Transl Med (2013); 215ra172 (215); glienke et al front Pharmacol (2015)6: 21; kakarla & Gottschalk 52cancer J (2014)20(2) 151-5; riddell et al cancer J (2014)20(2) 141-4; cancer J (2014)20(2) 127-33; cheadle et al. Immunol Rev (2014)257(1): 91-106; barrett et al Annu Rev Med (2014)65: 333-47; sadelain et al cancer Discov (2013)3(4): 388-98; cartellieri et al, J BiomedBiotechnol (2010) 956304; the disclosure of which is incorporated herein by reference in its entirety.

The term "multi-chain chimeric polypeptide" means a polypeptide comprising two or more single-chain polypeptides, wherein at least one of the two or more single-chain polypeptides is a single-chain chimeric polypeptide as defined and described herein. Non-limiting examples of multi-chain chimeric polypeptides (e.g., multi-chain chimeric antigen receptors) are described herein. Additional examples of multi-chain chimeric polypeptides (e.g., multi-chain chimeric antigen receptors) are known in the art.

The term "transmembrane domain" means a domain of a polypeptide comprising at least one contiguous amino acid sequence that, when expressed in a mammalian cell, traverses the lipid bilayer when present in a corresponding endogenous polypeptide. For example, the transmembrane domain may comprise one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid sequences that each traverse the lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell. As known in the art, a transmembrane domain may, for example, comprise at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) contiguous amino acid sequence (that traverses a lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell) having an alpha-helical secondary structure in the lipid bilayer. In some embodiments, the transmembrane domain may comprise two or more contiguous amino acid sequences (each of which traverses the lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell) that form a β -barrel secondary structure in the lipid bilayer. Non-limiting examples of transmembrane domains are described herein. Additional examples of transmembrane domains are known in the art.

The phrase "extracellular side of the plasma membrane" when used to describe the location of a polypeptide means that the polypeptide includes at least one transmembrane domain that traverses the plasma membrane and at least one domain (e.g., at least one antigen binding domain) that is located in the extracellular space.

The term "hormone receptor ligand binding domain" means a domain in a hormone receptor polypeptide that specifically binds to a hormone or hormone analog. Non-limiting examples of hormone receptor Ligand Binding Domains (LBDs) are described herein. In some cases, the LBD of a nuclear hormone receptor is selected from the group consisting of an estrogen receptor, an ecdysone receptor, a PPAR γ receptor, a glucocorticoid receptor, an androgen receptor, a thyroid hormone receptor, a mineralocorticoid receptor, a progesterone receptor, a vitamin D receptor, a PPAR receptor α, a PPAR β/δ receptor, a pregnane X receptor, a liver X receptor, a farnesoid X receptor, a retinoid X receptor, a RAR-related orphan receptor, and a retinoic acid receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises a single LBD of a nuclear hormone receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises multiple (e.g., two or more) LBDs of a nuclear hormone receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises two LBDs of a nuclear hormone receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises three LBDs of a nuclear hormone receptor. Where the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises multiple (e.g., two or more) LBDs of a nuclear hormone receptor, in some cases, the multiple LBDs comprise the same amino acid sequence. In some cases, two or more LBDs are in tandem, separated directly or by a linker (e.g., any of the linkers described herein).

The term "antigen binding domain" means a domain that specifically binds to a target antigen. In some examples, the antigen binding domain may be formed from amino acids present within a single chain polypeptide. In other examples, the antigen binding domain may be formed from amino acids present within a first single chain polypeptide and amino acids present within one or more additional single chain polypeptides (e.g., a second single chain polypeptide). Non-limiting examples of antigen binding domains are described herein, including but not limited to, scFv or LBD of a growth factor. Additional examples of antigen binding domains are known in the art.

As used herein, the term "antigen" generally refers to a binding partner specifically recognized by an antigen binding domain as described herein. Exemplary antigens include different classes of molecules such as, but not limited to, polypeptides and peptide fragments thereof, small molecules, lipids, carbohydrates, and nucleic acids. Non-limiting examples of one or more antigens that can be specifically bound by any antigen binding domain are described herein. Additional examples of one or more antigens that can be specifically bound by any antigen binding domain are known in the art.

The term "co-stimulatory domain" means a signaling domain from an endogenous co-stimulatory transmembrane polypeptide expressed in a T lymphocyte that promotes downstream T cell receptor signaling and/or T cell activation. Non-limiting examples of co-stimulatory domains are described herein. Additional costimulatory domains are known in the art. See, e.g., Chen et al, Naturereviews immunol.13: 227-.

The term "immunoreceptor tyrosine-based activation motif" or "ITAM" means an amino acid motif that includes a four-amino acid consensus sequence with two other amino acids separating one tyrosine from one leucine or isoleucine (YxxL/I). Tyrosine residues in the four amino acid consensus sequence are phosphorylated following interaction of a signaling pathway kinase (e.g., a lymphocyte signaling pathway kinase). Non-limiting examples of ITAMs are described herein. Additional examples of ITAMs are known in the art.

The term "hormone" means a naturally occurring molecule that specifically binds to the ligand binding domain of a hormone receptor and modulates the activity and/or location of the hormone receptor. Non-limiting examples of hormones are described herein. Additional examples of hormones are known in the art.

The term "hormone analog" means a molecule that mimics the structure of a hormone and specifically binds to the ligand binding domain of a hormone receptor. Non-limiting examples of hormone analogs are described herein. Additional examples of hormone analogs are known in the art.

The phrase "treatment of cancer" means reducing the number, frequency, or severity of one or more (e.g., two, three, four, or five) symptoms of cancer in a subject having cancer. Non-limiting symptoms of cancer are described herein. Additional symptoms of cancer are known in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Brief Description of Drawings

Figure 1 is a schematic of the different constructs tested. Extracellular and intracellular markers are understood to mean that they are depicted as grey boxes between these markers with respect to the cell membrane. The extracellular scFv domain and intracellular costimulatory ("Costim"), CD3zeta and mCherry domains are indicated.

Figure 2 is a set of flow cytometry dot plots of cells expressing one of the different constructs. Cells were either untreated or treated with 500ng/mL 4-hydroxytamoxifen ("4-OHT"). (A) Jurkat T cells transduced with lentiviral vectors encoding canonical CARs or with anti-CD 19(FMC63) ERa-LBD containing v2, v3 and v4CAR constructs. (B) Primary CD4 and CD8 human T cells were transduced with lentiviral vectors encoding v4CAR constructs.

FIG. 3 is a set of flow cytometry dot plots of HEK-293T cells transduced with lentiviral vectors encoding FMC63 CAR fusions containing ERA-LBD. Cells were untreated or treated with 500ng/mL 4-OHT.

Figure 4 is a set of flow cytometry dot plots of primary human CD 4T cells transduced with lentiviral vectors encoding FMC63 NHR LBD-CAR fusion and NHR LBD-CAR with J6M0 scFv. Cells were untreated or treated with 500ng/mL 4-OHT.

FIG. 5A is a schematic representation of the V5 nuclear hormone receptor ligand binding domain NHR-LBDCAR construct lacking the costimulatory and CD3zeta domain, and a set of flow cytometry dot plots for cells transduced with a lentiviral vector encoding the V5 construct. Cells were untreated or treated with 500ng/mL 4-OHT.

Figure 5B is a set of flow cytometry schematic of untransduced Jurkat T cells or Jurkat T cells transduced with lentiviral vectors encoding CARs in which the scFv domain was replaced with a FLAG tag. Transduced cells were untreated or treated with 500ng/mL 4-OHT.

Figure 6 is a set of schematic flow cytometric views of Jurkat T cells transduced with CAR constructs with progesterone receptor LBD. Cells were either untreated or treated with 500ng/mL mifepristone (mifepristone).

Figure 7A is a graph showing T cell activation of primary human T cells transduced with lentiviral vectors encoding the J6M0-ERa-LBD CAR construct after overnight co-culture with antigen negative (K562 cell line) and antigen positive (MM1S cell line) targets.

Figure 7B is a graph showing IFN- γ production by primary human T cells transduced with lentiviral vectors encoding the J6M0-ERa-LBD CAR construct after overnight co-culture with antigen negative (K562 cell line) and antigen positive (MM1S cell line) targets.

Figure 8A is a set of flow cytometry schematic showing proliferation of primary human CD 4T cells transduced with lentiviral vectors encoding V4 FMC63-ERa-LBD CARs or canonical CAR controls, co-cultured with antigen negative (K562 cell line) or antigen positive (Raji cell line) targets, and with increasing concentrations of ligand 4-OHT.

Figure 8B is a graph showing lysis of antigen positive targets (MM1S cell line) mediated by primary human T cells transduced with lentiviral vectors encoding the J6M0-ERa-LBD CAR construct, using increasing concentrations of 4-OHT.

Figure 9 is a graph showing surface expression of various CAR constructs with LBD at different positions relative to the cell membrane at increasing concentrations of 4-OHT.

Fig. 10A is a diagram showing a timeline of an experimental procedure.

Figure 10B is a bar graph showing surface expression of various CAR constructs before and after co-culture with 4-OHT.

FIG. 11A is a set of flow cytometry illustrations showing Jurkat T cells transduced with a FLAG-ERA-LBD CAR and treated with no or 4-OHT for 15 or 120 minutes.

Figure 11B is a set of flow cytometry schematic showing human T cells transduced with lentiviral vectors encoding FMC63-ERa-LBD CAR and untreated or treated with 4-OHT for 2 hours, 4 hours, or 24 hours.

Fig. 12 is a set of flow cytometry schematic of primary human T cells expressing FMC63-CAR with ERa LBD. Cells were untreated or treated with 500ng/mL 4-OHT. Some samples were pretreated with 1. mu.M of Brefeldin A.

Figure 13 is a schematic representation of the v2 CAR construct and ecd.v1 CAR (also known as LBD-ECD CAR) construct tested. Cell membranes are depicted as grey boxes. Depicted are the extracellular scFv domain, extracellular ERA LBD, transmembrane domain (embedded in the cell membrane), intracellular 4-1BB domain, and intracellular CD3 γ domain.

Figure 14 depicts a set of flow cytometry dot plots for cells expressing either the v2 CAR construct or the LBD-ECD CAR construct. Cells were either untreated or treated with 500ng/mL 4-hydroxytamoxifen ("4-OHT").

Figure 15 is a graph showing disruption (% lysis) of Nalm 6 tumor cells by T cells transduced with a lentiviral vector encoding the LBD-ECD CAR construct (ECD. v 1). Cells were treated with 1.5ng/mL to 500ng/mL 4-OHT. Positive controls were T cells transduced with lentiviral vectors encoding a histone CAR. Negative controls were T cells with LBD-ECD CAR without 4-OHT treatment.

Figure 16 depicts a set of flow cytometry dot plots for cells expressing constitutive CAR, LBD-ECD CAR, or v2 CAR. Cells were either untreated or stimulated with beads containing CD3/CD28 for 48 hours prior to flow cytometry. Beads, Dynabeads aCD3/aCD28, from ThermoFisher. See, tricokett et al, j.immunol.methods275: 251-. Each construct was fused to a cMyc tag.

Figure 17 is a graph showing IL-2 production from ecd. v1 CAR-T cells or conventional CAR-T cells after incubating CAR-bearing T cells with NALM-6 target overnight in the presence (500ng/mL) or absence of 4-hydroxytamoxifen.

Detailed Description

Provided herein are single-chain chimeric polypeptides, single-chain Chimeric Antigen Receptors (CARs), and multi-chain chimeric antigen receptors; a nucleic acid encoding the same; a vector comprising any of these nucleic acids; mammalian cells comprising any of these vectors; and methods of reversibly inducing membrane localization of the outer membrane of cells and methods of altering the membrane localization of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric polypeptides.

Also provided herein are genetically modified mammalian cells having a Chimeric Antigen Receptor (CAR). In some cases, the genetically modified mammalian cell is derived from a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell. In some cases, the mammalian cell is a T lymphocyte or an NK cell.

Non-limiting aspects of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric antigen receptors, nucleic acids, vectors, mammalian cells, and methods are described below, and can be used in any combination without limitation. Additional aspects of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors and multi-chain chimeric antigen receptors, nucleic acids, vectors, mammalian cells, and methods are known in the art.

In some embodiments, the compositions and/or methods disclosed herein can be used to selectively kill cancer cells in a subject in the presence of a hormone or hormone analog that binds to LBD present in a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, and/or a multi-chain chimeric antigen receptor). In some embodiments, the polypeptide (e.g., single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multi-chain chimeric antigen receptor) is localized to the outer surface of the cell, presenting an antigen binding domain (e.g., scFv or other antigen binding domain described herein or known in the art) on the exterior of the cell in the presence of the hormone or hormone analog. A polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor) can recognize an antigen bound thereto, and thus can selectively kill a target cell (e.g., a cancer cell) that expresses the antigen on its surface.

In some embodiments, localization of a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor) to the exterior surface of a cell can result in toxicity in a subject. In some embodiments, a subject who experiences toxicity due to hormone or hormone analog-induced polypeptide (e.g., single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor) being localized to the outer surface of a cell may be treated or ameliorated by reducing the dose of hormone or hormone analog, or eliminating hormone or hormone analog treatment of the subject altogether. Reduction of such doses or elimination of hormone or hormone analog therapy can result in removal of the polypeptide (e.g., single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric polypeptide) from the cell surface and reduction or elimination of antigen binding domain presentation on the cell surface, thereby reducing or eliminating toxicity in the subject.

Single chain chimeric polypeptides

Single chain chimeric polypeptide-intracellular hormone receptor LBD

Provided herein are single chain chimeric polypeptides comprising a transmembrane domain (e.g., any transmembrane domain described herein or known in the art) and a hormone receptor ligand binding domain (e.g., any hormone receptor ligand binding domain described herein or known in the art), wherein the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein), and both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same (single) endogenous single chain polypeptide in a mammal (e.g., a human).

In some examples of single-chain chimeric polypeptides, the transmembrane domain is directly adjacent to the hormone receptor ligand binding domain (i.e., there are no intervening amino acids between the transmembrane domain and the hormone receptor ligand binding domain). In some examples of single chain chimeric polypeptides, the transmembrane domain and the hormone receptor ligand binding domain are separated by: 1 amino acid to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 5 amino acids, about 4 amino acids, or about 3 amino acids (inclusive); about 2 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 5 amino acids, or about 4 amino acids (inclusive); about 3 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, or about 5 amino acids (inclusive); about 4 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, or about 6 amino acids, inclusive; about 5 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, or about 8 amino acids, inclusive; about 6 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, or about 8 amino acids, inclusive; about 8 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, or about 10 amino acids, inclusive; about 10 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, or about 12 amino acids (inclusive); about 12 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, or about 14 amino acids (inclusive); about 14 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, or about 16 amino acids (inclusive); about 16 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, or about 18 amino acids (inclusive); about 18 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids (inclusive); about 20 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids (inclusive); about 25 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, or about 30 amino acids (inclusive); about 30 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, or about 35 amino acids (inclusive); about 35 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, or about 40 amino acids (inclusive); about 40 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, about 50 amino acids, or about 45 amino acids (inclusive); about 45 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids, about 55 amino acids, or about 50 amino acids (inclusive); about 50 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about, About 60 amino acids or about 55 amino acids (inclusive); about 55 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, or about 60 amino acids (inclusive); about 60 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, or about 65 amino acids, inclusive; about 65 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, or about 70 amino acids (inclusive); about 70 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, or about 75 amino acids (inclusive); about 75 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, or about 80 amino acids, inclusive; about 80 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, or about 85 amino acids, inclusive; about 85 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, about 95 amino acids, or about 90 amino acids (inclusive); about 90 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, about 100 amino acids, or about 95 amino acids (inclusive); about 95 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, about 105 amino acids, or about 100 amino acids (inclusive); about 100 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, about 110 amino acids, or about 105 amino acids (inclusive); about 105 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, about 115 amino acids, or about 110 amino acids, inclusive; about 110 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, about 120 amino acids, or about 115 amino acids (inclusive); about 115 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, about 125 amino acids, or about 120 amino acids, inclusive; about 120 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, about 130 amino acids, or about 125 amino acids (inclusive); about 125 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, about 135 amino acids, or about 130 amino acids (inclusive); about 130 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, about 140 amino acids, or about 135 amino acids, inclusive; about 135 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, about 145 amino acids, or about 140 amino acids (inclusive); about 140 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, about 150 amino acids, or about 145 amino acids (inclusive); about 145 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, about 155 amino acids, or about 150 amino acids (inclusive); about 150 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, about 160 amino acids, or about 155 amino acids (inclusive); about 155 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, about 165 amino acids, or about 160 amino acids (inclusive); about 160 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, about 170 amino acids, or about 165 amino acids (inclusive); about 165 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, about 175 amino acids, or about 170 amino acids (inclusive); about 170 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, about 180 amino acids, or about 175 amino acids (inclusive); about 175 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, about 185 amino acids, or about 180 amino acids (inclusive); about 180 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, about 190 amino acids, or about 185 amino acids (inclusive); about 185 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, about 195 amino acids, or about 190 amino acids (inclusive); about 190 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, about 200 amino acids, or about 195 amino acids (inclusive); about 195 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, about 210 amino acids, or about 200 amino acids (inclusive); about 200 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, about 220 amino acids, or about 210 amino acids (inclusive); about 210 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, about 230 amino acids, or about 220 amino acids (inclusive); about 220 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 240 amino acids, or about 230 amino acids (inclusive); about 230 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, or about 240 amino acids (inclusive); about 240 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, or about 250 amino acids (inclusive); about 250 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, about 270 amino acids, or about 260 amino acids (inclusive); about 260 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids, about 280 amino acids, or about 270 amino acids (inclusive); about 270 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, about, About 290 amino acids or about 280 amino acids (inclusive); about 280 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, or about 290 amino acids (inclusive); about 290 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, or about 300 amino acids (inclusive); from about 300 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, about 300 amino acids, or about 290 amino acids (inclusive); about 290 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, about 310 amino acids, or about 300 amino acids (inclusive); from about 300 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, about 320 amino acids, or about 310 amino acids (inclusive); about 310 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, about 330 amino acids, or about 320 amino acids (inclusive); about 320 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, about 340 amino acids, or about 330 amino acids, inclusive; about 330 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, about 350 amino acids, or about 340 amino acids (inclusive); about 340 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, about 360 amino acids, or about 350 amino acids (inclusive); about 350 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids, or about 360 amino acids, inclusive; about 360 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, about 380 amino acids, about 370 amino acids (inclusive); about 370 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, about 390 amino acids, or about 380 amino acids (inclusive); about 380 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, about 400 amino acids, or about 390 amino acids, inclusive; about 390 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, about 410 amino acids, or about 400 amino acids (inclusive); about 400 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, about 420 amino acids, or about 410 amino acids, inclusive; about 410 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, about 440 amino acids, about 430 amino acids, or about 420 amino acids (inclusive); about 420 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, or about 440 amino acids (inclusive); about 430 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, about 450 amino acids, or about 440 amino acids (inclusive); about 440 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, about 460 amino acids, or about 450 amino acids (inclusive); about 450 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids, or about 460 amino acids (inclusive); about 460 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, about 480 amino acids, about 470 amino acids (inclusive); about 470 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, about 490 amino acids, or about 480 amino acids (inclusive); about 480 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, about 500 amino acids, or about 490 amino acids (inclusive); about 490 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, about 520 amino acids, or about 500 amino acids (inclusive); about 500 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, about 540 amino acids, or about 520 amino acids (inclusive); about 520 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, about 560 amino acids, or about 540 amino acids (inclusive); about 540 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, about 580 amino acids, or about 560 amino acids (inclusive); about 560 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, about 600 amino acids, or about 580 amino acids (inclusive); about 580 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, about 620 amino acids, or about 600 amino acids (inclusive); about 600 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, about 640 amino acids, or about 620 amino acids (inclusive); about 620 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, about 660 amino acids, or about 640 amino acids (inclusive); about 640 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, about 680 amino acids, or about 660 amino acids (inclusive); from about 660 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, about 700 amino acids, or about 680 amino acids (inclusive); about 680 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, about 720 amino acids, or about 700 amino acids (inclusive); from about 700 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, about 740 amino acids, or about 720 amino acids (inclusive); from about 720 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, or about 740 amino acids (inclusive); from about 740 amino acids to about 800 amino acids, about 780 amino acids, or about 760 amino acids (inclusive); from about 760 amino acids to about 800 amino acids or about 780 amino acids inclusive; or from about 780 amino acids to about 800 amino acids, inclusive.

In some embodiments, one or more intervening amino acids between the transmembrane domain and the hormone receptor ligand binding domain may be or include a linker peptide. In some embodiments, the linker peptide may comprise one or more of the following amino acid sequences: GSGSGSGSGS (SEQ ID NO:20), GSGSGSGS (SEQ ID NO:21), RSGSGSGS (SEQ ID NO:22) or GSGSGSGSGS (SEQ ID NO: 23). In some embodiments, the linker peptide may be encoded by one or more of the following nucleic acid sequences: GGATCCGGCAGCGGATCTGGCAGTGGAAGC (SEQ ID NO:24), GGATCTGGCTCTGGAAGCGGCAGC (SEQ ID NO:25), AGATCCGGATCTGGAAGTGGCTCC (SEQ ID NO:26) or GGAAGTGGATCTGGGAGCGGCTCT (SEQ ID NO: 27). In some embodiments, the linker peptide can, for example, comprise in tandem one or more (e.g., two, three, or four) copies of any of SEQ ID NOs: 20-23.

In some embodiments, one or more intervening amino acids between the transmembrane domain and the hormone receptor ligand binding domain may be or include an intracellular sequence that adjoins a transmembrane domain in the endogenous polypeptide from which the transmembrane domain (e.g., any of the transmembrane domains described herein) is derived. In some embodiments, one or more intervening amino acids between the transmembrane domain and the hormone receptor ligand binding domain may be or include one or more additional domains (e.g., any of the exemplary additional domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide can be a single-chain chimeric antigen receptor (e.g., any of the single-chain chimeric antigen receptors described herein). In some embodiments, the chimeric polypeptide may further comprise one or more additional domains. Non-limiting examples of additional domains that may be included in a single chain chimeric polypeptide include one or more of the following: an extracellular ligand-binding domain (e.g., an antigen-binding domain such as any of the antigen-binding domains described herein), an ITIM (e.g., any ITIM known in the art), an ITAM (e.g., any ITAM described herein or known in the art), a dimerization domain (e.g., capable of dimerizing with another dimerization domain present in another polypeptide (e.g., a recombinant polypeptide) expressed in a mammalian cell, such as any dimerization domain known in the art), and a peptide tag (e.g., a poly-His tag).

Single chain chimeric polypeptide-extracellular hormone receptor LBD

Provided herein are single-chain chimeric polypeptides comprising an extracellular hormone receptor ligand binding domain (e.g., any hormone receptor ligand binding domain described herein or known in the art) and a transmembrane domain (e.g., any transmembrane domain described herein or known in the art), wherein the transmembrane domain and extracellular hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., or any subrange of the range described herein), and both the transmembrane domain and hormone receptor ligand binding domain are not present in a single endogenous single-chain polypeptide in a mammal (e.g., a human).

In some examples of single-chain chimeric polypeptides, the transmembrane domain is directly adjacent to the hormone receptor ligand binding domain (i.e., there are no intervening amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain). In some examples of single chain chimeric polypeptides, the transmembrane domain and the extracellular hormone receptor ligand binding domain are separated by: from 1 amino acid to about 800 amino acids (e.g., any subrange of this range described herein).

In some embodiments, one or more intervening amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain may be or include a linker peptide. In some embodiments, the linker peptide may comprise one or more of the following amino acid sequences: GSGSGSGSGS (SEQ ID NO:20), GSGSGSGS (SEQ ID NO:21), RSGSGSGS (SEQ ID NO:22) or GSGSGSGSGS (SEQ ID NO: 23). In some embodiments, the linker peptide may be encoded by one or more of the following nucleic acid sequences: GGATCCGGCAGCGGATCTGGCAGTGGAAGC (SEQ ID NO:24), GGATCTGGCTCTGGAAGCGGCAGC (SEQ ID NO:25), AGATCCGGATCTGGAAGTGGCTCC (SEQ ID NO:26) or GGAAGTGGATCTGGGAGCGGCTCT (SEQ ID NO: 27). In some embodiments, the linker peptide can, for example, comprise in tandem one or more (e.g., two, three, or four) copies of any of SEQ ID NOs: 20-23. In some embodiments, the linker peptide may be or may include one or more of SEQ ID NOs 45, 47, 49, 58, 60, 62, and 106. In some embodiments, the linker peptide may be or include GS.

In some embodiments, one or more intervening amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain may be or include a sequence that borders a transmembrane domain in an endogenous polypeptide from which the transmembrane domain (e.g., any of the transmembrane domains described herein) is derived. In some embodiments, one or more intervening amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain may be or include one or more additional domains (e.g., any of the exemplary additional domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide can be a single-chain chimeric antigen receptor (e.g., any of the single-chain chimeric antigen receptors described herein). In some embodiments, the chimeric polypeptide may further comprise one or more (e.g., two, three, four, or five) additional domains. Non-limiting examples of additional domains that may be included in a single chain chimeric polypeptide include one or more of the following: an extracellular ligand-binding domain (e.g., an antigen-binding domain such as any of the antigen-binding domains described herein), an ITIM (e.g., any ITIM known in the art), an ITAM (e.g., any ITAM described herein or known in the art), a dimerization domain (e.g., capable of dimerizing with another dimerization domain present in another polypeptide (e.g., a recombinant polypeptide) expressed in a mammalian cell, such as any dimerization domain known in the art), and a peptide tag (e.g., a poly-His tag).

In some embodiments, the single-chain chimeric polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments, the single-chain chimeric polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction. In some embodiments, the single-chain chimeric polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments, the single-chain chimeric polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction.

Single-chain chimeric antigen receptors

Single-chain chimeric antigen receptor-intracellular hormone receptor LBD

Also provided herein are single-chain chimeric antigen receptors comprising: an extracellular antigen-binding domain (e.g., any antigen-binding domain described herein or known in the art), a transmembrane domain (e.g., any transmembrane domain described herein or known in the art), a hormone receptor ligand-binding domain (e.g., any hormone receptor ligand-binding domain described herein or known in the art), a costimulatory domain (e.g., any costimulatory domain described herein or known in the art), and an immunoreceptor tyrosine-based activation motif (ITAM), wherein: the transmembrane domain and hormone ligand binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein), and both the transmembrane domain and hormone receptor ligand binding domain are not present in the same (single) endogenous single chain polypeptide in a mammal. The single-chain chimeric antigen receptors described herein can bind to any of the exemplary antigens described herein or any other antigen known in the art. In some embodiments, the single-chain chimeric antigen receptor specifically binds to a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, a single-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of the exemplary antigens described herein).

In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain is contiguous with the hormone receptor ligand binding domain. In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein). In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain do not comprise a domain. In other embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain include one or more intervening domains (e.g., one or more costimulatory domains and/or one or more ITAMs). In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain comprise sequences from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain comprise a linker sequence.

Some embodiments of these single-chain chimeric antigen receptors may include one or more (e.g., two, three, four, or five) costimulatory domains (e.g., any combination of any of the exemplary costimulatory domains described herein or known in the art). Some embodiments of these single-chain chimeric antigen receptors include one or both of a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain.

Some embodiments of these single-chain chimeric antigen receptors may include one or more (e.g., two, three, four, or five) ITAMs (e.g., any ITAM described herein or known in the art). In some embodiments of these single chain chimeric antigen receptors, the ITAMs include cytoplasmic signaling sequences from CD3 ζ (e.g., human CD3 ζ).

In some embodiments of any of these single-chain chimeric antigen receptors, any two adjacent domains may be separated by: 1 amino acid to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 amino acids, or about 3 amino acids (inclusive); about 2 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, or about 4 amino acids (inclusive); about 3 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, or about 5 amino acids (inclusive); about 4 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, about 8 amino acids, or about 6 amino acids (inclusive); about 5 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, about 8 amino acids, or about 7 amino acids (inclusive); about 6 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, or about 8 amino acids (inclusive); about 8 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, or about 10 amino acids (inclusive); about 10 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, or about 15 amino acids (inclusive); about 15 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids (inclusive); about 20 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids (inclusive); about 25 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, about 35 amino acids, or about 30 amino acids (inclusive); about 30 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids, or about 35 amino acids (inclusive); about 35 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about, About 45 amino acids, about 40 amino acids inclusive; from about 40 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, or about 45 amino acids (inclusive); about 45 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, or about 50 amino acids (inclusive); from about 50 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, or about 55 amino acids (inclusive); about 55 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, or about 60 amino acids (inclusive); about 60 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, or about 65 amino acids, inclusive; about 65 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, or about 70 amino acids, inclusive; about 70 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, or about 75 amino acids (inclusive); about 75 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, or about 75 amino acids (inclusive); about 75 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, or about 80 amino acids, inclusive; about 80 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, or about 90 amino acids, inclusive; from about 100 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, or about 110 amino acids, inclusive; about 110 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, or about 120 amino acids (inclusive); from about 120 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, or about 120 amino acids (inclusive); from about 120 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, or about 130 amino acids, inclusive; about 130 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, or about 140 amino acids, inclusive; about 140 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, or about 150 amino acids (inclusive); from about 150 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, or about 160 amino acids, inclusive; from about 160 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, about 180 amino acids, or about 170 amino acids, inclusive; about 170 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, about 190 amino acids, or about 180 amino acids (inclusive); about 180 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, about 200 amino acids, or about 190 amino acids (inclusive); about 190 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, about 220 amino acids, or about 200 amino acids (inclusive); from about 200 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, about 240 amino acids, or about 220 amino acids (inclusive); about 220 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, about 260 amino acids, or about 240 amino acids, inclusive; about 240 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, about 280 amino acids, or about 260 amino acids (inclusive); about 260 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, about 300 amino acids, or about 280 amino acids (inclusive); about 280 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, about 320 amino acids, or about 300 amino acids (inclusive); from about 300 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, about 340 amino acids, or about 320 amino acids (inclusive); from about 320 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, about 360 amino acids, or about 340 amino acids (inclusive); about 340 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, about 380 amino acids, or about 360 amino acids (inclusive); from about 360 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, about 400 amino acids, or about 380 amino acids (inclusive); from about 380 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, about 420 amino acids, or about 400 amino acids (inclusive); from about 400 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, about 440 amino acids, or about 420 amino acids (inclusive); about 420 amino acids to about 500 amino acids, about 480 amino acids, about 460 amino acids, or about 440 amino acids (inclusive); about 440 amino acids to about 500 amino acids, about 480 amino acids, or about 460 amino acids (inclusive); from about 460 amino acids to about 500 amino acids or about 480 amino acids (inclusive); or about 480 amino acids to about 500 amino acids inclusive.

In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain are sequences from the same endogenous single-chain polypeptide from which the transmembrane domain is derived (e.g., a CD 8a hinge region, such as SEQ ID NO: 112). In some embodiments, the sequence comprising SEQ ID NO 42, 55, or 112 is positioned between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, the one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain are or include a hinge region sequence of an antibody, such as, but not limited to, a human antibody (e.g., IgG1, IgG2, IgG3, or IgG 4). In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain are or comprise a linker sequence (e.g., a non-naturally occurring linker sequence, such as GS or any other linker sequence described herein).

In some embodiments of the single-chain chimeric antigen receptor, the next domain is a co-stimulatory domain followed by an ITAM after the hormone receptor ligand binding domain. In some of these embodiments, the one or more amino acids between the hormone receptor ligand binding domain and the co-stimulatory domain are or include sequences from the same endogenous single chain polypeptide from which the co-stimulatory domain is derived or from which the ITAM is derived. In some of these embodiments, the one or more amino acids between the hormone receptor ligand binding domain and the co-stimulatory domain are or include a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the co-stimulatory domain and the ITAM are or include sequences from the same endogenous single chain polypeptide from which the co-stimulatory domain is derived or from which the ITAM is derived.

In some embodiments of the single-chain chimeric antigen receptor, the next domain is a co-stimulatory domain followed by an ITAM after the hormone receptor ligand binding domain. In some embodiments of the single-chain chimeric antigen receptor, the next domain is an ITAM followed by a co-stimulatory domain after the ligand binding domain. In some of these embodiments, the one or more amino acids between the hormone receptor ligand binding domain and the ITAM are or include sequences from the same endogenous single chain polypeptide from which the co-stimulatory domain is derived or from which the ITAM is derived. In some of these embodiments, one or more amino acids between the hormone receptor ligand binding domain and the ITAM is or includes a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between an ITAM and a co-stimulatory domain are or include sequences from the same endogenous single-chain polypeptide from which the ITAM or the co-stimulatory domain is derived.

In some embodiments, where two or more co-stimulatory domains are included in the single-chain chimeric antigen receptor, the two or more co-stimulatory domains may be placed between the hormone receptor ligand binding domain and one or more ITAMs. In some embodiments of the single-chain chimeric antigen receptors described herein, the co-stimulatory domain and ITAM may alternate in the primary amino acid sequence of the single-chain chimeric antigen receptor.

Some embodiments of any of the single-chain chimeric antigen receptors described herein may further comprise a dimerization domain and/or a peptide tag.

In some examples, the single-chain chimeric antigen receptor may comprise a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 31, 33, 35, 37, 39, or 52.

Single-chain chimeric antigen receptor-extracellular hormone receptor LBD

Also provided herein are single-chain chimeric antigen receptors comprising: an extracellular antigen-binding domain (e.g., any antigen-binding domain described herein or known in the art), an extracellular hormone receptor ligand-binding domain (e.g., any hormone receptor ligand-binding domain described herein or known in the art), a transmembrane domain (e.g., any transmembrane domain described herein or known in the art), a costimulatory domain (e.g., any costimulatory domain described herein or known in the art), and an immunoreceptor tyrosine-based activation motif (ITAM), wherein: the transmembrane domain and the extracellular hormone ligand-binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., any subrange of this range described herein), and the transmembrane domain and the extracellular hormone receptor ligand-binding domain are not present in the same endogenous single chain polypeptide in the mammal. The single-chain chimeric antigen receptors described herein can bind to any of the exemplary antigens described herein or any other antigen known in the art. In some embodiments, the single-chain chimeric antigen receptor specifically binds to a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, a single-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of the exemplary antigens described herein).

In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain is contiguous with the extracellular hormone receptor ligand binding domain. In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain and the extracellular hormone receptor ligand binding domain are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein). In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain do not comprise a domain. In other embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain include one or more intervening domains (e.g., an antigen binding domain or additional antigen binding domains). In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain include sequences from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain comprise a linker sequence.

In some embodiments of any of these single-chain chimeric antigen receptors, any two adjacent domains may be separated by 1 amino acid to about 500 amino acids (e.g., or any subrange of this range described herein).

In some embodiments, the extracellular hormone receptor ligand binding domain or one or more amino acids between the extracellular antigen binding domain and the transmembrane domain are from the same endogenous single chain polypeptide from which the transmembrane domain is derived (e.g., a hinge sequence from human CD 8a, e.g., SEQ ID NO: 112). In some embodiments, the sequence comprising SEQ ID NO 42, 55, or 112 is positioned between the extracellular hormone receptor ligand binding domain or extracellular antigen binding domain and the transmembrane domain. In some embodiments, the extracellular antigen-binding domain or one or more amino acids between the extracellular hormone receptor ligand binding domain and the transmembrane domain is or includes a hinge region sequence of an antibody, such as, but not limited to, a human antibody (e.g., IgG1, IgG2, IgG3, or IgG 4). In some embodiments, one or more amino acids between the extracellular antigen-binding domain or extracellular hormone receptor ligand-binding domain (e.g., any of the hormone receptor ligand-binding domains described herein or known in the art) and the transmembrane domain is or comprises a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the extracellular hormone receptor ligand-binding domain is or comprises a linker sequence (e.g., a non-naturally occurring linker sequence, such as GS or any other linker sequence described herein).

In some embodiments of the single-chain chimeric antigen receptor, the next domain is a co-stimulatory domain followed by an ITAM after the transmembrane domain. In some embodiments of the single-chain chimeric antigen receptor, the next domain is an ITAM followed by a costimulatory domain after the transmembrane domain. In some of these embodiments, the one or more amino acids between the transmembrane domain and the costimulatory domain or ITAM are or include sequences from the same endogenous single-chain polypeptide from which the costimulatory domain or from which the ITAM is derived. In some of these embodiments, one or more amino acids between the transmembrane domain and the costimulatory domain or ITAM is or includes a linker sequence (e.g., a non-naturally occurring linker sequence). In some of these embodiments, one or more amino acids between the co-stimulatory domain and the ITAM is or includes a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the co-stimulatory domain and the ITAM are or include sequences from the same endogenous single chain polypeptide from which the co-stimulatory domain is derived or from which the ITAM is derived.

In some embodiments, where two or more co-stimulatory domains are included in a single-chain chimeric antigen receptor, the two or more co-stimulatory domains may be placed between one or more ITAMs. In some embodiments, where two or more ITAMs are included in a single-chain chimeric antigen receptor, the two or more ITAMs may be placed between one or more co-stimulatory molecules. In some embodiments of the single-chain chimeric antigen receptors described herein, the co-stimulatory domain and ITAM may alternate in the primary amino acid sequence of the single-chain chimeric antigen receptor.

In some embodiments, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the N-terminal to C-terminal direction. In some embodiments, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the C-terminal to N-terminal direction. In some embodiments, the single-chain chimeric antigen receptor comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the N-terminal to C-terminal direction. In some embodiments, the single-chain chimeric polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen-binding domain, a transmembrane domain, an intracellular costimulatory domain, and ITAMs when moved in the C-terminal to N-terminal direction.

In some examples, a single-chain chimeric antigen receptor may comprise a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 98.

Multi-chain chimeric antigen receptors

Multi-chain chimeric antigen receptor-intracellular hormone receptor LBD

Also provided herein are multi-chain chimeric antigen receptors comprising at least one first polypeptide comprising: an extracellular antigen-binding domain (e.g., any antigen-binding domain described herein or known in the art), a transmembrane domain (e.g., any transmembrane domain described herein or known in the art), and a hormone receptor ligand-binding domain (e.g., any hormone receptor ligand-binding domain described herein or known in the art), wherein the transmembrane domain and the hormone receptor ligand-binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., or any subrange of the range described herein), and both the transmembrane domain and the hormone receptor ligand-binding domain are not present in the same (single) endogenous single-chain polypeptide. The antigen specifically bound by the antigen binding domain in any of these multi-chain chimeric antigen receptors may be any antigen described herein or known in the art. In some embodiments, the multi-chain chimeric antigen receptor specifically binds to a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, the multi-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of any of the exemplary antigens described herein).

In some embodiments of the multi-chain chimeric antigen receptor, the extracellular antigen-binding domain and the transmembrane domain of the at least one first polypeptide are contiguous with each other. In some embodiments of the multi-chain chimeric antigen receptor, 1 to about 500 amino acids (e.g., any subrange of this range described herein) are between the extracellular antigen-binding domain and the transmembrane domain in the at least one first polypeptide. In some embodiments, the one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain in the at least one first polypeptide are sequences from the same endogenous single-chain polypeptide from which the transmembrane domain is derived. In some embodiments, the one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain of the at least one first polypeptide is or includes a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG 4). In some embodiments, the one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain in the at least one first polypeptide is or includes a linker sequence (e.g., a non-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, at least one first polypeptide is adjoined by a hormone receptor ligand binding domain across a membrane domain. In some embodiments of these multi-chain chimeric antigen receptors, the transmembrane domain and the hormone receptor ligand binding domain in the at least one first polypeptide are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein). In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain in the at least one first polypeptide do not comprise a domain. In other embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain in at least one first polypeptide comprise one or more intervening domains (e.g., one or more co-stimulatory domains (e.g., any of the co-stimulatory domains described herein) and/or one or more ITAMs (e.g., any of the ITAMs described herein)). In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain in the at least one first polypeptide comprise sequences from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain in the at least one first polypeptide comprise a linker sequence (e.g., a non-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, the at least one first polypeptide may further comprise one or more (e.g., two or three) of the following: a dimerization domain (e.g., any dimerization domain known in the art), a costimulatory domain (e.g., any costimulatory domain described herein), and/or a peptide tag (e.g., any peptide tag known in the art).

Some embodiments of these multi-chain chimeric antigen receptors further include a second polypeptide, which may include one or more (e.g., two, three, or four) of the following: a transmembrane domain (e.g., any of the transmembrane domains described herein), a dimerization domain (e.g., a dimerization domain that can interact with a dimerization domain in at least one first polypeptide in a mammalian cell), one or more costimulatory domains (e.g., any of the costimulatory domains described herein), and one or more ITAMs (e.g., any of the ITAMs described herein). As will be appreciated by those skilled in the art, one or each pair of adjacent domains in the second polypeptide may be contiguous with each other or may be separated by 1 to about 800 amino acids (e.g., any subrange of this range described herein). One or more amino acids between a pair of adjacent domains in the second polypeptide may be a sequence from an endogenous single chain polypeptide from which the transmembrane, costimulatory domain, or ITAM present in the second polypeptide is derived.

Multi-chain chimeric antigen receptor-extracellular hormone receptor LBD

Also provided herein are multi-chain chimeric antigen receptors comprising at least one first polypeptide comprising: an extracellular antigen-binding domain (e.g., any antigen-binding domain described herein or known in the art), an extracellular hormone receptor ligand-binding domain (e.g., any hormone receptor ligand-binding domain described herein or known in the art), and a transmembrane domain (e.g., any transmembrane domain described herein or known in the art), wherein the transmembrane domain and extracellular hormone receptor ligand-binding domain are directly contiguous with each other or are separated by 1 to about 800 amino acids (e.g., any subrange of this range described herein), and both the transmembrane domain and hormone receptor ligand-binding domain are not present in a single endogenous single-chain polypeptide. The antigen specifically bound by the antigen binding domain in any of these multi-chain chimeric antigen receptors may be any antigen described herein or known in the art. In some embodiments, the multi-chain chimeric antigen receptor specifically binds to a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, the multi-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of any of the exemplary antigens described herein).

In some embodiments of the multi-chain chimeric antigen receptor, the extracellular hormone receptor ligand binding domain and the transmembrane domain are contiguous with each other in at least one first polypeptide. In some embodiments of the multi-chain chimeric antigen receptor, 1 to about 700 amino acids (e.g., any subrange of this range described herein) are between the extracellular hormone receptor ligand binding domain and the transmembrane domain in the at least one first polypeptide. In some embodiments, the one or more amino acids between the extracellular hormone receptor ligand binding domain and the transmembrane domain in the at least one first polypeptide are sequences from the same endogenous single chain polypeptide from which the transmembrane domain is derived. In some embodiments, the one or more amino acids between the extracellular hormone receptor ligand binding domain and the transmembrane domain of the at least one first polypeptide is or includes a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG 4). In some embodiments, the one or more amino acids between the extracellular hormone receptor ligand binding domain and the transmembrane domain in the at least one first polypeptide is or includes a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the extracellular hormone receptor ligand binding domain and the antigen binding domain is or includes a linker sequence (e.g., a non-naturally occurring linker sequence).

In some embodiments of these multi-chain chimeric antigen receptors, the at least one first polypeptide is adjoined by an extracellular hormone receptor ligand binding domain across a membrane domain. In some embodiments of these multi-chain chimeric antigen receptors, the transmembrane domain and the extracellular hormone receptor ligand binding domain in the at least one first polypeptide are separated by 1 to about 800 amino acids (e.g., or any subrange of this range described herein). In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain in the at least one first polypeptide do not comprise a domain.

In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand binding domain in the at least one first polypeptide comprise one or more intervening domains (e.g., antigen binding domains). In some embodiments, the amino acids between the transmembrane domain and the antigen-binding domain in at least one first polypeptide comprise sequences from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, the amino acids between the transmembrane domain and the antigen-binding domain in at least one first polypeptide comprise a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, the one or more amino acids between the antigen binding domain and the transmembrane domain of the at least one first polypeptide is or includes a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG 4). In some embodiments, one or more amino acids between the antigen-binding domain and the extracellular hormone receptor ligand-binding domain in the at least one first polypeptide is or includes a linker sequence (e.g., a non-naturally occurring linker sequence).

In some embodiments, the at least one first polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments, the at least one first polypeptide comprises an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction. In some embodiments, the at least one first polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen-binding domain, and a transmembrane domain when moved in an N-terminal to C-terminal direction. In some embodiments, the at least one first polypeptide comprises an extracellular hormone receptor ligand binding domain, an extracellular antigen-binding domain, and a transmembrane domain when moved in a C-terminal to N-terminal direction.

Some embodiments of these multi-chain chimeric antigen receptors further include a second polypeptide, which may include one or more (e.g., two, three, or four, in any order or combination) of: a transmembrane domain (e.g., any of the transmembrane domains described herein), a dimerization domain (e.g., a dimerization domain that can interact with a dimerization domain in at least one first polypeptide in a mammalian cell), one or more costimulatory domains (e.g., any of the costimulatory domains described herein), and one or more ITAMs (e.g., any of the ITAMs described herein). As will be appreciated by those skilled in the art, one or each pair of adjacent domains in the second polypeptide may be contiguous with each other or may be separated by 1 to about 800 amino acids (e.g., any subrange of this range described herein). One or more amino acids between a pair of adjacent domains in the second polypeptide may be a sequence from an endogenous single chain polypeptide from which the transmembrane, costimulatory domain, or ITAM present in the second polypeptide is derived. One or more amino acids between a pair of adjacent domains in the second polypeptide can be or can include a linker sequence (e.g., a non-naturally occurring linker sequence).

As can be appreciated in the art, two or more polypeptides present in a multi-chain chimeric antigen receptor may associate via a pair of domains that interact with each other (via a dimerization domain). In some embodiments, the interaction between dimerization domains may be triggered by the addition of small molecules. In some embodiments, two or more polypeptides present in a multimeric chimeric antigen receptor may associate through a non-covalent interaction (e.g., between associations between dimerization domains). In some embodiments, two or more polypeptides present in a multimeric chimeric antigen receptor may interact through covalent interactions (e.g., through disulfide bonds, through ester bonds, through amide bonds, through thioester bonds, or a combination thereof).

Transmembrane domain

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain or portion thereof from an endogenous polypeptide, wherein the endogenous polypeptide is selected from the group consisting of: alpha chain of T cell receptor, beta chain of T cell receptor, zeta chain of T cell receptor, CD28 (also known as Tp44), CD3 epsilon, CD3 delta, CD3gamma, CD33 (also known as GP 33-40 or TSPAN 33), CD33 (also known as FCGR1 33), CD33 (also known as B33, B33-1, B7.1, BB 33, CD28LG 33 and LAB 33), CD33 (also known as PTPRC, B220, CD45 33, GP180, L-CA, LCA, LY 33, T200 and protein tyrosine phosphatase, type C receptor), CD33 (also known as LEU 33 and T33), CD33 alpha (also known as Leu 33, MAL and P33), CD33 (also known as BTCC-1, DRAP-27, MRS-72, TSLAB 33 and T33), CD33 (also known as TNFR-33), CD 33-III family III, CD 33-III, CD137 (also known as TNFRSF9, 4-1BB, CDw137, ILA and tumor necrosis factor receptor superfamily member 9), CD27 (also known as S152, S152.LPFS2, T14, TNFRSF7 and Tp55), CD152 (also known as CTLA4, ALPS5, CELIAC3, CTLA-4, GRD4, GSE, IDDM12 and cytotoxic T lymphocyte-associated protein 4), PD1 (also known as PDCD1, CD279, PD-1, SLEB2, hPD-1, hPD-l, hSLE1 and programmed cell death 1), ICOS (also known as AILIM, CD278 and CVID1), CD272 (also known as BTLA and BTLA1), CD30 (also known as TNFRSF8, D1S166E and Ki-1), GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357 and GITR-D), HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHT TR and TR2), DAP10 and CD154 (also known as CD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L and CD40 ligand). The letter "CD" in the preceding sentence represents a "differentiated group". For example, CD3 represents "differentiation group 3". In some embodiments, a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor includes a transmembrane domain or portion thereof from an endogenous mammalian (e.g., human) polypeptide (e.g., a mammalian or human homolog of any of the polypeptides listed above).

Any transmembrane domain or portion thereof used to anchor an endogenous polypeptide in a lipid bilayer (e.g., plasma membrane) of a mammalian cell is suitable for use in accordance with the compositions and methods disclosed herein. In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain from human CD28 or a portion thereof, e.g., accession number P01747, e.g., amino acids 153 to 179 of SEQ ID No. 1. In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor includes a transmembrane domain that is at least 80% (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%) identical to amino acids 153 to 179, or a portion thereof, of SEQ ID No. 1.

In some embodiments, the transmembrane domain may comprise a sequence at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:114 (or a portion thereof).

SEQ ID NO:1

MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

In some embodiments, the single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain from human CD3 or a portion thereof, e.g., accession number P20963, e.g., amino acids 31 to 51 of SEQ ID No. 2. In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain that is or comprises the sequence: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical) to amino acids 31 to 51 of SEQ ID No. 2.

SEQ ID NO:2

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises the transmembrane domain of any one of SEQ ID nos. 3-9, or a portion thereof.

LGLLVAGVLVLLVSLGVAIHLCC(SEQ ID NO:3)；

VAAILGLGLVLGLLGPLAILLALYLL(SEQ ID NO:4)；

ALIVLGGVAGLLLFIGLGIFFCVRC(SEQ ID NO:5)；

LCYLLDGILFIYGVILTALFLRV(SEQ ID NO:6)；

WVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:7)；

IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 8); and

ALPAALAVISFLLGLGLGVACVLA(SEQ ID NO:9)。

in some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain that is or comprises the sequence: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical) to any one of SEQ ID nos. 3-9.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a transmembrane domain that is or comprises the sequence: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical) to SEQ ID No. 43, 56, or 114.

As will be understood by those of ordinary skill in the art, certain endogenous polypeptides have two or more isoforms that differ in at least their primary polypeptide sequence. The single-chain chimeric polypeptides, single-chain chimeric antigen receptors, or multi-chain chimeric antigen receptors disclosed herein can include a transmembrane domain comprising a sequence of amino acids from any isoform of an endogenous transmembrane protein (e.g., an endogenous mammalian, e.g., human transmembrane protein) including, for example, the following isoforms (e.g., human isoforms): an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD 154.

In some embodiments, the transmembrane domain of a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor, or portion thereof, comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the transmembrane domain from one or more of the following endogenous mammalian (e.g., human) transmembrane proteins, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, the zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD 154. In some embodiments, the transmembrane domain of a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor, or portion thereof, comprises a sequence having one or more amino acid substitutions, deletions, or additions compared to the transmembrane domain of an endogenous mammalian (e.g., human) transmembrane protein of: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD 154.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a synthetic transmembrane domain. In some cases, the synthetic transmembrane domain may include predominantly hydrophobic residues, such as, but not limited to, leucine and valine. In some embodiments, the synthetic transmembrane domain includes a triplet of phenylalanine, tryptophan, and valine at each end of the domain.

In some embodiments, a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor includes a transmembrane domain that is a chimeric transmembrane domain having portions from the transmembrane domains of two or more endogenous mammalian (e.g., human) transmembrane polypeptides, such as, but not limited to, the alpha chain of a T cell receptor, the beta chain of a T cell receptor, the zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, and CD154, e.g., two or more portions of the transmembrane domain together comprise a functional transmembrane domain. In some embodiments, portions of such chimeric transmembrane domains may include one or more amino acid substitutions, deletions, or additions compared to the corresponding portion of the wild-type transmembrane domain.

The transmembrane domain may comprise one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid sequences, each of which traverses the lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell. As known in the art, a transmembrane domain may, for example, comprise at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) contiguous amino acid sequence (that traverses a lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell) having an alpha-helical secondary structure in the lipid bilayer. In some embodiments, the transmembrane domain may comprise two or more contiguous amino acid sequences (each of which traverses the lipid bilayer when present in a corresponding endogenous polypeptide when expressed in a mammalian cell) that form a β -barrel secondary structure in the lipid bilayer. Additional examples and features of transmembrane domains are known in the art.

Hormone receptor ligand binding domains

In some embodiments, the hormone receptor ligand binding domain is or comprises an estrogen receptor ligand binding domain or a fragment thereof. Additional examples of hormone receptor ligand binding domains that can be used for any polypeptide described herein (e.g., single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor) include the following ligand binding domains: for example, estrogen receptors, ecdysone receptors, PPAR γ receptors, glucocorticoid receptors, androgen receptors, thyroid hormone receptors, mineralocorticoid receptors, progesterone receptors, vitamin D receptors, PPAR α, PPAR β/δ receptors, pregnane X receptors, liver X receptors, farnesoid X receptors, retinoid X receptors, RAR-related orphan receptors, and retinoic acid receptors.

A non-limiting example of a human estrogen receptor ligand binding domain is amino acids 10 to 247 of SEQ ID NO 10. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 10 to 247 of SEQ ID No. 10.

SEQ ID NO 10 (human)

Another non-limiting example of an estrogen receptor ligand binding domain is amino acids 4 to 241 of SEQ ID NO. 11. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 4 to 241 of SEQ ID No. 11.

SEQ ID NO 11 (human)

In some embodiments, the estrogen receptor ligand binding domain has a wild-type sequence except that it comprises one or more (e.g., two, three, four, five, or six) amino acid substitutions. In some embodiments, the estrogen receptor ligand binding domain has a wild-type sequence except that it comprises amino acid substitutions (each numbered relative to the full-length wild-type sequence of the human estrogen receptor, e.g., SEQ ID NO:119 below) at one or both of amino acid positions 521 (e.g., the G521R substitution) and 537 (e.g., the G537F substitution).

119 (human) SEQ ID NO

Non-limiting examples of estrogen receptor ligand binding domains may include the following sequences: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to seq id No. 110 (or a fragment thereof).

Further non-limiting examples of hormone receptor ligand binding domains are or include the following sequences: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 46, 59, or 110.

Another non-limiting example of an estrogen receptor ligand binding domain is amino acids 314 to 551 of SEQ ID NO: 88. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 314 to 551 of SEQ ID No. 88.

88 (mouse) of SEQ ID NO

Another non-limiting example of an estrogen receptor ligand binding domain is amino acids 315 to 552 of SEQ ID NO: 89. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 315 to 552 of SEQ ID No. 89.

89 (rat)

Another non-limiting example of an estrogen receptor ligand binding domain is amino acids 310 to 547 of SEQ ID NO: 90. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 310 to 547 of SEQ ID No. 90.

SEQ ID NO:90 (Baboon)

In some embodiments, the hormone receptor ligand binding domain is or comprises a progesterone receptor ligand binding domain or a fragment thereof. A non-limiting example of a human progesterone receptor ligand binding domain is amino acids 11 to 258 of SEQ ID NO 12. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 11 to 258 of SEQ ID No. 12.

SEQ ID NO 12 (human)

Another non-limiting example of a ligand binding domain of a progesterone receptor is amino acids 14 to 261 of SEQ ID NO 13. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 14 to 261 of SEQ ID No. 13.

SEQ ID NO 13 (human)

Another non-limiting example of a ligand binding domain of a progesterone receptor is amino acids 679 to 926 of SEQ ID NO 91. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 679 to 926 of SEQ ID No. 91.

91 (mouse) SEQ ID NO

Another non-limiting example of a ligand binding domain of the progesterone receptor is amino acids 676 to 923 of SEQ ID NO 92. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 676 to 923 of SEQ ID No. 92.

SEQ ID NO 92 (rat)

Another non-limiting example of a progesterone receptor ligand binding domain is amino acids 686 to 933 of SEQ ID NO: 93. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% identical to amino acids 686 to 933 of SEQ ID No. 93.

SEQ ID NO:93 (Baboon)

In some embodiments, the hormone receptor ligand binding domain is or comprises an androgen receptor ligand binding domain or fragment thereof. A non-limiting example of a human androgen receptor ligand binding domain is amino acids 2 through 247 of SEQ ID NO 14. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 2 to 247 of SEQ ID No. 14.

SEQ ID NO 14 (human)

Another non-limiting example of an androgen receptor ligand binding domain is amino acids 34 to 279 of SEQ ID NO 15. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 34 to 279 of SEQ ID No. 15.

SEQ ID NO 15 (human)

Another non-limiting example of an androgen receptor ligand binding domain is amino acids 19 to 264 of SEQ ID NO 16. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 19 to 264 of SEQ ID No. 16.

SEQ ID NO 16 (human)

Another non-limiting example of an androgen receptor ligand binding domain is amino acids 652 to 897 of SEQ ID NO 94. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 652 to 897 of SEQ ID No. 94.

94 (mouse)

Another non-limiting example of an androgen receptor ligand binding domain is amino acids 655 to 900 of SEQ ID NO 95. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 655 to 900 of SEQ ID No. 95.

SEQ ID NO 95 (rat)

Another non-limiting example of an androgen receptor ligand binding domain is amino acids 648 to 893 of SEQ ID NO 96. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: 96 is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% identical to amino acids 648 to 893 of SEQ ID NO.

SEQ ID NO 96 (Baboon)

Another non-limiting example of a ligand binding domain is SEQ ID NO 28. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the amino acid sequence of SEQ ID No. 28.

SEQ ID NO 28 (human)

Another non-limiting example of a ligand binding domain is SEQ ID NO. 29. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to the amino acid sequence of SEQ ID No. 29.

SEQ ID NO:29 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of ecdysone receptor. For example, the ligand binding domain of the ecdysone receptor may be amino acids 256 to 467 of SEQ ID NO: 74. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 256 to 467 of SEQ ID No. 74.

SEQ ID NO 74 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of the PPAR γ receptor. For example, the ligand binding domain of the PPAR γ receptor may be amino acids 237 to 504 of SEQ ID NO 75. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 237 to 504 of SEQ ID No. 75.

SEQ ID NO 75 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of the glucocorticoid receptor. For example, the ligand binding domain of the glucocorticoid receptor can be amino acids 531 to 777 of SEQ ID NO: 76. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 531 to 777 of SEQ id No. 76.

76 (human) SEQ ID NO

Another non-limiting example of a ligand binding domain is the ligand binding domain of thyroid hormone receptor. For example, the ligand binding domain of thyroid hormone receptor may be amino acids 162 to 370 of SEQ ID NO 77. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 162 to 370 of SEQ id No. 77.

SEQ ID NO 77 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of mineralocorticoid receptor. For example, the ligand binding domain of the mineralocorticoid receptor may be amino acids 737 to 984 of SEQ ID NO 78. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: 78, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%.

SEQ ID NO:78 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of vitamin D receptor. For example, the ligand binding domain of the vitamin D receptor may be amino acids 124 to 426 of SEQ ID NO. 79. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 124 to 426 of SEQ ID No. 79.

SEQ ID NO:79 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of the PPAR α receptor. For example, the ligand binding domain of the PPAR α receptor may be amino acids 201 to 467 of SEQ ID NO 80. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% identical to amino acids 201 to 467 of SEQ ID No. 80.

SEQ ID NO 80 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of the PPAR β/δ receptor. For example, the ligand binding domain of the PPAR β/δ receptor may be amino acids 173 to 440 of SEQ ID NO: 81. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 173 to 440 of SEQ ID No. 81.

SEQ ID NO:81 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of the pregnane X receptor. For example, the ligand binding domain of the pregnane X receptor may be amino acids 143 to 428 of SEQ ID NO: 82. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 143 to 428 of SEQ ID No. 82.

SEQ ID NO:82 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of liver X receptor. For example, the ligand binding domain of liver X receptor may be amino acids 20 to 255 of SEQ ID NO 83. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 20 to 255 of SEQ ID No. 83.

83 (human) SEQ ID NO

Another non-limiting example of a ligand binding domain is the ligand binding domain of farnesoid X receptor. For example, the ligand binding domain of the farnesoid X receptor can be amino acids 247 to 467 of SEQ ID NO 84. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% or at least 99% identical to amino acids 247 to 467 of SEQ ID No. 84.

84 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of RAR-related orphan receptor a. For example, the ligand binding domain of RAR-related orphan receptor A can be amino acids 217 through 456 of SEQ ID NO 85. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 217 to 456 of SEQ ID No. 85.

SEQ ID NO:85 (human)

Another non-limiting example of a ligand binding domain is the ligand binding domain of RAR-related orphan receptor C. For example, the ligand binding domain of RAR-associated orphan receptor C can be amino acids 268 to 506 of SEQ ID NO 86. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 268 to 506 of SEQ ID No. 86.

86 (human) SEQ ID NO

Another non-limiting example of a ligand binding domain is the ligand binding domain of a retinoic acid receptor. For example, the ligand binding domain of a retinoic acid receptor may be amino acids 156 to 385 of SEQ ID NO 87. In some embodiments, the hormone receptor ligand binding domain is or includes the following sequence: the sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identical to amino acids 156 to 385 of SEQ ID No. 87.

SEQ ID NO:87 (human)

As will be appreciated in the art, amino acids that are not conserved between different mammalian homologs of the hormone receptor ligand binding domain are more likely to play no significant role in the hormone or hormone analog binding activity of the ligand binding domain, while those amino acids that are conserved between different mammalian homologs of the hormone receptor ligand binding domain are more likely to play a significant role in the hormone or hormone analog binding activity of the ligand binding domain. Some ligand binding domains may include one or more (e.g., two, three, four, five, or six) amino acid substitutions of amino acids that are not conserved between different mammalian homologs of the hormone receptor ligand binding domain.

Antigen binding domains

In some embodiments of the single-chain or multi-chain chimeric antigen receptor, the antigen binding domain may be selected from the group consisting of scFv, scFv-Fc, VHH domain, VNAR domain, (scFv)₂And BiTE. Additional examples of antigen binding domains that can be used in single chain chimeric antigen receptors or multi-chain chimeric antigen receptors are known in the art.

Single chain Fv or scFv fragments comprising V in a single polypeptide chain_HDomains and V_LA domain. V_HAnd V_LTypically via a peptide linker. In other examples, the linker may be a single amino acid. In some casesIn an example, the linker can be a chemical bond. See, e.g., Pluckthun, Antibodies from E.&Moore G.P, (Eds.), the Pharmacology of Monoclonal Antibodies, Vol.113, pp.269-315, Spinger-Verlag, New York, 1994. Exemplary V that can be used in scFv_LAnd V_HThe sequences are shown in SEQ ID NO 104 and 108 in the examples. V which may be in scFv_LAnd V_HExemplary linkers used between domains can be any of the exemplary linkers described herein (e.g., a linker having the sequence of SEQ ID NO: 106).

Sc-Fv-Fc fragments include scFv attached to an Fc domain. For example, the Fc domain may be attached, e.g., to the C-terminus of the scFv. The Fc domain may follow V_LOr V_HAccording to the orientation of the variable domains in the scFv. The Fc domain may be any Fc domain known in the art. In some examples, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc domain).

BiTE is the inclusion of two V in a single polypeptide_LAnd two V_H(which together form two scfvs) that can each bind to a different epitope on the same antigen, or each bind to a different antigen. See, e.g., baeuuerle et al, curr. opin. mol. ther.11:22-30,2009; wolf et al, drug discovery Today 10:1237-1244, 2005; and Huehls et al, Immunol.cell biol.93: 290-.

V_HThe H domain is a single monomeric variable antibody domain found in camelids, while V_NARThe domain is a single monomeric variable antibody domain found in cartilaginous fish. V_HH domain and V_NARDomains are described, for example, in Vanaudehove et al, EBioMedicine 8:40-48,2016; krah et al, immunopharmacol.38: 21-28,2016; cromie et al, Curr. Top. Med. chem.15:2543-2557, 2016; kijanka et al, Nanomedicine 10:161-174, 2015; kovaleva et al, expert, Opin, biol, ther, 14: 1527-; de Meyer et al, Trends Biotechnol.32: 263-42, 2014; Mujic-Delic et al, Trends Pharmacol. Sci.35:247-255, 2014; muydermans, Ann.Rev.Biochem.82:775-797, 2013; vincke et al, methods mol.biol.911:15-26,2012; rahbarizadeh et al, Immunol. invest.40:299-338, 2011; van Bockstaele et al, curr, Opin, Investig, drugs10:1212-1224, 2009; wesolowski et, med, microbiol, immunol.198: 157-; de Genst et al, Dev. Comp. Immunol.30:187-198, 2006; muydermans, J.Biotechnol.74:277-302, 2001; and Muydermans et al, Trends biochem. Sci.26:230-235, 2001.

In some embodiments of the multi-chain chimeric antigen receptor, at least two of the polypeptides that make up the multi-chain chimeric antigen receptor can interact together to form an antigen binding domain. In such embodiments, the antigen binding domain may be selected from the group consisting of: antigen binding antibody fragments, amphipathic heavy targeting antibodies (DART), Fab-scFv-Fc, triomab, crossmab, ortho-Fab IgG, IgG-scFv, scFv₂-Fc, diabody, tanden antibody, DART-Fc, scFv-HAS-scFv, DNL-Fab₃DAF (two-in-one or four-in-one), Dutamab, DT-IgG, protrusion-entry-hole (knobs-in-hole) common LC, protrusion-entry-hole assembly, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, scDiabody-Fc, Fcab, k λ -body, orthogonal Fab, DVD-IgG, IgG (H) -scFv, scFv- (H) IgG, IgG (L) -scFv, scFv- (L) -IgG, IgG (L, H) -Fc, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody-HSA, bivalent antibody, a TandAb, diabody, scDiabody-CH3, Charge-pairing antibodies (charge pair antibodies), bivalent antibody-CH 3, Cov-X-Bod, trivalent antibody (Triple Body), minibody (minibody), DVD-Ig, minibody (minibody), two-in-one IgG TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F (ab')₂-scFV₂scFv-KIH, tetravalent HCAb, bivalent antibody-Fc, tandem scFv-Fc, intrabody (intrabody), docking and locking (dock and lock) bispecific antibody, ImmTAC, HSAbody, scDiabody-HAS, tandem scFv, IgG-IgG and scFv1-PEG-scFv₂。

Non-limiting examples of antigen-binding antibody fragments include Fv fragments, Fab fragments, F (ab')₂Fragments and Fab' fragments.Additional examples of antigen-binding antibody fragments are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG (e.g., human or humanized IgG1, IgG2, IgG3, or IgG 4)); an antigen-binding fragment of IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of human or humanized IgA (e.g., human or humanized IgA1 or IgA 2)); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).

Examples of DVD-Ig are described, for example, in Digiammarino et al, Methods mol. biol.899:145-156,2012; jakob et al, MABs 5:358-363, 2013; and U.S. patent No. 7,612,181; 8,258,268, respectively; 8,586,714, respectively; 8,716,450, respectively; 8,722,855, respectively; 8,735,546, respectively; and 8,822,645. Examples of DART are described, for example, in Garber, Nature Reviews Drug Discovery13:799-801, 2014. Examples of triomas, kihIgG with ordinary LC, crossmab, ortho-Fab IgG, two-in-one IgG, IgG-ScFv, scFv2-Fc, diabodies, tanden antibodies, DART-Fc, scFv-HAS-scFv and DNL-Fab3 are described, for example, in Kontermann et al, drug discovery Today 20:838-847, 2015. DAF (two-in-one or four-in-one), Dutamab, DT-IgG, protrusion-entry-hole consensus LC, protrusion-entry-hole assembly, charge-pairing antibody, Fab-arm exchange antibody, SEEDbody, Triomab, LUZ-Y, Fcab, k.lambda.body, orthogonal Fab, DVD-IgG, IgG (H) -scFv, scFv- (H) IgG, IgG (L) -scFv, scFv- (L) -IgG, IgG (L, H) -Fc, IgG (H) -V, V (H) -IgG, IgG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody-HSA, diabody, Tandbody, scDiabody, scABdy-CH 3, bivalent antibody-CH 3, trivalent antibody, and so-called scFv-Fc, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F (ab')₂-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, bivalent antibody-Fc, tandem scFv-Fc, intrabody, dock-and-lock bispecific antibody, ImmTAC, HSAbody, sExamples of cDiabody-HAS, tandem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFv2 are described, for example, in Spiess et al, mol.Immunol.67:95-106,2015.

Any of the antigen binding domains described herein can bind to an antigen with a dissociation equilibrium constant (K)_D) Is less than 1x10^-7M, less than 1x10^-8M, less than 1x10^-9M, less than 1x10^-10M, less than 1x10^-11M, less than 1x10^-12M or less than 1x10^-13And M. In some embodiments, the antigen binding protein complexes provided herein can bind to a first and/or second antigen, wherein K is_DIs about 1x10^-4M to about 1x10^-6M, about 1X10^-5M to about 1x10^-7M, about 1X10^-6M to about 1x10^- ⁸M, about 1X10^-7M to about 1x10^-9M, about 1X10^-8M to about 1x10^-10M or about 1x10^-9M to about 1x10^-11M (inclusive). K of an antigen binding domain can be determined using a variety of different methods known in the art_DValues (e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecule binding kinetics assays, etc.).

Antigens

In some embodiments, the antigen binding domains described herein can bind to a single antigen (e.g., any of the exemplary antigens described herein or known in the art). In some embodiments, the antigen binding domains described herein can bind to two or more different antigens (e.g., two or more of any of the exemplary antigens described herein or known in the art). Non-limiting examples of antigens include: HER2, A33 antigen, 9-0-acetyl-GD 3, CA19-9 marker, BhCG, CA-125 marker, carbonic anhydrase IX (MN/CA IX), calreticulin, CCR5, CCR8, CD2, CD3, CD5, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40L, CD44, CD44V6, CD63, CD70, CD84, CD96, CD100, CC123, CD133, CD137, CD138, CD150, CD152(CTLA-4), CD160, CRTAM, CR 1(CD319), CS M-1(CD226), CD229, CD244,CD272(BTLA), CD274(PDL-1, B7H1), CD279(PD-1), CD319, CD352, CRTAM (CD355), CD358, DR3, GITR (TNFRSF 18), HVEM, ICOS, LIGHT, LTBR, OX40, an activated form of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxic receptors, NTB-A, PEN-5, carcinoembryonic antigen (CEA; CD66E), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin (ephrin) A2(EphA2), Epidermal Growth Factor Receptor (EGFR), epithelial cell adhesion molecule (EpCAM), fucosyl GM1, GD2, GD3, GM2, ganglioside H2, Globo H, glycoprotein 100, HER 638/neu 3, HER4, HER-like factor, HER-like receptor, MCCP-II, LIGHT, LTBR, LG 40, KIR, NKG2, NKG 3, TNF-like receptor, TNF-6, LIKE, LIGU-like receptor, LISH-6, LI, MUC2, MUC3, MUC4, MUC5AC, MUC5_bMUC7, MUC16, Muller Inhibitory Substance (MIS) type II receptor, plasma cell antigen, poly SA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, 2B4(CD244), beta 2-integrin, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KLRGI, LAIR-1, NKG2A, NKR-P IA, Siglec-3, Siglec-7, Siglec-9, TCRa, TCRB, TCR5y, TIM1, LAG3, LAIR1, PD-1H, TIGIT, 2, and TIM 3. Additional examples of antigens are known in the art.

Co-stimulatory domains

In normal lymphocytes, T cell activation is mediated by two classes of intracellular signaling domains. Primary signaling is initiated via MHC-mediated antigen-dependent activation via a T cell receptor (e.g., TCR/CD3 complex). The secondary or costimulatory signal is provided by a distinct receptor that includes a costimulatory signaling domain that functions in an antigen-independent manner. The signal generated by the signaling domain of the TCR alone is insufficient for complete T cell activation; a co-stimulation signal is also required.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a co-stimulatory domain, or portion thereof, from an endogenous mammalian (e.g., human) transmembrane polypeptide selected from the group consisting of: CD27 (also referred to as S152, S152.LPFS2, T14, TNFRSF7 and Tp55), CD28 (also referred to as Tp44), 4-1BB (also referred to as TNFRSF9, CD137, CDw137, ILA and tumor necrosis factor receptor superfamily member 9), OX40 (also referred to as TNFRSF4, ACT35, RP5-902P8.3, IMD16, CD134, TXGP 116 and tumor necrosis factor receptor superfamily member 4), CD16 (also referred to as TNFRSF 16, D1S166 16 and Ki-1), CD40 16 (also referred to as CD40 16, CD154, HIGM 16, IGM, IMD16, T-BARM, TNFRSF 16, TRAP, gp 16, hCD40 16 and CD16 ligand), CD16 (also referred to as Bp 16, TNFRSF 16, CD16, PDF 72, PDL 16, PDL-16, SLEP-16, ICOS (also known as AILIM, CD278 and CVID1), LFA-1 (also known as lymphocyte function-associated antigen 1), CD1 (also known as LFA-2, SRBC, T1 and CD1 molecules), CD1 (also known as GP1, LEU-9, TP 1, Tp 1 and CD1 molecules), CD160 (also known as BY 1, NK1 and CD160 molecules), LIGHT (also known as TNFSF1, CD258, HVEML, LIGHT, LTg, TR 1, TNLG 11 and tumor necrosis factor superfamily member 14), BTLA (also known as CD272 and BTLA1), TIM1 (also known as CR 1, HACR-2, KIM-3, 1, TIMD 1, TIMM-3, TIM-3, CD 36366 and hepatitis A virus receptor 2), CD1 (also known as HAVLB 2, NACKB-72, NACKB-3, NKB-1, CD36, D12S2489E, KLR, NKG2-D, NKG2D and the killer lectin-like receptor K1), GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357 and GITR-D), HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR and TR2), TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRIO, CARDl, CD54(ICAM), CD83, DAP10, LAT, SLP76, TRIM, ZAP70 and B7-H3 (also known as CD276, 4-B7-H3, B7H3, B7 RP-H592 and CD276 molecules). The letter "CD" in the preceding sentence represents a "differentiated group". For example, CD3 represents "differentiation group 3". In some embodiments, a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor includes a co-stimulatory domain from an endogenous mammalian (e.g., human) transmembrane polypeptide (e.g., a mammalian or human homolog of any of the polypeptides listed above), or a portion thereof.

Any co-stimulatory domain or portion thereof used to provide a co-stimulatory signal is suitable for use in accordance with the compositions and methods disclosed herein. In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor includes a co-stimulatory domain from human CD28 or a portion thereof (e.g., accession number P01747, e.g., amino acids 180 to 220 from SEQ ID NO: 18). In some embodiments, the co-stimulatory domain is or includes the following sequence or fragment thereof: the sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical (or identical) to amino acids 180 to 220 of SEQ ID No. 18. In some embodiments, the co-stimulatory domain is or includes the following sequence or fragment thereof: the sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (or identical) to SEQ ID No. 116.

SEQ ID NO:18

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a co-stimulatory domain from human 4-1BB or a portion thereof (e.g., accession number Q07011, e.g., amino acids 214 through 255 from SEQ ID NO: 19). In some embodiments, the co-stimulatory domain is or includes the following sequence: the sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to amino acids 214 to 255 or a portion thereof of SEQ ID No. 19.

SEQ ID NO:19

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

In some embodiments, the co-stimulatory domain is or includes the following sequence: the sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 44, 57 or 116 or a portion thereof.

The single-chain chimeric polypeptides, single-chain chimeric antigen receptors, or multi-chain chimeric antigen receptors disclosed herein can include a co-stimulatory domain comprising a sequence of amino acids from any isoform of an endogenous mammalian (e.g., human) transmembrane polypeptide having a co-stimulatory domain, including, for example, the following isoforms: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C or B7-H3 (including but not limited to mammalian or human homologs of any of these polypeptides).

In some embodiments, the co-stimulatory domain of a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor, or portion thereof, comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to the co-stimulatory domain of: the co-stimulatory domain is from one or more of mammalian (e.g., human) CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3. In some embodiments, the co-stimulatory domain of a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor, or portion thereof, comprises a sequence of amino acids having one or more amino acid substitutions, deletions, or additions as compared to the co-stimulatory domain of one or more of the following endogenous mammalian (e.g., human) transmembrane polypeptides: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3 (including but not limited to a mammalian or human homolog of any of these polypeptides).

In some embodiments, a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor includes a co-stimulatory domain that is a chimeric co-stimulatory domain having portions from co-stimulatory domains of two or more endogenous mammalian (e.g., human) transmembrane polypeptides including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3 (including, but not limited to, mammalian or human homologs of any of these polypeptides), such that two or more portions of the transmembrane domain together comprise a functional co-stimulatory domain. In some embodiments, portions of such chimeric costimulatory domains may include one or more amino acid substitutions, deletions, or additions compared to the corresponding portions of the wild-type costimulatory domain.

The co-stimulatory domain of a single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor disclosed herein may be of any suitable length. For example, the co-stimulatory domain may have the following length: from about 20 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids, inclusive; about 25 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, or about 30 amino acids, inclusive; about 30 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, or about 35 amino acids (inclusive); about 35 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, or about 40 amino acids (inclusive); from about 40 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, or about 45 amino acids, inclusive; about 45 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, or about 50 amino acids, inclusive; about 50 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, or about 55 amino acids (inclusive); about 55 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, or about 60 amino acids (inclusive); about 60 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, or about 65 amino acids, inclusive; about 65 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, or about 70 amino acids (inclusive); about 70 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, or about 80 amino acids (inclusive); from about 80 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, about 100 amino acids, about 95 amino acids, or about 90 amino acids, inclusive; from about 90 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, about 110 amino acids, or about 100 amino acids, inclusive; from about 100 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, about 120 amino acids, or about 110 amino acids (inclusive); about 110 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, about 130 amino acids, or about 120 amino acids (inclusive); from about 120 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, about 140 amino acids, or about 130 amino acids (inclusive); about 130 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, about 150 amino acids, or about 140 amino acids (inclusive); from about 140 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, about 160 amino acids, or about 150 amino acids (inclusive); from about 150 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, about 170 amino acids, or about 160 amino acids (inclusive); from about 160 amino acids to about 200 amino acids, about 190 amino acids, about 180 amino acids, or about 170 amino acids (inclusive); from about 170 amino acids to about 200 amino acids, about 190 amino acids, or about 180 amino acids (inclusive); from about 180 amino acids to about 200 amino acids or about 190 amino acids (inclusive); or about 190 amino acids to about 200 amino acids, inclusive.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises two or more co-stimulatory domains, e.g., two, three, four, or five or more co-stimulatory domains. In some embodiments, the two or more co-stimulatory domains are identical (e.g., they have the same amino acid sequence). In some embodiments, the co-stimulatory domains are not identical. For example, the co-stimulatory domain may be selected from different endogenous mammalian (e.g., human) transmembrane polypeptides including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, or B7-H3 (including but not limited to mammalian or human homologs of any of these polypeptides). In some embodiments, the two or more co-stimulatory domains may differ from each other by one or more (e.g., two, three, four, or five) amino acid substitutions, deletions, or additions. In some embodiments, two or more co-stimulatory domains exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to each other.

Immune receptor tyrosine-based activation motifs (ITAM)

ITAMs include tyrosines separated from one leucine or isoleucine by any two other amino acids, and thus can be denoted as Tyr-X-Leu/Ile, for example. ITAMs are typically repeated (e.g., two or more times) in the cytoplasmic tail of certain cell surface proteins of the immune system and are typically separated by six to eight amino acids.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises an ITAM or portion thereof from an endogenous mammalian (e.g., human) polypeptide selected from the group consisting of: CD3 ζ (also known as CD3 zeta), CD3 Δ (CD3 delta), CD3 ε (CD3 epsilon), CD3 γ (CD3gamma), DAP12, FC ε R1 γ (Fc epsilon receptor Igama chain), FcFcFcRy, FcFcft, CD35, CD22, CD79A (antigen receptor complex associated protein alpha chain), CD79B (antigen receptor complex associated protein beta chain), and CD66 d. The letter "CD" in the preceding sentence represents a "differentiated group". For example, CD3 represents "differentiation group 3".

Any ITAM or portion thereof useful for mediating signaling in endogenous mammalian (e.g., human) transmembrane proteins is suitable for use in accordance with the compositions and methods disclosed herein. In some embodiments, the single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises an ITAM or portion thereof from human CD3zeta (e.g., accession number P20963, e.g., the ITAM or portion thereof present at amino acids 52-164 of SEQ ID NO: 17; or SEQ ID NO:118 or portion thereof). In some embodiments, the ITAM comprises a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to the sequence of amino acids 52-165 of SEQ ID NO:17 (or a portion thereof) or the sequence of SEQ ID NO:118 (or a portion thereof).

SEQ ID NO:17

In some embodiments, the ITAM comprises a sequence that is or includes: the sequence is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 48, 61, or 118, or a portion thereof.

As will be understood by those of ordinary skill in the art, certain polypeptides have two or more isoforms that differ at least in their primary polypeptide sequence. For example, different isoforms may be generated as a result of alternative splicing. The single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor disclosed herein can include an ITAM comprising a sequence of amino acids from any isoform of an endogenous mammalian transmembrane polypeptide having an ITAM, including, for example, the mammalian (e.g., human) isoforms of: CD3 ζ, CD3D, CD3E, CD3G, DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B, or CD66 d.

In some embodiments, the ITAMs of a single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor, or portion thereof, include sequences having one or more (e.g., two, three, four, or five) amino acid substitutions, deletions, or additions in comparison to the ITAMs of endogenous mammalian (e.g., human) transmembrane proteins (e.g., one or more of CD3 ζ, CD3D, CD3E, CD3G, DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B, or CD66 d). For example, tyrosine and leucine or isoleucine of ITAM may be retained, while the two amino acids separating them may be replaced with different amino acids.

In some embodiments, a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor includes an ITAM that is a chimeric ITAM having portions of ITAMs from two or more endogenous mammalian (e.g., human) transmembrane polypeptides including, but not limited to, CD3 ζ, CD3D, CD3E, CD3G, DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B, or CD66d (including, but not limited to, mammalian or human homologs of any of these polypeptides) such that the two or more ITAM portions together comprise a functional ITAM. In some embodiments, portions of such chimeric ITAMs may include one or more amino acid substitutions, deletions, or additions compared to the corresponding portions of wild-type ITAMs.

In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises two or more ITAMs, e.g., two, three, four, or five or more ITAMs. In some embodiments, the two or more ITAMs are identical (e.g., they have the same amino acid sequence). In some embodiments, the ITAMs are not identical. For example, an ITAM may be selected from different endogenous mammalian (e.g., human) transmembrane polypeptides including, but not limited to, CD3 ζ, CD3D, CD3E, CD3G, DAP12, FCER1G, FcRy, FcRft, CD35, CD22, CD79A, CD79B (including, but not limited to, mammalian or human homologs of any of these polypeptides). In some embodiments, the two or more ITAMs may differ from each other by one or more amino acid substitutions, deletions, or additions.

Nucleic acids

Also provided herein are nucleic acids encoding any of the single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric antigen receptors. Also provided herein is a set of nucleic acids encoding one or more single-chain polypeptides making up a multi-chain chimeric polypeptide (e.g., where at least one single-chain polypeptide making up the multi-chain chimeric polypeptide is encoded by each nucleic acid in the set).

Carrier

Provided herein are vectors comprising any of the nucleic acids provided herein. A "vector" according to the present disclosure is a polynucleotide capable of inducing expression of a recombinant protein (e.g., a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor) in a mammalian cell. The vectors provided herein may be, for example, in circular or linearized form. Non-limiting examples of vectors include plasmids, SV40 vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors. Non-limiting examples of vectors include lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. See, e.g., Carlens et al, exp.Hematol.28(10:1137-1146, 2000; Park et al, Trends Biotechnol.29(11):550-557, 2011; and Alonso-Camino et al, mol.Ther.nucleic Acids 2: E93, 2013. non-limiting examples of retroviral vectors include retroviral vectors derived from Moloney murine leukemia virus (Moloney leukemia virus), myeloproliferative sarcoma virus, murine embryonic stem cell virus, murine stem cell virus, spleen disease forming virus (spelen focus for virus) or adeno-associated virus. non-limiting examples of retroviral vectors are described, e.g., in U.S. Pat. Nos. 5,219,740 and 6,207,453; Miller et al, Bioques 7: 990, 1989; Miller et al, Humatol.28; 10: 1137-H.11, 2000; Park et al, J. 80. Nature virus, 1989; Nature virus, Inc. 849: 80-9; Gene vaccine et al, J.1993; example, Nature virus vectors, Val. 9: 80. promoter et al., Val. 10. 80. 10. 1989; and 9. J. 10. phage, 1989; and 3. 10. Nature. 10. animal Virus. 10. phage virus. animal leukemia. 10. animal leukemia Virus. 10. U.4. D. Virus. D. J.Immunother.35(9):689-701, 2003; cooper et al, Blood 101: 1637-; verhoeyen et al, Methods mol. biol.506:97-114,2009; and Cavalieri et al, Blood102(2): 497-reservoir 505, 2003.

Exemplary vectors into which any of the nucleic acids provided herein can be inserted are described, for example, in Ausubel et al, eds. "Current Protocols in Molecular Biology" Current Protocols, 1993; and Sambrook et al, eds, "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring harborPress, 1989.

In some embodiments, the vector further comprises a promoter and/or enhancer operably linked to any of the nucleic acids described herein. Non-limiting examples of promoters include promoters from: human Cytomegalovirus (CMV), mouse phosphoglycerate kinase 1, polyoma adenovirus, thyroid stimulating hormone alpha, vimentin, simian virus 40(SV40), tumor necrosis factor, beta-globulin, alpha-fetoprotein, gamma-globulin, beta-interferon, gamma-glutamyltransferase, human ubiquitin C (UBC), Mouse Mammary Tumor Virus (MMTV), rous sarcoma virus, 3-glyceraldehyde phosphate dehydrogenase, beta-actin, metallothionein II (MTII), amylase, human EF1 alpha, cathepsin, muscarinic receptor, retrovirus LTR (e.g., human T cell leukemia virus HTLV), AAV ITR, interleukin 2, collagenase, platelet derived growth factor, adenovirus E2, stromelysin, murine MX, rat insulin, glucose regulatory protein 78, human immunodeficiency virus, Glucose regulatory protein 94, alpha-2-macroglobulin, MHC class I, HSP70, a proliferative protein, an immunoglobulin light chain, a T cell receptor, HLA DQ alpha, HLA DQ beta, interleukin 2 receptor, MHC class II, prealbumin (transthyretin), elastase I, albumin, c-fos, a Neuronal Cell Adhesion Molecule (NCAM), H2B histone, rat growth hormone, human serum amyloid (SAA), muscle creatine kinase, troponin I (tn I), and Gibbon Ape Leukemia Virus (GALV). In some embodiments, the promoter may be an inducible promoter or a constitutive promoter. Additional examples of promoters are known in the art.

In some examples, the vectors provided herein further comprise a poly (a) sequence operably linked to and positioned 3' of the sequence encoding the single chain chimeric polypeptide, the single chain chimeric antigen receptor, or any polypeptide comprising a multi-chain chimeric antigen receptor. Non-limiting examples of poly (A) sequences include poly (A) sequences derived from: bovine growth hormone (Wychik et al, Proc. Natl. Acad. Sci. U.S. A.81(13):3944-3948,1984 and U.S. Pat. No. 5,122,458), mouse beta-globin, mouse alpha-globin (Orkin et al, EMBO J.4(2): 453-1985), human collagen, polyoma virus (Batt et al, mol.cell biol 15(9):4783-4790,1995), herpes simplex virus thymidine kinase gene (HSVTK), IgG heavy chain gene polyadenylation signal (U.S. patent application publication No. 2006/0040354), human growth hormone (hGH) (Szymann ski et al, mol.Therapy 15(7):1340-1347,2007), SV40poly (A) sites, such as SV40 and Schpoly (A) early sites (Szynski et al, mol.12. cell 539 12, Biok et al, 539 12). In some embodiments, the poly (a) sequence includes a highly conserved upstream element (AATAAA). The AATAAA sequence may be substituted, for example, with other hexanucleotide sequences homologous to AATAAA that are capable of signaling polyadenylation, including, for example, ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, and AATAAG. See, for example, WO 06012414a 2).

The poly (A) sequence may, for example, be a synthetic polyadenylation site. See, e.g., Levitt el al, GenesDev.3(7):1019-1025, 1989). In some examples, the poly (a) sequence may be a polyadenylation signal of soluble neuropilin (neuropilin) -1: AAATAAAATACGAAATG are provided. Additional examples of poly (A) sequences are known in the art. Additional examples and aspects of vectors are also known in the art.

Method for introducing nucleic acid or vector into mammalian cell

Any of the nucleic acids and vectors disclosed herein can be introduced into a mammalian cell (e.g., any of the mammalian cells described herein, such as any of the T cells described herein (e.g., human T cells)) using a variety of different methods known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids or vectors into mammalian cells include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer based transfection, cationic polymer transfection, cell extrusion, ultrasonic perforation, optical transfection, immunopathion, hydrodynamic delivery, magnetic transfection, viral transduction (e.g., adenovirus and lentivirus transduction), and nanoparticle transfection. Additional methods of introducing nucleic acids or vectors into mammalian cells are known in the art.

Mammalian cell

Also provided herein are mammalian cells comprising any of the nucleic acids or vectors described herein. In some embodiments, the mammalian cell was previously obtained from a subject (e.g., a human subject, e.g., a human subject identified or diagnosed as having cancer).

Non-limiting examples of mammalian cells include T cells (e.g., human T cells). Non-limiting examples of T cells (e.g., human T cells) include, for example, immature thymocytes, peripheral blood lymphocytes, naive T cells, pluripotent T cells_HCell precursor, lymphoid progenitor cell, T_regCells, memory T cells, T_H17 cells, T_H22 cells, T_H9 cells, T_H2 cells, T_H1 cell, T_H3 cells, γ δ T cells, α β T cells, Treg cells and tumor infiltrating T cells. Additional examples of T cells (e.g., human T cells) include CD8⁺T cell, CD4⁺T cells and natural killer T cells. Additional examples of mammalian cells include mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells.

Hormones and hormone analogs

Non-limiting examples of hormones include estrogen (17 β -estradiol), progesterone, testosterone, and dihydrotestosterone. Non-limiting examples of hormone analogs include estrogen hormone analogs, progestin hormone analogs, and androgen hormone analogs.

Non-limiting examples of estrogen analogs include: afifene (afimoxifene) (also known as 4-hydroxyttamoxifen, 4-OHT, 4-HT, and OHT); selective Estrogen Receptor Modulators (SERMs), such as clomiphene (clomifene), oxymetaxifene (ormeloxifene), raloxifene (raloxifene), tamoxifen (tamoxifen), toremifene (toremifene), lasofoxifene (lasofoxifene), and ospemifene (ospemifene); estradiol; estrone; raloxifene; estriol; genistein (genistein); phytoestrogens (e.g., isoflavones such as genistein, daidzein (daidzein), formononetin (formononetin) and glycitein (glycitein); coumestans such as coumestrol (coumestrol) and repensol andfoliol, and lignans (lignans) such as larch (larciresinol), matairesinol (matairesinol), pinoresinol (pinoresinol), secoisolariciresinol (secoisolariciresinol), podophyllotoxin (podophyllotoxin) and araliphenyl hojaponin (stepanacardin)); and exogenous estrogens (xenoestrogen) (e.g., diethylstilbestrol, ethinylestradiol, butylated hydroxyanisole, erythrosine (erythrosine), camphor (e.g., 4-methylbenzylidene camphor), parabens (parabens) (e.g., methyl, ethyl, and propyl parabens), atrazine (atrazine), dichlorodiphenyldichloroethylene, dichlorodiphenyltrichloroethane, methoxychloride, dieldrin (dieldrin), endosulfan (endosulfan), heptachloro, lindane (lindane), phenols (e.g., bisphenol a and nonylphenol), biphenyls (e.g., monochlorobiphenyl and dichlorobiphenyl), phthalates (e.g., di-2-ethylhexyl phthalate, diisodecyl phthalate, and diisononyl phthalate), and metallic estrogens). In some embodiments, the estrogen analog can be, for example, mifepristone (also known as RU-486). In some embodiments, the estrogen analog may be a Selective Progesterone Receptor Modulator (SPRM), such as onapristone (onapristone) (ZK98299), lonaprisan (lonaprisan) (ZK230211, BAY86-5044), APR19, EC304, WAY-255348, ORG31710, Solprionil (Asoprisnil) (J867), terapristone (telastoprene) (Proellex, CDB-4124), and CDB-2914 (ulipristal acetate).

Non-limiting examples of progesterone hormone analogs include Selective Progesterone Receptor Modulators (SPRMs) such as onapristone (ZK98299), lonelin (ZK230211, BAY86-5044), APR19, EC304, WAY-255348, ORG31710, Soprinib (J867), terapristone (Proellex, CDB-4124), and CDB-2914 (ulipristal acetate).

Non-limiting examples of androgen analogues include enoboxate (also known AS ostarine, MK-2866, GTx-024, and S-22), BMS-564,929, LGD-4033(ligandrol), AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, S-23, RAD140, acetothiolutimide, Andarine, LG-121071, TFM-4AS-1, and YK-11.

As will be appreciated in the art, estrogen or estrogen hormone analogs can be used with single chain chimeric polypeptides, single chain chimeric antigen receptors, or multi-chain chimeric antigen receptors that include an estrogen receptor ligand binding domain.

Compositions and kits

Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the nucleic acids, vectors, sets of nucleic acids, sets of vectors, or mammalian cells described herein. For example, provided herein are compositions comprising any nucleic acid or group of nucleic acids described herein, or any vector or group of vectors provided herein, and a pharmaceutically acceptable solvent or carrier.

In some embodiments, a composition can be any mammalian cell described herein (e.g., any mammalian cell described herein previously obtained from a subject (e.g., a subject identified or diagnosed as having cancer) comprising a nucleic acid encoding any of a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor described herein. In a composition comprising any of the mammalian cells described herein, the composition can further comprise a cell culture medium or a pharmaceutically acceptable buffer (e.g., phosphate buffered saline). Compositions comprising any of the mammalian cells described herein can be formulated for intravenous or intra-arterial administration.

Also provided are kits that include one or more of any of the compositions described herein. For example, a kit can include any of the compositions described herein and one or more doses of a hormone or hormone analog formulated for administration to a subject (e.g., formulated for oral, intravenous, intramuscular, or subcutaneous administration). In some embodiments, the kit may further comprise instructions for performing any of the methods described herein.

Method for inducing membrane localization of single-chain chimeric polypeptides, single-chain chimeric antigen receptors, or multi-chain chimeric polypeptides in mammalian cells

Also provided herein are methods of inducing membrane localization of a single-chain chimeric polypeptide (e.g., any single-chain chimeric polypeptide described herein), a single-chain chimeric antigen receptor (e.g., any single-chain chimeric antigen receptor described herein), or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein) in a mammalian cell (e.g., any mammalian cell described herein), comprising: contacting a mammalian cell expressing a single-chain chimeric polypeptide (e.g., any of the single-chain chimeric polypeptides described herein), a single-chain chimeric antigen receptor (e.g., any of the single-chain chimeric antigen receptors described herein), or a multi-chain chimeric antigen receptor (e.g., any of the multi-chain chimeric antigen receptors described herein) with a hormone or hormone analog in an amount sufficient to induce the single-chain chimeric polypeptide, the single-chain chimeric antigen-binding receptor, or the multi-chain chimeric antigen receptor, respectively, to localize to the extracellular side of the plasma membrane of the cell.

Some embodiments of these methods further comprise introducing a nucleic acid encoding a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor into a mammalian cell (e.g., any mammalian cell described herein or known in the art) to generate a mammalian cell expressing a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor, respectively. In some examples of these methods, the mammalian cell is a T cell (e.g., a CD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, and a natural killer T cell). In some examples, a mammalian cell (e.g., any of the mammalian cells described herein) is a mammalian cell previously obtained from a subject (e.g., a subject that has been identified or diagnosed as having a cancer, e.g., any of the cancers described herein). Some embodiments of these methods further comprise obtaining the mammalian cell from a subject.

In some embodiments of any of these methods, the contacting step is performed in vitro. In some embodiments of any of these methods, the contacting step is performed in a mammal, such as a mammal (e.g., a human) having a cancer (e.g., any of the cancers described herein). In some embodiments, the contacting step results in the treatment of cancer in a mammal (e.g., a human).

Non-limiting examples of cancers that can be treated using any of the methods provided herein include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, kaposi's sarcoma, lymphoma, anal carcinoma, appendiceal carcinoma, teratoid/rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain carcinoma, breast carcinoma, bronchial carcinoma, carcinoid tumor, cardiac tumor, cervical carcinoma, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasms, colon carcinoma, colorectal carcinoma, craniopharyngiocarcinoma, cholangiocarcinoma, endometrial carcinoma, ependymoma, esophageal carcinoma, neuroblastoma (estheoneuroblastoma), ewing's sarcoma, eye carcinoma, fallopian tube carcinoma, gallbladder carcinoma, gastric carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck carcinoma, heart carcinoma, liver carcinoma, hypopharynx (hypopharyngeal carcinoma), pancreatic carcinoma, kidney carcinoma, laryngeal carcinoma, chronic myelogenous leukemia, lip and oral carcinoma, lip and lip cancer, Lung cancer, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer (mouth cancer), oral cancer (oral cancer), osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, throat cancer, thyroid cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulval cancer.

In some embodiments of any of these methods, the method results in a reduction in tumor burden (e.g., tumor mass and/or volume) in the subject. For example, any of the methods described herein can result in a reduction in tumor burden in the subject (e.g., as compared to tumor burden in a pre-treatment subject) of at least about 1% to about 99% (e.g., about 1% to about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 2% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60% >, or, About 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 3% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 5% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% (inclusive); about 10% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 15% (inclusive); about 15% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, or about 20% (inclusive); about 20% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25% (inclusive); about 25% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, or about 30% (inclusive); about 30% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, or about 35% (inclusive); about 35% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, or about 40% (inclusive); about 40% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, or about 45% (inclusive); about 45% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, or about 50% (inclusive); about 50% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, or about 55% (inclusive); about 55% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, or about 60% (inclusive); about 60% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, or about 65% (inclusive); about 65% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, or about 70% (inclusive); about 70% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, or about 72% (inclusive); about 72% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, or about 74% (inclusive); about 74% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, or about 76% (inclusive); about 76% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, or about 78% (inclusive); about 78% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, or about 80% (inclusive); about 80% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, or about 82% (inclusive); about 82% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, or about 84% (inclusive); about 84% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, or about 86% (inclusive); about 86% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, or about 88% (inclusive); about 88% to about 99%, about 98%, about 96%, about 94%, about 92%, or about 90% (inclusive); about 90% to about 99%, about 98%, about 96%, about 94%, or about 92% (inclusive); about 92% to about 99%, about 98%, about 96%, or about 94% (inclusive); about 94% to about 99%, about 98%, or about 96% (inclusive); or from about 96% to about 99% or about 98% (inclusive)).

In some embodiments, the method results in a decrease in the rate of progression of the cancer in the subject. For example, any of the methods described herein can result in a decrease in the rate of progression of the cancer in the subject (e.g., as compared to the rate of progression of the cancer in a pre-treatment subject or in a control subject or a control population of subjects having the same cancer and not administered a treatment or administered a different treatment) by at least about 1% to about 99% (e.g., about 1% to about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 2% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88% >, or, About 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 3% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% (inclusive); about 5% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% (inclusive); about 10% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 15% (inclusive); about 15% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, or about 20% (inclusive); about 20% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, or about 25% (inclusive); about 25% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, or about 30% (inclusive); about 30% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, or about 35% (inclusive); about 35% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, or about 40% (inclusive); about 40% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, or about 45% (inclusive); about 45% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, or about 50% (inclusive); about 50% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, or about 55% (inclusive); about 55% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, or about 60% (inclusive); about 60% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, or about 65% (inclusive); about 65% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, or about 70% (inclusive); about 70% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, or about 72% (inclusive); about 72% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, or about 74% (inclusive); about 74% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, or about 76% (inclusive); about 76% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, or about 78% (inclusive); about 78% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, or about 80% (inclusive); about 80% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, or about 82% (inclusive); about 82% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, or about 84% (inclusive); about 84% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, or about 86% (inclusive); about 86% to about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, or about 88% (inclusive); about 88% to about 99%, about 98%, about 96%, about 94%, about 92%, or about 90% (inclusive); about 90% to about 99%, about 98%, about 96%, about 94%, or about 92% (inclusive); about 92% to about 99%, about 98%, about 96%, or about 94% (inclusive); about 94% to about 99%, about 98%, or about 96% (inclusive); or from about 96% to about 99% or about 98% (inclusive)).

In some embodiments of any of these methods, the method results in an increase in survival time of the cancer in the subject. For example, any of the methods described herein can result in an increase in survival time of the cancer in the subject (e.g., as compared to survival time of a control subject or population of control subjects having the same cancer and not receiving treatment or receiving a different treatment) by about 1% to about 800% (e.g., about 1% to about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, about 20%, about 10%, or about 5% (inclusive); about 5% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, about 20%, or about 10% (inclusive); about 10% to about 800%; or a population of control subjects having the same cancer and not receiving treatment or a different treatment, About 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, or about 20% (inclusive); about 20% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, or about 40% (inclusive); about 40% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, or about 60% (inclusive); about 60% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80% (inclusive); about 80% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% (inclusive); about 100% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, or about 150% (inclusive); about 150% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, or about 200% (inclusive); about 200% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, or about 250% (inclusive); about 250% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, or about 300% (inclusive); about 300% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, or about 350% (inclusive); about 350% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, or about 400% (inclusive); about 400% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, or about 450% (inclusive); about 450% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, or about 500% (inclusive); about 500% to about 800%, about 750%, about 700%, about 650%, about 600%, or about 550% (inclusive); about 550% to about 800%, about 750%, about 700%, about 650%, or about 600% (inclusive); about 600% to about 800%, about 750%, about 700%, or about 650% (inclusive); about 650% to about 800%, about 750%, or about 700% (inclusive); from about 700% to about 800% or about 750% (inclusive); or from about 750% to about 800% (inclusive)).

Method for reversibly altering the membrane localization of a single-chain chimeric polypeptide, a single-chain chimeric antigen receptor or a multi-chain chimeric polypeptide in mammalian cells

Also provided herein is a method of reversibly altering the membrane localization of a single-chain chimeric polypeptide (e.g., any single-chain chimeric polypeptide described herein), a single-chain chimeric antigen receptor (e.g., any single-chain chimeric antigen receptor described herein), or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein) in a mammalian cell, comprising: (a) contacting a mammalian cell (e.g., any mammalian cell described herein) that expresses a single-chain chimeric polypeptide (e.g., any single-chain chimeric polypeptide described herein), a single-chain chimeric antigen receptor (e.g., any single-chain chimeric antigen receptor described herein), or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein) with a hormone or hormone analog in an amount sufficient to induce the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor to localize to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with a reduced amount of a hormone or hormone analog that results in a reduced level of single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the mammalian cell (e.g., as compared to the level on the extracellular side of the plasma membrane in step (a)). In some embodiments, the level reduced in step (b) is at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least an 80% reduction, at least an 85% reduction, at least a 90% reduction, at least a 95% reduction, or at least a 99% reduction in the level of the extracellular side of the plasma membrane in step (a). In some embodiments, the level reduced in step (b) is a 100% reduction in the level of the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor on the extracellular side of the plasma membrane in step (a). In some embodiments, the mammalian cells in step (b) are not contacted with any hormone or hormone analog hormone. Some embodiments of these methods further comprise: (c) contacting a mammalian cell (e.g., any of the mammalian cells described herein) with an increased amount of a hormone or hormone analog that results in an increased level of single chain chimeric polypeptide, single chain chimeric antigen receptor, or multi-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell as compared to the level in step (b).

Some embodiments of any of these methods further comprise introducing a nucleic acid encoding a single-chain chimeric polypeptide into a mammalian cell (e.g., any of the mammalian cells described herein) to generate a mammalian cell that expresses the single-chain chimeric polypeptide, the single-chain chimeric antigen receptor, or the multi-chain chimeric antigen receptor. In some embodiments of any of the methods described herein, the mammalian cell is a T cell (e.g., a human T cell). In some embodiments, the T cell (e.g., human T cell) is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells. In some embodiments, the mammalian cell is a mammalian cell previously obtained from a subject (e.g., a human). In some examples, a subject (e.g., a human) has been identified or diagnosed as having a cancer (e.g., any type of cancer described herein). Some embodiments further comprise testing from a subjectObtaining mammalian cells.

In some embodiments of any of the methods described herein, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in a mammal (e.g., a subject). In some embodiments, the contacting step results in the treatment of cancer in a mammal (e.g., a human).

Non-limiting examples of cancers that can be treated using any of the methods provided herein include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, kaposi's sarcoma, lymphoma, anal carcinoma, appendiceal carcinoma, teratoid/rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain carcinoma, breast carcinoma, bronchial carcinoma, carcinoid tumor, cardiac tumor, cervical carcinoma, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasms, colon carcinoma, colorectal carcinoma, craniopharyngiocarcinoma, cholangiocarcinoma, endometrial carcinoma, ependymoma, esophageal carcinoma, neuroblastoma (estheoneuroblastoma), ewing's sarcoma, eye carcinoma, fallopian tube carcinoma, gallbladder carcinoma, gastric carcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck carcinoma, heart carcinoma, liver carcinoma, hypopharynx (hypopharyngeal carcinoma), pancreatic carcinoma, kidney carcinoma, laryngeal carcinoma, chronic myelogenous leukemia, lip and oral carcinoma, lip and lip cancer, Lung cancer, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, throat cancer, thyroid cancer, urinary tract cancer, uterine cancer, vaginal cancer, and vulvar cancer.

Method for controlling toxicity of single-chain chimeric polypeptide, single-chain chimeric antigen receptor or multi-chain chimeric polypeptide in mammalian cells by reversibly changing membrane localization thereof

In some embodiments, the compositions and/or methods disclosed herein can be used to selectively kill cancer cells in a subject (e.g., a human) in the presence of a hormone or hormone analog that binds to LBD present in a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor). In some embodiments, a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor) is localized to the outer surface of a cell of a mammal in the presence of a hormone or hormone analog, presenting its antigen binding domain (e.g., an scFv or other antigen binding domain described herein or known in the art) on the exterior of the cell. The polypeptide can then recognize the antigen to which it binds and can therefore selectively kill target cells expressing the antigen on their surface.

In some embodiments, localization of a polypeptide (e.g., a single chain chimeric polypeptide, a single chain chimeric antigen receptor, or a multi-chain chimeric antigen receptor) to the exterior surface of a cell can result in toxicity in a subject. Such toxicity may be caused by, for example, cytokine storm (also known as hypercytokinemia or cytokine release syndrome). In some embodiments, a subject who has experienced toxicity due to localization of hormone or hormone analog-induced polypeptides to the extracellular surface may be treated by reducing the dose of hormone or hormone analog administered to the subject, or eliminating hormone or hormone analog therapy altogether. Reduction of such doses or elimination of hormone or hormone analog therapy can result in removal of the polypeptide from the cell surface and reduction or elimination of antigen binding domain presentation on the cell surface, thereby reducing or eliminating toxicity in the subject.

Exemplary symptoms of toxicity include, but are not limited to, high fever, swelling, chills, hypotension, tachycardia, weakness, headache, rash, itchy throat, dyspnea, redness, extreme fatigue, nausea, and cerebral edema (brain swelling). In some embodiments, toxicity in a subject can be determined by assessing the presence or severity of one or more of these symptoms in a subject following administration of one or more doses of a hormone or hormone analog to the subject. In some embodiments, one or more of these or other symptoms associated with toxicity may be determined after introducing mammalian cells (e.g., T cells) comprising a single chain chimeric polypeptide having LBD (e.g., any single chain polypeptide described herein), a single chain chimeric antigen receptor (e.g., any single chain chimeric antigen receptor described herein), or a multiple chain chimeric antigen receptor (e.g., any multiple chain chimeric antigen receptor described herein), and a hormone or hormone analog that binds LBD into a subject. In some embodiments, the determined level of one or more of these or other symptoms associated with toxicity may be compared to a baseline level associated with lack of toxicity or a reduced level of toxicity. Such baseline levels may be, for example, levels of one or more of these or other symptoms in the subject prior to administration of the mammalian cells, the hormone or hormone analog, or both. Additionally or alternatively, such baseline levels may be levels of one or more of these or other symptoms in a different subject that is not administered mammalian cells, hormones or hormone analogs, or both. One of ordinary skill in the art will know of appropriate clinical and laboratory methods to determine the level of one or more symptoms associated with toxicity.

In some embodiments, toxicity may be the result of a cytokine storm. Both pro-inflammatory and anti-inflammatory cytokines are elevated in the serum of subjects undergoing cytokine storm. Non-limiting examples of cytokines that are elevated in the serum of a subject experiencing a cytokine storm include tumor necrosis factor alpha, IFN γ, IL-10, IL-1 β, IL-2, IL-6, IL-8, and IL-10, granulocyte macrophage colony stimulating factor (GM-CSF), and IL-5. In some embodiments, one or more of these or other cytokines associated with a cytokine storm is determined after introducing mammalian cells (e.g., T cells) comprising a single chain chimeric polypeptide having an LBD (e.g., any single chain polypeptide described herein), a single chain chimeric antigen receptor (e.g., any single chain chimeric antigen receptor described herein), or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein), and a hormone or hormone analog that binds to the LBD into a subject. In some embodiments, the determined level of one or more of these or other cytokines associated with a cytokine storm may be compared to a baseline level associated with a lack of a cytokine storm or a reduction in the level of a cytokine storm. Such baseline levels may be, for example, the levels of one or more of these or other cytokines in the subject prior to administration of the mammalian cells, the hormone or hormone analog, or both. Additionally or alternatively, such baseline levels may be levels of one or more of these or other cytokines in a different subject that is not administered mammalian cells, hormones or hormone analogs, or both. The level of the cytokine (e.g., the level of the cytokine after administration of mammalian cells and hormone or hormone analog to the subject and/or the baseline level of the cytokine) can be determined by any of a variety of methods and techniques, including but not limited to ELISA, Western blot, antibody array plates, multiplex arrays, Elispot assays, immunohistochemistry, or any of a variety of commercial assays useful for determining cytokine levels.

In some embodiments, methods of treating cancer (e.g., any of the various cancers described herein) in a subject (e.g., a mammal, e.g., a human) are provided. In some embodiments, a method of treating cancer comprises: (a) administering to a subject a mammalian cell (e.g., a T cell) comprising a nucleic acid encoding a single-chain chimeric polypeptide having a hormone receptor LBD (e.g., any single-chain polypeptide described herein), a single-chain chimeric antigen receptor (e.g., any single-chain chimeric antigen receptor described herein), or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein), (b) administering to the subject a hormone or hormone analog that binds to LBD; (c) determining the level of one or more symptoms associated with toxicity and/or the level of one or more cytokines associated with a cytokine storm, (d) comparing the determined level of one or more symptoms associated with toxicity and/or the level of one or more cytokines associated with a cytokine storm to a baseline level of symptoms or cytokines and (e) reducing the amount of hormone or hormone analog administered to the subject such that the determined level of one or more symptoms associated with toxicity and/or the level of one or more cytokines associated with a cytokine storm is reduced. In some embodiments, the level of hormone or hormone analogue administered to the subject in step (e) is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In some embodiments, the level of hormone or hormone analog administered to the subject in step (e) is reduced by 100% (e.g., no mammalian cells are administered to the subject in step (e)). In some embodiments, after the hormone or hormone analog is administered in step (b), the single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multi-chain chimeric antigen receptor localize to the extracellular surface of the mammalian cell in a reversible manner. In some embodiments, after reducing the amount of hormone or hormone analog administered to the subject in step (e), the single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multi-chain chimeric antigen receptor is removed from the outer surface of the cell, thereby reducing toxicity (e.g., one or more symptoms associated with toxicity and/or one or more cytokines associated with a cytokine storm). In some embodiments, toxicity (e.g., one or more symptoms associated with toxicity and/or one or more cytokines associated with a cytokine storm) is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% after reducing the amount of the hormone or hormone analog administered to the subject in step (e). In some embodiments, after reducing the amount of hormone or hormone analog administered to the subject in step (e), toxicity (e.g., one or more symptoms associated with toxicity and/or one or more cytokines associated with a cytokine storm) is reduced by 100% (e.g., the subject does not experience toxicity or experiences only baseline levels of toxicity). In some embodiments, after reducing the amount of hormone or hormone analog administered to the subject in step (e), toxicity (e.g., one or more symptoms associated with toxicity and/or one or more cytokines associated with a cytokine storm) is normalized to a baseline level of toxicity (e.g., a level associated with a lack of a cytokine storm or a reduction in the level of a cytokine storm). For example, after reducing the amount of hormone or hormone analogue administered to the subject in step (e), toxicity may be normalized to the following levels: less than 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold or less than the baseline level of toxicity.

In some embodiments, a subject may be treated by administering to the subject a mammalian cell (e.g., a T cell) comprising a single chain chimeric polypeptide having an LBD (e.g., any single chain polypeptide described herein), a single chain chimeric antigen receptor (e.g., any single chain chimeric antigen receptor described herein), and/or a multi-chain chimeric antigen receptor (e.g., any multi-chain chimeric antigen receptor described herein), and a hormone or hormone analog that binds the LBD. The level of toxicity in the subject can be monitored, and toxicity can be reduced by any of the methods described herein, e.g., by reducing the level of a hormone or hormone analog. After toxicity is reduced (e.g., to a baseline level, or a sufficiently low level above the baseline level), the level of hormone or hormone analog can then be increased to improve the efficacy of the treatment. In some embodiments, the level of hormone or hormone analog is reduced multiple times and then increased to control toxicity, while still providing an effective course of treatment. In some embodiments, the level of the hormone is subsequently increased to 100% of the level at which the hormone or hormone analog was initially administered. In some embodiments, the level of the hormone or hormone analog is subsequently increased to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the level at which the hormone or hormone analog was initially administered. In some embodiments, the level of the hormone or hormone analog is subsequently increased to a level that is higher than the level at which the hormone or hormone analog was initially administered. For example, the subsequently administered hormone or hormone analog may be administered at a level that is at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 300%, at least 400%, or at least 500% of the level at which the hormone or hormone analog was initially administered.

In some embodiments, toxicity generated after administration of a first hormone or hormone analog to a subject may be reduced by administering a second hormone or hormone analog to the subject. For example, the second hormone or hormone analog may have a reduced affinity for LBD present in the single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multiple chain chimeric antigen receptor such that plasma membrane localization of the single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multiple chain chimeric antigen receptor is reduced as compared to plasma membrane localization facilitated by the first hormone or hormone analog. In some embodiments, the second hormone or hormone analog may be less effective than the first hormone or hormone analog in promoting plasma membrane localization of the single chain chimeric polypeptide, single chain chimeric antigen receptor, and/or multi-chain chimeric antigen receptor. In some embodiments, the second hormone or hormone analog competes with the first hormone or hormone analog for binding to LBD.

In some embodiments, in addition to or as an alternative to reducing the level of hormone or hormone analog to reduce toxicity, toxicity may be further reduced by administering a composition that inactivates or sequesters (sequester) the hormone or hormone analog. For example, exogenous LBD may be administered to a subject (e.g., as a pharmaceutically acceptable formulation) to sequester a hormone or hormone analog previously administered to the subject. As another example, a composition that inactivates, degrades, or otherwise isolates an administered hormone or hormone analog can be administered to a subject. Non-limiting examples of such compositions are antibodies or antibody fragments that bind the administered hormone or hormone analog and render it unable to penetrate the cell membrane, rendering it unable to bind LBD or facilitate its degradation.

Examples

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLE 1 Generation of constructs

Canonical chimeric antigen receptors ("canonical CARs", fig. 1, left) were modified to include an estrogen receptor alpha ("ERa") ligand binding domain ("ERa-LBD", green and indicated by arrows) at various locations within the protein. ERa-LBD was introduced at different positions in CAR (fig. 1, right) to create different versions of ERa-LBD, respectively expressed as: "v.2" (also denoted as "V2" or "V2"), "v.3" (also denoted as "V3" or "V3") and "v.4" (also denoted as "V4" or "V4"). mCherry fusion proteins are also included to allow tracking of cells containing engineered proteins. The nucleic acid and polypeptide sequences of v.2 (SEQ ID NOS: 30 and 31, respectively), v.3 (SEQ ID NOS: 32 and 33, respectively) and v.4 (SEQ ID NOS: 34 and 35, respectively) are provided below.

SEQ ID NO:30

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGAAGTGGATCTGGGAGCGGCTCTATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG

SEQ ID NO:31

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

SEQ ID NO:32

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG

SEQ ID NO:33

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

SEQ ID NO:34

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCAAGCGGGGCAGAAAGAAACTGCTGTACATCTTTAAGCAGCCCTTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGATCCGGATCTGGAAGTGGCTCCCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGAAGTGGATCTGGGAGCGGCTCTATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTA

SEQ ID NO:35

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

Example 2-NHR-LBD-containing anti-CD 19 chimeric antigen receptor exhibits a surface Table of ligand-induced CAR So as to achieve the purpose.

Jurkat T cells were transduced with lentiviral vectors encoding canonical CARs or with anti-CD 19(FMC63) ERa-LBD containing v2, v3 and v4CAR constructs described in example 1. The cells were then untreated or treated with ligands for ERa-LBD. One hour after drug treatment, cells were stained with protein L-biotin (protein L bound to extracellular scFv) and then stained with streptavidin-BV 421 to detect surface expression of CAR. Cells were then analyzed by flow cytometry for mCherry expression and the presence of CAR on the cell surface (figure 2A). Canonical CARs were expressed at the same level on the surface, whether the drug was present or absent (figure 2A, dot plot under the "canonical CAR" heading). In contrast, ERa-LBD containing v2 and v4 CARs (exemplary single chain chimeric antigen receptors described herein) were expressed in the presence of ligand (as determined by mCherry positive expression), but in the absence of ligand, no CARs were detected on the cell surface (fig. 2A, top row, protein L staining, dot plots under the "v 2" and "v 4" headings). CAR was detected on the cell surface at levels comparable to the control CAR after treatment with 500ng/mL4-OHT (figure 2A, bottom row, protein L staining). Notably, not all designs behave identically: in the absence of ligand, v3 had some basal surface expression of CAR (top dot plot under the "v 3" heading of fig. 2A), and expression was further increased after addition of ligand (bottom dot plot under the "v 3" heading of fig. 2A).

Primary CD4 and CD8 human T cells were transduced with lentiviral vectors encoding a v4CAR construct. Cells were treated as described in this example and stained with anti-cMyc antibody to detect cMyc-labeled scFv on the cell surface. Surface induction was observed in both CD4 and CD 8T cells after co-culturing the T cells with 500ng/mL4-OHT ligand (fig. 2B), indicating that the surface-induced phenotype extends from Jurkat to primary human T cells.

Example 3-NHR-LBD fusion protein shows ligand-induced surface expression in a variety of cell types.

HEK-293T cells were transduced with the lentiviral vectors described in example 1 encoding FMC63 CAR fusions containing ERa-LBD. Cells were either untreated or incubated with 500ng/mL4-OHT for 2 hours before staining for surface CAR expression. Consistent with the ERa-LBD results in Jurkat cells and primary T cells demonstrated in example 2, several ERa-LBD-CAR fusions exhibited minimal CAR surface expression in the absence of ligand and strongly upregulated surface CARs after ligand treatment (figure 3). These data indicate that ligand-induced surface expression of NHR-LBD CAR fusions plays a role in a variety of cell types, including immortalized (Jurkat and HEK-293T) and primary (human CD4 and CD 8T cells), as well as lymphoid (Jurkat and human T cells) and non-lymphoid (HEK-293T) cell types.

Example 4-ligand induced surface expression of NHR LBD-CAR fusions is conserved between different scFv In (1).

To demonstrate the conserved function and modularity of NHR LBD-CAR fusion proteins, the FMC63 scFv in V4 was replaced with a different scFv (J6M0) that recognizes B cell maturation antigen ("BCMA"). Primary human CD 4T cells were transduced with lentiviral vectors encoding NHR-LBD CAR fusions and either untreated or treated with 500ng/mL of ERA ligand 4-OHT. Similar to the FMC63NHR LBD-CAR fusion, NHR LBD-CAR with J6M0 scFv showed ligand-induced surface expression (figure 4).

Example 5-ligand-induced surface trafficking of NHR LBD fusion proteins does not require scFv or scFv from canonical CAR An intracellular domain.

The LBD-CAR fusions tested in example 2 contained several protein motifs in addition to the NHR LBD domain, including a CD137 intracellular domain, a CD3z intracellular signaling domain, and a scFv domain. To determine whether the intracellular portions of the CAR constructs (CD137 endodomain and CD3z endodomain) were involved in ligand-induced surface trafficking, constructs lacking these regions were generated ("v 5", fig. 5A, left) and tested in primary human T cells. Construct v5 included a scFv domain and a mCherry domain. Construct v5 showed comparable ligand-induced trafficking to constructs v2 and v4 in example 2, indicating that the CAR intracellular motif is not responsible for ligand-induced trafficking (figure 5A, right). The nucleotide (SEQ ID NO:36) and amino acid (SEQ ID NO:37) sequences of v5 are shown below.

SEQ ID NO:36

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCATGCCGCTAGACCTGAGCAGAAGCTGATCTCCGAAGAGGACCTGGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGATCTGAAGTGAAACTGCAGGAAAGCGGCCCTGGCCTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCACAACCACCCCTGCCCCTAGACCTCCAACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCCGAGGCTTGTAGACCAGCTGCTGGCGGAGCCGTGCACACCAGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCCGGCACATGTGGCGTGCTGCTGCTGAGCCTCGTGATCACCCTGTACTGCGGATCCGGCAGCGGATCTGGCAGTGGAAGCGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCGGATCTGGCTCTGGAAGCGGCAGCATGGTGTCTAAGGGGGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCTCCGTGAATGGCCACGAATTCGAGATCGAGGGGGAGGGCGAGGGCAGACCTTATGAGGGAACCCAGACCGCCAAGCTGAAAGTGACCAAGGGCGGACCCCTGCCTTTCGCCTGGGATATCCTGTCTCCCCAGTTTATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCTGAGGGCTTCAAGTGGGAGAGAGTGATGAATTTCGAGGACGGCGGAGTCGTGACAGTGACCCAGGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGACCCGTGATGCAGAAAAAGACCATGGGCTGGGAGGCCAGCTCCGAGAGAATGTACCCAGAGGACGGGGCCCTGAAGGGGGAGATCAAGCAGCGGCTGAAACTGAAGGATGGCGGCCACTACGACGCAGAAGTGAAAACCACCTACAAGGCCAAGAAACCTGTGCAGCTGCCTGGCGCCTACAATGTGAACATCAAGCTGGACATTACCAGCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGGGCCGAGGGCAGGCATTCTACAGGCGGAATGGATGAACTGTATAAGTGCGTGACCGACTAG

SEQ ID NO:37

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCGSGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

To test whether extracellular scFv are involved in ligand-induced surface expression, constructs lacking scFv were generated; a FLAG tag was inserted in its place to allow surface detection of the protein (the nucleic acid and polypeptide sequences of this construct are shown as SEQ ID NO:38 and 39, respectively). Jurkat T cells were transduced with lentiviral vectors encoding ERA-LBD fusion proteins and stained for surface expression of the polypeptide in the presence and absence of ERA ligand 4-OHT. Ligand treatment induced robust surface detection of the polypeptides (fig. 5B), indicating that NHR-LBD mediated surface expression did not require scFv, and the technique can be generalized to a wide variety of polypeptides.

SEQ ID NO:38

ATGATCCACCTGGGACACATCCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCCgattacaaagacgacgatgataaaCAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCCatctatatttgggcacccctggctggaacctgcggagtgctgctgctgtctctcgtgattacactgtattgcAAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTGggatcaggcagtggctctggcagcGATAGAAGAGGCGGCAGAATGCTGAAACACAAGCGGCAGAGGGACGACGGGGAAGGCAGAGGCGAAGTGGGATCTGCCGGCGATATGAGAGCCGCCAACCTGTGGCCTAGCCCCCTGATGATCAAGCGGAGCAAGAAGAACTCCCTGGCCCTGAGCCTGACCGCCGACCAGATGGTGTCTGCCCTGCTGGATGCCGAGCCCCCCATCCTGTACAGCGAGTACGACCCCACCAGACCCTTCAGCGAGGCCAGCATGATGGGCCTGCTGACCAACCTGGCCGACCGGGAACTGGTGCACATGATCAACTGGGCCAAGCGGGTGCCCGGCTTCGTGGATCTGACACTGCACGACCAGGTGCACCTGCTGGAATGCGCTTGGCTGGAAATCCTGATGATCGGCCTCGTGTGGCGGAGCATGGAACACCCTGGCAAGCTGCTGTTCGCCCCCAACCTGCTGCTGGACCGGAACCAGGGCAAATGCGTGGAAGGCATGGTGGAAATCTTCGACATGCTGCTGGCCACCTCCAGCCGGTTCCGGATGATGAACCTGCAGGGCGAAGAGTTCGTGTGTCTGAAGTCCATCATCCTGCTGAATAGCGGCGTGTACACCTTCCTGAGCAGCACCCTGAAAAGCCTGGAAGAAAAGGACCACATCCACCGGGTGCTGGACAAGATCACCGACACCCTGATTCACCTGATGGCCAAGGCCGGACTGACCCTGCAGCAGCAGCATCAGAGACTGGCTCAGCTGCTGCTGATCCTGTCCCACATCCGGCACATGAGCAACAAGCGGATGGAACATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCTCTGTTCGATCTGCTGCTGGAAATGCTGGACGCCCACAGGCTGCACGCCCCAACATCCaGATCCggatctggaagtggctccCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAggaagtggatctgggagcggctctatggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagcttccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtataagtgcgtgaccgacTAG

SEQ ID NO:39

MIHLGHILFLLLLPVAAADYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

39 comprises the following domains: dap10 signal peptide (MIHLGHILFLLLLPVAAA, SEQID NO:40), flag tag (DYKDDDDK, SEQ ID NO:41), Dap10 extracellular domain (QTTPGERSSLPAFYPGTSGSCSGCGSLSLP, SEQ ID NO:42), CD8 transmembrane domain (IYIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO:43), 4-1BB costimulatory domain (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, SEQ ID NO:44), first linker (GSGSGSGS, SEQ ID NO:45), modified ERA domain (DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTS, SEQ ID NO:46), second linker (RSGSGS, SEQ ID NO:47), CD3zeta signaling domain (LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, SEQ ID NO:48), third linker (GSGSGSGS, SEQ ID NO:49), mCheerry (MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD, SEQ ID NO: 50).

Example 6 ligand-induced surface expression of NHR-LBD fusion constructs extended to include Progesterone Receptor (PR) Different NHR within

To test whether progesterone receptor ("PR") LBD fusions exhibited ligand-induced surface expression, construct (v33) was generated (the nucleic acid and polypeptide sequences of the construct are shown as SEQ ID NOs 51 and 52, respectively). The tested constructs contained an N-terminal FLAG tag for surface detection. Jurkat T cells were transduced with lentiviral vectors encoding this construct and either untreated or treated with 500ng/mL mifepristone (PR ligand). After two hours of drug treatment, cells were stained for surface protein expression. The construct exhibited ligand-induced surface expression of the polypeptide as detected by flow cytometry (fig. 6), indicating that other NHR-LBDs (other than ERa) can be used to generate constructs with ligand-induced surface expression, and that ligand-induced surface expression of NHR-LBD fusion proteins is a generalizable phenomenon.

SEQ ID NO:51

ATGATCCACCTGGGACACATCCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCCgattacaaagacgacgatgataaaCAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCCatctatatttgggcacccctggctggaacctgcggagtgctgctgctgtctctcgtgattacactgtattgcAAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTGggatcaggcagtggctctggcagcGGGCAAGACATTCAGCTCATACCTCCTTTGATAAATTTGCTGATGTCTATAGAACCAGATGTCATATACGCTGGTCACGACAATACGAAACCGGACACATCTTCATCTTTGCTTACCTCTCTGAATCAACTGGGTGAACGACAGCTCCTGAGTGTTGTTAAGTGGTCTAAAAGCCTCCCGGGCTTCAGGAATTTGCACATAGACGACCAAATCACGCTCATCCAATATTCCTGGATGAGTCTCATGGTCTTTGGTCTCGGTTGGCGCAGCTATAAGCACGTCTCTGGCCAGATGTTGTATTTCGCACCAGACCTGATCCTGAACGAACAGAGGATGAAGGAATCAAGCTTTTACTCTCTCTGCTTGACTATGTGGCAAATCCCCCAAGAATTCGTGAAACTTCAAGTTTCCCAAGAAGAATTCCTCTGCATGAAAGTCCTTCTTTTGCTCAACACGATTCCCCTGGAAGGCTTGAGGTCTCAAACGCAATTCGAGGAGATGCGGAGTAGCTATATACGCGAACTCATCAAGGCCATCGGTTTGCGGCAAAAGGGAGTGGTCTCTAGTAGCCAACGATTTTACCAGCTGACTAAGCTCCTTGACAACCTTCACGATCTCGTCAAACAACTGCACCTGTACTGTCTTAACACATTTATACAATCACGGGCACTTTCTGTAGAGTTCCCAGAGATGATGTCTGAGGTCATCGCAGCCCAACTTCCGAAAATTCTTGCAGGAATGGTGAAGCCACTTCTGTTCCATAAGAAAaGATCCggatctggaagtggctccCTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAggaagtggatctgggagcggctctatggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagcttccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtataagtgcgtgaccgac

SEQ ID NO:52

MIHLGHILFLLLLPVAAADYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKKRSGSGSGSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD

52 comprises the following domains: dap10 signal peptide (MIHLGHILFLLLLPVAAA, SEQID NO:53), flag tag (DYKDDDDK, SEQ ID NO:54), Dap10 extracellular domain (QTTPGERSSLPAFYPGTSGSCSGCGSLSLP, SEQ ID NO:55), CD8 transmembrane domain (IYIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO:56), 4-1BB costimulatory domain (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, SEQ ID NO:57), first linker (GSGSGSGSGS, SEQ ID NO:58), progesterone receptor LBD (GQDIQLIPPLINLLMSIEPDVIYAGHDNTKPDTSSSLLTSLNQLGERQLLSVVKWSKSLPGFRNLHIDDQITLIQYSWMSLMVFGLGWRSYKHVSGQMLYFAPDLILNEQRMKESSFYSLCLTMWQIPQEFVKLQVSQEEFLCMKVLLLLNTIPLEGLRSQTQFEEMRSSYIRELIKAIGLRQKGVVSSSQRFYQLTKLLDNLHDLVKQLHLYCLNTFIQSRALSVEFPEMMSEVIAAQLPKILAGMVKPLLFHKK, SEQ ID NO:59), second linker (RSGSGS, SEQ ID NO:60), CD3zeta signaling domain (LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, SEQ ID NO:61), third linker (GSGSGSGS, SEQ ID NO:62) and mChery (MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD, SEQ ID NO: 63).

51 includes the following sequences encoding the indicated domains: dap10 signal peptide (ATGATCCACCTGGGACACATCCTGTTTTTGCTGCTGCTGCCAGTGGCTGCCGCC, SEQ ID NO:63), flag tag (gattacaaagacgacgatgataaa, SEQ ID NO:64), Dap10 extracellular domain (CAGACAACACCAGGCGAGAGATCTAGCCTGCCCGCCTTCTACCCTGGCACCAGCGGCTCTTGTTCTGGCTGTGGCAGCCTGTCTCTGCCC, SEQ ID NO:65), CD8 transmembrane domain (atctatatttgggcacccctggctggaacctgcggagtgctgctgctgtctctcgtgattacactgtattgc, SEQ ID NO:66), 4-1BB costimulatory domain (AAAAGGGGCCGGAAAAAGCTGCTGTATATTTTCAAACAGCCTTTTATGAGGCCTGTGCAGACAACACAGGAAGAGGACGGCTGTAGCTGTCGGTTCCCCGAAGAGGAAGAGGGGGGCTGCGAACTG, SEQ ID NO:67), first linker (ggatcaggcagtggctctggcagc, SEQ ID NO:68), progesterone receptor LBD (GGGCAAGACATTCAGCTCATACCTCCTTTGATAAATTTGCTGATGTCTATAGAACCAGATGTCATATACGCTGGTCACGACAATACGAAACCGGACACATCTTCATCTTTGCTTACCTCTCTGAATCAACTGGGTGAACGACAGCTCCTGAGTGTTGTTAAGTGGTCTAAAAGCCTCCCGGGCTTCAGGAATTTGCACATAGACGACCAAATCACGCTCATCCAATATTCCTGGATGAGTCTCATGGTCTTTGGTCTCGGTTGGCGCAGCTATAAGCACGTCTCTGGCCAGATGTTGTATTTCGCACCAGACCTGATCCTGAACGAACAGAGGATGAAGGAATCAAGCTTTTACTCTCTCTGCTTGACTATGTGGCAAATCCCCCAAGAATTCGTGAAACTTCAAGTTTCCCAAGAAGAATTCCTCTGCATGAAAGTCCTTCTTTTGCTCAACACGATTCCCCTGGAAGGCTTGAGGTCTCAAACGCAATTCGAGGAGATGCGGAGTAGCTATATACGCGAACTCATCAAGGCCATCGGTTTGCGGCAAAAGGGAGTGGTCTCTAGTAGCCAACGATTTTACCAGCTGACTAAGCTCCTTGACAACCTTCACGATCTCGTCAAACAACTGCACCTGTACTGTCTTAACACATTTATACAATCACGGGCACTTTCTGTAGAGTTCCCAGAGATGATGTCTGAGGTCATCGCAGCCCAACTTCCGAAAATTCTTGCAGGAATGGTGAAGCCACTTCTGTTCCATAAGAAA, SEQ ID NO:69), second linker (ggatctggaagtggctcc, SEQ ID NO:70), CD3zeta signaling domain (CTGAGAGTGAAGTTTAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAACCTGGGCAGGCGGGAAGAGTACGACGTGCTGGATAAGAGGCGGGGCAGGGACCCTGAAATGGGCGGCAAACCCAGACGGAAGAACCCCCAGGAAGGCCTGTACAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGCAAGGGACATGATGGCCTGTACCAGGGCCTGTCCACCGCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGA, SEQ ID NO:71), third linker (ggaagtggatctgggagcggctct, SEQ ID NO:72), and mCherry (atggtgtctaagggggaagaggacaacatggccatcatcaaagaattcatgcggttcaaggtgcacatggaaggctccgtgaatggccacgaattcgagatcgagggggagggcgagggcagaccttatgagggaacccagaccgccaagctgaaagtgaccaagggcggacccctgcctttcgcctgggatatcctgtctccccagtttatgtacggcagcaaggcctacgtgaagcaccccgccgacatccccgactacctgaagctgagcttccctgagggcttcaagtgggagagagtgatgaatttcgaggacggcggagtcgtgacagtgacccaggatagctctctgcaggacggcgagttcatctacaaagtgaagctgcggggcaccaacttccccagcgacggacccgtgatgcagaaaaagaccatgggctgggaggccagctccgagagaatgtacccagaggacggggccctgaagggggagatcaagcagcggctgaaactgaaggatggcggccactacgacgcagaagtgaaaaccacctacaaggccaagaaacctgtgcagctgcctggcgcctacaatgtgaacatcaagctggacattaccagccacaacgaggactacaccatcgtggaacagtacgagcgggccgagggcaggcattctacaggcggaatggatgaactgtataagtgcgtgaccgac, SEQ ID NO: 73).

Example 7-ligand-induced NHR-LBD CAR T cell activation is antigen and drug dependent.

Primary human T cells were transduced with lentiviral vectors encoding the J6M0-ERa-LBD CAR construct. T cell activation was assessed by surface staining for the canonical T cell activation marker CD69 after overnight co-culture with antigen negative (K562 cell line) and antigen positive (MM1S cell line) targets in the presence of increasing concentrations of 4-OHT (fig. 7A). For each construct, a unique dose-dependent T cell response was observed, where the proximity of LBD to the cell membrane correlated with background activation (activation in the absence of ligand) and sensitivity to ligand. With increasing drug concentrations, all constructs showed activation levels comparable to the positive control (canonical CAR).

The ability of J6M0-ERa-LBD CAR cells to produce inflammatory cytokines was assessed via IFN- γ ELISA on co-culture supernatants isolated from overnight co-cultures of T cells with antigen negative (K562 cell line) and antigen positive (MM1S cell line) targets in the presence of increasing concentrations of 4-OHT. IFN- γ production was shown to be dose-dependent, with all NHR-LBD constructs producing cytokines with increasing ligand concentrations (FIG. 7B).

Example 8-ligand-induced NHR-LBD CAR T cell proliferation and killing is antigen and ligand dependent.

Primary human CD 4T cells were transduced with lentiviral vectors encoding V4 FMC63-ERa-LBD CAR or canonical CAR controls. Cells were labeled with carboxyfluorescein succinimidyl ester ("CFSE") prior to co-culture with antigen negative (K562 cell line) or antigen positive (Raji cell line) targets. Cells were also co-cultured with increasing concentrations of ligand (4-OHT). Three days after co-cultivation, cells were analyzed via flow cytometry and proliferation was assessed by CFSE dye dilution on CD4+ T cells. FMC63-ERa-LBD CAR T cells proliferated only in the presence of antigen and ligand (fig. 8A).

Primary human T cells were transduced with lentiviral vectors encoding the J6M0-ERa-LBD CAR construct described in example 7. The ability of T cells carrying the J6M0-ERa-LBD CAR construct to kill antigen positive targets (MM1S cell line) in a drug-dependent manner was evaluated via flow cytometry-based killing assay. Target cells were labeled with CFSE prior to co-culture, and the number of cells remaining in each well after co-culture was determined by quantifying a fixed volume using an intellichyt flow cytometer. Specific lysis was determined by comparing the number of targets remaining per experimental well with the number of targets remaining in control wells containing target and non-antigen specific conventional CAR T cells (-CTRL). Specific lysis is reported as a percentage derived from the following equation: 1- (# events per experimental well/# mean events-CTRL wells). Target cell killing was dependent on the dose of ligand, with all constructs being able to eliminate antigen positive targets at comparable levels to conventional CARs (+ CTRL) at higher drug doses (fig. 8B).

Example 9-location of LBD within CAR polypeptide determines surface expression of ligand-induced NHR-LBD CAR The sensitivity of (2).

The dose responsiveness of the V2, V3, and V4 constructs shown in figure 1 were compared in Jurkat T cells. Detection of surface expression of NHR-LBD CAR protein on Jurkat T cells was performed after 1 hour incubation with 4-OHT ligand at the concentrations indicated on the x-axis of FIG. 9. Surface receptor expression was detected using anti-cMyc antibody and reported as the percentage of reporter-positive cells (determined by mCherry expression). For each construct, the detected surface-expressed ligand dose corresponds to the location of the LBD of each construct relative to the surface membrane. Construct V4 with LBD in close proximity to the surface membrane showed no detectable surface expression in the absence of ligand, with surface expression detected only at higher concentrations of 4-OHT. In contrast, when the LBD is located further away from the membrane (see e.g. V2), a higher basal level (-20%) of surface expression is detected and an increased sensitivity to 4-OHT is observed. Activation of Jurkat T cells with PMA/ionomycin did not result in ligand-independent NHR-LBD CAR surface expression, indicating that activation of T cells alone (e.g., via an endogenous TCR) did not result in receptor trafficking to the cell surface.

Example 10-duration of surface expression of NHR-LBD CAR depends on the location of the LBD in the polypeptide.

T cells were transduced with FMC63-ERa-LBD CAR and exposed to 35ng/mL of 4-OHT for 1 hour, then stained for surface expression with anti-cMyc antibody, or washed and cultured overnight in the absence of drug (time lines are shown in figure 10A).

The number of cells expressing the surface protein was reported as the percentage of reporter positive cells (determined by mCherry expression). For constructs V2, V3 and V4, the duration of NHR-LBD CAR expression on the cell surface was determined. 24 hours after ligand removal, the construct with the LBD closest to the membrane (V4) showed the fastest and most complete return to baseline (FIG. 10B, bar labeled "V4").

Example 11-kinetics of surface expression of NHR-LBD-CAR constructs after ligand addition.

Jurkat T cells transduced with the FLAG-ERA-LBD construct used in example 5 were untreated or treated with 500ng/mL4-OHT for 15 min or 120 min and surface expression was detected after ligand treatment. Ligand treatment induced surface expression within 15 minutes, and further upregulation was observed over time (fig. 11A).

Human T cells were transduced with a lentiviral vector encoding FMC63-ERA-LBD CAR (v2 as shown in FIG. 1). Cells were treated with ERa ligand 4-OHT for the indicated duration, and then stained for surface expression of CARs (figure 11B). The data in fig. 11B show kinetics of ligand treatment-induced surface expression similar to that observed for Jurkat T cells.

Example 12-brefeldin A treatment blocked ligand-induced surface trafficking of NHR LBD fusions.

Primary human T cells expressing FMC63-CAR with ERa LBD fusion were either untreated or treated with 500ng/mL4-OHT ligand for 2 hours to induce surface expression of the CAR. Half of the cells were pretreated with 1uM brefeldin a (fig. 12, bottom two panels) to inhibit protein export from golgi prior to 4-OHT treatment. Brefeldin a pretreatment prevented 4-OHT-induced increase in surface protein expression. Without wishing to be bound by theory, these data suggest that increased surface expression results from the relocation of pools of intracellular sequestered polypeptides in the absence of ligand.

EXAMPLE 13 Generation of the construct

Conventional chimeric antigen receptors ("conventional CARs", fig. 13, left) were modified to include the estrogen receptor alpha ("ERa") ligand binding domain ("ERa LBD," fig. 1, green) in the intracellular portion of the protein (v 2CAR as described in example 1) or in the extracellular portion of the protein ("cryptic carrying carecd. v 1" or "LBD-ECDCAR"). Point mutations were introduced into the ERa LBD domains of both v2CAR and ecd. v1 CAR. The nucleic acid and polypeptide sequences of the v2CAR (shown above as SEQ ID NOS: 30 and 31) and ECD. v1(SEQ ID NOS: 97 and 98) are provided below.

SEQ ID NO:97(ECD v.1CAR)

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgattcctgaacagaagctgataagtgaggaggacttggacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggacatcagcaagtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgcccagcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgctacctacttctgtcagcaaggcaacaccctgccctacaccttcggcggaggcaccaagctggaaatcacaggcggcggaggatctggcggaggcggaagtggcggagggggatctgaagtgaaactgcaggaaagcggccctggcctggtggccccatctcagtctctgagcgtgacctgtaccgtgtccggcgtgtccctgcctgactatggcgtgtcctggatcagacagccccccagaaagggcctggaatggctgggagtgatctggggcagcgagacaacctactacaacagcgccctgaagtcccggctgaccatcatcaaggacaactccaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcacaagcgtgaccgtgtctagcggatccgatagaagaggcggcagaatgctgaaacacaagcggcagagggacgacggggaaggcagaggcgaagtgggatctgccggcgatatgagagccgccaacctgtggcctagccccctgatgatcaagcggagcaagaagaactccctggccctgagcctgaccgccgaccagatggtgtctgccctgctggatgccgagccccccatcctgtacagcgagtacgaccccaccagacccttcagcgaggccagcatgatgggcctgctgaccaacctggccgaccgggaactggtgcacatgatcaactgggccaagcgggtgcccggcttcgtggatctgacactgcacgaccaggtgcacctgctggaatgcgcttggctggaaatcctgatgatcggcctcgtgtggcggagcatggaacaccctggcaagctgctgttcgcccccaacctgctgctggaccggaaccagggcaaatgcgtggaaggcatggtggaaatcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaagagttcgtgtgtctgaagtccatcatcctgctgaatagcggcgtgtacaccttcctgagcagcaccctgaaaagcctggaagaaaaggaccacatccaccgggtgctggacaagatcaccgacaccctgattcacctgatggccaaggccggactgaccctgcagcagcagcatcagagactggctcagctgctgctgatcctgtcccacatccggcacatgagcaacaagcggatggaacatctgtacagcatgaagtgcaagaacgtggtgcctctgttcgatctgctgctggaaatgctggacgcccacaggctgcacgccccaacatccggatccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctag

SEQ ID NO:98(ECD v.1CAR)

MLLLVTSLLLCELPHPAFLLIPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTSGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The protein and nucleic acid sequences of each domain in ecd.v1 from N-terminus to C-terminus are shown below.

99(CSF2RA signal peptide)

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgattcct

100(CSF2RA signal peptide) SEQ ID NO

MLLLVTSLLLCELPHPAFLLIP

SEQ ID NO 101(cMyc tag)

gaacagaagctgataagtgaggaggacttg

SEQ ID NO 102(cMyc tag)

EQKLISEEDL

SEQ ID NO:103(FMC63 V_L)

gacatccagatgacccagaccaccagcagcctgagcgccagcctgggcgatagagtgaccatcagctgcagagccagccaggacatcagcaagtacctgaactggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccacaccagcagactgcacagcggcgtgcccagcagattttctggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgctacctacttctgtcagcaaggcaacaccctgccctacaccttcggcggaggcaccaagctggaaatcaca

SEQ ID NO:104(FMC63 V_H)

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

SEQ ID NO:105(V_LAnd V_HIn (G) of₄S)₃Joint)

ggcggcggaggatctggcggaggcggaagtggcggagggggatct

SEQ ID NO:106(V_LAnd V_HIn (G) of₄S)₃Joint)

GGGGSGGGGSGGGGS

SEQ ID NO:107(FMC63 V_H)

gaagtgaaactgcaggaaagcggccctggcctggtggccccatctcagtctctgagcgtgacctgtaccgtgtccggcgtgtccctgcctgactatggcgtgtcctggatcagacagccccccagaaagggcctggaatggctgggagtgatctggggcagcgagacaacctactacaacagcgccctgaagtcccggctgaccatcatcaaggacaactccaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcacaagcgtgaccgtgtctagc

SEQ ID NO:108(FMC63 V_H)

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

V_HGS linker between domain and estrogen receptor alpha ligand binding domain

ggatcc

V_HGS linker between domain and estrogen receptor alpha ligand binding domain

GS

109 (Estrogen receptor alpha ligand binding Domain) (hinge-encoding sequence underlined, helix 12-encoding sequence in bold)

gatagaagaggcggcagaatgctgaaacacaagcggcagagggacgacggggaaggcagaggcgaagt gggatctgccggcgatatgagagccgccaacctgtggcctagccccctgatgatcaagcggagcaagaagaactccctggccctgagcctgaccgccgaccagatggtgtctgccctgctggatgccgagccccccatcctgtacagcgagtacgaccccaccagacccttcagcgaggccagcatgatgggcctgctgaccaacctggccgaccgggaactggtgcacatgatcaactgggccaagcgggtgcccggcttcgtggatctgacactgcacgaccaggtgcacctgctggaatgcgcttggctggaaatcctgatgatcggcctcgtgtggcggagcatggaacaccctggcaagctgctgttcgcccccaacctgctgctggaccggaaccagggcaaatgcgtggaaggcatggtggaaatcttcgacatgctgctggccacctccagccggttccggatgatgaacctgcagggcgaagagttcgtgtgtctgaagtccatcatcctgctgaatagcggcgtgtacaccttcctgagcagcaccctgaaaagcctggaagaaaaggaccacatccaccgggtgctggacaagatcaccgacaccctgattcacctgatggccaaggccggactgaccctgcagcagcagcatcagagactggctcagctgctgctgatcctgtcccacatccggcacatgagcaacaagcggatggaacatctgtacagcatgaagtgcaagaac

110 (Estrogen receptor alpha ligand binding Domain) (hinge underlined, helix 12 in bold)

DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKN

GS linker between estrogen receptor alpha ligand binding domain and CD8 alpha hinge region

ggatcc

GS

111(CD8 alpha hinge) of SEQ ID NO

accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

112(CD8 alpha hinge)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC

113(CD8 alpha transmembrane domain)

gatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

114(CD8 alpha transmembrane domain)

DIYIWAPLAGTCGVLLLSLVITLYC

115(4-1BB costimulatory domain)

aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaa

116(4-1BB costimulatory domain)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

117(CD 3zeta signaling domain)

ctgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

118(CD 3zeta signaling domain)

LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 14-LBD-ECD reduces background localization of CAR on cell surface in the absence of ligand

As shown, primary human T cells were transduced with lentiviral vectors encoding either cc.v2 or LBD ecd.v1 constructs. Cells were either untreated or treated with 500ng/mL 4-OHT. The duration of the treatment is shown in fig. 14. Cells were subsequently stained for surface expression of the chimeric receptor using an anti-MycAlexaFluor 647 antibody.

Placement of the LBD in the extracellular portion of the CAR reduced the background level of CAR surface expression when no ligand was added relative to when the LBD was placed in the intracellular portion of the CAR (figure 14). V2CAR and ecd. v1 are both expressed on the surface of cells when 500ng/ml4-OHT is added.

Example 15-LBD-ECD CAR killing of cells in a drug-dependent manner

Primary human T cells were transduced with lentiviral vectors encoding either LBD ecd. v1 or control CAR constructs as shown. After expansion, the T cells were allowed to rest and then co-cultured with luciferase-expressing Nalm6 tumor cells at effector to target ratios as shown in figure 15. After 24 hours of co-culture, cells were lysed and luciferase substrate was added to quantify the number of surviving tumor cells.

LBD ecd. v1 CAR kills cells in a drug-dependent manner. Addition of increasing amounts of 4-OHT resulted in higher levels of cell lysis (approximately 50% lysis at 500ng/mL 4-OHT) (FIG. 15).

Example 16 localization of LBD-ECD CAR in activated T cells

Primary human T cells were transduced with lentiviral vectors encoding LBD _ ECD, cc. v2 or constitutive control CAR constructs as shown. After cell rest, cells were restimulated with aCD3/aCD28 dynabeads for 48 hours. Cells were then stained with anti-Myc antibody to detect surface expression of CAR and analyzed via flow cytometry.

CARs with extracellular LBD (LBD ECD v1) were localized intracellularly in T cells that were activated when exposed to CD3/CD28 beads. CARs with intracellular LBD (v2 CARs) showed some localization to the cell surface in activated T cells (figure 16).

Example 17-ECD v1 CAR (LBD _ ECD) production of the cytokine IL-2

IL-2 production from ecd. v1 CAR T cells was compared to conventional CAR + control after overnight co-culture of CAR-bearing T cells with NALM-6 target in the presence (500ng/mL) or in the absence of 4-hydroxytamoxifen (figure 16). IL-2 production from ecd. v1 CAR receptor (figure 13) was comparable to conventional CAR controls and was undetectable in the absence of drug, depending on 4-hydroxytamoxifen.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence listing

<110> cell design laboratory Ltd

<120> chimeric polypeptides and methods for altering their localization in cell membranes

<130>43159-0064WO1

<150>US 62/587,262

<151>2017-11-16

<150>US 62/477,733

<151>2017-03-28

<160>119

<170>PatentIn version 3.5

<210>1

<211>220

<212>PRT

<213> human

<400>1

Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val

1 5 10 15

Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr

20 25 30

Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser

35 40 45

Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu

50 55 60

Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser

65 70 75 80

Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr

85 90 95

Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys

100 105 110

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

115 120 125

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

130 135 140

Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly

145 150 155 160

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

165 170 175

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

180 185 190

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

195 200 205

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

210 215 220

<210>2

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<212>PRT

<213> human

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Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly ArgAsp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210>3

<211>23

<212>PRT

<213> human

<400>3

Leu Gly Leu Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly

1 5 10 15

Val Ala Ile His Leu Cys Cys

20

<210>4

<211>26

<212>PRT

<213> human

<400>4

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

15 10 15

Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

20 25

<210>5

<211>25

<212>PRT

<213> human

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Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly

1 5 10 15

Leu Gly Ile Phe Phe Cys Val Arg Cys

20 25

<210>6

<211>23

<212>PRT

<213> human

<400>6

Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu

1 5 10 15

Thr Ala Leu Phe Leu Arg Val

20

<210>7

<211>26

<212>PRT

<213> human

<400>7

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

1 5 10 15

Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210>8

<211>24

<212>PRT

<213> human

<400>8

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 Leu Tyr Cys

20

<210>9

<211>24

<212>PRT

<213> human

<400>9

Ala Leu Pro Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly

1 5 10 15

Leu Gly Val Ala Cys Val Leu Ala

20

<210>10

<211>250

<212>PRT

<213> human

<400>10

Ser Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val

1 5 10 15

Ser Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp

20 25 30

Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn

35 40 45

Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val

50 55 60

Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu

65 70 75 80

Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met

85 90 95

Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg

100 105 110

Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu

115 120 125

Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu

130 135 140

Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr

145 150 155 160

Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His

165 170 175

Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys

180 185 190

Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu

195 200 205

Leu Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu His

210 215 220

Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu

225 230 235 240

Leu Glu Met Leu Asp Ala His Arg Leu His

245 250

<210>11

<211>248

<212>PRT

<213> human

<400>11

Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp Ala

1 5 10 15

Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser

20 25 30

Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu Leu

35 40 45

Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp Leu

50 55 60

Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu Ile

65 70 75 80

Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys Leu

85 90 95

Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys Val

100 105 110

Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser Arg

115 120 125

Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys Ser

130 135 140

Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu

145 150 155 160

Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys Ile

165 170 175

Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu Gln

180 185 190

Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile

195 200 205

Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met Lys Cys

210 215 220

Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala

225 230 235 240

His Arg Leu His Ala Pro Thr Ser

245

<210>12

<211>200

<212>PRT

<213> human

<400>12

Ser Pro Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile Asn Leu Leu

1 5 10 15

Met Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp Asn Thr Lys

20 25 30

Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu

35 40 45

Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly Phe

50 55 60

Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser Trp

65 70 75 80

Met Ser Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr Lys His Val

85 90 95

Ser Gly Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu Asn Glu Gln

100 105 110

Arg Met Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr Met Trp Gln

115 120 125

Ile Pro Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu Glu Phe Leu

130 135 140

Cys Met Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu

145 150 155 160

Arg Ser Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu

165 170 175

Leu Ile Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val Ser Ser Ser

180 185 190

Gln Arg Phe Tyr Gln Leu Thr Lys

195 200

<210>13

<211>261

<212>PRT

<213> human

<400>13

Phe Thr Phe Ser Pro Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile

1 5 10 15

Asn Leu Leu Met Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp

20 25 30

Asn Thr Lys Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln

35 40 45

Leu Gly Glu Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu

50 55 60

Pro Gly Phe Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln

65 70 75 80

Tyr Ser Trp Met Ser Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr

85 90 95

Lys His Val Ser Gly Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu

100 105 110

Asn Glu Gln Arg Met Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr

115 120 125

Met Trp Gln Ile Pro Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu

130 135 140

Glu Phe Leu Cys Met Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu

145 150 155 160

Glu Gly Leu Arg Ser Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr

165 170 175

Ile Arg Glu Leu Ile Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val

180 185 190

Ser Ser Ser Gln Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Asn Leu

195 200 205

His Asp Leu Val Lys Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile

210 215 220

Gln Ser Arg Ala Leu Ser Val Glu Phe Pro Glu Met Met Ser Glu Val

225 230 235 240

Ile Ala Ala Gln Leu Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu

245 250 255

Leu Phe His Lys Lys

260

<210>14

<211>249

<212>PRT

<213> human

<400>14

Pro Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys

1 5 10 15

Ala Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser

20 25 30

Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp

35 40 45

Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met

50 55 60

Ala Val Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly

65 70 75 80

Trp Arg Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro

85 90 95

Asp Leu Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser

100 105 110

Gln Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln

115 120 125

Ile Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser

130 135 140

Ile Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu

145 150 155 160

Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg

165 170 175

Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu

180 185 190

Leu Asp Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe

195 200 205

Asp Leu Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met

210 215 220

Met Ala Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys

225 230 235 240

Val Lys Pro Ile Tyr Phe His Thr Gln

245

<210>15

<211>283

<212>PRT

<213> human

<400>15

Ser Leu Glu Glu Gly Glu Ala Ser Ser Thr Thr Ser Pro Thr Glu Glu

1 5 10 15

Thr Thr Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln

20 25 30

Pro Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys

35 40 45

Ala Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser

50 55 60

Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp

65 70 75 80

Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met

85 90 95

Ala Val Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly

100 105 110

Trp Arg Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro

115 120 125

Asp Leu Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser

130 135 140

Gln Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln

145 150 155 160

Ile Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser

165 170 175

Ile Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu

180 185 190

Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg

195 200 205

Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu

210 215 220

Leu Asp Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe

225 230 235 240

Asp Leu Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met

245 250 255

Met Ala Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys

260 265 270

Val Lys Pro Ile Tyr Phe His Thr Gln Glu Gly

275 280

<210>16

<211>266

<212>PRT

<213> human

<400>16

Glu Thr Thr Gln Lys Leu Thr Val Ser His Ile Glu Gly Tyr Glu Cys

1 5 10 15

Gln Pro Ile Phe Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val

20 25 30

Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu

35 40 45

Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys

50 55 60

Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln

65 70 75 80

Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met

85 90 95

Gly Trp Arg Ser Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala

100 105 110

Pro Asp Leu Val Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr

115 120 125

Ser Gln Cys Val Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu

130 135 140

Gln Ile Thr Pro Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe

145 150 155160

Ser Ile Ile Pro Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu

165 170 175

Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys

180 185 190

Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys

195 200 205

Leu Leu Asp Ser Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr

210 215 220

Phe Asp Leu Leu Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu

225 230 235 240

Met Met Ala Glu Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly

245 250 255

Lys Val Lys Pro Ile Tyr Phe His Thr Gln

260 265

<210>17

<211>164

<212>PRT

<213> human

<400>17

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210>18

<211>220

<212>PRT

<213> human

<400>18

Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val

1 5 10 15

Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr

20 25 30

Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser

35 40 45

Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu

50 55 60

Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser

65 70 75 80

Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr

85 90 95

Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys

100 105 110

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

115 120 125

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

130 135 140

Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly

145 150 155 160

Gly ValLeu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

165 170 175

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

180 185 190

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

195 200 205

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

210 215 220

<210>19

<211>255

<212>PRT

<213> human

<400>19

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210>20

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>20

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210>21

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>21

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>22

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>22

Arg Ser Gly Ser Gly Ser Gly Ser

1 5

<210>23

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>23

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>24

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>24

ggatccggca gcggatctgg cagtggaagc 30

<210>25

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>25

ggatctggct ctggaagcgg cagc 24

<210>26

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>26

agatccggat ctggaagtgg ctcc 24

<210>27

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>27

ggaagtggat ctgggagcgg ctct 24

<210>28

<211>297

<212>PRT

<213> human

<400>28

Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp

1 5 10 15

Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala

20 25 30

Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser

35 40 45

Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp

50 55 60

Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe

65 70 75 80

Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu

85 90 95

Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp

100 105 110

LeuThr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu

115 120 125

Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys

130 135 140

Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys

145 150 155 160

Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser

165 170 175

Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys

180 185 190

Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr

195 200 205

Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys

210 215 220

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu

225 230 235 240

Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His

245 250 255

Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser Met Lys

260 265 270

Cys Lys AsnVal Val Pro Leu Phe Asp Leu Leu Leu Glu Met Leu Asp

275 280 285

Ala His Arg Leu His Ala Pro Thr Ser

290 295

<210>29

<211>256

<212>PRT

<213> human

<400>29

Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile Asn Leu Leu Met Ser

1 5 10 15

Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp Asn Thr Lys Pro Asp

20 25 30

Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu Arg Gln

35 40 45

Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly Phe Arg Asn

50 55 60

Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser Trp Met Ser

65 70 75 80

Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr Lys His Val Ser Gly

85 90 95

Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu Asn Glu Gln Arg Met

100 105 110

Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr Met Trp Gln Ile Pro

115 120 125

Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu Glu Phe Leu Cys Met

130 135 140

Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu Arg Ser

145 150 155 160

Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu Leu Ile

165 170 175

Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val Ser Ser Ser Gln Arg

180 185 190

Phe Tyr Gln Leu Thr Lys Leu Leu Asp Asn Leu His Asp Leu Val Lys

195 200 205

Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile Gln Ser Arg Ala Leu

210 215 220

Ser Val Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala Gln Leu

225 230 235 240

Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu Leu Phe His Lys Lys

245 250 255

<210>30

<211>3183

<212>DNA

<213> Artificial sequence

<220>

<223> v.2 constructs

<400>30

atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgccgctaga 60

cctgagcaga agctgatctc cgaagaggac ctggacatcc agatgaccca gaccaccagc 120

agcctgagcg ccagcctggg cgatagagtg accatcagct gcagagccag ccaggacatc 180

agcaagtacc tgaactggta tcagcagaaa cccgacggca ccgtgaagct gctgatctac 240

cacaccagca gactgcacag cggcgtgccc agcagatttt ctggcagcgg ctccggcacc 300

gactacagcc tgaccatctc caacctggaa caggaagata tcgctaccta cttctgtcag 360

caaggcaaca ccctgcccta caccttcggc ggaggcacca agctggaaat cacaggcggc 420

ggaggatctg gcggaggcgg aagtggcgga gggggatctg aagtgaaact gcaggaaagc 480

ggccctggcc tggtggcccc atctcagtct ctgagcgtga cctgtaccgt gtccggcgtg 540

tccctgcctg actatggcgt gtcctggatc agacagcccc ccagaaaggg cctggaatgg 600

ctgggagtga tctggggcag cgagacaacc tactacaaca gcgccctgaa gtcccggctg 660

accatcatca aggacaactc caagagccag gtgttcctga agatgaacag cctgcagacc 720

gacgacaccg ccatctacta ctgcgccaag cactactact acggcggcag ctacgccatg 780

gactactggg gccagggcac aagcgtgacc gtgtctagca caaccacccc tgcccctaga 840

cctccaaccc cagcccctac aatcgccagc cagcctctgt ctctgaggcc cgaggcttgt 900

agaccagctg ctggcggagc cgtgcacacc agaggactgg atttcgcctg cgacatctac 960

atctgggccc ctctggccgg cacatgtggc gtgctgctgc tgagcctcgt gatcaccctg 1020

tactgcggat ccaagcgggg cagaaagaaa ctgctgtaca tctttaagca gcccttcatg 1080

cggcccgtgc agaccaccca ggaagaggac ggctgctcct gcagattccc cgaggaagaa 1140

gaaggcggct gcgagctggg cagcggatct ggcagtggaa gcgatagaag aggcggcaga 1200

atgctgaaac acaagcggca gagggacgac ggggaaggca gaggcgaagt gggatctgcc 1260

ggcgatatga gagccgccaa cctgtggcct agccccctga tgatcaagcg gagcaagaag 1320

aactccctgg ccctgagcct gaccgccgac cagatggtgt ctgccctgct ggatgccgag 1380

ccccccatcc tgtacagcga gtacgacccc accagaccct tcagcgaggc cagcatgatg 1440

ggcctgctga ccaacctggc cgaccgggaa ctggtgcaca tgatcaactg ggccaagcgg 1500

gtgcccggct tcgtggatct gacactgcac gaccaggtgc acctgctgga atgcgcttgg 1560

ctggaaatcc tgatgatcgg cctcgtgtgg cggagcatgg aacaccctgg caagctgctg 1620

ttcgccccca acctgctgct ggaccggaac cagggcaaat gcgtggaagg catggtggaa 1680

atcttcgaca tgctgctggc cacctccagc cggttccgga tgatgaacct gcagggcgaa 1740

gagttcgtgt gtctgaagtc catcatcctg ctgaatagcg gcgtgtacac cttcctgagc 1800

agcaccctga aaagcctgga agaaaaggac cacatccacc gggtgctgga caagatcacc 1860

gacaccctga ttcacctgat ggccaaggcc ggactgaccc tgcagcagca gcatcagaga 1920

ctggctcagc tgctgctgat cctgtcccac atccggcaca tgagcaacaa gcggatggaa 1980

catctgtaca gcatgaagtg caagaacgtg gtgcctctgt tcgatctgct gctggaaatg 2040

ctggacgccc acaggctgca cgccccaaca tccagatccg gatctggaag tggctccctg 2100

agagtgaagt ttagcagaag cgccgacgcc cctgcctatc agcagggaca gaaccagctg 2160

tataacgagc tgaacctggg caggcgggaa gagtacgacg tgctggataa gaggcggggc 2220

agggaccctg aaatgggcgg caaacccaga cggaagaacc cccaggaagg cctgtacaac 2280

gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgg 2340

cggagaggca agggacatga tggcctgtac cagggcctgt ccaccgccac caaggacacc 2400

tatgacgccc tgcacatgca ggccctgcct ccaagaggaa gtggatctgg gagcggctct 2460

atggtgtcta agggggaaga ggacaacatg gccatcatca aagaattcat gcggttcaag 2520

gtgcacatgg aaggctccgt gaatggccac gaattcgaga tcgaggggga gggcgagggc 2580

agaccttatg agggaaccca gaccgccaag ctgaaagtga ccaagggcgg acccctgcct 2640

ttcgcctggg atatcctgtc tccccagttt atgtacggca gcaaggccta cgtgaagcac 2700

cccgccgaca tccccgacta cctgaagctg agcttccctg agggcttcaa gtgggagaga 2760

gtgatgaatt tcgaggacgg cggagtcgtg acagtgaccc aggatagctc tctgcaggac 2820

ggcgagttca tctacaaagt gaagctgcgg ggcaccaact tccccagcga cggacccgtg 2880

atgcagaaaa agaccatggg ctgggaggcc agctccgaga gaatgtaccc agaggacggg 2940

gccctgaagg gggagatcaa gcagcggctg aaactgaagg atggcggcca ctacgacgca 3000

gaagtgaaaa ccacctacaa ggccaagaaa cctgtgcagc tgcctggcgc ctacaatgtg 3060

aacatcaagc tggacattac cagccacaac gaggactaca ccatcgtgga acagtacgag 3120

cgggccgagg gcaggcattc tacaggcgga atggatgaac tgtataagtg cgtgaccgac 3180

tag 3183

<210>31

<211>1060

<212>PRT

<213> Artificial sequence

<220>

<223> v.2 constructs

<400>31

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp

35 40 45

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu

50 55 60

Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

65 70 75 80

His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

85 90 95

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu

100 105 110

Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr

115 120 125

Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser

145 150 155 160

Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr

165 170 175

Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln

180 185 190

Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu

195 200 205

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys

210 215 220

Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr

225 230 235 240

Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly

245 250 255

Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

260 265 270

Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Gly Ser Lys Arg Gly Arg Lys Lys Leu Leu

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

370 375 380

Glu Leu Gly Ser Gly Ser Gly Ser Gly Ser Asp Arg Arg Gly Gly Arg

385 390 395 400

Met Leu Lys His Lys Arg Gln Arg Asp Asp Gly Glu Gly Arg Gly Glu

405 410 415

Val Gly Ser Ala Gly Asp Met Arg Ala Ala Asn Leu Trp Pro Ser Pro

420 425 430

Leu Met Ile Lys Arg Ser Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr

435440 445

Ala Asp Gln Met Val Ser Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu

450 455 460

Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met

465 470 475 480

Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn

485 490 495

Trp Ala Lys Arg Val Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln

500 505 510

Val His Leu Leu Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu

515 520 525

Val Trp Arg Ser Met Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn

530 535 540

Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu

545 550 555 560

Ile Phe Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn

565 570 575

Leu Gln Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn

580 585 590

Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu

595 600605

Lys Asp His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile

610 615 620

His Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg

625 630 635 640

Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Asn

645 650 655

Lys Arg Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro

660 665 670

Leu Phe Asp Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu His Ala

675 680 685

Pro Thr Ser Arg Ser Gly Ser Gly Ser Gly Ser Leu Arg Val Lys Phe

690 695 700

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

705 710 715 720

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

725 730 735

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

740 745 750

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

755 760765

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

770 775 780

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

785 790 795 800

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ser

805 810 815

Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile

820 825 830

Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn

835 840 845

Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu

850 855 860

Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro

865 870 875 880

Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala

885 890 895

Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe

900 905 910

Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly

915 920925

Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile

930 935 940

Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val

945 950 955 960

Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr

965 970 975

Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu

980 985 990

Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala

995 1000 1005

Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys

1010 1015 1020

Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln

1025 1030 1035

Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu

1040 1045 1050

Leu Tyr Lys Cys Val Thr Asp

1055 1060

<210>32

<211>3183

<212>DNA

<213> Artificial sequence

<220>

<223> v.3 constructs

<400>32

atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgccgctaga 60

cctgagcaga agctgatctc cgaagaggac ctggacatcc agatgaccca gaccaccagc 120

agcctgagcg ccagcctggg cgatagagtg accatcagct gcagagccag ccaggacatc 180

agcaagtacc tgaactggta tcagcagaaa cccgacggca ccgtgaagct gctgatctac 240

cacaccagca gactgcacag cggcgtgccc agcagatttt ctggcagcgg ctccggcacc 300

gactacagcc tgaccatctc caacctggaa caggaagata tcgctaccta cttctgtcag 360

caaggcaaca ccctgcccta caccttcggc ggaggcacca agctggaaat cacaggcggc 420

ggaggatctg gcggaggcgg aagtggcgga gggggatctg aagtgaaact gcaggaaagc 480

ggccctggcc tggtggcccc atctcagtct ctgagcgtga cctgtaccgt gtccggcgtg 540

tccctgcctg actatggcgt gtcctggatc agacagcccc ccagaaaggg cctggaatgg 600

ctgggagtga tctggggcag cgagacaacc tactacaaca gcgccctgaa gtcccggctg 660

accatcatca aggacaactc caagagccag gtgttcctga agatgaacag cctgcagacc 720

gacgacaccg ccatctacta ctgcgccaag cactactact acggcggcag ctacgccatg 780

gactactggg gccagggcac aagcgtgacc gtgtctagca caaccacccc tgcccctaga 840

cctccaaccc cagcccctac aatcgccagc cagcctctgt ctctgaggcc cgaggcttgt 900

agaccagctg ctggcggagc cgtgcacacc agaggactgg atttcgcctg cgacatctac 960

atctgggccc ctctggccgg cacatgtggc gtgctgctgc tgagcctcgt gatcaccctg 1020

tactgcggat ccaagcgggg cagaaagaaa ctgctgtaca tctttaagca gcccttcatg 1080

cggcccgtgc agaccaccca ggaagaggac ggctgctcct gcagattccc cgaggaagaa 1140

gaaggcggct gcgagctgag atccggatct ggaagtggct ccctgagagt gaagtttagc 1200

agaagcgccg acgcccctgc ctatcagcag ggacagaacc agctgtataa cgagctgaac 1260

ctgggcaggc gggaagagta cgacgtgctg gataagaggc ggggcaggga ccctgaaatg 1320

ggcggcaaac ccagacggaa gaacccccag gaaggcctgt acaacgaact gcagaaagac 1380

aagatggccg aggcctacag cgagatcgga atgaagggcg agcggcggag aggcaaggga 1440

catgatggcc tgtaccaggg cctgtccacc gccaccaagg acacctatga cgccctgcac 1500

atgcaggccc tgcctccaag aggcagcgga tctggcagtg gaagcgatag aagaggcggc 1560

agaatgctga aacacaagcg gcagagggac gacggggaag gcagaggcga agtgggatct 1620

gccggcgata tgagagccgc caacctgtgg cctagccccc tgatgatcaa gcggagcaag 1680

aagaactccc tggccctgag cctgaccgcc gaccagatgg tgtctgccct gctggatgcc 1740

gagcccccca tcctgtacag cgagtacgac cccaccagac ccttcagcga ggccagcatg 1800

atgggcctgc tgaccaacct ggccgaccgg gaactggtgc acatgatcaa ctgggccaag 1860

cgggtgcccg gcttcgtgga tctgacactg cacgaccagg tgcacctgct ggaatgcgct 1920

tggctggaaa tcctgatgat cggcctcgtg tggcggagca tggaacaccc tggcaagctg 1980

ctgttcgccc ccaacctgct gctggaccgg aaccagggca aatgcgtgga aggcatggtg 2040

gaaatcttcg acatgctgct ggccacctcc agccggttcc ggatgatgaa cctgcagggc 2100

gaagagttcg tgtgtctgaa gtccatcatc ctgctgaata gcggcgtgta caccttcctg 2160

agcagcaccc tgaaaagcct ggaagaaaag gaccacatcc accgggtgct ggacaagatc 2220

accgacaccc tgattcacct gatggccaag gccggactga ccctgcagca gcagcatcag 2280

agactggctc agctgctgct gatcctgtcc cacatccggc acatgagcaa caagcggatg 2340

gaacatctgt acagcatgaa gtgcaagaac gtggtgcctc tgttcgatct gctgctggaa 2400

atgctggacg cccacaggct gcacgcccca acatccggat ctggctctgg aagcggcagc 2460

atggtgtcta agggggaaga ggacaacatg gccatcatca aagaattcat gcggttcaag 2520

gtgcacatgg aaggctccgt gaatggccac gaattcgaga tcgaggggga gggcgagggc 2580

agaccttatg agggaaccca gaccgccaag ctgaaagtga ccaagggcgg acccctgcct 2640

ttcgcctggg atatcctgtc tccccagttt atgtacggca gcaaggccta cgtgaagcac 2700

cccgccgaca tccccgacta cctgaagctg agcttccctg agggcttcaa gtgggagaga 2760

gtgatgaatt tcgaggacgg cggagtcgtg acagtgaccc aggatagctc tctgcaggac 2820

ggcgagttca tctacaaagt gaagctgcgg ggcaccaact tccccagcga cggacccgtg 2880

atgcagaaaa agaccatggg ctgggaggcc agctccgaga gaatgtaccc agaggacggg 2940

gccctgaagg gggagatcaa gcagcggctg aaactgaagg atggcggcca ctacgacgca 3000

gaagtgaaaa ccacctacaa ggccaagaaa cctgtgcagc tgcctggcgc ctacaatgtg 3060

aacatcaagc tggacattac cagccacaac gaggactaca ccatcgtgga acagtacgag 3120

cgggccgagg gcaggcattc tacaggcgga atggatgaac tgtataagtg cgtgaccgac 3180

tag 3183

<210>33

<211>1060

<212>PRT

<213> Artificial sequence

<220>

<223> v.3 constructs

<400>33

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp

35 40 45

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu

50 55 60

Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

65 70 75 80

His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

85 90 95

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu

100 105 110

Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr

115 120 125

Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser

145 150 155 160

Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr

165 170 175

Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln

180 185 190

Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu

195 200 205

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys

210 215 220

Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr

225 230 235 240

Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly

245 250 255

Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

260 265 270

Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Gly Ser Lys Arg Gly Arg Lys Lys Leu Leu

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

370 375 380

Glu Leu Arg Ser Gly Ser Gly Ser Gly Ser Leu Arg Val Lys Phe Ser

385 390 395 400

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

405 410 415

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

420 425 430

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

435 440 445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ser Gly

500 505 510

Ser Gly Ser Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln

515 520 525

Arg Asp Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met

530 535 540

Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys

545 550 555 560

Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala

565 570 575

Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr

580 585 590

Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala

595 600 605

Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly

610 615 620

Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala

625 630 635 640

Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His

645 650 655

Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln

660 665 670

Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala

675 680 685

Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val

690 695 700

Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu

705 710 715 720

Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val

725 730 735

Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly

740 745 750

Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile

755 760 765

Leu Ser His Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr

770 775 780

Ser Met Lys Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu

785 790 795 800

Met Leu Asp Ala His Arg Leu His Ala Pro Thr Ser Gly Ser Gly Ser

805 810 815

Gly Ser Gly Ser Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile

820 825 830

Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn

835 840 845

Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu

850 855 860

Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro

865 870 875 880

Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala

885 890 895

Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe

900 905 910

Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly

915 920 925

Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile

930 935 940

Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val

945 950 955 960

Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr

965 970 975

Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu

980 985 990

Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala

995 1000 1005

Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys

1010 1015 1020

Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln

1025 1030 1035

Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu

1040 1045 1050

Leu Tyr Lys Cys Val Thr Asp

1055 1060

<210>34

<211>3206

<212>DNA

<213> Artificial sequence

<220>

<223> v.4 constructs

<400>34

atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgccgctaga 60

cctgagcaga agctgatctc cgaagaggac ctggacatcc agatgaccca gaccaccagc 120

agcctgagcg ccagcctggg cgatagagtg accatcagct gcagagccag ccaggacatc 180

agcaagtacc tgaactggta tcagcagaaa cccgacggca ccgtgaagct gctgatctac 240

cacaccagca gactgcacag cggcgtgccc agcagatttt ctggcagcgg ctccggcacc 300

gactacagcc tgaccatctc caacctggaa caggaagata tcgctaccta cttctgtcag 360

caaggcaaca ccctgcccta caccttcggc ggaggcacca agctggaaat cacaggcggc 420

ggaggatctg gcggaggcgg aagtggcgga gggggatctg aagtgaaact gcaggaaagc 480

ggccctggcc tggtggcccc atctcagtct ctgagcgtga cctgtaccgt gtccggcgtg 540

tccctgcctg actatggcgt gtcctggatc agacagcccc ccagaaaggg cctggaatgg 600

ctgggagtga tctggggcag cgagacaacc tactacaaca gcgccctgaa gtcccggctg 660

accatcatca aggacaactc caagagccag gtgttcctga agatgaacag cctgcagacc 720

gacgacaccg ccatctacta ctgcgccaag cactactact acggcggcag ctacgccatg 780

gactactggg gccagggcac aagcgtgacc gtgtctagca caaccacccc tgcccctaga 840

cctccaaccc cagcccctac aatcgccagc cagcctctgt ctctgaggcc cgaggcttgt 900

agaccagctg ctggcggagc cgtgcacacc agaggactgg atttcgcctg cgacatctac 960

atctgggccc ctctggccgg cacatgtggc gtgctgctgc tgagcctcgt gatcaccctg 1020

tactgcggat ccggcagcgg atctggcagt ggaagcgata gaagaggcgg cagaatgctg 1080

aaacacaagc ggcagaggga cgacggggaa ggcagaggcg aagtgggatc tgccggcgat 1140

atgagagccg ccaacctgtg gcctagcccc ctgatgatca agcggagcaa gaagaactcc 1200

ctggccctga gcctgaccgc cgaccagatg gtgtctgccc tgctggatgc cgagcccccc 1260

atcctgtaca gcgagtacga ccccaccaga cccttcagcg aggccagcat gatgggcctg 1320

ctgaccaacc tggccgaccg ggaactggtg cacatgatca actgggccaa gcgggtgccc 1380

ggcttcgtgg atctgacact gcacgaccag gtgcacctgc tggaatgcgc ttggctggaa 1440

atcctgatga tcggcctcgt gtggcggagc atggaacacc ctggcaagct gctgttcgcc 1500

cccaacctgc tgctggaccg gaaccagggc aaatgcgtgg aaggcatggt ggaaatcttc 1560

gacatgctgc tggccacctc cagccggttc cggatgatga acctgcaggg cgaagagttc 1620

gtgtgtctga agtccatcat cctgctgaat agcggcgtgt acaccttcct gagcagcacc 1680

ctgaaaagcc tggaagaaaa ggaccacatc caccgggtgc tggacaagat caccgacacc 1740

ctgattcacc tgatggccaa ggccggactg accctgcagc agcagcatca gagactggct 1800

cagctgctgc tgatcctgtc ccacatccgg cacatgagca acaagcggat ggaacatctg 1860

tacagcatga agtgcaagaa cgtggtgcct ctgttcgatc tgctgctgga aatgctggac 1920

gcccacaggc tgcacgcccc aacatccgga tctggctctg gaagcggcag caagcggggc 1980

agaaagaaac tgctgtacat ctttaagcag cccttcatgc ggcccgtgca gaccacccag 2040

gaagaggacg gctgctcctg cagattcccc gaggaagaag aaggcggctg cgagctgaga 2100

tccggatctg gaagtggctc cctgagagtg aagtttagca gaagcgccga cgcccctgcc 2160

tatcagcagg gacagaacca gctgtataac gagctgaacc tgggcaggcg ggaagagtac 2220

gacgtgctgg ataagaggcg gggcagggac cctgaaatgg gcggcaaacc cagacggaag 2280

aacccccagg aaggcctgta caacgaactg cagaaagaca agatggccga ggcctacagc 2340

gagatcggaa tgaagggcga gcggcggaga ggcaagggac atgatggcct gtaccagggc 2400

ctgtccaccg ccaccaagga cacctatgac gccctgcaca tgcaggccct gcctccaaga 2460

ggaagtggat ctgggagcgg ctctatggtg tctaaggggg aagaggacaa catggccatc 2520

atcaaagaat tcatgcggtt caaggtgcac atggaaggct ccgtgaatgg ccacgaattc 2580

gagatcgagg gggagggcga gggcagacct tatgagggaa cccagaccgc caagctgaaa 2640

gtgaccaagg gcggacccct gcctttcgcc tgggatatcc tgtctcccca gtttatgtac 2700

ggcagcaagg cctacgtgaa gcaccccgcc gacatccccg actacctgaa gctgagcttc 2760

cctgagggct tcaagtggga gagagtgatg aatttcgagg acggcggagt cgtgacagtg 2820

acccaggata gctctctgca ggacggcgag ttcatctaca aagtgaagct gcggggcacc 2880

aacttcccca gcgacggacc cgtgatgcag aaaaagacca tgggctggga ggccagctcc 2940

gagagaatgt acccagagga cggggccctg aagggggaga tcaagcagcg gctgaaactg 3000

aaggatggcg gccactacga cgcagaagtg aaaaccacct acaaggccaa gaaacctgtg 3060

cagctgcctg gcgcctacaa tgtgaacatc aagctggaca ttaccagcca caacgaggac 3120

tacaccatcg tggaacagta cgagcgggcc gagggcaggc attctacagg cggaatggat 3180

gaactgtata agtgcgtgac cgacta 3206

<210>35

<211>1068

<212>PRT

<213> Artificial sequence

<220>

<223> v.4 constructs

<400>35

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp

35 40 45

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu

50 55 60

Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

65 70 75 80

His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

85 90 95

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu

100 105 110

Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr

115 120 125

Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser

145 150 155 160

Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr

165 170 175

Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln

180 185 190

Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu

195 200 205

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys

210 215 220

Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr

225 230 235 240

Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly

245 250 255

Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

260 265 270

Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

340 345 350

Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp

355 360 365

Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala

370 375 380

Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser

385 390 395 400

Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp

405 410 415

Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe

420 425 430

Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu

435 440 445

Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp

450 455 460

Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu

465 470 475 480

Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys

485 490 495

Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys

500 505 510

Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser

515 520 525

Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys

530 535 540

Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr

545 550 555 560

Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys

565 570 575

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu

580 585 590

Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His

595 600 605

Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser Met Lys

610 615 620

Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu Met Leu Asp

625 630 635 640

Ala His Arg Leu His Ala Pro Thr Ser Gly Ser Gly Ser Gly Ser Gly

645 650 655

Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

660 665 670

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

675 680 685

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Ser Gly Ser Gly

690 695 700

Ser Gly Ser Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

705 710 715 720

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

725 730 735

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

740 745 750

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

755 760 765

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

770 775 780

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

785 790 795 800

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

805 810 815

Leu Pro Pro Arg Gly Ser Gly Ser Gly Ser Gly Ser Met Val Ser Lys

820 825 830

Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

835 840 845

Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

850 855 860

Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys

865 870 875 880

Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

885 890 895

Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile

900 905 910

Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg

915 920 925

Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser

930 935 940

Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

945 950 955 960

Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

965 970 975

Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly

980 985 990

Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala

995 1000 1005

Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro

1010 1015 1020

Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn

1025 1030 1035

Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg

1040 1045 1050

His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Cys Val Thr Asp

1055 1060 1065

<210>36

<211>2694

<212>DNA

<213> Artificial sequence

<220>

<223> v.5 constructs

<400>36

atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgccgctaga 60

cctgagcaga agctgatctc cgaagaggac ctggacatcc agatgaccca gaccaccagc 120

agcctgagcg ccagcctggg cgatagagtg accatcagct gcagagccag ccaggacatc 180

agcaagtacc tgaactggta tcagcagaaa cccgacggca ccgtgaagct gctgatctac 240

cacaccagca gactgcacag cggcgtgccc agcagatttt ctggcagcgg ctccggcacc 300

gactacagcc tgaccatctc caacctggaa caggaagata tcgctaccta cttctgtcag 360

caaggcaaca ccctgcccta caccttcggc ggaggcacca agctggaaat cacaggcggc 420

ggaggatctg gcggaggcgg aagtggcgga gggggatctg aagtgaaact gcaggaaagc 480

ggccctggcc tggtggcccc atctcagtct ctgagcgtga cctgtaccgt gtccggcgtg 540

tccctgcctg actatggcgt gtcctggatc agacagcccc ccagaaaggg cctggaatgg 600

ctgggagtga tctggggcag cgagacaacc tactacaaca gcgccctgaa gtcccggctg 660

accatcatca aggacaactc caagagccag gtgttcctga agatgaacag cctgcagacc 720

gacgacaccg ccatctacta ctgcgccaag cactactact acggcggcag ctacgccatg 780

gactactggg gccagggcac aagcgtgacc gtgtctagca caaccacccc tgcccctaga 840

cctccaaccc cagcccctac aatcgccagc cagcctctgt ctctgaggcc cgaggcttgt 900

agaccagctg ctggcggagc cgtgcacacc agaggactgg atttcgcctg cgacatctac 960

atctgggccc ctctggccgg cacatgtggc gtgctgctgc tgagcctcgt gatcaccctg 1020

tactgcggat ccggcagcgg atctggcagt ggaagcgata gaagaggcgg cagaatgctg 1080

aaacacaagc ggcagaggga cgacggggaa ggcagaggcg aagtgggatc tgccggcgat 1140

atgagagccg ccaacctgtg gcctagcccc ctgatgatca agcggagcaa gaagaactcc 1200

ctggccctga gcctgaccgc cgaccagatg gtgtctgccc tgctggatgc cgagcccccc 1260

atcctgtaca gcgagtacga ccccaccaga cccttcagcg aggccagcat gatgggcctg 1320

ctgaccaacc tggccgaccg ggaactggtg cacatgatca actgggccaa gcgggtgccc 1380

ggcttcgtgg atctgacact gcacgaccag gtgcacctgc tggaatgcgc ttggctggaa 1440

atcctgatga tcggcctcgt gtggcggagc atggaacacc ctggcaagct gctgttcgcc 1500

cccaacctgc tgctggaccg gaaccagggc aaatgcgtgg aaggcatggt ggaaatcttc 1560

gacatgctgc tggccacctc cagccggttc cggatgatga acctgcaggg cgaagagttc 1620

gtgtgtctga agtccatcat cctgctgaat agcggcgtgt acaccttcct gagcagcacc 1680

ctgaaaagcc tggaagaaaa ggaccacatc caccgggtgc tggacaagat caccgacacc 1740

ctgattcacc tgatggccaa ggccggactg accctgcagc agcagcatca gagactggct 1800

cagctgctgc tgatcctgtc ccacatccgg cacatgagca acaagcggat ggaacatctg 1860

tacagcatga agtgcaagaa cgtggtgcct ctgttcgatc tgctgctgga aatgctggac 1920

gcccacaggc tgcacgcccc aacatccgga tctggctctg gaagcggcag catggtgtct 1980

aagggggaag aggacaacat ggccatcatc aaagaattca tgcggttcaa ggtgcacatg 2040

gaaggctccg tgaatggcca cgaattcgag atcgaggggg agggcgaggg cagaccttat 2100

gagggaaccc agaccgccaa gctgaaagtg accaagggcg gacccctgcc tttcgcctgg 2160

gatatcctgt ctccccagtt tatgtacggc agcaaggcct acgtgaagca ccccgccgac 2220

atccccgact acctgaagct gagcttccct gagggcttca agtgggagag agtgatgaat 2280

ttcgaggacg gcggagtcgt gacagtgacc caggatagct ctctgcagga cggcgagttc 2340

atctacaaag tgaagctgcg gggcaccaac ttccccagcg acggacccgt gatgcagaaa 2400

aagaccatgg gctgggaggc cagctccgag agaatgtacc cagaggacgg ggccctgaag 2460

ggggagatca agcagcggct gaaactgaag gatggcggcc actacgacgc agaagtgaaa 2520

accacctaca aggccaagaa acctgtgcag ctgcctggcg cctacaatgt gaacatcaag 2580

ctggacatta ccagccacaa cgaggactac accatcgtgg aacagtacga gcgggccgag 2640

ggcaggcatt ctacaggcgg aatggatgaa ctgtataagt gcgtgaccga ctag 2694

<210>37

<211>897

<212>PRT

<213> Artificial sequence

<220>

<223> v.5 constructs

<400>37

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp

20 25 30

Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp

3540 45

Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu

50 55 60

Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

65 70 75 80

His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

85 90 95

Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu

100 105 110

Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr

115 120 125

Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser

145 150 155 160

Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr

165 170 175

Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln

180 185 190

Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu

195200 205

Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys

210 215 220

Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr

225 230 235 240

Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly

245 250 255

Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

260 265 270

Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

340 345 350

Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp

355 360365

Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala

370 375 380

Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser

385 390 395 400

Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp

405 410 415

Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe

420 425 430

Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu

435 440 445

Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp

450 455 460

Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu

465 470 475 480

Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys

485 490 495

Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys

500 505 510

Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser

515 520525

Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys

530 535 540

Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr

545 550 555 560

Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys

565 570 575

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu

580 585 590

Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His

595 600 605

Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser Met Lys

610 615 620

Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu Met Leu Asp

625 630 635 640

Ala His Arg Leu His Ala Pro Thr Ser Gly Ser Gly Ser Gly Ser Gly

645 650 655

Ser Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu

660 665 670

Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu

675 680 685

Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln

690 695 700

Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp

705 710 715 720

Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys

725 730 735

His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly

740 745 750

Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr

755 760 765

Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val

770 775 780

Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys

785 790 795 800

Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp

805 810 815

Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly

820 825 830

Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro

835 840 845

Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr

850 855 860

Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu

865 870 875 880

Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Cys Val Thr

885 890 895

Asp

<210>38

<211>2391

<212>DNA

<213> Artificial sequence

<220>

<223> ERA-LBD fusion protein with FLAG construct

<400>38

atgatccacc tgggacacat cctgtttttg ctgctgctgc cagtggctgc cgccgattac 60

aaagacgacg atgataaaca gacaacacca ggcgagagat ctagcctgcc cgccttctac 120

cctggcacca gcggctcttg ttctggctgt ggcagcctgt ctctgcccat ctatatttgg 180

gcacccctgg ctggaacctg cggagtgctg ctgctgtctc tcgtgattac actgtattgc 240

aaaaggggcc ggaaaaagct gctgtatatt ttcaaacagc cttttatgag gcctgtgcag 300

acaacacagg aagaggacgg ctgtagctgt cggttccccg aagaggaaga ggggggctgc 360

gaactgggat caggcagtgg ctctggcagc gatagaagag gcggcagaat gctgaaacac 420

aagcggcaga gggacgacgg ggaaggcaga ggcgaagtgg gatctgccgg cgatatgaga 480

gccgccaacc tgtggcctag ccccctgatg atcaagcgga gcaagaagaa ctccctggcc 540

ctgagcctga ccgccgacca gatggtgtct gccctgctgg atgccgagcc ccccatcctg 600

tacagcgagt acgaccccac cagacccttc agcgaggcca gcatgatggg cctgctgacc 660

aacctggccg accgggaact ggtgcacatg atcaactggg ccaagcgggt gcccggcttc 720

gtggatctga cactgcacga ccaggtgcac ctgctggaat gcgcttggct ggaaatcctg 780

atgatcggcc tcgtgtggcg gagcatggaa caccctggca agctgctgtt cgcccccaac 840

ctgctgctgg accggaacca gggcaaatgc gtggaaggca tggtggaaat cttcgacatg 900

ctgctggcca cctccagccg gttccggatg atgaacctgc agggcgaaga gttcgtgtgt 960

ctgaagtcca tcatcctgct gaatagcggc gtgtacacct tcctgagcag caccctgaaa 1020

agcctggaag aaaaggacca catccaccgg gtgctggaca agatcaccga caccctgatt 1080

cacctgatgg ccaaggccgg actgaccctg cagcagcagc atcagagact ggctcagctg 1140

ctgctgatcc tgtcccacat ccggcacatg agcaacaagc ggatggaaca tctgtacagc 1200

atgaagtgca agaacgtggt gcctctgttc gatctgctgc tggaaatgct ggacgcccac 1260

aggctgcacg ccccaacatc cagatccgga tctggaagtg gctccctgag agtgaagttt 1320

agcagaagcg ccgacgcccc tgcctatcag cagggacaga accagctgta taacgagctg 1380

aacctgggca ggcgggaaga gtacgacgtg ctggataaga ggcggggcag ggaccctgaa 1440

atgggcggca aacccagacg gaagaacccc caggaaggcc tgtacaacga actgcagaaa 1500

gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcggcg gagaggcaag 1560

ggacatgatg gcctgtacca gggcctgtcc accgccacca aggacaccta tgacgccctg 1620

cacatgcagg ccctgcctcc aagaggaagt ggatctggga gcggctctat ggtgtctaag 1680

ggggaagagg acaacatggc catcatcaaa gaattcatgc ggttcaaggt gcacatggaa 1740

ggctccgtga atggccacga attcgagatc gagggggagg gcgagggcag accttatgag 1800

ggaacccaga ccgccaagct gaaagtgacc aagggcggac ccctgccttt cgcctgggat 1860

atcctgtctc cccagtttat gtacggcagc aaggcctacg tgaagcaccc cgccgacatc 1920

cccgactacc tgaagctgag cttccctgag ggcttcaagt gggagagagt gatgaatttc 1980

gaggacggcg gagtcgtgac agtgacccag gatagctctc tgcaggacgg cgagttcatc 2040

tacaaagtga agctgcgggg caccaacttc cccagcgacg gacccgtgat gcagaaaaag 2100

accatgggct gggaggccag ctccgagaga atgtacccag aggacggggc cctgaagggg 2160

gagatcaagc agcggctgaa actgaaggat ggcggccact acgacgcaga agtgaaaacc 2220

acctacaagg ccaagaaacc tgtgcagctg cctggcgcct acaatgtgaa catcaagctg 2280

gacattacca gccacaacga ggactacacc atcgtggaac agtacgagcg ggccgagggc 2340

aggcattcta caggcggaat ggatgaactg tataagtgcg tgaccgacta g 2391

<210>39

<211>796

<212>PRT

<213> Artificial sequence

<220>

<223> ERA-LBD fusion protein with FLAG construct

<400>39

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu ProVal Ala

1 5 10 15

Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr Pro Gly Glu

20 25 30

Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly Ser Cys Ser

35 40 45

Gly Cys Gly Ser Leu Ser Leu Pro Ile Tyr Ile Trp Ala Pro Leu Ala

50 55 60

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

65 70 75 80

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

85 90 95

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

100 105 110

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Ser Gly Ser Gly Ser

115 120 125

Gly Ser Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg

130 135 140

Asp Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg

145 150 155 160

Ala Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys

165 170 175

Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu

180 185 190

Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg

195 200 205

Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp

210 215 220

Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe

225 230 235 240

Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp

245 250 255

Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro

260 265 270

Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly

275 280 285

Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr

290 295 300

Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys

305 310 315 320

Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser

325 330 335

Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu

340 345 350

Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu

355 360 365

Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu

370 375 380

Ser His Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser

385 390 395 400

Met Lys Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu Met

405 410 415

Leu Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Ser Gly Ser Gly

420 425 430

Ser Gly Ser Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

435 440 445

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

450 455 460

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

465 470 475 480

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

485 490 495

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

500 505 510

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

515 520 525

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

530 535 540

Leu Pro Pro Arg Gly Ser Gly Ser Gly Ser Gly Ser Met Val Ser Lys

545 550 555 560

Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

565 570 575

Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

580 585 590

Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys

595 600 605

Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

610 615 620

Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp Ile

625 630 635 640

Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg

645 650 655

Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser

660 665 670

Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

675 680 685

Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

690 695 700

Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly

705 710 715 720

Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp Ala

725 730 735

Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly

740 745 750

Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn Glu Asp

755 760 765

Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser Thr

770 775 780

Gly Gly Met Asp Glu Leu Tyr Lys Cys Val Thr Asp

785 790 795

<210>40

<211>18

<212>PRT

<213> human

<400>40

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

Ala Ala

<210>41

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> FLAG tag

<400>41

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210>42

<211>30

<212>PRT

<213> human

<400>42

Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly

1 5 10 15

Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro

20 25 30

<210>43

<211>24

<212>PRT

<213> human

<400>43

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

15 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210>44

<211>42

<212>PRT

<213> human

<400>44

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210>45

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>45

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>46

<211>297

<212>PRT

<213> Artificial sequence

<220>

<223> modified ERa ligand binding domain

<400>46

Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp

1 5 10 15

Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala

20 25 30

Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser

35 40 45

Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp

50 55 60

Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe

65 70 75 80

Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu

85 90 95

Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp

100 105 110

Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu

115 120 125

Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys

130 135 140

Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys

145 150 155 160

Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser

165 170 175

Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys

180 185 190

Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr

195 200 205

Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys

210 215 220

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu

225 230 235 240

Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His

245 250 255

Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser Met Lys

260 265 270

Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu Met Leu Asp

275 280 285

Ala His Arg Leu His Ala Pro Thr Ser

290 295

<210>47

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>47

Arg Ser Gly Ser Gly Ser Gly Ser

1 5

<210>48

<211>113

<212>PRT

<213> human

<400>48

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210>49

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>49

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>50

<211>240

<212>PRT

<213> Artificial sequence

<220>

<223>mCherry

<400>50

Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe

1 5 10 15

Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe

20 25 30

Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr

35 40 45

Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp

50 55 60

Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His

65 70 75 80

Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe

85 90 95

Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val

100 105 110

Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys

115 120 125

Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

130 135 140

Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly

145 150 155 160

Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly

165 170 175

His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val

180 185 190

Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser

195 200 205

His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly

210 215 220

Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Cys Val Thr Asp

225 230 235 240

<210>51

<211>2265

<212>DNA

<213> Artificial sequence

<220>

<223> PR ligand binding domain constructs

<400>51

atgatccacc tgggacacat cctgtttttg ctgctgctgc cagtggctgc cgccgattac 60

aaagacgacg atgataaaca gacaacacca ggcgagagat ctagcctgcc cgccttctac 120

cctggcacca gcggctcttg ttctggctgt ggcagcctgt ctctgcccat ctatatttgg 180

gcacccctgg ctggaacctg cggagtgctg ctgctgtctc tcgtgattac actgtattgc 240

aaaaggggcc ggaaaaagct gctgtatatt ttcaaacagc cttttatgag gcctgtgcag 300

acaacacagg aagaggacgg ctgtagctgt cggttccccg aagaggaaga ggggggctgc 360

gaactgggat caggcagtgg ctctggcagc gggcaagaca ttcagctcat acctcctttg 420

ataaatttgc tgatgtctat agaaccagat gtcatatacg ctggtcacga caatacgaaa 480

ccggacacat cttcatcttt gcttacctct ctgaatcaac tgggtgaacg acagctcctg 540

agtgttgtta agtggtctaa aagcctcccg ggcttcagga atttgcacat agacgaccaa 600

atcacgctca tccaatattc ctggatgagt ctcatggtct ttggtctcgg ttggcgcagc 660

tataagcacg tctctggcca gatgttgtat ttcgcaccag acctgatcct gaacgaacag 720

aggatgaagg aatcaagctt ttactctctc tgcttgacta tgtggcaaat cccccaagaa 780

ttcgtgaaac ttcaagtttc ccaagaagaa ttcctctgca tgaaagtcct tcttttgctc 840

aacacgattc ccctggaagg cttgaggtct caaacgcaat tcgaggagat gcggagtagc 900

tatatacgcgaactcatcaa ggccatcggt ttgcggcaaa agggagtggt ctctagtagc 960

caacgatttt accagctgac taagctcctt gacaaccttc acgatctcgt caaacaactg 1020

cacctgtact gtcttaacac atttatacaa tcacgggcac tttctgtaga gttcccagag 1080

atgatgtctg aggtcatcgc agcccaactt ccgaaaattc ttgcaggaat ggtgaagcca 1140

cttctgttcc ataagaaaag atccggatct ggaagtggct ccctgagagt gaagtttagc 1200

agaagcgccg acgcccctgc ctatcagcag ggacagaacc agctgtataa cgagctgaac 1260

ctgggcaggc gggaagagta cgacgtgctg gataagaggc ggggcaggga ccctgaaatg 1320

ggcggcaaac ccagacggaa gaacccccag gaaggcctgt acaacgaact gcagaaagac 1380

aagatggccg aggcctacag cgagatcgga atgaagggcg agcggcggag aggcaaggga 1440

catgatggcc tgtaccaggg cctgtccacc gccaccaagg acacctatga cgccctgcac 1500

atgcaggccc tgcctccaag aggaagtgga tctgggagcg gctctatggt gtctaagggg 1560

gaagaggaca acatggccat catcaaagaa ttcatgcggt tcaaggtgca catggaaggc 1620

tccgtgaatg gccacgaatt cgagatcgag ggggagggcg agggcagacc ttatgaggga 1680

acccagaccg ccaagctgaa agtgaccaag ggcggacccc tgcctttcgc ctgggatatc 1740

ctgtctcccc agtttatgta cggcagcaag gcctacgtga agcaccccgc cgacatcccc 1800

gactacctga agctgagctt ccctgagggc ttcaagtggg agagagtgat gaatttcgag 1860

gacggcggag tcgtgacagt gacccaggat agctctctgc aggacggcga gttcatctac 1920

aaagtgaagc tgcggggcac caacttcccc agcgacggac ccgtgatgca gaaaaagacc 1980

atgggctggg aggccagctc cgagagaatg tacccagagg acggggccct gaagggggag 2040

atcaagcagc ggctgaaact gaaggatggc ggccactacg acgcagaagt gaaaaccacc 2100

tacaaggcca agaaacctgt gcagctgcct ggcgcctaca atgtgaacat caagctggac 2160

attaccagcc acaacgagga ctacaccatc gtggaacagt acgagcgggc cgagggcagg 2220

cattctacag gcggaatgga tgaactgtat aagtgcgtga ccgac 2265

<210>52

<211>755

<212>PRT

<213> Artificial sequence

<220>

<223> PR ligand binding domain constructs

<400>52

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gln Thr Thr Pro Gly Glu

20 25 30

Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly Thr Ser Gly Ser Cys Ser

35 40 45

Gly Cys Gly Ser Leu Ser Leu Pro Ile Tyr Ile Trp Ala Pro Leu Ala

50 55 60

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

65 70 75 80

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

85 90 95

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

100 105 110

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Ser Gly Ser Gly Ser

115 120 125

Gly Ser Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile Asn Leu Leu

130 135 140

Met Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp Asn Thr Lys

145 150 155 160

Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu

165 170 175

Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly Phe

180 185 190

Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser Trp

195 200 205

Met Ser Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr Lys His Val

210 215 220

Ser Gly Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu Asn Glu Gln

225 230 235 240

Arg Met Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr Met Trp Gln

245 250 255

Ile Pro Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu Glu Phe Leu

260 265 270

Cys Met Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu

275 280 285

Arg Ser Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu

290 295 300

Leu Ile Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val Ser Ser Ser

305 310 315 320

Gln Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Asn Leu His Asp Leu

325 330 335

Val Lys Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile Gln Ser Arg

340 345 350

Ala Leu Ser Val Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala

355 360 365

Gln Leu Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu Leu Phe His

370 375 380

Lys Lys Arg Ser Gly Ser Gly Ser Gly Ser Leu Arg Val Lys Phe Ser

385 390 395 400

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

405 410 415

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

420 425 430

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

435 440 445

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

450 455 460

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

465 470 475 480

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

485 490 495

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ser Gly

500 505 510

Ser Gly Ser Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile

515 520 525

Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly

530 535 540

His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly

545 550 555 560

Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe

565 570 575

Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr

580 585 590

Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro

595 600 605

Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val

610 615 620

Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr

625 630 635 640

Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met

645 650 655

Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro

660 665 670

Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys

675 680 685

Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys

690 695 700

Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp

705 710 715 720

Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg

725 730 735

Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Cys

740 745 750

Val Thr Asp

755

<210>53

<211>18

<212>PRT

<213> human

<400>53

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

Ala Ala

<210>54

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> FLAG tag

<400>54

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210>55

<211>30

<212>PRT

<213> human

<400>55

Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly

1 5 10 15

Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro

20 25 30

<210>56

<211>24

<212>PRT

<213> human

<400>56

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 Leu Tyr Cys

20

<210>57

<211>42

<212>PRT

<213> human

<400>57

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210>58

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>58

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>59

<211>256

<212>PRT

<213> human

<400>59

Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile Asn Leu Leu Met Ser

1 5 10 15

Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp Asn Thr Lys Pro Asp

20 25 30

Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu Arg Gln

35 40 45

Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly Phe Arg Asn

50 55 60

Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser Trp Met Ser

65 70 75 80

Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr Lys His Val Ser Gly

85 90 95

Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu Asn Glu Gln Arg Met

100 105 110

Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr Met Trp Gln Ile Pro

115 120 125

Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu Glu Phe Leu Cys Met

130 135 140

Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu Arg Ser

145 150 155 160

Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu Leu Ile

165 170 175

Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val Ser Ser Ser Gln Arg

180 185 190

Phe Tyr Gln Leu Thr Lys Leu Leu Asp Asn Leu His Asp Leu Val Lys

195 200 205

Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile Gln Ser Arg Ala Leu

210 215 220

Ser Val Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala Gln Leu

225 230 235 240

Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu Leu Phe His Lys Lys

245 250 255

<210>60

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>60

Arg Ser Gly Ser Gly Ser Gly Ser

1 5

<210>61

<211>113

<212>PRT

<213> human

<400>61

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210>62

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>62

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210>63

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> Dap10 Signal

<400>63

atgatccacc tgggacacat cctgtttttg ctgctgctgc cagtggctgc cgcc 54

<210>64

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> FLAG tag

<400>64

gattacaaag acgacgatga taaa 24

<210>65

<211>90

<212>DNA

<213> Artificial sequence

<220>

<223> Dap10 extracellular domain

<400>65

cagacaacac caggcgagag atctagcctg cccgccttct accctggcac cagcggctct 60

tgttctggct gtggcagcct gtctctgccc 90

<210>66

<211>72

<212>DNA

<213> Artificial sequence

<220>

<223> transmembrane domain of CD8

<400>66

atctatattt gggcacccct ggctggaacc tgcggagtgc tgctgctgtc tctcgtgatt 60

acactgtatt gc 72

<210>67

<211>126

<212>DNA

<213> Artificial sequence

<220>

<223>4-1BB Co-stimulatory Domain

<400>67

aaaaggggcc ggaaaaagct gctgtatatt ttcaaacagc cttttatgag gcctgtgcag 60

acaacacagg aagaggacgg ctgtagctgt cggttccccg aagaggaaga ggggggctgc 120

gaactg 126

<210>68

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>68

ggatcaggca gtggctctgg cagc 24

<210>69

<211>768

<212>DNA

<213> Artificial sequence

<220>

<223> ligand binding domain of progesterone receptor

<400>69

gggcaagaca ttcagctcat acctcctttg ataaatttgc tgatgtctat agaaccagat 60

gtcatatacg ctggtcacga caatacgaaa ccggacacat cttcatcttt gcttacctct 120

ctgaatcaac tgggtgaacg acagctcctg agtgttgtta agtggtctaa aagcctcccg 180

ggcttcagga atttgcacat agacgaccaa atcacgctca tccaatattc ctggatgagt 240

ctcatggtct ttggtctcgg ttggcgcagc tataagcacg tctctggcca gatgttgtat 300

ttcgcaccag acctgatcct gaacgaacag aggatgaagg aatcaagctt ttactctctc 360

tgcttgacta tgtggcaaat cccccaagaa ttcgtgaaac ttcaagtttc ccaagaagaa 420

ttcctctgca tgaaagtcct tcttttgctc aacacgattc ccctggaagg cttgaggtct 480

caaacgcaat tcgaggagat gcggagtagc tatatacgcg aactcatcaa ggccatcggt 540

ttgcggcaaa agggagtggt ctctagtagc caacgatttt accagctgac taagctcctt 600

gacaaccttc acgatctcgt caaacaactg cacctgtact gtcttaacac atttatacaa 660

tcacgggcac tttctgtaga gttcccagag atgatgtctg aggtcatcgc agcccaactt 720

ccgaaaattc ttgcaggaat ggtgaagcca cttctgttcc ataagaaa 768

<210>70

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>70

ggatctggaa gtggctcc 18

<210>71

<211>339

<212>DNA

<213> Artificial sequence

<220>

<223> sequence encoding CD3zeta signaling domain

<400>71

ctgagagtga agtttagcag aagcgccgac gcccctgcct atcagcaggg acagaaccag 60

ctgtataacg agctgaacct gggcaggcgg gaagagtacg acgtgctgga taagaggcgg 120

ggcagggacc ctgaaatggg cggcaaaccc agacggaaga acccccagga aggcctgtac 180

aacgaactgc agaaagacaa gatggccgag gcctacagcg agatcggaat gaagggcgag 240

cggcggagag gcaagggaca tgatggcctg taccagggcc tgtccaccgc caccaaggac 300

acctatgacg ccctgcacat gcaggccctg cctccaaga 339

<210>72

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>72

ggaagtggat ctgggagcgg ctct 24

<210>73

<211>720

<212>DNA

<213> Artificial sequence

<220>

<223>mCherry

<400>73

atggtgtcta agggggaaga ggacaacatg gccatcatca aagaattcat gcggttcaag 60

gtgcacatgg aaggctccgt gaatggccac gaattcgaga tcgaggggga gggcgagggc 120

agaccttatg agggaaccca gaccgccaag ctgaaagtga ccaagggcgg acccctgcct 180

ttcgcctggg atatcctgtc tccccagttt atgtacggca gcaaggccta cgtgaagcac 240

cccgccgaca tccccgacta cctgaagctg agcttccctg agggcttcaa gtgggagaga 300

gtgatgaatt tcgaggacgg cggagtcgtg acagtgaccc aggatagctc tctgcaggac 360

ggcgagttca tctacaaagt gaagctgcgg ggcaccaact tccccagcga cggacccgtg 420

atgcagaaaa agaccatggg ctgggaggcc agctccgaga gaatgtaccc agaggacggg 480

gccctgaagg gggagatcaa gcagcggctg aaactgaagg atggcggcca ctacgacgca 540

gaagtgaaaa ccacctacaa ggccaagaaa cctgtgcagc tgcctggcgc ctacaatgtg 600

aacatcaagc tggacattac cagccacaac gaggactaca ccatcgtgga acagtacgag 660

cgggccgagg gcaggcattc tacaggcgga atggatgaac tgtataagtg cgtgaccgac 720

<210>74

<211>480

<212>PRT

<213> human

<400>74

Met Glu Arg Asp Glu Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly

1 5 10 15

Ser Ala Gly Phe Leu Glu Pro Pro Ala Ala Leu Pro Pro Pro Pro Arg

20 25 30

Asn Gly Phe Cys Gln Asp GluLeu Ala Glu Leu Asp Pro Gly Thr Ile

35 40 45

Ser Val Ser Asp Asp Arg Ala Glu Gln Arg Thr Cys Leu Ile Cys Gly

50 55 60

Asp Arg Ala Thr Gly Leu His Tyr Gly Ile Ile Ser Cys Glu Gly Cys

65 70 75 80

Lys Gly Phe Phe Lys Arg Ser Ile Cys Asn Lys Arg Val Tyr Arg Cys

85 90 95

Ser Arg Asp Lys Asn Cys Val Met Ser Arg Lys Gln Arg Asn Arg Cys

100 105 110

Gln Tyr Cys Arg Leu Leu Lys Cys Leu Gln Met Gly Met Asn Arg Lys

115 120 125

Ala Ile Arg Glu Asp Gly Met Pro Gly Gly Arg Asn Lys Ser Ile Gly

130 135 140

Pro Val Gln Ile Ser Glu Glu Glu Ile Glu Arg Ile Met Ser Gly Gln

145 150 155 160

Glu Phe Glu Glu Glu Ala Asn His Trp Ser Asn His Gly Asp Ser Asp

165 170 175

His Ser Ser Pro Gly Asn Arg Ala Ser Glu Ser Asn Gln Pro Ser Pro

180 185 190

Gly Ser Thr Leu Ser Ser Ser Arg Ser ValGlu Leu Asn Gly Phe Met

195 200 205

Ala Phe Arg Glu Gln Tyr Met Gly Met Ser Val Pro Pro His Tyr Gln

210 215 220

Tyr Ile Pro His Leu Phe Ser Tyr Ser Gly His Ser Pro Leu Leu Pro

225 230 235 240

Gln Gln Ala Arg Ser Leu Asp Pro Gln Ser Tyr Ser Leu Ile His Gln

245 250 255

Leu Leu Ser Ala Glu Asp Leu Glu Pro Leu Gly Thr Pro Met Leu Ile

260 265 270

Glu Asp Gly Tyr Ala Val Thr Gln Ala Glu Leu Phe Ala Leu Leu Cys

275 280 285

Arg Leu Ala Asp Glu Leu Leu Phe Arg Gln Ile Ala Trp Ile Lys Lys

290 295 300

Leu Pro Phe Phe Cys Glu Leu Ser Ile Lys Asp Tyr Thr Cys Leu Leu

305 310 315 320

Ser Ser Thr Trp Gln Glu Leu Ile Leu Leu Ser Ser Leu Thr Val Tyr

325 330 335

Ser Lys Gln Ile Phe Gly Glu Leu Ala Asp Val Thr Ala Lys Tyr Ser

340 345 350

Pro Ser Asp Glu Glu Leu His Arg Phe Ser Asp GluGly Met Glu Val

355 360 365

Ile Glu Arg Leu Ile Tyr Leu Tyr His Lys Phe His Gln Leu Lys Val

370 375 380

Ser Asn Glu Glu Tyr Ala Cys Met Lys Ala Ile Asn Phe Leu Asn Gln

385 390 395 400

Asp Ile Arg Gly Leu Thr Ser Ala Ser Gln Leu Glu Gln Leu Asn Lys

405 410 415

Arg Tyr Trp Tyr Ile Cys Gln Asp Phe Thr Glu Tyr Lys Tyr Thr His

420 425 430

Gln Pro Asn Arg Phe Pro Asp Leu Met Met Cys Leu Pro Glu Ile Arg

435 440 445

Tyr Ile Ala Gly Lys Met Val Asn Val Pro Leu Glu Gln Leu Pro Leu

450 455 460

Leu Phe Lys Val Val Leu His Ser Cys Lys Thr Ser Val Gly Lys Glu

465 470 475 480

<210>75

<211>505

<212>PRT

<213> human

<400>75

Met Gly Glu Thr Leu Gly Asp Ser Pro Ile Asp Pro Glu Ser Asp Ser

1 5 10 15

Phe Thr Asp Thr Leu Ser Ala Asn Ile Ser Gln Glu Met Thr Met Val

20 25 30

Asp Thr Glu Met Pro Phe Trp Pro Thr Asn Phe Gly Ile Ser Ser Val

35 40 45

Asp Leu Ser Val Met Glu Asp His Ser His Ser Phe Asp Ile Lys Pro

50 55 60

Phe Thr Thr Val Asp Phe Ser Ser Ile Ser Thr Pro His Tyr Glu Asp

65 70 75 80

Ile Pro Phe Thr Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp

85 90 95

Leu Lys Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser

100 105 110

Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu

115 120 125

Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp

130 135 140

Lys Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys

145 150 155 160

Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys

165 170 175

Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln Tyr

180 185 190

Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn Ala Ile

195 200 205

Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu

210 215 220

Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp Leu Arg

225 230 235 240

Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys Ser Phe Pro Leu

245 250 255

Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr Gly Lys Thr Thr Asp Lys

260 265 270

Ser Pro Phe Val Ile Tyr Asp Met Asn Ser Leu Met Met Gly Glu Asp

275 280 285

Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu

290 295 300

Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala

305 310 315 320

Val Gln Glu Ile Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn

325 330 335

Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu

340 345 350

Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu

355 360 365

Ile Ser Glu Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu

370 375 380

Arg Lys Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val

385 390 395 400

Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile

405 410 415

Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys

420 425 430

Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln

435 440 445

Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys Leu Leu

450 455 460

Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu His Val Gln Leu

465 470 475 480

Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met Ser Leu His Pro Leu

485 490 495

Leu Gln Glu Ile Tyr Lys Asp Leu Tyr

500 505

<210>76

<211>777

<212>PRT

<213> human

<400>76

Met Asp Ser Lys Glu Ser Leu Thr Pro Gly Arg Glu Glu Asn Pro Ser

1 5 10 15

Ser Val Leu Ala Gln Glu Arg Gly Asp Val Met Asp Phe Tyr Lys Thr

20 25 30

Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Leu

35 40 45

Ala Val Ala Ser Gln Ser Asp Ser Lys Gln Arg Arg Leu Leu Val Asp

50 55 60

Phe Pro Lys Gly Ser Val Ser Asn Ala Gln Gln Pro Asp Leu Ser Lys

65 70 75 80

Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr Lys

85 90 95

Val Met Gly Asn Asp Leu Gly Phe Pro Gln Gln Gly Gln Ile Ser Leu

100 105 110

Ser Ser Gly Glu Thr Asp Leu Lys Leu Leu Glu Glu Ser Ile Ala Asn

115 120 125

Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Ala Ser

130 135 140

Thr Ala Val Ser Ala Ala Pro Thr Glu Lys Glu Phe Pro Lys Thr His

145 150 155 160

Ser Asp Val Ser Ser Glu Gln Gln His Leu Lys Gly Gln Thr Gly Thr

165 170 175

Asn Gly Gly Asn Val Lys Leu Tyr Thr Thr Asp Gln Ser Thr Phe Asp

180 185 190

Ile Leu Gln Asp Leu Glu Phe Ser Ser Gly Ser Pro Gly Lys Glu Thr

195 200 205

Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Cys Leu

210 215 220

Leu Ser Pro Leu Ala Gly Glu Asp Asp Ser Phe Leu Leu Glu Gly Asn

225 230 235 240

Ser Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys

245 250 255

Ile Lys Asp Asn Gly Asp Leu Val Leu Ser Ser Pro Ser Asn Val Thr

260 265 270

Leu Pro Gln Val Lys Thr Glu Lys Glu Asp Phe Ile Glu Leu Cys Thr

275 280 285

Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Thr Val Tyr Cys Gln Ala

290 295 300

Ser Phe Pro Gly Ala Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser

305 310 315 320

Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met

325 330 335

Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Ile Phe Asn

340 345 350

Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln

355 360 365

Gly Ser Gly Asp Asp Asn Leu Thr Ser Leu Gly Thr Leu Asn Phe Pro

370 375 380

Gly Arg Thr Val Phe Ser Asn Gly Tyr Ser Ser Pro Ser Met Arg Pro

385 390 395 400

Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Thr Ala Thr Thr Gly Pro

405 410 415

Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His

420 425 430

Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala

435 440 445

Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile

450 455 460

Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys

465 470 475 480

Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys

485 490 495

Ile Lys Gly Ile Gln Gln Ala Thr Thr Gly Val Ser Gln Glu Thr Ser

500 505 510

Glu Asn Pro Gly Asn Lys Thr Ile Val Pro Ala Thr Leu Pro Gln Leu

515 520 525

Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu

530 535 540

Tyr Ala Gly Tyr Asp Ser Ser Val Pro Asp Ser Thr Trp Arg Ile Met

545 550 555 560

Thr Thr Leu Asn Met Leu Gly Gly Arg Gln Val Ile Ala Ala Val Lys

565 570 575

Trp Ala Lys Ala Ile Pro Gly Phe Arg Asn Leu His Leu Asp Asp Gln

580 585 590

Met Thr Leu Leu Gln Tyr Ser Trp Met Phe Leu Met Ala Phe Ala Leu

595 600 605

Gly Trp Arg Ser Tyr Arg Gln Ser Ser Ala Asn Leu Leu Cys Phe Ala

610 615 620

Pro Asp Leu Ile Ile Asn Glu Gln Arg Met Thr Leu Pro Cys Met Tyr

625 630 635 640

Asp Gln Cys Lys His Met Leu Tyr Val Ser Ser Glu Leu His Arg Leu

645 650 655

Gln Val Ser Tyr Glu Glu Tyr Leu Cys Met Lys Thr Leu Leu Leu Leu

660 665 670

Ser Ser Val Pro Lys Asp Gly Leu Lys Ser Gln Glu Leu Phe Asp Glu

675 680 685

Ile Arg Met Thr Tyr Ile Lys Glu Leu Gly Lys Ala Ile Val Lys Arg

690 695 700

Glu Gly Asn Ser Ser Gln Asn Trp Gln Arg Phe Tyr Gln Leu Thr Lys

705 710 715 720

Leu Leu Asp Ser Met His Glu Val Val Glu Asn Leu Leu Asn Tyr Cys

725 730 735

Phe Gln Thr Phe Leu Asp Lys Thr Met Ser Ile Glu Phe Pro Glu Met

740 745 750

Leu Ala Glu Ile Ile Thr Asn Gln Ile Pro Lys Tyr Ser Asn Gly Asn

755 760 765

Ile Lys Lys Leu Leu Phe His Gln Lys

770 775

<210>77

<211>490

<212>PRT

<213> human

<400>77

Met Glu Gln Lys Pro Ser Lys Val Glu Cys Gly Ser Asp Pro Glu Glu

1 5 10 15

Asn Ser Ala Arg Ser Pro Asp Gly Lys Arg Lys Arg Lys Asn Gly Gln

20 25 30

Cys Ser Leu Lys Thr Ser Met Ser Gly Tyr Ile Pro Ser Tyr Leu Asp

35 40 45

Lys Asp Glu Gln Cys Val Val Cys Gly Asp Lys Ala Thr Gly Tyr His

50 55 60

Tyr Arg Cys Ile Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr

65 70 75 80

Ile Gln Lys Asn Leu His Pro Thr Tyr Ser Cys Lys Tyr Asp Ser Cys

85 90 95

Cys Val Ile Asp Lys Ile Thr Arg Asn Gln Cys Gln Leu Cys Arg Phe

100 105 110

Lys Lys Cys Ile Ala Val Gly Met Ala Met Asp Leu Val Leu Asp Asp

115 120 125

Ser Lys Arg Val Ala Lys Arg Lys Leu Ile Glu Gln Asn Arg Glu Arg

130 135 140

Arg Arg Lys Glu Glu Met Ile Arg Ser Leu Gln Gln Arg Pro Glu Pro

145 150 155 160

Thr Pro Glu Glu Trp Asp Leu Ile His Ile Ala Thr Glu Ala His Arg

165 170 175

Ser Thr Asn Ala Gln Gly Ser His Trp Lys Gln Arg Arg Lys Phe Leu

180 185 190

Pro Asp Asp Ile Gly Gln Ser Pro Ile Val Ser Met Pro Asp Gly Asp

195 200 205

Lys Val Asp Leu Glu Ala Phe Ser Glu Phe Thr Lys Ile Ile Thr Pro

210 215 220

Ala Ile Thr Arg Val Val Asp Phe Ala Lys Lys Leu Pro Met Phe Ser

225 230 235 240

Glu Leu Pro Cys Glu Asp Gln Ile Ile Leu Leu Lys Gly Cys Cys Met

245 250 255

Glu Ile Met Ser Leu Arg Ala Ala Val Arg Tyr Asp Pro Glu Ser Asp

260 265 270

Thr Leu Thr Leu Ser Gly Glu Met Ala Val Lys Arg Glu Gln Leu Lys

275 280 285

AsnGly Gly Leu Gly Val Val Ser Asp Ala Ile Phe Glu Leu Gly Lys

290 295 300

Ser Leu Ser Ala Phe Asn Leu Asp Asp Thr Glu Val Ala Leu Leu Gln

305 310 315 320

Ala Val Leu Leu Met Ser Thr Asp Arg Ser Gly Leu Leu Cys Val Asp

325 330 335

Lys Ile Glu Lys Ser Gln Glu Ala Tyr Leu Leu Ala Phe Glu His Tyr

340 345 350

Val Asn His Arg Lys His Asn Ile Pro His Phe Trp Pro Lys Leu Leu

355 360 365

Met Lys Glu Arg Glu Val Gln Ser Ser Ile Leu Tyr Lys Gly Ala Ala

370 375 380

Ala Glu Gly Arg Pro Gly Gly Ser Leu Gly Val His Pro Glu Gly Gln

385 390 395 400

Gln Leu Leu Gly Met His Val Val Gln Gly Pro Gln Val Arg Gln Leu

405 410 415

Glu Gln Gln Leu Gly Glu Ala Gly Ser Leu Gln Gly Pro Val Leu Gln

420 425 430

His Gln Ser Pro Lys Ser Pro Gln Gln Arg Leu Leu Glu Leu Leu His

435 440 445

Arg Ser GlyIle Leu His Ala Arg Ala Val Cys Gly Glu Asp Asp Ser

450 455 460

Ser Glu Ala Asp Ser Pro Ser Ser Ser Glu Glu Glu Pro Glu Val Cys

465 470 475 480

Glu Asp Leu Ala Gly Asn Ala Ala Ser Pro

485 490

<210>78

<211>924

<212>PRT

<213> human

<400>78

Met Glu Thr Lys Gly Tyr His Ser Leu Pro Glu Gly Leu Asp Met Glu

1 5 10 15

Arg Arg Trp Gly Gln Val Ser Gln Ala Val Glu Arg Ser Ser Leu Gly

20 25 30

Pro Thr Glu Arg Thr Asp Glu Asn Asn Tyr Met Glu Ile Val Asn Val

35 40 45

Ser Cys Val Ser Gly Ala Ile Pro Asn Asn Ser Thr Gln Gly Ser Ser

50 55 60

Lys Glu Lys Gln Glu Leu Leu Pro Cys Leu Gln Gln Asp Asn Asn Arg

65 70 75 80

Pro Gly Ile Leu Thr Ser Asp Ile Lys Thr Glu Leu Glu Ser Lys Glu

85 90 95

Leu Ser Ala Thr Val Ala Glu Ser Met Gly Leu Tyr Met Asp Ser Val

100 105 110

Arg Asp Ala Asp Tyr Ser Tyr Glu Gln Gln Asn Gln Gln Gly Ser Met

115 120 125

Ser Pro Ala Lys Ile Tyr Gln Asn Val Glu Gln Leu Val Lys Phe Tyr

130 135 140

Lys Gly Asn Gly His Arg Pro Ser Thr Leu Ser Cys Val Asn Thr Pro

145 150 155 160

Leu Arg Ser Phe Met Ser Asp Ser Gly Ser Ser Val Asn Gly Gly Val

165 170 175

Met Arg Ala Ile Val Lys Ser Pro Ile Met Cys His Glu Lys Ser Pro

180 185 190

Ser Val Cys Ser Pro Leu Asn Met Thr Ser Ser Val Cys Ser Pro Ala

195 200 205

Gly Ile Asn Ser Val Ser Ser Thr Thr Ala Ser Phe Gly Ser Phe Pro

210 215 220

Val His Ser Pro Ile Thr Gln Gly Thr Pro Leu Thr Cys Ser Pro Asn

225 230 235 240

Ala Glu Asn Arg Gly Ser Arg Ser His Ser Pro Ala His Ala Ser Asn

245 250 255

Val Gly Ser Pro Leu Ser Ser Pro Leu Ser Ser Met Lys Ser Ser Ile

260 265 270

Ser Ser Pro Pro Ser His Cys Ser Val Lys Ser Pro Val Ser Ser Pro

275 280 285

Asn Asn Val Thr Leu Arg Ser Ser Val Ser Ser Pro Ala Asn Ile Asn

290 295 300

Asn Ser Arg Cys Ser Val Ser Ser Pro Ser Asn Thr Asn Asn Arg Ser

305 310 315 320

Thr Leu Ser Ser Pro Ala Ala Ser Thr Val Gly Ser Ile Cys Ser Pro

325 330 335

Val Asn Asn Ala Phe Ser Tyr Thr Ala Ser Gly Thr Ser Ala Gly Ser

340 345 350

Ser Thr Leu Arg Asp Val Val Pro Ser Pro Asp Thr Gln Glu Lys Gly

355 360 365

Ala Gln Glu Val Pro Phe Pro Lys Thr Glu Glu Val Glu Ser Ala Ile

370 375 380

Ser Asn Gly Val Thr Gly Gln Leu Asn Ile Val Gln Tyr Ile Lys Pro

385 390 395 400

Glu Pro Asp Gly Ala Phe Ser Ser Ser Cys Leu Gly Gly Asn Ser Lys

405 410 415

Ile Asn Ser Asp Ser Ser Phe Ser Val Pro Ile Lys Gln Glu Ser Thr

420 425 430

Lys His Ser Cys Ser Gly Thr Ser Phe Lys Gly Asn Pro Thr Val Asn

435 440 445

Pro Phe Pro Phe Met Asp Gly Ser Tyr Phe Ser Phe Met Asp Asp Lys

450 455 460

Asp Tyr Tyr Ser Leu Ser Gly Ile Leu Gly Pro Pro Val Pro Gly Phe

465 470 475 480

Asp Gly Asn Cys Glu Gly Ser Gly Phe Pro Val Gly Ile Lys Gln Glu

485 490 495

Pro Asp Asp Gly Ser Tyr Tyr Pro Glu Ala Ser Ile Pro Ser Ser Ala

500 505 510

Ile Val Gly Val Asn Ser Gly Gly Gln Ser Phe His Tyr Arg Ile Gly

515 520 525

Ala Gln Gly Thr Ile Ser Leu Ser Arg Ser Ala Arg Asp Gln Ser Phe

530 535 540

Gln His Leu Ser Ser Phe Pro Pro Val Asn Thr Leu Val Glu Ser Trp

545 550 555 560

Lys Ser His Gly Asp Leu Ser Ser Arg Arg Ser Asp Gly Tyr Pro Val

565 570 575

Leu Glu Tyr Ile Pro Glu Asn Val Ser Ser Ser Thr Leu Arg Ser Val

580 585 590

Ser Thr Gly Ser Ser Arg Pro Ser Lys Ile Cys Leu Val Cys Gly Asp

595 600 605

Glu Ala Ser Gly Cys His Tyr Gly Val Val Thr Cys Gly Ser Cys Lys

610 615 620

Val Phe Phe Lys Arg Ala Val Glu Gly Gln His Asn Tyr Leu Cys Ala

625 630 635 640

Gly Arg Asn Asp Cys Ile Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro

645 650 655

Ala Cys Arg Leu Gln Lys Cys Leu Gln Ala Gly Met Asn Leu Gly Ala

660 665 670

Arg Lys Ser Lys Lys Leu Gly Lys Leu Lys Gly Ile His Glu Glu Gln

675 680 685

Pro Gln Gln Gln Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Ser Pro

690 695 700

Glu Glu Gly Thr Thr Tyr Ile Ala Pro Ala Lys Glu Pro Ser Val Asn

705 710 715 720

Thr Ala Leu Val Pro Gln Leu Ser Thr Ile Ser Arg Ala Leu Thr Pro

725 730 735

Ser Pro Val Met Val Leu Glu Asn Ile Glu Pro Glu Ile Val Tyr Ala

740 745 750

Gly Tyr Asp Ser Ser Lys Pro Asp Thr Ala Glu Asn Leu Leu Ser Thr

755 760 765

Leu Asn Arg Leu Ala Gly Lys Gln Met Ile Gln Val Val Lys Trp Ala

770 775 780

Lys Val Leu Pro Gly Phe Lys Asn Leu Pro Leu Glu Asp Gln Ile Thr

785 790 795 800

Leu Ile Gln Tyr Ser Trp Met Cys Leu Ser Ser Phe Ala Leu Ser Trp

805 810 815

Arg Ser Tyr Lys His Thr Asn Ser Gln Phe Leu Tyr Phe Ala Pro Asp

820 825 830

Leu Val Phe Asn Glu Glu Lys Met Lys Glu Leu Arg Lys Met Val Thr

835 840 845

Lys Cys Pro Asn Asn Ser Gly Gln Ser Trp Gln Arg Phe Tyr Gln Leu

850 855 860

Thr Lys Leu Leu Asp Ser Met His Asp Leu Val Ser Asp Leu Leu Glu

865 870 875 880

Phe Cys Phe Tyr Thr Phe Arg Glu Ser His Ala Leu Lys Val Glu Phe

885 890 895

Pro Ala Met Leu Val Glu Ile Ile Ser Asp Gln Leu Pro Lys Val Glu

900 905 910

Ser Gly Asn Ala Lys Pro Leu Tyr Phe His Arg Lys

915 920

<210>79

<211>427

<212>PRT

<213> human

<400>79

Met Glu Ala Met Ala Ala Ser Thr Ser Leu Pro Asp Pro Gly Asp Phe

1 5 10 15

Asp Arg Asn Val Pro Arg Ile Cys Gly Val Cys Gly Asp Arg Ala Thr

20 25 30

Gly Phe His Phe Asn Ala Met Thr Cys Glu Gly Cys Lys Gly Phe Phe

35 40 45

Arg Arg Ser Met Lys Arg Lys Ala Leu Phe Thr Cys Pro Phe Asn Gly

50 55 60

Asp Cys Arg Ile Thr Lys Asp Asn Arg Arg His Cys Gln Ala Cys Arg

65 70 75 80

Leu Lys Arg Cys Val Asp Ile Gly Met Met Lys Glu Phe Ile Leu Thr

85 90 95

Asp Glu Glu Val Gln Arg Lys Arg Glu Met Ile Leu Lys Arg Lys Glu

100 105 110

Glu Glu Ala Leu Lys Asp Ser Leu Arg Pro Lys Leu Ser Glu Glu Gln

115 120 125

Gln Arg Ile Ile Ala Ile Leu Leu Asp Ala His His Lys Thr Tyr Asp

130 135 140

Pro Thr Tyr Ser Asp Phe Cys Gln Phe Arg Pro Pro Val Arg Val Asn

145 150 155 160

Asp Gly Gly Gly Ser His Pro Ser Arg Pro Asn Ser Arg His Thr Pro

165 170 175

Ser Phe Ser Gly Asp Ser Ser Ser Ser Cys Ser Asp His Cys Ile Thr

180 185 190

Ser Ser Asp Met Met Asp Ser Ser Ser Phe Ser Asn Leu Asp Leu Ser

195 200 205

Glu Glu Asp Ser Asp Asp Pro Ser Val Thr Leu Glu Leu Ser Gln Leu

210 215 220

Ser Met Leu Pro His Leu Ala Asp Leu Val Ser Tyr Ser Ile Gln Lys

225 230 235 240

Val Ile Gly Phe Ala Lys Met Ile Pro Gly Phe Arg Asp Leu Thr Ser

245 250 255

Glu Asp Gln Ile Val Leu Leu Lys Ser Ser Ala Ile Glu Val Ile Met

260 265 270

Leu Arg Ser Asn Glu Ser Phe Thr Met Asp Asp Met Ser Trp Thr Cys

275 280 285

Gly Asn Gln Asp Tyr Lys Tyr Arg Val Ser Asp Val Thr Lys Ala Gly

290 295 300

His Ser Leu Glu Leu Ile Glu Pro Leu Ile Lys Phe Gln Val Gly Leu

305 310 315 320

Lys Lys Leu Asn Leu His Glu Glu Glu His Val Leu Leu Met Ala Ile

325 330 335

Cys Ile Val Ser Pro Asp Arg Pro Gly Val Gln Asp Ala Ala Leu Ile

340 345 350

Glu Ala Ile Gln Asp Arg Leu Ser Asn Thr Leu Gln Thr Tyr Ile Arg

355 360 365

Cys Arg His Pro Pro Pro Gly Ser His Leu Leu Tyr Ala Lys Met Ile

370 375 380

Gln Lys Leu Ala Asp Leu Arg Ser Leu Asn Glu Glu His Ser Lys Gln

385 390 395 400

Tyr Arg Cys Leu Ser Phe Gln Pro Glu Cys Ser Met Lys Leu Thr Pro

405 410 415

Leu Val Leu Glu Val Phe Gly Asn Glu Ile Ser

420 425

<210>80

<211>468

<212>PRT

<213> human

<400>80

Met Val Asp Thr Glu Ser Pro Leu Cys Pro Leu Ser Pro Leu Glu Ala

1 5 10 15

Gly Asp Leu Glu Ser Pro Leu Ser Glu Glu Phe Leu Gln Glu Met Gly

20 25 30

Asn Ile Gln Glu Ile Ser Gln Ser Ile Gly Glu Asp Ser Ser Gly Ser

35 40 45

Phe Gly Phe Thr Glu Tyr Gln Tyr Leu Gly Ser Cys Pro Gly Ser Asp

50 55 60

Gly Ser Val Ile Thr Asp Thr Leu Ser Pro Ala Ser Ser Pro Ser Ser

65 70 75 80

Val Thr Tyr Pro Val Val Pro Gly Ser Val Asp Glu Ser Pro Ser Gly

85 90 95

Ala Leu Asn Ile Glu Cys Arg Ile Cys Gly Asp Lys Ala Ser Gly Tyr

100 105 110

His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg

115 120 125

Thr Ile Arg Leu Lys Leu Val Tyr Asp Lys Cys Asp Arg Ser Cys Lys

130 135 140

Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg Phe His Lys

145 150 155 160

Cys Leu Ser Val Gly Met Ser His Asn Ala Ile Arg Phe Gly Arg Met

165 170 175

Pro Arg Ser Glu Lys Ala Lys Leu Lys Ala Glu Ile Leu Thr Cys Glu

180 185 190

His Asp Ile Glu Asp Ser Glu Thr Ala Asp Leu Lys Ser Leu Ala Lys

195 200 205

Arg Ile Tyr Glu Ala Tyr Leu Lys Asn Phe Asn Met Asn Lys Val Lys

210 215 220

Ala Arg Val Ile Leu Ser Gly Lys Ala Ser Asn Asn Pro Pro Phe Val

225 230 235 240

Ile His Asp Met Glu Thr Leu Cys Met Ala Glu Lys Thr Leu Val Ala

245 250 255

Lys Leu Val Ala Asn Gly Ile Gln Asn Lys Glu Ala Glu Val Arg Ile

260 265 270

Phe His Cys Cys Gln Cys Thr Ser Val Glu Thr Val Thr Glu Leu Thr

275 280 285

Glu Phe Ala Lys Ala Ile Pro Gly Phe Ala Asn Leu Asp Leu Asn Asp

290 295 300

Gln Val Thr Leu Leu Lys Tyr Gly Val Tyr Glu Ala Ile Phe Ala Met

305 310 315 320

Leu Ser Ser Val Met Asn Lys Asp Gly Met Leu Val Ala Tyr Gly Asn

325 330 335

Gly Phe Ile Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Cys

340 345 350

Asp Ile Met Glu Pro Lys Phe Asp Phe Ala Met Lys Phe Asn Ala Leu

355 360 365

Glu Leu Asp Asp Ser Asp Ile Ser Leu Phe Val Ala Ala Ile Ile Cys

370 375 380

Cys Gly Asp Arg Pro Gly Leu Leu Asn Val Gly His Ile Glu Lys Met

385 390 395 400

Gln Glu Gly Ile Val His Val Leu Arg Leu His Leu Gln Ser Asn His

405 410 415

Pro Asp Asp Ile Phe Leu Phe Pro Lys Leu Leu Gln Lys Met Ala Asp

420 425 430

Leu Arg Gln Leu Val Thr Glu His Ala Gln Leu Val Gln Ile Ile Lys

435 440 445

Lys Thr Glu Ser Asp Ala Ala Leu His Pro Leu Leu Gln Glu Ile Tyr

450 455 460

Arg Asp Met Tyr

465

<210>81

<211>441

<212>PRT

<213> human

<400>81

Met Glu Gln Pro Gln Glu Glu Ala Pro Glu Val Arg Glu Glu Glu Glu

1 5 10 15

Lys Glu Glu Val Ala Glu Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly

20 25 30

Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser

35 40 45

Ser Ser Pro Pro Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly

50 55 60

Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp Lys

65 70 75 80

Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly

85 90 95

Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu

100 105 110

Arg Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys

115 120 125

Arg Phe Gln Lys CysLeu Ala Leu Gly Met Ser His Asn Ala Ile Arg

130 135 140

Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu

145 150 155 160

Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys

165 170 175

Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met

180 185 190

Thr Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr

195 200 205

Ala Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys

210 215 220

Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu

225 230 235 240

Ile Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr

245 250 255

Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser

260 265 270

Leu Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu

275 280 285

Ala Ile Phe Ala Met Leu AlaSer Ile Val Asn Lys Asp Gly Leu Leu

290 295 300

Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu

305 310 315 320

Arg Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val

325 330 335

Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile

340 345 350

Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro

355 360 365

Arg Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His

370 375 380

Leu Gln Ala Asn His Pro Asp Ala Gln Tyr Leu Phe Pro Lys Leu Leu

385 390 395 400

Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met

405 410 415

Met Gln Arg Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His Pro Leu

420 425 430

Leu Gln Glu Ile Tyr Lys Asp Met Tyr

435 440

<210>82

<211>434

<212>PRT

<213> human

<400>82

Met Glu Val Arg Pro Lys Glu Ser Trp Asn His Ala Asp Phe Val His

1 5 10 15

Cys Glu Asp Thr Glu Ser Val Pro Gly Lys Pro Ser Val Asn Ala Asp

20 25 30

Glu Glu Val Gly Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala

35 40 45

Thr Gly Tyr His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe

50 55 60

Phe Arg Arg Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg

65 70 75 80

Lys Gly Ala Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala

85 90 95

Cys Arg Leu Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met Ile

100 105 110

Met Ser Asp Glu Ala Val Glu Glu Arg Arg Ala Leu Ile Lys Arg Lys

115 120 125

Lys Ser Glu Arg Thr Gly Thr Gln Pro Leu Gly Val Gln Gly Leu Thr

130 135 140

Glu Glu Gln Arg MetMet Ile Arg Glu Leu Met Asp Ala Gln Met Lys

145 150 155 160

Thr Phe Asp Thr Thr Phe Ser His Phe Lys Asn Phe Arg Leu Pro Gly

165 170 175

Val Leu Ser Ser Gly Cys Glu Leu Pro Glu Ser Leu Gln Ala Pro Ser

180 185 190

Arg Glu Glu Ala Ala Lys Trp Ser Gln Val Arg Lys Asp Leu Cys Ser

195 200 205

Leu Lys Val Ser Leu Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn

210 215 220

Tyr Lys Pro Pro Ala Asp Ser Gly Gly Lys Glu Ile Phe Ser Leu Leu

225 230 235 240

Pro His Met Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Ile Ile Ser

245 250 255

Phe Ala Lys Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln

260 265 270

Ile Ser Leu Leu Lys Gly Ala Ala Phe Glu Leu Cys Gln Leu Arg Phe

275 280 285

Asn Thr Val Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys Gly Arg Leu

290 295 300

Ser Tyr Cys Leu Glu Asp ThrAla Gly Gly Phe Gln Gln Leu Leu Leu

305 310 315 320

Glu Pro Met Leu Lys Phe His Tyr Met Leu Lys Lys Leu Gln Leu His

325 330 335

Glu Glu Glu Tyr Val Leu Met Gln Ala Ile Ser Leu Phe Ser Pro Asp

340 345 350

Arg Pro Gly Val Leu Gln His Arg Val Val Asp Gln Leu Gln Glu Gln

355 360 365

Phe Ala Ile Thr Leu Lys Ser Tyr Ile Glu Cys Asn Arg Pro Gln Pro

370 375 380

Ala His Arg Phe Leu Phe Leu Lys Ile Met Ala Met Leu Thr Glu Leu

385 390 395 400

Arg Ser Ile Asn Ala Gln His Thr Gln Arg Leu Leu Arg Ile Gln Asp

405 410 415

Ile His Pro Phe Ala Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr

420 425 430

Gly Ser

<210>83

<211>257

<212>PRT

<213> human

<400>83

Gly Ser His Met Ser Gln Gly Ser Gly Glu Gly Glu Gly Val Gln Leu

1 5 10 15

Thr Ala Ala Gln Glu Leu Met Ile Gln Gln Leu Val Ala Ala Gln Leu

20 25 30

Gln Cys Asn Lys Arg Ser Phe Ser Asp Gln Pro Lys Val Thr Pro Trp

35 40 45

Pro Leu Gly Ala Asp Pro Gln Ser Arg Asp Ala Arg Gln Gln Arg Phe

50 55 60

Ala His Phe Thr Glu Leu Ala Ile Ile Ser Val Gln Glu Ile Val Asp

65 70 75 80

Phe Ala Lys Gln Val Pro Gly Phe Leu Gln Leu Gly Arg Glu Asp Gln

85 90 95

Ile Ala Leu Leu Lys Ala Ser Thr Ile Glu Ile Met Leu Leu Glu Thr

100 105 110

Ala Arg Arg Tyr Asn His Glu Thr Glu Cys Ile Thr Phe Leu Lys Asp

115 120 125

Phe Thr Tyr Ser Lys Asp Asp Phe His Arg Ala Gly Leu Gln Val Glu

130 135 140

Phe Ile Asn Pro Ile Phe Glu Phe Ser Arg Ala Met Arg Arg Leu Gly

145 150 155 160

Leu Asp Asp Ala Glu Tyr Ala Leu Leu Ile Ala Ile Asn Ile Phe Ser

165 170 175

Ala Asp Arg Pro Asn Val Gln Glu Pro Gly Arg Val Glu Ala Leu Gln

180 185 190

Gln Pro Tyr Val Glu Ala Leu Leu Ser Tyr Thr Arg Ile Lys Arg Pro

195 200 205

Gln Asp Gln Leu Arg Phe Pro Arg Met Leu Met Lys Leu Val Ser Leu

210 215 220

Arg Thr Leu Ser Ser Val His Ser Glu Gln Val Phe Ala Leu Arg Leu

225 230 235 240

Gln Asp Lys Lys Leu Pro Pro Leu Leu Ser Glu Ile Trp Asp Val His

245 250 255

Glu

<210>84

<211>472

<212>PRT

<213> human

<400>84

Met Gly Ser Lys Met Asn Leu Ile Glu His Ser His Leu Pro Thr Thr

1 5 10 15

Asp Glu Phe Ser Phe Ser Glu Asn Leu Phe Gly Val Leu Thr Glu Gln

20 25 30

Val Ala Gly Pro Leu Gly Gln Asn Leu Glu Val Glu Pro Tyr Ser Gln

35 40 45

Tyr Ser Asn Val Gln Phe Pro Gln Val Gln Pro Gln Ile Ser Ser Ser

50 55 60

Ser Tyr Tyr Ser Asn Leu Gly Phe Tyr Pro Gln Gln Pro Glu Glu Trp

65 70 75 80

Tyr Ser Pro Gly Ile Tyr Glu Leu Arg Arg Met Pro Ala Glu Thr Leu

85 90 95

Tyr Gln Gly Glu Thr Glu Val Ala Glu Met Pro Val Thr Lys Lys Pro

100 105 110

Arg Met Gly Ala Ser Ala Gly Arg Ile Lys Gly Asp Glu Leu Cys Val

115 120 125

Val Cys Gly Asp Arg Ala Ser Gly Tyr His Tyr Asn Ala Leu Thr Cys

130 135 140

Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Ile Thr Lys Asn Ala Val

145 150 155 160

Tyr Lys Cys Lys Asn Gly Gly Asn Cys Val Met Asp Met Tyr Met Arg

165 170 175

Arg Lys Cys Gln Glu Cys Arg Leu Arg Lys Cys Lys Glu Met Gly Met

180 185 190

Leu Ala Glu Cys Leu Leu Thr Glu Ile Gln Cys Lys Ser Lys Arg Leu

195 200 205

Arg Lys Asn Val Lys Gln His Ala Asp Gln Thr Val Asn Glu Asp Ser

210 215 220

Glu Gly Arg Asp Leu Arg Gln Val Thr Ser Thr Thr Lys Ser Cys Arg

225 230 235 240

Glu Lys Thr Glu Leu Thr Pro Asp Gln Gln Thr Leu Leu His Phe Ile

245 250 255

Met Asp Ser Tyr Asn Lys Gln Arg Met Pro Gln Glu Ile Thr Asn Lys

260 265 270

Ile Leu Lys Glu Glu Phe Ser Ala Glu Glu Asn Phe Leu Ile Leu Thr

275 280 285

Glu Met Ala Thr Asn His Val Gln Val Leu Val Glu Phe Thr Lys Lys

290 295 300

Leu Pro Gly Phe Gln Thr Leu Asp His Glu Asp Gln Ile Ala Leu Leu

305 310 315 320

Lys Gly Ser Ala Val Glu Ala Met Phe Leu Arg Ser Ala Glu Ile Phe

325 330 335

Asn Lys Lys Leu Pro Ser Gly His Ser Asp Leu Leu Glu Glu Arg Ile

340 345 350

Arg Asn Ser Gly Ile Ser Asp Glu Tyr Ile Thr Pro Met Phe Ser Phe

355 360 365

TyrLys Ser Ile Gly Glu Leu Lys Met Thr Gln Glu Glu Tyr Ala Leu

370 375 380

Leu Thr Ala Ile Val Ile Leu Ser Pro Asp Arg Gln Tyr Ile Lys Asp

385 390 395 400

Arg Glu Ala Val Glu Lys Leu Gln Glu Pro Leu Leu Asp Val Leu Gln

405 410 415

Lys Leu Cys Lys Ile His Gln Pro Glu Asn Pro Gln His Phe Ala Cys

420 425 430

Leu Leu Gly Arg Leu Thr Glu Leu Arg Thr Phe Asn His His His Ala

435 440 445

Glu Met Leu Met Ser Trp Arg Val Asn Asp His Lys Phe Thr Pro Leu

450 455 460

Leu Cys Glu Ile Trp Asp Val Gln

465 470

<210>85

<211>468

<212>PRT

<213> human

<400>85

Met Met Tyr Phe Val Ile Ala Ala Met Lys Ala Gln Ile Glu Ile Ile

1 5 10 15

Pro Cys Lys Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr Gly Val

20 25 30

Ile Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln Gln Ser

35 40 45

Asn Ala Thr Tyr Ser Cys Pro Arg Gln Lys Asn Cys Leu Ile Asp Arg

50 55 60

Thr Ser Arg Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys Leu Ala

65 70 75 80

Val Gly Met Ser Arg Asp Ala Val Lys Phe Gly Arg Met Ser Lys Lys

85 90 95

Gln Arg Asp Ser Leu Tyr Ala Glu Val Gln Lys His Arg Met Gln Gln

100 105 110

Gln Gln Arg Asp His Gln Gln Gln Pro Gly Glu Ala Glu Pro Leu Thr

115 120 125

Pro Thr Tyr Asn Ile Ser Ala Asn Gly Leu Thr Glu Leu His Asp Asp

130 135 140

Leu Ser Asn Tyr Ile Asp Gly His Thr Pro Glu Gly Ser Lys Ala Asp

145 150 155 160

Ser Ala Val Ser Ser Phe Tyr Leu Asp Ile Gln Pro Ser Pro Asp Gln

165 170 175

Ser Gly Leu Asp Ile Asn Gly Ile Lys Pro Glu Pro Ile Cys Asp Tyr

180 185 190

Thr Pro Ala Ser Gly Phe Phe Pro Tyr Cys Ser Phe Thr Asn Gly Glu

195 200 205

Thr Ser Pro Thr Val Ser Met Ala Glu Leu Glu His Leu Ala Gln Asn

210 215 220

Ile Ser Lys Ser His Leu Glu Thr Cys Gln Tyr Leu Arg Glu Glu Leu

225 230 235 240

Gln Gln Ile Thr Trp Gln Thr Phe Leu Gln Glu Glu Ile Glu Asn Tyr

245 250 255

Gln Asn Lys Gln Arg Glu Val Met Trp Gln Leu Cys Ala Ile Lys Ile

260 265 270

Thr Glu Ala Ile Gln Tyr Val Val Glu Phe Ala Lys Arg Ile Asp Gly

275 280 285

Phe Met Glu Leu Cys Gln Asn Asp Gln Ile Val Leu Leu Lys Ala Gly

290 295 300

Ser Leu Glu Val Val Phe Ile Arg Met Cys Arg Ala Phe Asp Ser Gln

305 310 315 320

Asn Asn Thr Val Tyr Phe Asp Gly Lys Tyr Ala Ser Pro Asp Val Phe

325 330 335

Lys Ser Leu Gly Cys Glu Asp Phe Ile Ser Phe Val Phe Glu Phe Gly

340 345 350

Lys Ser Leu Cys Ser Met His Leu Thr Glu Asp Glu Ile Ala Leu Phe

355 360 365

Ser Ala Phe Val Leu Met Ser Ala Asp Arg Ser Trp Leu Gln Glu Lys

370 375 380

Val Lys Ile Glu Lys Leu Gln Gln Lys Ile Gln Leu Ala Leu Gln His

385 390 395 400

Val Leu Gln Lys Asn His Arg Glu Asp Gly Ile Leu Thr Lys Leu Ile

405 410 415

Cys Lys Val Ser Thr Leu Arg Ala Leu Cys Gly Arg His Thr Glu Lys

420 425 430

Leu Met Ala Phe Lys Ala Ile Tyr Pro Asp Ile Val Arg Leu His Phe

435 440 445

Pro Pro Leu Tyr Lys Glu Leu Phe Thr Ser Glu Phe Glu Pro Ala Met

450 455 460

Gln Ile Asp Gly

465

<210>86

<211>518

<212>PRT

<213> human

<400>86

Met Asp Arg Ala Pro Gln Arg Gln His Arg Ala Ser Arg Glu Leu Leu

1 5 10 15

Ala Ala Lys Lys Thr His Thr Ser Gln Ile Glu Val IlePro Cys Lys

20 25 30

Ile Cys Gly Asp Lys Ser Ser Gly Ile His Tyr Gly Val Ile Thr Cys

35 40 45

Glu Gly Cys Lys Gly Phe Phe Arg Arg Ser Gln Arg Cys Asn Ala Ala

50 55 60

Tyr Ser Cys Thr Arg Gln Gln Asn Cys Pro Ile Asp Arg Thr Ser Arg

65 70 75 80

Asn Arg Cys Gln His Cys Arg Leu Gln Lys Cys Leu Ala Leu Gly Met

85 90 95

Ser Arg Asp Ala Val Lys Phe Gly Arg Met Ser Lys Lys Gln Arg Asp

100 105 110

Ser Leu His Ala Glu Val Gln Lys Gln Leu Gln Gln Arg Gln Gln Gln

115 120 125

Gln Gln Glu Pro Val Val Lys Thr Pro Pro Ala Gly Ala Gln Gly Ala

130 135 140

Asp Thr Leu Thr Tyr Thr Leu Gly Leu Pro Asp Gly Gln Leu Pro Leu

145 150 155 160

Gly Ser Ser Pro Asp Leu Pro Glu Ala Ser Ala Cys Pro Pro Gly Leu

165 170 175

Leu Lys Ala Ser Gly Ser Gly Pro Ser Tyr Ser Asn Asn Leu Ala Lys

180 185 190

Ala Gly Leu Asn Gly Ala Ser Cys His Leu Glu Tyr Ser Pro Glu Arg

195 200 205

Gly Lys Ala Glu Gly Arg Glu Ser Phe Tyr Ser Thr Gly Ser Gln Leu

210 215 220

Thr Pro Asp Arg Cys Gly Leu Arg Phe Glu Glu His Arg His Pro Gly

225 230 235 240

Leu Gly Glu Leu Gly Gln Gly Pro Asp Ser Tyr Gly Ser Pro Ser Phe

245 250 255

Arg Ser Thr Pro Glu Ala Pro Tyr Ala Ser Leu Thr Glu Ile Glu His

260 265 270

Leu Val Gln Ser Val Cys Lys Ser Tyr Arg Glu Thr Cys Gln Leu Arg

275 280 285

Leu Glu Asp Leu Leu Arg Gln Arg Ser Asn Ile Phe Ser Arg Glu Glu

290 295 300

Val Thr Gly Tyr Gln Arg Lys Ser Met Trp Glu Met Trp Glu Arg Cys

305 310 315 320

Ala His His Leu Thr Glu Ala Ile Gln Tyr Val Val Glu Phe Ala Lys

325 330 335

Arg Leu Ser Gly Phe Met Glu Leu Cys Gln Asn Asp Gln Ile Val Leu

340 345 350

Leu Lys Ala Gly Ala Met Glu Val Val Leu Val Arg Met Cys Arg Ala

355 360 365

Tyr Asn Ala Asp Asn Arg Thr Val Phe Phe Glu Gly Lys Tyr Gly Gly

370 375 380

Met Glu Leu Phe Arg Ala Leu Gly Cys Ser Glu Leu Ile Ser Ser Ile

385 390 395 400

Phe Asp Phe Ser His Ser Leu Ser Ala Leu His Phe Ser Glu Asp Glu

405 410 415

Ile Ala Leu Tyr Thr Ala Leu Val Leu Ile Asn Ala His Arg Pro Gly

420 425 430

Leu Gln Glu Lys Arg Lys Val Glu Gln Leu Gln Tyr Asn Leu Glu Leu

435 440 445

Ala Phe His His His Leu Cys Lys Thr His Arg Gln Ser Ile Leu Ala

450 455 460

Lys Leu Pro Pro Lys Gly Lys Leu Arg Ser Leu Cys Ser Gln His Val

465 470 475 480

Glu Arg Leu Gln Ile Phe Gln His Leu His Pro Ile Val Val Gln Ala

485 490 495

Ala Phe Pro Pro Leu Tyr Lys Glu Leu Phe Ser Thr Glu Thr Glu Ser

500 505 510

Pro Val Gly Leu Ser Lys

515

<210>87

<211>431

<212>PRT

<213> human

<400>87

Met Leu Gly Gly Leu Ser Pro Pro Gly Ala Leu Thr Thr Leu Gln His

1 5 10 15

Gln Leu Pro Val Ser Gly Tyr Ser Thr Pro Ser Pro Ala Thr Ile Glu

20 25 30

Thr Gln Ser Ser Ser Ser Glu Glu Ile Val Pro Ser Pro Pro Ser Pro

35 40 45

Pro Pro Leu Pro Arg Ile Tyr Lys Pro Cys Phe Val Cys Gln Asp Lys

50 55 60

Ser Ser Gly Tyr His Tyr Gly Val Ser Ala Cys Glu Gly Val Lys Gly

65 70 75 80

Phe Phe Arg Arg Ser Ile Gln Lys Asn Met Val Tyr Thr Val His Arg

85 90 95

Asp Lys Asn Cys Ile Ile Asn Lys Val Thr Arg Asn Arg Cys Gln Tyr

100 105 110

Cys Arg Leu Gln Lys Cys Phe Glu Val Gly Met Ser Lys Glu Ser Val

115 120 125

Arg Asn Asp Arg Asn Lys Lys Lys Lys Glu Val Pro Lys Pro Glu Cys

130 135 140

Ser Glu Ser Tyr Thr Val Thr Pro Glu Val Gly Glu Leu Ile Glu Lys

145 150 155 160

Val Arg Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys Gln Leu Gly

165 170 175

Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser Leu Asp Ile

180 185 190

Asp Leu Trp Asp Lys Phe Ser Glu Leu Ser Thr Lys Cys Ile Ile Lys

195 200 205

Thr Val Glu Phe Ala Lys Gln Leu Pro Gly Phe Thr Thr Leu Thr Ile

210 215 220

Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala Cys Leu Asp Ile Leu Ile

225 230 235 240

Leu Arg Ile Cys Thr Arg Tyr Thr Pro Glu Gln Asp Thr Met Thr Phe

245 250 255

Ser Asp Gly Leu Thr Leu Asn Arg Thr Gln Met His Asn Ala Gly Phe

260 265 270

Gly Pro Leu Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu Leu Pro

275 280 285

Leu Glu Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile Cys Leu

290 295 300

Ile Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val Asp Met

305 310 315 320

Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg Lys Arg

325 330 335

Arg Pro Ser Arg His Met Phe Pro Lys Met Leu Met Lys Ile Thr Asp

340 345 350

Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Val Ile Thr Leu Lys

355 360 365

Met Glu Ile Pro Gly Ser Met Pro Pro Leu Ile Gln Glu Met Leu Glu

370 375 380

Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly Gln Pro Gly Gly Gly Gly

385 390 395 400

Arg Asp Gly Gly Gly Leu Ala Pro Pro Pro Gly Ser Cys Ser Pro Ser

405 410 415

Leu Ser Pro Ser Ser Asn Arg Ser Ser Pro Ala Thr His Ser Pro

420 425 430

<210>88

<211>599

<212>PRT

<213> mice

<400>88

Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His

1 5 10 15

Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys

20 25 30

Met Pro Met Glu Arg Ala Leu Gly Glu Val Tyr Val Asp Asn Ser Lys

35 40 45

Pro Thr Val Phe Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala

50 55 60

Ala Ala Ala Ala Ala Ala Ala Ala Ser Ala Pro Val Tyr Gly Gln Ser

65 70 75 80

Gly Ile Ala Tyr Gly Pro Gly Ser Glu Ala Ala Ala Phe Ser Ala Asn

85 90 95

Ser Leu Gly Ala Phe Pro Gln Leu Asn Ser Val Ser Pro Ser Pro Leu

100 105 110

Met Leu Leu His Pro Pro Pro Gln Leu Ser Pro Phe Leu His Pro His

115 120 125

Gly Gln Gln Val Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Ala Tyr Ala

130 135 140

Val Arg Asp Thr Gly Pro Pro Ala Phe TyrArg Ser Asn Ser Asp Asn

145 150 155 160

Arg Arg Gln Asn Gly Arg Glu Arg Leu Ser Ser Ser Asn Glu Lys Gly

165 170 175

Asn Met Ile Met Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys

180 185 190

Asn Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly

195 200 205

Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met

210 215 220

Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser

225 230 235 240

Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys

245 250 255

Gly Gly Ile Arg Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys

260 265 270

Arg Gln Arg Asp Asp Leu Glu Gly Arg Asn Glu Met Gly Ala Ser Gly

275 280 285

Asp Met Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Val Ile Lys His

290 295 300

Thr Lys Lys Asn Ser Pro Ala Leu Ser Leu Thr AlaAsp Gln Met Val

305 310 315 320

Ser Ala Leu Leu Asp Ala Glu Pro Pro Met Ile Tyr Ser Glu Tyr Asp

325 330 335

Pro Ser Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn

340 345 350

Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val

355 360 365

Pro Gly Phe Gly Asp Leu Asn Leu His Asp Gln Val His Leu Leu Glu

370 375 380

Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met

385 390 395 400

Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg

405 410 415

Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu

420 425 430

Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu

435 440 445

Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr

450 455 460

Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp HisIle His

465 470 475 480

Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys

485 490 495

Ala Gly Leu Thr Leu Gln Gln Gln His Arg Arg Leu Ala Gln Leu Leu

500 505 510

Leu Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu His

515 520 525

Leu Tyr Asn Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu

530 535 540

Leu Glu Met Leu Asp Ala His Arg Leu His Ala Pro Ala Ser Arg Met

545 550 555 560

Gly Val Pro Pro Glu Glu Pro Ser Gln Thr Gln Leu Ala Thr Thr Ser

565 570 575

Ser Thr Ser Ala His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu Ala

580 585 590

Glu Gly Phe Pro Asn Thr Ile

595

<210>89

<211>600

<212>PRT

<213> rat

<400>89

Met Thr Met Thr Leu His Thr Lys Ala Ser GlyMet Ala Leu Leu His

1 5 10 15

Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys

20 25 30

Met Pro Met Glu Arg Ala Leu Gly Glu Val Tyr Val Asp Asn Ser Lys

35 40 45

Pro Ala Val Phe Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala

50 55 60

Ala Ala Ala Ala Ala Ala Ala Gly Ala Ser Ala Pro Val Tyr Gly Gln

65 70 75 80

Ser Ser Ile Thr Tyr Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ala

85 90 95

Asn Ser Leu Gly Ala Phe Pro Gln Leu Asn Ser Val Ser Pro Ser Pro

100 105 110

Leu Met Leu Leu His Pro Pro Pro His Val Ser Pro Phe Leu His Pro

115 120 125

His Gly His Gln Val Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Ala Tyr

130 135 140

Ala Val Arg Asp Thr Gly Pro Pro Ala Phe Tyr Arg Ser Asn Ser Asp

145 150 155 160

Asn Arg Arg Gln Asn Gly Arg Glu Arg Leu Ser Ser Ser Ser Glu Lys

165 170 175

Gly Asn Met Ile Met Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val

180 185 190

Cys Asn Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu

195 200 205

Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr

210 215 220

Met Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys

225 230 235 240

Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met

245 250 255

Lys Gly Gly Ile Arg Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His

260 265 270

Lys Arg Gln Arg Asp Asp Leu Glu Gly Arg Asn Glu Met Gly Thr Ser

275 280 285

Gly Asp Met Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Val Ile Lys

290 295 300

His Thr Lys Lys Asn Ser Pro Ala Leu Ser Leu Thr Ala Asp Gln Met

305 310 315 320

Val Ser Ala Leu Leu Asp Ala Glu Pro Pro Leu Ile Tyr Ser Glu Tyr

325 330 335

Asp Pro Ser Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr

340 345 350

Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg

355 360 365

Val Pro Gly Phe Gly Asp Leu Asn Leu His Asp Gln Val His Leu Leu

370 375 380

Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser

385 390 395 400

Met Glu His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp

405 410 415

Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met

420 425 430

Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu

435 440 445

Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr

450 455 460

Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile

465 470 475 480

His Arg Val Leu Asp Lys Ile Asn Asp Thr Leu Ile His Leu Met Ala

485 490 495

Lys Ala Gly Leu Thr Leu Gln Gln Gln His Arg Arg Leu Ala Gln Leu

500 505 510

Leu Leu Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu

515 520 525

His Leu Tyr Asn Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu

530 535 540

Leu Leu Glu Met Leu Asp Ala His Arg Leu His Ala Pro Ala Ser Arg

545 550 555 560

Met Gly Val Pro Pro Glu Glu Pro Ser Gln Ser Gln Leu Thr Thr Thr

565 570 575

Ser Ser Thr Ser Ala His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu

580 585 590

Ala Glu Gly Phe Pro Asn Thr Ile

595 600

<210>90

<211>595

<212>PRT

<213> Baboon of Dongfei

<400>90

Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His

1 5 10 15

Gln Ile Gln Gly Asn Glu Leu Glu ProLeu Asn Arg Pro Gln Leu Lys

20 25 30

Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Val Asp Ser Ser Lys

35 40 45

Pro Ala Val Tyr Ser Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala

50 55 60

Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr

65 70 75 80

Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly

85 90 95

Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His

100 105 110

Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val

115 120 125

Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala

130 135 140

Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly

145 150 155 160

Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met

165 170 175

Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala

180 185 190

Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe

195 200 205

Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr

210 215 220

Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys

225 230 235 240

Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg

245 250 255

Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp

260 265 270

Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala

275 280 285

Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys His Ser Lys Lys Asn

290 295 300

Ser Pro Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu

305 310 315 320

Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro

325 330 335

Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg

340 345 350

Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val

355 360 365

Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu

370 375 380

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly

385 390 395 400

Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys

405 410 415

Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser

420 425 430

Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu

435 440 445

Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser

450 455 460

Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp

465 470 475 480

Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr

485 490 495

Leu Gln Gln Gln His Arg Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser

500 505 510

His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met

515 520 525

Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu

530 535 540

Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Pro Met

545 550 555 560

Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser

565 570 575

His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Asp Ala Glu Gly Phe Pro

580 585 590

Ala Thr Val

595

<210>91

<211>866

<212>PRT

<213> mice

<400>91

Met Thr Glu Leu Gln Ala Lys Asp Pro Gln Val Leu His Thr Ser Gly

1 5 10 15

Ala Ser Pro Ser Pro Pro His Ile Gly Ser Pro Leu Leu Ala Arg Leu

20 25 30

Asp Ser Gly Pro Phe Gln Gly Ser Gln His Ser Asp Val Ser Ser Val

35 40 45

Val Ser Pro Ile Pro Ile Ser Leu Asp Gly Leu Leu Phe Pro Arg Ser

50 55 60

Cys Arg Gly Pro Glu Leu Pro Asp Gly Lys Thr Gly Asp Gln Gln Ser

65 70 75 80

Leu Ser Asp Val Glu Gly Ala Phe Ser Gly Val Glu Ala Thr His Arg

85 90 95

Glu Gly Gly Arg Asn Ser Arg Ala Pro Glu Lys Asp Ser Arg Leu Leu

100 105 110

Asp Ser Val Leu Asp Ser Leu Leu Thr Pro Ser Gly Thr Glu Gln Ser

115 120 125

His Ala Ser Pro Pro Ala Cys Glu Ala Ile Thr Ser Trp Cys Leu Phe

130 135 140

Gly Pro Glu Leu Pro Glu Asp Pro Arg Ser Val Pro Ala Thr Lys Gly

145 150 155 160

Leu Leu Ser Pro Leu Met Ser Arg Pro Glu Ile Lys Ala Gly Asp Ser

165 170 175

Ser Gly Thr Gly Ala Gly Gln Lys Val Leu Pro Lys Gly Leu Ser Pro

180 185 190

Pro Arg Gln Leu Leu Leu Pro Thr Ser Gly Ser Ala His Trp Pro Gly

195 200 205

Ala Gly Val Lys Pro Ser Pro Gln Pro Ala Ala Gly Glu Val Glu Glu

210 215 220

Asp Ser Gly Leu Glu Thr Glu Gly Ser Ala Ala Pro Leu Leu Lys Ser

225 230 235 240

Lys Pro Arg Ala Leu Glu Gly Thr Gly Ser Gly Gly Gly Val Ala Ala

245 250 255

Asn Ala Ala Ser Ala Ala Pro Gly Gly Val Thr Leu Val Pro Lys Glu

260 265 270

Asp Ser Arg Phe Ser Ala Pro Arg Val Ser Leu Glu Gln Asp Ser Pro

275 280 285

Ile Ala Pro Gly Arg Ser Pro Leu Ala Thr Thr Val Val Asp Phe Ile

290 295 300

His Val Pro Ile Leu Pro Leu Asn His Ala Leu Leu Ala Ala Arg Thr

305 310 315 320

Arg Gln Leu Leu Glu Gly Asp Ser Tyr Asp Gly Gly Ala Thr Ala Gln

325 330 335

Gly Pro Phe Ala Pro Pro Arg Gly Ser Pro Ser Ala Pro Ser Pro Pro

340 345 350

Val Pro Cys Gly Asp Phe Pro Asp Cys Thr Tyr Pro Leu Glu Gly Asp

355 360 365

Pro Lys Glu Asp Val Phe Pro Leu Tyr Gly Asp Phe Gln Thr Pro Gly

370 375 380

Leu Lys Ile Lys Glu Glu Glu Glu Gly Ala Asp Ala Ala Val Arg Ser

385 390 395 400

Pro Arg Pro Tyr Leu Ser Ala Gly Ala Ser Ser Ser Thr Phe Pro Asp

405 410 415

Phe Pro Leu Ala Pro Ala Pro Gln Arg Ala Pro Ser Ser Arg Pro Gly

420 425 430

Glu Ala Ala Val Ala Gly Gly Pro Ser Ser Ala Ala Val Ser Pro Ala

435 440 445

Ser Ser Ser Gly Ser Ala Leu Glu Cys Ile Leu Tyr Lys Ala Glu Gly

450 455 460

Ala Pro Pro Thr Gln Gly Ser Phe Ala Pro Leu Pro Cys Lys Pro Pro

465 470 475 480

Ala Ala Gly Ser Cys Leu Leu Pro Arg Asp Ser Leu Pro Ala Ala Pro

485 490 495

Ala Thr Ala Ala Ala Pro Ala Ile Tyr Gln Pro Leu Gly Leu Asn Gly

500 505 510

Leu Pro Gln Leu Gly Tyr Gln Ala Ala Val Leu Lys Asp Ser Leu Pro

515 520 525

Gln Val Tyr Pro Pro Tyr Leu Asn Tyr Leu Arg Pro Asp Ser Glu Ala

530 535 540

Ser Gln Ser Pro Gln Tyr Gly Phe Asp Ser Leu Pro Gln Lys Ile Cys

545 550 555 560

Leu Ile Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Val Leu Thr

565 570 575

Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Met Glu Gly Gln His

580 585 590

Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile Val Asp Lys Ile Arg

595 600 605

Arg Lys Asn Cys Pro Ala Cys Arg Leu Arg Lys Cys Cys Gln Ala Gly

610 615 620

Met Val Leu Gly Gly Arg Lys Phe Lys Lys Phe Asn Lys Val Arg Val

625 630 635 640

Met Arg Thr Leu Asp Gly Val Ala Leu Pro Gln Ser Val Gly Leu Pro

645 650 655

Asn Glu Ser Gln Ala Leu Gly Gln Arg Ile Thr Phe Ser Pro Asn Gln

660 665 670

Glu Ile Gln Leu Val Pro Pro Leu Ile Asn Leu Leu Met Ser Ile Glu

675 680 685

Pro Asp Val Val Tyr Ala Gly His Asp Asn Thr Lys Pro Asp Thr Ser

690 695 700

Ser Ser Leu Leu Thr Ser Leu Asn Gln Leu Gly Glu Arg Gln Leu Leu

705 710 715 720

Arg Met Lys Glu Leu Ser Phe Tyr Ser Leu Cys Leu Thr Met Trp Gln

725 730 735

Ile Pro Gln Glu Phe Val Lys Leu Gln Val Thr His Glu Glu Phe Leu

740 745 750

Cys Met Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu

755 760 765

Arg Ser Gln Ser Gln Phe Glu Glu Met Arg Ser Ser Tyr Ile Arg Glu

770 775 780

Leu Ile Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val Pro Ser Ser

785 790 795 800

Gln Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Leu His Asp Leu

805 810 815

Val Lys Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile Gln Ser Arg

820 825 830

Thr Leu Ala Val Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala

835840 845

Gln Leu Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu Leu Phe His

850 855 860

Lys Lys

865

<210>92

<211>923

<212>PRT

<213> rat

<400>92

Met Thr Glu Leu Gln Ala Lys Asp Pro Arg Thr Leu His Thr Ser Gly

1 5 10 15

Ala Ala Pro Ser Pro Thr His Val Gly Ser Pro Leu Leu Ala Arg Leu

20 25 30

Asp Pro Asp Pro Phe Gln Gly Ser Gln His Ser Asp Ala Ser Ser Val

35 40 45

Val Ser Pro Ile Pro Ile Ser Leu Asp Arg Leu Leu Phe Ser Arg Ser

50 55 60

Cys Gln Ala Gln Glu Leu Pro Asp Glu Lys Thr Gln Asn Gln Gln Ser

65 70 75 80

Leu Ser Asp Val Glu Gly Ala Phe Ser Gly Val Glu Ala Ser Arg Arg

85 90 95

Arg Ser Arg Asn Pro Arg Ala Pro Glu Lys Asp Ser Arg Leu Leu Asp

100 105 110

Ser Val Leu Asp Thr Leu Leu Ala Pro Ser Gly Pro Glu Gln Ser Gln

115 120 125

Thr Ser Pro Pro Ala Cys Glu Ala Ile Thr Ser Trp Cys Leu Phe Gly

130 135 140

Pro Glu Leu Pro Glu Asp Pro Arg Ser Val Pro Ala Thr Lys Gly Leu

145 150 155 160

Leu Ser Pro Leu Met Ser Arg Pro Glu Ser Lys Ala Gly Asp Ser Ser

165 170 175

Gly Thr Gly Ala Gly Gln Lys Val Leu Pro Lys Ala Val Ser Pro Pro

180 185 190

Arg Gln Leu Leu Leu Pro Thr Ser Gly Ser Ala His Trp Pro Gly Ala

195 200 205

Gly Val Lys Pro Ser Gln Gln Pro Ala Thr Val Glu Val Glu Glu Asp

210 215 220

Gly Gly Leu Glu Thr Glu Gly Ser Ala Gly Pro Leu Leu Lys Ser Lys

225 230 235 240

Pro Arg Ala Leu Glu Gly Met Cys Ser Gly Gly Gly Val Thr Ala Asn

245 250 255

Ala Pro Gly Ala Ala Pro Gly Gly Val Thr Leu Val Pro Lys Glu Asp

260 265 270

Ser Arg Phe Ser Ala Pro Arg Val Ser Leu Glu Gln Asp Ala Pro Val

275 280 285

Ala Pro Gly Arg Ser Pro Leu Ala Thr Thr Val Val Asp Phe Ile His

290 295 300

Val Pro Ile Leu Pro Leu Asn His Ala Leu Leu Ala Ala Arg Thr Arg

305 310 315 320

Gln Leu Leu Glu Gly Asp Ser Tyr Asp Gly Gly Ala Ala Ala Gln Val

325 330 335

Pro Phe Ala Pro Pro Arg Gly Ser Pro Ser Ala Pro Ser Pro Pro Val

340 345 350

Pro Cys Gly Asp Phe Pro Asp Cys Thr Tyr Pro Pro Glu Gly Asp Pro

355 360 365

Lys Glu Asp Gly Phe Pro Val Tyr Gly Glu Phe Gln Pro Pro Gly Leu

370 375 380

Lys Ile Lys Glu Glu Glu Glu Gly Thr Glu Ala Ala Ser Arg Ser Pro

385 390 395 400

Arg Pro Tyr Leu Leu Ala Gly Ala Ser Ala Ala Thr Phe Pro Asp Phe

405 410 415

Pro Leu Pro Pro Arg Pro Pro Arg Ala Pro Pro Ser Arg Pro Gly Glu

420 425 430

Ala Ala Val Ala Ala Pro Ser Ala Ala Val Ser Pro Val Ser Ser Ser

435 440 445

Gly Ser Ala Leu Glu Cys Ile Leu Tyr Lys Ala Glu Gly Ala Pro Pro

450 455 460

Thr Gln Gly Ser Phe Ala Pro Leu Pro Cys Lys Pro Pro Ala Ala Ser

465 470 475 480

Ser Cys Leu Leu Pro Arg Asp Ser Leu Pro Ala Ala Pro Thr Ser Ser

485 490 495

Ala Ala Pro Ala Ile Tyr Pro Pro Leu Gly Leu Asn Gly Leu Pro Gln

500 505 510

Leu Gly Tyr Gln Ala Ala Val Leu Lys Asp Ser Leu Pro Gln Val Tyr

515 520 525

Pro Pro Tyr Leu Asn Tyr Leu Arg Pro Asp Ser Glu Ala Ser Gln Ser

530 535 540

Pro Gln Tyr Gly Phe Asp Ser Leu Pro Gln Lys Ile Cys Leu Ile Cys

545 550 555 560

Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Val Leu Thr Cys Gly Ser

565 570 575

Cys Lys Val Phe Phe Lys Arg Ala Met Glu Gly Gln His Asn Tyr Leu

580 585 590

Cys Ala Gly Arg Asn Asp Cys Ile Val Asp Lys Ile Arg Arg Lys Asn

595 600 605

Cys Pro Ala Cys Arg Leu Arg Lys Cys Cys Gln Ala Gly Met Val Leu

610 615 620

Gly Gly Arg Lys Phe Lys Lys Phe Asn Lys Val Arg Val Met Arg Ala

625 630 635 640

Leu Asp Gly Val Ala Leu Pro Gln Ser Val Ala Phe Pro Asn Glu Ser

645 650 655

Gln Thr Leu Gly Gln Arg Ile Thr Phe Ser Pro Asn Gln Glu Ile Gln

660 665 670

Leu Val Pro Pro Leu Ile Asn Leu Leu Met Ser Ile Glu Pro Asp Val

675 680 685

Val Tyr Ala Gly His Asp Asn Thr Lys Pro Asp Thr Ser Ser Ser Leu

690 695 700

Leu Thr Ser Leu Asn Gln Leu Gly Glu Arg Gln Leu Leu Ser Val Val

705 710 715 720

Lys Trp Ser Lys Ser Leu Pro Gly Phe Arg Asn Leu His Ile Asp Asp

725 730 735

Gln Ile Thr Leu Ile Gln Tyr Ser Trp Met Ser Leu Met Val Phe Gly

740 745 750

Leu Gly Trp Arg Ser Tyr Lys His Val Ser Gly Gln Met Leu Tyr Phe

755 760 765

Ala Pro Asp Leu Ile Leu Asn Glu Gln Arg Met Lys Glu Leu Ser Phe

770 775 780

Tyr Ser Leu Cys Leu Thr Met Trp Gln Ile Pro Gln Glu Phe Val Lys

785 790 795 800

Leu Gln Val Thr His Glu Glu Phe Leu Cys Met Lys Val Leu Leu Leu

805 810 815

Leu Asn Thr Ile Pro Leu Glu Gly Leu Arg Ser Gln Ser Gln Phe Glu

820 825 830

Glu Met Arg Ser Ser Tyr Ile Arg Glu Leu Ile Lys Ala Ile Gly Leu

835 840 845

Arg Gln Lys Gly Val Val Pro Ser Ser Gln Arg Phe Tyr Gln Leu Thr

850 855 860

Lys Leu Leu Asp Ser Leu His Asp Leu Val Lys Gln Leu His Leu Tyr

865 870 875 880

Cys Leu Asn Thr Phe Ile Gln Ser Arg Ala Leu Ala Val Glu Phe Pro

885 890 895

Glu Met Met Ser Glu Val Ile Ala Ala Gln Leu Pro Lys Ile Leu Ala

900 905 910

Gly Met Val Lys Pro Leu Leu Phe His Lys Lys

915 920

<210>93

<211>933

<212>PRT

<213> Baboon of Dongfei

<400>93

Met Thr Glu Leu Lys Ala Lys Gly Pro Arg Ala Pro His Val Ala Gly

1 5 10 15

Gly Pro Pro Ser Pro Glu Val Gly Ser Pro Leu Leu Cys Arg Pro Ala

20 25 30

Ala Gly Pro Phe Gln Gly Ser Gln Thr Ser Asp Thr Leu Pro Glu Val

35 40 45

Ser Ala Ile Pro Ile Ser Leu Asp Gly Leu Leu Phe Pro Arg Pro Cys

50 55 60

Gln Gly Gln Asp Pro Leu Asp Glu Lys Thr Gln Asp Gln Gln Ser Leu

65 70 75 80

Ser Asp Val Glu Gly Ala Tyr Ser Arg Ala Glu Ala Thr Arg Gly Thr

85 90 95

Gly Gly Ser Ser Ser Arg Pro Pro Glu Lys Asp Ser Gly Leu Leu His

100 105 110

Ser Val Leu Asp Thr Leu Leu Ala Pro Ser Gly Pro Gly Gln Ser Gln

115 120 125

Pro Ser Pro Pro Ala Cys Glu Val Thr Ser Ser Trp Cys Leu Phe Gly

130 135 140

Pro Glu Leu Pro Glu Asp Pro Pro Ala Ala Pro Ala Thr Gln Gly Val

145 150 155 160

Leu Ser Pro Leu Met Ser Arg Ser Gly Cys Lys Ala Gly Asp Ser Ser

165 170 175

Gly Thr Ala Ala Ala His Lys Val Leu Pro Arg Gly Leu Ser Pro Ser

180 185 190

Arg Gln Leu Leu Leu Pro Ala Ser Gly Ser Pro His Trp Ser Gly Ala

195 200 205

Pro Val Lys Pro Ser Pro Gln Pro Ala Ala Val Glu Val Glu Glu Glu

210 215 220

Asp Gly Ser Glu Ser Glu Glu Ser Ala Gly Pro Leu Leu Lys Gly Lys

225 230 235 240

Pro Arg Ala Leu Gly Gly Ala Ala Ala Gly Gly Gly Ala Ala Ala Val

245 250 255

Pro Pro Gly Ala Ala Ala Gly Gly Val Ala Leu Val Pro Lys Glu Asp

260 265 270

Ser Arg Phe Ser Ala Pro Arg Val Ala Leu Val Glu Gln Asp Ala Pro

275 280 285

Met Ala Pro Gly Arg Ser Pro Leu Ala Thr Thr Thr Met Asp Phe Thr

290 295 300

His Val Pro Ile Leu Pro Leu Ser His Ala Leu Leu Ala Ala Arg Thr

305 310 315 320

Arg Gln Leu Leu Glu Glu Glu Ser Tyr Asp Gly Gly Ala Gly Ala Ala

325 330 335

Ser Ala Phe Ala Pro Pro Arg Ser Ser Pro Ser Ala Ser Ser Thr Pro

340 345 350

Val Ala Val Gly Asp Phe Pro Asp Cys Ala Tyr Pro Pro Asp Ala Asp

355 360 365

Pro Lys Asp Asp Ala Tyr Pro Leu Tyr Gly Asp Phe Gln Pro Pro Ala

370 375 380

Leu Lys Ile Lys Glu Glu Glu Glu Gly Ala Glu Val Ser Ala Arg Ser

385 390 395 400

Pro Arg Ser Tyr Leu Val Ala Gly Ala Asn Pro Ala Ala Phe Pro Asp

405 410 415

Phe Pro Leu Gly Pro Pro Pro Pro Leu Pro Pro Arg Ala Pro Pro Ser

420 425 430

Arg Pro Gly Glu Ala Ala Val Thr Ala Ala Pro Ala Gly Ala Ser Val

435 440 445

Ser Ser Ala Ser Ser Ser Gly Ser Thr Leu Glu Cys Ile Leu Tyr Lys

450 455 460

Ala Glu Gly Ala Pro Pro Gln Gln Gly Pro Phe Ala Pro Pro Pro Cys

465 470 475 480

Lys Ala Pro Gly Ala Gly Gly Cys Leu Leu Pro Arg Asp Gly Leu Pro

485 490 495

Ser Thr Ser Ala Ser Ala Ala Ala Ala Gly Ala Ala Pro Ala Leu Tyr

500 505 510

Pro Ala Leu Gly Leu Asn Gly Leu Pro Gln Leu Gly Tyr Gln Ala Ala

515 520 525

Val Leu Lys Glu Gly Leu Gln Gln Val Tyr Pro Pro Tyr Leu Asn Tyr

530 535 540

Leu Arg Pro Asp Ser Glu Ala Ser Gln Ser Pro Gln Tyr Ser Phe Glu

545 550 555 560

Ser Leu Pro Gln Lys Ile Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly

565 570 575

Cys His Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys

580 585 590

Arg Ala Met Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp

595 600 605

Cys Ile Val Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Leu

610 615 620

Arg Lys Cys Cys Gln Ala Gly Met Val Leu Gly Gly Arg Lys Phe Lys

625 630 635 640

Lys Phe Asn Lys Val Arg Val Met Arg Ala Leu Asp Ala Val Ala Leu

645 650 655

Pro Gln Pro Val Gly Ile Pro Asn Glu Ser Gln Ala Leu Ser Gln Arg

660 665 670

Phe Thr Phe Pro Pro Gly Gln Asp Ile Gln Leu Ile Pro Pro Leu Ile

675 680 685

Asn Leu Leu Val Ser Ile Glu Pro Asp Val Ile Tyr Ala Gly His Asp

690 695 700

Asn Ser Lys Pro Asp Thr Ser Ser Ser Leu Leu Thr Ser Leu Asn Gln

705 710 715 720

Leu Gly Glu Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Leu Leu

725 730 735

Pro Gly Phe Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln

740 745 750

Tyr Ser Trp Met Ser Leu Met Val Phe Gly Leu Gly Trp Arg Ser Tyr

755 760 765

Lys His Val Ser Gly Gln Met Leu Tyr Phe Ala Pro Asp Leu Ile Leu

770 775 780

Asn Glu Gln Arg Met Lys Glu Ser Ser Phe Tyr Ser Leu Cys Leu Thr

785 790 795 800

Met Trp Gln Ile Pro Gln Glu Phe Val Lys Leu Gln Val Ser Gln Glu

805 810 815

Glu Phe Leu Cys Met Lys Val Leu Leu Leu Leu Asn Thr Ile Pro Leu

820 825 830

Glu Gly Leu Arg Ser Gln Thr Gln Phe Glu Glu Met Arg Ser Ser Tyr

835 840 845

Ile Arg Glu Leu Ile Lys Ala Ile Gly Leu Arg Gln Lys Gly Val Val

850 855 860

Ser Ser Ser Gln Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Asn Leu

865 870 875 880

His Asp Leu Val Lys Gln Leu His Leu Tyr Cys Leu Asn Thr Phe Ile

885 890 895

Gln Ser Arg Ala Leu Ser Val Glu Phe Pro Glu Met Met Ser Glu Val

900 905 910

Ile Ala Ala Gln Leu Pro Lys Ile Leu Ala Gly Met Val Lys Pro Leu

915 920 925

Leu Phe His Lys Lys

930

<210>94

<211>899

<212>PRT

<213> mice

<400>94

Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser

1 5 10 15

Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu

20 25 30

Ala Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Asn Ile Ala

35 40 45

Pro Pro Gly Ala Cys Leu Gln Gln Arg Gln Glu Thr Ser Pro Arg Arg

50 55 60

Arg Arg Arg Gln Gln His Thr Glu Asp Gly Ser Pro Gln Ala His Ile

65 70 75 80

Arg Gly Pro Thr Gly Tyr Leu Ala Leu Glu Glu Glu Gln Gln Pro Ser

85 90 95

Gln Gln Gln Ala Ala Ser Glu Gly His Pro Glu Ser Ser Cys Leu Pro

100 105 110

Glu Pro Gly Ala Ala Thr Ala Pro Gly Lys Gly Leu Pro Gln Gln Pro

115 120 125

Pro Ala Pro ProAsp Gln Asp Asp Ser Ala Ala Pro Ser Thr Leu Ser

130 135 140

Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser Ala Asp Ile

145 150 155 160

Lys Asp Ile Leu Asn Glu Ala Gly Thr Met Gln Leu Leu Gln Gln Gln

165 170 175

Gln Gln Gln Gln Gln His Gln Gln Gln His Gln Gln His Gln Gln Gln

180 185 190

Gln Glu Val Ile Ser Glu Gly Ser Ser Ala Arg Ala Arg Glu Ala Thr

195 200 205

Gly Ala Pro Ser Ser Ser Lys Asp Ser Tyr Leu Gly Gly Asn Ser Thr

210 215 220

Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys Ala Val Ser Val Ser Met

225 230 235 240

Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly Glu Gln Leu

245 250 255

Arg Gly Asp Cys Met Tyr Ala Ser Leu Leu Gly Gly Pro Pro Ala Val

260 265 270

Arg Pro Thr Pro Cys Ala Pro Leu Pro Glu Cys Lys Gly Leu Pro Leu

275 280 285

Asp Glu Gly Pro Gly LysSer Thr Glu Glu Thr Ala Glu Tyr Ser Ser

290 295 300

Phe Lys Gly Gly Tyr Ala Lys Gly Leu Glu Gly Glu Ser Leu Gly Cys

305 310 315 320

Ser Gly Ser Ser Glu Ala Gly Ser Ser Gly Thr Leu Glu Ile Pro Ser

325 330 335

Ser Leu Ser Leu Tyr Lys Ser Gly Ala Leu Asp Glu Ala Ala Ala Tyr

340 345 350

Gln Asn Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu Ser Gly Pro Pro

355 360 365

His Pro Pro Pro Pro Thr His Pro His Ala Arg Ile Lys Leu Glu Asn

370 375 380

Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala Gln Cys Arg

385 390 395 400

Tyr Gly Asp Leu Gly Ser Leu His Gly Gly Ser Val Ala Gly Pro Ser

405 410 415

Thr Gly Ser Pro Pro Ala Thr Thr Ser Ser Ser Trp His Thr Leu Phe

420 425 430

Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Gly Gly Gly Gly Gly Ser

435 440 445

Ser Ser Pro Ser Asp Ala Gly ProVal Ala Pro Tyr Gly Tyr Thr Arg

450 455 460

Pro Pro Gln Gly Leu Thr Ser Gln Glu Ser Asp Tyr Ser Ala Ser Glu

465 470 475 480

Val Trp Tyr Pro Gly Gly Val Val Asn Arg Val Pro Tyr Pro Ser Pro

485 490 495

Asn Cys Val Lys Ser Glu Met Gly Pro Trp Met Glu Asn Tyr Ser Gly

500 505 510

Pro Tyr Gly Asp Met Arg Leu Asp Ser Thr Arg Asp His Val Leu Pro

515 520 525

Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp

530 535 540

Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys

545 550 555 560

Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala

565 570 575

Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro

580 585 590

Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala

595 600 605

Arg Lys Leu Lys Lys Leu Gly Asn Leu LysLeu Gln Glu Glu Gly Glu

610 615 620

Asn Ser Asn Ala Gly Ser Pro Thr Glu Asp Pro Ser Gln Lys Met Thr

625 630 635 640

Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val

645 650 655

Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly His Asp Asn Asn

660 665 670

Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu Gly

675 680 685

Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly

690 695 700

Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser

705 710 715 720

Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn

725 730 735

Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu

740 745 750

Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg

755 760 765

His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile ThrPro Gln Glu Phe

770 775 780

Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro Val Asp Gly

785 790 795 800

Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile Lys

805 810 815

Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys

820 825 830

Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro

835 840 845

Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser

850 855 860

His Met Val Ser Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser

865 870 875 880

Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe

885 890 895

His Thr Gln

<210>95

<211>902

<212>PRT

<213> rat

<400>95

Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser

15 10 15

Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu

20 25 30

Ala Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ile Ala

35 40 45

Pro Pro Gly Ala Cys Leu Gln Gln Arg Gln Glu Thr Ser Pro Arg Arg

50 55 60

Arg Arg Arg Gln Gln His Pro Glu Asp Gly Ser Pro Gln Ala His Ile

65 70 75 80

Arg Gly Thr Thr Gly Tyr Leu Ala Leu Glu Glu Glu Gln Gln Pro Ser

85 90 95

Gln Gln Gln Ser Ala Ser Glu Gly His Pro Glu Ser Gly Cys Leu Pro

100 105 110

Glu Pro Gly Ala Ala Thr Ala Pro Gly Lys Gly Leu Pro Gln Gln Pro

115 120 125

Pro Ala Pro Pro Asp Gln Asp Asp Ser Ala Ala Pro Ser Thr Leu Ser

130 135 140

Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys Ser Ala Asp Ile

145 150 155 160

Lys Asp Ile Leu Ser Glu Ala Gly Thr Met Gln Leu Leu Gln Gln Gln

165170 175

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

180 185 190

Gln Gln Gln Glu Val Ile Ser Glu Gly Ser Ser Ser Val Arg Ala Arg

195 200 205

Glu Ala Thr Gly Ala Pro Ser Ser Ser Lys Asp Ser Tyr Leu Gly Gly

210 215 220

Asn Ser Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys Ala Val Ser

225 230 235 240

Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu Ser Pro Gly

245 250 255

Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Ser Leu Leu Gly Gly Pro

260 265 270

Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu Cys Lys Gly

275 280 285

Leu Ser Leu Asp Glu Gly Pro Gly Lys Gly Thr Glu Glu Thr Ala Glu

290 295 300

Tyr Ser Ser Phe Lys Gly Gly Tyr Ala Lys Gly Leu Glu Gly Glu Ser

305 310 315 320

Leu Gly Cys Ser Gly Ser Ser Glu Ala Gly Ser Ser Gly Thr Leu Glu

325330 335

Ile Pro Ser Ser Leu Ser Leu Tyr Lys Ser Gly Ala Val Asp Glu Ala

340 345 350

Ala Ala Tyr Gln Asn Arg Asp Tyr Tyr Asn Phe Pro Leu Ala Leu Ser

355 360 365

Gly Pro Pro His Pro Pro Pro Pro Thr His Pro His Ala Arg Ile Lys

370 375 380

Leu Glu Asn Pro Ser Asp Tyr Gly Ser Ala Trp Ala Ala Ala Ala Ala

385 390 395 400

Gln Cys Arg Tyr Gly Asp Leu Ala Ser Leu His Gly Gly Ser Val Ala

405 410 415

Gly Pro Ser Thr Gly Ser Pro Pro Ala Thr Ala Ser Ser Ser Trp His

420 425 430

Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro Gly Gly Gly

435 440 445

Gly Gly Ser Ser Ser Pro Ser Asp Ala Gly Pro Val Ala Pro Tyr Gly

450 455 460

Tyr Thr Arg Pro Pro Gln Gly Leu Ala Ser Gln Glu Gly Asp Phe Ser

465 470 475 480

Ala Ser Glu Val Trp Tyr Pro Gly Gly Val Val Asn Arg Val Pro Tyr

485 490495

Pro Ser Pro Ser Cys Val Lys Ser Glu Met Gly Pro Trp Met Glu Asn

500 505 510

Tyr Ser Gly Pro Tyr Gly Asp Met Arg Leu Asp Ser Thr Arg Asp His

515 520 525

Val Leu Pro Ile Asp Tyr Tyr Phe Pro Pro Gln Lys Thr Cys Leu Ile

530 535 540

Cys Gly Asp Glu Ala Ser Gly Cys His Tyr Gly Ala Leu Thr Cys Gly

545 550 555 560

Ser Cys Lys Val Phe Phe Lys Arg Ala Ala Glu Gly Lys Gln Lys Tyr

565 570 575

Leu Cys Ala Ser Arg Asn Asp Cys Thr Ile Asp Lys Phe Arg Arg Lys

580 585 590

Asn Cys Pro Ser Cys Arg Leu Arg Lys Cys Tyr Glu Ala Gly Met Thr

595 600 605

Leu Gly Ala Arg Lys Leu Lys Lys Leu Gly Asn Leu Lys Leu Gln Glu

610 615 620

Glu Gly Glu Asn Ser Ser Ala Gly Ser Pro Thr Glu Asp Pro Ser Gln

625 630 635 640

Lys Met Thr Val Ser His Ile Glu Gly Tyr Glu Cys Gln Pro Ile Phe

645 650655

Leu Asn Val Leu Glu Ala Ile Glu Pro Gly Val Val Cys Ala Gly His

660 665 670

Asp Asn Asn Gln Pro Asp Ser Phe Ala Ala Leu Leu Ser Ser Leu Asn

675 680 685

Glu Leu Gly Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala

690 695 700

Leu Pro Gly Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile

705 710 715 720

Gln Tyr Ser Trp Met Gly Leu Met Val Phe Ala Met Gly Trp Arg Ser

725 730 735

Phe Thr Asn Val Asn Ser Arg Met Leu Tyr Phe Ala Pro Asp Leu Val

740 745 750

Phe Asn Glu Tyr Arg Met His Lys Ser Arg Met Tyr Ser Gln Cys Val

755 760 765

Arg Met Arg His Leu Ser Gln Glu Phe Gly Trp Leu Gln Ile Thr Pro

770 775 780

Gln Glu Phe Leu Cys Met Lys Ala Leu Leu Leu Phe Ser Ile Ile Pro

785 790 795 800

Val Asp Gly Leu Lys Asn Gln Lys Phe Phe Asp Glu Leu Arg Met Asn

805 810815

Tyr Ile Lys Glu Leu Asp Arg Ile Ile Ala Cys Lys Arg Lys Asn Pro

820 825 830

Thr Ser Cys Ser Arg Arg Phe Tyr Gln Leu Thr Lys Leu Leu Asp Ser

835 840 845

Val Gln Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu

850 855 860

Ile Lys Ser His Met Val Ser Val Asp Phe Pro Glu Met Met Ala Glu

865 870 875 880

Ile Ile Ser Val Gln Val Pro Lys Ile Leu Ser Gly Lys Val Lys Pro

885 890 895

Ile Tyr Phe His Thr Gln

900

<210>96

<211>895

<212>PRT

<213> Arababoon

<400>96

Met Glu Val Gln Leu Gly Leu Gly Arg Val Tyr Pro Arg Pro Pro Ser

1 5 10 15

Lys Thr Tyr Arg Gly Ala Phe Gln Asn Leu Phe Gln Ser Val Arg Glu

20 25 30

Val Ile Gln Asn Pro Gly Pro Arg His Pro Glu Ala Ala Ser Ala Ala

35 4045

Pro Pro Gly Ala Ser Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Glu

50 55 60

Thr Ser Pro Arg Gln Gln Gln Gln Gln Gln Gly Glu Asp Gly Ser Pro

65 70 75 80

Gln Ala His Arg Arg Gly Pro Thr Gly Tyr Leu Val Leu Asp Glu Glu

85 90 95

Gln Gln Pro Ser Gln Pro Gln Ser Ala Pro Glu Cys His Pro Glu Arg

100 105 110

Gly Cys Val Pro Glu Pro Gly Ala Ala Val Ala Ala Gly Lys Gly Leu

115 120 125

Pro Gln Gln Leu Pro Ala Pro Pro Asp Glu Asp Asp Ser Ala Ala Pro

130 135 140

Ser Thr Leu Ser Leu Leu Gly Pro Thr Phe Pro Gly Leu Ser Ser Cys

145 150 155 160

Ser Ala Asp Leu Lys Asp Ile Leu Ser Glu Ala Ser Thr Met Gln Leu

165 170 175

Leu Gln Gln Gln Gln Gln Glu Ala Val Ser Glu Gly Ser Ser Ser Gly

180 185 190

Arg Ala Arg Glu Ala Ser Gly Ala Pro Thr Ser Ser Lys Asp Asn Tyr

195 200 205

Leu Gly Gly Thr Ser Thr Ile Ser Asp Ser Ala Lys Glu Leu Cys Lys

210 215 220

Ala Val Ser Val Ser Met Gly Leu Gly Val Glu Ala Leu Glu His Leu

225 230 235 240

Ser Pro Gly Glu Gln Leu Arg Gly Asp Cys Met Tyr Ala Pro Val Leu

245 250 255

Gly Val Pro Pro Ala Val Arg Pro Thr Pro Cys Ala Pro Leu Ala Glu

260 265 270

Cys Lys Gly Ser Leu Leu Asp Asp Ser Ala Gly Lys Ser Thr Glu Asp

275 280 285

Thr Ala Glu Tyr Ser Pro Phe Lys Gly Gly Tyr Thr Lys Gly Leu Glu

290 295 300

Gly Glu Ser Leu Gly Cys Ser Gly Ser Ala Ala Ala Gly Ser Ser Gly

305 310 315 320

Thr Leu Glu Leu Pro Ser Thr Leu Ser Leu Tyr Lys Ser Gly Ala Leu

325 330 335

Asp Glu Ala Ala Ala Tyr Gln Ser Arg Asp Tyr Tyr Asn Phe Pro Leu

340 345 350

Ala Leu Ala Gly Pro Pro Pro Pro Pro Pro Pro Pro His Pro His Ala

355 360 365

Arg Ile Lys Leu Glu Asn Pro Leu Asp Tyr Gly Ser Ala Trp Ala Ala

370 375 380

Ala Ala Ala Gln Cys Arg Tyr Gly Glu Leu Ala Ser Leu His Gly Ala

385 390 395 400

Gly Ala Ala Gly Pro Gly Ser Gly Ser Pro Ser Ala Ala Ala Ser Ser

405 410 415

Ser Trp His Thr Leu Phe Thr Ala Glu Glu Gly Gln Leu Tyr Gly Pro

420 425 430

Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly

435 440 445

Glu Ala Gly Ala Val Ala Pro Tyr Gly Tyr Thr Arg Pro Pro Gln Gly

450 455 460

Leu Ala Gly Gln Glu Gly Asp Phe Thr Ala Pro Asp Val Trp Tyr Pro

465 470 475 480

Gly Gly Met Val Ser Arg Val Pro Tyr Pro Ser Pro Thr Cys Val Lys

485 490 495

Ser Glu Met Gly Pro Trp Met Asp Ser Tyr Ser Gly Pro Tyr Gly Asp

500 505 510

Met Arg Leu Glu Thr Ala Arg Asp His Val Leu Pro Ile Asp Tyr Tyr

515 520 525

Phe Pro Pro Gln Lys Thr Cys Leu Ile Cys Gly Asp Glu Ala Ser Gly

530 535 540

Cys His Tyr Gly Ala Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys

545 550 555 560

Arg Ala Ala Glu Gly Lys Gln Lys Tyr Leu Cys Ala Ser Arg Asn Asp

565 570 575

Cys Thr Ile Asp Lys Phe Arg Arg Lys Asn Cys Pro Ser Cys Arg Leu

580 585 590

Arg Lys Cys Tyr Glu Ala Gly Met Thr Leu Gly Ala Arg Lys Leu Lys

595 600 605

Lys Leu Gly Asn Leu Lys Leu Gln Glu Glu Gly Glu Ala Ser Ser Thr

610 615 620

Thr Ser Pro Thr Glu Glu Thr Ala Gln Lys Leu Thr Val Ser His Ile

625 630 635 640

Glu Gly Tyr Glu Cys Gln Pro Ile Phe Leu Asn Val Leu Glu Ala Ile

645 650 655

Glu Pro Gly Val Val Cys Ala Gly His Asp Asn Asn Gln Pro Asp Ser

660 665 670

Phe Ala Ala Leu Leu Ser Ser Leu Asn Glu Leu Gly Glu Arg Gln Leu

675 680 685

Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly Phe Arg Asn Leu

690 695 700

His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser Trp Met Gly Leu

705 710 715 720

Met Val Phe Ala Met Gly Trp Arg Ser Phe Thr Asn Val Asn Ser Arg

725 730 735

Met Leu Tyr Phe Ala Pro Asp Leu Val Phe Asn Glu Tyr Arg Met His

740 745 750

Lys Ser Arg Met Tyr Ser Gln Cys Val Arg Met Arg His Leu Ser Gln

755 760 765

Glu Phe Gly Trp Leu Gln Ile Thr Pro Gln Glu Phe Leu Cys Met Lys

770 775 780

Ala Leu Leu Leu Phe Ser Ile Ile Pro Val Asp Gly Leu Lys Asn Gln

785 790 795 800

Lys Phe Phe Asp Glu Leu Arg Met Asn Tyr Ile Lys Glu Leu Asp Arg

805 810 815

Ile Ile Ala Cys Lys Arg Lys Asn Pro Thr Ser Cys Ser Arg Arg Phe

820 825 830

Tyr Gln Leu Thr Lys Leu Leu Asp Ser Val Gln Pro Ile Ala Arg Glu

835 840 845

Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys Ser His Met Val Ser

850 855 860

Val Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser Val Gln Val Pro

865 870 875 880

Lys Ile Leu Ser Gly Lys Val Lys Pro Ile Tyr Phe His Thr Gln

885 890 895

<210>97

<211>2397

<212>DNA

<213> Artificial sequence

<220>

<223>ECD v.1 CAR

<400>97

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

attcctgaac agaagctgat aagtgaggag gacttggaca tccagatgac ccagaccacc 120

agcagcctga gcgccagcct gggcgataga gtgaccatca gctgcagagc cagccaggac 180

atcagcaagt acctgaactg gtatcagcag aaacccgacg gcaccgtgaa gctgctgatc 240

taccacacca gcagactgca cagcggcgtg cccagcagat tttctggcag cggctccggc 300

accgactaca gcctgaccat ctccaacctg gaacaggaag atatcgctac ctacttctgt 360

cagcaaggca acaccctgcc ctacaccttc ggcggaggca ccaagctgga aatcacaggc 420

ggcggaggat ctggcggagg cggaagtggc ggagggggat ctgaagtgaa actgcaggaa 480

agcggccctg gcctggtggc cccatctcag tctctgagcg tgacctgtac cgtgtccggc 540

gtgtccctgc ctgactatgg cgtgtcctgg atcagacagc cccccagaaa gggcctggaa 600

tggctgggag tgatctgggg cagcgagaca acctactaca acagcgccct gaagtcccgg 660

ctgaccatca tcaaggacaa ctccaagagc caggtgttcc tgaagatgaa cagcctgcag 720

accgacgaca ccgccatcta ctactgcgcc aagcactact actacggcgg cagctacgcc 780

atggactact ggggccaggg cacaagcgtg accgtgtcta gcggatccga Label aagaggc 840

ggcagaatgc tgaaacacaa gcggcagagg gacgacgggg aaggcagagg cgaagtggga 900

tctgccggcg atatgagagc cgccaacctg tggcctagcc ccctgatgat caagcggagc 960

aagaagaact ccctggccct gagcctgacc gccgaccaga tggtgtctgc cctgctggat 1020

gccgagcccc ccatcctgta cagcgagtac gaccccacca gacccttcag cgaggccagc 1080

atgatgggcc tgctgaccaa cctggccgac cgggaactgg tgcacatgat caactgggcc 1140

aagcgggtgc ccggcttcgt ggatctgaca ctgcacgacc aggtgcacct gctggaatgc 1200

gcttggctgg aaatcctgat gatcggcctc gtgtggcgga gcatggaaca ccctggcaag 1260

ctgctgttcg cccccaacct gctgctggac cggaaccagg gcaaatgcgt ggaaggcatg 1320

gtggaaatct tcgacatgct gctggccacc tccagccggt tccggatgat gaacctgcag 1380

ggcgaagagt tcgtgtgtct gaagtccatc atcctgctga atagcggcgt gtacaccttc 1440

ctgagcagca ccctgaaaag cctggaagaa aaggaccaca tccaccgggt gctggacaag 1500

atcaccgaca ccctgattca cctgatggcc aaggccggac tgaccctgca gcagcagcat 1560

cagagactgg ctcagctgct gctgatcctg tcccacatcc ggcacatgag caacaagcgg 1620

atggaacatc tgtacagcat gaagtgcaag aacgtggtgc ctctgttcga tctgctgctg 1680

gaaatgctgg acgcccacag gctgcacgcc ccaacatccg gatccaccac gacgccagcg 1740

ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 1800

gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1860

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1920

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1980

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 2040

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 2100

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 2160

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 2220

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 2280

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 2340

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctag 2397

<210>98

<211>798

<212>PRT

<213> Artificial sequence

<220>

<223>ECD v.1 CAR

<400>98

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 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

20 25 30

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

35 40 45

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

50 55 60

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

65 70 75 80

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

85 90 95

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

100 105 110

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

115 120 125

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

145 150 155 160

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

165 170 175

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

180 185 190

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

195 200 205

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

210 215 220

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

225 230 235 240

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

245 250 255

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

260 265 270

Ser Ser Gly Ser Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg

275 280 285

Gln Arg Asp Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp

290 295 300

Met Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser

305 310 315 320

Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser

325 330 335

Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro

340 345 350

Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu

355 360 365

Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro

370 375 380

Gly Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys

385 390 395 400

Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu

405 410 415

His Pro Gly Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn

420 425 430

Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu

435 440 445

Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe

450 455 460

Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe

465 470 475 480

Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg

485 490 495

Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala

500 505 510

Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu

515 520 525

Ile Leu Ser His Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu

530 535 540

Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu

545 550 555 560

Glu Met Leu Asp Ala His Arg Leu His Ala Pro Thr Ser Gly Ser Thr

565 570 575

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

580 585 590

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

595 600 605

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

610 615 620

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

625 630 635 640

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

645 650 655

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

660 665 670

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

675 680 685

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

690 695 700

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

705 710 715 720

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

725 730 735

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

740 745 750

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

755 760 765

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

770 775 780

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

785 790 795

<210>99

<211>66

<212>DNA

<213> Artificial sequence

<220>

<223> CSF2RA Signal sequence

<400>99

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

attcct 66

<210>100

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> CSF2RA Signal sequence

<400>100

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>101

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> cMyc tag

<400>101

gaacagaagc tgataagtga ggaggacttg 30

<210>102

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> cMyc tag

<400>102

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210>103

<211>321

<212>DNA

<213> Artificial sequence

<220>

<223>FMC63 VL

<400>103

gacatccaga tgacccagac caccagcagc ctgagcgcca gcctgggcga Label agtgacc 60

atcagctgca gagccagcca ggacatcagc aagtacctga actggtatca gcagaaaccc 120

gacggcaccg tgaagctgct gatctaccac accagcagac tgcacagcgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ctacctactt ctgtcagcaa ggcaacaccc tgccctacac cttcggcgga 300

ggcaccaagc tggaaatcac a 321

<210>104

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>FMC63 VL

<400>104

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210>105

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> linker sequence

<400>105

ggcggcggag gatctggcgg aggcggaagt ggcggagggg gatc 44

<210>106

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400>106

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>107

<211>360

<212>DNA

<213> Artificial sequence

<220>

<223>FMC63 VH

<400>107

gaagtgaaac tgcaggaaag cggccctggc ctggtggccc catctcagtc tctgagcgtg 60

acctgtaccg tgtccggcgt gtccctgcct gactatggcg tgtcctggat cagacagccc 120

cccagaaagg gcctggaatg gctgggagtg atctggggca gcgagacaac ctactacaac 180

agcgccctga agtcccggct gaccatcatc aaggacaact ccaagagcca ggtgttcctg 240

aagatgaaca gcctgcagac cgacgacacc gccatctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggactactgg ggccagggca caagcgtgac cgtgtctagc 360

<210>108

<211>120

<212>PRT

<213> Artificial sequence

<220>

<223>FMC63 VH

<400>108

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>109

<211>891

<212>DNA

<213> Artificial sequence

<220>

<223> Estrogen receptor alpha ligand binding Domain

<400>109

gatagaagag gcggcagaat gctgaaacac aagcggcaga gggacgacgg ggaaggcaga 60

ggcgaagtgg gatctgccgg cgatatgaga gccgccaacc tgtggcctag ccccctgatg 120

atcaagcgga gcaagaagaa ctccctggcc ctgagcctga ccgccgacca gatggtgtct 180

gccctgctgg atgccgagcc ccccatcctg tacagcgagt acgaccccac cagacccttc 240

agcgaggcca gcatgatggg cctgctgacc aacctggccg accgggaact ggtgcacatg 300

atcaactgggccaagcgggt gcccggcttc gtggatctga cactgcacga ccaggtgcac 360

ctgctggaat gcgcttggct ggaaatcctg atgatcggcc tcgtgtggcg gagcatggaa 420

caccctggca agctgctgtt cgcccccaac ctgctgctgg accggaacca gggcaaatgc 480

gtggaaggca tggtggaaat cttcgacatg ctgctggcca cctccagccg gttccggatg 540

atgaacctgc agggcgaaga gttcgtgtgt ctgaagtcca tcatcctgct gaatagcggc 600

gtgtacacct tcctgagcag caccctgaaa agcctggaag aaaaggacca catccaccgg 660

gtgctggaca agatcaccga caccctgatt cacctgatgg ccaaggccgg actgaccctg 720

cagcagcagc atcagagact ggctcagctg ctgctgatcc tgtcccacat ccggcacatg 780

agcaacaagc ggatggaaca tctgtacagc atgaagtgca agaacgtggt gcctctgttc 840

gatctgctgc tggaaatgct ggacgcccac aggctgcacg ccccaacatc c 891

<210>110

<211>297

<212>PRT

<213> Artificial sequence

<220>

<223> Estrogen receptor alpha ligand binding Domain

<400>110

Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp

1 5 10 15

Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala

20 25 30

Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser

35 40 45

Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp

50 55 60

Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe

65 70 75 80

Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu

85 90 95

Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp

100 105 110

Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu

115 120 125

Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys

130 135 140

Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys

145 150 155 160

Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser

165 170 175

Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys

180 185 190

Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr

195 200 205

Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys

210 215 220

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu

225 230 235 240

Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His

245 250 255

Ile Arg His Met Ser Asn Lys Arg Met Glu His Leu Tyr Ser Met Lys

260 265 270

Cys Lys Asn Val Val Pro Leu Phe Asp Leu Leu Leu Glu Met Leu Asp

275 280 285

Ala His Arg Leu His Ala Pro Thr Ser

290 295

<210>111

<211>207

<212>DNA

<213> human

<400>111

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210>112

<211>44

<212>PRT

<213> human

<400>112

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

35 40

<210>113

<211>75

<212>DNA

<213> human

<400>113

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 60

atcacccttt actgc 75

<210>114

<211>25

<212>PRT

<213> human

<400>114

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

1 5 10 15

Leu Ser Leu Val Ile Thr Leu Tyr Cys

20 25

<210>115

<211>123

<212>DNA

<213> human

<400>115

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaa 123

<210>116

<211>41

<212>PRT

<213> human

<400>116

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu

35 40

<210>117

<211>339

<212>DNA

<213> human

<400>117

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300

acctacgacg cccttcacat gcaggccctg ccccctcgc 339

<210>118

<211>113

<212>PRT

<213> human

<400>118

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210>119

<211>595

<212>PRT

<213> Artificial sequence

<220>

<223> Estrogen receptor ligand binding Domain

<400>119

Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His

1 5 10 15

Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys

20 25 30

Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys

35 40 45

Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala

50 55 60

Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr

65 70 75 80

Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly

85 90 95

Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His

100 105 110

Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val

115 120 125

Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala

130135 140

Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly

145 150 155 160

Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met

165 170 175

Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala

180 185 190

Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe

195 200 205

Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr

210 215 220

Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys

225 230 235 240

Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg

245 250 255

Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp

260 265 270

Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala

275 280 285

Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn

290295 300

Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu

305 310 315 320

Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro

325 330 335

Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg

340 345 350

Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val

355 360 365

Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu

370 375 380

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly

385 390 395 400

Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys

405 410 415

Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser

420 425 430

Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu

435 440 445

Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser

450455 460

Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp

465 470 475 480

Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr

485 490 495

Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser

500 505 510

His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met

515 520 525

Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu

530 535 540

Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val

545 550 555 560

Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser

565 570 575

His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro

580 585 590

Ala Thr Val

595

Claims

1. A single chain chimeric polypeptide, wherein said single chain chimeric polypeptide comprises:

a transmembrane domain; and

a hormone receptor ligand binding domain which is capable of binding to a hormone receptor,

wherein:

the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 500 amino acids; and

both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single chain polypeptide in a mammal.

2. The single chain chimeric polypeptide of claim 1, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

3. The single chain chimeric polypeptide of claim 1, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

4. The single chain chimeric polypeptide of claim 3, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

5. The single chain chimeric polypeptide of any one of claims 1-4, wherein the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other.

6. The single chain chimeric polypeptide of any one of claims 1-4, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids.

7. The single chain chimeric polypeptide of claim 6, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 250 amino acids.

8. The single chain chimeric polypeptide of claim 7, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 50 amino acids.

9. A nucleic acid comprising a nucleotide sequence encoding the single-chain chimeric polypeptide of any one of claims 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. A mammalian cell comprising the vector of claim 10.

12. A method of inducing membrane localization of a single chain chimeric polypeptide in a mammalian cell, the method comprising:

contacting a mammalian cell expressing the single chain chimeric polypeptide of any of claims 1-8 with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell.

13. A method of reversibly altering the membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, said method comprising:

(a) contacting a mammalian cell expressing the single chain chimeric polypeptide of any one of claims 1 to 8 with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell; and

(b) contacting said mammalian cell with a reduced amount of said hormone or said hormone analog, said reduced amount of hormone or hormone analog resulting in a reduced level of said single chain chimeric polypeptide on the extracellular side of the plasma membrane of said mammalian cell.

14. The method of claim 13, further comprising:

(c) contacting said mammalian cell with an increased amount of said hormone or said hormone analog, said increased amount of hormone or hormone analog resulting in an increased level of said single chain chimeric polypeptide on the extracellular side of the plasma membrane of said cell as compared to the level in step (b).

15. The method of any one of claims 12-14, further comprising:

introducing a nucleic acid encoding the single-chain chimeric polypeptide into a mammalian cell to produce a mammalian cell expressing the single-chain chimeric polypeptide.

16. The method of any one of claims 12-15, wherein the mammalian cell is a T cell.

17. The method of claim 16, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

18. The method of any one of claims 12-17, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

19. The method of claim 18, wherein the subject has been identified or diagnosed as having cancer.

20. The method of claim 18 or 19, further comprising:

obtaining the mammalian cell from the subject.

21. The method of any one of claims 12-20, wherein the contacting step is performed in vitro.

22. The method of any one of claims 12-20, wherein the contacting step is performed in a mammal.

23. A single-chain chimeric antigen receptor, wherein the single-chain chimeric antigen receptor comprises:

an extracellular antigen-binding domain;

a transmembrane domain;

a hormone receptor ligand binding domain;

a co-stimulatory domain; and

an immunoreceptor tyrosine-based activation motif (ITAM);

wherein:

24. The single-chain chimeric antigen receptor of claim 23, wherein the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain.

25. The single-chain chimeric antigen receptor of claim 24 wherein the extracellular antigen-binding domain is an scFv.

26. The single-chain chimeric antigen receptor of any one of claims 23-25, wherein the extracellular antigen-binding domain specifically binds to a single antigen.

27. The single-chain chimeric antigen receptor of claim 26, wherein the single antigen is a tumor antigen.

28. The single-chain chimeric antigen receptor of claim 27, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

29. The single-chain chimeric antigen receptor of claim 28, wherein the tumor antigen is CD 19.

30. The single-chain chimeric antigen receptor of claim 23 wherein the extracellular antigen-binding domain specifically binds to two different antigens.

31. The single-chain chimeric antigen receptor of any one of claims 23-30, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

32. The single-chain chimeric antigen receptor of any one of claims 23-31, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

33. The single-chain chimeric antigen receptor of claim 32, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

34. The single-chain chimeric antigen receptor of any one of claims 23-33, wherein the co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C and B7-H3.

35. The single-chain chimeric antigen receptor of claim 34, wherein the co-stimulatory domain is 4-1BB or CD 28.

36. The single-chain chimeric antigen receptor of any one of claims 23-35, wherein the ITAM comprises a cytoplasmic signaling sequence from CD3 ζ.

37. The single-chain chimeric antigen receptor of any one of claims 23-36, wherein the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other.

38. The single-chain chimeric antigen receptor of any one of claims 23-36, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids.

39. The single-chain chimeric antigen receptor of claim 38, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 250 amino acids.

40. The single-chain chimeric antigen receptor of claim 39, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 50 amino acids.

41. A nucleic acid comprising a nucleotide sequence encoding the single-chain chimeric antigen receptor of any one of claims 23-40.

42. A vector comprising the nucleic acid of claim 41.

43. A mammalian cell comprising the vector of claim 42.

44. A method of inducing membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, the method comprising:

contacting a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 23 to 40 with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell.

45. A method of altering the membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 23 to 40 with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell; and

(b) contacting said mammalian cell with an amount of said hormone or said hormone analog which results in a decrease in the level of said single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of said cell.

46. The method of claim 45, further comprising:

(c) contacting said mammalian cell with an amount of said hormone or said hormone analog which results in an increase in the level of said single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of said cell as compared to the level in (b).

47. The method of any one of claims 44-46, further comprising:

introducing a nucleic acid encoding the single-chain chimeric antigen receptor into a mammalian cell to generate a mammalian cell that expresses the single-chain chimeric antigen receptor.

48. The method of claim 47, wherein the mammalian cell is a T cell.

49. The method of claim 48, wherein said T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

50. The method of any one of claims 44-49, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

51. The method of claim 50, wherein the subject has been identified or diagnosed as having cancer.

52. The method of claim 50 or 51, further comprising:

obtaining the mammalian cell from the subject.

53. The method of any one of claims 44-52, wherein the contacting step is performed in vitro.

54. The method of any one of claims 44-52, wherein the contacting step is performed in a mammal.

55. The method of claim 54, wherein the mammal has cancer.

56. The method of claim 55, wherein said contacting step results in the treatment of said cancer in said mammal.

57. A multi-chain chimeric antigen receptor, wherein the multi-chain chimeric antigen receptor comprises at least one first polypeptide comprising:

an extracellular antigen-binding domain;

a transmembrane domain; and

a hormone receptor ligand binding domain;

wherein:

58. The multi-chain chimeric antigen receptor of claim 57, wherein the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain.

59. The multi-chain chimeric antigen receptor of claim 58 wherein the extracellular antigen-binding domain is an scFv.

60. The multi-chain chimeric antigen receptor of any one of claims 57-59, wherein the extracellular antigen-binding domain specifically binds to a single antigen.

61. The multi-chain chimeric antigen receptor of claim 60, wherein the single antigen is a tumor antigen.

62. The multi-chain chimeric antigen receptor of claim 61, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

63. The multi-chain chimeric antigen receptor of claim 62, wherein the tumor antigen is CD 19.

64. The multi-chain chimeric antigen receptor of claim 57 wherein the extracellular antigen-binding domain specifically binds to two different antigens.

65. The multi-chain chimeric antigen receptor of any one of claims 57-64, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

66. The multi-chain chimeric antigen receptor of any one of claims 57-65, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

67. The multi-chain chimeric antigen receptor of claim 66, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

68. The multi-chain chimeric antigen receptor of any one of claims 57-67, wherein the transmembrane domain and the hormone receptor ligand binding domain are directly contiguous with each other.

69. The multi-chain chimeric antigen receptor of any one of claims 57-67, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 500 amino acids.

70. The multi-chain chimeric antigen receptor of claim 69, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 250 amino acids.

71. The multi-chain chimeric antigen receptor of claim 70, wherein the transmembrane domain and the hormone receptor ligand binding domain are separated by 1 to 50 amino acids.

72. The multi-chain chimeric antigen receptor of any one of claims 57-71, wherein the at least one polypeptide further comprises a co-stimulatory domain.

73. The multi-chain chimeric antigen receptor of claim 72, wherein the co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C and B7-H3.

74. The multi-chain chimeric antigen receptor of claim 33, wherein the co-stimulatory domain is 4-1BB or CD 28.

75. A method of inducing membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, the method comprising:

contacting a mammalian cell expressing a multi-chain chimeric antigen receptor of any one of claims 57-74 with an amount of a hormone or hormone analog sufficient to induce localization of said multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of said cell.

76. A method of altering the membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing a multi-chain chimeric antigen receptor of any one of claims 57-74 with an amount of a hormone or hormone analog sufficient to induce localization of said multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of said cell; and

(b) contacting said mammalian cell with an amount of said hormone or said hormone analog which results in a decrease in the level of said multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of said cell.

77. The method of claim 76, further comprising:

(c) contacting said mammalian cell with an amount of said hormone or said hormone analog which results in an increase in the level of said multi-chain chimeric antigen-binding receptor on the extracellular side of the plasma membrane of said cell.

78. The method of any one of claims 75-77, further comprising:

79. The method of claim 78, wherein said mammalian cell is a T cell.

80. The method of claim 79, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

81. The method of any one of claims 75-80, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

82. The method of claim 81, wherein the subject has been identified or diagnosed as having cancer.

83. The method of claim 81 or 82, further comprising:

obtaining the mammalian cell from the subject.

84. The method of any one of claims 75-83, wherein the contacting step is performed in vitro.

85. The method of any one of claims 75-83, wherein the contacting step is performed in a mammal.

86. The method of claim 85, wherein the mammal has cancer.

87. The method of claim 86, wherein said contacting step results in treatment of said cancer in said mammal.

88. A method of treating cancer in a subject, comprising:

(a) administering to the subject a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 23 to 40 or the multi-chain chimeric antigen receptor of any one of claims 57 to 74, wherein the antigen binding domain specifically binds to an antigen expressed by a cancer in the subject;

(b) administering to the subject an amount of a hormone or hormone sufficient to induce the single-or multi-chain chimeric antigen receptor to localize to the extracellular side of the plasma membrane of the cell;

(c) determining the presence or severity of one or more symptoms associated with toxicity, the level of one or more cytokines associated with a cytokine storm, or both in the subject after (b);

(d) comparing the presence or severity of the determined one or more symptoms associated with toxicity, the determined level of one or more cytokines associated with a cytokine storm, or both, to a control; and

(e) administering to said subject an amount of said hormone or hormone analog sufficient to reduce the level of single-chain or multi-chain chimeric antigen receptors on the extracellular side of the plasma membrane of said cell such that the presence or severity of said one or more symptoms associated with toxicity, the level of said one or more cytokines associated with a cytokine storm, or both, is reduced in said subject following (e).

89. The method of claim 88, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 25% as compared to the amount of said hormone or hormone analog administered to said subject in step (b).

90. The method of claim 89, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 50% as compared to the amount of said hormone or hormone analog administered to said subject in step (b).

91. The method of claim 90, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 75% compared to the level of said hormone or hormone analog administered to said subject in step (b).

92. The method of claim 91, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by 100% compared to the level of said hormone or hormone analog administered to said subject in step (b).

93. The method of claim 88, wherein the one or more symptoms associated with toxicity are selected from the group consisting of: high fever, swelling, chills, hypotension, tachycardia, weakness, headache, rash, itching throat, dyspnea, redness, extreme fatigue, nausea and encephaledema.

94. The method of claim 88, wherein the one or more cytokines associated with a cytokine storm are selected from the group consisting of: including tumor necrosis factor alpha, IFN gamma, IL-10, IL-1 beta, IL-2, IL-6, IL-8 and IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5.

95. A single chain chimeric polypeptide, wherein said single chain chimeric polypeptide comprises:

an extracellular hormone receptor ligand binding domain; and

(ii) a transmembrane domain which is capable of,

wherein:

the hormone receptor ligand binding domain and the extracellular transmembrane domain are directly contiguous with each other or are separated by 1 to 700 amino acids; and

both the transmembrane domain and the extracellular hormone receptor ligand binding domain are absent from a single endogenous single chain polypeptide in a mammal.

96. The single chain chimeric polypeptide of claim 95, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

97. The single chain chimeric polypeptide of claim 95, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

98. The single chain chimeric polypeptide of claim 97, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

99. The single chain chimeric polypeptide of claim 98, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor.

100. The single chain chimeric polypeptide of claim 99, wherein the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it comprises one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

101. The single chain chimeric polypeptide of any one of claims 95-100, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other.

102. The single chain chimeric polypeptide of any one of claims 95-100, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids.

103. The single chain chimeric polypeptide of claim 102, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 500 amino acids.

104. The single chain chimeric polypeptide of claim 103, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 250 amino acids.

105. The single chain chimeric polypeptide of claim 104, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 100 amino acids.

106. The single chain chimeric polypeptide of claim 105, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 50 amino acids.

107. The single chain chimeric polypeptide of claim 106, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 25 amino acids.

108. The single chain chimeric polypeptide of any one of claims 95-100, wherein the single chain chimeric polypeptide comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain, and the transmembrane domain when moved in an N-terminal to C-terminal direction.

109. The single chain chimeric polypeptide of any one of claims 95-100, wherein the single chain chimeric polypeptide comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain, and the transmembrane domain when moved in the C-terminus to N-terminus direction.

110. The single chain chimeric polypeptide of any one of claims 95-100, wherein the single chain chimeric polypeptide comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain, and the transmembrane domain when moved in an N-terminal to C-terminal direction.

111. The single chain chimeric polypeptide of any one of claims 95-100, wherein the single chain chimeric polypeptide comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain, and the transmembrane domain when moved in a C-terminal to N-terminal direction.

112. A nucleic acid comprising a nucleotide sequence encoding the single-chain chimeric polypeptide of any one of claims 95-111.

113. A vector comprising the nucleic acid of claim 112.

114. A mammalian cell comprising the vector of claim 113.

115. The mammalian cell of claim 114, wherein the mammalian cell is a T cell.

116. The mammalian cell of claim 115, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

117. A method of inducing plasma membrane localization of a single chain chimeric polypeptide in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing the single chain chimeric polypeptide of any of claims 95-111 with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell.

118. A method of reversibly altering plasma membrane localization of a single-stranded chimeric polypeptide in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing the single chain chimeric polypeptide of any one of claims 95-111 with an amount of a hormone or hormone analog sufficient to induce the single chain chimeric polypeptide to localize to the extracellular side of the plasma membrane of the cell; and

119. The method of claim 118, further comprising:

120. The method of any one of claims 117-119, further comprising, prior to step (a):

121. The method of any one of claims 117-120, wherein the mammalian cell is a T cell.

122. The method of claim 121, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

123. The method of any one of claims 117-122, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

124. The method of claim 123, wherein the subject has been identified or diagnosed as having cancer.

125. The method of claim 123 or 124, further comprising:

obtaining the mammalian cell from the subject.

126. The method of any one of claims 117-125, wherein the contacting step is performed in vitro.

127. The method of any one of claims 117-125, wherein the contacting step is performed in a mammal.

128. A single-chain chimeric antigen receptor, wherein the single-chain chimeric antigen receptor comprises:

an extracellular antigen-binding domain;

an extracellular hormone receptor ligand binding domain;

a transmembrane domain;

an intracellular co-stimulatory domain; and

an intracellular immunoreceptor tyrosine-based activation motif (ITAM);

wherein:

the transmembrane domain and the extracellular hormone receptor ligand binding domain are directly contiguous with each other or are separated by 1 to 700 amino acids; and

129. The single-chain chimeric antigen receptor of claim 128, wherein the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain.

130. The single-chain chimeric antigen receptor of claim 129, wherein the extracellular antigen-binding domain is an scFv.

131. The single-chain chimeric antigen receptor of any one of claims 128-130, wherein the extracellular antigen-binding domain specifically binds to a single antigen.

132. The single-chain chimeric antigen receptor of claim 131, wherein the single antigen is a tumor antigen.

133. The single-chain chimeric antigen receptor of claim 132, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

134. The single-chain chimeric antigen receptor of claim 133, wherein the tumor antigen is CD 19.

135. The single-chain chimeric antigen receptor of claim 128, wherein the extracellular antigen-binding domain specifically binds to two different antigens.

136. The single-chain chimeric antigen receptor of claim 135, wherein at least one of the two different antigens is a tumor antigen.

137. The single-chain chimeric antigen receptor of claim 136, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

138. The single-chain chimeric antigen receptor of claim 137, wherein the tumor antigen is CD 19.

139. The single-chain chimeric antigen receptor of any one of claims 128-138, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

140. The single-chain chimeric antigen receptor of any one of claims 128-139, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

141. The single-chain chimeric antigen receptor of claim 140, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

142. The single-chain chimeric antigen receptor of claim 141, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor.

143. The single-chain chimeric antigen receptor of claim 142, wherein the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it comprises one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

144. The single-chain chimeric antigen receptor of any one of claims 128-143, wherein the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C and B7-H3.

145. The single-chain chimeric antigen receptor of claim 144, wherein the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from 4-1BB or CD 28.

146. The single-chain chimeric antigen receptor of any one of claims 128-145, wherein the intracellular ITAM comprises a cytoplasmic signaling sequence from CD3 ζ.

147. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other.

148. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids.

149. The single-chain chimeric antigen receptor of claim 148, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 500 amino acids.

150. The single-chain chimeric antigen receptor of claim 149, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 250 amino acids.

151. The single-chain chimeric antigen receptor of claim 150, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 100 amino acids.

152. The single-chain chimeric antigen receptor of claim 151, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 50 amino acids.

153. The single-chain chimeric antigen receptor of claim 152, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 25 amino acids.

154. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the single-chain chimeric antigen receptor comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain, the transmembrane domain, the intracellular costimulatory domain, and the ITAM when moved in the N-terminal to C-terminal direction.

155. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the single-chain chimeric antigen receptor comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain, the transmembrane domain, the intracellular costimulatory domain, and the ITAM when moved in the C-terminal to N-terminal direction.

156. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the single-chain chimeric antigen receptor comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain, the transmembrane domain, the intracellular costimulatory domain, and the ITAM when moved in the N-terminal to C-terminal direction.

157. The single-chain chimeric antigen receptor of any one of claims 128-146, wherein the single-chain chimeric polypeptide comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain, the transmembrane domain, the intracellular costimulatory domain, and the ITAM, when shifted in the C-terminus to N-terminus direction.

158. A nucleic acid comprising a nucleotide sequence encoding the single-chain chimeric antigen receptor of any one of claims 128-157.

159. A vector comprising the nucleic acid of claim 158.

160. A mammalian cell comprising the vector of claim 159.

161. The mammalian cell of claim 160, wherein the mammalian cell is a T cell.

162. The mammalian cell of claim 161, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

163. A method of inducing plasma membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 128-157 with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell.

164. A method of altering plasma membrane localization of a single-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 128-157 with an amount of a hormone or hormone analog sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell; and

165. The method of claim 164, further comprising:

166. The method of any one of claims 163-165, further comprising, prior to step (a):

167. The method of claim 166, wherein the mammalian cell is a T cell.

168. The method of claim 167, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

169. The method of any one of claims 163-168, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

170. The method of claim 169, wherein the subject has been identified or diagnosed as having cancer.

171. The method of claim 169 or 170, further comprising:

obtaining the mammalian cell from the subject.

172. The method of any one of claims 163-171, wherein the contacting step is performed in vitro.

173. The method of any one of claims 163-171 wherein the contacting step is performed in a mammal.

174. The method of claim 173, wherein the mammal has cancer.

175. The method of claim 174, wherein said contacting step results in treatment of said cancer in said mammal.

176. A multi-chain chimeric antigen receptor, wherein the multi-chain chimeric antigen receptor comprises at least one first polypeptide comprising:

an extracellular antigen-binding domain;

an extracellular hormone receptor ligand binding domain; and

a transmembrane domain;

wherein:

177. The multi-chain chimeric antigen receptor of claim 176, wherein the extracellular antigen-binding domain is selected from the group consisting of: scFv, (scFv)₂、V_HH domain and V_NARA domain.

178. The multi-chain chimeric antigen receptor of claim 177, wherein the extracellular antigen-binding domain is an scFv.

179. The multi-chain chimeric antigen receptor of any one of claims 176-178 wherein the extracellular antigen-binding domain specifically binds to a single antigen.

180. The multi-chain chimeric antigen receptor of claim 179, wherein the single antigen is a tumor antigen.

181. The multi-chain chimeric antigen receptor of claim 180, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

182. The multi-chain chimeric antigen receptor of claim 181, wherein the tumor antigen is CD 19.

183. The multi-chain chimeric antigen receptor of claim 179, wherein the extracellular antigen-binding domain specifically binds to two different antigens.

184. The multi-chain chimeric antigen receptor of claim 183, wherein at least one of said two different antigens is a tumor antigen.

185. The multi-chain chimeric antigen receptor of claim 184, wherein the tumor antigen is selected from the group consisting of: CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE and MUC 16.

186. The multi-chain chimeric antigen receptor of claim 185, wherein the tumor antigen is CD 19.

187. The multi-chain chimeric antigen receptor of any one of claims 176-186, wherein the transmembrane domain is a transmembrane domain from: an alpha chain of a T cell receptor, a beta chain of a T cell receptor, a zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD 154.

188. The multi-chain chimeric antigen receptor of any one of claims 176-187, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor, a progesterone receptor, or an androgen receptor.

189. The multi-chain chimeric antigen receptor of claim 188, wherein the hormone receptor ligand binding domain is a ligand binding domain from an estrogen receptor.

190. The multi-chain chimeric antigen receptor of claim 189, wherein the extracellular hormone receptor ligand binding domain is a ligand binding domain from a human estrogen receptor.

191. The multi-chain chimeric antigen receptor of claim 190, wherein the ligand binding domain of the human estrogen receptor has a wild-type sequence, except that it comprises one or both of the substitution at amino acid position 521 and the substitution at amino acid position 537, each numbered relative to the full-length wild-type sequence of the human hormone receptor.

192. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are directly contiguous with each other.

193. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 700 amino acids.

194. The multi-chain chimeric antigen receptor of claim 193, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 500 amino acids.

195. The multi-chain chimeric antigen receptor of claim 194, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 250 amino acids.

196. The multi-chain chimeric antigen receptor of claim 195, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 100 amino acids.

197. The multi-chain chimeric antigen receptor of claim 196, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 50 amino acids.

198. The multi-chain chimeric antigen receptor of claim 197, wherein the extracellular hormone receptor ligand binding domain and the transmembrane domain are separated by 1 to 25 amino acids.

199. The multi-chain chimeric antigen receptor of any one of claims 176-198 wherein the at least one polypeptide further comprises an intracellular co-stimulatory domain.

200. The multi-chain chimeric antigen receptor of claim 199, wherein the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from: CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C and B7-H3.

201. The multi-chain chimeric antigen receptor of claim 200, wherein the intracellular co-stimulatory domain is a cytoplasmic co-stimulatory domain from 4-1BB or CD 28.

202. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the at least one first polypeptide comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain and the transmembrane domain when moved in the N-terminal to C-terminal direction.

203. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the at least one first polypeptide comprises the extracellular antigen-binding domain, the extracellular hormone receptor ligand-binding domain and the transmembrane domain when moved in the C-terminal to N-terminal direction.

204. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the at least one first polypeptide comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain and the transmembrane domain when moved in an N-terminal to C-terminal direction.

205. The multi-chain chimeric antigen receptor of any one of claims 176-191, wherein the at least one first polypeptide comprises the extracellular hormone receptor ligand binding domain, the extracellular antigen binding domain and the transmembrane domain when moved in the C-terminal to N-terminal direction.

206. A nucleic acid encoding the multi-chain chimeric receptor of any one of claims 176-205.

207. A set of nucleic acids which together encode the multi-stranded chimeric receptor of any one of claims 176-205.

208. A mammalian cell comprising the nucleic acid of claim 206.

209. A mammalian cell comprising the set of nucleic acids of claim 207.

210. The mammalian cell of claim 208 or 209, wherein the mammalian cell is a T cell.

211. The mammalian cell of claim 210, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells and natural killerT cells.

212. A method of inducing plasma membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing a multi-chain chimeric antigen receptor of any one of claims 176-205 with an amount of a hormone or hormone analog sufficient to induce localization of said multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of said cell.

213. A method of altering plasma membrane localization of a multi-chain chimeric antigen receptor in a mammalian cell, the method comprising:

(a) contacting a mammalian cell expressing a multi-chain chimeric antigen receptor of any one of claims 176-205 with an amount of a hormone or hormone analog sufficient to induce localization of said multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of said cell; and

214. The method of claim 213, further comprising:

215. The method of any one of claims 212-214, further comprising, prior to step (a):

introducing a nucleic acid encoding the multi-chain chimeric antigen receptor, or a set of nucleic acids that together encode the multi-chain chimeric receptor, into a mammalian cell to generate a mammalian cell that expresses the multi-chain chimeric antigen receptor.

216. The method of claim 215, wherein the mammalian cell is a T cell.

217. The method of claim 216, wherein the T cell is selected from the group consisting of: CD8⁺T cell, CD4⁺T cells, memory T cells, Treg cells, and natural killer T cells.

218. The method of any one of claims 212-217, wherein the mammalian cell is a mammalian cell previously obtained from a subject.

219. The method of claim 218, wherein the subject has been identified or diagnosed as having cancer.

220. The method of claim 218 or 219, further comprising:

obtaining the mammalian cell from the subject.

221. The method of any one of claims 212-220, wherein the contacting step is performed in vitro.

222. The method of any one of claims 212-220, wherein the contacting step is performed in a mammal.

223. The method of claim 222, wherein the mammal has cancer.

224. The method of claim 223, wherein said contacting step results in treatment of said cancer in said mammal.

225. A method of treating cancer in a subject, comprising:

(a) administering to the subject a mammalian cell expressing the single-chain chimeric antigen receptor of any one of claims 128-157 or the multi-chain chimeric antigen receptor of any one of claims 176-205, wherein the antigen binding domain specifically binds to an antigen expressed by a cancer in the subject;

(d) comparing the presence or severity of the determined one or more symptoms associated with toxicity or the determined level of one or more cytokines associated with a cytokine storm, or both, to a control; and

(e) administering to said subject an amount of said hormone or hormone analog sufficient to reduce the level of single-chain or multi-chain chimeric antigen receptors on the extracellular side of the plasma membrane of said cell such that the presence or severity of said one or more symptoms associated with toxicity or the level of said one or more cytokines associated with a cytokine storm, or both, is reduced in said subject following (e).

226. The method of claim 225, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 25% as compared to the amount of said hormone or hormone analog administered to said subject in step (b).

227. The method of claim 226, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 50% as compared to the amount of said hormone or hormone analog administered to said subject in step (b).

228. The method of claim 227, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by at least 75% as compared to the level of said hormone or hormone analog administered to said subject in step (b).

229. The method of claim 228, wherein the amount of said hormone or hormone analog administered to said subject in step (e) is reduced by 100% as compared to the level of said hormone or hormone analog administered to said subject in step (b).

230. The method of claim 225, wherein the one or more symptoms associated with toxicity are selected from the group consisting of: high fever, swelling, chills, hypotension, tachycardia, weakness, headache, rash, itching throat, dyspnea, redness, extreme fatigue, nausea and encephaledema.

231. The method of claim 225, wherein the one or more cytokines associated with a cytokine storm are selected from the group consisting of: including tumor necrosis factor alpha, IFN gamma, IL-10, IL-1 beta, IL-2, IL-6, IL-8 and IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IL-5.