Chimeric polypeptides and methods for altering their localization in the cell membrane

Abstract

To provide a single-chain chimeric polypeptide, and a method for inducing the membrane localization of a single-chain chimeric polypeptide in a mammalian cell.SOLUTION: A single-chain chimeric polypeptide, as one aspect, comprises a transmembrane domain and a hormone receptor ligand binding domain. The transmembrane domain and the hormone receptor ligand binding domain directly abut on each other or are separated by 1 to 500 amino acids, and both of the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single-chain polypeptide in a mammal.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 62 / 477,733, filed March 28, 2017, and U.S. Provisional Patent Application No. 62 / 587,262, filed November 16, 2017, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to the field of biotechnology, and more particularly to modulating the cellular localization of chimeric polypeptides. [Background technology]

[0003] Chimeric antigen receptors (CARs) are single-chain and multi-chain polypeptides. Chimeric antigen receptors (CARs) are a subclass of peptides. Chimeric antigen receptors typically include, for example, an antigen-binding domain, a transmembrane domain, a costimulatory domain, and a CD3ζ or other signaling domain. CARs have been investigated for use in the clinical treatment of various cancers in mammals, including blood cancers, with considerable success. However, uncontrollable toxicity associated with CAR anticancer activity has been a limiting factor.

[0004] Nuclear hormone receptors are a group of polypeptides containing several domains, including DNA-binding motifs and ligand-binding domains. In the absence of ligand, native nuclear hormone receptors associate with various chaperones and other interacting proteins that capture the nuclear hormone receptor outside the nucleus. Ligand binding induces changes in the nuclear hormone receptor's conformation and interaction partners, allowing for nuclear translocation and transcriptional activation or repression of nuclear hormone target genes. The classical view of steroid signaling is that these hormones bind intracellular receptors that act as ligand-activated transcription factors; however, it has been reported that steroids can also act via non-genomic mechanisms at the cell surface. Summary of the Invention

[0005] The present disclosure is based, at least in part, on the discovery that reversible extracellular membrane localization of chimeric polypeptides comprising a transmembrane domain can be utilized in medical applications where it is desirable to reversibly express a protein or peptide of interest on the extracellular membrane. An exemplary feature of the present disclosure is that toxicity associated with CAR T cells can be modulated by reversibly expressing the antigen-binding domain of the CAR on the surface of the T cells.

[0006] The present disclosure is further based, at least in part, on the discovery that when the transmembrane domain and the hormone receptor ligand-binding domain are directly adjacent to each other or are 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., a single-chain chimeric antigen receptor) in a mammal, the plasma membrane localization or extracellular membrane localization of a chimeric polypeptide comprising the transmembrane domain and the hormone receptor ligand-binding domain can be modulated in a mammalian cell by contacting the mammalian cell with a hormone or hormone analog. In light of this discovery, provided herein are single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric antigen receptors; nucleic acids encoding them; vectors comprising any of these nucleic acids; mammalian cells comprising any of these vectors; and methods for reversibly inducing extracellular membrane localization and methods for altering the membrane localization of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric polypeptides.

[0007] In some embodiments, reversible induction of plasma membrane or extracellular membrane localization of polypeptides (e.g., single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and / or multi-chain chimeric antigen receptors) using one or more compositions or methods disclosed herein is useful in the area of ​​cell-based adoptive immunotherapy, in which immune cells isolated from a subject can be engineered to express synthetic proteins, enabling them to perform new therapeutic functions after subsequent return to the subject. Non-limiting examples of such synthetic proteins are chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs).

[0008] In some embodiments, reversibly inducing plasma membrane 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 for controlling the toxic effects of chimeric antigen receptor T cells (CAR-T cells), which are involved in the elimination of cancer cells by regulating the presence or absence of antigen-binding moieties on the plasma membrane or extracellular membrane of the cell.

[0009] 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 either directly adjacent to each other or separated by 1 to 500 amino acids, and wherein both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same 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 derived from a T cell receptor α chain, a T cell receptor β chain, a T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. In some embodiments of any of the single-chain chimeric polypeptides described herein, the hormone receptor ligand binding domain is a ligand binding domain derived 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 derived from an estrogen receptor.

[0010] 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 adjacent to 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 between 1 and 500 amino acids (e.g., between 1 and 250 amino acids, or between about 1 and 50 amino acids).

[0011] 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 any of the nucleic acids 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.

[0012] Also provided herein are methods for inducing membrane localization of a single-chain chimeric polypeptide in a mammalian cell, comprising contacting a mammalian cell expressing any of the single-chain chimeric polypeptides described herein with a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric polypeptide to the extracellular side of the plasma membrane of the cell.

[0013] Also provided are methods for reversibly altering membrane localization of a single-chain chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric polypeptides described herein with a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric polypeptide to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with a decreased amount of the hormone or hormone analog, thereby resulting in a decreased level of the single-chain chimeric polypeptide on the extracellular side 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 the hormone or hormone analog, thereby resulting in an increased level of the single-chain chimeric polypeptide on the extracellular side of the plasma membrane of the cell compared to the level in step (b).

[0014] Some embodiments of any of the methods described herein further include introducing into a mammalian cell a nucleic acid encoding the single-chain chimeric polypeptide to generate a mammalian cell that expresses the single-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the mammalian cell is a T cell (e.g., CD8 + T cells, CD4 +T cells selected from the group consisting of T cells, memory T cells, Treg cells, and natural killer T cells.

[0015] In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from a subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed with cancer. Some embodiments of any of the methods described herein further include obtaining the mammalian cell from the 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.

[0016] Also provided herein is a single-chain chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, a hormone receptor ligand-binding domain, a costimulatory domain, and an immunoreceptor tyrosine-based activation motif (ITAM), wherein the transmembrane domain and the hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to 500 amino acids, and wherein both the transmembrane domain and the hormone receptor ligand-binding domain are not present in the same 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), V, and the like. H H domain and V NAR In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

[0017] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD19. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds two different antigens.

[0018] In some embodiments of any of the single chain chimeric antigen receptors described herein, the transmembrane domain is selected from the group consisting of a T cell receptor alpha chain, a T cell receptor beta chain, a T cell receptor zeta chain, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD 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 derived from estrogen receptor, progesterone receptor, or 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 derived from estrogen receptor.

[0019] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the costimulatory domain is a cytoplasmic costimulatory domain derived 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 costimulatory domain is 4-1BB or CD28. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the ITAM comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0020] 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 adjacent to 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).

[0021] 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 nucleic acids 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.

[0022] Also provided is a method for 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 a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell.

[0023] Also provided herein are methods for 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 a hormone or hormone analog in an amount 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 the hormone or hormone analog in an amount 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 the mammalian cell with the hormone or hormone analog in an amount that results in an increase in the level of the single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell, compared to the level in (b).

[0024] Some embodiments of any of the methods described herein further comprise 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. 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 a CD8 + T cells, CD4 + The cells are selected from the group of T cells, memory T cells, Treg cells, and natural killer T cells.

[0025] In some embodiments of any of the methods described herein, the mammalian cell is a mammalian cell previously obtained from a subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed with cancer. Some embodiments of any of the methods described herein further include obtaining the mammalian cell from the 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 treatment of cancer in the mammal.

[0026] Also provided herein is a multi-chain chimeric antigen receptor 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 either directly adjacent to each other or are separated by 1 to 500 amino acids, and wherein both the transmembrane domain and the hormone receptor ligand-binding domain are not present in the same 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), V, and VI. H H domain and V NAR In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

[0027] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD19. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds two different antigens.

[0028] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain is derived from a T cell receptor alpha chain, a T cell receptor beta chain, a T cell receptor zeta chain, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. 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 derived 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 derived from an estrogen receptor.

[0029] 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 adjacent to 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).

[0030] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one polypeptide further comprises a costimulatory domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the costimulatory domain is a cytoplasmic costimulatory domain derived 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 costimulatory domain is 4-1BB or CD28.

[0031] Also provided herein are methods for 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 a hormone or hormone analog in an amount sufficient to induce localization of the multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell.

[0032] Also provided herein are methods for 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 a hormone or hormone analog in an amount sufficient to induce localization of the multi-chain chimeric antigen-binding receptor on the extracellular side of the cell's plasma membrane; and (b) contacting the mammalian cell with the hormone or hormone analog in an amount that results in a decrease in the level of the multi-chain chimeric antigen-binding receptor on the extracellular side of the cell's plasma membrane. Some embodiments of any of the methods described herein further comprise: (c) contacting the mammalian cell with the hormone or hormone analog in an amount that results in an increase in the level of the multi-chain chimeric antigen-binding receptor on the extracellular side of the cell's plasma membrane. Some embodiments of any of the methods described herein further comprise introducing a nucleic acid encoding the single-chain chimeric antigen receptor into the mammalian cell to generate a mammalian cell that expresses 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 cells are CD8 + T cells, CD4 + The cells are selected from the group of T cells, memory T cells, Treg cells, and natural killer T cells.

[0033] In some embodiments of any of the methods described herein, the mammalian cells are mammalian cells previously obtained from a subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed with cancer. In some embodiments of any of the methods described herein, further comprising obtaining the mammalian cells from the 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 treatment of cancer in the mammal.

[0034] Also provided herein are methods of administering to a subject: (a) administering to the subject mammalian cells 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 a hormone or hormones in an amount sufficient to induce localization of the single-chain or multi-chain chimeric antigen receptor to the extracellular side of the plasma membrane of the cell; (c) identifying in the subject after (b) the presence or severity of one or more symptoms associated with toxicity, the level of one or more cytokines associated with cytokine storm, or both; and (d) the presence or severity of the identified one or more symptoms associated with toxicity, the level of the identified one or more cytokines associated with cytokine storm. and (e) after (e), administering to the subject a hormone or hormone analog in an amount sufficient to decrease the level of a single-chain or multi-chain chimeric antigen receptor 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 cytokine storm, or both, is decreased in the subject.

[0035] In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 25% compared to the amount of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 50% compared to the amount of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 75% compared to the level of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by 100% compared to the level of hormone or hormone analog administered to the subject in step (b).

[0036] 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, low blood pressure, tachycardia, weakness, headache, rash, scratchy throat, difficulty breathing, redness, extreme fatigue, nausea, and cerebral edema. In some embodiments of these methods, the one or more cytokines associated with cytokine storm are selected from the group consisting of tumor necrosis factor alpha, IFNγ, IL-10, IL-1β, IL-2, IL-6, IL-8, and IL-10, granulocyte-macrophage-colony stimulating factor (GM-CSF), and IL-5.

[0037] Also provided herein are single-chain chimeric polypeptides 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 either directly adjacent to each other or are separated by 1 to 700 amino acids, and wherein both the transmembrane domain and the extracellular hormone receptor ligand-binding domain are not present in 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 derived from a T cell receptor α chain, a T cell receptor β chain, a T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. 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 derived 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 derived 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 derived 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 the wild-type sequence except for including one or both of a substitution at amino acid position 521 and a substitution at amino acid position 537, each numbered relative to the wild-type full-length sequence of the human hormone receptor.

[0038] 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 adjacent to 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).

[0039] In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide comprises, from N-terminus to C-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain. In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain.

[0040] Also provided herein is a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein is a vector comprising a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric polypeptides described herein. Also provided herein is a mammalian cell 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 a CD8 + T cells, CD4 + The cells are selected from the group of T cells, memory T cells, Treg cells, and natural killer T cells.

[0041] Also provided herein are methods for inducing plasma membrane localization of a single-chain chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric polypeptides described herein with a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric polypeptide to the extracellular side of the plasma membrane of the cell.

[0042] Also provided herein are methods for reversibly altering the plasma membrane localization of a single-chain chimeric polypeptide in a mammalian cell, comprising: (a) contacting a mammalian cell expressing any of the single-chain chimeric polypeptides described herein with a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric polypeptide to the extracellular side of the plasma membrane of the cell; and (b) contacting the mammalian cell with a decreased amount of the hormone or hormone analog, thereby resulting in a decrease in the level of the single-chain chimeric polypeptide on the extracellular side 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 the hormone or hormone analog, thereby resulting in an increase in the level of the single-chain chimeric polypeptide on the extracellular side of the plasma membrane of the cell 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 the single-chain chimeric polypeptide into the mammalian cell to produce a mammalian cell that expresses the single-chain chimeric polypeptide.

[0043] 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 CD8 + T cells, CD4 + The mammalian cells are selected from the group of T cells, memory T cells, Treg cells, and natural killer T cells. In some embodiments of any of the methods described herein, the mammalian cells are mammalian cells previously obtained from a 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 include obtaining the mammalian cells from the subject.

[0044] In some embodiments of any of the methods described herein, the contacting step(s) is / are performed in vitro. In some embodiments of any of the methods described herein, the contacting step(s) is / are performed in a mammal.

[0045] Also provided herein is a single-chain chimeric antigen receptor comprising an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an intracellular immunoreceptor tyrosine-based activation motif (ITAM), wherein the transmembrane domain and the extracellular hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to 700 amino acids, and wherein both the transmembrane domain and the extracellular hormone receptor ligand-binding domain are not present in 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), V, and V. H H domain and V NAR In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

[0046] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD19. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the tumor antigen is CD19.

[0047] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the transmembrane domain is a transmembrane domain derived from a T cell receptor α chain, a T cell receptor β chain, a T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. 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 derived 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 derived from an estrogen receptor. In some embodiments of any of the chimeric antigen receptors, the extracellular hormone receptor ligand-binding domain is a ligand-binding domain derived from the 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 the wild-type sequence except that it contains one or both of a substitution at amino acid position 521 and a substitution at amino acid position 537, each numbered relative to the wild-type full-length sequence of the human hormone receptor.

[0048] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the intracellular costimulatory domain is a cytoplasmic costimulatory domain derived 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 costimulatory domain is a cytoplasmic costimulatory domain derived from 4-1BB or CD28. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the intracellular ITAM comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0049] 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 adjacent to 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).

[0050] In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises, from N-terminus to C-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises, from C-terminus to N-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric antigen receptor comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments of any of the single-chain chimeric antigen receptors described herein, the single-chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM.

[0051] Also provided herein is a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided herein is a vector comprising a nucleic acid comprising a nucleotide sequence encoding any of the single-chain chimeric antigen receptors described herein. Also provided is a mammalian cell 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 a CD8 + T cells, CD4 + The cells are selected from the group of T cells, memory T cells, Treg cells, and natural killer T cells.

[0052] Also, (a) a method for inducing localization of a single-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of a mammalian cell expressing any of the single-chain chimeric antigen receptors described herein. A method for inducing plasma membrane localization of a single-chain chimeric antigen receptor in a mammalian cell is provided, comprising contacting the cell with a sufficient amount of a hormone or hormone analog.

[0053] Also provided herein are methods for altering the 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 a hormone or hormone analog in an amount 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 the hormone or hormone analog in an amount 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 the mammalian cell with the hormone or hormone analog in an amount that results in an increase in the level of the single-chain chimeric antigen receptor on the extracellular side of the plasma membrane of the cell, 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 the single-chain chimeric antigen receptor into the mammalian cell to produce a mammalian cell that expresses the single-chain chimeric antigen receptor.

[0054] In some embodiments of any of the methods described herein, the mammalian cells are T cells. In some embodiments of any of the methods described herein, the T cells are CD8 + T cells, CD4 +The mammalian cell is selected from the group of 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 a subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed with cancer. Some embodiments of any of the methods described herein further include obtaining the mammalian cell from the subject. In some embodiments of any of the methods described herein, the contacting step(s) is / are performed in vitro. In some embodiments of any of the methods described herein, the contacting step(s) is / are 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 treatment of cancer in the mammal.

[0055] Also provided herein is a multi-chain chimeric antigen receptor 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 either directly adjacent to each other or are separated by 1 to 700 amino acids, and wherein both the transmembrane domain and the extracellular hormone receptor ligand-binding domain are not present in 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), V, and the like. H H domain and V NAR In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain is an scFv.

[0056] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD19. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD19. In some embodiments, the extracellular antigen-binding domain specifically binds 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 of CD19, WT-1, CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, and MUC16. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the tumor antigen is CD19.

[0057] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the transmembrane domain is derived from a T cell receptor α chain, a T cell receptor β chain, a T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, or CD154. 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 derived 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 derived 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 derived 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 the wild-type sequence except that it contains one or both of a substitution at amino acid position 521 and a substitution at amino acid position 537, each numbered relative to the wild-type full-length sequence of the human hormone receptor.

[0058] 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 adjacent to 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).

[0059] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one polypeptide further comprises an intracellular costimulatory domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the intracellular costimulatory domain is a cytoplasmic costimulatory domain derived 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 costimulatory domain is a cytoplasmic costimulatory domain derived from 4-1BB or CD28.

[0060] In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises, from N-terminus to C-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, the at least one first polypeptide comprises, from C-terminus to N-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least At least one first polypeptide comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain. In some embodiments of any of the multi-chain chimeric antigen receptors described herein, at least one first polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain.

[0061] Also provided herein is a nucleic acid encoding any of the multi-chain chimeric antigen receptors described herein. Also provided herein is a set of nucleic acids that together encode any of the multi-chain chimeric antigen receptors described herein. Also provided herein is a mammalian cell comprising a nucleic acid encoding any of the multi-chain chimeric antigen receptors described herein. Also provided herein is a mammalian cell comprising a set of nucleic acids that together encode any of the multi-chain chimeric antigen receptors 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 a CD8 + T cells, CD4 + The cells are selected from the group consisting of T cells, memory T cells, Treg cells, and natural killer T cells.

[0062] Also provided herein are methods for 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 a hormone or hormone analog in an amount sufficient to induce localization of the multi-chain chimeric antigen-binding receptor to the extracellular side of the plasma membrane of the cell.

[0063] Also provided herein are methods for altering the 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 a hormone or hormone analog in an amount 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 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 the mammalian cell with an amount of hormone or hormone analog that results in an increase 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, 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 the mammalian cell to produce a mammalian cell that expresses the multi-chain chimeric antigen receptor. In some embodiments of any of the methods described herein, the mammalian cells are T cells. In some embodiments of any of the methods described herein, the T cells are CD8 + T cells, CD4 +The mammalian cell is selected from the group of 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 a subject. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed with cancer. Some embodiments of any of the methods described herein further include obtaining the mammalian cell from the 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 treatment of cancer in the mammal.

[0064] Also provided herein are methods for producing a mammalian cell that expresses any of the single-chain chimeric antigen receptors described herein or any of the multi-chain chimeric antigen receptors described herein, (b) administering to the subject a hormone or hormone analog in an amount sufficient to induce localization of a single- or multi-chain chimeric antigen receptor to the extracellular side of the plasma membrane of the cell; (c) after (b), identifying in the subject the presence or severity of one or more symptoms associated with toxicity, or the levels of one or more cytokines associated with cytokine storm, or both; (d) comparing the presence or severity of the identified one or more symptoms associated with toxicity, or the levels of one or more cytokines associated with cytokine storm, or both, with control(s); and (e) after (e), administering to the subject a hormone or hormone analog in an amount sufficient to reduce the level of the single- or multi-chain chimeric antigen receptor 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 levels of one or more cytokines associated with cytokine storm, or both, is reduced in the subject.

[0065] In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 25% compared to the amount of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 50% compared to the amount of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by at least 75% compared to the level of hormone or hormone analog administered to the subject in step (b). In some embodiments of any of the methods described herein, the amount of hormone or hormone analog administered to the subject in step (e) is reduced by 100% compared to the level of hormone or hormone analog administered to the subject in step (b).

[0066] 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, low blood pressure, tachycardia, weakness, headache, rash, scratchy throat, difficulty breathing, redness, extreme fatigue, nausea, and cerebral edema. In some embodiments of any of the methods described herein, the one or more cytokines associated with cytokine storm are selected from the group consisting of tumor necrosis factor alpha, IFNγ, IL-10, IL-1β, IL-2, IL-6, IL-8, and IL-10, granulocyte-macrophage-colony stimulating factor (GM-CSF), and IL-5.

[0067] The use of the term "a" before a noun means "one or more" of that particular noun. Examples: For example, the phrase "a mammalian cell" means "one or more mammalian cells."

[0068] The term "single-chain chimeric polypeptide" refers to 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 meant to limit in any way the relative arrangement of the first contiguous amino acid sequence and the second contiguous sequence in the single-chain polypeptide. Non-limiting examples of single-chain chimeric polypeptides (e.g., single-chain chimeric antigen receptors) are described herein. Further examples of single-chain chimeric polypeptides (e.g., single-chain chimeric antigen receptors) are described in the art. It is known in the field.

[0069] The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein and generally, but not exclusively, refer to an artificial, multi-modular molecule capable of inducing or inhibiting immune cell activation, comprising an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. CAR molecules and their derivatives (e.g., CAR variants) are described, for example, in International Application No. PCT / US2014 / 016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172. ;Glienke et al. Front Pharmacol (2015) 6:21;Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5;Riddell et al. Cancer J (2014) 20(2):141-4;Pegram et al. 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; These disclosures are incorporated herein by reference in their entirety.

[0070] 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.

[0071] The term "transmembrane domain" refers to a domain of a polypeptide that comprises at least one contiguous amino acid sequence that spans a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. For example, a transmembrane domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid sequences that each span the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. As known in the art, a transmembrane domain may comprise, for example, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) contiguous amino acid sequence that has an α-helical secondary structure in the lipid bilayer (that spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). In some embodiments, a transmembrane domain may comprise two or more consecutive amino acid sequences that form a β-barrel secondary structure in a lipid bilayer (each spanning the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). Non-limiting examples of transmembrane domains are described herein. Further examples of transmembrane domains are known in the art.

[0072] The phrase "extracellular side of the plasma membrane," when used to describe the location of a polypeptide, means that the polypeptide contains at least one transmembrane domain that crosses the plasma membrane and at least one domain (e.g., at least one antigen-binding domain) that is located in the extracellular space.

[0073] The term "hormone receptor ligand binding domain" refers to 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 estrogen receptor, ecdysone receptor, PPARγ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR receptor α, PPARβ / δ receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, retinoid X receptor, RAR-related orphan receptor, and 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. When 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, the two or more LBDs are in tandem, either directly or separated by a linker (e.g., any of the linkers described herein).

[0074] The term "antigen-binding domain" refers to a domain that specifically binds to a target antigen. In some instances, an antigen-binding domain can be formed from amino acids present in a single-chain polypeptide. In other instances, an antigen-binding domain can be formed from amino acids present in a first single-chain polypeptide and amino acids present in 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, an scFv or an LBD of a growth factor. Further examples of antigen-binding domains are known in the art.

[0075] As used herein, the term "antigen" generally refers to the binding partner specifically recognized by the antigen-binding domains described herein. Exemplary antigens include various 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 antigens that can be specifically bound by either antigens or antigen-binding domains are described herein. Additional examples of antigens that can be specifically bound by either antigens or antigen-binding domains are known in the art.

[0076] The term "costimulatory domain" refers to a signaling domain derived from an endogenous costimulatory transmembrane polypeptide expressed in T lymphocytes that promotes downstream T cell receptor signaling and / or T cell activation. Non-limiting examples of costimulatory domains are described herein. Additional examples of costimulatory domains are known in the art. See, e.g., Chen et al., Nature Reviews Immunol. 13:227-242, 2013.

[0077] The term "immunoreceptor tyrosine-based activation motif" (ITAM) refers to an amino acid motif that contains a four amino acid consensus sequence (YxxL / I) in which a tyrosine is separated from a leucine or isoleucine by two other amino acids. The tyrosine residue in the four amino acid consensus sequence becomes phosphorylated after interaction with a signal transduction pathway kinase (e.g., lymphocyte signaling pathway kinase). Non-limiting examples of ITAMs are described herein. Additional examples of ITAMs are known in the art.

[0078] The term "hormone" refers to 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.

[0079] The term "hormone analog" refers to 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.

[0080] The phrase "treating cancer" refers to a reduction in the number, frequency, or severity of one or more (e.g., two, three, four, or five) symptoms of cancer in a subject with cancer. Non-limiting symptoms of cancer are described herein. Additional symptoms of cancer are known in the art.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are 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.

[0082] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0083] [Figure 1] Diagram of the various constructs tested. The extracellular and intracellular labels refer to the cell membrane, which is represented as a gray box between these labels. The extracellular scFv domain and the intracellular costimulatory domain ("Costim"), CD3 zeta domain, and mCherry domain are shown. [Figure 2] A set of flow cytometry dot plots for cells expressing one of the various constructs. Cells were left untreated or treated with 500 ng / mL 4-hydroxytamoxifen ("4-OHT"). (A) Jurkat T cells transduced with a lentiviral vector encoding the canonical CAR or with v2, v3, and v4 CAR constructs containing the anti-CD19 (FMC63) ERa-LBD. (B) Primary CD4 and CD8 human T cells were transduced with a lentiviral vector encoding the v4 CAR construct. [Figure 3]

[0023] Figure 1 shows a set of flow cytometry dot plots of HEK-293T cells transduced with a lentiviral vector encoding an ERa-LBD-containing FMC63 CAR fusion. Cells were left untreated or treated with 500 ng / mL of 4-OHT. [Figure 4]

[0033] Figure 1 is a set of flow cytometry dot plots of primary human CD4 T cells transduced with lentiviral vectors encoding the FMC63 NHR LBD-CAR fusion and the NHR LBD-CAR with the J6M0 scFv. Cells were left untreated or treated with 500 ng / mL of 4-OHT. [Figure 5A]

[0023] Figure 1 shows a diagram of the V5 nuclear hormone receptor ligand binding domain NHR-LBD CAR construct lacking the costimulatory domain and CD3 zeta domain, and a set of flow cytometry dot plots for cells transduced with a lentiviral vector encoding the V5 construct. Cells were left untreated or treated with 500 ng / mL of 4-OHT. [Figure 5B] Figure 1 shows a set of flow cytometry images of untransduced Jurkat T cells or Jurkat T cells transduced with a lentiviral vector encoding a CAR in which the scFv domain has been replaced with a FLAG tag. Transduced cells were left untreated or treated with 500 ng / mL of 4-OHT. [Figure 6] A set of flow cytometry images of Jurkat T cells transduced with a CAR construct bearing the progesterone receptor LBD. Cells were left untreated or treated with 500 ng / mL mifepristone. [Figure 7A] Graph showing T cell activation of primary human T cells transduced with lentiviral vector encoding the J6M0-ERa-LBD CAR construct after overnight co-culture with antigen-negative targets (K562 cell line) and antigen-positive targets (MM1S cell line). [Figure 7B]Graph showing IFN-γ production by primary human T cells transduced with a lentiviral vector encoding the J6M0-ERa-LBD CAR construct after overnight co-culture with antigen-negative targets (K562 cell line) and antigen-positive targets (MM1S cell line). [Figure 8A] FIG. 10 is a set of flow cytometry images showing the proliferation of primary human CD4 T cells transduced with lentiviral vectors encoding the V4 FMC63-ERa-LBD CAR or a canonical CAR control and co-cultured with either antigen-negative targets (K562 cell line) or antigen-positive targets (Raji cell line) with increasing concentrations of the ligand 4-OHT. [Figure 8B] Graph showing lysis of antigen-positive targets (MM1S cell line) mediated by primary human T cells transduced with a lentiviral vector encoding the J6M0-ERa-LBD CAR construct with increasing concentrations of 4-OHT. [Figure 9] Graph showing surface expression of various CAR constructs with LBDs at different locations relative to the cell membrane at increasing concentrations of 4-OHT. [Figure 10A] FIG. 1 is a schematic diagram showing the timeline of the experimental procedure. [Figure 10B] 1 is a bar graph showing the surface expression of various CAR constructs before and after co-culture with 4-OHT. [Figure 11A] FIG. 1 is a set of flow cytometry images showing Jurkat T cells transduced with FLAG-ERa-LBD CAR and left untreated or treated with 4-OHT for 15 or 120 minutes. [Figure 11B] FIG. 1 is a set of flow cytometry images showing human T cells transduced with a lentiviral vector encoding the FMC63-ERa-LBD CAR and left untreated or treated with 4-OHT for 2, 4, or 24 hours. [Figure 12]A set of flow cytometry images of primary human T cells expressing FMC63-CAR with ERa LBD. Cells were left untreated or treated with 500 ng / mL 4-OHT. Some samples were pretreated with 1 μM Brefeldin A. [Figure 13] Schematic of the v2 CAR and ECD.v1 CAR (also referred to as LBD-ECD CAR) constructs tested. The cell membrane is represented as a gray rectangle. The extracellular scFv domain, extracellular ERa LBD, transmembrane domain (embedded in the cell membrane), intracellular 4-1BB domain, and intracellular CD3γ domain are shown. [Figure 14] Figure 1 shows a set of flow cytometry dot plots for cells expressing the v2 CAR construct or the LBD-ECD CAR construct. Cells were left untreated or treated with 500 ng / mL of 4-hydroxytamoxifen ("4-OHT"). [Figure 15] Figure 1 shows the destruction (% lysis) of Nalm 6 tumor cells by T cells transduced with a lentiviral vector encoding the LBD-ECD CAR construct (ECD.v1). Cells were treated with 1.5 ng / mL to 500 ng / mL of 4-OHT. The positive control was T cells transduced with a lentiviral vector encoding a constitutive CAR. The negative control was T cells bearing the LBD-ECD CAR that were not treated with 4-OHT. [Figure 16] Figure 1 shows a set of flow cytometry dot plots for cells expressing a constitutive CAR, an LBD-ECD CAR, or a v2 CAR. Cells were left untreated or stimulated with CD3 / CD28-containing beads for 48 hours prior to flow cytometry. Dynabeads aCD3 / aCD28 beads were from ThermoFisher. See Trickett et al., J. Immunol. Methods 275:251-255, 2003. Each construct was fused to a cMyc tag. [Figure 17]Graph showing IL-2 production from ECD.v1 CAR-T cells or conventional CAR-T cells after overnight co-culture of T cells bearing CARs with NALM-6 targets in the presence (500 ng / mL) or absence of 4-hydroxytamoxifen. DETAILED DESCRIPTION OF THE INVENTION

[0084] Provided herein are single-chain chimeric polypeptides, single-chain chimeric antigen receptors (CARs), and multi-chain chimeric antigen receptors; nucleic acids encoding them; vectors comprising any of these nucleic acids; mammalian cells comprising any of these vectors; and methods for reversibly inducing and altering the extracellular membrane localization of these single-chain chimeric polypeptides, single-chain chimeric antigen receptors, and multi-chain chimeric polypeptides.

[0085] Also provided herein are genetically modified mammalian cells having a chimeric antigen receptor (CAR). In some cases, the genetically modified mammalian cells are derived from stem cells, progenitor cells, or cells derived from stem or progenitor cells. In some cases, the mammalian cells are T lymphocytes or NK cells.

[0086] 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. Further 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.

[0087] 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 the LBD present in the polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, and / or multi-chain chimeric antigen receptor). In some embodiments, in the presence of a hormone or hormone analog, the polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, and / or multi-chain chimeric antigen receptor) localizes to the extracellular surface, displaying an extracellular antigen-binding domain (e.g., an scFv or other antigen-binding domain described herein or known in the art). The polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor) can recognize the antigen to which it binds, and thus selectively kill target cells (e.g., cancer cells) that express the antigen on their surface.

[0088] In some embodiments, localization of the polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor) to the extracellular surface may cause toxicity in a subject. In some embodiments, a subject experiencing toxicity due to hormone- or hormone analog-induced localization of the polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor) to the extracellular surface can be treated or ameliorated by reducing the dose of hormone or hormone analog or by eliminating the subject's hormone or hormone analog treatment altogether. Such reduction in the dose or elimination of hormone or hormone analog treatment may reduce or eliminate toxicity in the subject by removing the polypeptide (e.g., single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric polypeptide) from the cell surface and reducing or eliminating the presentation of the antigen-binding domain on the cell surface.

[0089] Single-chain chimeric polypeptides Single-chain chimeric polypeptide-intracellular hormone receptor LBD As used herein, a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art) and a hormone receptor ligand binding domain (e.g., any of the hormone receptor ligand binding domains described herein or known in the art) are used. and a hormone receptor ligand binding domain (either a transmembrane domain or a hormone receptor ligand binding domain), wherein the transmembrane domain and the hormone receptor ligand binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., or any of the subranges of this range described herein), and wherein 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).

[0090] 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 can be from 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, or 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 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 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 4 50 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 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, about 24 0 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 11 5 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 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; amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 5 amino acids, or about 4 amino acids (including both ends); 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 48 0 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 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 an amino acid, 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, 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 4 40 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 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 2 20 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 1 00 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, 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 290 amino acids, about 280 amino acids, about 270 amino acids, about 2; 60 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 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 39 0 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 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 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 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 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, 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 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 (including both ends); 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 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 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 12 5 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 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 4 50 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 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 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 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 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 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 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids 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 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 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, 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, 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 5; 60 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 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 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 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 7 60 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 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 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 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 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 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 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, 60 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 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 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 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 no 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 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 1; 95 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 60 amino acids, about 55 amino acids, or about 50 amino acids (including both ends); 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 4 30 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 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 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 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 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 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; 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 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 17 5 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 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 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 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 72 0 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 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 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 (including both ends); 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, 520 amino acids, approximately 500 amino acids, approximately 490 amino acids, approximately 480 amino acids, approximately 470 amino acids, approximately 460 amino acids, approximately 450 amino acids, approximately 440 amino acids, approximately 430 amino acids, approximately 420 amino acids, approximately 410 amino acids, approximately 400 amino acids, approximately 390 amino acids, approximately 380 amino acids, approximately 370 amino acids, approximately 360 amino acids, approximately 350 amino acids, approximately 340 amino acids, approximately 330 amino acids, approximately 320 amino acids, approximately 310 amino acids, approximately 300 amino acids, approximately 290 amino acids, approximately 280 amino acids, approximately 270 amino acids, approximately 260 amino acids, approximately 250 amino acids, approximately 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 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 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 (including both ends); 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 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 2 20 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 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 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 Acid, 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 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 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 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 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, approximately 580 amino acids, approximately 560 amino acids, approximately 540 amino acids, approximately 520 amino acids, approximately 500 amino acids, approximately 490 amino acids, approximately 480 amino acids, approximately 470 amino acids, approximately 460 amino acids, approximately 450 amino acids, approximately 440 amino acids, approximately 430 amino acids, approximately 420 amino acids, approximately 410 amino acids, approximately 400 amino acids, approximately 390 amino acids, approximately 380 amino acids, approximately 370 amino acids, approximately 360 amino acids, approximately 350 amino acids, approximately 340 amino acids, approximately 330 amino acids, approximately 320 amino acids, approximately 310 amino acids, approximately 300 amino acids amino acids, 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 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 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 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, 55 amino acids, about 150 amino acids, about 145 amino acids, or about 140 amino acids (including both ends); 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 acid, 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 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 18; 0 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 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 17 0 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 5 20 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 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 45 0 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 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; 0 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 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, 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 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 4 00 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 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 (both ends) 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 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 290 amino acids; 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 (including both ends); 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 4 80 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 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 4 20 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 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 (both ends) 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 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 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 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 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 2 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 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 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 290 amino acids, about 280 amino acids, about 270 amino acids, about 260 amino acids, about 250 amino acids, or about 240 amino acids (both ends) 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 4 50 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 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 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; 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 290 amino acids, about 280 amino acids, or about 270 amino acids (inclusive); about 270 amino acids to about 80 0 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 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 (both ends) 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);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 (including both ends); 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 54 0 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 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 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 (including both ends); 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 62 0 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 (including both ends); 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, approximately 480 amino acids, approximately 470 amino acids, approximately 460 amino acids, approximately 450 amino acids, approximately 440 amino acids, approximately 430 amino acids, approximately 420 amino acids, approximately 410 amino acids, approximately 400 amino acids, approximately 390 amino acids, approximately 380 amino acids, approximately 370 amino acids (including both ends); approximately 370 amino acids to approximately 800 amino acids, approximately 780 amino acids, approximately 760 amino acids, approximately 740 amino acids, approximately 720 amino acids, approximately 700 amino acids, approximately 680 amino acids, approximately 660 amino acids, approximately 640 amino acids, approximately 620 amino acids 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 (including both ends); 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 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 (including both ends); 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 6 60 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 (including both ends); 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); 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); about 700 amino acids to about 800 amino acids, about 780 amino acids, about The amino acids are separated by 760 amino acids, about 740 amino acids, or about 720 amino acids (inclusive); about 720 amino acids to about 800 amino acids, about 780 amino acids, about 760 amino acids, or about 740 amino acids (inclusive); about 740 amino acids to about 800 amino acids, about 780 amino acids, or about 760 amino acids (inclusive); about 760 amino acids to about 800 amino acids, or about 780 amino acids (inclusive); or about 780 amino acids to about 800 amino acids (inclusive).

[0091] In some embodiments, one or more amino acids intervening 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 include 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 GSGSGSGS (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 may include one or more (e.g., two, three, or four) copies of any one of SEQ ID NOs:20-23, e.g., in tandem.

[0092] In some embodiments, one or more amino acids intervening between the transmembrane domain and the hormone receptor ligand-binding domain may be or include an intracellular sequence that abuts the 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 amino acids intervening 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 may 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 an extracellular ligand-binding domain (e.g., an antigen-binding domain, e.g., any of the antigen-binding domains described herein), an ITIM (e.g., any of the ITIMs known in the art), an ITAM (e.g., any of the ITAMs described herein or known in the art), a dimerization domain (e.g., any of the dimerization domains known in the art that can dimerize with another dimerization domain present in another polypeptide (e.g., a recombinant polypeptide) expressed in a mammalian cell), and a peptide tag (e.g., a poly-His tag).

[0093] Single-chain chimeric polypeptide - extracellular hormone receptor LBD Provided herein is a single-chain chimeric polypeptide comprising an extracellular hormone receptor ligand binding domain (e.g., any of the hormone receptor ligand binding domains described herein or known in the art) and a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art), wherein the transmembrane domain and the extracellular hormone receptor ligand binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., or any of the subranges within this range described herein), and wherein both the transmembrane domain and the hormone receptor ligand binding domain are not present in a single endogenous single-chain polypeptide in a mammal (e.g., a human).

[0094] 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 1 to about 800 amino acids (e.g., any of the subranges within this range described herein).

[0095] In some embodiments, one or more amino acids intervening 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 include 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 GSGSGSGS (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 may include one or more (e.g., two, three, or four) copies of any one of SEQ ID NOs: 20-23, e.g., in tandem. In some embodiments, the linker peptide may be or include one or more of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 106. In some embodiments, the linker peptide may be or include GS.

[0096] In some embodiments, one or more amino acids intervening between the transmembrane domain and the extracellular hormone receptor ligand-binding domain may be or include a sequence that abuts the 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 amino acids intervening 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 may 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 the single-chain chimeric polypeptide include an extracellular ligand-binding domain (e.g., an antigen-binding domain, e.g., any of the antigen-binding domains described herein), an ITIM (e.g., any of the ITIMs known in the art), an ITAM (e.g., any of the ITAMs described herein or known in the art), a dimerization domain (e.g., a dimerization domain present in another polypeptide (e.g., a recombinant polypeptide) expressed in a mammalian cell), a nucleotide sequence encoding ... The dimerization domains may include one or more of: a dimerization domain capable of dimerizing with another dimerization domain (e.g., any dimerization domain known in the art); and a peptide tag (e.g., a poly-His tag).

[0097] In some embodiments, the single-chain chimeric polypeptide comprises, from N-terminus to C-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments, the single-chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular antigen binding domain, an extracellular hormone receptor ligand binding domain, and a transmembrane domain. In some embodiments, the single-chain chimeric polypeptide comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain. In some embodiments, the single-chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand binding domain, an extracellular antigen binding domain, and a transmembrane domain.

[0098] Single-chain chimeric antigen receptor Single-chain chimeric antigen receptor-intracellular hormone receptor LBD Also provided herein is a single-chain chimeric antigen receptor comprising an extracellular antigen-binding domain (e.g., any of the antigen-binding domains described herein or known in the art), a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art), a hormone receptor ligand-binding domain (e.g., any of the hormone receptor ligand-binding domains described herein or known in the art), a costimulatory domain (e.g., any of the costimulatory domains described herein or known in the art), and an immunoreceptor tyrosine-based activation motif (ITAM), wherein the transmembrane domain and the hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., any of the subranges within this range described herein), and wherein 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. The single-chain chimeric antigen receptor 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, the single-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of the exemplary antigens described herein).

[0099] In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain is adjacent to 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 between 1 and about 800 amino acids (e.g., or any of the subranges within 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 comprise 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 a sequence derived 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.

[0100] 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 comprise one or both of the 4-1BB and CD28 costimulatory domains.

[0101] Some embodiments of these single-chain chimeric antigen receptors may comprise one or more (e.g., two, three, four, or five) ITAMs (e.g., any of the ITAMs described herein or known in the art). In some embodiments of these single-chain chimeric antigen receptors, the ITAMs comprise a cytoplasmic signaling sequence derived from CD3ζ (e.g., human CD3ζ).

[0102] In some embodiments of any of these single-chain chimeric antigen receptors, any two adjacent domains may be between 1 amino acid and 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 17 ... 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, 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 (including both ends); 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 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, 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, 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 45 amino acids, about 40 amino acids, about 3; 5 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 (including both ends); 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 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, 15 amino acids, about 10 amino acids, about 8 amino acids, or about 7 amino acids (including both ends); 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 16 0 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, 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 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 (both ends 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 15 0 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, 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 45 amino acids, about 40 amino acids, about 35; amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids (including both ends); 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 19 0 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 3 0 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 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 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 45 amino acids, about 40 amino acids (inclusive); 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 13 0 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, or 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 amino acids, about 60 amino acids, about 55 amino acids, or about 50 amino acids (including both ends); 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 (including both ends); 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 (including both ends); 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 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; 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 (including both ends); 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, or 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); about 100 amino acids to about 500 amino acids, about 480 amino acids 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 ( 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 1 20 amino acids (inclusive); 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 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); 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); 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 19 0 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); 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 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); 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);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); 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); 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); 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); 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).

[0103] In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain is a sequence derived from the same endogenous single-chain polypeptide from which the transmembrane domain is derived (e.g., the CD8α hinge region, e.g., SEQ ID NO: 112). In some embodiments, a sequence comprising SEQ ID NO: 42, SEQ ID NO: 55, or SEQ ID NO: 112 is located between the extracellular antigen-binding domain and the transmembrane domain. In some embodiments, one or more amino acids between the extracellular antigen-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 IgG4). In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain is or includes a linker sequence (e.g., a non-naturally occurring linker sequence, such as GS, or any of the other linker sequences described herein).

[0104] In some embodiments of the single-chain chimeric antigen receptor, after the hormone receptor ligand-binding domain, the next domain is a costimulatory domain followed by an ITAM. In some of these embodiments, one or more amino acids between the hormone receptor ligand-binding domain and the costimulatory domain are or include a sequence derived from the same endogenous single-chain polypeptide from which the costimulatory domain or ITAM is derived. In some of these embodiments, one or more amino acids between the hormone receptor ligand-binding domain and the costimulatory 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 costimulatory domain and the ITAM are or include a sequence derived from the same endogenous single-chain polypeptide from which the costimulatory domain or ITAM is derived.

[0105] In some embodiments of the single-chain chimeric antigen receptor, after the hormone receptor ligand-binding domain, the next domain is a costimulatory domain followed by an ITAM. In some embodiments of the single-chain chimeric antigen receptor, after the ligand-binding domain, the next domain is an ITAM followed by a costimulatory domain. In some of these embodiments, one or more amino acids between the hormone receptor ligand-binding domain and the ITAM are or include a sequence derived from the same endogenous single-chain polypeptide from which the costimulatory domain or ITAM is derived. In some of these embodiments, one or more amino acids between the hormone receptor ligand-binding domain and the ITAM are or include a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the ITAM and the costimulatory domain are or include a sequence derived from the same endogenous single-chain polypeptide from which the ITAM or costimulatory domain is derived.

[0106] In some embodiments, when two or more costimulatory domains are included in the single-chain chimeric antigen receptor, the two or more costimulatory domains may be located 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 costimulatory domains and ITAMs are located within the primary amino acid sequence of the single-chain chimeric antigen receptor. They may alternate in rows.

[0107] Some embodiments of any of the single-chain chimeric antigen receptors described herein may further comprise a dimerization domain and / or a peptide tag.

[0108] In some examples, the single-chain chimeric antigen receptor 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:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:52.

[0109] Single-chain chimeric antigen receptor-extracellular hormone receptor LBD Also provided herein is a single-chain chimeric antigen receptor comprising an extracellular antigen-binding domain (e.g., any of the antigen-binding domains described herein or known in the art), an extracellular hormone receptor ligand-binding domain (e.g., any of the hormone receptor ligand-binding domains described herein or known in the art), a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art), a costimulatory domain (e.g., any of the costimulatory domains described herein or known in the art), and an immunoreceptor tyrosine-based activation motif (ITAM), wherein the transmembrane domain and the extracellular hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., any of the subranges within 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 a mammal. The single-chain chimeric antigen receptor 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, the single-chain chimeric antigen receptor specifically binds to two different antigens (e.g., any combination of the exemplary antigens described herein).

[0110] In some embodiments of these single-chain chimeric antigen receptors, the transmembrane domain is adjacent to 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 between 1 and about 800 amino acids (e.g., or any of the subranges within 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 comprise one or more intervening domains (e.g., an antigen-binding domain and / or additional antigen-binding domains). In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand-binding domain comprise a sequence derived 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.

[0111] Some embodiments of these single-chain chimeric antigen receptors may comprise 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 comprise one or both of a 4-1BB costimulatory domain and a CD28 costimulatory domain.

[0112] Some embodiments of these single-chain chimeric antigen receptors comprise one or more (e.g., two, three, In some embodiments of these single-chain chimeric antigen receptors, the ITAM comprises a cytoplasmic signaling sequence derived from CD3ζ (e.g., human CD3ζ).

[0113] In some embodiments of any of these single-chain chimeric antigen receptors, any two adjacent domains may be separated by between 1 amino acid and about 500 amino acids (e.g., or any of the subranges within this range described herein).

[0114] In some embodiments, one or more amino acids between the extracellular hormone receptor ligand binding domain or extracellular antigen binding domain and the transmembrane domain is a sequence derived from the same endogenous single-chain polypeptide from which the transmembrane domain is derived (e.g., a hinge sequence derived from human CD8α, e.g., SEQ ID NO: 112). In some embodiments, a sequence comprising SEQ ID NO: 42, SEQ ID NO: 55, or SEQ ID NO: 112 is disposed between the extracellular hormone receptor ligand binding domain or extracellular antigen binding domain and the transmembrane domain. In some embodiments, one or more amino acids between the extracellular antigen binding domain or 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 IgG4). 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 includes 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 includes a linker sequence (e.g., a non-naturally occurring linker sequence, such as GS, or any of the other linker sequences described herein).

[0115] In some embodiments of the single-chain chimeric antigen receptor, after the transmembrane domain, the next domain is a costimulatory domain, followed by an ITAM. In some embodiments of the single-chain chimeric antigen receptor, after the transmembrane domain, the next domain is an ITAM, followed by a costimulatory domain. In some of these embodiments, one or more amino acids between the transmembrane domain and the costimulatory domain or ITAM are or include a sequence derived from the same endogenous single-chain polypeptide from which the costimulatory domain or ITAM is derived, respectively. In some of these embodiments, one or more amino acids between the transmembrane domain and the costimulatory domain or ITAM are or include a linker sequence (e.g., a non-naturally occurring linker sequence). In some of these embodiments, one or more amino acids between the costimulatory domain and the ITAM are or include a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the costimulatory domain and the ITAM are or include a sequence derived from the same endogenous single-chain polypeptide from which the costimulatory domain or ITAM is derived.

[0116] In some embodiments, when two or more costimulatory domains are included in the single-chain chimeric antigen receptor, the two or more costimulatory domains may be interposed between one or more ITAMs. In some embodiments, when two or more ITAMs are included in the single-chain chimeric antigen receptor, the two or more ITAMs may be interposed between one or more costimulatory molecules. In some embodiments of the single-chain chimeric antigen receptors described herein, the costimulatory domains and ITAMs may alternate in the primary amino acid sequence of the single-chain chimeric antigen receptor.

[0117] Some embodiments of any of the single-chain chimeric antigen receptors described herein may further comprise a dimerization domain and / or a peptide tag.

[0118] In some embodiments, the single-chain chimeric antigen receptor comprises, from N-terminus to C-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments, the single-chain chimeric antigen receptor comprises, from C-terminus to N-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments, the single-chain chimeric antigen receptor comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM. In some embodiments, the single-chain chimeric polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, a transmembrane domain, an intracellular costimulatory domain, and an ITAM.

[0119] In some examples, the single-chain chimeric antigen receptor 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:98.

[0120] Multichain chimeric antigen receptor Multichain 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 of the antigen-binding domains described herein or known in the art), a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art), and a hormone receptor ligand-binding domain (e.g., any of the hormone receptor ligand-binding domains described herein or known in the art), wherein the transmembrane domain and the hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., any of the subranges within this range described herein), and wherein 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 of any of these multi-chain chimeric antigen receptors may be any of the antigens described herein or known in the art. In some embodiments, the multi-chain chimeric antigen receptor specifically binds only a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, the multi-chain chimeric antigen receptor specifically binds two different antigens (e.g., any combination of any of the exemplary antigens described herein).

[0121] In some embodiments of the multi-chain chimeric antigen receptor, the extracellular antigen-binding domain and the transmembrane domain are adjacent to each other in at least one first polypeptide. In some embodiments of the multi-chain chimeric antigen receptor, between 1 and about 500 amino acids (e.g., any of the subranges within this range described herein) are located between the extracellular antigen-binding domain and the transmembrane domain in at least one first polypeptide. In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain in at least one first polypeptide are sequences derived from the same endogenous single-chain polypeptide from which the transmembrane domain is derived. In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain in at least one first polypeptide are or include a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain in at least one first polypeptide are or include a linker sequence (e.g., a non-naturally occurring linker sequence).

[0122] In some embodiments of these multi-chain chimeric antigen receptors, in at least one first polypeptide, the transmembrane domain is adjacent to the hormone receptor ligand-binding domain. In some embodiments of these multi-chain chimeric antigen receptors, in at least one first polypeptide, the transmembrane domain and the hormone receptor ligand-binding domain are separated by 1 to about 800 amino acids (e.g., or any of the subranges within this range described herein). In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the hormone receptor ligand-binding domain do not comprise a domain. In other embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the hormone receptor ligand-binding domain comprise one or more intervening domains (e.g., one or more costimulatory domains (e.g., any of the costimulatory domains described herein) and / or one or more ITAMs (e.g., any of the ITAMs described herein)). In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the hormone receptor ligand-binding domain comprise a sequence derived from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the hormone receptor ligand binding domain comprise a linker sequence (eg, a non-naturally occurring linker sequence).

[0123] In some embodiments of these multi-chain chimeric antigen receptors, at least one first polypeptide may further comprise one or more (e.g., two or three) of a dimerization domain (e.g., any dimerization domain known in the art), a costimulatory domain (e.g., any of the costimulatory domains described herein), and / or a peptide tag (e.g., any peptide tag known in the art).

[0124] Some embodiments of these multi-chain chimeric antigen receptors further include a second polypeptide that may include one or more (e.g., two, three, or four) of a transmembrane domain (e.g., any of the transmembrane domains described herein), a dimerization domain (e.g., a dimerization domain capable of interacting 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, the or each pair of adjacent domains in the second polypeptide may be adjacent to each other or may be separated by from 1 to about 800 amino acids (e.g., any of the subranges within this range described herein). One or more amino acids between a pair of adjacent domains in the second polypeptide may be a sequence derived from the endogenous single-chain polypeptide from which the transmembrane domain, costimulatory domain, or ITAM present in the second polypeptide is derived.

[0125] Multichain 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 of the antigen-binding domains described herein or known in the art), an extracellular hormone receptor ligand-binding domain (e.g., any of the hormone receptor ligand-binding domains described herein or known in the art), and a transmembrane domain (e.g., any of the transmembrane domains described herein or known in the art), wherein the transmembrane domain and the extracellular hormone receptor ligand-binding domain are either directly adjacent to each other or are separated by 1 to about 800 amino acids (e.g., any of the subranges within this range described herein), and both the transmembrane domain and the hormone receptor ligand-binding domain are not present in a single endogenous single-chain polypeptide. Multi-chain chimeric antigen receptors specifically bound by the antigen-binding domain of any of these multi-chain chimeric antigen receptors are also provided. The antigen may be any of the antigens described herein or known in the art. In some embodiments, the multi-chain chimeric antigen receptor specifically binds only a single antigen (e.g., any of the exemplary antigens described herein). In some embodiments, the multi-chain chimeric antigen receptor specifically binds two different antigens (e.g., any combination of any of the exemplary antigens described herein).

[0126] In some embodiments of the multi-chain chimeric antigen receptor, in at least one first polypeptide, the extracellular hormone receptor ligand-binding domain and the transmembrane domain are adjacent to each other. In some embodiments of the multi-chain chimeric antigen receptor, in at least one first polypeptide, between 1 and about 700 amino acids (e.g., any of the subranges within this range described herein) are located between the extracellular hormone receptor ligand-binding domain and the transmembrane domain. In some embodiments, in at least one first polypeptide, one or more amino acids between the extracellular hormone receptor ligand-binding domain and the transmembrane domain are sequences derived from the same endogenous single-chain polypeptide from which the transmembrane domain is derived. In some embodiments, one or more amino acids between the extracellular hormone receptor ligand-binding domain and the transmembrane domain of at least one first polypeptide are or include a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, in at least one first polypeptide, one or more amino acids between the extracellular hormone receptor ligand-binding domain and the transmembrane domain are or include a linker sequence (e.g., a non-naturally occurring linker sequence), hi some embodiments, one or more amino acids between the extracellular hormone receptor ligand-binding domain and the antigen-binding domain are or include a linker sequence (e.g., a non-naturally occurring linker sequence).

[0127] In some embodiments of these multi-chain chimeric antigen receptors, the transmembrane domain is adjacent to the extracellular hormone receptor ligand-binding domain in at least one first polypeptide. In some embodiments of these multi-chain chimeric antigen receptors, the transmembrane domain and the extracellular hormone receptor ligand-binding domain in at least one first polypeptide are separated by between 1 and about 800 amino acids (e.g., or any of the subranges within this range described herein). In some embodiments, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand-binding domain in at least one first polypeptide do not comprise a domain.

[0128] In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the extracellular hormone receptor ligand-binding domain comprise one or more intervening domains (e.g., antigen-binding domains). In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the antigen-binding domain comprise a sequence derived from the same endogenous transmembrane protein from which the transmembrane domain is derived. In some embodiments, in at least one first polypeptide, the amino acids between the transmembrane domain and the antigen-binding domain comprise a linker sequence (e.g., a non-naturally occurring linker sequence). In some embodiments, one or more amino acids between the antigen-binding domain and the transmembrane domain of at least one first polypeptide are or comprise a hinge region sequence of a human antibody (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, in at least one first polypeptide, one or more amino acids between the antigen-binding domain and the extracellular hormone receptor ligand-binding domain are or comprise a linker sequence (e.g., a non-naturally occurring linker sequence).

[0129] In some embodiments, the at least one first polypeptide comprises, from N-terminus to C-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain. comprises, from C-terminus to N-terminus, an extracellular antigen-binding domain, an extracellular hormone receptor ligand-binding domain, and a transmembrane domain. In some embodiments, at least one first polypeptide comprises, from N-terminus to C-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, and a transmembrane domain. In some embodiments, at least one first polypeptide comprises, from C-terminus to N-terminus, an extracellular hormone receptor ligand-binding domain, an extracellular antigen-binding domain, and a transmembrane domain.

[0130] In some embodiments of these multi-chain chimeric antigen receptors, at least one first polypeptide may further comprise one or more (e.g., two or three) of a dimerization domain (e.g., any dimerization domain known in the art), a costimulatory domain (e.g., any of the costimulatory domains described herein), and / or a peptide tag (e.g., any peptide tag known in the art).

[0131] Some embodiments of these multi-chain chimeric antigen receptors further include a second polypeptide that 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 capable of interacting in a mammalian cell with a dimerization domain in at least one first polypeptide), 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, the or each pair of adjacent domains in the second polypeptide may be adjacent to each other or may be separated by from 1 to about 800 amino acids (e.g., any of the subranges within this range described herein). One or more amino acids between a pair of adjacent domains in the second polypeptide may be a sequence derived from the endogenous single-chain polypeptide from which the transmembrane domain, costimulatory domain, or ITAM present in the second polypeptide is derived. In the second polypeptide, one or more amino acids between a pair of adjacent domains may be or include a linker sequence (eg, a non-naturally occurring linker sequence).

[0132] As is well understood in the art, two or more polypeptides present in a multi-chain chimeric antigen receptor can associate via a pair of domains that interact with each other (via dimerization domains). In some embodiments, the interaction between the dimerization domains can be induced by the addition of a small molecule. In some embodiments, two or more polypeptides present in a multimeric chimeric antigen receptor can associate via non-covalent interactions (e.g., association between dimerization domains). In some embodiments, two or more polypeptides present in a multimeric chimeric antigen receptor can also associate via covalent interactions (e.g., via disulfide bonds, ester bonds, amide bonds, thioester bonds, or combinations thereof).

[0133] Transmembrane domain In some embodiments, the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor is selected from the group consisting of T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28 (also known as Tp44), CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37 (also known as GP52-40 or TSPAN26), CD64 (also known as FCGR1A), CD80 (also known as B7, B7-1, B7.1, B1, CD28LG, CD28LG1, and LAB7), CD45 (also known as PTPRC, B220, CD45R, GP180, L-CA, LCA, LY5, T200, and protein tyrosine phosphatase, receptor type C), CD4, CD5 (also known as LEU1 and T1), CD8 alpha (also known as Leu2, MAL, and p32). , CD9 (also known as BTCC-1, DRAP-27, MIC3, MRP-1, TSPAN-29, and TSPAN29), CD16 (also known as FCGR3 and FCG3), CD22 (also known as SIGLEC-2 and SIGLEC2), CD86 (also known as B7-2, B7.2, B70, CD28LG2, and LAB72), CD134 (also known as TNFRSF4, ACT35, RP5-902P8.3, IMD16, OX40, TXGP1L, and tumor Necrosis factor receptor superfamily member 4), 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-1, 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 (TNFRSF18, RP5-902P8.2, AITR, CD357, and GIT The term "CD" includes a transmembrane domain or portion thereof derived from an endogenous polypeptide selected from the group g of: CD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, and CD40 ligand. The letters "CD" are preamble for "cluster of differentiation." For example, CD3 stands for "cluster of differentiation 3."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 derived from an endogenous mammalian (e.g., human) polypeptide (e.g., a mammalian or human homolog of any of the above-listed polypeptides).

[0134] Any transmembrane domain or portion thereof that serves to anchor an endogenous polypeptide in the lipid bilayer (e.g., the plasma membrane) of a mammalian cell is suitable for use 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 or portion thereof derived from human CD28, e.g., Accession No. 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 comprises a transmembrane domain or portion thereof 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 of SEQ ID NO: 1.

[0135] 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).

[0136] SEQ ID NO: 1 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIY FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0137] 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 derived from human CD3, e.g., Accession No. 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 a sequence 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.

[0138] SEQ ID NO: 2 MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0139] 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 to 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).

[0140] 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 a sequence 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 of SEQ ID NOs: 3 to 9.

[0141] 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 a sequence 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, SEQ ID NO:56, or SEQ ID NO:114.

[0142] As will be appreciated by those skilled 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 may be transmembrane polypeptides that contain a sequence of amino acids derived from any isoform of an endogenous transmembrane protein (e.g., an endogenous mammalian, e.g., human, transmembrane protein), including, for example, an isoform (e.g., human isoform) of the T cell receptor α chain, the T cell receptor β chain, the T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD154. It may also include a domain.

[0143] In some embodiments, the transmembrane domain or portion thereof of the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor 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 a transmembrane domain derived from one or more of the following endogenous mammalian (e.g., human) transmembrane proteins: T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD154. In some embodiments, the transmembrane domain or portion thereof of the single-chain chimeric polypeptide, single-chain chimeric antigen receptor, or multi-chain chimeric antigen receptor comprises a sequence of amino acids that has one or more amino acid substitutions, deletions, or additions compared to the transmembrane domain of an endogenous mammalian (e.g., human) transmembrane protein: T cell receptor alpha chain, T cell receptor beta chain, T cell receptor zeta chain, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, or CD154.

[0144] 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 comprise primarily hydrophobic residues such as, but not limited to, leucine and valine. In some embodiments, the synthetic transmembrane domain comprises a triplet of phenylalanine, tryptophan, and valine at each end of the domain.

[0145] 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 a chimeric transmembrane domain having portions of a transmembrane domain derived from two or more endogenous mammalian (e.g., human) transmembrane polypeptides, such as, but not limited to, the T cell receptor α chain, the T cell receptor β chain, the T cell receptor ζ chain, CD28, CD3ε, CD3δ, CD3γ, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD86, CD134, CD137, CD27, CD152, PD1, and CD154, such that the two or more portions of the transmembrane domain together constitute a functional transmembrane domain. In some embodiments, such portions of the chimeric transmembrane domain may comprise one or more amino acid substitutions, deletions, or additions compared to the corresponding portions of the wild-type transmembrane domain.

[0146] A transmembrane domain may comprise one, two, three, four, five, six, seven, eight, nine, or ten consecutive amino acid sequences that each span a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. As known in the art, a transmembrane domain may comprise, for example, at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) consecutive amino acid sequence that has an α-helical secondary structure in the lipid bilayer (which spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). In some embodiments, a transmembrane domain may comprise two or more consecutive amino acid sequences that form a β-barrel secondary structure in the lipid bilayer (which each spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). Further examples and characteristics of transmembrane domains are known in the art.

[0147] Hormone receptor ligand binding domain In some embodiments, the hormone receptor ligand binding domain is an estrogen receptor The ligand-binding domain may be or comprise a ligand-binding domain, or a fragment thereof. Further examples of hormone receptor ligand-binding domains that may be used in any of the polypeptides described herein (e.g., single-chain chimeric polypeptides, single-chain chimeric antigen receptors, or multi-chain chimeric antigen receptors) include, for example, the ligand-binding domains of estrogen receptor, ecdysone receptor, PPARγ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR receptor α, PPARβ / δ receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, retinoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

[0148] 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 comprises a sequence that 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.

[0149] SEQ ID NO: 10 (human) 1 skknslalsl tadqmvsall daeppilyse ydptrpfsea smmglltnla drelvhminw 61 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpg kllfapnlll drnqgkcveg 121 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvyt flsstlksle ekdhihrvld 181 kitdtlihlm akagltlqqq hqrlaqllli lshirhmsnk gmehlysmkc knvvplydll 241 lemldahrlh

[0150] Another non-limiting example of an estrogen receptor ligand binding domain is amino acids 4 through 241 of SEQ ID NO: 11. In some embodiments, the hormone receptor ligand binding domain is or comprises a sequence 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 through 241 of SEQ ID NO: 11.

[0151] SEQ ID NO: 11 (human) 1 alsltadqmv salldaeppi lyseydptrp fseasmmgll tnladrelvh minwakrvpg 61 fvdltlhdqv hllecawlei lmiglvwrsm ehpgkllfap nllldrnqgk cvegmveifd 121 mllatssrfr mmnlqgeefv clksiillns gvytflsstl ksleekdhih rvldkitdtl 181 ihlmakaglt lqqqhqrlaq lllilshirh msnkgmehly smkcknvvpl ydlllemlda 241 hrlhapts

[0152] In some embodiments, the estrogen receptor ligand binding domain has a wild-type sequence except for one or more (e.g., 2, 3, 4, 5, or 6) amino acid substitutions. In some embodiments, the estrogen receptor ligand binding domain has a wild-type sequence except for one or both amino acid substitutions at amino acid positions 521 (e.g., a G521R substitution) and 537 (e.g., a G537F substitution) (each numbered with respect to the wild-type full-length sequence of a human estrogen receptor, e.g., SEQ ID NO: 119 below).

[0153] SEQ ID NO: 119 (human) 1 mtmtlhtkas gmallhqiqg neleplnrpq lkiplerplg evyldsskpa vynypegaay 61 efnaaaaana qvygqtglpy gpgseaaafg snglggfppl nsvspsplml lhpppqlspf 121 lqphgqqvpy ylenepsgyt vreagppafy rpnsdnrrqg grerlastnd kgsmamesak 181 etrycavcnd yasgyhygvw scegckaffk rsiqghndym cpatnqctid knrrkscqac 241 rlrkcyevgm mkggirkdrr ggrmlkhkrq rddgegrgev gsagdmraan lwpsplmikr 301 skknslalsl tadqmvsall daeppilyse ydptrpfsea smmglltnla drelvhminw 361 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpg kllfapnllll drnqgkcveg 421 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvyt flsstlksle ekdhihrvld 481 kitdtlihlm akagltlqqq hqrlaqllli lshirhmsnk gmehlysmkc knvvplydll 541 lemldahrlh aptsrggasv eetdqshlat agstsshslq kyyitgeaeg fpatv

[0154] Non-limiting examples of estrogen receptor ligand binding domains include sequences 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).

[0155] Further non-limiting examples of hormone receptor ligand binding domains are or 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:46, SEQ ID NO:59, or SEQ ID NO:110.

[0156] 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 comprises a sequence 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.

[0157] SEQ ID NO: 88 (mouse) 1 mtmtlhtkas gmallhqiqg neleplnrpq lkmpmeralg evyvdnskpt vfnypegaay 61 efnaaaaaaa aasapvygqs giaygpgsea aafsanslga fpqlnsvsps plmllhpppq 121 lspflhphgq qvpyylenep sayavrdtgp pafyrsnsdn rrqngrerls ssnekgnmim 181 esaketryca vcndyasgyh ygvwscegck affkrsiqgh ndymcpatnq ctidknrrks 241 cqacrlrkcy evgmmkggir kdrrggrmlk hkrqrddleg rnemgasgdm raanlwpspl 301 vikhtkknsp alsltadqmv salldaeppm iyseydpsrp fseasmmgll tnladrelvh 361 minwakrvpg fgdlnlhdqv hllecawlei lmiglvwrsm ehpgkllfap nllldrnqgk 421 cvegmveifd mllatssrfr mmnlqgeefv clksiillns gvytflsstl ksleekdhih 481 rvldkitdtl ihlmakaglt lqqqhrrlaq lllilshirh msnkgmehly nmkcknvvpl 541 ydlllemlda hrlhapasrm gvppeepsqt qlattsstsa hslqtyyipp eaegfpnti

[0158] 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 has a sequence identity that 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%, It may be at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or comprise a sequence.

[0159] SEQ ID NO: 89 (rat) 1 mtmtlhtkas gmallhqiqg neleplnrpq lkmpmeralg evyvdnskpa vfnypegaay 61 efnaaaaaaa agasapvygq ssitygpgse aaafganslg afpqlnsvsp splmllhppp 121 hvspflhphg hqvpyylene psayavrdtg ppafyrsnsd nrrqngrerl ssssekgnmi 181 mesaketryc avcndyasgy hygvwscegc kaffkrsiqg hndymcpatn qctidknrrk 241 scqacrlrkc yevgmmkggi rkdrrggrml khkrqrddle grnemgtsgd mraanlwpsp 301 lvikhtkkns palsltadqm vsalldaepp liyseydpsr pfseasmmgl ltnladrelv 361 hminwakrvp gfgdlnlhdq vhllcawle ilmiglvwrs mehpgkllfa pnllldrnqg 421 kcvegmveif dmllatssrf rmmnlqgeef vclksiilln sgvytflsst lksleekdhi 481 hrvldkindt lihlmakagl tlqqqhrrla qlllilshir hmsnkgmehl ynmkcknvvp 541 lydlllemld ahrlhapasr mgvppeepsq sqltttssts ahslqtyyip peaegfpnti

[0160] 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 comprises a sequence 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.

[0161] SEQ ID NO: 90 (baboon) 1 mtmtlhtkas gmallhqiqg neleplnrpq lkiplerplg evyvdsskpa vysypegaay 61 efnaaaaana qvygqtglpy gpgseaaafg snglggfppl nsvspsplml lhpppqlspf 121 lqphgqqvpy ylenepsgyt vreagppafy rpnsdnrrqg grerlastnd kgsmamesak 181 etrycavcnd yasgyhygvw scegckaffk rsiqghndym cpatnqctid knrrkscqac 241 rlrkcyevgm mkggirkdrr ggrmlkhkrq rddgegrgev gsagdmraan lwpsplmikh 301 skknspalsl tadqmvsall daeppilyse ydptrpfsea smmglltnla drelvhminw 361 akrvpgfvdl tlhdqvhlle cawleilmig lvwrsmehpg kllfapnllll drnqgkcveg 421 mveifdmlla tssrfrmmnl qgeefvclks iillnsgvyt flsstlksle ekdhihrvld 481 kitdtlihlm akagltlqqq hrrlaqllli lshirhmsnk gmehlysmkc knvvplydll 541 lemldahrlh aptsrggapm eetdqshlat agstsshslq kyyitgdaeg fpatv

[0162] In some embodiments, the hormone receptor ligand binding domain is or comprises a progesterone receptor ligand binding domain, or a fragment thereof. Another 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 comprises a sequence that 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.

[0163] SEQ ID NO: 12 (human) 1 spgqdiqlip plinllmsie pdviyaghdn tkpdtsssll tslnqlgerq llsvvkwsks 61 lpgfrnlhid dqitliqysw mslmvfglgw rsykhvsgqm lyfapdliln eqrmkessfy 121 slcltmwqip qefvklqvsq eeflcmkvll llntiplegl rsqtqfeemr ssyirelika 181 iglrqkgvvs ssqrfyqltk lldnlhdlvk qlhlyclntf iqsralsvef pemmseviaa 241 qlpkilagmv kpllfhkk

[0164] Another non-limiting example of a progesterone receptor ligand binding domain is amino acids 14 to 261 of SEQ ID NO: 13. In some embodiments, the hormone receptor ligand binding domain is or comprises a sequence 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.

[0165] SEQ ID NO: 13 (human) 1 ftfspgqdiq lipplinllm siepdviyag hdntkpdtss slltslnqlg erqllsvvkw 61 skslpgfrnl hiddqitliq yswmslmvfg lgwrsykhvs gqmlyfapdl ilneqrmkes 121 sfyslcltmw qipqefvklq vsqeeflcmk vllllntipl eglrsqtqfe emrssyirel 181 ikaiglrqkg vvsssqrfyq ltklldnlhd lvkqlhlycl ntfiqsrals vefpemmsev 241 iaaqlpkila gmvkpllfhk k

[0166] Another non-limiting example of a progesterone receptor ligand binding domain is amino acids 679 to 926 of SEQ ID NO: 91. In some embodiments, the hormone receptor ligand binding domain is or comprises a sequence 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.

[0167] SEQ ID NO: 91 (mouse) 1 mtelqakdpq vlhtsgasps pphigsplla rldsgpfqgs qhsdvssvvs pipisldgll 61 fprscrgpel pdgktgdqqs lsdvegafsg veathreggr nsrapekdsr lldsvldsll 121 tpsgteqsha sppaceaits wclfgpelpe dprsvpatkg llsplmsrpe ikagdssgtg 181 agqkvlpkgl spprqllllpt sgsahwpgag vkpspqpaag eveedsglet egsaapllks 241 kpralegtgs gggvaanaas aapggvtlvp kedsrfsapr vsleqdspia pgrsplattv 301 vdfihvpilp lnhallaart rqllegdsyd ggataqgpfa pprgspsaps ppvpcgdfpd 361 ctyplegdpk edvfplygdf qtpglkikee eegadaavrs prpylsagas sstfpdfpla 421 papqrapssr pgeaavaggp ssaavspass sgsalecily kaegapptqg sfaplpckpp 481 aagscllprd slpaapataa apaiyqplgl nglpqlgyqa avlkdslpqv yppylnylrp 541 dseasqspqy gfdslpqkic licgdeasgc hygvltcgsc kvffkrameg qhnylcagrn 601 dcivdkirrk ncpacrlrkc cqagmvlggr kfkkfnkvrv mrtldgvalp qsvglpnesq 661 algqritfsp nqeiqlvppl inllmsiepd vvyaghdntk pdtsssllts lnqlgerqll 721 svvkwskslp gfrnlhiddq itliqyswms lmvfglgwrs ykhvsgqmly fapdlilneq 781 rmkelsfysl cltmwqipqe fvklqvthee flcmkvllll ntipleglrs qsqfeemrss 841 yirelikaig lrqkgvvpss qrfyqltkll dslhdlvkql hlyclntfiq srtlavefpe 901 mmseviaaql pkilagmvkp llfhkk

[0168] Another non-limiting example of a progesterone receptor ligand binding domain is amino acids 676 to 923 of SEQ ID NO: 92. In some embodiments, the hormone receptor ligand binding domain contains at least be or comprise a sequence that is 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 sequence.

[0169] SEQ ID NO: 92 (rat) 1 mtelqakdpr tlhtsgaaps pthvgsplla rldpdpfqgs qhsdassvvs pipisldrll 61 fsrscqaqel pdektqnqqs lsdvegafsg veasrrrsrn prapekdsrl ldsvldtlla 121 psgpeqsqts ppaceaitsw clfgpelped prsvpatkgl lsplmsrpes kagdssgtga 181 gqkvlpkavs pprqlllpts gsahwpgagv kpsqqpatve veedgglete gsagpllksk 241 pralegmcsg ggvtanapga apggvtlvpk edsrfsaprv sleqdapvap grsplattvv 301 dfihvpilpl nhallaartr qllegdsydg gaaaqvpfap prgspsapsp pvpcgdfpdc 361 typpegdpke dgfpvygefq ppglkikeee egteaasrsp rpyllagasa atfpdfplpp 421 rpprappsrp geaavaapsa avspvsssgs alecilykae gapptqgsfa plpckppaas 481 scllprdslp aaptssaapa iypplglngl pqlgyqaavl kdslpqvypp ylnylrpdse 541 asqspqygfd slpqkiclic gdeasgchyg vltcgsckvf fkramegqhn ylcagrndci 601 vdkirrkncp acrlrkccqa gmvlggrkfk kfnkvrvmra ldgvalpqsv afpnesqtlg 661 qritfspnqe iqlvpplinl lmsiepdvvy aghdntkpdt ssslltslnq lgerqllsvv 721 kwskslpgfr nlhiddqitl iqyswmslmv fglgwrsykh vsgqmlyfap dlilneqrmk 781 elsfyslclt mwqipqefvk lqvtheeflc mkvllllnti pleglrsqsq feemrssyir 841 elikaiglrq kgvvpssqrf yqltklldsl hdlvkqlhly clntfiqsra lavefpemms 901 eviaaqlpki lagmvkpllf hkk

[0170] 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 comprises a sequence 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.

[0171] SEQ ID NO: 93 (baboon) 1 mtelkakgpr aphvaggpps pevgspllcr paagpfqgsq tsdtlpevsa ipisldgllf 61 prpcqgqdpl dektqdqqsl sdvegaysra eatrgtggss srppekdsgl lhsvldtlla 121 psgpgqsqps ppacevtssw clfgpelped ppaapatqgv lsplmsrsgc kagdssgtaa 181 ahkvlprgls psrqlllpas gsphwsgapv kpspqpaave veeedgsese esagpllkgk 241 pralggaaag ggaaavppga aaggvalvpk edsrfsaprv alveqdapma pgrsplattt 301 mdfthvpilp lshallaart rqlleeesyd ggagaasafa pprsspsass tpvavgdfpd 361 cayppdadpk ddayplygdf qppalkikee eegaevsars prsylvagan paafpdfplg 421 pppplpprap psrpgeaavt aapagasvss asssgstlec ilykaegapp qqgpfapppc 481 kapgaggcll prdglpstsa saaaagaapa lypalglngl pqlgyqaavl keglqqvypp 541 ylnylrpdse asqspqysfe slpqkiclic gdeasgchyg vltcgsckvf fkramegqhn 601 ylcagrndci vdkirrkncp acrlrkccqa gmvlggrkfk kfnkvrvmra ldavalpqpv 661 gipnesqals qrftfppgqd iqlipplinl lvsiepdviy aghdnskpdt ssslltslnq 721 lgerqllsvv kwskllpgfr nlhiddqitl iqyswmslmv fglgwrsykh vsgqmlyfap 781 dlilneqrmk essfyslclt mwqipqefvk lqvsqeeflc mkvllllnti pleglrsqtq 841 feemrssyir elikaiglrq kgvvsssqrf yqltklldnl hdlvkqlhly clntfiqsra 901 lsvefpemms eviaaqlpki lagmvkpllf hkk

[0172] In some embodiments, the hormone receptor ligand binding domain is or comprises an androgen receptor ligand binding domain, or a fragment thereof. A non-limiting example of a human androgen receptor ligand binding domain is amino acids 2 to 247 of SEQ ID NO: 14. In some embodiments, the hormone receptor ligand binding domain is or comprises a sequence that 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.

[0173] SEQ ID NO: 14 (human) 1 piflnvleai epgvvcaghd nnqpdsfaal lsslnelger qlvhvvkwak alpgfrnlhv 61 ddqmaviqys wmglmvfamg wrsftnvnsr mlyfapdlvf neyrmhksrm ysqcvrmrhl 121 sqefgwlqit pqeflcmkal llfsiipvdg lknqkffdel rmnyikeldr iiackrknpt 181 scsrrfyqlt klldsvqpia relhqftfdl likshmvsvd fpemmaeiis vqvpkilsgk 241 vkpiyfhtq

[0174] 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 comprises a sequence 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.

[0175] SEQ ID NO: 15 (human) 1 sleegeasst tspteettqk ltvshiegye cqpiflnvle aiepgvvcag hdnnqpdsfa 61 allsslnelg erqlvhvvkw akalpgfrnl hvddqmaviq yswmglmvfa mgwrsftnvn 121 srmlyfapdl vfneyrmhks rmysqcvrmr hlsqefgwlq itpqeflcmk alllfsiipv 181 dglknqkffd elrmnyikel driiackrkn ptscsrrfyq ltklldsvqp iarelhqftf 241 dllikshmvs vdfpemmaei isvqvpkils gkvkpiyfht qeg

[0176] 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 comprises a sequence 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.

[0177] SEQ ID NO: 16 (human) 1 ettqkltvsh iegyecqpif lnvleaiepg vvcaghdnnq pdsfaallss lnelgerqlv 61 hvvkwakalp gfrnlhvddq maviqyswmg lmvfamgwrs ftnvnsrmly fapdlvfney 121 rmhksrmysq cvrmrhlsqe fgwlqitpqe flcmkalllf siipvdglkn qkffdelrmn 181 yikeldriia ckrknptscs rrfyqltkll dsvqpiarel hqftfdllik shmvsvdfpe 241 mmaeiisvqv pkilsgkvkp iyfhtq

[0178] 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 comprises a sequence 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.

[0179] SEQ ID NO: 94 (mouse) 1 mevqlglgrv yprppsktyr gafqnlfqsv reaiqnpgpr hpeaaniapp gaclqqrqet 61 sprrrrrqqh tedgspqahi rgptgylale eeqqpsqqqa aseghpessc lpepgaatap 121 gkglpqqppa ppdqddsaap stlsllgptf pglsscsadi kdilneagtm qllqqqqqqq 181 qhqqqhqqhq qqqevisegs sarareatga pssskdsylg gnstisdsak elckavsvsm 241 glgvealehl spgeqlrgdc myasllggpp avrptpcapl peckglplde gpgksteeta 301 eyssfkggya kglegeslgc sgsseagssg tleipsslsl yksgaldeaa ayqnrdyynf 361 plalsgpphp pppthphari klenpldygs awaaaaaqcr ygdlgslhgg svagpstgsp 421 pattssswht lftaeegqly gpgggggsss psdagpvapy gytrppqglt sqesdysase 481 vwypggvvnr vpypspncvk semgpwmeny sgpygdmrld strdhvlpid yyfppqktcl 541 icgdeasgch ygaltcgsck vffkraaegk qkylcasrnd ctidkfrrkn cpscrlrkcy 601 eagmtlgark lkklgnlklq eegensnags ptedpsqkmt vshiegyecq piflnvleai 661 epgvvcaghd nnqpdsfaal lsslnelger qlvhvvkwak alpgfrnlhv ddqmaviqys 721 wmglmvfamg wrsftnvnsr mlyfapdlvf neyrmhksrm ysqcvrmrhl sqefgwlqit 781 pqeflcmkal llfsiipvdg lknqkffdel rmnyikeldr iiackrknpt scsrrfyqlt 841 klldsvqpia relhqftfdl likshmvsvd fpemmaeiis vqvpkilsgk vkpiyfhtq

[0180] 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 comprises a sequence 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.

[0181] SEQ ID NO: 95 (rat) 1 mevqlglgrv yprppsktyr gafqnlfqsv reaiqnpgpr hpeaasiapp gaclqqrqet 61 sprrrrrqqh pedgspqahi rgttgylale eeqqpsqqqs aseghpesgc lpepgaatap 121 gkglpqqppa ppdqddsaap stlsllgptf pglsscsadi kdilseagtm qllqqqqqqq 181 qqqqqqqqqq qqqqqevise gsssvrarea tgapssskds ylggnstisd sakelckavs 241 vsmglgveal ehlspgeqlr gdcmyasllg gppavrptpc aplaeckgls ldegpgkgte 301 etaeyssfkg gyakgleges lgcsgsseag ssgtleipss lslyksgavd eaaayqnrdy 361 ynfplalsgp phppppthph ariklenpsd ygsawaaaaa qcrygdlasl hggsvagpst 421 gsppatasss whtlftaeeg qlygpggggg ssspsdagpv apygytrppq glasqegdfs 481 asevwypggv vnrvpypsps cvksemgpwm enysgpygdm rldstrdhvl pidyyfppqk 541 tclicgdeas gchygaltcg sckvffkraa egkqkylcas rndctidkfr rkncpscrlr 601 kcyeagmtlg arklkklgnl klqeegenss agsptedpsq kmtvshiegy ecqpiflnvl 661 eaiepgvvca ghdnnqpdsf aallsslnel gerqlvhvvk wakalpgfrn lhvddqmavi 721 qyswmglmvf amgwrsftnv nsrmlyfapd lvfneyrmhk srmysqcvrm rhlsqefgwl 781 qitpqeflcm kalllfsiip vdglknqkff delrmnyike ldriiackrk nptscsrrfy 841 qltklldsvq piarelhqft fdllikshmv svdfpemmae iisvqvpkil sgkvkpiyfh 901 tq

[0182] 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 comprises a sequence 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:96.

[0183] SEQ ID NO: 96 (baboon) 1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gaslqqqqqq 61 qqqetsprqq qqqqgedgsp qahrrgptgy lvldeeqqps qpqsapechp ergcvpepga 121 avaagkglpq qlpappdedd saapstlsll gptfpglssc sadlkdilse astmqllqqq 181 qqeavsegss sgrareasga ptsskdnylg gtstisdsak elckavsvsm glgvealehl 241 spgeqlrgdc myapvlgvpp avrptpcapl aeckgslldd sagkstedta eyspfkggyt 301 kglegeslgc sgsaaagssg tlelpstlsl yksgaldeaa ayqsrdyynf plalagpppp 361 pppphphari klenpldygs awaaaaaqcr ygelaslhga gaagpgsgsp saaassswht 421 lftaeegqly gpcggggggg ggggggagea gavapygytr ppqglagqeg dftapdvwyp 481 ggmvsrvpyp sptcvksemg pwmdsysgpy gdmrletard hvlpidyyfp pqktclicgd 541 easgchygal tcgsckvffk raaegkqkyl casrndctid kfrrkncpsc rlrkcyeagm 601 tlgarklkkl gnlklqeege assttsptee taqkltvshi egyecqpifl nvleaiepgv 661 vcaghdnnqp dsfaallssl nelgerqlvh vvkwakalpg frnlhvddqm aviqyswmgl 721 mvfamgwrsf tnvnsrmlyf apdlvfneyr mhksrmysqc vrmrhlsqef gwlqitpqef 781 lcmkalllfs iipvdglknq kffdelrmny ikeldriiac krknptscsr rfyqltklld 841 svqpiarelh qftfdlliks hmvsvdfpem maeiisvqvp kilsgkvkpi yfhtq

[0184] 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 comprises a sequence that 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.

[0185] SEQ ID NO: 28 (human) 1 drrggrmlkh krqrddgegr gevgsagdmr aanlwpsplm ikrskknsla lsltadqmvs 61 alldaeppil yseydptrpf seasmmgllt nladrelvhm inwakrvpgf vdltlhdqvh 121 llecawleil miglvwrsme hpgkllfapn llldrnqgkc vegmveifdm llatssrfrm 181 mnlqgeefvc lksiillnsg vytflsstlk sleekdhihr vldkitdtli hlmakagltl 241 qqqhqrlaql llilshirhm snkrmehlys mkcknvvplf dlllemldah rlhapts

[0186] Another non-limiting example of a ligand binding domain is SEQ ID NO: 29. In some embodiments, the hormone receptor ligand binding domain has 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%, or at least 93% identity to the amino acid sequence of SEQ ID NO: 29. The sequence may be or comprise a sequence that is 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the original.

[0187] SEQ ID NO: 29 (human) 1 gqdiqlippl inllmsiepd viyaghdntk pdtsssllts lnqlgerqll svvkwskslp 61 gfrnlhiddq itliqyswms lmvfglgwrs ykhvsgqmly fapdlilneq rmkessfysl 121 cltmwqipqe fvklqvsqee flcmkvllll ntipleglrs qtqfeemrss yirelikaig 181 lrqkgvvsss qrfyqltkll dnlhdlvkql hlyclntfiq sralsvefpe mmseviaaql 241 pkilagmvkp llfhkk

[0188] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of an ecdysone receptor. For example, the ligand-binding domain of an ecdysone receptor can be amino acids 256 to 467 of SEQ ID NO: 74. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence 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.

[0189] SEQ ID NO: 74 (human) 1 merdepppsg ggggggsagf leppaalppp prngfcqdel aeldpgtisv sddraeqrtc 61 licgdratgl hygiiscegc kgffkrsicn krvyrcsrdk ncvmsrkqrn rcqycrllkc 121 lqmgmnrkai redgmpggrn ksigpvqise eeierimsgq efeeeanhws nhgdsdhssp 181 gnrasesnqp spgstlsssr svelngfmaf reqymgmsvp phyqyiphlf sysghspllp 241 qqarsldpqs yslihqllsa edleplgtpm liedgyavtq aelfallcrl adellfrqia 301 wikklpffce lsikdytcll sstwqelill ssltvyskqi fgeladvtak yspsdeelhr 361 fsdegmevie rliylyhkfh qlkvsneeya cmkainflnq dirgltsasq leqlnkrywy 421 icqdfteyky thqpnrfpdl mmclpeiryi agkmvnvple qlpllfkvvl hscktsvgke

[0190] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of a PPARγ receptor. For example, the ligand-binding domain of a PPARγ receptor can be amino acids 237 to 504 of SEQ ID NO: 75. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence 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.

[0191] SEQ ID NO: 75 (human) 1 mgetlgdspi dpesdsftdt lsanisqemt mvdtempfwp tnfgissvdl svmedhshsf 61 dikpfttvdf ssistphyed ipftrtdpvv adykydlklq eyqsaikvep asppyysekt 121 qlynkpheep snslmaiecr vcgdkasgfh ygvhacegck gffrrtirlk liydrcdlnc 181 rihkksrnc qycrfqkcla vgmshnairf grmpqaekek llaeissdid qlnpesadlr 241 alakhlydsy iksfpltkak arailtgktt dkspfviydm nslmmgedki kfkhitplqe 301 qskevairif qgcqfrsvea vqeiteyaks ipgfvnldln dqvtllkygv heiiytmlas 361 lmnkdgvlis egqgfmtref lkslrkpfgd fmepkfefav kfnaleldds dlaifiavii 421 lsgdrpglln vkpiediqdn llqalelqlk lnhpessqlf akllqkmtdl rqivtehvql 481 lqvikktetd mslhpllqei ykdly

[0192] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of a glucocorticoid receptor. For example, the ligand-binding domain of a 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 comprises a sequence 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.

[0193] SEQ ID NO: 76 (human) 1 mdskesltpg reenpssvla qergdvmdfy ktlrggatvk vsasspslav asqsdskqrr 61 llvdfpkgsv snaqqpdlsk avslsmglym getetkvmgn dlgfpqqgqi slssgetdlk 121 lleesianln rstsvpenpk ssastavsaa ptekefpkth sdvsseqqhl kgqtgtnggn 181 vklyttdqst fdilqdlefs sgspgketne spwrsdllid encllsplag eddsfllegn 241 snedckplil pdtkpkikdn gdlvlsspsn vtlpqvktek edfielctpg vikqeklgtv 301 ycqasfpgan iignkmsais vhgvstsggq myhydmntas lsqqqdqkpi fnvippipvg 361 senwnrcqgs gddnltslgt lnfpgrtvfs ngysspsmrp dvssppssss tattgpppkl 421 clvcsdeasg chygvltcgs ckvffkrave gqhnylcagr ndciidkirr kncpacryrk 481 clqagmnlea rktkkkikgi qqattgvsqe tsenpgnkti vpatlpqltp tlvsllevie 541 pevlyagyds svpdstwrim ttlnmlggrq viaavkwaka ipgfrnlhld dqmtllqysw 601 mflmafalgw rsyrqssanl lcfapdliin eqrmtlpcmy dqckhmlyvs selhrlqvsy 661 eeylcmktll llssvpkdgl ksqelfdeir mtyikelgka ivkregnssq nwqrfyqltk 721 lldsmhevve nllnycfqtf ldktmsiefp emlaeiitnq ipkysngnik kllfhqk

[0194] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of a thyroid hormone receptor. For example, the ligand-binding domain of a glucocorticoid receptor can be amino acids 162 to 370 of SEQ ID NO: 77. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence 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.

[0195] SEQ ID NO: 77 (human) 1 meqkpskvec gsdpeensar spdgkrkrkn gqcslktsms gyipsyldkd eqcvvcgdka 61 tgyhyrcitc egckgffrrt iqknlhptys ckydsccvid kitrnqcqlc rfkkciavgm 121 amdlvlddsk rvakrklieq nrerrrkeem irslqqrpep tpeewdlihi ateahrstna 181 qgshwkqrrk flpddigqsp ivsmpdgdkv dleafseftk iitpaitrvv dfakklpmfs 241 elpcedqiil lkgccmeims lraavrydpe sdtltlsgem avkreqlkng glgvvsdaif 301 elgkslsafn lddtevallq avllmstdrs gllcvdkiek sqeayllafe hyvnhrkhni 361 phfwpkllmk erevqssily kgaaaegrpg gslgvhpegq qllgmhvvqg pqvrqleqql 421 geagslqgpv lqhqspkspq qrllellhrs gilharavcg eddsseadsp ssseeepevc 481 edlagnaasp

[0196] Another non-limiting example of a ligand binding domain is a ligand for the mineralocorticoid receptor. For example, the ligand binding domain of a mineralocorticoid receptor can be amino acids 737 to 984 of SEQ ID NO: 78. In some embodiments, the hormone receptor ligand binding domain is or comprises a sequence that 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 737 to 984 of SEQ ID NO: 78.

[0197] SEQ ID NO: 78 (human) 1 metkgyhslp egldmerrwg qvsqaverss lgptertden nymeivnvsc vsgaipnnst 61 qgsskekqel lpclqqdnnr pgiltsdikt eleskelsat vaesmglymd svrdadysye 121 qqnqqgsmsp akiyqnveql vkfykgnghr pstlscvntp lrsfmsdsgs svnggvmrai 181 vkspimchek spsvcsplnm tssvcspagi nsvssttasf gsfpvhspit qgtpltcspn 241 aenrgsrshs pahasnvgsp lssplssmks sissppshcs vkspvsspnn vtlrssvssp 301 aninnsrcsv sspsntnnrs tlsspaastv gsicspvnna fsytasgtsa gsstlrdvvp 361 spdtqekgaq evpfpkteev esaisngvtg qlnivqyikp epdgafsssc lggnskinsd 421 ssfsvpikqe stkhscsgts fkgnptvnpf pfmdgsyfsf mddkdyysls gilgppvpgf 481 dgncegsgfp vgikqepddg syypeasips saivgvnsgg qsfhyrigaq gtislsrsar 541 dqsfqhlssf ppvntlvesw kshgdlssrr sdgypvleyi penvssstlr svstgssrps 601 kiclvcgdea sgchygvvtc gsckvffkra vegqhnylca grndciidki rrkncpacrl 661 qkclqagmnl garkskklgk lkgiheeqpq qqqppppppp pqspeegtty iapakepsvn 721 talvpqlsti sraltpspvm vleniepeiv yagydsskpd taenllstln rlagkqmiqv 781 vkwakvlpgf knlpledqit liqyswmcls sfalswrsyk htnsqflyfa pdlvfneekm 841 hqsamyelcq gmhqislqfv rlqltfeeyt imkvllllst ipkdglksqa afeemrtnyi 901 kelrkmvtkc pnnsgqswqr fyqltkllds mhdlvsdlle fcfytfresh alkvefpaml 961 veiisdqlpk vesgnakply fhrk

[0198] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of a vitamin D receptor. For example, the ligand-binding domain of a vitamin D receptor can be amino acids 124 to 426 of SEQ ID NO: 79. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence 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.

[0199] SEQ ID NO: 79 (human) 1 meamaastsl pdpgdfdrnv pricgvcgdr atgfhfnamt cegckgffrr smkrkalftc 61 pfngdcritk dnrrhcqacr lkrcvdigmm kefiltdeev qrkremilkr keeealkdsl 121 rpklseeqqr iiailldahh ktydptysdf cqfrppvrvn dgggshpsrp nsrhtpsfsg 181 dsssscsdhc itssdmmdss sfsnldlsee dsddpsvtle lsqlsmlphl adlvsysiqk 241 vigfakmipg frdltsedqi vllkssaiev imlrsnesft mddmswtcgn qdykyrvsdv 301 tkaghsleli eplikfqvgl kklnlheeeh vllmaicivs pdrpgvqdaa lieaiqdrls 361 ntlqtyircr hpppgshlly akmiqkladl rslneehskq yrclsfqpec smkltplvle 421 vfgneis

[0200] Another non-limiting example of a ligand binding domain is the ligand binding domain of the PPARα receptor. For example, the ligand-binding domain of a PPARα receptor can be amino acids 201 to 467 of SEQ ID NO: 80. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence 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.

[0201] SEQ ID NO: 80 (human) 1 mvdtesplcp lspleagdle splseeflqe mgniqeisqs igedssgsfg fteyqylgsc 61 pgsdgsvitd tlspasspss vtypvvpgsv despsgalni ecricgdkas gyhygvhace 121 gckgffrrti rlklvydkcd rsckiqkknr nkcqycrfhk clsvgmshna irfgrmprse 181 kaklkaeilt cehdiedset adlkslakri yeaylknfnm nkvkarvils gkasnnppfv 241 ihdmetlcma ektlvaklva ngiqnkeaev rifhccqcts vetvteltef akaipgfanl 301 dlndqvtllk ygvyeaifam lssvmnkdgm lvaygngfit reflkslrkp fcdimepkfd 361 famkfnalel ddsdislfva aiiccgdrpg llnvghiekm qegivhvlrl hlqsnhpddi 421 flfpkllqkm adlrqlvteh aqlvqiikkt esdaalhpll qeiyrdmy

[0202] Another non-limiting example of a ligand-binding domain is the ligand-binding domain of a PPARβ / δ receptor. For example, the ligand-binding domain of a PPARβ / δ receptor can be amino acids 173 to 440 of SEQ ID NO: 81. In some embodiments, the hormone receptor ligand-binding domain is or comprises a sequence that 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.

[0203] SEQ ID NO: 81 (human) 1 meqpqeeape vreeeekeev aeaegapeln ggpqhalpss sytdlsrsss ppslldqlqm 61 gcdgascgsl nmecrvcgdk asgfhygvha cegckgffrr tirmkleyek cersckiqkk 121 nrnkcqycrf qkclalgmsh nairfgrmpe aekrklvagl tanegsqynp qvadlkafsk 181 hiynaylknf nmtkkkarsi ltgkashtap fvih...

Claims

1. A single-chain chimeric antigen receptor, the single-chain chimeric antigen receptor comprising: (I) an extracellular antigen-binding domain that specifically binds to a tumor-associated antigen; (II) a transmembrane domain derived from the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the zeta chain of the T cell receptor, CD28, CD3, CD35, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD86, CD134, CD137, or CD154; (III) a hormone receptor ligand binding domain derived from an estrogen receptor, a progesterone receptor, or an androgen receptor; (IV) an intracellular costimulatory domain; and (V) an intracellular immunoreceptor tyrosine-based activation motif (ITAM); and Including, the hormone receptor ligand-binding domain is present in the intracellular domain of the single-chain chimeric antigen receptor or is present between the extracellular antigen-binding domain and the transmembrane domain of the single-chain chimeric antigen receptor; the transmembrane domain and the hormone receptor ligand binding domain are directly adjacent to each other or are separated by 1 to 500 amino acids; When the hormone receptor ligand-binding domain is present in the intracellular domain of the single-chain chimeric antigen receptor, the hormone receptor ligand-binding domain is linked to (i) the C-terminus of the intracellular costimulatory domain, or (ii) the C-terminus of the transmembrane domain; The intracellular costimulatory domain is located on the N-terminal side of the intracellular ITAM. Single-chain chimeric polypeptides.

2. The single-chain chimeric antigen receptor of claim 1, wherein the hormone receptor ligand-binding domain is located between the extracellular antigen-binding domain and the transmembrane domain of the single-chain chimeric antigen receptor.

3. The single-chain chimeric antigen receptor of claim 2, comprising, from N-terminus to C-terminus, an extracellular antigen-binding domain that is an scFv, a linker, a hormone receptor ligand-binding domain, a linker, a hinge region, the transmembrane domain, the intracellular costimulatory domain, and the intracellular ITAM.

4. The single-chain chimeric antigen receptor of claim 1 , wherein the hormone receptor ligand-binding domain is present within the intracellular domain of the single-chain chimeric antigen receptor.

5. (i) from N-terminus to C-terminus, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain that is an scFv, a hinge region, a transmembrane domain, an optional linker, an intracellular costimulatory domain, an optional linker, an intracellular hormone receptor ligand-binding domain, an optional linker, and an intracellular ITAM; (ii) from N-terminus to C-terminus, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain that is an scFv, a hinge, a transmembrane domain, an optional linker, an intracellular costimulatory domain, an optional linker, an intracellular ITAM, an optional linker, and an intracellular hormone receptor ligand-binding domain; (iii) from N-terminus to C-terminus, the single-chain chimeric antigen receptor comprises an extracellular antigen-binding domain that is an scFv, a hinge region, a transmembrane domain, an optional linker, an intracellular hormone receptor ligand-binding domain, an optional linker, an intracellular costimulatory domain, an optional linker, and an intracellular ITAM; The single-chain chimeric antigen receptor of claim 4.

6. The single-chain chimeric antigen receptor of claim 1, wherein the tumor-associated antigen is human CD19.

7. The single-chain chimeric antigen receptor of claim 6, wherein the extracellular antigen-binding domain is an scFV that specifically binds to human CD19.

8. The single-chain chimeric antigen receptor of claim 7, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 104 and the amino acid sequence set forth in SEQ ID NO:

108.

9. The single-chain chimeric antigen receptor of claim 1 , wherein the transmembrane domain is a human CD8α transmembrane domain.

10. The single-chain chimeric antigen receptor of claim 1 , wherein the transmembrane domain comprises or consists solely of the amino acid sequence of SEQ ID NO: 43, 56, or 114.

11. The single-chain chimeric antigen receptor according to claim 1, wherein the transmembrane domain comprises or consists solely of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 9.

12. The single-chain chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises or consists solely of the amino acid sequence of amino acids 153 to 179 of SEQ ID NO:

1.

13. The single-chain chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises or consists solely of the amino acid sequence of amino acids 31 to 51 of SEQ ID NO:

2.

14. The single-chain chimeric antigen receptor of claim 1, wherein the hormone receptor ligand-binding domain is a ligand-binding domain derived from a human estrogen receptor.

15. 15. The single-chain chimeric antigen receptor of claim 14, wherein the hormone receptor ligand binding domain is (i) SEQ ID NO: 110; (ii) amino acids 10-247 of SEQ ID NO: 10; (iii) amino acids 4-241 of SEQ ID NO: 11; or (iv) a wild-type estrogen receptor ligand binding domain except for having a mutation at one or both of positions 521 and 537 of SEQ ID NO: 119, wherein optionally the mutation is G521R and / or Y537F.

16. The single-chain chimeric antigen receptor of claim 1, wherein the hormone receptor ligand-binding domain is a ligand-binding domain derived from a human progesterone receptor.

17. 17. The single-chain chimeric antigen receptor of claim 16, wherein the hormone receptor ligand binding domain comprises the amino acid sequence of: (i) amino acids 11 to 258 of SEQ ID NO: 12; or (ii) amino acids 14 to 261 of SEQ ID NO:

13.

18. The single-chain chimeric antigen receptor of claim 1, wherein the hormone receptor ligand-binding domain is a ligand-binding domain derived from the human androgen receptor.

19. The single-chain chimeric antigen receptor of claim 18, wherein the hormone receptor ligand-binding domain comprises the amino acid sequence of amino acids 19 to 264 of SEQ ID NO:

16.

20. The single-chain chimeric antigen receptor of claim 1, wherein the intracellular costimulatory domain is a cytoplasmic costimulatory domain derived from a human protein selected from the group consisting of 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.

21. The single-chain chimeric antigen receptor of claim 1, wherein the intracellular costimulatory domain is (i) a 4-1BB costimulatory domain comprising or consisting of the amino acid sequence of SEQ ID NO: 44 or 116, or (ii) a CD28 costimulatory domain comprising or consisting of amino acids 180 to 220 of SEQ ID NO:

18.

22. The single-chain chimeric antigen receptor of claim 1, wherein the intracellular ITAM comprises a cytoplasmic signal sequence derived from human CD3ζ.

23. 23. The single-chain chimeric antigen receptor of claim 22, wherein the intracellular ITAM comprises or consists solely of the sequence of SEQ ID NO:

48.

24. The single-chain chimeric antigen receptor of claim 3 or 5, wherein the hinge region comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 42, 55, or 112.

25. The single-chain chimeric antigen receptor of claim 3 or 5, wherein the linker comprises or consists solely of GS and / or the amino acid sequence of SEQ ID NO:

106.

26. 2. The single-chain chimeric antigen receptor of claim 1, wherein the hormone receptor ligand-binding domain and the transmembrane domain are separated by 1 to 500 amino acids.

27. The single-chain chimeric antigen receptor of claim 1, comprising an amino acid sequence that is at least 95% identical to any one of the amino acid sequences set forth in SEQ ID NO: 31 (i) amino acids 22 to 812 of SEQ ID NO: 33; (ii) amino acids 22 to 812 of SEQ ID NO: 33; (iii) amino acids 22 to 820 of amino acid sequence 35; (iv) amino acids 27 to 507 of SEQ ID NO: 52; or (v) amino acids 33 to 798 of amino acid sequence 98.

28. The single-chain chimeric antigen receptor of claim 1, comprising an amino acid sequence represented by any one of: (i) amino acids 22 to 812 of SEQ ID NO: 31; (ii) amino acids 22 to 812 of SEQ ID NO: 33; (iii) amino acids 22 to 820 of amino acid sequence 35; (iv) amino acids 27 to 507 of SEQ ID NO: 52; or (v) amino acids 33 to 798 of amino acid sequence 98.

29. A multi-chain chimeric antigen receptor comprising at least one first polypeptide which is the single-chain chimeric antigen receptor of any one of claims 1 to 28.

30. A nucleic acid(s) comprising a nucleotide sequence encoding the single-chain chimeric antigen receptor of any one of claims 1 to 28 or the multi-chain chimeric antigen receptor of claim 29.

31. A vector comprising the nucleic acid(s) of claim 30.

32. 32. A human cell comprising the nucleic acid(s) of claim 31 or the vector of claim 31.

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

34. An in vitro method for inducing plasma membrane localization of a single-chain chimeric antigen receptor or a multi-chain chimeric antigen receptor in human cells, comprising: A method comprising contacting a human cell expressing (i) the single-chain chimeric antigen receptor of any one of claims 1 to 28, or (ii) the multi-chain chimeric antigen receptor of claim 29, with a hormone or hormone analog in an amount sufficient to induce localization of the single-chain chimeric antigen receptor or the multi-chain chimeric antigen receptor to the extracellular side of the plasma membrane of the human cell.

35. 35. The method of claim 34, wherein the human cell is a T cell.

36. 30. A pharmaceutical composition for treating cancer in a human subject, comprising a T cell expressing the single-chain chimeric antigen receptor of any one of claims 1 to 28 or the multi-chain chimeric antigen receptor of claim 29, wherein the antigen-binding domain specifically binds to a tumor-associated antigen expressed by cancer in the human subject, and optionally the tumor-associated antigen is human CD19.

37. 37. The pharmaceutical composition of claim 36, wherein the T cell expresses the single-chain chimeric antigen receptor of claim 27 or 28.

38. a transmembrane domain derived from the human T cell receptor α chain, the human T cell receptor β chain, the human T cell receptor ζ chain, human CD28, human CD3, human CD35, human CD33, human CD37, human CD64, human CD80, human CD45, human CD4, human CD5, human CD8, human CD9, human CD16, human CD22, human CD86, human CD134, human CD137, or human CD154; a hormone receptor ligand binding domain that is a human estrogen receptor ligand binding domain, a human progesterone receptor ligand binding domain, or a human androgen receptor ligand binding domain, and that is present intracellularly or extracellularly; A single-chain chimeric polypeptide comprising: the transmembrane domain and the hormone receptor ligand binding domain are either directly adjacent to each other or are separated by 1 to 500 amino acids; the transmembrane domain and the hormone receptor ligand binding domain are not both present in the same endogenous single polypeptide chain in humans; Single-chain chimeric polypeptides.

39. an extracellular antigen-binding domain that specifically binds to a tumor antigen; a transmembrane domain derived from the human T cell receptor α chain, the human T cell receptor β chain, the human T cell receptor ζ chain, human CD28, human CD3, human CD35, human CD33, human CD37, human CD64, human CD80, human CD45, human CD4, human CD5, human CD8, human CD9, human CD16, human CD22, human CD86, human CD134, human CD137, or human CD154; a hormone receptor ligand binding domain that is a human estrogen receptor ligand binding domain, a human progesterone receptor ligand binding domain, or a human androgen receptor ligand binding domain, and that is present intracellularly or extracellularly; an intracellular costimulatory domain; a human intracellular immunoreceptor tyrosine-based activation motif (ITAM); A single-chain chimeric antigen receptor comprising: the transmembrane domain and the hormone receptor ligand binding domain are either directly adjacent to each other or are separated by 1 to 500 amino acids; wherein both the transmembrane domain and the hormone receptor ligand binding domain are not present in the same endogenous single polypeptide chain in humans; the intracellular costimulatory domain is located on the N-terminal side of the human intracellular ITAM; The hormone receptor ligand-binding domain is located C-terminal to the extracellular antigen-binding domain, or C-terminal to the transmembrane domain or the intracellular costimulatory domain. Single-chain chimeric antigen receptor polypeptides.

40. The single-chain chimeric antigen receptor of claim 39, wherein the intracellular antigen-binding domain specifically binds to human CD19.

41. The single-chain chimeric antigen receptor of claim 39 or 40, wherein the extracellular antigen-binding domain is an scFv.

Images