HK40000445A

HK40000445A - Conditionally active heterodimeric polypeptides and methods of use thereof

Info

Publication number: HK40000445A
Application number: HK19123816.1A
Authority: HK
Inventors: J·W·汤顿; W·A·利姆; C－Y·吴
Original assignee: 加利福尼亚大学董事会
Priority date: 2016-01-08
Filing date: 2017-01-06
Publication date: 2020-02-07

Description

Conditionally active heterodimeric polypeptides and methods of use thereof

Cross-referencing

This application claims the benefit of U.S. provisional patent application No. 62/276,725 filed on 8/1/2016, which is hereby incorporated by reference in its entirety.

Statement regarding federally sponsored research

The invention was accomplished with government support under grant numbers R01 CA196277, P50 GM081879, and R01 GM055040 awarded by the National Institutes of Health. The government has certain rights in the invention.

Incorporation by reference of sequence listing provided in text file

A sequence listing of the text file "UCSF-524 WO _ seqlist _ st25. txt" created as 2017, 1, 6, and having a size of 2,005KB is hereby provided. The contents of the text file are incorporated by reference herein in their entirety.

Introduction to the design reside in

In cell-based adoptive immunotherapy, immune cells isolated from a patient may be modified to express synthetic proteins that enable the immune cells to perform new therapeutic functions after subsequent transfer back into the patient. Examples of such synthetic proteins are Chimeric Antigen Receptors (CARs) and engineered T Cell Receptors (TCRs). Examples of currently used CARs are fusions of an extracellular recognition domain (e.g., an antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen engagement, the intracellular signaling portion of the CAR can elicit an activation-related response in the immune cell, such as the release of cytolytic molecules that induce tumor cell death, and the like. However, such CARs cannot be pharmacologically controlled. Safe human testing and clinical use of such CARs and engineered TCRs requires good control over the powerful stimulatory activity of these highly engineered proteins and protein complexes. In some cases, control is required to inhibit, prevent, or otherwise modulate immune cell activation when activation of the stimulation receptor is designed to be undesirable, becomes undesirable, or is no longer necessary.

SUMMARY

The present disclosure provides conditionally active heterodimeric polypeptides. Conditionally active heterodimeric polypeptides are active in the presence of a dimerizing agent that induces dimerization of the heterodimeric polypeptides. The conditionally active heterodimeric polypeptides of the present disclosure are useful in a variety of research and therapeutic methods, which are also provided.

The disclosure provides a heterodimeric conditionally active polypeptide comprising a first chimeric polypeptide comprising a first member of a dimerization pair and a first heterologous polypeptide, and B) a second chimeric polypeptide comprising a second member of a dimerization pair and a second heterologous polypeptide, wherein the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor (SRC) and the second member of the dimerization pair comprises a coresin receptor (LBD) or a coregulatory receptor (SRC) 140, and the second member of the dimerization pair comprises a coresin receptor (SRC) 140, or a chimeric polypeptide of SEQ ID NO) (SEQ ID NO) 140, a chimeric polypeptide of SEQ ID NO) (SEQ ID NO) (NO: 7, SEQ ID NO) (SEQ ID NO:14) NO) (SEQ ID NO:14, NO) (SEQ ID NO:14) NO) (SEQ ID NO:14, NO) (SEQ ID NO: 14: 10: 140) or 140: 140), and a [ SEQ ID NO), and a [ SEQ ID NO) a [ 10: 140, a [ 10 ] a [ SEQ ID NO: 140, a No), and a ] a [ SEQ ID NO: 140, a [ SEQ ID NO), and a [ SEQ ID NO) a [ 10 ] a [ 10 ] a [ SEQ ID NO:10 ] a [ SEQ ID NO:10 ] a [ SEQ ID NO:10 ] a [ SEQ ID NO:10, a [ 1 [ SEQ ID ] a [ 10 ] a [ 1 [ a ] a [ 1 [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a [ a ] a.

The invention provides a heterodimeric conditionally active receptor comprising a first chimeric polypeptide comprising i a first member of a specific binding pair, ii a first regulatory domain, iii a first member of a dimerization pair, iv a transmembrane domain inserted between the first member of the specific binding pair and the first regulatory domain, and B) a second chimeric polypeptide comprising i a transmembrane domain, ii a second regulatory domain, iii a second member of a dimerization pair, iii a second member of the dimerization pair, and iv a first intracellular signaling domain or comprising a first member of a specific binding pair, i a first member of a specific binding pair, ii a regulatory domain, iii a first member of a dimerization pair, iv a first member of a specific binding pair, and a second polypeptide comprising 16a transmembrane domain, 140a receptor, 12, 140, a chimeric polypeptide comprising 16a second receptor, 140, 12 a transmembrane domain, 12 a chimeric polypeptide, 140, 12 a transmembrane domain, 140, a receptor, 12 a receptor, 140, a receptor, 12, a receptor-12, a receptor-motif, a polypeptide, a receptor-motif, a polypeptide, such as a polypeptide, a receptor-12, a receptor-motif, a receptor-motif, a receptor-polypeptide, a receptor-motif, a receptor-10, a receptor-motif, a receptor-motif, a receptor-motif, a polypeptide, a motif, a polypeptide, a motif, a receptor-specific receptor-motif, a receptor-polypeptide, a motif, a polypeptide, a receptor-polypeptide, a motif, a polypeptide, a receptor-.

The expression cassette is a heterodimeric conditionally-repressed synthetic Immune Cell Receptor (ICR) including a dimerization domain thereof (SEQ ID NO:16) when the receptor is secreted by a receptor such as SEQ ID NO:51) when the receptor is secreted by a receptor such as SEQ ID NO:35, when the receptor is secreted by a receptor such as SEQ ID NO: 140, when the receptor is secreted by a receptor such as SEQ ID NO:51, when the receptor is secreted by a receptor such as SEQ ID NO: 140, when the receptor is secreted by a receptor such as SEQ ID NO:12, when the receptor is secreted by a receptor such as SEQ ID NO:51, when the receptor is secreted by a receptor such as SEQ ID NO:7, when the receptor is secreted by a receptor such as SEQ ID NO:12, when the receptor such as SEQ ID NO:11, when the receptor is secreted by a receptor such as SEQ ID NO:12, when the receptor is secreted by a receptor, when the receptor is secreted by a receptor such as SEQ ID NO:12, when the receptor, or when the receptor is secreted by a receptor such as SEQ ID NO:11, or when the receptor is secreted by a receptor such as SEQ ID NO:11, or 140, or when the receptor is secreted by a receptor such as SEQ ID NO:11, when the receptor such as SEQ ID NO:11, or 140, when the receptor is secreted by a receptor, or when the receptor is secreted by a receptor such as SEQ ID NO:11, or 140, or when the receptor such as a receptor is secreted by a receptor such as SEQ ID NO: 140, or when the receptor such as a receptor such as SEQ ID NO:11, or such as a receptor, or as a receptor such as SEQ ID NO: 140, or a receptor such as SEQ ID NO: 140, or a receptor such as SEQ ID NO: 150 as a receptor such as SEQ ID NO:7, or a receptor such as SEQ ID NO: 140, or a receptor such as SEQ ID NO: 150 as a receptor such as SEQ ID NO: 140, or a receptor such as SEQ ID NO:12, or a receptor such as SEQ ID NO: 150 as SEQ ID NO:12, or a receptor such as SEQ ID NO: 150 as SEQ ID NO: 140, or a receptor such as SEQ ID NO: 140, or SEQ ID NO: 150 as a receptor such as SEQ ID NO: 150 as a receptor, or SEQ ID NO: 150 as SEQ ID NO: 140, or SEQ ID NO:11, or SEQ ID NO:7, or SEQ ID NO: 150 as a receptor such as SEQ ID NO: 140, or SEQ ID NO: 150 as SEQ ID NO: 140, or SEQ ID NO:11, or 140, or SEQ ID NO: 150 as a receptor, or SEQ ID NO:11, or SEQ ID NO: 150 as SEQ ID NO: 140, or a receptor, or SEQ ID NO: 150 as a receptor, or SEQ ID NO: 140, or SEQ ID NO: 150 as a receptor such as a receptor, or SEQ ID NO: 150 as a receptor, or SEQ ID NO: 140, or SEQ ID NO: 150 as a receptor, or SEQ ID NO: 150 as a receptor, or SEQ ID NO: 150 as a receptor, or 140, or SEQ ID NO: 150 as a receptor, or SEQ ID NO: 140, or SEQ ID NO: 150 as a receptor, or 140, or SEQ ID NO: 150 as a receptor, or SEQ ID.

The present disclosure provides a heterodimeric conditionally-repressed Chimeric Antigen Receptor (CAR) comprising a receptor I) extracellular recognition domain (I) a receptor II (I) a receptor III (II) a receptor III (III) a receptor III (II) a receptor III (III) a receptor III (II) a receptor III (III) a receptor III (II) a receptor III (III) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III (II) a receptor III, a receptor III) a receptor III (II) a receptor III, a receptor III) a receptor III.

The present disclosure provides a heterodimeric conditionally-repressed T Cell Receptor (TCR) comprising a synthetic stimulatory TCR comprising a transmembrane domain, ii) a first member of a dimerization pair linked to the transmembrane domain, iii) an engineered TCR polypeptide comprising at least one TCR or chain, wherein the at least one TCR or chain is linked to the transmembrane domain or a first member of the dimerization pair, B) a synthetic TCR repressor comprising a second member of the dimerization pair, iii) an intracellular inhibitory domain linked to a second member of the dimerization pair, wherein the first member of the dimerization pair comprises a nuclear hormone receptor Ligand Binding Domain (LBD) and the second member of the dimerization pair (LBJ) is a nuclear hormone receptor (SEQ ID NO) 14, 140, 12, 140, 12, 140, 35, 140, 12, 140, 8, 140, 12, 140, 7, 8, 140, 8, 7, 8, III, 8, III.

The invention provides a heterodimeric conditionally active Chimeric Antigen Receptor (CAR) comprising a first polypeptide comprising a first member of a specific binding pair SEQ ID NO, ii) a first regulatory domain, iii) a first member of a dimerization pair SEQ ID NO, iv) a transmembrane domain inserted between the first member of the specific binding pair and the first regulatory domain, B) a second polypeptide comprising i) a transmembrane domain, ii) a second regulatory domain, iii) a second member of a dimerization pair SEQ ID NO, 140, a receptor specific receptor SEQ ID NO, a receptor specific receptor SEQ ID NO, a receptor ID NO, such as a receptor ID NO, such as a receptor ID NO, a receptor NO, such as a receptor No, SEQ ID NO, NO.

The present disclosure provides mammalian cells genetically modified to produce conditionally active polypeptides or receptors of heterodimers as described above or elsewhere herein. In some cases, the cell is a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell. In some cases, the cell is a T lymphocyte or an NK cell.

The present disclosure provides nucleic acids comprising nucleotide sequences encoding conditionally active receptors or polypeptides of heterodimers as described above or elsewhere herein. In some cases, the nucleotide sequence is operably linked to a promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a cell type-specific promoter or a tissue-specific promoter. In some cases, the promoter is a T lymphocyte-specific promoter or an NK cell-specific promoter. In some cases, the nucleic acid is an in vitro transcribed RNA. The present disclosure provides recombinant expression vectors comprising nucleic acids. In some cases, the recombinant expression vector is a viral vector, such as a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

The present disclosure provides a method of modulating eukaryotic cell activity, the method comprising: a) expressing a conditionally active polypeptide or receptor of a heterodimer as described above or elsewhere herein in a eukaryotic cell; and b) contacting the cell with the ligand.

The present disclosure provides a method of modulating T lymphocyte activity, the method comprising contacting a T lymphocyte with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a conditionally active receptor for a heterodimer as described above or elsewhere herein, and wherein in the presence of the dimerizing agent and the second member of the specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates the activity of the T lymphocyte, thereby producing a modulated T lymphocyte. In some cases, the second member of the specific binding pair is an antigen. In some cases, the contacting occurs in vivo. In some cases, the T lymphocytes are activated, thereby producing activated T lymphocytes. In some cases, activated T lymphocytes mediate killing of target cells. In some cases, the activated T lymphocytes produce IL-2 and/or IFN- γ. In some cases, the target cell is a cancer cell.

The present disclosure provides a method of making the cell of any one of claims 66-68, the method comprising genetically modifying a mammalian cell with an expression vector comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of a heterodimer as described above or elsewhere herein, or genetically modifying a mammalian cell with an RNA comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of a heterodimer as described above or elsewhere herein. In some cases, the genetic modification is performed ex vivo. In some cases, the cell is a T lymphocyte, a stem cell, an NK cell, a progenitor cell, a cell derived from a stem cell, or a cell derived from a progenitor cell.

The present disclosure provides a method of treating cancer in an individual, the method comprising: i) genetically modifying T lymphocytes obtained from an individual with an expression vector comprising a nucleotide sequence encoding a heterodimeric, conditionally active Chimeric Antigen Receptor (CAR) as described above or elsewhere herein, wherein the antigen-binding domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell of the individual, and wherein the genetic modification is performed ex vivo; ii) introducing genetically modified T lymphocytes into the individual; and iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of conditionally active receptors of heterodimers, wherein the dimerization provides activation of genetically modified T lymphocytes and killing of cancer cells, thereby treating the cancer. In some cases, the dimerizing agent is a nuclear hormone that binds to the LBD of the nuclear hormone receptor and a co-regulator.

The present disclosure provides methods of modulating host cell activity, the methods comprising contacting a host cell with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a conditionally active receptor for a heterodimer as described above or elsewhere herein, and wherein in the presence of the dimerizing agent and the second member of the specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates at least one activity of the host cell. In some cases, the activity is proliferation, cell survival, apoptosis, gene expression, or immune activation. In some cases, the second member of the specific binding pair is an antigen.

Drawings

FIGS. 1A-1F show the amino acid sequence of the Mineralocorticoid Receptor (MR). FIG. 1A: a full-length MR amino acid sequence; FIGS. 1B-1E: the amino acid sequence of the Ligand Binding Domain (LBD) of MR; FIG. 1F: multiple sequence alignments of the LBD of MR of various species. (SEQ ID NO: 70-84).

FIGS. 2A-2I show the amino acid sequence of the Androgen Receptor (AR). FIG. 2A: a full-length AR amino acid sequence; FIGS. 2B-2I: the amino acid sequence of the LBD of AR. (SEQ ID NO: 85-93).

Fig. 3A-3D show the amino acid sequence of the Progesterone Receptor (PR). FIG. 3A: a full length PR amino acid sequence; FIGS. 3B-3C: the amino acid sequence of LBD of PR; FIG. 3D: multiple sequence alignments of LBD of PR of various species. (SEQ ID NO: 94-103).

FIGS. 4A-4D show the amino acid sequence of thyroid hormone receptor β (TR β), FIG. 3A the full length PR amino acid sequence, FIGS. 3B-3C the amino acid sequence of LBD of TR β, FIG. 3D multiple sequence alignments of LBD of TR β of various species (SEQ ID NO: 104-118).

FIGS. 5A-5H show the amino acid sequence of estrogen receptor α (ER α), FIG. 5A: full-length ER α amino acid sequence, FIGS. 5B-5G: the amino acid sequence of LBD of ER α, FIG. 5H: multiple sequence alignment of LBD of ER α of various species (SEQ ID NO: 119-.

FIGS. 6A-6C show the amino acid sequence of estrogen receptor β (ER β), FIG. 6A: full-length ER β amino acid sequence, FIG. 6B: the amino acid sequence of LBD of ER β, FIG. 6C: multiple sequence alignment of LBD of ER β of various species (SEQ ID NO: 134-143).

FIGS. 7A-7E depict amino acid sequences of peroxisome proliferator-activated receptor- γ (PPAR- γ). FIG. 7A: a full length PPAR-gamma amino acid sequence; FIGS. 7B-7D: the amino acid sequence of the LBD of PPAR-gamma; FIG. 7E: multiple sequence alignments of the LBD of PPAR-gamma of various species. (SEQ ID NO: 144-157).

FIGS. 8A-8C show the amino acid sequences of Glucocorticoid Receptor (GR). FIG. 8A: a full length GR amino acid sequence; FIG. 8B: the amino acid sequence of the LBD of GR; FIG. 8C: multiple sequence alignments of LBDs of GR of various species. (SEQ ID NO: 158-166).

FIGS. 9A-9C show the amino acid sequence of Vitamin D Receptor (VDR). FIG. 9A: a full-length VDR amino acid sequence; FIG. 9B: the amino acid sequence of LBD of VDR; FIG. 9C: multiple sequence alignments of LBD of VDR of various species. (SEQ ID NO: 167-172).

FIGS. 10A-10C show the amino acid sequence of thyroid hormone receptor α (TR α), FIG. 10A the full length TR α amino acid sequence, FIG. 10B the amino acid sequence of LBD of TR α, FIG. 10C multiple sequence alignment of LBD of TR α for various species (SEQ ID NO: 173-185).

FIGS. 11A-11C show the amino acid sequence of retinoic acid receptor β (RAR β). FIG. 11A: full-length RAR β amino acid sequence, FIG. 11B: the amino acid sequence of LBD of RAR β, FIG. 11C: multiple sequence alignment of LBD of RAR β of various species (SEQ ID NO: 186-196).

Figure 12 is a schematic of a CAR with the switch open, characterized by the Ligand Binding Domain (LBD) of the nuclear hormone receptor, a co-regulatory peptide and a small molecule as a ternary heterodimerization module.

Figure 13 is a schematic of a PPAR γ -based CAR construct with an open switch.

Figure 14 shows the production of IL-2 cytokines by CAR + Jurkat cells (transduced with the lentiviral constructs shown) with the switch open after 18 hours of co-culture with K562 target cells (+/-CD19Ag, as shown) in the presence or absence of rosiglitazone dimerizer (10 micromolar).

Figure 15 is a schematic of CAR constructs based on the open switch of estrogen receptor α.

Figures 16A-16B show up-regulation of CD69 of ER CAR + Jurkat cells after co-culture with K562 target cells expressing CD19 or an unrelated antigen ("meso") in the presence or absence of a small molecule dimerizing agent (4-hydroxytamoxifen or rapamycin analogue AP21967) for 24 hours. CD69 expression in ER CAR + Jurkat cells was strongly induced by 4-hydroxytamoxifen (EC 50-100 nM) only in the presence of K562 cells expressing CD 19. Rapamycin CARs and dead rapamycin CARs were tested as positive and negative controls, respectively. Dead rapamycin CARs contain mutations in the ITAM domain that render them unable to signal. The data sets presented from back to front in FIG. 16B are CD19 antigen + 4-hydroxytamoxifen 10 μ M, 1 μ M, 100nM, 10nM, 1nM, 0nM, meso antigen +10 μ M rapamycin and meso antigen +0 μ M rapamycin analogue.

Figure 17 provides a schematic of CAR constructs based on the opening switch of estrogen receptor β.

FIGS. 18A-18B show measured upregulation of CD69 in primary human T cells expressing CAR based on the open switch of ER- β/CoRNR after 24 hours of co-culture with K562 target cells expressing CD19 or an unrelated antigen ("meso") in the presence or absence of a small molecule dimerizing agent (4-hydroxytamoxifen or rapamycin analogue). CD69 expression is induced in a dimerizing agent dose-dependent manner. the data series presented from back to front in FIG. 18B are CD19 antigen + 4-hydroxytamoxifen 10. mu.M, 1. mu.M, 100nM, 10nM, 1nM, 0nM, meso antigen + 10. mu.M rapamycin and meso antigen + 0. mu.M rapamycin analogue.

Figure 19 provides a table showing various possible combinations of LBD, co-regulators and dimerizers.

FIGS. 20A-20C show the amino acid sequence of Lyn kinase (FIG. 20A), the N-terminal portion of Lyn kinase (FIG. 20B) and the C-terminal portion of Lyn kinase (FIG. 20C). (SEQ ID NO:197-199)

Figure 21 provides the amino acid sequence of Fak kinase. (SEQ ID NO:200)

FIGS. 22A-22B show the amino acid sequence of FXR (FIG. 22A) and the LBD of FXR (FIG. 22B). (SEQ ID NO:201-202)

FIGS. 23A-23B show the amino acid sequence of LXR α (FIG. 23A) and the LBD of LXR α (FIG. 23B) (SEQ ID NO:203-204)

FIGS. 24A-24B show the amino acid sequence of ROR γ (FIG. 24A) and the LBD of ROR γ (FIG. 24B). (SEQ ID NO:205-206)

FIGS. 25A-25B show the amino acid sequence of RXR α (FIG. 25A) and the LBD of RXR α (FIG. 25B) (SEQ ID NO:207-

FIGS. 26A-26B show the amino acid sequence of PXR (FIG. 26A) and the LBD of PXR (FIG. 26B). (SEQ ID NO:209-210)

FIG. 27 provides Table 1(SEQ ID NO: 211-278).

FIG. 28 provides the amino acid sequence of Streptococcus pyogenes (S. pyogenes) Cas9 (SEQ ID NO: 279).

FIGS. 29-51B provide the amino acid sequences of various co-regulatory polypeptides (SEQ ID NO: 280-302).

Figure 52 shows target cell killing of primary human CD8+ T cells expressing CAR of CD 19-specific ER- β/cockr open switch.

FIG. 53 provides the amino acid sequences of exemplary IL-2 family receptors (SEQ ID NO: 303-313).

FIG. 54 provides the amino acid sequences of exemplary IL-3 family receptors (SEQ ID NO: 314-317).

FIG. 55 provides the amino acid sequences of exemplary IL-6 family receptors (SEQ ID NO: 318-326).

FIG. 56 provides the amino acid sequences of exemplary IL-12 family receptors (SEQ ID NO: 327-329).

FIG. 57 provides the amino acid sequences of exemplary prolactin family receptors (SEQ ID NO: 330-334).

FIG. 58 provides the amino acid sequences of exemplary interferon family receptors (SEQ ID NO: 335-338).

FIG. 59 provides the amino acid sequences of exemplary IL-10 family receptors (SEQ ID NO: 339-345).

FIG. 60 provides the amino acid sequence of an exemplary IL-17 family receptor (SEQ ID NO: 346-350).

FIG. 61 provides the amino acid sequence of an exemplary immunoglobulin-like superfamily receptor (SEQ ID NO: 351-361).

FIG. 62 provides the amino acid sequence of an exemplary tumor necrosis factor family receptor (SEQ ID NO: 362-392).

FIG. 63 provides the amino acid sequences of exemplary chemokine receptors (SEQ ID NO: 393-409).

FIG. 64 provides the amino acid sequence of an exemplary TGF- β family receptor (SEQ ID NO: 410-412).

FIG. 65 provides the amino acid sequence of an exemplary Receptor Tyrosine Kinase (RTK) (SEQ ID NO: 413-469).

Definition of

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length (ribonucleotides or deoxyribonucleotides). Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

"operably linked" refers to juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it affects its transcription or expression.

A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment (i.e., an "insert") may be attached in order to cause replication of the attached segment in a cell.

As used herein, "heterologous" means a nucleotide or polypeptide sequence not present in the native (naturally occurring) nucleic acid or protein, respectively.

The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen (including but not limited to Fab, Fv, scFv, and Fd fragments), chimeric antibodies, humanized antibodies, single chain antibodies (scabs), single domain antibodies (dabs), single domain heavy chain antibodies, single domain light chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, or the like. The antibody may also be conjugated to other moieties, such as members of a specific binding pair, e.g., biotin (a member of a biotin-avidin specific binding pair), and the like. The antibody may also be conjugatedBound to a solid support including, but not limited to, polystyrene plates or beads, and the like. The term also encompasses Fab ', Fv, F (ab')₂And/or other antibody fragments that retain specific binding to the antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which are produced by repeated cell replication from a single cell. That is, the cloning of cells produces only a single antibody species. Although monoclonal antibodies can be produced using hybridoma production techniques, other production methods known to those of skill in the art (e.g., antibodies derived from antibody phage display libraries) can also be used. The antibody may be monovalent or bivalent. An antibody may be an Ig monomer, which is a "Y-shaped" molecule consisting of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

The term "humanized immunoglobulin" as used herein refers to an immunoglobulin comprising immunoglobulin parts of different origin, wherein at least one part comprises an amino acid sequence of human origin. For example, a humanized antibody may comprise portions derived from an immunoglobulin of non-human (such as mouse) origin having the requisite specificity, and immunoglobulin sequences derived from human origin (e.g., chimeric immunoglobulins) that are chemically linked together by conventional techniques (e.g., synthesis) or prepared as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portion of the chimeric antibody may be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising CDRs derived from an antibody of non-human origin and framework regions derived from a light and/or heavy chain of human origin (e.g., a CDR-grafted antibody with or without framework changes). The term humanized immunoglobulin also encompasses chimeric or CDR-grafted single chain antibodies. See, e.g., Cabilly et al, U.S. Pat. Nos. 4,816,567; cabilly et al, European patent No. 0,125,023B 1; boss et al, U.S. Pat. Nos. 4,816,397; boss et al, European patent No. 0,120,694B 1; neuberger, m.s., et al, WO 86/01533; neuberger, m.s. et al, european patent No. 0,194,276B 1; winter, U.S. Pat. No. 5,225,539; winter, european patent No. 0,239,400B 1; padlan, e.a. et al, european patent application No. 0,519,596 a 1. See also Ladner et al, U.S. Pat. nos. 4,946,778; huston, U.S. patent No. 5,476,786; and Bird, R.E., et al, Science,242: 423-.

As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally-occurring heavy chain antibody_HH) And are known to those skilled in the art. They are derived from heavy chain-only antibodies, as described in camelids (Hamers-Casterman et al, 1993; Desmyter et al, 1996). Immunoglobulins lacking light polypeptide chains are found in the family "camelids". "Camelidae" includes old world camelids (Bacillario camel (Camelus bactrianus) and dromedarius) and new world camelids (e.g. alpaca (Llama paccos), Llama (Llama glama), alpaca (Llama guanicoe) and alpaca minor (Llamavicula)). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V_HHAn antibody.

An "antibody fragment" comprises a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (Zapata et al, Protein Eng.8(10):1057-1062 (1995)); domain antibodies (dAb; Holt et al (2003) Trends Biotechnol.21: 484); a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. Pepsin treatment to yield F (ab')₂A fragment that has two antigen combining sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of dimers of a heavy chain variable domain in close, non-covalent association with a light chain variable domain. Therein, theIn the configuration, the three CDRs of each variable domain interact to define the antigen binding site at V_H-V_LOn the surface of the dimer. The six CDRs collectively confer antigen binding specificity to the antibody. However, although a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, it is at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy Chain (CH)₁). Fab' fragment is due to heavy chain CH₁The carboxy terminus of the domain is distinguished from the Fab' fragment by the addition of a small number of residues, including one or more cysteines from the antibody hinge region. Fab '-SH is the name for Fab' herein, in which the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different types (termed κ and λ) based on the amino acid sequence of its constant domains. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA 2. Subclasses can be further divided into various types, such as IgG2a and IgG2 b.

"Single chain Fv" or "sFv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide is further comprised in V_HDomains with V_LA polypeptide linker between the domains that enables the sFv to form the structure required for antigen binding. For a review of sFvs, see Pluckthun in the pharmacology of Monoclonal Antibodies, Vol.113, eds Rosenburg and Moore, Springer-Verlag, N.York, page 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are contained in the same polypeptide chain (V)_H-V_L) Neutralizing light chain variable domain (V)_L) Linked heavy chain variable domains (V)_H). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and two antigen binding sites are created. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161 and Hollinger et al, (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

As used herein, the term "hinge region" refers to a flexible polypeptide connecting region (also referred to herein as a "hinge" or "spacer") that provides structural flexibility and spacing to the flanking polypeptide regions, and may be composed of a natural polypeptide or a synthetic polypeptide. A "hinge region" derived from an immunoglobulin such as IgG1 is generally defined as extending from Glu216 of human IgG1 to Pro230(Burton (1985) molecular. Immunol.,22: 161-206). The hinge region of other IgG isotypes can be aligned to the IgG1 sequence by placing the first cysteine residue and the last cysteine residue at the same position to form an inter-heavy chain disulfide (S-S) bond. The hinge region may be naturally occurring or non-naturally occurring, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425. The hinge region may comprise the complete hinge region derived from antibodies of different classes or subclasses of the CH1 domain. The term "hinge region" may also include regions derived from CD8 and other receptors that have similar functions of providing flexibility and spacing in the flanking regions.

As used herein, the term "affinity" refers to the equilibrium constant for reversible binding of two reagents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (K)_D) And (4) showing. The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 5-fold greater than the affinity of an antibody of an unrelated amino acid sequence0 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 1,000 times or more. The affinity of an antibody for a target protein can be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactivity" and "preferential binding" with respect to antibodies and/or antigen binding fragments are used interchangeably herein.

The term "binding" refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. In some cases, a first member of a specific binding pair present in the extracellular domain of a conditionally active heterodimeric polypeptide of the present disclosure specifically binds to a second member of the specific binding pair. "specifically binds" means to at least about 10^-7M or greater (e.g., 5x 10)^-7M、10^-8M、5x10^-8M and greater). By "non-specific binding" is meant binding at less than about 10^-7Binding by affinity of M, e.g. having 10^-6M、10^-5M、10^-4Binding of affinity of M.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes: fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; an immunolabeling protein; and so on.

An "isolated" polypeptide is one that has been identified and separated from and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the polypeptide will: (1) purified to greater than 90 wt.%, greater than 95 wt.%, or greater than 98 wt.%, e.g., greater than 99 wt.% of the protein as determined by the labor method (Lowry method); (2) purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue (Coomassie blue) or silver staining under reducing or non-reducing conditions. An isolated polypeptide includes an in situ polypeptide within a recombinant cell, as at least one component of the polypeptide's natural environment will not be present. In some cases, the isolated polypeptide will be prepared by at least one purification step.

As used herein, the term "module" refers to a contiguous polypeptide sequence or fragment thereof associated with some function, particularly a biological function.

The terms "domain" and "motif, used interchangeably herein, refer to an unstructured fragment of a structured domain and polypeptide having one or more specific functions, which, although unstructured, retain one or more specific functions. For example, a structural domain may encompass, but is not limited to, a plurality of amino acids, or portions thereof, that are contiguous or non-contiguous in a folded polypeptide, comprising a three-dimensional structure that contributes to a particular function of the polypeptide. In other cases, a domain may comprise an unstructured segment of a polypeptide that contains multiple two or more amino acids or portions thereof and maintains the specific function of the unfolded or disordered polypeptide. Also encompassed within this definition are domains that may be disordered or unstructured, but which become structured or ordered upon association with a target or binding partner. Non-limiting examples of domains that are unstructured in nature and domains that are unstructured in nature are described, for example, in Dyson and Wright.

The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein to refer to a synthetic, multi-modular molecule capable of triggering or inhibiting immune cell activation, typically, but not exclusively, comprising an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term "CAR" is not specifically limited to CAR molecules, but also includes CAR variants. CAR variants include split CARs in which the extracellular portion (e.g., ligand binding portion) and the intracellular portion (e.g., intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include switch-on CARs that are conditionally activatable CARs, e.g., comprising a split CAR, wherein the conditional heterodimerization of the two parts of the split CAR is under pharmacological control. CAR variants also include bispecific CARs that include a secondary CAR binding domain that can amplify or inhibit the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (icars), which can be used, for example, as components of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules or derivatives thereof (i.e., CAR variants) are described, for example, in PCT application nos. US 2014/016527; fedorov et al Sci Transl Med (2013); 215ra172 (215); glienke et al Front Pharmacol (2015)6: 21; kakarla and Gottschalk 52Cancer J (2014)20(2) 151-5; riddell et al Cancer J (2014)20(2) 141-4; cancer J (2014)20(2) 127-33 of Pegram et al; chemale et al Immunol Rev (2014)257(1): 91-106; barrett et al Annu RevMed (2014)65: 333-47; sacelain et al Cancer Discov (2013)3(4): 388-98; cartellieri et al, JBiomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

As used herein, the term "immune cell" generally includes white blood cells (leukocytes) derived from Hematopoietic Stem Cells (HSCs) produced in the bone marrow. "immune cells" include, for example, lymphocytes (T cells, B cells, Natural Killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

"T cells" include all types of immune cells expressing CD3, whichIncluding helper T cells (CD 4)⁺Cells), cytotoxic T cells (CD 8)⁺Cells), regulatory T cells (tregs), and γ - δ T cells.

"cytotoxic cells" include CD8⁺T cells, Natural Killer (NK) cells and neutrophils, which are capable of mediating a cytotoxic response.

As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES); mesenchymal Stem Cells (MSCs); induced pluripotent stem cells (iPS); and committed progenitors (hematopoietic stem cells (HSCs); bone marrow derived cells, neural progenitors, etc.).

As used herein, the terms "treatment," treating, "and the like" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or a side effect attributable to the disease. As used herein, "treatment" covers any treatment of a disease in a mammal (particularly a human) and includes: (a) preventing a disease from occurring in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e. arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murine (rat, mouse), non-human primate, human, canine, feline, ungulate (e.g., equine, bovine, ovine, porcine, caprine), lagomorpha, and the like. In some cases, the subject is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, such as a rat or a mouse. In some cases, the individual is an animal of the order lagomorpha, e.g., a rabbit.

"therapeutically effective amount" or "effective amount" refers to the amount of an agent or a combined amount of two agents that, when administered to a mammal or other subject to treat a disease, is sufficient to effect such treatment of the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject to be treated.

The term "heteromeric" as used herein refers to a polypeptide or protein that contains more than one subunit. Such heteromeric polypeptides may be referred to as "heteromers" in some instances. A heteromer polypeptide can contain two or more different polypeptides, wherein a different polypeptide is defined as at least two different polypeptides, however, such different polypeptides may or may not comprise one or more portions of similar and/or identical amino acid sequences. In some cases, two or more polypeptides of a heteromer do not share the same amino acid sequence or do not share the same domain. In some cases, a heteromer may consist of two different polypeptides or two different types of polypeptides, and may be referred to as a heterodimer. In some cases, a heteromer can be composed of three different polypeptides or three different types of polypeptides, and can be referred to as a heterotrimer. In some cases, a heteromer can be composed of two or more different polypeptides, or two or more different types of polypeptides, including, but not limited to, for example, three or more different polypeptides, four or more different polypeptides, five or more different polypeptides, six or more different polypeptides, seven or more different polypeptides, eight or more different polypeptides, and the like.

The term "synthetic" as used herein generally refers to a non-naturally occurring artificially derived polypeptide or a nucleic acid encoding a polypeptide. Such synthetic polypeptides and/or nucleic acids may be reassembled from basic subunits (including, e.g., single amino acids, single nucleotides, etc.) or may be derived, e.g., by recombinant methods, from pre-existing polypeptides or polynucleotides (whether naturally-occurring or artificially-derived).

As used herein, the term "recombinant" describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, due to the origin or manipulation of the polynucleotide, is not associated with all or part of the polynucleotide sequence with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term recombinant term as used with respect to a host cell or virus is intended to mean a host cell or virus into which a recombinant polynucleotide has been introduced. Recombination is also used herein with respect to a material (e.g., a cell, nucleic acid, protein, or vector) by which is meant that the material has been modified by the introduction of a heterologous material (e.g., a cell, nucleic acid, protein, or vector).

"biological samples" encompass a variety of sample types obtained from an individual or population of individuals and may be used in diagnostic, monitoring, or screening assays. The definition encompasses liquid samples of blood and other biological origin, solid tissue samples (such as biopsy specimens or tissue cultures) or cells derived therefrom and progeny thereof. The definition also includes samples that have been manipulated in any manner after being obtained, such as by mixing or pooling individual samples, treating with reagents, solubilizing or enriching for certain components (such as cells, polynucleotides, polypeptides, etc.). The term "biological sample" encompasses clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood components (such as plasma and serum), and the like. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

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

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in which the publications are cited.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a dimerizing agent" includes a plurality of such dimerizing agents, and reference to "the antigen binding domain" includes reference to one or more antigen binding domains and equivalents thereof known to those skilled in the art, and so forth. It is also noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," etc., or use of a "negative" limitation in reciting claim elements.

It is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations that are embodiments of the invention are expressly contemplated herein and disclosed herein as if each and every combination were individually and specifically disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly contemplated and are disclosed herein as if each and every such subcombination was individually and specifically disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication date provided may be different from the actual publication date that may require independent confirmation.

Detailed Description

Conditionally active heterodimeric polypeptides

The present invention provides conditionally active polypeptides of a heterodimer comprising: a) a first chimeric polypeptide comprising a first member of a dimerization pair and a first heterologous polypeptide; and b) a second chimeric polypeptide comprising a second member of a dimerization pair and a second heterologous polypeptide. In some cases, the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor. In other cases, the first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor. The first and second chimeric polypeptides dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulatory factor.

In some cases, the function of the conditionally active heterodimeric polypeptides of the present disclosure does not utilize the DNA binding function or transcriptional activation function of the nuclear hormone receptor from which the LBD is derived. In other words, the conditionally active heterodimeric polypeptides of the present disclosure may function independently of any DNA binding function of the nuclear hormone receptor from which the LBD is derived. In some cases, activation of a conditionally active heterodimeric polypeptide of the present disclosure does not result in transcriptional activation functions attributable to a domain (e.g., DNA binding domain) of the nuclear hormone receptor from which the LBD of the conditionally active heterodimeric polypeptide is derived. In some cases, the conditionally active heterodimeric polypeptides of the present disclosure do not contain a DNA binding domain of a nuclear hormone receptor, including, for example, the DNA binding domain of the nuclear hormone receptor from which the LBD of the conditionally active heterodimeric polypeptide is derived.

Conditionally active heterodimeric polypeptides of the present disclosure can be activated in some cases. Conditionally active heterodimeric polypeptides of the present disclosure may have repressor activity in some cases. For example, when the conditionally active heterodimeric polypeptide of the present disclosure is present in a cell membrane and the cell is contacted with a dimerizing agent that induces LBD binding to a co-regulatory factor, in some cases, the conditionally active heterodimeric polypeptide activates the cell. For example, where the conditionally active heterodimeric polypeptide of the present disclosure is present in a cell membrane and the cell is contacted with a dimerizing agent that induces LBD binding to a co-regulatory factor, in some cases, the conditionally active heterodimeric polypeptide suppresses an activity within or of the cell. In some cases, activation of the conditionally active heterodimeric polypeptides of the present disclosure is dependent on the presence of a dimerizing agent. In some cases, activation of a conditionally active heterodimeric polypeptide of the present disclosure is dependent on the presence of a dimerizing agent and a second agent (e.g., an antigen). Depending on the nature of the conditionally active heterodimeric polypeptide and/or depending on the nature of the dimerizing agent, activation of the conditionally active heterodimeric polypeptide of the present disclosure can result in activation of cellular activity (where the conditionally active heterodimeric polypeptide is present in the cell) or repression of cellular activity (where the conditionally active heterodimeric polypeptide is present in the cell). Depending on the nature of the conditionally active heterodimeric polypeptide and/or depending on the nature of the dimerizing agent, activation of a conditionally active heterodimeric polypeptide of the present disclosure may result in activation of the activity of a heterologous polypeptide present in the conditionally active heterodimeric polypeptide, or may result in inhibition of the activity of a heterologous polypeptide present in the conditionally active heterodimeric polypeptide.

By "conditionally active" is meant that the activity attributable to the relevant heterodimeric polypeptide depends on a condition, such as, for example, dimerization of the heterodimeric portion of the polypeptide. For example, the activity of the heterodimeric polypeptides of the disclosure may be conditionally low dependent on the presence of the dimerizing agent. In such cases, the heterodimeric polypeptide is "activatable," i.e., can be activated (e.g., by binding a second member of a specific binding pair of the polypeptides, such as an antigen), in the presence of the dimerizing agent. Conditionally activatable polypeptides will generally not be activatable in the absence of a dimerizing agent, i.e., the relevant polypeptide will not be activatable in the absence of a dimerizing agent, whether or not some additional functional condition is met, such as the presence and/or binding of a second member of a specific binding pair for the polypeptide (e.g., an antigen).

The activity attributable to the relevant polypeptide, which may be conditionally dependent on, for example, the presence of the relevant dimerizing agent, may vary and will generally include any activity, including activating and repressing activities as described above and in more detail below. As an example, as described in more detail below, a conditionally active open switch Chimeric Antigen Receptor (CAR) can be activated in the presence of a relevant dimerizing agent, such that in the presence of the dimerizing agent and a relevant member of a specific binding pair that binds to the polypeptide, signal transduction produced by the open switch CAR can activate a cell (e.g., an immune cell) in which the open switch CAR resides. In the opposite case, in the absence of the relevant dimerizing agent, no signal transduction resulting in activation of the cell expressing the open-switched CAR occurs, regardless of the presence or absence of the relevant member of the specific binding pair to which the open-switched CAR binds. As another example, as described in more detail below, repression of a CAR attributable to a conditionally active off-switch can be activated in the presence of an associated dimerizing agent, such that in the presence of the dimerizing agent and an associated member of a specific binding pair that binds to a polypeptide, the off-switch CAR can repress activation of a cell (e.g., an immune cell) in which the off-switch CAR resides. In the opposite case, in the absence of the relevant dimerizing agent, activation of the off-switch CAR, whether or not the relevant member of the specific binding pair to which the off-switch CAR binds is present, does not occur, which results in repression of activation of the cell expressing the off-switch CAR.

Interacting polypeptides

The first heterologous polypeptide and the second heterologous polypeptide of the conditionally active heterodimeric polypeptides of the invention can be any polypeptide that exhibits activity when in proximity to each other, e.g., when the first chimeric polypeptide and the second chimeric polypeptide dimerize by inducing LBD binding to a co-regulatory factor binding dimerizing agent, the first heterologous polypeptide and the second heterologous polypeptide alone (e.g., when not in proximity to each other) do not exhibit activity, in other words, the activity of the first heterologous polypeptide and the second heterologous polypeptide results from the assembly of the two polypeptides, e.g., in some cases, the first heterologous polypeptide is a receptor and the second heterologous polypeptide is a co-receptor, in some cases, the first heterologous polypeptide is a T Cell Receptor (TCR) α chain, and the second heterologous polypeptide is a TCR β chain.

In some cases, the first heterologous polypeptide and the second heterologous polypeptide have the same amino acid sequence. In some cases, the first heterologous polypeptide and the second heterologous polypeptide have different amino acid sequences.

Cleavage products

In some cases, the first heterologous polypeptide and the second heterologous polypeptide are split products of a single parent polypeptide that exhibits parent polypeptide activity when in proximity to each other, e.g., when the first chimeric polypeptide and the second chimeric polypeptide dimerize via a dimerizing agent that induces LBD binding to a co-regulatory factor. For example, in some cases, the first heterologous polypeptide is an N-terminal portion of the parent polypeptide; and the second heterologous polypeptide is the C-terminal portion of the parent polypeptide. The N-terminal portion and the C-terminal portion of the parent polypeptide do not independently exhibit the activity of the parent polypeptide when split such that they are not in proximity to each other. For example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a kinase. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a protease. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a fluorescent protein; see, e.g., Ghosh et al (2000) J.Am.chem.Soc.122: 5658. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are split products of ubiquitin; see, e.g., Johnsson and Varshavsky (1994) Proc. Natl. Acad. Sci. USA 91: 10340. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a lactamase polypeptide; see, e.g., Galarneau et al (2002) nat. Biotechnol.20: 619. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are split products of luciferase; see, e.g., Luker et al (2004) Proc.Natl.Acad.Sci.USA 101: 12288; remy and Michnick (2006) nat. methods 3: 977; and Paulmurugan and Gambrir (2003) anal. chem.75: 1584. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a caspase. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a phosphatase. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of an endonuclease. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of an RNA-guided endonuclease. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of Tobacco Etch Virus (TEV) protease; see, e.g., Gray et al (2010) Cell 142: 637. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a tyrosine phosphatase; see, e.g., Camacho-Soto et al (2014) J.Am.chem.Soc.136: 17078. As another example, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a Cas9 polypeptide; see, e.g., Zetche et al (2015) nat. Biotechnol.33: 139.

As mentioned above, in some cases the first and second heterologous polypeptides are cleavage products of kinases, tyrosine kinases may be parent polypeptides, and the cleavage products of parent tyrosine kinases may be first and second heterologous polypeptides, for example, tyrosine kinases may be cleaved between amino acids E393 and D394 of the amino acid sequence shown in fig. 20A or between corresponding sites in different tyrosine kinases, such that the first heterologous polypeptide is an N-terminal portion of the tyrosine kinase ending with E393, and the second heterologous polypeptide is a C-terminal portion of the tyrosine kinase starting with D, as examples, the first heterologous polypeptide may comprise the amino acid sequence shown in fig. 20B, and the second heterologous polypeptide may comprise the amino acid sequence shown in fig. 20C, as another embodiment, tyrosine kinase may be cleaved between amino acids E572 and D573 of the amino acid sequence shown in fig. 21 or between corresponding sites in different tyrosine kinases, such that the first heterologous polypeptide is an N-terminal portion of the tyrosine kinase ending with E572, and the second heterologous polypeptide is a tyrosine kinase ending with e.g. 3 e.g. kinase, e.g. 3, K, kinase, PDGF, etc., including all receptor subtypes (including PDGF, K-3, etc., PDGF, etc., PDGF, K-receptor subtypes, etc., including rat, K, PDGF, K-receptor subtypes, etc., including rat, K-receptor subtypes (including rat, PDGF, K-1, PDGF, K-kinase, PDGF, K-receptor subtypes, etc., PDGF, etc., including rat, PDGF, K, PDGF, K, etc. 1, K, PDGF, K, etc. 1, K-kinase, etc. 1, K-kinase, K-kinase including all subtypes (including tyrosine kinase, K-kinase, K-kinase, K, etc. 1, K, etc. 1, K, etc. 1, K.

As described above, in some cases, the first heterologous polypeptide and the second heterologous polypeptide are cleavage products of a caspase. Chelur and Chalfie (2007) Proc.Natl.Acad.Sci.USA 104: 2283. In some cases, the caspase is an apoptotic caspase.

As described above, in some cases, the first and second heterologous polypeptides are cleavage products of a Cas9 polypeptide. The Cas9 polypeptide can comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in fig. 28. In some cases, Cas9 is catalytically inactive Cas9 ("dCas 9"); for example, where dCas9 comprises D10A and H840A substitutions relative to the amino acid sequence shown in figure 28 or the corresponding amino acids of another Cas9 polypeptide.

Specific binding pairs and extracellular recognition domains

The present disclosure provides conditionally active heterodimeric polypeptides. When expressed on the plasma membrane of a cell, the conditionally active heterodimeric polypeptide is active in the presence of: 1) an antigen or other moiety that interacts with an extracellular domain; and 2) a dimerizing agent that induces dimerization of heterodimeric polypeptides.

In some cases, conditionally active heterodimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain. In some cases, the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor. In some cases, the first member of the dimerization pair comprises a co-regulator of a nuclear hormone receptor and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor.

Thus, in some cases, conditionally active heterodimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair, wherein the first member of the dimerization pair comprises the LBD of a nuclear hormone receptor; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; wherein the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain. In other cases, conditionally active heterodimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair, wherein the first member of the dimerization pair comprises a coregulator of a nuclear hormone receptor; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair, wherein the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor; and iv) an intracellular signaling domain.

In some cases, conditionally active heterodimeric polypeptides of the present disclosure comprise: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a regulatory domain; iii) a first member of a dimerization pair; iv) a transmembrane domain interposed between the first member of the specific binding pair and the regulatory domain; and b) a second polypeptide comprising: i) a second member of a dimerization pair; and ii) an intracellular signaling domain. In some cases, the first member of the dimerization pair comprises the LBD of the nuclear hormone receptor and the second member of the dimerization pair is a co-regulator of the nuclear hormone receptor. In some cases, the first member of the dimerization pair is a co-regulator of a nuclear hormone receptor, and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor.

In some cases, a conditionally active heterodimeric polypeptide of the present disclosure is a conditionally active open-switched Chimeric Antigen Receptor (CAR). In some cases, a conditionally active heterodimeric polypeptide of the present disclosure is a conditionally active off-switch CAR. In some cases, a conditionally active heterodimeric polypeptide of the present disclosure is a conditionally active T Cell Receptor (TCR).

The present disclosure provides a conditionally blocked synthetic Immune Cell Receptor (ICR) of a heterodimer, comprising: a synthetic stimulatory ICR comprising a first member of a dimerization pair linked to the synthetic stimulatory ICR; and a synthetic ICR repressor factor comprising a second member of a dimerization pair linked to an intracellular inhibitory domain, wherein the first member of the dimerization pair comprises a LBD of a nuclear hormone receptor and the second member of the dimerization pair comprises a coactivator of the nuclear hormone receptor, or wherein the first member of the dimerization pair is a coactivator of a nuclear hormone receptor and the second member of the dimerization pair comprises a LBD of the nuclear hormone receptor. The synthetic stimulatory ICR and synthetic ICR repressor dimerize in the presence of a ligand (dimerizer) that induces binding of LBD to the co-regulator.

The present disclosure provides a conditionally-repressed synthetic Chimeric Antigen Receptor (CAR) of a heterodimer, comprising: a) a synthetic stimulatory CAR comprising: i) an extracellular recognition domain; ii) a transmembrane domain linked to an extracellular recognition domain; iii) a first member of a dimerization pair linked to a transmembrane domain; and iv) an intracellular stimulatory domain; and b) a synthetic CAR repressor comprising: i) a second member of a dimerization pair; and ii) an intracellular inhibitory domain attached to the second member of the dimerization pair. The first member of the dimerization pair comprises the LBD of the nuclear hormone receptor and the second member of the dimerization pair comprises the coactivator of the indicated nuclear hormone receptor; or the first member of the dimerization pair is a coactivator of a nuclear hormone receptor and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor. The synthetic stimulatory CAR and synthetic CAR repressor dimerize in the presence of a dimerizing agent that induces binding of LBD to a co-regulatory factor.

The present disclosure provides a conditionally-repressed synthetic T-cell receptor (TCR) of a heterodimer comprising a) a synthetic stimulatory TCR comprising i) a transmembrane domain, ii) a first member of a dimerization pair linked to the transmembrane domain, iii) an engineered TCR polypeptide comprising at least one TCR α or β chain, wherein the at least one TCR α or β chain is linked to the indicated transmembrane domain or the indicated first member of the dimerization pair, and b) a synthetic TCR repressor comprising i) a second member of the dimerization pair, and ii) an intracellular inhibitory domain linked to the second member of the dimerization pair.

The present disclosure provides a heterodimeric, conditionally active Chimeric Antigen Receptor (CAR) comprising: I) a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain; or II) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a regulatory domain; iii) a first member of a dimerization pair; iv) a transmembrane domain interposed between the first member of the specific binding pair and the regulatory domain; and b) a second polypeptide comprising: i) a second member of a dimerization pair; and ii) an intracellular signaling domain. The first member of the dimerization pair comprises the LBD of the nuclear hormone receptor and the second member of the dimerization pair comprises the coactivator of the indicated nuclear hormone receptor; or the first member of the dimerization pair is a coactivator of a nuclear hormone receptor and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor. The first polypeptide and the second polypeptide dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulatory factor.

Conditionally active opening switch CAR

In some cases, a conditionally active heterodimeric polypeptide of the present disclosure is a conditionally active open-switched Chimeric Antigen Receptor (CAR).

Members of specific binding pairs

As described above, conditionally active heterodimeric polypeptides of the present disclosure comprise a first polypeptide comprising a first member of a specific binding pair. The second member of the specific binding pair may be present on the cell surface. The second member of the specific binding pair can be immobilized on an insoluble support expressed on the surface of a cell (e.g., target cell, non-target cell, etc.), or the like. The second member of the specific binding pair may be soluble. The second member of the specific binding pair may be present in an intracellular environment (e.g., extracellular matrix). The second member of the specific binding pair may be present in an artificial matrix. The second member of the specific binding pair may be present in an acellular environment.

Suitable first members of specific binding pairs include, but are not limited to, antibody-based recognition scaffolds; antibodies (i.e., antibody-based recognition scaffolds, including antigen-binding antibody fragments); a non-antibody based recognition scaffold; antigens (e.g., endogenous antigens; exogenous antigens, etc.); a ligand for a receptor; a receptor; a non-antibody based target that recognizes the scaffold; fc receptors (e.g., Fc γ RIIIa; Fc γ RIIIb, etc.); an extracellular matrix component; and so on.

Specific binding pairs include, for example, antigen-antibody specific binding pairs, wherein a first member is an antibody (or antibody-based recognition scaffold) that specifically binds to a second member that is an antigen, or wherein a first member is an antigen and a second member is an antibody (or antibody-based recognition scaffold) that specifically binds to an antigen; a ligand-receptor specific binding pair, wherein the first member is a ligand and the second member is a receptor that binds to the ligand, or wherein the first member is a receptor and the second member is a binding ligand to the receptor; a non-antibody based recognition scaffold-target specific binding pair, wherein the first member is a non-antibody based recognition scaffold and the second member is a target that binds to the non-antibody based recognition scaffold, or wherein the first member is a target and the second member is a non-antibody based recognition scaffold that binds to the target; adhesion molecule-extracellular matrix binding pair; an Fc receptor-Fc binding pair, wherein a first member comprises an immunoglobulin Fc bound to a second member, said second member being an Fc receptor, or wherein a first member is an Fc receptor bound to a second member, said second member comprising an immunoglobulin Fc; and a receptor-co-receptor binding pair, wherein the first member is a receptor that specifically binds to a second member that is a co-receptor, or wherein the first member is a co-receptor that specifically binds to a second member that is a receptor.

Antigen binding domains

In some cases, the antigen binding domain is a single chain fv (scFv). it is suitable to use other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized forms, IgNAR VH (shark antibody variable domain) and humanized forms, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains.

The antigen binding domains in conditionally active heterodimeric polypeptides suitable for use in the present disclosure can have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific for an epitope present in an antigen expressed (synthesized) by a cancer cell (i.e., a cancer cell-associated antigen). The cancer cell-associated antigen can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, and the like. Cancer cell-associated antigens may also be expressed by non-cancerous cells.

Non-limiting examples of antigens to which the antigen-binding domain of conditionally active heterodimeric polypeptides of the present disclosure can bind include, for example, CD19, CD20, CD38, CD30, Her2/neu/ERBB2, CA125, MUC-1, Prostate Specific Membrane Antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2(VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-a1, IL-13R-a2, GD2, and the like.

In some cases, the antigen binding domains of conditionally active heterodimeric polypeptides of the present disclosure can target cancer-associated antigens, in some cases, the antigen binding domains of the present disclosure can include antibodies or portions thereof specific for cancer-associated antigens non-limiting examples of cancer-associated antigens include, but are not limited to, e.g., CD, Her/neu, ERBB, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD surface adhesion molecules, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2 (VEGFR), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A, IL-13R-a, GD, and the like, cancer-associated antigens also include, e.g., 4-1BB, 5T, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphocytes, C242 antigen, pancreatic islet-CA 125, carbonic anhydrase 9(CA-IX), C-MET, TRAIL 152, TRAIL, CD221, VEGF, CD200, VEGF-13-LR, VEGF-binding protein, VEGF-binding protein receptor, VEGF-binding protein.

In some cases, the antigen binding domain can specifically bind to a target comprising a Major Histocompatibility Complex (MHC) molecule as well as a fragment of a protein (e.g., a peptide). Since MHC molecules present peptide fragments of proteins expressed intracellularly and extracellularly, specific binding members directed against MHC-peptide complexes allow targeting of intracellular as well as extracellularly expressed antigens.

Target proteins expressed intracellularly (e.g., cytoplasmic expressed proteins (i.e., cytoplasmic proteins), nuclear expressed proteins (i.e., nuclear proteins), etc.) can be referred to as intracellular antigens (e.g., cytoplasmic antigens, nuclear antigens, etc.). Thus, in some cases, the antigen binding domains of the present disclosure may have specificity for intracellular antigen fragments complexed with MHC, such as peptide-MHC complexes, also described in some cases as Human Leukocyte Antigen (HLA) -peptide complexes.

Exemplary protein targets to which the antigen binding domain of the targeting peptide-MHC complex may be directed as well as exemplary peptides in the context of the MHC of each protein target are provided in table 2 below.

Table 2: anti-peptide-MHC targets

In some cases, the antigen binding domain of a conditionally active heterodimeric polypeptide of the present disclosure is or includes a portion of an antibody (e.g., an scFv) that specifically binds to a peptide-MHC having an intracellular cancer antigen peptide of table 2.

In some cases, the antigen binding domain of the conditionally active heterodimeric polypeptides of the present disclosure is or includes a portion of an antibody (e.g., an scFv) that specifically binds to a peptide-MHC as described in Dhanik et al BMC Bioinformatics (2016)17:286, the disclosures of which are incorporated herein by reference in their entirety, including, but not limited to, e.g., an NLRP4 peptide (e.g., HLSPIDCEV (SEQ ID NO:496)) -MHC complex, UMODL1 peptide (e.g., LTSMWSPAV (SEQ ID NO:497)) -MHC complex, NLRP4 peptide (e.g., HLDHPHPAV (SEQ ID NO:498)) -MHC complex, MAGEC2 peptide (e.g., SLSVMSSNV (SEQ ID NO:499)) -MHC complex), NLRP4 peptide (e.g., MMAWSDNKI (SEQ ID NO:500)) -MHC complex, COX7B2 peptide (e.g., TQIGIEWNL (SEQ ID NO:501)) -MHC complex, dhandler) MHC complex, NLRP4 peptide (e.g., CLFEMQDPA (SEQ ID NO:502)) -MHC complex, UMODL1 peptide (e.g., YLSHPSCNV (SEQ ID NO:503)) -MHC complex, COX7B2 peptide (e.g., GIEWNLSPV (SEQ ID NO:504)) -MHC complex, MAGEA11 peptide (e.g., GLGCSPASI (SEQ ID NO:505)) -MHC complex, RPE65 peptide (e.g., RQAFEFPQI (SEQ ID NO:506)) -MHC complex, RPE65 peptide (e.g., RQAFEFPQI (SEQ ID NO:507)) -MHC complex, NLRP4 peptide (e.g., GMWTDTFEF (SEQ ID NO:508)) -MHC complex, TRIM51 peptide (e.g., YLNWQDTAV (SEQ ID NO:509)) -MHC complex, MAGEA11 peptide (e.g., VLWGPITQI (SEQ ID NO:510)) -MHC complex), NL 4 peptide (e.g., TLDHTGVVV (SEQ ID NO:511)) -MHC 65 peptide (e.g., RPE65 ID NO: TMGVWLHIA)) -MHC complex, NL 4 peptide (e.g., SEQ ID NO: 512-MHC complex), RPE65 peptide (e.g., TMGVWLHIA (SEQ ID NO:513)) -MHC complex, MAGEC2 peptide (e.g., KVWVQGHYL (SEQ ID NO:514)) -MHC complex, UMODL1 peptide (e.g., KINCNNFRL (SEQ ID NO:515)) -MHC complex, and the like.

Ligands

In some cases, a member of a specific binding pair suitable for use in the conditionally active heterodimeric polypeptides of the present invention is a ligand for a receptor. Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; Vascular Endothelial Growth Factor (VEGF), etc.); integrin binding peptides (e.g., peptides comprising the sequence Arg-Gly-Asp); and so on.

In the case where the member of the specific binding pair in the conditionally active heterodimeric polypeptides of the present invention is a ligand, the conditionally active heterodimeric polypeptide can be activated in the presence of both a dimerizing agent and a second member of the specific binding pair, wherein the second member of the specific binding pair is a receptor for the ligand. For example, where the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor, including a soluble VEGF receptor. As another example, where the ligand is heregulin, the second member of the specific binding pair may be Her 2.

Receptors

As noted above, in some instances, the member of a specific binding pair included in a conditionally active heterodimeric polypeptide of the invention is a receptor, e.g., a receptor for a ligand, a co-receptor, and the like. The receptor may be a ligand binding fragment of the receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptors); killer lectin-like receptor subfamily K, member 1(NKG2D) polypeptide (receptor for MICA, MICB and ULB 6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); her 2; CD 27; natural Cytotoxic Receptors (NCRs) (e.g., NKP30(NCR3/CD337) polypeptides (receptors for HLA-B-related transcript 3(BAT3) and B7-H6), etc.); and so on.

Antibody-based recognition scaffolds

In some cases, the antigen binding domain is a single chain fv (scFv). it is suitable for use with other antibody-based recognition domains (cAb VHH and humanized forms, IgNAR VH and humanized forms, sdAb VH and "camelized" antibody variable domains.in some cases, a T Cell Receptor (TCR) -based recognition domain, such as a single chain TCR (scTv, a single chain double domain TCR containing V α V β), is also suitable for use.

In the case where the member of the specific binding pair in the conditionally active heterodimeric polypeptides of the present disclosure is an antibody-based recognition scaffold, the conditionally active heterodimeric polypeptide can be activated in the presence of the second member of the specific binding pair, wherein the second member of the specific binding pair is an antigen bound to the antibody-based recognition scaffold.

Antibodies suitable for inclusion in the conditionally active heterodimeric polypeptides of the present disclosure can have a variety of antigen binding specificities.

In some cases, the antigen binding domain is specific for an epitope present in an antigen expressed (synthesized) by a cancer cell (i.e., a cancer cell-associated antigen). The cancer cell-associated antigen can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a pancreatic cancer, a hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, and the like. Cancer cell-associated antigens may also be expressed by non-cancerous cells.

In some cases, the antigen binding domain is specific for an epitope present in a tissue-specific antigen (including, e.g., those antigens described herein). In some cases, the antigen binding domain is specific for an epitope present in a disease-associated antigen (including, e.g., those antigens described herein).

Non-limiting examples of antigens to which the antigen-binding domain of the conditionally active heterodimeric polypeptides of the invention can bind include, for example, CD19, CD20, CD38, CD30, Her2/neu/ERBB2, CA125, MUC-1, Prostate Specific Membrane Antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2(VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-a1, IL-13R-a2, GD2, and the like.

Non-limiting examples of antigens to which the antigen-binding domain of the conditionally active heterodimeric polypeptides of the invention can bind include, for example, cadherin (CDH1-20), integrins (α and β isoforms), ephrin, NCAM, connexin, CD44, syndecans, CD47, DG α/β, SV2, procalcitonin, Fas, Dectin-1, CD7, CD40, neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4, CD28, TIGIT, and ICOS.

In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell surface receptor.

In some cases, the antibody is specific for a cell surface target, wherein non-limiting examples of cell surface targets include CD19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD11a, and α -integrin.

In some cases, the antigen (the second member of the specific binding pair) to which the antibody-based scaffold binds is soluble. In some cases, the antigen is membrane-bound, e.g., in some cases, the antigen is present on the cell surface. In some cases, the antigen is immobilized on an insoluble support, wherein the insoluble support may comprise any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); and wherein the insoluble support may take various forms, such as a plate, tissue culture dish, column, and the like. In some cases, the antigen is present in an extracellular matrix (ECM) (e.g., the antigen is an ECM component). In some cases, the antigen is present in an artificial matrix. In some cases, the antigen is present in the extracellular environment.

Non-antibody based recognition scaffolds

In some cases, the first member of the specific binding pair is a non-antibody based recognition scaffold. Where the member of the specific binding pair in the conditionally active heterodimeric polypeptides of the present disclosure is a non-antibody based recognition scaffold, the conditionally active heterodimeric polypeptides can be activated in the presence of a second member of the specific binding pair, wherein the second member of the specific binding pair is the target to which the non-antibody based recognition scaffold binds.

Non-antibody based recognition scaffolds include, for example, affibodies; engineering a Kunitz domain; monomer (adnectin); anti-transporter proteins (anticalin); designed ankyrin repeat domain (DARPin); a binding site for a cysteine-rich polypeptide (e.g., a cysteine-rich knottin peptide); affinity multimers (avimers); afflin; and so on. See, e.g., Gebauer and Skerra (2009) curr. opin. chem. biol.13: 245.

Non-antibody based scaffolds (also referred to herein as "antibody mimetic molecules") can be identified by selecting or isolating target binding variants from a library of binding molecules with artificially diversified binding sites. Diversified libraries can be generated using entirely random methods (e.g., error-prone Polymerase Chain Reaction (PCR), exon shuffling, or directed evolution) or with the aid of art-recognized design strategies. For example, when a binding site interacts with its cognate target molecule, the amino acid positions typically involved can be randomized by inserting degenerate codons, trinucleotides, random peptides or complete loops at the corresponding positions within the nucleic acid encoding the binding site (see, e.g., U.S. publication No. 20040132028). The location of the amino acid position can be identified by studying the crystal structure of the binding site for the target molecule in the proteinaceous entity. Candidate positions for randomization include loops, flat surfaces, helices, and binding cavities for binding sites. In certain embodiments, it is possible to identify amino acids within a binding site that are likely candidates for diversification by homology to immunoglobulin folding. For example, residues within the CDR-like loops of fibronectin can be randomized to generate a library of fibronectin binding molecules (see, e.g., Koide et al, J.Mol.biol.,284:1141-1151 (1998)). Other portions of the randomizable binding sites include flat surfaces. After randomization, the variegated library can then be subjected to a selection or screening process to obtain binding molecules having the desired binding characteristics. For example, selection can be achieved by art-recognized methods such as phage display, yeast display, or ribosome display.

For example, in some cases, the non-antibody based scaffold comprises a binding site from a fibronectin binding molecule. Fibronectin binding molecules (e.g., molecules comprising fibronectin type I, type II, or type III domains) exhibit CDR-like loops that are independent of intrachain disulfide bonds as opposed to immunoglobulins. The FnIII loop contains regions that may undergo random mutation as well as directed evolution protocols that target iterative cycles of binding, selection, and further mutation to develop useful therapeutic tools. Fibronectin-based "addressable" therapeutic binding molecules ("FATBIMs") can be developed to specifically bind to a target antigen or epitope. Methods for making fibronectin binding polypeptides are described, for example, in WO 01/64942 and U.S. patent nos. 6,673,901, 6,703,199, 7,078,490, and 7,119,171.

As another example, in some cases, non-antibody based scaffolds comprise binding sites from affibodies derived from immunoglobulin binding domains of Staphylococcal Protein A (SPA) (see, e.g., Nord et al, nat. biotechnol.,15: 772-.

As another example, in some cases, non-antibody based scaffolds contain binding sites from anti-transporters.an antibody functional mimetic derived from human lipocalins.lipocalins is a family of naturally occurring binding proteins that bind and transport small hydrophobic molecules (such as steroids, hind bile pigments, retinoids, and lipids). The main structure of anti-transporters is similar to wild-type lipocalins.the central element of this protein architecture is the β -barrel structure of eight antiparallel chains, which supports four loops at the open ends.

As another example, in some cases, non-antibody based scaffolds comprise binding sites from cysteine-rich polypeptides in some cases, the cysteine-rich domains do not form α -helices, β -folds or β -barrel structures in some cases, disulfide bonds facilitate domain folding into three-dimensional structures in some cases, cysteine-rich domains have at least two disulfide bonds, e.g., at least three disulfide bonds.an exemplary cysteine-rich polypeptide is an A-domain protein.A domain (sometimes referred to as a "complement-like repeat") contains about 30-50 or 30-65 amino acids in some cases, the domain contains about 35-45 amino acids and in some cases about 40 amino acids within 30-50 amino acids.about 6 cysteine residues are present.6 within 30-50 amino acids.in six cysteine residues.in six cysteine residues, disulfide bonds are typically found between C1 and C3, C2 and C5, C39 4 and transmembrane domain constitute binding portions of the binding portion of the transmembrane domain of a ligand binding domain of a LRP.LRP binding domain, such as a LRP binding domain for the binding of a protein with a polypeptide binding protein binding specificity, including the binding domain of a polypeptide binding protein binding to a polypeptide binding protein binding to a protein binding protein (e.g. LRP binding to a protein binding.

As another example, in some cases, the non-antibody based scaffold comprises binding sites from repeat proteins. Repetitive proteins are proteins that contain successive copies of small (e.g., about 20 to about 40 amino acid residues) building blocks or repeats that are stacked together to form a continuous domain. By adjusting the number of repeats in a protein, the repeat protein can be modified to accommodate a particular target binding site. Exemplary repeat proteins include the designed ankyrin repeat protein (i.e., DARPin) (see, e.g., Binz et al, nat. Biotechnol.,22: 575-. As another example, in some cases, the non-antibody based scaffold comprises darpins.

As used herein, the term "DARPin" refers to a genetically engineered antibody mimetic protein that generally exhibits high specificity and high affinity for target protein binding DARPin is first derived from a native ankyrin in some cases, DARPin contains three, four, or five repeating motifs of ankyrin in some cases, ankyrin repeat units consist of 30-34 amino acid residues and function to mediate protein-protein interactions in some cases, each ankyrin repeat exhibits a helix-turn-helix conformation, and such series repeat strings of tandem repeats are assembled into an approximately linear array to form helix-turn-helix bundles connected by relatively flexible loops in some cases, the global structure of ankyrin repeat proteins is stabilized by intra-and inter-repeat hydrophobicity and hydrogen bonding interactions in some cases, the repeat and elongation properties of ankyrin repeats provide the molecular basis for the unique features of ankyrin repeat proteins in terms of protein stability, folding and unfolding and binding specificity darp repeat domains for four or five repeats, respectively, DARPin 14 or about 18, and WO 19, darp 19, DARPin repeat units are constructed as a loop repeat unit from a DARPin, darp, DARPin, darp, a.

As another example, in some cases, Non-Antibody based scaffolds comprise Binding sites derived from Src homology domains (e.g., SH2 or SH3 domain), PDZ domains, β -lactamase, high affinity protease inhibitors or disulfide bond Binding Protein scaffolds (such as scorpion toxin). A method for preparing Binding sites derived from these molecules is disclosed in the art, see, for example, Panni et al, J.biol.chem.,277:21666- -.

As another example, in some cases, a non-antibody based scaffold comprises a Kunitz domain as used herein, the term "Kunitz domain" refers to a conserved protein domain that inhibits certain proteases (e.g., serine proteases). the Kunitz domain is relatively small, typically about 50 to 60 amino acids long and has a molecular weight of about 6 kDa.

The Kunitz domain has a pear-shaped structure that is stabilized by, for example, three disulfide bonds and contains a reactive site region characterized by the major determinant P1 residue in a rigid conformation. These inhibitors competitively prevent access of a target protein (e.g., serine protease) to its physiologically relevant macromolecular substrate by inserting the P1 residue into the active site cleft. The P1 residue in the protease inhibitory loop provides the primary determinant of specificity and defines the many inhibitory activities a particular Kunitz protein has on the targeted protease. Generally, the N-terminal side of the reactive site (P) is more energetically important than the P' C-terminal side. In most cases, lysine or arginine occupy position P1 to inhibit proteases that cleave adjacent to those residues in the protein substrate. Other residues, particularly in the zone of the inhibition loop, contribute to the strength of the binding. Typically, about 10-12 amino acid residues in the target protein and 20-25 residues in the protease are in direct contact during formation of the stable protease inhibitor protein entity and provide buried regions of about 600 to 900A. By modifying residues in and around the P site, Kunitz domains can be designed to target selected proteins. Kunitz domains are described, for example, in U.S. patent No. 6,057,287.

As another example, in some cases, non-antibody based scaffolds are avidin (affilin), avidin is a small antibody mimetic protein designed for specific affinity of proteins and small molecule compounds new avidin can be rapidly selected from two libraries, each based on a different human-derived scaffold protein avidin does not show any structural homology to immunoglobulins.

As another example, in some cases, the non-antibody based scaffold is an affinity multimer. Affinity multimers have evolved from a large family of human extracellular receptor domains by exon shuffling and phage display in vitro, resulting in multi-domain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to generate avidity and result in improved affinity and specificity compared to conventional single epitope binding proteins. In certain embodiments, the affinity multimer consists of two or more peptide sequences of 30 to 35 amino acids each linked by a spacer peptide. Each sequence is derived from the a domain of a variety of membrane receptors and has a rigid structure that is stabilized by disulfide bonds and calcium. Each a domain may bind to a certain epitope of the target protein. The combination of domains binding different epitopes of the same protein increases the affinity of this protein, an effect known as avidity (hence the name). Avimers with sub-nanomolar affinities have been obtained for a variety of targets. Alternatively, the domains may be directed against epitopes on different target proteins. Additional information regarding affinity polymers can be found in U.S. patent application publication nos. 2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512, 2004/0175756.

Suitable targets for non-antibody based scaffolds include any of the above antigens to which an antibody based scaffold can bind.

In some cases, the target (second member of the specific binding pair) to which the non-antibody based scaffold binds is soluble. In some cases, the target is membrane-bound, e.g., in some cases, the target is present on the surface of a cell. In some cases, the target is immobilized on an insoluble support, wherein the insoluble support may comprise any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); and wherein the insoluble support may take various forms, such as a plate, tissue culture dish, column, and the like. In some cases, the target is present in an extracellular matrix (ECM) (e.g., the antigen is an ECM component). In some cases, the target is present in an artificial matrix. In some cases, the target is present in an extracellular environment.

Cell adhesion molecules

In some cases, the first member of the specific binding pair is a Cell Adhesion Molecule (CAM), i.e., a polypeptide that binds to a component of an extracellular matrix (ECM) or binds to a cell surface molecule. For example, in some cases, the first member of the specific binding pair is an extracellular region of a CAM. In some cases, the CAM is a non-calcium dependent adhesion molecule; for example, in some cases, the CAM is an immunoglobulin superfamily CAM. In some cases, the CAM is a calcium-dependent adhesion molecule; for example, the CAM is an integrin, cadherin or selectin. In some cases, the first member of the specific binding pair is an integrin. In some cases, the first member of the specific binding pair is a cadherin, e.g., E-cadherin, P-cadherin, N-cadherin, R-cadherin, M-cadherin, and the like. In some cases, the first member of the specific binding pair is a selectin, e.g., an E-selectin, an L-selectin, or a P-selectin. The binding fragment of the CAM can be used as a first member of a specific binding pair.

Where the first member of the specific binding pair is a CAM, the second member of the specific binding pair is a component of the ECM or a cell surface molecule that binds the CAM. For example, where the first member of the specific binding pair is an integrin, the second member of the specific binding pair is a component of collagen, fibrinogen, fibronectin, or vitronectin. As another example, where the first member of the specific binding pair is cadherin, the second member of the specific binding pair is a cadherin-bound cell surface antigen. As another example, where the first member of the specific binding pair is a selectin, the second member of the specific binding pair is a fucosylated carbohydrate.

Ligands

In some cases, the first member of the specific binding pair is a ligand of a receptor. Ligands include polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, and the like. In some cases, the ligand is soluble.

Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; Vascular Endothelial Growth Factor (VEGF), etc.); a peptide hormone; integrin binding peptides (e.g., peptides comprising the sequence Arg-Gly-Asp); an N-glycan; and so on.

Where a member of a specific binding pair in the conditionally active heterodimeric polypeptides of the present disclosure is a ligand, the conditionally active heterodimeric polypeptide can be activated in the presence of a second member of the specific binding pair, wherein the second member of the specific binding pair is a receptor for the ligand. For example, where the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor, including a soluble VEGF receptor. Alternatively, the first member of the specific binding pair may be a VEGF receptor; and the first member of the specific binding pair may be VEGF. As another example, where the ligand is heregulin, the second member of the specific binding pair may be Her 2.

Where the first member of the specific binding pair is a ligand, the second member of the specific binding pair is a ligand-binding molecule, e.g., the second member of the specific binding pair is an antibody that specifically binds the ligand, a receptor for the ligand, etc.

Where the first member of the specific binding pair is a ligand, in some cases, the second member of the specific binding pair (the ligand-binding molecule) is soluble. In some cases, the second member of the specific binding pair is membrane-bound, e.g., in some cases, the second member of the specific binding pair is present on the surface of the cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support, wherein the insoluble support may comprise any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); and wherein the insoluble support may take various forms, such as a plate, tissue culture dish, column, and the like. In some cases, the second member of the specific binding pair is present in an acellular environment.

Antigens

In some cases, the first member of the specific binding pair is an antigen that specifically binds to an antibody. The antigen may be any antigen, such as a naturally occurring (endogenous) antigen; synthetic (e.g., modified in no longer the same manner as a naturally occurring antigen; modified from its natural state; etc.) antigen, and the like.

In the event that a member of a specific binding pair in the conditionally active heterodimeric polypeptides of the present disclosure is an antigen, the conditionally active heterodimeric polypeptides can be activated in the presence of a second member of the specific binding pair, wherein the second member of the specific binding pair is an antibody that binds to the antigen (antibody-based recognition scaffold).

In some cases, the antigen is a disease-associated antigen, such as a cancer-associated antigen, an autoimmune disease-associated antigen, a pathogen-associated antigen, an inflammation-associated antigen, and the like.

For example, where the second member of the specific binding pair is an antibody specific for a cancer-associated antigen, the antigen can be a cancer-associated antigen, wherein the cancer-associated antigen includes, for example, CD, Her/neu/ERBB, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD surface adhesion molecules, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2 (VEGFR), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A, IL-13R-a, GD, and the like, cancer-associated antigens also include, for example, 4-1BB, 5T, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphocytes, C242 antigen, CA-125, carbonic anhydrase 9(CA-IX), C-MET, CCR, CD152, CD200, CD221, CD (CD receptor), CD (TNFR, CD, TRAIL, CD44, CTDV-binding protein, VEGF-binding protein receptor binding protein, VEGF-binding protein receptor binding protein, VEGF-binding protein, VEGF-binding protein domain, VEGF-binding protein, VEGF-binding protein, protein binding protein.

Non-limiting examples of antigens associated with inflammatory diseases include, for example, AOC3(VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basic immunoglobulin), CD154(CD40L), CD2, CD20, CD23(IgE receptor), CD25(α chain of IL-2 receptor), CD3, CD4, CD5, IFN- α, IFN- γ, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17A, IL-22, IL-4, IL-5, IL-6 receptor, integrin α 4, integrin α 4 β 7, LFA-1(CD11a), myostatin, OX-40, sclerostin, SOST, β 1, TGF- α, and VEGF-A.

Where the first member of the specific binding pair is an antigen, the second member of the specific binding pair may be an antibody-based scaffold (e.g., an antibody) or a non-antibody-based scaffold. In some cases, the second member of the specific binding pair is present on the surface of the cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support. In some cases, the second member of the specific binding pair is soluble. In some cases, the second member of the specific binding pair is present in an extracellular environment (e.g., extracellular matrix). In some cases, the second member of the specific binding pair is present in an artificial matrix. In some cases, the second member of the specific binding pair is present in an acellular environment.

Non-antibody based recognition scaffold targets

In some cases, the first member of the specific binding pair is a target of a non-antibody based scaffold. Targets include, for example, polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, and the like.

Where the first member of the specific binding pair is the target of a non-antibody based scaffold, the second member of the specific binding pair is a non-antibody based scaffold.

Receptors

In some cases, the first member of the specific binding pair is a receptor. In some cases, the receptor is a growth factor receptor. In some cases, the receptor is a cytokine receptor. In some cases, the receptor is a cell surface receptor that binds to a co-receptor on a cell. In some cases, the receptor is a neurotransmitter receptor. In some cases, the receptor binds to an extracellular matrix component. In some cases, the receptor is an immunoglobulin receptor.

Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptors), the killer lectin-like receptor subfamily K member 1(NKG2D) polypeptides (receptors for MICA, MICB, and ULB 6), cytokine receptors (e.g., IL-13 receptors; IL-2 receptors, etc.), Epidermal Growth Factor (EGF) receptors, Her2, CD27, Natural Cytotoxic Receptors (NCR) (e.g., NKP30(NCR3/CD337) polypeptides (receptors for HLA-B related transcript 3(BAT3) and B7-H6), etc.), T cell antigen receptors, dihydrofolate receptors, chimeric cytokine receptors, Fc receptors, extracellular matrix receptors (e.g., integrins), cell adhesion receptors (e.g., cadherins), immunomodulatory receptors including positive co-receptors (e.g., CD28) and negative (immunosuppressive) co-receptors (e.g., PD1), cytokine receptors, and receptors for immunomodulatory molecules (e.g., TGF β), etc. in some cases, wild-type receptors are present.

Where the first member of the specific binding pair is a receptor, the second member of the specific binding pair is a target of the receptor, where the target can be a ligand for the receptor or co-receptor. In some cases, the second member of the specific binding pair is present on the surface of the cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support. In some cases, the second member of the specific binding pair is soluble. In some cases, the second member of the specific binding pair is present in an extracellular environment (e.g., extracellular matrix). In some cases, the second member of the specific binding pair is present in an artificial matrix. In some cases, the second member of the specific binding pair is present in an acellular environment.

Hinge region

In some cases, the first polypeptide of the conditionally active heterodimeric polypeptides of the invention comprises a hinge region (also referred to herein as a "spacer"), wherein the hinge region is interposed between the antigen-binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD 8-derived hinge region).

The hinge region may be about 4 amino acids to about 50 amino acids in length, such as about 4 aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25 aa, about 25 aa to about 30aa, about 30aa to about 40aa, or about 40aa to about 50 aa.

Suitable spacers can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to8 amino acids, or 7 amino acids to8 amino acids, and can be 1, 2, 3,4, 5,6, or 7 amino acids.

Exemplary spacers include glycine polymer (G)_nGlycine-serine polymers (including, for example, (GS)_n、(GSGGS)_n(SEQ ID NO:516) and (GGGS)_n(SEQ ID NO:517) wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used; gly and Ser are relatively unstructured and therefore can serve as neutral chains between the components. Glycine polymers may be used; glycine approaches even significantly more phi-psi spacing than alanine and is much less restricted than residues with longer side chains (see Scheraga, rev. comparative chem.11173-142 (1992)). Exemplary spacers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:518), GGSGG (SEQ ID NO:519), GSGSGSG (SEQ ID NO:520), GSGGG (SEQ ID NO:521), GGGSG (SEQ ID NO:522), GSSSG (SEQ ID NO:523), and the like.

In some cases, the hinge region in the first polypeptide of the conditionally active heterodimeric polypeptides of the invention comprises at least one cysteine. For example, in some cases, the hinge region may include the sequence Cys-Pro-Pro-Cys. If present, cysteines in the hinge region of the first conditionally active heterodimeric polypeptide can be used to form disulfide bonds with the hinge region of the second conditionally active heterodimeric polypeptide.

Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al (1990) Proc.Natl.Acad.Sci.USA 87: 162; and Huck et al (1986) nucleic acids Res.14: 1779. As a non-limiting example, the immunoglobulin hinge region may comprise one of the following amino acid sequences: DKKHT (SEQ ID NO: 524); CPPC (SEQ ID NO: 525); CPEPKSCDTPPPCPR (SEQ ID NO:526) (see, e.g., Glaser et al (2005) J.biol.chem.280: 41494); ELKTPLGDTTHT (SEQ ID NO: 527); KSCDKTHTCP (SEQ ID NO: 528); KCCVDCP (SEQ ID NO: 529); KYGPPCP (SEQ ID NO: 530); EPKSCDKTHTCPPCP (SEQ ID NO:531) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:532) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:533) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:534) (human IgG4 hinge); and so on.

The hinge region may comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. The hinge region may comprise one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally occurring) hinge region. For example, His of human IgG1 hinge₂₂₉Can be substituted with Tyr such that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 535); see, e.g., Yan et al (2012) j.biol.chem.287: 5891.

The hinge region may comprise an amino acid sequence derived from human CD 8; for example, the hinge region may comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:536) or a variant thereof.

Transmembrane domain

The first and second polypeptides of the CAR of the present disclosure include a transmembrane domain for insertion into a eukaryotic cell membrane. The transmembrane domain of the first polypeptide is interposed between the antigen binding domain and the co-stimulatory domain. Where the first polypeptide comprises a hinge region, the transmembrane domain is interposed between the hinge region and the costimulatory domain such that the first polypeptide comprises the antigen-binding domains in order from the amino-terminus (N-terminus) to the carboxy-terminus (C-terminus); a hinge region; a transmembrane domain; a first co-stimulatory domain; and a first member of a dimeric binding pair.

The transmembrane domain of the second polypeptide is located at or near the N-terminus of the polypeptide such that the second polypeptide comprises, in order from N-terminus to C-terminus: a transmembrane domain; a second costimulatory domain; a second member of a dimeric binding pair; and an intracellular signaling domain.

As a non-limiting example, TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:537) may be used additional non-limiting examples of suitable TM sequences include a) CD8 β -derived LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:538), b) CD 4-derived ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:539), c) CD3 zeta-derived LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:540), d) CD 28-derived WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:541), e) CD134(OX40) -derived VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:542), and f) CD 7-derived ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 543).

Joint

In some cases, the first polypeptide of a CAR of the invention comprises a linker between any two adjacent domains. For example, a linker may be disposed between the transmembrane domain of the first polypeptide and the first costimulatory domain. As another example, a linker may be disposed between the first co-stimulatory domain of the first polypeptide and the first member of the dimeric binding pair. As another example, a linker may be disposed between the transmembrane domain of the second polypeptide and the second costimulatory domain. As another example, a linker may be disposed between the second co-stimulatory domain of the second polypeptide and the second member of the dimeric binding pair. As another example, a linker may be disposed between the second member of the dimeric binding pair of the second polypeptide and the intracellular signaling domain.

The linker peptide may have any of a variety of amino acid sequences. Proteins may be linked by spacer peptides, generally of a flexible nature, but other chemical linkages are not excluded. The linker may be a peptide having a length of between about 6 and about 40 amino acids or a length of between about 6 and about 25 amino acids. These linkers can be generated by coupling proteins using synthetic linker-encoding oligonucleotides. Peptide linkers with a certain degree of flexibility may be used. The linking peptide may have virtually any amino acid sequence, bearing in mind that a suitable linker will have a sequence that typically results in a flexible peptide. The use of small amino acids (such as glycine and alanine) can be used to generate flexible peptides. It is routine for a person skilled in the art to generate such sequences.

Suitable linkers can be readily selected and can be any of various lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to8 amino acids, or 7 amino acids to8 amino acids, and can be 1, 2, 3,4, 5,6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (including, for example, (GS)_n、GSGGS_n(SEQ ID NO:516) and GGGS_n(SEQ ID NO:517) wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Of interest are glycine and glycine-serine polymers; since these amino acids are relatively unstructured and therefore can act as neutral chains between the components. Of particular interest are glycine polymers; since glycine approaches even significantly more phi-psi spacing than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:518), GGSGG (SEQ ID NO:519), GSGSGSG (SEQ ID NO:520), GSGGG (SEQ ID NO:521), GGGSG (SEQ ID NO:522), GSSSG (SEQ ID NO:523), and the like. One of ordinary skill will recognize that the design of a peptide conjugated to any of the elements described above may include a linker that is fully or partially flexible, such that the linker may include a flexible linker as well asOne or more portions of less flexible structure are imparted.

Regulatory domains

Regulatory domains suitable for use in the CARs of the present disclosure include co-stimulatory domains. The regulatory domains may or may not be present in the conditionally active heterodimeric polypeptides of the invention disclosed herein. Thus, a particular conditionally active heterodimeric polypeptide of the present disclosure (or one or more polypeptide chains of a multi-chain conditionally active heterodimeric polypeptide of the present disclosure) can include anywhere from 0 to 6 or more regulatory domains. For example, in some cases, the polypeptide chains of conditionally active heterodimeric polypeptides of the present disclosure can collectively comprise 0,1, 2, 3,4, 5,6, or more regulatory domains. In some cases, the polypeptide chains of conditionally active heterodimeric polypeptides of the present disclosure can each comprise 0,1, 2, 3,4, 5,6, or more regulatory domains. A particular regulatory domain may be present in one polypeptide chain of a conditionally active heterodimeric polypeptide but absent in another polypeptide chain. In some cases, a particular regulatory domain may be present in both strands of a two-stranded conditionally active heterodimeric polypeptide. Furthermore, the location of the regulatory domain on or within the polypeptide can vary widely, as a particular regulatory domain can be located at the end (e.g., N-terminus or C-terminus) of the polypeptide or substantially any suitable location within the polypeptide, e.g., adjacent to a transmembrane domain, adjacent to an intracellular signaling domain, adjacent to one or more other regulatory domains, etc.

In some cases, the regulatory domain on the first polypeptide of the CAR of the invention has substantially the same amino acid sequence as the regulatory domain on the second polypeptide of the CAR. For example, in some cases, the regulatory domain on the first polypeptide of the CAR comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the regulatory domain on the second polypeptide of the CAR. The regulatory domain of the first polypeptide of the CAR of the invention may be substantially the same length as the regulatory domain of the second polypeptide of the CAR of the invention; for example, the lengths of the first and second regulatory domains may differ from each other by less than 10 amino acids or less than 5 amino acids. In some cases, the first regulatory domain and the second regulatory domain have the same length.

The length of the regulatory domain suitable for inclusion in the first and second polypeptides of the CAR of the invention may be from about 30 amino acids to about 70 amino acids (aa), e.g., the length of the regulatory domain may be from about 30aa to about 35aa, from about 35aa to about 40aa, from about 40aa to about 45aa, from about 45aa to about 50aa, from about 50aa to about 55aa, from about 55aa to about 60aa, from about 60aa to about 65aa, or from about 65aa to about 70 aa. In other cases, the conditioning domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200aa in length.

The co-stimulatory domain of a CAR suitable for use in the present disclosure is typically a polypeptide derived from a receptor. In some embodiments, the co-stimulatory domains homodimerize. The co-stimulatory domain of the invention may be the intracellular portion of the transmembrane protein (i.e., the co-stimulatory domain may be derived from the transmembrane protein). Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

In some cases, the co-stimulatory domain on the first polypeptide of a CAR of the invention has substantially the same amino acid sequence as the co-stimulatory domain on the second polypeptide of the CAR. For example, in some cases, the co-stimulatory domain on the first polypeptide of the CAR comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the co-stimulatory domain on the second polypeptide of the CAR. The co-stimulatory domain of the first polypeptide of the CAR of the invention may be substantially the same length as the co-stimulatory domain of the second polypeptide of the CAR of the invention; for example, the lengths of the first and second co-stimulatory domains may differ from each other by less than 10 amino acids or less than 5 amino acids. In some cases, the first co-stimulatory domain and the second co-stimulatory domain have the same length.

The co-stimulatory domain suitable for inclusion in the first and second polypeptides of the CAR of the invention may be from about 30 amino acids to about 70 amino acids (aa) in length, e.g., the co-stimulatory domain may be from about 30aa to about 35aa, from about 35aa to about 40aa, from about 40aa to about 45aa, from about 45aa to about 50aa, from about 50aa to about 55aa, from about 55aa to about 60aa, from about 60aa to about 65aa, or from about 65aa to about 70aa in length. In other cases, the length of the co-stimulatory domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200 aa.

In some cases, the costimulatory domain is derived from the intracellular portion of transmembrane protein 4-1BB (also known as TNFRSF 9; CD 137; 4-1 BB; CDw 137; ILA, etc.). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 544). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp 44). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 545). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID 1). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 546). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP 1L). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 547). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD 272). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 548).

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp 55). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 549). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, or about 160aa to about 185aa of the amino acid sequence: RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 550).

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane proteins GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 551). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, light and TR 2). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 552). In some of these embodiments, the co-stimulatory domain in the first polypeptide and the second polypeptide is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa in length.

Dimeric pair

As noted above, the conditionally active heterodimeric polypeptides of the present disclosure comprise two polypeptide chains, one of which comprises a first member of a dimerization pair, and the second of which comprises a second member of a dimerization pair. One of the members of the dimerization pair will comprise the Ligand Binding Domain (LBD) of the nuclear hormone receptor and the other member of the dimerization pair will comprise the co-regulatory peptide of the same nuclear hormone receptor. In the presence of a dimerizing agent (e.g., a nuclear hormone or a functional derivative or analog of a nuclear hormone; also referred to herein as a "dimerizer"), the first member and the second member of the dimerization pair will bind to each other and will effect dimerization of the two polypeptide chains of the conditionally active heterodimeric polypeptides of the present disclosure. The first member of a dimerization pair or the second member of a dimerization pair may also be referred to as a "dimerization domain".

Ligand Binding Domain (LBD)

The ligand binding domain of a nuclear hormone receptor may be from any of a variety of nuclear hormone receptors including, but not limited to, ER α, ER β, PR, AR, GR, MR, RAR β 0, RAR β 1, RAR γ, TR β 2, TR β 3, VDR, EcR, RXR β 4, RXR β 5, RXR γ, PPAR β 6, PPAR β 7, PPAR γ, LXR β 8, LXR β 9, FXR, UP R, SXR, CAR, SF-1, LRH-1, PXDAX-1, SHP, TLX, PNR, NGF1-B α, NGF1-B β, NGF1-B γ, UP R α, ROR β, ROR γ, ERR α, ERR β, ERR γ, NGF, GCNF, TR2/4, HNF- α, COTF- β, and COTF- β.

The abbreviations of nuclear hormone receptors are ER: estrogen receptor, PR: progesterone receptor, AR: androgen receptor, GR: glucocorticoid receptor, MR: mineralocorticoid receptor, RAR: retinoic acid receptor, TR α, β: thyroid receptor, VDR: vitamin D3 receptor, EcR: ecdysone receptor, RXR: retinoic acid X receptor, PPAR: peroxisome proliferator activated receptor, LXR: liver X receptor, FXR: farnesol X receptor, PXR/SXR: pregnane X receptor/steroid and xenobiotic receptor, CAR: constitutive androstane receptor, SF-1: steroidogenic factor 1, DAX-1: dose-sensitive critical region of adrenocorticotropic-hypoplasia on X chromosome, gene 1, LRH-1: liver receptor homolog 1, SHP: small heterodimer, TLX: tailless gene, partner R: NGF specific nuclear receptor, RAR 1-B: neuroorphan orphan receptor, GCR: congenital growth factor, GCR: chick receptor related receptor, nuclear receptor related factor, nuclear factor receptor, nuclear factor receptor related to estrogen receptor, nuclear factor receptor related factor, nuclear.

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 (two or more) LBDs of a nuclear hormone receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises two LBDs of a nuclear hormone receptor. In some cases, the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises three LBDs of a nuclear hormone receptor. Where the polypeptide chain of a heterodimeric polypeptide of the present disclosure comprises multiple (two or more) LBDs of a nuclear hormone receptor, in some cases, the multiple LBDs comprise the same amino acid sequence. In some cases, two or more LBDs are in tandem either directly or separately through a linker.

Mineralocorticoid receptors

In some cases, an LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of the Mineralocorticoid Receptor (MR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of an MR having the amino acid sequence set forth in fig. 1A.

As one non-limiting example, the LBD of MR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 1F; and is about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 250 amino acids in length; e.g., 248 amino acids in length).

As one non-limiting example, the LBD of MR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 1B; and is about 250 amino acids to 299 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 299 amino acids in length).

As one non-limiting example, the LBD of MR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 1C; and is about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 250 amino acids in length; e.g., 248 amino acids in length).

As another non-limiting example, the LBD of MR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 1D; and having an S810L substitution (wherein the amino acid numbering is based on the amino acid sequence shown in figure 1A); and is about 250 amino acids to 299 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 299 amino acids in length).

As one non-limiting example, the LBD of MR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 1C; and having an S810L substitution (wherein the amino acid numbering is based on the amino acid sequence shown in figure 1A); and is about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 250 amino acids in length; e.g., 248 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of MR, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence SLTARHKILHRLLQEGSPSDI (SEQ ID NO:2), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of MR, the first member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence SLTARHKILHRLLQEGSPSDI (SEQ ID NO:2), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of MR, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence QEAEEPSLLKKLLLAPANTQL (SEQ ID NO:6), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of MR, the first member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence QEAEEPSLLKKLLLAPANTQL (SEQ ID NO:6), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of MR, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence SKVSQNPILTSLLQITGNGGS (SEQ ID NO:7), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of MR, the first member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence SKVSQNPILTSLLQITGNGGS (SEQ ID NO:7), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length).

Androgen receptor

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of the Androgen Receptor (AR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of an AR having the amino acid sequence set forth in figure 2A.

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2B; and is about 250 amino acids to 301 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 301 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2C; and is about 190 amino acids to 230 amino acids in length (e.g., 190 amino acids to 210 amino acids or 210 amino acids to 230 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2B; and having a T877A substitution (wherein the amino acid numbering is based on the amino acid sequence shown in figure 2A); and is about 250 amino acids to 301 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 301 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2C; and having a T877A substitution (wherein the amino acid numbering is based on the amino acid sequence shown in figure 2A); and is about 190 amino acids to 230 amino acids in length (e.g., 190 amino acids to 210 amino acids or 210 amino acids to 230 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2B; and having a F876L substitution (wherein the amino acid numbering is based on the amino acid sequence shown in FIG. 2A); and is about 250 amino acids to 301 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 301 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2C; and having a F876L substitution (wherein the amino acid numbering is based on the amino acid sequence shown in FIG. 2A); and is about 190 amino acids to 230 amino acids in length (e.g., 190 amino acids to 210 amino acids or 210 amino acids to 230 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2B; and having T877A and F876L substitutions (wherein the amino acid numbering is based on the amino acid sequence shown in figure 2A); and is about 250 amino acids to 301 amino acids in length (e.g., 250 amino acids to 275 amino acids or 275 amino acids to 301 amino acids in length).

As one non-limiting example, the LBD of an AR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 2C; and having T877A and F876L substitutions (wherein the amino acid numbering is based on the amino acid sequence shown in figure 2A); and is about 190 amino acids to 230 amino acids in length (e.g., 190 amino acids to 210 amino acids or 210 amino acids to 230 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of AR, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence ESKGHKKLLQLLTCSSDDR (SEQ ID NO:3), wherein the co-regulatory peptide is from about 19 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 19 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of AR, the first member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence ESKGHKKLLQLLTCSSDDR (SEQ ID NO:3), wherein the co-regulatory peptide is from about 19 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 19 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length).

Progesterone receptors

In some cases, an LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of the Progesterone Receptor (PR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of a PR having the amino acid sequence set forth in fig. 3A.

As one non-limiting example, the LBD of a PR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 3D; and is about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 250 amino acids in length; e.g., 248 amino acids in length).

As one non-limiting example, the LBD of a PR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 3B; and is about 200 amino acids to 256 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 256 amino acids in length; e.g., 256 amino acids in length).

As one non-limiting example, the LBD of a PR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 3C; and is about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 250 amino acids in length; e.g., 248 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of a PR, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence GHSFADPASNLGLEDIIRKALMGSF (SEQ id no:8), wherein the co-regulatory peptide is from about 25 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a PR, the first member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence GHSFADPASNLGLEDIIRKALMGSF (SEQ ID NO:8), wherein the co-regulatory peptide is from about 25 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length).

Thyroid hormone receptor β

In some cases, a LBD suitable as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure comprises a LBD that is that of thyroid hormone receptor β (TR β), e.g., in some cases, the LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the LBD of TR β having the amino acid sequence set forth in fig. 4A.

As one non-limiting example, the LBD of TR β may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in FIG. 4D, and be about 200 amino acids to 250 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 230 amino acids, 230 amino acids to 240 amino acids, or 240 amino acids to 250 amino acids in length).

As one non-limiting example, the LBD of TR β may comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 4B, and is about 200 amino acids to 260 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 260 amino acids in length; e.g., 260 amino acids in length).

As one non-limiting example, the LBD of TR β may comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 4B, and is about 200 amino acids to 246 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 246 amino acids in length; e.g., 246 amino acids in length).

In some cases, when the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of TR β, the second member of the dimerization pair is an NCOA3/SRC3 polypeptide.

For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence STAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO:555) in some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the first member of the dimerization pair is a NCOA2/SRC2 polypeptide.

Estrogen receptor α

In some cases, a LBD suitable as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure comprises a LBD that is that of estrogen receptor α (ER α), e.g., in some cases, the LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the LBD of ER α having the amino acid sequence set forth in fig. 5A.

As one non-limiting example, the LBD of ER α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5H, and be about 200 amino acids to 240 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 230 amino acids, 230 amino acids to 235 amino acids, or 235 amino acids to 240 amino acids in length).

As one non-limiting example, the LBD of ER α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5B, and be about 180 amino acids to 229 amino acids in length (e.g., 180 amino acids to 200 amino acids or 200 amino acids to 229 amino acids in length; e.g., 229 amino acids in length).

As one non-limiting example, the LBD of ER α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5C, and be about 250 amino acids to 314 amino acids in length (e.g., 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 314 amino acids in length; e.g., 314 amino acids in length).

As a non-limiting example, the LBD of ER α can comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5D, and is about 190 amino acids to 238 amino acids in length (e.g., 190 amino acids to 220 amino acids or 220 amino acids to 238 amino acids in length; e.g., 238 amino acids in length).

As a non-limiting example, the LBD of ER α can comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5E, and have a D351Y substitution (where the amino acid numbering is based on the amino acid sequence set forth in FIG. 5A), and be about 180 amino acids to 229 amino acids in length (e.g., 180 amino acids to 200 amino acids or 200 amino acids to 229 amino acids in length; e.g., 229 amino acids in length).

As one non-limiting example, the LBD of ER α can comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5F, and have a D351Y substitution (where the amino acid numbering is based on the amino acid sequence set forth in FIG. 5A), and be about 250 amino acids to 314 amino acids in length (e.g., 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 314 amino acids in length; e.g., 314 amino acids in length).

As a non-limiting example, the LBD of ER α can comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 5G, and have a D351Y substitution (where the amino acid numbering is based on the amino acid sequence set forth in FIG. 5A), and be about 190 amino acids to 238 amino acids in length (e.g., 190 amino acids to 220 amino acids or 220 amino acids to 238 amino acids in length; e.g., 238 amino acids in length).

In some cases, when the first member of a dimerization pair of the conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER α, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence DAFQLRQLILRGLQDD (SEQ ID NO:12), wherein the co-regulatory peptide is from about 16 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 16 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length). in some cases, when the second member of a dimerization pair of the conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER α, the first member of a dimerization pair is a co-regulatory peptide comprising amino acid sequence DAFQLRQLILRGLQDD (SEQ ID NO:12), wherein the co-regulatory peptide is from about 16 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 16 amino acids to about 50 amino acids, 30 amino acids, 40 amino acids, 35 amino acids, or 45 amino acids).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER α, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence SPGSREWFKDMLS (SEQ ID NO:13), wherein the co-regulatory peptide is from about 13 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 13 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length), in some cases, where the second member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER α, the first member of a dimerization pair is a co-regulatory peptide comprising amino acid sequence SPGSREWFKDMLS (SEQ ID NO:13), wherein the co-regulatory peptide is from about 13 amino acids to 25 amino acids in length (e.g., from about 13 amino acids to 20 amino acids, 30 amino acids, 35 amino acids, 15 amino acids, 25 amino acids, 15 amino acids, or 45 amino acids in length).

Estrogen receptor β (ER β)

In some cases, a LBD suitable as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure comprises a LBD that is that of estrogen receptor α (ER β), e.g., in some cases, the LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the LBD of ER β having the amino acid sequence set forth in fig. 6A.

As one non-limiting example, the LBD of ER β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 6C, and be about 200 amino acids to 243 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 230 amino acids, 230 amino acids to 235 amino acids, or 235 amino acids to 243 amino acids in length).

As one non-limiting example, the LBD of ER β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 6B, and be about 200 amino acids to 243 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 230 amino acids, 230 amino acids to 235 amino acids, or 235 amino acids to 243 amino acids in length).

In some cases, when the first member of a dimerization pair of the conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER β, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence PRQGSILYSMLTSAKQT (SEQ ID NO:9), wherein the co-regulatory peptide is from about 17 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 17 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length). in some cases, when the second member of a dimerization pair of the conditionally active heterodimeric polypeptides of the present disclosure is an LBD of ER β, the first member of a dimerization pair is a co-regulatory peptide comprising amino acid sequence PRQGSILYSMLTSAKQT (SEQ ID NO:9), wherein the co-regulatory peptide is from about 17 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 17 amino acids to about 50 amino acids, from 17 amino acids to 30 amino acids, 40 amino acids, 35 amino acids, or 45 amino acids to 50 amino acids in length).

Peroxisome proliferator activated receptor-gamma

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of peroxisome proliferator-activated receptor-gamma (PPAR- γ). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of a PPAR- γ having the amino acid sequence set forth in figure 7A.

As one non-limiting example, the LBD of PPAR- γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences depicted in figure 7E; and is about 200 amino acids to 269 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 269 amino acids in length).

As one non-limiting example, the LBD of PPAR- γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in figure 7B; and is about 150 amino acids to 202 amino acids in length (e.g., 150 amino acids to 160 amino acids, 160 amino acids to 170 amino acids, 170 amino acids to 190 amino acids, or 190 amino acids to 202 amino acids in length).

As one non-limiting example, the LBD of PPAR- γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in figure 7C; and is about 200 amino acids to 269 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 269 amino acids in length).

As one non-limiting example, the LBD of PPAR- γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in figure 7D; and is about 200 amino acids to 269 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 271 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of PPAR- γ, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence CPSSHSSLTERHKILHRLLQEGSPS (SEQ ID NO:1), wherein the co-regulatory peptide is from about 25 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 25 amino acids to 28 amino acids, 28 amino acids to 29 amino acids, 29 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerized pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of PPAR- γ, the first member of the dimerized pair is a co-regulatory peptide comprising amino acid sequence CPSSHSSLTERHKILHRLLQEGSPS (SEQ ID NO:1), wherein the co-regulatory peptide is from about 17 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from about 25 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 25 amino acids to 28 amino acids, 28 amino acids to 29 amino acids, 29 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of PPAR- γ, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence PKKENNALLRYLLDRDDPSDV (SEQ id no:4), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 23 amino acids, from 23 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerized pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of PPAR- γ, the first member of the dimerized pair is a co-regulatory peptide comprising amino acid sequence PKKENNALLRYLLDRDDPSDV (SEQ ID NO:4), wherein the co-regulatory peptide is from about 17 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 23 amino acids, 23 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of PPAR- γ, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence PKKKENALLRYLLDKDDTKDI (SEQ id no:11), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 23 amino acids, from 23 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerized pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of PPAR- γ, the first member of the dimerized pair is a co-regulatory peptide comprising amino acid sequence PKKKENALLRYLLDKDDTKDI (SEQ ID NO:11), wherein the co-regulatory peptide is from about 21 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from 21 amino acids to 23 amino acids, 23 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length).

Glucocorticoid receptor

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of Glucocorticoid Receptor (GR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD having a GR of the amino acid sequence set forth in fig. 8A.

As one non-limiting example, the LBD of a GR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in fig. 8C; and is about 200 amino acids to 247 amino acids in length (e.g., 200 amino acids to 225 amino acids, 225 amino acids to 230 amino acids, 230 amino acids to 240 amino acids, or 240 amino acids to 247 amino acids in length).

As one non-limiting example, the LBD of a GR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 8B; and is about 200 amino acids to 247 amino acids in length (e.g., 200 amino acids to 225 amino acids or 225 amino acids to 247 amino acids in length; e.g., 247 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA1/SRC1 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA1/SRC1 polypeptide comprising amino acid sequence NYGTNPGTPPASTSPFSQLAANPEASLANRNSMVSRGMTGNIGGQFGTGINPQMQQNVFQYPGAGMVPQGEANFAPSLSPGSSMVPMPIPPPQSSLLQQTPPASGYQSPDMKAWQQGAIGNNNVFSQAVQNQPTPAQPGVYNNMSITVSMAGGNTNVQNMNPMMAQMQMSSLQMPGMNTVCPEQINDPALRHTGLYCNQLSSTDLLKTEADGTQQVQQVQVFADVQCTVNLVGGDPYLNQPGPLGTQKPTSGPQTPQAQQKSLLQQLLTE (SEQ ID NO:556) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the first member of the dimerization pair is a NCOA1/SRC1 polypeptide.

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of GR, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide comprising amino acid sequence KRHHHEVLRQGLAFSQIYRFSLSDGTLVAAQTKSKLIRSQTTNEPQLVISLHMLHREQNVCVMNPDLTGQTMGKPLNPISSNSPAHQALCSGNPGQDMTLSSNINFPINGPKEQMGMPMGRFGGSGGMNHVSGMQATTPQGSNYALKMNSPSQSSPGMNPGQPTSMLSPRHRMSPGVAGSPRIPPSQFSPAGSLHSPVGVCSSTGNSHSYTNSSLNALQALSEGHGVSLGSSLASPDLKMGNLQNSPVNMNPPPLSKMGSLDSKDCFGLYGEPSEGTTGQAESSCHPGEQKETNDPNLPPAVSSERADGQSRLHDSKGQTKLLQLLTTKSDQMEPSPLASSLSDTNKDSTGSLPGSGSTHGTSLKEKHKILHRLLQDSSSPVDLAKLTAEATGKDLSQESSSTAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO:557) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the first member of the dimerization pair is a NCOA2/SRC2 polypeptide.

Vitamin D receptors

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a Vitamin D Receptor (VDR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of a VDR having the amino acid sequence set forth in fig. 9A.

As one non-limiting example, the LBD of a VDR can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in fig. 9C; and is about 250 to 310 amino acids in length (e.g., 250 to 275 amino acids, 275 to 300 amino acids, or 300 to 310 amino acids in length).

As one non-limiting example, the LBD of a VDR can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 9B; and is about 250 to 303 amino acids in length (e.g., 250 to 275 amino acids, 275 to 300 amino acids, or 300 to 303 amino acids in length).

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA1/SRC1 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA1/SRC1 polypeptide comprising amino acid sequence NYGTNPGTPPASTSPFSQLAANPEASLANRNSMVSRGMTGNIGGQFGTGINPQMQQNVFQYPGAGMVPQGEANFAPSLSPGSSMVPMPIPPPQSSLLQQTPPASGYQSPDMKAWQQGAIGNNNVFSQAVQNQPTPAQPGVYNNMSITVSMAGGNTNVQNMNPMMAQMQMSSLQMPGMNTVCPEQINDPALRHTGLYCNQLSSTDLLKTEADGTQQVQQVQVFADVQCTVNLVGGDPYLNQPGPLGTQKPTSGPQTPQAQQKSLLQQLLTE (SEQ ID NO:558) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the first member of the dimerization pair is an NCOA1/SRC1 polypeptide.

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA2/SRC2 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA2/SRC2 polypeptide comprising amino acid sequence KRHHHEVLRQGLAFSQIYRFSLSDGTLVAAQTKSKLIRSQTTNEPQLVISLHMLHREQNVCVMNPDLTGQTMGKPLNPISSNSPAHQALCSGNPGQDMTLSSNINFPINGPKEQMGMPMGRFGGSGGMNHVSGMQATTPQGSNYALKMNSPSQSSPGMNPGQPTSMLSPRHRMSPGVAGSPRIPPSQFSPAGSLHSPVGVCSSTGNSHSYTNSSLNALQALSEGHGVSLGSSLASPDLKMGNLQNSPVNMNPPPLSKMGSLDSKDCFGLYGEPSEGTTGQAESSCHPGEQKETNDPNLPPAVSSERADGQSRLHDSKGQTKLLQLLTTKSDQMEPSPLASSLSDTNKDSTGSLPGSGSTHGTSLKEKHKILHRLLQDSSSPVDLAKLTAEATGKDLSQESSSTAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO:559) or a fragment thereof. For example, in some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of a VDR, the second member of the dimerization pair is an NCOA2/SRC2 polypeptide comprising the amino acid sequence LLRYLLDK (SEQ ID NO:560), wherein the co-regulatory peptide is from about 8 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from about 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 23 amino acids, 23 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the first member of the dimerization pair is an NCOA2/SRC2 polypeptide.

Thyroid hormone receptor α

In some cases, a LBD suitable as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure comprises a LBD that is that of thyroid hormone receptor α (TR α), e.g., in some cases, the LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the LBD of TR α having the amino acid sequence set forth in fig. 10A.

As one non-limiting example, the LBD of TR α may comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in FIG. 10C, and is about 190 amino acids to about 245 amino acids in length (e.g., 190 amino acids to 210 amino acids, 210 amino acids to 230 amino acids, or 230 amino acids to 245 amino acids in length).

As one non-limiting example, the LBD of TR α may comprise an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in FIG. 10B, and is about 190 amino acids to about 243 amino acids in length (e.g., 190 amino acids to 210 amino acids, 210 amino acids to 230 amino acids, or 230 amino acids to 243 amino acids in length).

A suitable co-modulatory peptide of TR α is a SRC1 polypeptide or fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

Retinoic acid receptor β

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is that of retinoic acid receptor β (RAR β) -e.g., in some cases, a LBD includes an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the LBD of RAR β having the amino acid sequence set forth in fig. 11A.

As one non-limiting example, the LBD of the RAR β may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in figure 11C, and from about 180 amino acids to about 235 amino acids in length (e.g., from 180 amino acids to 200 amino acids, from 200 amino acids to 220 amino acids, or from 220 amino acids to 235 amino acids in length).

As one non-limiting example, the LBD of the RAR β may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 11B, and from about 180 amino acids to about 231 amino acids in length (e.g., from 180 amino acids to 200 amino acids, from 200 amino acids to 220 amino acids, or from 220 amino acids to 231 amino acids in length).

A suitable co-modulatory peptide of RAR β is SRC1 polypeptide or a fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

In some cases, when the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of RAR β, the second member of the dimerization pair is a NCOA1/SRC1 polypeptide.

In some cases, when the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of RAR β, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide.

Farnesoid X receptor

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of Farnesoid X Receptor (FXR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of an FXR having the amino acid sequence set forth in figure 22A.

As one non-limiting example, the LBD of the FXR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 22B; and from about 100 amino acids to about 136 amino acids in length (e.g., from 100 amino acids to 110 amino acids, from 110 amino acids to 120 amino acids, or from 120 amino acids to 136 amino acids in length).

A suitable co-modulatory peptide of FXR is a SRC1 polypeptide or fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

LXRα

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of liver X receptor α (LRX α), e.g., in some cases, a LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a LBD of LRX α having the amino acid sequence set forth in fig. 23A.

As one non-limiting example, the LBD of LRX α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 23B, and be about 200 amino acids to about 266 amino acids in length (e.g., 200 amino acids to 220 amino acids, 220 amino acids to 240 amino acids, or 240 amino acids to 266 amino acids in length).

A suitable co-modulatory peptide of LRX α is a SRC1 polypeptide or fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

RORγ

In some cases, an LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is an LBD of retinoid-related orphan receptor gamma (ROR γ). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD having ROR γ of the amino acid sequence set forth in figure 24A.

As one non-limiting example, the LBD of rory may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 24B; and from about 200 amino acids to about 261 amino acids in length (e.g., from 200 amino acids to 220 amino acids, from 220 amino acids to 240 amino acids, or from 240 amino acids to 261 amino acids in length).

A suitable co-regulator of ROR γ is the NCORNR peptide (CDPASNLGLEDIIRKALMGSFDDK, SEQ ID NO: 563).

A suitable co-modulatory peptide of ROR γ is a SRC1 polypeptide or fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

RXRα

In some cases, a LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of retinoid X receptor α (RXR α) -for example, in some cases, a LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a LBD of RXR α having the amino acid sequence set forth in fig. 25A.

As one non-limiting example, the LBD of rory may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 25B; and from about 190 amino acids to about 238 amino acids in length (e.g., from 190 amino acids to 200 amino acids, from 200 amino acids to 210 amino acids, or from 210 amino acids to 238 amino acids in length).

A suitable co-modulatory peptide for RXR α is a SRC1 polypeptide or fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from a SRC1 polypeptide).

PXR

In some cases, an LBD suitable for inclusion as a member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of the Pregnane X Receptor (PXR). For example, in some cases, an LBD comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an LBD of a PXR having the amino acid sequence set forth in fig. 26A. In some cases, the LBD comprises an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 143 and 428 having the amino acid sequences set forth in FIG. 26A. In some cases, the LBD comprises an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acid 205-434 having the amino acid sequence set forth in FIG. 26A.

As one non-limiting example, the LBD of PXR may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in fig. 26B; and from about 250 amino acids to about 302 amino acids in length (e.g., from 250 amino acids to 275 amino acids, from 275 amino acids to 290 amino acids, or from 290 amino acids to 302 amino acids in length).

A suitable co-modulatory peptide of PXR is the SRC1 polypeptide or a fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from the SRC1 polypeptide).

Co-regulatory polypeptides

Suitable co-regulatory polypeptides include full-length naturally occurring nuclear hormone co-regulatory polypeptides. Suitable co-regulatory polypeptides include fragments of naturally occurring nuclear hormone co-regulatory polypeptides. Suitable co-regulatory polypeptides include synthetic or recombinant nuclear hormone co-regulatory polypeptides.

Suitable co-modulating polypeptides may be 8 amino acids to 2000 amino acids in length. Suitable co-modulating polypeptides may be 8 amino acids to 50 amino acids in length, for example 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids. Suitable co-modulating polypeptides may be from 50 amino acids to 100 amino acids in length, for example from 50 amino acids to 60 amino acids, from 60 amino acids to 70 amino acids, from 70 amino acids to 80 amino acids, from 80 amino acids to 90 amino acids or from 90 amino acids to 100 amino acids. Suitable co-modulating polypeptides may be 100 amino acids to 200 amino acids, 200 amino acids to 300 amino acids, 300 amino acids to 400 amino acids, 400 amino acids to 500 amino acids, 500 amino acids to 600 amino acids, 600 amino acids to 700 amino acids, 700 amino acids to 800 amino acids, 800 amino acids to 900 amino acids, or 900 amino acids to 1000 amino acids in length. Suitable co-modulating polypeptides may be 1000 amino acids to 2000 amino acids in length.

Suitable co-modulatory polypeptides include, but are not limited to, SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

The accession numbers of the National Center for Biotechnology Information (NCBI) for such co-regulators include the following: SRC1(NP _003734), GRIP1(NP _006531), AIB1(NP _006525), PGC1a (NP _037393), PGC1b (NP _573570), PRC (NP _055877), TRAP220(NP _004765), ASC2(NP _054790), CBP (NP _004371), P300(NP _001420), CIA (NP _066018), ARA70(NP _005428), TIF1(NP _003843), NSD1(NP _071900), SMAP (NP _006687), Tip60(NP _006379), ERAP140(NP _861447), Nix1(NP _113662), LCoR (NP _115816), N-CoR (NP _006302), SMRT (NP _006303), RIP140(NP _003480), and PRIC285(NP _ 208384).

Examples of suitable co-regulatory polypeptides are provided in FIGS. 29-51B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 29.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 30.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 31.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 32.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 33.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 34.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 35.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in fig. 36A-36B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in fig. 37A-37B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 39.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 40.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 41.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 42A-42B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 43.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 44.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 45.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 46.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 47.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 48A-48B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 49A-49B.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 50.

In some cases, suitable co-modulating polypeptides are 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length; and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a segment of 8 to 50 consecutive amino acids of the amino acid sequence set forth in figure 51A-51B.

Suitable co-modulatory peptides include, but are not limited to, steroid receptor co-activating factor (SRC) -1, SRC-2, SRC-3, TRAP220-1, TRAP220-2, NR0B1, NRIP1, CoRNR cassette, αβ V, TIF1, TIF2, SRC2, TA1, EAB1, SRC1-1, SRC1-2, SRC1-3, SRC1-4a, SRC1-4B, GRIP1-1, GRIP1-2, GRIP1-3, AIB1-1, AIB1-2, AIB1-3, PGC 11, PGC1, PRC, ASC 1-1, ASC 1-2, CBP-1, CBP-2, P300, CIA, ARA 1-1, ARA 1-2, PRNSD 1, PRAP, TAP, SMAP, ERP 285-140, RIC 285-140, RIP 285-140, RIC285-3, RIC 1, RIC 140, RIC285-3, RIP1, RIC 140, RIC285-3, RIC 140, RIP140, and RIC 285-3.

In some cases, suitable co-regulatory peptides comprise the LXXLL motif, wherein X is any amino acid; wherein the co-regulatory peptide is 8 amino acids to 50 amino acids in length, e.g., 8 amino acids to 10 amino acids, 10 amino acids to about 12 amino acids, 12 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids.

Non-limiting examples of suitable co-regulatory peptides are as follows:

additional examples are provided in example 1, example 2 and example 3.

For example, as shown in fig. 19, PPAR γ may be paired with SRC1, SRC2, SRC3, or TRAP220 as another example, ER α may be paired with cockr, αβ V, or TA1 as another example, ER β may be paired with cockr, αβ V, or TA1 as another example, AR may be paired with SRC1, SRC2, SRC3, or TRAP220 as another example, PR may be paired with SRC1, SRC2, SRC3, TRAP220, NR0B1, PGC1B, NRIP1, EA2, or EAB1 as another example, TR β may be paired with SRC1, SRC2, SRC3, or TRAP 220.

In some cases, heterodimeric polypeptides of the present disclosure comprise a polypeptide chain comprising a plurality (two or more) of co-regulatory peptides. Where the heterodimeric polypeptides of the present disclosure comprise polypeptide chains comprising multiple (two or more) co-regulatory peptides, the multiple co-regulatory peptides can be directly linked in series or separately linked in series via a linker. In some cases, two or more co-regulatory peptides present in a polypeptide chain are identical to each other in amino acid sequence. In some cases, where a heterodimeric polypeptide of the present disclosure comprises a polypeptide chain comprising multiple (two or more) co-regulatory peptides, the polypeptide chain comprises two co-regulatory peptides. In some cases, where a heterodimeric polypeptide of the present disclosure comprises a polypeptide chain comprising multiple (two or more) co-regulatory peptides, the polypeptide chain comprises three co-regulatory peptides. In such cases, the second polypeptide chain can comprise multiple (two or more) LBDs of the nuclear hormone receptor. For example, when the second polypeptide chain comprises two LBDs of a nuclear hormone receptor, the two LBDs may be identical to each other in amino acid sequence.

Intracellular signaling domains

Intracellular signaling domains suitable for use in the conditionally active heterodimeric polypeptides of the present disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., an increase in production of one or more cytokines by a cell, a change in transcription of a target gene, a change in protein activity, a change in cellular behavior, such as cell death, cell proliferation, cell differentiation, cell survival, modulation of a cellular signaling response, etc.) in response to activation of the conditionally active heterodimeric polypeptide (i.e., by activation of an antigen and a dimerizing agent). In some cases, the intracellular signaling domain of the conditionally active heterodimeric polypeptides of the present disclosure provides a signaling function when activated, and thus may be referred to as a signaling domain in some cases. In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10/CD 28-type signaling strand. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane to which the conditionally active heterodimeric polypeptide binds, but diffuses into the cytoplasm.

ITAM

The intracellular signaling domains of conditionally active heterodimeric polypeptides suitable for use in the present disclosure include the immunoreceptor tyrosine-based activation motif (ITAM) comprising an intracellular signaling polypeptide. Motif of ITAM is YX₁X₂L/I, wherein X₁And X₂Independently any amino acid (SEQ ID NO: 564). In some cases, the intracellular signaling domain of a CAR of the invention comprises 1,2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are spaced 6 to 8 amino acids apart from each other, e.g., (YX)₁X₂L/I)(X₃)_n(YX₁X₂L/I) where n is an integer of 6 to 8, and 6-8X₃Can be any amino acid (SEQ ID NO: 565). In some cases, the intracellular signaling domain of a CAR of the invention comprises 3 ITAM motifs.

Suitable intracellular signaling domains may contain portions of the ITAM motif derived from polypeptides containing the ITAM motif for example, suitable intracellular signaling domains may be domains containing the ITAM motif from any protein containing the ITAM motif therefore suitable intracellular signaling domains need not contain the complete sequence of the complete protein from which they are derived examples of suitable polypeptide containing the ITAM motif include, but are not limited to, DAP12, FCER1G (fcepsilon receptor I γ chain), CD3D (CD3 δ), CD3E (CD3 ε), CD3G (CD3 γ), CD3Z (CD3 ζ), and CD79A (α chain of antigen receptor complex related proteins).

In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR related protein; TYRO protein tyrosine kinase binding protein; killer cell activating receptor related protein; etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any one of the following amino acid sequences (4 isoforms):orWherein the ITAM motif is shown in bold and underlined.

Likewise, suitable intracellular signaling domain polypeptides may comprise the ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor Igamma chain; Fc receptor gamma chain; Fc-epsilon RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor high affinity gamma chain, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain polypeptide may comprise the portion of the full-length FCER1G amino acid sequence that contains the ITAM motif. Thus, suitable intracellular signaling domainsThe polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 DELTA chain (also known as CD 3D; CD 3-DELTA; T3D; CD3 antigen DELTA subunit; CD3 DELTA; CD3d antigen; DELTA polypeptide (TiT3 complex); OKT 3; DELTA chain; T cell receptor T3 DELTA chain; T cell surface glycoprotein CD3 DELTA chain; etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 170aa of any of the following amino acid sequences (2 isoforms):orWherein the ITAM motif is shown in bold and underlined.

Likewise, suitable intracellular signaling domain polypeptides may comprise the full-length CD3 delta amino acid sequence containingPart of the ITAM motif. Thus, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 205aa of the following amino acid sequence:wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain polypeptide may comprise the portion of the full length CD3 epsilon amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also referred to as CD3G, T cell receptor T3 gamma chain, CD3 gamma, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 180aa of the following amino acid sequence:wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain polypeptide may comprise the portion of the full-length CD3 γ amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3 zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 160aa of any of the following amino acid sequences (2 isoforms):orWherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain polypeptide may comprise the portion of the full-length CD3 ζ amino acid sequence that comprises the ITAM motif. Thus, suitable intracellular signaling domain polypeptides may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more amino acid sequence identity to any one of the following amino acid sequencesAmino acid sequence of about 98% or 100% amino acid sequence identity:orWherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from CD79A (also referred to as B cell antigen receptor complex-related protein α chain; CD79a antigen (immunoglobulin-related α); MB-1 membrane glycoprotein; ig α; membrane-bound immunoglobulin-related protein; surface IgM-related protein, etc.) for example, suitable intracellular signaling domain polypeptides may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 150aa, about 150aa to about 200aa, or about 200aa to about 220aa of any of the following amino acid sequences (2 isoforms):or

MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:587), wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain polypeptide may comprise the portion of the full length CD79A amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain polypeptide may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

DAP10/CD28

Intracellular signaling domains suitable for use in the conditionally active heterodimeric polypeptides of the present disclosure include DAP10/CD 28-type signaling chains.

Examples of amino acid sequences for DAP10 signaling chains are:in some embodiments, suitable intracellular signaling domains comprise amino acid sequencesHas at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity.

An example of an amino acid sequence for the CD28 signaling chain isIn some embodiments, suitable intracellular signaling domains comprise amino acid sequencesHas at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity.

ZAP70

The intracellular signaling domains of conditionally active heterodimeric polypeptides suitable for use in the present disclosure include ZAP70 polypeptides, e.g., polypeptides comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous segment of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to 619 amino acids of the amino acid sequence:

MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA(SEQ ID NO:593)。

additional sequences

The first polypeptide and/or the second polypeptide of the conditionally active heterodimeric polypeptides of the present invention may further comprise one or more additional polypeptide domains, wherein such domains include, but are not limited to, signal sequences; an epitope tag; an affinity domain; and a polypeptide that produces a detectable signal.

Signal sequence

Signal sequences suitable for use in a CAR of the invention (e.g., the first polypeptide of a CAR of the invention) include any eukaryotic signal sequence, including naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, and the like.

Epitope tag

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 594)), FLAG (e.g., DYKDDDDK (SEQ ID NO: 595)), c-myc (e.g., EQKLISEEDL; SEQ ID NO:596), and the like.

Affinity domains

Affinity domains include peptide sequences that can interact with a binding partner, such as, for example, peptide sequences that can be identified or purified, immobilized on a solid support. When fused to an expressed protein, DNA sequences encoding multiple contiguous single amino acids (such as histidine) can be used to purify the recombinant protein in one step by high affinity binding to a resin column (such as nickel sepharose). Exemplary affinity domains include His5(HHHHH) (SEQ ID NO:597), HisX6 (HHHHHHH) (SEQ ID NO:598), C-myc (EQKLISEEDL) (SEQ ID NO:599), flag (DYKDDDDK) (SEQ ID NO:600), Streptag (WSHPQFEK) (SEQ ID NO:601), hemagglutinin (e.g., HA tag (YPYDDYA) (SEQ ID NO:602)), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:603), Phe-His-Thr (SEQ ID NO:064), chitin binding domain, S peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:605), metal binding domain (e.g., zinc-binding domain or calcium binding domain, e.g., from calponin (e.g., calponin, troponin B, troponin C-light chain myosin, troponin B, and troponin B, S regulatory protein, cone protein (visinin), VILIP, calcineurin, hippocampal calbindin, agrin, kallikrein, the large subunit of calpain, S100 protein, parvalbumin, calbindin D9K, calbindin D28K and calomenine) those calcium binding domains), intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences and maltose binding protein.

Polypeptide producing detectable signal

Suitable proteins that produce a detectable signal include, for example, fluorescent proteins; an enzyme that catalyzes a reaction to produce a detectable signal as a product; and so on.

Suitable fluorescent proteins include, but are not necessarily limited to, Green Fluorescent Protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), Enhanced GFP (EGFP), Enhanced CFP (ECFP), Enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFpm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, T-HcRed, DsRed2, DsRed-monomer, J-Red, dimer 2, T-dimer 2(12), mRFP1, gobicillotoxin (porcelloporin), Renilla GFP, MonsterGFP, paGFP, Kaede protein and kindling protein, phycobiliprotein and phycobiliprotein conjugates (including B-phycoerythrin, R-phycoerythrin and allophycocyanin). Other examples of fluorescent proteins include mHoneydev, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al (2005) nat. methods 2:905-909) and the like. Any of a variety of fluorescent and colored proteins from species of the class Corallium as described, for example, in Matz et al (1999) Nature Biotechnol.17:969-973 are suitable for use.

Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), Alkaline Phosphatase (AP), β -Galactosidase (GAL), glucose-6-phosphate dehydrogenase, β -N-acetylglucosaminidase, β -glucuronidase, invertase, xanthine oxidase, firefly luciferase, Glucose Oxidase (GO), and the like.

Exemplary conditionally active heterodimeric polypeptides

In some cases, conditionally active heterodimeric polypeptides of the present disclosure comprise: a) a first chimeric polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second chimeric polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain, wherein i) the first regulatory domain and the second regulatory domain are derived from 4-1 BB; ii) the first and second members of the dimerization pair are SRC3 and PPAR γ LBD, respectively; and iii) the signaling domain comprises ITAM. In some cases, the first member of the dimerization pair comprises one copy of the SRC3 co-regulatory peptide. In some cases, the SRC3 co-regulator peptide is 20 amino acids to 25 amino acids in length. In some cases, the SRC3 co-regulator peptide is 75 amino acids to 80 amino acids in length. In some cases, the first chimeric polypeptide comprises a single SRC3 co-regulatory peptide. In some cases, the first chimeric polypeptide comprises 2 copies of the SRC3 co-regulatory peptide. In some cases, the first chimeric polypeptide comprises 3 copies of the SRC3 co-regulatory peptide. In some cases, each copy of the SRC3 co-regulated peptide is 20 amino acids to 25 amino acids in length.

For example, in some cases, a conditionally active heterodimeric polypeptide of the present disclosure comprises a first polypeptide chain comprising: i) single-chain Fv; ii) a transmembrane polypeptide; and iii) a polypeptide comprising the amino acid sequence:wherein the 4-1BB sequence is shown in italics and bold text, and the co-regulatory peptide is underlined (single underline). In some cases, the second polypeptide chain comprises the following amino acid sequence:

wherein PPAR γ LBD is underlined (single underlined), the CD8 α TM domain is double underlined, 4-1BB is shown in bold text, and CD3 ζ is shown in bold and italics.

In some cases, the first member of the specific binding pair is an scFv specific for CD19, and the first polypeptide chain comprises a transmembrane domain between the anti-CD 19scFv and a first regulatory domain, wherein the first regulatory domain is 4-1 BB. For example, in some cases, a conditionally active heterodimeric polypeptide of the present disclosure comprises a first polypeptide chain comprising the following amino acid sequence:

wherein the anti-CD 19 sequence is shown in bold, the CD8 α Transmembrane (TM) domain is double underlined, the 4-1BB sequence is shown in italics and bold text, and the co-regulatory peptide is underlined (single underlined).

As another example, in some cases, conditionally active heterodimeric polypeptides of the present disclosure comprise a first polypeptide chain comprising the following amino acid sequence:wherein the anti-CD 19 sequence is shown in bold, the CD8 α Transmembrane (TM) domain is double underlined, the 4-1BB sequence is shown in italics and bold text, and the co-regulatory peptide is underlined (single underlined).

As another example, in some cases conditionally active heterodimeric polypeptides of the present disclosure comprise a) a first chimeric polypeptide comprising i) a first member of a specific binding pair, ii) a first regulatory domain, iii) a first member of a dimerization pair, and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain, and b) a second chimeric polypeptide comprising i) a transmembrane domain, ii) a second regulatory domain, iii) a second member of the dimerization pair, and iv) an intracellular signaling domain, wherein i) the first regulatory domain and the second regulatory domain are derived from 4-1BB, ii) the first member and the second member of the dimerization pair are respectively a cognr and an α LBD, and iii) the signaling domain comprises itam, hi some cases, the first member of the pair comprises one copy of a cognr corer co-regulatory peptide, in some cases, the cognr co-regulatory peptide is 60 amino acids in some cases, the first amino acid co-regulatory peptide comprises a single copy of a cognr co-regulatory peptide, in some cases, the first chimeric polypeptide comprises a single copy of a cognr co-regulatory peptide, and in some cases, the first chimeric polypeptide comprises a single amino acid, and in some cases, the second regulatory peptide, and in some cases, the chimeric polypeptide comprises a single copy of a chimeric polypeptide, and a chimeric polypeptide comprising a chimeric polypeptide.

For example, in some cases, a conditionally active heterodimeric polypeptide of the present disclosure comprises a first polypeptide chain comprising: i) single-chain Fv; ii) a transmembrane polypeptide; and iii) a polypeptide comprising the amino acid sequence:wherein the 4-1BB amino acid sequence is shown in bold and italics, and the 3x CoR NR co-regulatory peptide is underlined. In some cases, the second polypeptide chain comprises the following amino acid sequence:wherein the TM polypeptide is drawn down by twoLine, the 4-1BB amino acid sequence is shown in bold and italics, and ER LBD is underlined (single underline).

wherein the anti-CD 19scFv is shown in bold, the TM region is double underlined, the 4-1BB polypeptide is shown in bold and italics, and the 3x corrnr co-regulatory peptide is underlined (single underlined). In some cases, the second polypeptide chain comprises the following amino acid sequence:wherein the TM polypeptide is double underlined, the 4-1BB amino acid sequence is shown in bold and italics, and the ER LBD is underlined (single underlined).

Dimerizing agent

Suitable dimerizing agents (also referred to as dimerizing agents) bind to the LBD of the nuclear hormone receptor in a first polypeptide of a conditionally active heterodimeric polypeptide of the present disclosure and to a co-regulatory peptide in a second polypeptide of the conditionally active heterodimeric polypeptide. Binding of a dimerizing agent to the LBD and the co-regulatory peptide functions to dimerize the first polypeptide and the second polypeptide of the conditionally active heterodimeric polypeptides of the present disclosure.

Suitable dimerizing agents are known in the art; any known dimerizing agent may be used.

Examples of dimerizing agents include corticosterone (11, 21-dihydroxy-4-pregnene-3, 20-dione), deoxycorticosterone (21-hydroxy-4-pregnene-3, 20-dione), cortisol (11, 17, 21-trihydroxy-4-pregnene-3, 20-dione), 11-deoxycorticosterol (17, 21-dihydroxy-4-pregnene-3, 20-dione), cortisone (17, 21-dihydroxy-4-pregnene-3, 11, 20-trione), 18-hydroxycorticosterone (11, 18, 21-trihydroxy-4-pregnene-3, 20-dione), 1.1-hydroxycorticosterone (1, 11, 21-trihydroxy-4-pregnene-3, 20-dione), aldosterone acetal of 11, 21-dihydroxy-3, 20-dioxo-4-androstene-18-aldehyde, 11-hemigestrel, androstenedione (4-androstenedione-3, 17-dione), testosterone (5, 17-dehydroandrostenedione-androstenedione (1, 5, 17-dihydroxy-4-pregnene-3, 17-testosterone), testosterone (1, 5-dehydroandrostenedione-4-hydroxyprogesterone-3, 17-androstenedione (1, 17-5-hydroxy-4-pregnene-3, 17-androstenedione), testosterone-5-androstenediol (1, 17-5-androstenediol), 5-hydroxy-androstenediol, 17-5-androstenediol, 5-androstenediol (1, 5-2-androstenediol, 17-androstenediol, 5-estrene-androstenediol, 5-2-5-estrene-5-4-estrene-3, 5-estrene-3, 17-estrene-3, 17-estradone-5-estradone-3, 17-estradone-5-estradone-3, 17-estradone-5-estradone-.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise the LBD of the Mineralocorticoid Receptor (MR) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, spironolactone and eplerenone.

Spironolactones are compounds having the following structure:

spironolactone may be administered at a dose in the range of 10 to 35mg per day (e.g. 25mg per day).

Eplerenone is a compound having the structure:

where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of the Androgen Receptor (AR) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, cyproterone acetate, hydroxyflutamide, enzalutamide, ARN-509, 3' -Diindolylmethane (DIM), beclomede, bicalutamide, N-butylbenzene-sulfonamide (NBBS), dutasteride, epristeride, finasteride, flutamide, isoxazolemide (izonsteride), ketoconazole, N-butylbenzene-sulfonamide, nilutamide, megestrol, steroidal antiandrogen, and tolongeurea.

Cyproterone acetate is a compound having the formula:

flutamide is a compound having the formula:

hydroxyflutamide is a compound having the formula:

enzalutamide is a compound having the formula:

ARN-509 is a compound having the formula:

in the case of the conditionally active heterodimeric polypeptides disclosed herein comprising an LBD of the Progesterone Receptor (PR) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, mifepristone (RU-486; 11- [4N, N-dimethylaminophenyl ] -17-hydroxy-17- (1-propynyl) -estra-4, 9-dien-3-one), rilospirone (11- (4N, N-dimethylaminophenyl) -17-hydroxy-17- ((Z) -3-hydroxypropenyl) -estra-4, 9-dien-3-one; linaloone (11- (4N, N-dimethylaminophenyl) -17-0-hydroxy-17- (3-hydroxypropyl) -13-estra-4, 9-dien-3-one), esocinone (11- (3-methoxy-1, 17-yl) -17- (11, 17-methoxy-17-methyl) -3-oxo-4, 9-dien-11-yl ] -1- (E) -17-yl ] -1- (11, 17-7, 17-methoxy-17-oxo-4, 9-dien-11-yl ] -1- (E) -1, 17-11-oxo-4, 9-11-dien-11-yl-17-oxo-17-4, 9-17-5- (11-hydroxy-17-4-5- (4-propenyl) -5- (4-methoxy-1- (4-1, N-dimethylaminophenyl) -17-5-4- ((Z-5-3-hydroxy-2-3-2-hydroxypropyl) -4- ((Z-hydroxy-3-hydroxy-propenyl) -13- ((Z) -estra-3-hydroxy-3-hydroxy-4- ((Z) -3-hydroxy-3-4- ((Z-hydroxy-4, 9-propenyl) -13-4, 9-hydroxy-4, 9-4-hydroxy-4, 9-4-hydroxy-4, 9-4-hydroxy-4, 9-propenyl) estra-4, 9-4, 9-hydroxy-4, 9-4, 9-4-hydroxy-4, 9-4-3-hydroxy-9-4-hydroxy-4, 9-hydroxy-9-hydroxy-ethyl-4, 9-4, 9-hydroxy-4, 9-4-3-9-4, 9-4, 9-ethyl-4-9-5-4-5-4-9-4, 9-hydroxy-4-hydroxy-3-ethyl-4, 9-4, 9-ethyl-4, 9-4-ethyl-4-3-4, 9-hydroxy-ethyl-hydroxy-ethyl-hydroxy-4-hydroxy-3-9-2-9-4-9-4, 9-4, 9-hydroxy-4-9-4, 9-5-hydroxy-2-4-hydroxy-4, 9-3-4-hydroxy-4, 9-hydroxy-4-hydroxy-4-hydroxy-3-4-3-hydroxy-3-4-3-5-ethyl-2-3-hydroxy-4-3-4-hydroxy-2-hydroxy-13-2-hydroxy-2-4, 9-one, 9-2-4-2-hydroxy-3-one, 9-4-3-hydroxy-one, 9-3-one; 9-4-one; 9-2-.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of thyroid receptor- β (TR β) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, T₃(3,5,3' -triiodo-L-thyronine); KB-141(3, 5-dichloro-4- (4-hydroxy-3-isopropylphenoxy) phenylacetic acid); sobetim (also known as GC-1) (3, 5-dimethyl-4- (4 '-hydroxy-3' -isopropylbenzyl) -phenoxyacetic acid); GC-24(3, 5-dimethyl-4- (4 '-hydroxy-3' -benzyl) benzylphenoxyacetic acid); 4-OH-PCB106(4-OH-2',3,3',4',5' -pentachlorodiphenyl); eprotirome (eprotirome); MB07811((2R,4S) -4- (3-chlorophenyl) -2- [3, 5-dimethyl-4- (4 '-hydroxy-3' -isopropylbenzyl) phenoxy) methyl]-2-oxo- [1,3,2]-dioxolanes); QH 2; and (3, 5-dimethyl-4- (4 '-hydroxy-3' -isopropylbenzyl) phenoxy) methylphosphonic acid (MB 07344).

Ilotirol has the following structure:

QH2 has the following structure:

where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of estrogen receptor α (ER α) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, tamoxifen, 4-OH-tamoxifen, raloxifene, lasofoxifene, bazedoxifene, fulvestrant, clomiphene, vetenabao (femarelle), oxymetaxifene, toremifene, ospemifene, and ethinyl estradiol.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of estrogen receptor β (ER β) and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, estradiol (E2; or 17 β estradiol) and ethinyl estradiol.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of PPAR γ and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, thiazolidinediones (e.g., rosiglitazone, pioglitazone, lobeglitazone, troglitazone), faglitazar, argazazar, and fenofibric acid.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of GR and a corresponding co-regulatory peptide, a suitable dimerizing agent may be a selective GR agonist (SEGRA) or a selective GR modulator (SEGRM). Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of GR and a corresponding co-modulatory peptide, suitable dimerizing agents include, but are not limited to, benzopyran quinoline a 276575, mapracoat, ZK216348, 55D1E1, dexamethasone, prednisolone, prednisone, methylprednisolone, fluticasone propionate, beclomethasone-17-monopropionate, betamethasone, rimexolone, paramethasone, and hydrocortisone.

Non-limiting examples are shown below.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise LBD of VDR and the corresponding co-modulatory peptide, suitable dimerizing agents may be 1, 25-dihydroxyvitamin D3 (calcitriol), paricalcitol, doxercalciferol, 25-hydroxyvitamin D3 (calcifediol), cholecalciferol, ergocalciferol, tacalcitol, 22-dihydroergocalciferol, (6Z) -tacalcitol, 2-methylene-19-nor-20 (S) -1 α -hydroxy-bishomopregnacalcitol, 19-nor-26, 27-dimethylene-20 (S) -2-methylene-1 α, 25-dihydroxyvitamin D3, 2-methylene-1 α, 25-dihydroxy- (17E) -17(20) -dehydro-19-vitamin D3, 2-methylene-19-nor- (24R) -1 α, 25-dihydroxyvitamin D2, 2-methylene- (20R) -19-nor- (2R) -2-hydroxy-19-nor-19, 2-nor-vitamin D-2-hydroxy-pregnacalcifernacalcitol, 2-methylene-1-2-methylene-2-methylene-1 3619, 2-nor-2-methylvitamin D2, 2-methylene-2-norpregnacalcitol, 2-norpregnacalciferol, 2-norpregnacalcitol, 2-norpregnacin-2-norvitamin D-2-norpregnacin-2-norvitamin D-2, 2-9-2.

Where the conditionally active heterodimeric polypeptide of the present disclosure comprises LBD of RAR β and a corresponding co-regulatory peptide, suitable dimerizing agents may be retinoic acid, all-trans retinoic acid, 9-cis-retinoic acid, tamibarotene, 13-cis-retinoic acid, (2E,4E,6Z,8E) -3, 7-dimethyl-9- (2,6, 6-trimethyl-1-cyclohexenyl) non-2, 4,6, 8-tetraenoic acid, 9- (4-methoxy-2, 3, 6-trimethyl-phenyl) -3, 7-dimethyl-non-2, 4,6, 8-tetraenoic acid, 6- [3- (1-adamantyl) -4-methoxyphenyl ] -2-naphthoic acid, 4- [1- (3,5,5,8, 8-pentamethyl-tetralin-2-yl) vinyl ] benzoic acid, retinyl benzoic acid, ethyl 6- [2- (4, 4-dimethylbenzothianidin-6-yl) pyridine-3-yl ] pyridine-carboxylate, retinol, and acetyl cholesterol.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of FXR and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, obeticholic acid, LY2562175(6- (4- ((5-cyclopropyl-3- (2, 6-dichlorophenyl) isoxazol-4-yl) methoxy) piperidin-1-yl) -1-methyl-1H-indole-3-carboxylic acid) and GW4064(3- [2- [ 2-chloro-4- [ [3- (2, 6-dichlorophenyl) -5- (1-methylethyl) -4-isoxazolyl ] methoxy ] phenyl ] vinyl ] benzoic acid).

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of LXR α and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, T0901317(N- (2,2, 2-trifluoroethyl) -N- [4- [2,2, 2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl ] phenyl ] benzenesulfonamide), GW3965(3- [3- [ [ [ 2-chloro-3- (trifluoromethyl) phenyl ] methyl ] (2, 2-diphenylethyl) amino ] propoxy ] phenylacetic acid hydrochloride), and LXR-623.

LXR-632 has the following structure:

where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of ROR γ and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to GNE-3500(27,1- {4- [ 3-fluoro-4- ((3S,6R) -3-methyl-1, 1-dioxo-6-phenyl- [1,2] thiazin-2-ylmethyl) -phenyl ] -piperazin-1-yl } -ethanone).

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise an LBD of rory and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, 7 β, 27-dihydrocholesterol and 7 α, 27-dihydrocholesterol.

Where the conditionally active heterodimeric polypeptides of the present disclosure comprise LBD of RXR α and a corresponding co-regulatory peptide, suitable dimerizing agents include, but are not limited to, 9-cis retinoic acid, LGD100268, CD3254(3- [ 4-hydroxy-3- (5,6,7, 8-tetrahydro-3, 5,5,8, 8-pentamethyl-2-naphthyl) phenyl ] -2-propenoic acid), and CD2915(Sorensen et al (1997) skinpharmacol.10: 144).

LGD100268 has the following structure:

where the conditionally active heterodimeric polypeptides of the present disclosure comprise the LBD of PXR and a corresponding co-regulatory peptide, suitable dimerizing agents may be rifampicin, clotrimazole, and lovastatin.

Conditionally active off-switch CAR

In some cases, a conditionally active heterodimeric polypeptide of the present disclosure is a conditionally active off-switch CAR. The conditionally active off-switch CARs of the present disclosure are also referred to as heteromeric conditionally-repressed synthetic Immune Cell Receptors (ICRs). The conditionally active off switch CARs of the present disclosure comprise: a synthetic stimulatory ICR comprising a first member of a dimerization pair linked to the synthetic stimulatory ICR; and a synthetic ICR repressor comprising a second member of the dimerization pair linked to an intracellular inhibitory domain. The heteromerically conditionally repressed synthetic ICRs of the present disclosure will generally include a synthetic stimulatory ICR and a synthetic ICR repressor configured such that, upon introduction of a dimerizing agent, the synthetic ICR repressor dimerizes with the synthetic stimulatory ICR to repress activation due to the synthetic stimulatory ICR. Examples of heterodimeric, conditionally active off-switch CAR polypeptides include, but are not limited to, those described, for example, in PCT international application No. PCT/US2016/062612, the disclosure of which is incorporated herein by reference in its entirety.

The configuration of the heteromerically conditionally repressed synthetic ICR will vary depending on the particular environment in which the synthetic stimulatory ICR is desired to be repressed. In some cases, the stimulatory portion of a heteromerically conditionally repressed synthetic ICR may be referred to as portion 1 of the heteromerically conditionally repressed synthetic ICR. In some cases, the repressing portion of a heteromerically conditionally repressed synthetic ICR may be referred to as heteromerically conditionally repressed portion 2 of the synthetic ICR. Thus, heteromerically conditionally blocked synthetic ICRs collectively refer to a multi-modular protein or protein complex comprising various modules including a stimulatory moiety (e.g., synthetic stimulatory ICRs, moiety 1, etc.) and a repressor moiety (e.g., synthetic ICR repressor, moiety 2, etc.), whether or not the various modules are present at a certain point within the same protein and whether or not the various modules are expressed from the same or different nucleic acid constructs.

One of skill in the art will readily recognize from this disclosure that the first and second portions (e.g., stimulatory and inhibitory portions) will each comprise a first and second portion of a dimerization pair, and that such portions of the dimerization pair may be interchanged between the first and second portions of a synthetic ICR that is repressed under heteromeric conditions. One of skill in the art will also readily recognize from this disclosure that, in many cases, the individual domains of a heteromeric-conditionally-repressed synthetic ICR can be rearranged in order and/or orientation while maintaining the activatable and repressible functions as described herein. Thus, the description herein of a particular configuration of a heteromeric conditionally-repressed synthetic ICR also includes where the modules of the heteromeric conditionally-repressed synthetic ICR are rearranged without eliminating the primary function of the heteromeric conditionally-repressed synthetic ICR. Such rearrangements may also include the inclusion or exclusion of certain optional modules (including, for example, linkers, reporter genes, etc.) that do not result in the elimination of the primary function of the heteromerically conditionally repressed synthetic ICR, as they are included in or excluded from the heteromerically conditionally repressed synthetic ICR.

Synthetic stimulatory ICR

As described herein, for simplicity, synthetic ICRs that are repressed by heteromeric conditions include synthetic stimulatory ICRs, which are also referred to herein as "stimulatory ICRs" or "stimulatory moieties. Such stimulatory ICRs will vary depending on the particular circumstances of immune cell stimulation to which the construct is directed, and will generally function to mediate activation of immune cells expressing the stimulatory ICRs. Thus, stimulatory ICRs include an extracellular domain that functions upon receipt of a specified signal to transduce the signal into the cell to activate immune cells expressing the stimulatory ICRs.

In some cases, the extracellular component of a stimulatory ICR may therefore include an extracellular recognition domain, described in more detail below, that contains one member of a specific binding pair. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; a ligand-receptor binding pair; and so on. Thus, members of a specific binding pair suitable for use in an extracellular recognition domain of the present disclosure include antigens; an antibody; a ligand; and a receptor that binds to the ligand. Suitable extracellular recognition domains for use in stimulating ICRs include, but are not limited to, for example, members of a specific binding pair (e.g., antigen-binding domain-containing members of a specific binding pair), including, for example, those described above with respect to the above conditionally active heterodimeric open-switch CARs.

The stimulatory ICR also includes one or more intracellular stimulatory domains that mediate intracellular signaling upon activation of the one or more extracellular domains, resulting in activation of immune cells expressing the stimulatory ICR. The domain used as a signaling domain will vary depending on the particular circumstances of immune cell activation, including, for example, the particular type of cell to be activated and the degree of activation desired. Illustrative, non-limiting examples of stimulatory domains described in more detail below include, but are not limited to, domains derived from immunostimulatory molecules (including, e.g., co-stimulatory molecules, immunoreceptors, and the like) and motifs thereof. Suitable intracellular stimulation domains for stimulating ICRs include, but are not limited to, e.g., regulatory domains and portions thereof, including, e.g., those described above with respect to the above conditionally active heterodimeric open switch CARs.

In some cases, the stimulatory ICRs may be or may be derived from engineered or synthetic immunomodulatory constructs designed for therapeutic immune system modulation, including but not limited to, e.g., Chimeric Antigen Receptors (CARs) and derivatives, engineered T Cell Receptors (TCRs) and derivatives, and the like. Engineered CARs, TCRs, and derivatives thereof that can be used as the basis for the synthesis of ICRs include those activatable CARs, TCRs, and derivatives thereof that activate, e.g., when a binding partner binds to the CAR, TCR, or derivative thereof, and upon activation transduce a signal intracellularly to activate immune cells expressing the CAR, TCR, or derivative thereof. In some cases, the stimulatory ICR may be conditionally activatable such that activation upon binding of a partner to the stimulatory ICR requires additional events to transduce activation signals, including, for example, dimerization of components of the stimulatory ICR.

As described in more detail below, the stimulatory ICRs also include a domain (e.g., a first member or a second member) of the dimerization pair. Useful dimerization pairs will vary depending on the desired dimerizing agent and the relative position of the desired members of the dimerization pair within the stimulatory ICR. Typically, the presence of a first member of a dimerization pair within a stimulatory ICR mediates, upon introduction of a dimerizing agent, dimerization with a second member of a dimerization pair present in an ICR repressor such that, upon dimerization, the ICR repressor represses any immune cell activation due to the stimulatory ICR.

In some cases, the stimulatory ICR may also include additional domains. Such further domains may be functional, e.g. they directly contribute to the immune cell activation function of stimulatory ICRs, or may be non-functional, e.g. they do not directly contribute to the activation function of stimulatory ICRs. Non-functional additional domains may include domains with various purposes that do not directly affect the ability of the stimulatory ICR to activate immune cell functions (including, but not limited to, e.g., structural functions, linker functions, etc.).

Chimeric Antigen Receptor (CAR)

In some cases, a heteromerically conditionally repressed synthetic ICR may comprise a CAR in part or in whole or may be a substantially modified CAR such that the CAR is conditionally repressible by the modification. In such cases, a CAR containing a heteromeric conditionally-repressed synthetic ICR may be referred to as a heterodimeric conditionally-repressed synthetic CAR, or for simplicity, as a repressible CAR. Any CAR having immune cell activation function can be used in the heteromeric conditionally-repressed synthetic ICRs described herein, including but not limited to, e.g., those CAR variants described herein.

In some cases, a CAR can be modified to serve as a component of a conditionally-repressed synthetic ICR of a heterodimer by introducing or inserting a dimerization domain (e.g., a member of a dimerization pair) into the CAR, and in such cases, after modification, the CAR can be referred to as a dimerization domain-containing CAR or a dimerizable CAR.

The dimerization domain can be inserted into the CAR amino acid sequence, e.g., by introducing the coding sequence for the dimerization domain into the coding sequence of the CAR at any convenient location, so long as the insertion does not negatively affect the primary functional domain of the CAR (including, e.g., the extracellular recognition domain, the immune activation domain, etc.) and/or negatively affect the dimerization function of the dimerization domain.

In some cases, the dimer can be inserted into the extracellular portion of the CAR. In some cases, the dimer may be inserted into the intracellular portion of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer connects to the extracellular recognition domain of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer connects to the transmembrane domain of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer is linked to the extracellular side of the transmembrane domain of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer connects to the intracellular side of the transmembrane domain of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer connects to the immunostimulatory domain of the CAR. In some cases, the dimer can be inserted such that after insertion, the dimer connects to the co-stimulatory domain of the CAR. In some cases, the dimer may be inserted such that after insertion, the dimer is located at the N-terminus of the CAR. In some cases, the dimer may be inserted such that after insertion, the dimer is located at the C-terminus of the CAR.

In the case where part or all of the heteromerically conditionally-repressed synthetic ICR comprises a modified CAR or the heteromerically conditionally-repressed synthetic ICR is substantially a modified CAR, the CAR may contain an extracellular recognition domain, a stimulatory domain, and a transmembrane domain. Such CARs can optionally include a linker region and/or a hinge region. CARs may be encompassed within a single polypeptide or may be "split" across two or more polypeptides as part of a synthetic ICR that is repressed by heteromeric conditions.

Extracellular recognition domain

The repressible CAR includes a member of a specific binding pair. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; a ligand-receptor binding pair; and so on. Thus, members of a specific binding pair suitable for use in a repressible CAR of the present disclosure include antigens; an antibody; a ligand; and a receptor that binds to the ligand.

Antigen binding domains

Antigen binding domains suitable for use in the repressible CARs of the present disclosure can have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific for an epitope present in an antigen expressed (synthesized) by a cancer cell (i.e., a cancer cell-associated antigen). The cancer cell-associated antigen can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, and the like. Cancer cell-associated antigens may also be expressed by non-cancerous cells.

Non-limiting examples of antigens to which the antigen-binding domain of a suppressible CAR of the invention can bind include, for example, CD19, CD20, CD38, CD30, Her2/neu/ERBB2, CA125, MUC-1, Prostate Specific Membrane Antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2(VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Additional examples of antigens to which the antigen binding domain of the repressible CAR of the invention can bind include, but are not limited to, those described above with respect to conditionally active, open-switch CARs.

Ligands

In some cases, a member of a specific binding pair suitable for use in a repressible CAR of the invention is a ligand of a receptor. Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; Vascular Endothelial Growth Factor (VEGF), etc.); integrin binding peptides (e.g., peptides comprising the sequence Arg-Gly-Asp); and so on.

Where the member of a specific binding pair in a repressible CAR of the invention is a ligand, the repressible CAR can be activated in the presence of both a dimerizing agent and a second member of the specific binding pair, wherein the second member of the specific binding pair is a receptor for the ligand. For example, where the ligand is VEGF, the second member of the specific binding pair may be a VEGF receptor, including a soluble VEGF receptor. As another example, where the ligand is heregulin, the second member of the specific binding pair may be Her 2.

Receptors

As noted above, in some cases, the member of a specific binding pair included in a repressible CAR of the invention is a receptor, e.g., a receptor for a ligand, a co-receptor, etc. The receptor may be a ligand binding fragment of the receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptors); killer lectin-like receptor subfamily K, member 1(NKG2D) polypeptide (receptor for MICA, MICB and ULB 6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); her 2; CD 27; natural Cytotoxic Receptors (NCRs) (e.g., NKP30(NCR3/CD337) polypeptides (receptors for HLA-B-related transcript 3(BAT3) and B7-H6), etc.); and so on.

Stimulatory domains

The stimulatory domain of a stimulatory CAR suitable for use in the repressible ICR of the present invention can be any functional unit of a polypeptide as short as a3 amino acid linear motif and as long as the entire protein, with the size of the stimulatory domain being limited only, the domain must be large enough to retain its function and small enough to be compatible with the other components of the repressible CAR. Thus, the stimulatory domain may range in size from about 3 amino acids to about 100 amino acids or more in length, in some cases it may be from about 30 amino acids to about 70 amino acids (aa) in length, for example, the stimulatory domain may be from about 30aa to about 35aa, from about 35aa to about 40aa, from about 40aa to about 45aa, from about 45aa to about 50aa, from about 50aa to about 55aa, from about 55aa to about 60aa, from about 60aa to about 65aa, or from about 65aa to about 70aa in length. In other cases, the stimulatory domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200aa in length.

In some cases, a "co-stimulatory domain" can be used as a stimulatory domain of a repressible CAR of the present disclosure. Co-stimulation generally refers to a secondary non-specific activation mechanism by which a primary specific stimulus is propagated. Examples of co-stimulation include antigen-non-specific T cell co-stimulation based on antigen-specific signaling through T cell receptors and antigen-non-specific B cell co-stimulation based on signaling through B cell receptors. Costimulation (e.g., T cell costimulation) and the factors involved have been described in Chen and Flies. nat Rev Immunol (2013)13(4):227-42, the disclosure of which is incorporated herein by reference in its entirety. The co-stimulatory domain is typically a polypeptide derived from a receptor. In some embodiments, the co-stimulatory domains homodimerize. The co-stimulatory domain of the invention may be the intracellular portion of the transmembrane protein (i.e., the co-stimulatory domain may be derived from the transmembrane protein). Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In some cases, for example, the co-stimulatory domains used in the repressible CARs of the present disclosure can include the co-stimulatory domains listed in table 1. In some cases, the co-stimulatory domain of a repressible CAR comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the co-stimulatory domain described herein.

In some cases, a stimulatory CAR may contain an intracellular signaling domain, e.g., a costimulatory domain, derived from the intracellular portion of a transmembrane protein listed in table 1. For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to an amino acid sequence listed in table 1. In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 170aa to about 180aa, about 175aa to about 170aa, about 180aa to about 180aa, From about 180aa to about 185aa or from about 185aa to about 190 aa.

In some cases, a repressible CAR can contain more than two stimulatory domains, either on the same or different polypeptides. In some cases, where the repressible CAR contains more than two stimulatory domains, the stimulatory domains can have substantially the same amino acid sequence. For example, in some cases, the first stimulatory domain comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the second stimulatory domain. In some cases, where the repressible CAR contains more than two stimulatory domains, the stimulatory structures of the repressible CARs of the invention can be of substantially the same length; for example, the lengths of the first stimulatory domain and the second stimulatory domain may differ from each other by less than 10 amino acids or less than 5 amino acids. In some cases, where the repressible CAR contains more than two stimulatory domains, the first stimulatory domain and the second stimulatory domain are the same length. In some cases, where the repressible CAR contains more than two stimulatory domains, the two stimulatory domains are the same.

In some cases, a repressible CAR can contain an intracellular signaling domain (e.g., a co-stimulatory domain) derived from the intracellular portion of transmembrane protein 4-1BB (also known as TNFRSF 9; CD 137; 4-1 BB; CDw 137; ILA, etc.). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 618). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp 44). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 619). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane proteins ICOS (also known as AILIM, CD278, and CVID 1). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 620). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP 1L). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 621). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD 272). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS (SEQ ID NO: 622).

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp 55). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 623). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, or about 160aa to about 185aa of the amino acid sequence: RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO: 624).

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO: 625). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain, e.g., a co-stimulatory domain, derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, light TR, and TR 2). For example, a suitable co-stimulatory domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 626). In some of these embodiments, the co-stimulatory domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a repressible CAR can contain an intracellular signaling domain that includes at least one (e.g., 1,2, 3, 4, 5,6, etc.) intracellular signaling motif. In some cases, the intracellular signaling motif can be an immunoreceptor tyrosine-based activation motif (ITAM). In some cases, an intracellular signaling motif (e.g., an ITAM motif) is located within an intracellular signaling domain that is derived from a signaling molecule containing one or more ITAM motifs. In other cases, the ITAMs are derived, e.g., synthetically produced, within the amino acid sequence, e.g., by mutation of the amino acid sequence.

Motif of ITAM is YX₁X₂L/I, wherein X₁And X₂Independently any amino acid (SEQ ID NO: 564). In some cases, the intracellular signaling domain of a repressible CAR of the invention comprises 1,2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are spaced 6 to 8 amino acids apart from each other, e.g., (YX)₁X₂L/I)(X₃)_n(YX₁X₂L/I), where n is an integer from 6 to 8, and 6-8X₃Can be any amino acid (SEQ ID NO: 565). In some cases, the intracellular signaling domain of a repressible CAR of the invention comprises 3 ITAM motifs.

In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR related protein; TYRO protein tyrosine kinase binding protein; killer cell activating receptor related protein; etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any one of the following amino acid sequences (4 isoforms):orWherein the ITAM motif is shown in bold and underlined.

Likewise, appropriate intracellular signalingThe leader domain may comprise the ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor Igamma chain; Fc receptor gamma chain; Fc-epsilon RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor high affinity gamma chain, etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length FCER1G amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 DELTA chain (also known as CD 3D; CD 3-DELTA; T3D; CD3 antigen DELTA subunit; CD3 DELTA; CD3d antigen DELTA polypeptide (TiT3 complex); OKT3 DELTA chain; T cell receptor T3 DELTA chain; T cell surface glycoprotein CD3 DELTA chain; etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 170aa of any of the following amino acid sequences (2 isoforms):orWherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length CD3 δ amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity toAmino acid sequence for amino acid sequence identity:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 205aa of the following amino acid sequence:

wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length CD3 epsilon amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also referred to as CD3G, T cell receptor T3 gamma chain, CD3 gamma, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 180aa of the following amino acid sequence:wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length CD3 γ amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3 zeta, CD3H, CD3 zeta chainQ, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 160aa of any of the following amino acid sequences (2 isoforms):wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length CD3 ζ amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any one of the following amino acid sequences:orWherein the ITAM motif is shown in bold and underlined.

In some cases, an intracellular signaling domainDerived from CD79A (also referred to as B cell antigen receptor complex-related protein α chain; CD79a antigen (immunoglobulin-related α); MB-1 membrane glycoprotein; ig α; membrane-bound immunoglobulin-related protein; surface IgM-related protein, etc.) for example, suitable intracellular signaling domains may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a continuous segment of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 150aa, about 150aa to about 200aa, or about 200aa to about 220aa of any of the following amino acid sequences (2 isoforms):wherein the ITAM motif is shown in bold and underlined.

Likewise, a suitable intracellular signaling domain may comprise the portion of the full-length CD79A amino acid sequence that contains the ITAM motif. Thus, a suitable intracellular signaling domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence of seq id no:wherein the ITAM motif is shown in bold and underlined.

In some cases, a repressible CAR can contain an intracellular signaling domain derived from a DAP10/CD 28-type signaling chain. Intracellular signaling domains suitable for use in the repressible CARs of the present disclosure include DAP10/CD 28-type signaling chains.

The intracellular signaling domains of CARs suitable for use in the present disclosure include ZAP70 polypeptides, e.g., polypeptides comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous segment of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to 619 amino acids of the amino acid sequence: MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA (SEQ ID NO: 654).

Transmembrane domain

As a non-limiting example, TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:655) can be used additional non-limiting examples of suitable TM sequences include a) CD8 β derived LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:656), b) CD4 derived ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:657), c) CD3 ζ derived LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:658), d) CD28 derived WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:659), e) CD134(OX40) derived VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:660), and f) CD7 derived ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 661).

Joint

In some cases, the repressible CAR of the invention comprises a linker between any two adjacent domains. For example, a linker can be disposed between the transmembrane domain and a first intracellular functional domain (e.g., a co-stimulatory domain) of the repressible CAR. As another example, a linker can be disposed between the first intracellular functional domain of the repressible CAR and a member of the dimerization domain. As another example, a linker may be disposed between a member of the dimerization domain and a second intracellular functional domain (e.g., an immune cell activation domain). As another example, a linker can be disposed between any domain of the repressible CAR and any additional domain of the granite, including, for example, domains not involved in the primary immune activation function of the CAR, including, but not limited to, e.g., a reporter domain, a tag domain, and the like.

Linkers can be used in a repressible CAR in a suitable configuration so long as they do not eliminate the primary activity of the repressible CAR, including, for example, the ability of the repressible CAR to be activated upon extracellular binding, the ability of the repressible CAR's dimerization domain to bind to the dimerization domain of the ICR repressor.

Suitable linkers can be readily selected and can be any of various lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1,2, 3, 4, 5,6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (including, for example, (GS)_n、GSGGS_n(SEQ ID NO:516) and GGGS_n(SEQ ID NO:517) wherein n is an integer of at least 1), a glycine-alanine polymer,Alanine-serine polymers and other flexible linkers known in the art. Of interest are glycine and glycine-serine polymers; since these amino acids are relatively unstructured and therefore can act as neutral chains between the components. Of particular interest are glycine polymers; since glycine approaches even significantly more phi-psi spacing than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. comparative chem.11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGSG (SEQ ID NO:518), GGSGG (SEQ ID NO:519), GSGSGSG (SEQ ID NO:520), GSGGG (SEQ ID NO:521), GGGSG (SEQ ID NO:522), GSSSG (SEQ ID NO:523), and the like. One of ordinary skill will recognize that the design of the peptide conjugated to any of the elements described above may include a linker that is fully or partially flexible, such that the linker may include a flexible linker and one or more moieties that impart a less flexible structure.

Hinge region

In some cases, the first polypeptide of the repressible CAR of the invention comprises a hinge region (also referred to herein as a "spacer"), wherein the hinge region is interposed between the antigen-binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD 8-derived hinge region).

The hinge region can be from about 4 amino acids to about 50 amino acids in length, for example from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

Suitable spacers can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1,2, 3, 4, 5,6, or 7 amino acids.

In some cases, for example, when the stimulatory ICR portion of a repressible CAR is cleaved between two or more polypeptides, the repressible CAR can include a hinge region containing at least one cysteine. For example, in some cases, the hinge region may include the sequence Cys-Pro-Pro-Cys. If present, cysteines in the hinge region of the first polypeptide (e.g., the first portion of the repressible CAR) can be used to form disulfide bonds with the hinge region of the second polypeptide (e.g., the second portion of the repressible CAR).

Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al (1990) Proc.Natl.Acad.Sci.USA 87: 162; and Huck et al (1986) Nucl. acids Res.14: 1779. As a non-limiting example, the immunoglobulin hinge region may comprise one of the following amino acid sequences: DKKHT (SEQ ID NO: 524); CPPC (SEQ ID NO: 525); CPEPKSCDTPPPCPR (SEQ ID NO:526) (see, e.g., Glaser et al (2005) J.biol.chem.280: 41494); ELKTPLGDTTHT (SEQ ID NO: 527); KSCDKTHTCP (SEQ ID NO: 528); KCCVDCP (SEQ ID NO: 529); KYGPPCP (SEQ ID NO: 530); EPKSCDKTHTCPPCP (SEQ ID NO:531) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:532) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:533) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:534) (human IgG4 hinge); and so on.

The hinge region may comprise an amino acid sequence derived from human CD 8; for example, the hinge region may comprise the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACD (SEQ ID NO:536) or a variant thereof.

Dimerization domains

As noted above, the conditionally-repressed synthetic Immune Cell Receptor (ICR) of the heterodimers of the present disclosure comprises a first member of a dimerization pair and a second member of the dimerization pair. One of the members of the dimerization pair will comprise the LBD of the nuclear hormone receptor and the other member of the dimerization pair will comprise the co-regulatory peptide of the same nuclear hormone receptor. In the presence of a dimerizing agent (e.g., a nuclear hormone or a functional derivative or analog of a nuclear hormone), the members of the dimerization pair will bind to each other and will effect dimerization of the two polypeptide chains of the conditionally-repressed synthetic ICR of the present disclosure. The first member of a dimerization pair or the second member of a dimerization pair may also be referred to as a "dimerization domain".

Ligand Binding Domain (LBD)

The abbreviations of nuclear hormone receptors are ER: estrogen receptor α, PR: progesterone receptor, AR: androgen receptor, GR: glucocorticoid receptor, MR: mineralocorticoid receptor, RAR: retinoic acid receptor, TR α, β: thyroid receptor, VDR: vitamin D3 receptor, EcR: ecdysone receptor, RXR: retinoic acid X receptor, PPAR: peroxisome proliferator activated receptor, LXR: liver X receptor, FXR: farnesol X receptor, PXR/R: pregnane X receptor/steroid and xenobiotic receptor, CAR: constitutive androstane receptor, SF-1: steroidogenesis factor 1, DAX-1: dose-sensitive congenital critical area of sex reversal-adrenal insufficiency on X chromosome, gene 1, LRUP H-1: liver receptor homolog 1, SHP: small heterodimer, TLX: hairless gene partner, PNR: photoreceptor-specific nuclear receptor RAR, NGF 1-B: nerve growth factor, GCR: liver receptor homolog 1, nuclear receptor related factor receptor, nuclear estrogen receptor related receptor, nuclear receptor related factor receptor, nuclear factor receptor related receptor, nuclear factor receptor related factor, nuclear factor 364-receptor related factor, nuclear factor.

Mineralocorticoid receptors

Androgen receptor

Progesterone receptors

Thyroid hormone receptor β

For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the second member of the dimerization pair is a co-regulatory peptide comprising amino acid sequence CSSDDRGHSSLTNSPLDSSCKESSVSVTSPSGVSSSTSGGVSSTSNMHGSLLQEKHRILHKLLQNGNSPAEVAKITAEATGKDTSSITSCGDGNVVKQEQLSPKKKENNALLRYLLDRDDPSDALSKELQPQVEGVDNKMSQCTSSTIPSSSQEKDPKIKTETSEEGSGDLDNLDAILGDLTSSDFYNNSISSNGSHLGTKQQ (SEQ ID NO:662) in other cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the first member of the dimerization pair is a NCOA3/SRC3 polypeptide.

In some cases, when the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the second member of the dimerization pair is the NCOA2/SRC2 polypeptide, for example, in some cases, when the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of TR β, the second member of the dimerization pair is a co-regulatory peptide comprising the amino acid sequence STAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO: 663).

Estrogen receptor α

Estrogen receptor β

Peroxisome proliferator activated receptor-gamma

Glucocorticoid receptor

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA1/SRC1 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA1/SRC1 polypeptide comprising amino acid sequence NYGTNPGTPPASTSPFSQLAANPEASLANRNSMVSRGMTGNIGGQFGTGINPQMQQNVFQYPGAGMVPQGEANFAPSLSPGSSMVPMPIPPPQSSLLQQTPPASGYQSPDMKAWQQGAIGNNNVFSQAVQNQPTPAQPGVYNNMSITVSMAGGNTNVQNMNPMMAQMQMSSLQMPGMNTVCPEQINDPALRHTGLYCNQLSSTDLLKTEADGTQQVQQVQVFADVQCTVNLVGGDPYLNQPGPLGTQKPTSGPQTPQAQQKSLLQQLLTE (SEQ ID NO:664) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the first member of the dimerization pair is a NCOA1/SRC1 polypeptide.

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide comprising amino acid sequence KRHHHEVLRQGLAFSQIYRFSLSDGTLVAAQTKSKLIRSQTTNEPQLVISLHMLHREQNVCVMNPDLTGQTMGKPLNPISSNSPAHQALCSGNPGQDMTLSSNINFPINGPKEQMGMPMGRFGGSGGMNHVSGMQATTPQGSNYALKMNSPSQSSPGMNPGQPTSMLSPRHRMSPGVAGSPRIPPSQFSPAGSLHSPVGVCSSTGNSHSYTNSSLNALQALSEGHGVSLGSSLASPDLKMGNLQNSPVNMNPPPLSKMGSLDSKDCFGLYGEPSEGTTGQAESSCHPGEQKETNDPNLPPAVSSERADGQSRLHDSKGQTKLLQLLTTKSDQMEPSPLASSLSDTNKDSTGSLPGSGSTHGTSLKEKHKILHRLLQDSSSPVDLAKLTAEATGKDLSQESSSTAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO:665) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of GR, the first member of the dimerization pair is a NCOA2/SRC2 polypeptide.

Vitamin D receptors

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA1/SRC1 polypeptide. For example, in some cases, where the first member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA1/SRC1 polypeptide comprising amino acid sequence NYGTNPGTPPASTSPFSQLAANPEASLANRNSMVSRGMTGNIGGQFGTGINPQMQQNVFQYPGAGMVPQGEANFAPSLSPGSSMVPMPIPPPQSSLLQQTPPASGYQSPDMKAWQQGAIGNNNVFSQAVQNQPTPAQPGVYNNMSITVSMAGGNTNVQNMNPMMAQMQMSSLQMPGMNTVCPEQINDPALRHTGLYCNQLSSTDLLKTEADGTQQVQQVQVFADVQCTVNLVGGDPYLNQPGPLGTQKPTSGPQTPQAQQKSLLQQLLTE (SEQ ID NO:666) or a fragment thereof. In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the first member of the dimerization pair is an NCOA1/SRC1 polypeptide.

In some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA2/SRC2 polypeptide. For example, in some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the second member of the dimerization pair is an NCOA2/SRC2 polypeptide comprising amino acid sequence KRHHHEVLRQGLAFSQIYRFSLSDGTLVAAQTKSKLIRSQTTNEPQLVISLHMLHREQNVCVMNPDLTGQTMGKPLNPISSNSPAHQALCSGNPGQDMTLSSNINFPINGPKEQMGMPMGRFGGSGGMNHVSGMQATTPQGSNYALKMNSPSQSSPGMNPGQPTSMLSPRHRMSPGVAGSPRIPPSQFSPAGSLHSPVGVCSSTGNSHSYTNSSLNALQALSEGHGVSLGSSLASPDLKMGNLQNSPVNMNPPPLSKMGSLDSKDCFGLYGEPSEGTTGQAESSCHPGEQKETNDPNLPPAVSSERADGQSRLHDSKGQTKLLQLLTTKSDQMEPSPLASSLSDTNKDSTGSLPGSGSTHGTSLKEKHKILHRLLQDSSSPVDLAKLTAEATGKDLSQESSSTAPGSEVTIKQEPVSPKKKENALLRYLLDKDDTKDIGLPEITPKLERLDSKTDPASNTKLIAMKTEKEEMSFEPGDQPGSELDNLEEILDDLQNSQLPQLFPDTRPGAPAGSVDKQAIINDLMQLTAENSPVTPVGAQKTALRISQSTFNNPRPGQLGRLLPNQNLPLDITLQSPTGAGPFPPIRNSSPYSVIPQPGMMGNQGMIGNQGNLGNSSTGMIGNSASRPTMPSGEWAPQSSAVRVTCAATTSAMNRPVQGGMIRNPAASIPMRPSSQPGQRQTLQSQVMNIGPSELEMNMGGP (SEQ ID NO:667) or a fragment thereof. For example, in some cases, where the first member of a dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is a LBD of a VDR, the second member of the dimerization pair is a NCOA2/SRC2 polypeptide comprising the amino acid sequence LLRYLLDK (SEQ ID NO:668), wherein the co-regulatory peptide is from about 8 amino acids to about 50 amino acids in length (e.g., the co-regulatory peptide is from about 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 23 amino acids, 23 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length). In some cases, where the second member of the dimerization pair of conditionally active heterodimeric polypeptides of the present disclosure is the LBD of a VDR, the first member of the dimerization pair is an NCOA2/SRC2 polypeptide.

Thyroid hormone receptor α

Retinoic acid receptor β

Farnesoid X receptor

LXRα

A suitable co-modulatory peptide of LXR α is SRC1 polypeptide or a fragment thereof (e.g., a peptide 8 amino acids to 50 amino acids in length derived from SRC1 polypeptide).

RORγ

A suitable co-regulator of ROR γ is the NCORNR peptide (CDPASNLGLEDIIRKALMGSFDDK, SEQ ID NO: 669).

RXRα

PXR

Co-regulatory peptides

Examples of suitable co-regulatory polypeptides are provided in FIGS. 29-51B.

Suitable co-modulatory peptides include, but are not limited to, steroid receptor co-activating factor (SRC) -1, SRC-2, SRC-3, TRAP220-1, TRAP220-2, NR0B1, NRIP1, CoRNR cassette, abV, TIF1, TIF2, EA2, TA1, EAB1, SRC1-1, SRC1-2, SRC1-3, SRC1-4a, SRC1-4B, GRIP1-1, GRIP1-2, GRIP1-3, AIB1-1, AIB1-2, AIB1-3, PGC 11, PGC1, PRC, ASC 1-1, ASC 1-2, CBP-1, CBP-2, P300, CIA, ARA 1-1, ARA 1-2, TID 1, SMRIP 72, SMRIP 140, RIP 140-RIP 140, SMRIP 140, RIP 140-140, RIP140, CRP 140, RIP140, and CRP 140-140, and CRP 140, RIP140-8, RIP140-9, PRIC285-1, PRIC285-2, PRIC285-3, PRIC285-4, and PRIC 285-5.

In some cases, suitable co-regulatory peptides comprise the LXXLL motif, wherein X is any amino acid; wherein the length of the co-regulatory peptide is from 12 amino acids to 50 amino acids, such as from 12 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids.

Non-limiting examples of suitable co-regulatory peptides are as follows:

Suitable dimerizing agents are described above.

Engineered T Cell Receptor (TCR)

In some cases, heteromerically conditionally repressed synthetic ICRs may partially or fully comprise engineered T Cell Receptors (TCRs) or may be substantially modified engineered TCRs such that the engineered TCRs are conditionally repressible by the modification. In such cases, an engineered TCR containing a heteromeric conditionally-repressed synthetic ICR may be referred to as a heteromeric conditionally-repressed synthetic TCR, or for simplicity, as a repressible TCR.

Any engineered TCR with immune cell activation function may be used for heteromeric conditionally-repressed synthetic ICRs as described herein, including, but not limited to, for example, antigen-specific TCRs, Monoclonal TCRs (MTCR), single chain MTCR, high affinity CDR2 mutant TCRs, MTCR binding CD1, high affinity NY-ESO TCRs, VYG HLA-a24 telomerase TCRs, including those described in: PCT publication nos. WO 2003/020763, WO 2004/033685, WO 2004/044004, WO 2005/114215, WO 2006/000830, WO 2008/038002, WO 2008/039818, WO 2004/074322, WO 2005/113595, WO 2006/125962; strommes et al Immunol rev.2014; 145-64 parts of (257); schmitt et al blood.2013; 122(3) 348 to 56; chapuls et al Sci Transl Med.2013; 174ra27 (5) (174); thaxton et al Hum vaccine Immunother.2014; 3313-21(PMID:25483644) 10 (11); gschweng et al Immunol Rev.2014; 237-49(PMID:24329801) in (257); hinrichs et al Immunol Rev.2014; 56-71(PMID:24329789) in 257 (1); zoete et al Front immunol.2013; 4:268(PMID: 24062738); marr et al, Clin ExpImmunol.2012; 167(2):216-25(PMID: 22235997); zhang et al Adv Drug Deliv Rev.2012; 64(8) 756-62(PMID 22178904); chhabra et al Scientific World journal.2011; 11:121-9(PMID: 21218269); boulter et al Clin Exp immunol.2005; 142(3) 454-60(PMID: 16297157); sami et al Protein Eng Des Sel.2007; 20(8) 397-403; boulter et al Protein eng.2003; 16, (9) 707-11; ashfield et al IDrugs.2006; 9(8) 554-9; li et al Nat Biotechnol.2005; 23(3) 349-54; dunn et al Protein Sci.2006; 15(4) 710-21; liddy et al Mol Biotechnol.2010; 45 (2); liddy et al Nat Med.2012; 18, (6) 980-7; oats et al Oncoimmunology.2013; 2, (2) e 22891; cancer Immunol Immunother such as McCormac.2013 for 4 months; 773-85 in 62 (4); cancer Immunol Immunother.2014, Bossi et al; 437-48 and Mol Immunol.2015, Oates, etc. in 63(5) for 10 months; 67(2Pt A) 67-74; the disclosures of which are incorporated herein by reference in their entirety.

In some cases, engineered TCRs useful for heteromeric condition-suppressed synthetic ICRs as described herein may include, for example, a TCR that binds to NY-ESO-1 or a peptide derived therefrom. For example, in some cases, a TCR that binds to NY-ESO-1 can be a TCR that binds to a peptide having the amino acid sequence: SLLMWITQC (SEQ ID NO:670) peptide-bound engineered TCRs.

In some cases, engineered TCRs useful for heteromeric conditionally-repressed synthetic ICRs as described herein can be or can be derived from engineered TCRs having high affinity for their ligands, including but not limited to, for example, a K of less than or equal to 100 μ Μ_DIncluding but not limited to, for example, K less than or equal to 10 μ M_DOr less than or equal to 1 μ M K_D. In some cases, an engineered TCR useful for heteromeric condition-suppressed synthetic ICRs as described herein can be or can be derived from an engineered TCR having high affinity for peptide SLLMWITQC (SEQ ID NO:671), including but not limited to, for example, a K of less than or equal to 100 μ M for peptide SLLMWITQC (SEQ ID NO:672)_DIncluding but not limited to, for example, K less than or equal to 10 μ M_DOr less than or equal to 1 μ M K_D。K_DThe measurement may be performed by any known method, including but not limited to, for example, surface plasmon resonance (Biacore).

In some cases, engineered TCRs useful in heteromeric conditionally-repressed synthetic ICRs as described herein can be or can be derived from engineered TCRs that have slow off-rates for their ligands, including but not limited to, for example, 0.1S^-1Or slower k_offIncluding but not limited to, for example, 1x10^-2S^-1Or slower k_offOr 1x10^-3S^-1Or slower k_off. In some cases, engineered TCRs useful in heteromeric conditionally-repressed synthetic ICRs as described herein can be or can be derived from engineered TCRs that have slow off-rates, including but not limited to, for example, less than or equal to that of peptide SLLMWITQC (SEQ ID NO:673)At 0.1S^-1Or slower k_offIncluding but not limited to, for example, 1x10^-2S^-1Or slower k_offOr 1x10^-3S^-1Or slower k_off。k_offThe measurement may be performed by any known method, including but not limited to, for example, surface plasmon resonance (Biacore).

In some cases, the engineered TCRs can be modified to serve as components of synthetic ICRs that are blocked under heteromeric conditions by introducing or inserting a dimerization domain (e.g., a member of a dimerization pair) into the engineered TCR, and in such cases, after modification, the engineered TCR can be referred to as a TCR containing the dimerization domain or a dimerizable TCR.

The dimerization domain may be inserted into the engineered TCR amino acid sequence, for example, by introducing the coding sequence for the dimerization domain into the coding sequence of the engineered TCR at any convenient location, so long as the insertion does not negatively affect the primary functional domain of the engineered TCR (including, for example, the TCR α chain domain, TCR β chain domain, TCRCD3 chain domain, TCR zeta chain domain, TCR CD 3-zeta chain domain, TCR extracellular domain, TCR intracellular domain, TCR variable region domain, TCR constant region domain, TCR IgSF domain, etc., or functional domains thereof) and/or negatively affect the dimerization function of the dimerization domain.

The engineered TCR may comprise one or more epsilon, sigma or gamma chains, or in some cases, the engineered TCR may not comprise one or more epsilon, sigma or gamma chains and may instead rely on an endogenously expressed epsilon, sigma or gamma chain. In some cases, an engineered TCR may not comprise one or more CD 3-zeta chains and may instead rely on endogenously expressed CD 3-zeta.

In some cases, the dimerization domain may be inserted into the extracellular portion of the engineered TCR. In some cases, a dimerization domain (either the first member or the second member of a dimerization pair) may be inserted into the intracellular portion of the engineered TCR.

In some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked intracellularly to the α chain after insertion or linking, including, for example, where the dimerization domain is linked to the cytoplasmic side of the α chain transmembrane domain.

In some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked intracellularly to the β chain after insertion or linking, including, for example, where the dimerization domain is linked to the cytoplasmic side of the β chain transmembrane, in some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked intracellularly to the β chain after insertion or linking, including, for example, where the dimerization domain is linked to the extracellular side of the β chain transmembrane domain, where the dimerization domain is inserted between the β chain transmembrane domain and the β chain constant region domain, and so forth.

In some cases, the dimerization domain may be inserted into or linked to a fused α -CD 3-zeta chain, e.g., where the CD 3-zeta chain is the full length CD 3-zeta (e.g., TCR: zeta fusion) of an engineered TCR.

In some cases, the dimerization domain may be inserted into or linked to a fused β -CD 3-zeta chain, e.g., where the CD 3-zeta chain is the full length CD 3-zeta (e.g., TCR: zeta fusion) of an engineered TCR.

In some cases, the dimerization domain may be inserted or linked to the fused α -CD 3-zeta domain of the engineered TCR (e.g., in an engineered TCR α -zeta + β -zeta fusion.) in some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked intracellularly to the fused α -CD 3-zeta domain upon insertion or linking, including for example where the dimerization domain is inserted between one or more of the dimerization domains of the CD 3-zeta domain and the dimerization domain of the α chain.

In some cases, the dimerization domain may be inserted or linked to the fused β -CD 3-zeta domain of the engineered TCR (e.g., in an engineered TCR α -zeta + β -zeta fusion.) in some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked intracellularly to the fused β -CD 3-zeta domain upon insertion or linking, including for example where the dimerization domain is inserted between one or more domains of the CD 3-zeta domain and the β chain's dimerization domain.

In some cases, the dimerization domain may be inserted or linked to a chain of an engineered single chain TCR (e.g., in an engineered single chain TCR: zeta fusion, such as a TCR α chain variable domain linked to a TCR β chain fused to a full length CD 3-zeta chain) in some cases, the dimerization domain is inserted or linked such that the dimerization domain is linked to the engineered single chain TCR intracellularly upon insertion or linking, including, for example, where the dimerization domain is inserted between one or more domains of a CD 3-zeta chain and the transmembrane domain of a CD 3-zeta chain.

In some cases, there may be only a single dimerization domain present in the conditionally-repressed engineered TCR, e.g., where the single dimerization domain is linked or inserted to the α chain of the engineered TCR, where the single dimerization domain is linked or inserted to the β chain of the engineered TCR, where the single dimerization domain is linked or inserted to the CD 3-zeta chain of the engineered TCR, etc.

For example, two dimerization domains may be present in a conditionally-repressed engineered TCR, e.g., where a first dimerization domain is linked or inserted into the α chain of the engineered TCR and a second dimerization domain is linked or inserted into the β chain of the engineered TCR, where the first dimerization domain is linked or inserted into the first CD 3-zeta chain and the second dimerization domain is linked or inserted into the second CD 3-zeta chain of the engineered TCR, etc.

In some cases, the engineered TCR of the conditionally-repressed TCR may be an engineered TCR variant including, but not limited to, for example, a TCR variant comprising one or more variant or mutant TCR chains the engineered TCR of the conditionally-repressed TCR may comprise one or more unmodified chains including, but not limited to, an unmodified α chain, an unmodified β chain, etc. in some cases, the engineered TCR of the conditionally-repressed TCR may comprise one or more murine chains including, but not limited to, for example, a murine β chain, a murine β 0 chain, etc. in some cases, the engineered TCR of the conditionally-repressed TCR may comprise one or more cysteine-modified chains including, but not limited to, for example, a cysteine-modified α chain, a cysteine-modified 362 chain, etc. in some cases, the engineered TCR of the conditionally-repressed TCR may comprise a combination of mutated TCR chains including, but not limited to, a humanized mouse and cysteine-modified mouse chains including, cysteine-modified mouse chains β, mouse chains, mouse.

Where the heteromerically conditionally-repressed synthetic ICR comprises partially or wholly a modified TCR or the heteromerically conditionally-repressed synthetic ICR is a modified TCR in nature, the TCR may comprise an unmodified TRC chain having an extracellular domain or extracellular domains present in a modified TCR chain therefor, one or more intracellular stimulatory domains present in an unmodified or modified TCR chain, and a transmembrane domain of a chain comprising such extracellular or intracellular domains. Such TCRs may optionally include a linker region and/or a hinge region. The TCR as part of a heteromeric conditionally-repressed synthetic ICR may be encompassed within a single polypeptide (e.g., as in an engineered single-chain TCR) or the various chains and portions thereof may be "split" across two or more polypeptides.

TCR chain

In some embodiments, the engineered TCR that can suppress ICR includes both a α chain variable domain and a TCR β chain variable domain.

It will be apparent to those skilled in the art that mutations in the TCR chain sequences, including for example the α chain sequence and/or the TCR β chain sequence, may be one or more substitutions, deletions or insertions these mutations may be performed using any suitable method, including but not limited to those based on the Polymerase Chain Reaction (PCR), restriction enzyme based Cloning or Ligation Independent Cloning (LIC) program these methods are detailed in many standard Molecular biology texts, including but not limited to, for example, Sambrook and Russell, (2001) Molecular Cloning-a Laboratory Manual (3 rd edition) CSHL Press and ratchian, (1995) Curr Opin Biotechnol 6(1): 30-6.

As used herein, the term "variable domain" is understood to encompass all amino acids of a given TCR, which are not included in the constant domains encoded by either the TRAC gene of the TCR α chain and the TRBC1 or TRBC2 of the TCR β chain, as described, for example, in the Tcell receiver facetsbook, (2001) LeFranc and LeFranc, Academic Press.

In some cases, the engineered TCR has at least one TCR α chain domain that has or is derived from an amino acid sequence that is at least 70% identical, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical to the IG4 α chain amino acid sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 675).

In some cases, the engineered TCR has at least one TCR α chain domain that has or is derived from an amino acid sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:676) that is at least 70% identical, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical to the IG4 α chain a95: LY mutant amino acid sequence.

In some cases, the engineered TCR has at least one TCR β chain domain that has or is derived from an amino acid sequence at least 70% identical, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical to the IG4 β chain amino acid sequence MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ id no: 677).

In some cases, an engineered TCR has at least one TCR β chain domain that has or is derived from an amino acid sequence that is at least 70% identical, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical to the IG4 β chain G51A mutant amino acid sequence MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 678).

In some cases, TCRs that bind NY-ESO-1 have at least one TCR α chain variable domain that has an amino acid sequence that is at least 70% identical to α chain extracellular sequence, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 679). in some cases, α chain extracellular sequence contains one or more of the following amino acid substitutions T95L and S96Y.

In some cases, a TCR that binds NY-ESO-1 has at least one TCR β chain variable domain having an amino acid sequence at least 70% identical to β chain extracellular sequence, including at least 75% identical, including at least 80% identical, including at least 85% identical, including at least 90% identical, including at least 95% identical, or 100% identical MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 680).

In some cases, the engineered TCR may comprise a disulfide bond between cysteines introduced between cysteines-for example, a disulfide bond between cysteines may be introduced between substituted amino acids of two engineered chains, including but not limited to, for example, the α chain and the β chain of the TCR.

Joint

In some cases, a linker may be disposed between the intracellular portion of the transmembrane domain of the α chain of the conditionally-repressed TCR and the dimerization domain.

Linkers can be used in the conditionally repressed TCR in an appropriate configuration so long as they do not eliminate the primary activity of the conditionally repressed TCR, including, for example, the ability of the conditionally repressed TCR to activate an immune cell, the ability of the dimerization domain of the conditionally repressed TCR to bind to the dimerization domain of a synthetic ICR repressor, and the like.

The conditionally-repressed TCRs of the invention can use any suitable linker, including two or more linkers (e.g., where two or more linkers are the same or different, and including mixtures where a plurality of linkers are three or more, four or more, five or more, six or more, etc., and including where all linkers are different, and where a plurality of linkers includes some linkers for more than one location and some linkers specifically used at only one location, etc.), including those linkers acceptable for use with a CAR, such as those described herein.

Synthesis of ICR repressor

As described herein, for simplicity, a heteromeric conditionally-repressed synthetic ICR includes a synthetic ICR repressor, which is also referred to herein as an "ICR repressor" or "inhibitory moiety". Such inhibitory ICRs will vary depending on the particular circumstances of immune cell suppression to which the construct is directed, and will generally function to mediate the suppression of activated or activatable immune cells expressing stimulatory ICRs and ICR suppressors. Thus, the ICR repressor factor includes an inhibitory domain that functions to repress immune cell activation due to the stimulatory ICR when a complementary dimerization domain present in the ICR repressor dimerizes and the stimulatory ICR when a dimerizing agent is present.

Thus, ICR repressor factors include one or more intracellular inhibitory domains that mediate intracellular signaling, thereby inhibiting immune cell activation in immune cells expressing stimulatory ICRs. The domain used as an inhibitory domain will vary depending on the particular circumstances of immune cell activation and suppression, including, for example, the particular type of activated cell to be suppressed and the degree of suppression desired. Illustrative, non-limiting examples of inhibitory domains described in more detail below include, but are not limited to, domains derived from immune receptors (including, e.g., co-inhibitory molecules, immune checkpoint molecules, immune tolerance molecules, etc.) and motifs thereof.

As described in more detail below, ICR repressor also includes the domain of the dimerization pair. Useful dimerization domains will vary depending on the desired dimerizing agent and the desired relative position of the dimerization domain within the ICR repressor. Typically, the presence of the first domain of the dimerization pair within the stimulatory ICR mediates, upon introduction of the dimerizing agent, dimerization with the second domain of the dimerization pair present in the ICR repressor such that, upon dimerization, the ICR repressor represses immune cell activation due to the stimulatory ICR.

The ICR repressor may optionally include a transmembrane domain. Thus, ICR repressor factors as described herein may or may not be membrane bound. As such, the ICR repressor may contain a transmembrane domain or portion thereof, and thus may be a membrane-bound ICR repressor. In other cases, the ICR repressor may lack a transmembrane domain, and thus may be a cytoplasmic ICR repressor. Also described herein are such transmembrane domains useful for ICR repressors of the present disclosure.

In some cases, ICR repressor factors may also include additional domains. Such additional domains may be functional, e.g. they contribute directly to the immune cell activation inhibitory function of the ICR repressor, or may be non-functional, e.g. they do not contribute directly to the repressive function of the ICR repressor. Non-functional additional domains may include domains with various purposes that do not directly affect the ability of the ICR repressor to repress immune cell activation (including, but not limited to, e.g., structural function, linker function, etc.).

Intracellular inhibitory domains

The co-suppression domain of the synthetic ICR repressor suitable for repressible ICRs of the present invention can be any functional unit of polypeptide as short as a3 amino acid linear motif and as long as the entire protein, with the size of the stimulatory domain limited only, the domain must be large enough to maintain its function and small enough to be compatible with the other components of the repressible ICR. Thus, the size of the inhibitory domain may range from about 3 amino acids to about 1000 amino acids or more in length, in some cases it may be from about 30 amino acids to about 70 amino acids (aa) in length, for example, the length of the inhibitory domain may be from about 30aa to about 35aa, from about 35aa to about 40aa, from about 40aa to about 45aa, from about 45aa to about 50aa, from about 50aa to about 55aa, from about 55aa to about 60aa, from about 60aa to about 65aa, or from about 65aa to about 70 aa. In other cases, the stimulatory domain may be from about 70aa to about 100aa, from about 100aa to about 200aa, or greater than 200aa in length.

In some cases, a "co-suppression domain" may be used as a synthetic ICR repressor of the present disclosure. Such co-inhibitory domains are typically polypeptides derived from a receptor. Co-suppression generally refers to secondary inhibition of the primary antigen-specific activation mechanism that prevents co-stimulation. Cosuppression (e.g., T Cell cosuppression) and the factors involved have been described in Chen and flies. nat rev Immunol (2013)13(4):227-42 and tharenthiran et al J Clin Cell Immunol (2012) S12, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the co-suppression domain homodimerizes. The co-suppression domain of the present invention may be the intracellular portion of the transmembrane protein (i.e., the co-suppression domain may be derived from the transmembrane protein). Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1. In some cases, for example, the co-suppression domains used in the synthetic ICR repressors of the present disclosure may include the co-suppression domains listed in figure 27, which provides table 1. In some cases, the co-suppression domain of a synthetic ICR repressor comprises an amino acid sequence that has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the co-suppression domain described herein.

In some cases, synthetic ICR suppressors may contain an intracellular signaling domain (e.g., a co-suppression domain) derived from the intracellular portion of the transmembrane protein PD-1 (also known as CD279, programmed cell death 1; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: ICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 681). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, or about 95aa to about 100 aa.

In some cases, synthetic ICR repressor factors may contain an intracellular signaling domain (e.g., a co-suppression domain) derived from the intracellular portion of the transmembrane protein CTLA4 (also known as CD152, cytotoxic T-lymphocyte protein 4, cytotoxic T-lymphocyte-associated antigen 4; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: SLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 682). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70 aa.

In some cases, a synthetic ICR repressor may contain an intracellular signaling domain (e.g., a co-inhibitory domain) derived from the intracellular portion of the transmembrane protein HPK1 (also known as MAP4K1, mitogen-activated protein kinase 1, hematopoietic progenitor cell kinase, MAPK/ERK kinase 1, MEK kinase 1, MEKK 1; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: YDLLQRLGGGTYGEVFKARDKVSGDLVALKMVKMEPDDDVSTLQKEILILKTCRHANIVAYHGSYLWLQKLWICMEFCGAGSLQDIYQVTGSLSELQISYVCREVLQGLAYLHSQKKIHRDIKGANILINDAGEVRLADFGISAQIGATLARRLSFIGTPYWMAPEVAAVALKGGYNELCDIWSLGITAIELAELQPPLFDVHPLRVLFLMTKSGYQPPRLKEKGKWSAAFHNFIKVTLTKSPKKRPSATKMLSHQLV (SEQ ID NO: 683). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 175aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 175aa, about 170aa to about 180aa, About 180aa to about 185aa, about 185aa to about 190aa, about 190aa to about 195aa, about 195aa to about 200aa, about 200aa to about 205aa, about 205aa to about 210aa, about 210aa to about 215aa, about 215aa to about 220aa, about 220aa to about 225aa, about 225aa to about 230aa, about 230aa to about 235aa, about 235aa to about 240aa, about 240aa to about 245aa, about 245aa to about 250aa, about 250aa to about 255aa, or about 255aa to about 258 aa.

In some cases, synthetic ICR repressor factors may contain an intracellular signaling domain (e.g., a co-suppression domain) derived from the intracellular portion of the transmembrane protein SHP1 (also known as PTN6, tyrosine protein phosphatase non-receptor type 6, hematopoietic cell protein tyrosine phosphatase, protein tyrosine phosphatase 1C, PTP-1C, SH-PTP1, HCP, PTP 1C; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQF (SEQ ID NO: 684). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 170aa to about 180aa, about 175aa, about 180aa to about 180aa, About 180aa to about 185aa, about 185aa to about 190aa, about 190aa to about 195aa, about 195aa to about 200aa, about 200aa to about 205aa, about 205aa to about 210aa, about 210aa to about 215aa, about 215aa to about 220aa, about 220aa to about 225aa, about 225aa to about 230aa, about 230aa to about 235aa, about 235aa to about 240aa, about 240aa to about 245aa, about 245aa to about 250aa, about 250aa to about 255aa, about 255aa to about 260aa, about 260aa to about 265aa, about 265aa to about 270aa, or about 270aa to about 272 aa.

In some cases, synthetic ICR repressor factors may contain an intracellular signaling domain (e.g., a co-inhibitory domain) derived from the intracellular portion of the transmembrane protein SHP2 (also known as PTN11, tyrosine protein phosphatase non-receptor type 11, protein tyrosine phosphatase 1D, PTP-1D, protein tyrosine phosphatase 2C, PTP-2C, SH-PTP2, SHP-2, SH-PTP3, PTP2C, SHPTP 2; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: FWEEFETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDPNEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENSRVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYTLRELKLSKVGQALLQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVHHKQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQMVRSQRSGMVQTEAQYRFIYMA (SEQ ID NO: 685). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 170aa to about 180aa, about 175aa, about 180aa to about 180aa, About 180aa to about 185aa, about 185aa to about 190aa, about 190aa to about 195aa, about 195aa to about 200aa, about 200aa to about 205aa, about 205aa to about 210aa, about 210aa to about 215aa, about 215aa to about 220aa, about 220aa to about 225aa, about 225aa to about 230aa, about 230aa to about 235aa, about 235aa to about 240aa, about 240aa to about 245aa, about 245aa to about 250aa, about 250aa to about 255aa, about 255aa to about 260aa, about 260aa to about 265aa, about 265aa to about 270aa, or about 270aa to about 275 aa.

In some cases, synthetic ICR suppressors may contain an intracellular signaling domain (e.g., a co-suppression domain) derived from the intracellular portion of transmembrane protein St1 (also known as UBS3B, ubiquitin-related and SH3 domain-containing protein B, Cbl interacting protein p70, suppressor of T cell receptor signaling 1, STS-1, T cell ubiquitin ligand 2, TULA-2, tyrosine protein phosphatase STS1/TULA2, UBASH3B, KIAA 1959; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: GPQKRCLFVCRHGERMDVVFGKYWLSQCFDAKGRYIRTNLNMPHSLPQRSGGFRDYEKDAPITVFGCMQARLVGEALLESNTIIDHVYCSPSLRCVQTAHNILKGLQQENHLKIRVEPGLFEWTKWVAGSTLPAWIPPSELAAANLSVDTTYRPHIPISKLVVSESYDTYISRSFQVTKEIISECKSKGNNILIVAHASSLEACTCQLQGLSPQNSKDFVQMVRKIPYLGFCSCEELGETGIWQLTDPPILPLTHGPTGGFNWRETLLQE (SEQ ID NO: 686). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 170aa to about 180aa, about 175aa, about 180aa to about 180aa, About 180aa to about 185aa, about 185aa to about 190aa, about 190aa to about 195aa, about 195aa to about 200aa, about 200aa to about 205aa, about 205aa to about 210aa, about 210aa to about 215aa, about 215aa to about 220aa, about 220aa to about 225aa, about 225aa to about 230aa, about 230aa to about 235aa, about 235aa to about 240aa, about 240aa to about 245aa, about 245aa to about 250aa, about 250aa to about 255aa, about 255aa to about 260aa, about 260aa to about 265aa, or about 265aa to about 270 aa.

In some cases, a synthetic ICR repressor may contain an intracellular signaling domain (e.g., a co-inhibitory domain) that is derived from the intracellular portion of the transmembrane protein Csk (also known as tyrosine protein kinase CSK, C-Src kinase, protein tyrosine kinase CYL; etc.). For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: LKLLQTIGKGEFGDVMLGDYRGNKVAVKCIKNDATAQAFLAEASVMTQLRHSNLVQLLGVIVEEKGGLYIVTEYMAKGSLVDYLRSRGRSVLGGDCLLKFSLDVCEAMEYLEGNNFVHRDLAARNVLVSEDNVAKVSDFGLTKEASSTQDTGKLPVKWTAPEALREKKFSTKSDVWSFGILLWEIYSFGRVPYPRIPLKDVVPRVEKGYKMDAPDGCPPAVYEVMKNCWHLDAAMRPSFLQLREQLEHIKTHELH (SEQ ID NO: 687). In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 170aa to about 180aa, about 175aa, about 180aa to about 180aa, About 180aa to about 185aa, about 185aa to about 190aa, about 190aa to about 195aa, about 195aa to about 200aa, about 200aa to about 205aa, about 205aa to about 210aa, about 210aa to about 215aa, about 215aa to about 220aa, about 220aa to about 225aa, about 225aa to about 230aa, about 230aa to about 235aa, about 235aa to about 240aa, about 240aa to about 245aa, about 245aa to about 250aa, or about 250aa to about 255 aa.

In some cases, the synthetic ICR repressor may contain an intracellular signaling domain, such as a co-suppression domain, derived from the intracellular portion of the transmembrane protein listed in table 1. For example, a suitable co-suppression domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to an amino acid sequence set forth in table 1. In some of these embodiments, the co-suppression domain has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa, about 70aa to about 75aa, about 75aa to about 80aa, about 80aa to about 85aa, about 85aa to about 90aa, about 90aa to about 95aa, about 95aa to about 100aa, about 100aa to about 105aa, about 105aa to about 110aa, about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 125aa, about 125aa to about 130aa, about 130aa to about 135aa, about 135aa to about 140aa, about 140aa to about 145aa, about 145aa to about 150aa, about 150aa to about 155aa, about 160aa to about 165aa, about 170aa to about 175aa, about 175aa to about 170aa, about 175aa to about 180aa, about 170aa to about 180aa, about 175aa, about 180aa to about 180aa, From about 180aa to about 185aa or from about 185aa to about 190 aa.

Transmembrane domain

As a non-limiting example, TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:688) may be used, additional non-limiting examples of suitable TM sequences include a) CD8 β -derived LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:689), b) CD 4-derived ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:690), c) CD3 ζ -derived LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:691), d) CD 28-derived WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:692), e) CD134(OX40) -derived VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:693), and f) CD 7-derived ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 694).

Joint

In some cases, a synthetic ICR repressor of the invention comprises a linker between any two adjacent domains. For example, a linker may be disposed between the transmembrane domain (when present) and the first intracellular functional domain (e.g., co-suppression domain) of the synthetic ICR repressor. As another example, a linker may be disposed between the first intracellular functional domain of the synthetic ICR repressor and a member of the dimerization domain. As another example, a linker may be disposed between the transmembrane domain (when present) of the synthetic ICR repressor and a member of the dimerization domain. As another example, a linker may be disposed between a member of the dimerization domain and a second intracellular functional domain (e.g., an immune cell negative regulatory domain). As another example, a linker may be disposed between any domain of a synthetic ICR repressor and any additional domain of granite, including, for example, domains not involved in the primary immune repression function of the synthetic ICR repressor, including, but not limited to, for example, reporter domains, tag domains, and the like.

Linkers may be used in the synthesis of ICR repressor factors in a suitable configuration as long as they do not eliminate the primary activity of the synthetic ICR repressor factor, including, for example, the ability of the synthetic ICR repressor factor to repress an activated ICR, the ability of the dimerization domain of the synthetic ICR repressor to bind to the dimerization domain of the repressible ICR.

The synthetic ICR repressor of the invention can be used with any suitable linker, including two or more linkers (e.g., where two or more linkers are the same or different, and including mixtures where a plurality of linkers are three or more, four or more, five or more, six or more, etc., and including where all linkers are different, and where a plurality of linkers includes some linkers for more than one location and some linkers specifically used at only one location, etc.), including for example those linkers acceptable for CAR as described herein.

Additional sequences

The heteromerically conditionally repressed synthetic ICRs of the present disclosure may further comprise one or more additional polypeptide domains, wherein such domains include, but are not limited to, signal sequences; an epitope tag; an affinity domain; and a polypeptide that produces a detectable signal.

Signal sequence

Signal sequences suitable for use in a repressible synthetic ICR (e.g., a stimulatory ICR or ICR repressor) of the invention include any eukaryotic signal sequence, including naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, and the like.

Epitope tag

Affinity domains

Affinity domains include peptide sequences that can interact with a binding partner, such as, for example, peptide sequences that can be identified or purified, immobilized on a solid support. When fused to an expressed protein, DNA sequences encoding multiple contiguous single amino acids (such as histidine) can be used to purify the recombinant protein in one step by high affinity binding to a resin column (such as nickel sepharose). Exemplary affinity domains include His5(HHHHH) (SEQ ID NO:597), HisX6 (HHHHHHH) (SEQ ID NO:598), C-myc (EQKLISEEDL) (SEQ ID NO:599), flag (DYKDDDDK) (SEQ ID NO:600), Streptag (WSHPQFEK) (SEQ ID NO:601), hemagglutinin (e.g., HA tag (YPYDVPDYA) (SEQ ID NO:602)), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:603), Phe-His-Thr (SEQ ID NO:604), chitin binding domain, S peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:605), metal binding domain (e.g., zinc binding domain or calcium binding domain, e.g., from calponin (e.g., calmodulin, troponin C, troponin B, troponin C-light chain, troponin B, and recoverin C-D, S regulatory protein, cone protein (visinin), VILIP, calcineurin, hippocampal calbindin, agrin, kallikrein, the large subunit of calpain, S100 protein, parvalbumin, calbindin D9K, calbindin D28K and calomenine) those calcium binding domains), intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences and maltose binding protein.

Polypeptide producing detectable signal

Suitable fluorescent proteins include, but are not necessarily limited to, Green Fluorescent Protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), Enhanced GFP (EGFP), Enhanced CFP (ECFP), Enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilizing EGFP (dEGFP), destabilizing ECFP (dECFP), destabilizing EYFP (dEYFP), mCFPM, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, T-HcRed, DsRed2, DsRed-monomer, J-Red, dimer 2, T-dimer 2(12), mRFP1, goblet sarcodictyin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein (kindling protein), phycobiliprotein and phycobiliprotein conjugates (including B-phycoerythrin, R-phycoerythrin and allophycocyanin). Other examples of fluorescent proteins include mHoneydev, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al (2005) nat. methods 2:905-909) and the like. Any of a variety of fluorescent and colored proteins from species of the class Corallium as described, for example, in Matz et al (1999) Nature Biotechnol.17:969-973 are suitable for use.

Conditionally active dimerization-dependent cell surface receptors

In some cases, the conditionally active heterodimeric polypeptides of the present disclosure are conditionally active dimerization-dependent cell surface receptors. By "conditionally active dimerization-dependent cell surface receptor" is meant a variant of a cell surface receptor that naturally depends on dimerization for signal propagation, e.g., dimerization induced by ligand binding, wherein the variant comprises a modification (e.g., of a naturally occurring cell surface receptor or other parent cell surface receptor) such that it comprises one member of a dimerization pair and conditionally depends on dimerization of the dimerization pair induced by the presence of a dimerizing agent. Thus, a conditionally active dimerization-dependent cell surface receptor of the present disclosure will typically comprise a cell surface receptor polypeptide comprising a first member of a dimerization pair paired with a cell surface receptor polypeptide comprising a second member of the dimerization pair. For example, in some embodiments, a first dimerization-dependent cell surface receptor that naturally forms a dimer in the presence of a ligand is modified to include the LBD of a nuclear hormone receptor, and a second dimerization-dependent cell surface receptor is modified to include a co-regulator of a nuclear hormone receptor, such that the first dimerization-dependent cell surface receptor and the second dimerization-dependent cell surface receptor dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

In some cases, the first dimerization-dependent cell surface receptor and the second dimerization-dependent cell surface receptor may be the same dimerization-dependent cell surface receptor, i.e., the first dimerization-dependent cell surface receptor and the second dimerization-dependent cell surface receptor naturally form a homodimer in the presence of the dimerization ligand. Thus, in some cases, the first conditionally active dimerization-dependent cell surface receptor and the second conditionally active dimerization-dependent cell surface receptor may differ only in that one comprises the LBD of the nuclear hormone receptor and the other comprises the co-regulator of the nuclear hormone receptor.

In some cases, the first dimerization-dependent cell surface receptor and the second dimerization-dependent cell surface receptor may be different dimerization-dependent cell surface receptors, i.e., the first dimerization-dependent cell surface receptor and the second dimerization-dependent cell surface receptor naturally form heterodimers in the presence of a dimerization ligand. Thus, in some cases, the first conditionally active dimerization-dependent cell surface receptor and the second conditionally active dimerization-dependent cell surface receptor differ by one or more domains other than the LBD of the nuclear hormone receptor present in one member and the co-regulator of the nuclear hormone receptor present in the other member.

By "dimerization-dependent cell surface receptor" is meant any cell surface receptor polypeptide that relies on dimerization with a second polypeptide to propagate an intracellular signal. In its natural environment, dimerization-dependent cell surface receptors will typically dimerize in response to ligand binding of the receptor. Useful dimerization-dependent cell surface receptors include those that dimerize to form homodimers, those that dimerize to form heterodimers, and, depending on the context, those that dimerize to form homodimers or heterodimers.

The conditionally active dimerization-dependent cell surface receptors of the present disclosure may be constructed in various ways. In some cases, the LBD of the nuclear hormone receptor or a co-regulator of the nuclear hormone receptor is appended or recombinantly inserted into a dimerization-dependent cell surface receptor that is not otherwise modified. In some cases, one or more domains of a dimerization-dependent cell surface receptor are replaced with a nuclear hormone receptor or a co-regulator of a nuclear hormone receptor. In some cases, the LBD of a nuclear hormone receptor or a co-regulator of a nuclear hormone receptor may be appended to or recombinantly inserted into a modified dimerization-dependent cell surface receptor, such as a dimerization-dependent cell surface receptor that has been modified to remove its natural ligand binding activity or render it incapable of binding other activities of its natural receptor ligand (e.g., by deleting all or a portion of the endogenous ligand binding domain, obtaining a mutation in the ligand binding domain, etc.). In some cases, the endogenous ligand binding domain of the dimerization-dependent cell surface receptor may be replaced by the LBD of the nuclear hormone receptor or a co-regulator of the nuclear hormone receptor.

For example, in some embodiments, the endogenous ligand binding domain of a cytokine receptor may be replaced by the LBD of a nuclear hormone receptor or a co-modulator of a nuclear hormone receptor of the present disclosure to produce a conditionally active dimerization-dependent cytokine receptor. In some embodiments, the endogenous ligand binding domain of a cytokine receptor may be completely or partially deleted, and the LBD of a nuclear hormone receptor or a co-regulator of a nuclear hormone receptor of the present disclosure may be appended to or inserted into the modified cytokine receptor to produce a conditionally active dimerization-dependent cytokine receptor.

In some embodiments, the endogenous ligand binding domain of the RTK may be replaced by the LBD of the nuclear hormone receptor or a co-regulator of the nuclear hormone receptor of the present disclosure to produce a conditionally active dimerization-dependent RTK. In some embodiments, the endogenous ligand binding domain of the RTK may be completely or partially deleted, and the LBD of the nuclear hormone receptor or the co-regulator of the nuclear hormone receptor of the present disclosure may be added to or inserted into the modified RTK to produce a conditionally active dimerization-dependent RTK.

Cytokine receptors

As noted above, non-limiting examples of useful dimerization-dependent cell surface receptors include cytokine receptors including, for example, homodimer-forming cytokine receptors and heterodimer-forming cytokine receptors useful cytokine receptors include, but are not limited to, for example, IL-2 family receptors, IL-3 family receptors, IL-6 family receptors, IL-12 family receptors, prolactin family receptors, interferon family receptors, IL-10 family receptors, IL-17 family receptors, immunoglobulin-like superfamily receptors, tumor necrosis factor family receptors, chemokine receptors, TGF- β family receptors, and the like.

Non-limiting examples of IL-2 family receptors include interleukin 13 receptor α 2, interleukin-2 receptor subunit α, interleukin-2 receptor subunit β, interleukin-2 receptor subunit gamma, interleukin-4 receptor subunit α, interleukin-7 receptor subunit α, interleukin 9 receptor, interleukin-13 receptor subunit α 1, interleukin-15 receptor subunit α, interleukin 21 receptor, cytokine receptor-like factor 2, and the like the amino acid sequences of such examples are provided in FIG. 53.

Non-limiting examples of IL-3 family receptors include interleukin 3 receptor α subunit, interleukin 5 receptor α subunit, GM-CSF receptor α subunit, cytokine receptor common β subunit, and the like, the amino acid sequences of such examples are provided in FIG. 54.

Non-limiting examples of IL-6 family receptors include leptin receptor, IL6R (interleukin-6 receptor α subunit/interleukin 6 receptor), IL6ST (interleukin-6 receptor β subunit/interleukin 6 signal transducer), interleukin-11 receptor α subunit, interleukin 27 receptor α, interleukin-31 receptor α subunit, ciliary neurotrophic factor receptor α subunit, leukemia inhibitory factor receptor, oncostatin M-specific receptor β subunit, and the like.

Non-limiting examples of IL-12 family receptors include interleukin-12 receptor β 1 subunit, interleukin-12 receptor β 2 subunit, interleukin 23 receptor, and the like, the amino acid sequences of such examples are provided in FIG. 56.

Non-limiting examples of prolactin family receptors include erythropoietin receptor; granulocyte colony stimulating factor receptor; a growth hormone receptor; a prolactin receptor; thrombopoietin receptor; and so on. The amino acid sequences of such examples are provided in figure 57.

Non-limiting examples of interferon family receptors include interferon α/β receptor 1, interferon α/β receptor 2, interferon gamma receptor 1, interferon gamma receptor 2, and the like, the amino acid sequences of such examples are provided in FIG. 58.

Non-limiting examples of IL-10 family receptors include interleukin-22 receptor α 2, interleukin 10 receptor α subunit, interleukin 10 receptor β subunit, interleukin 20 receptor α subunit, interleukin 20 receptor β subunit, interleukin 22 receptor α 1 subunit, interferon-lambda receptor subunit 1, and the amino acid sequences of such examples are provided in FIG. 59.

Non-limiting examples of IL-17 family receptors include interleukin 17 receptor A; interleukin 17 receptor B; interleukin 17 receptor C; interleukin-17 receptor D; interleukin 17 receptor E; and so on. The amino acid sequences of such examples are provided in figure 60.

Non-limiting examples of immunoglobulin-like superfamily receptors include type I interleukin-1 receptor, type II interleukin-1 receptor, interleukin-1 receptor-like 1, interleukin-1 receptor-like 2, interleukin-181, IL-1 receptor accessory protein, IL-18 receptor accessory protein, PDGFR α (platelet-derived growth factor receptor α), PDGFR β (platelet-derived growth factor receptor β), KIT proto-oncogene receptor tyrosine kinase, CSFR (colony stimulating factor 1 receptor), and the like.

Non-limiting examples of tumor necrosis factor family receptors include TNFR1 (tumor necrosis factor receptor 1/TNFRSF1A), TNFR2 (tumor necrosis factor receptor 2/TNFRSF1B), lymphotoxin β receptor/TNFRSF 3; OX40/TNFRSF 4; CD40/TNFRSF 5; Fas/TNFRSF 6; decoy receptor 3/TNFRSF 6B; CD27/TNFRSF 7; CD30/TNFRSF 8; 4-1 BB/TNFRRSF 8; DR8 (death receptor 4/TNFRRSF 10 8), DR8 (death receptor 5/TNFRRSF 10 8), decoy receptor 1/TNFRSF10 8; receptor activator of TNFRRSF 2/TNFRSF10 8; RANK (receptor activator of TNFRSF 11/TNFRSF 8; TNFRSF8 receptor activator of TNFRSF receptor (TNFRSF receptor activator/receptor activator of TNFRSF 8; TNFRSF receptor effector of TNFRSF 8; TNFRSF receptor effector of TNFRSF 8; TNFRSF8, TNFRSF receptor effector genes of TNFRSF 8; TNFRSF8, TNFRSF receptor effector genes of TNFRSF 8; TNFRSF8, TNFRSF receptor effector genes of TNFRSF 8; TNFRSF receptor effector genes of TNFRSF 8; TNFRSF 8.

Non-limiting examples of chemokine receptors include CCR 1; CCR 2; CCR 3; CCR 4; CCR 5; CCR 6; CCR 7; CCR 8; CCR 9; CCR 10; CXCR1(IL8 Ra); CXCR2(IL8 Rb); CXCR 3; CXCR 4; CXCR 5; CXCR 6; CX3CR 1; and so on. The amino acid sequences of such examples are provided in fig. 63.

Non-limiting examples of TGF- β family receptors include transforming growth factor β type I receptor (TGFBR1(ALK5)), transforming growth factor β type II receptor (TGFBR2(MFS2)) and transforming growth factor β type III receptor (TGFBR3(β -glycan)), the amino acid sequences of such examples are provided in figure 64.

Receptor tyrosine kinases

As noted above, non-limiting examples of useful dimerization-dependent cell surface receptors include Receptor Tyrosine Kinases (RTKs), including, for example, homodimer-forming RTKs and heterodimer-forming RTKs. Useful RTKs include, but are not limited to, e.g., merks (RefSeq accession number NP _ 006334); LMTK3(RefSeq accession number NP-001073903); CSF1R (RefSeq accession No. NP _ 001275634); EGFR (RefSeq accession No. NP _ 005219); EPHA2(RefSeq accession number NP-004422); EPHA1(RefSeq accession No. NP _ 005223); EPHA3(RefSeq accession No. NP _ 005224); EPHA4(RefSeq accession No. NP _ 001291465); EPHA5(RefSeq accession number NP-004430); EPHA7(RefSeq accession No. NP _ 004431); EPHA8(RefSeq accession No. NP _ 065387); EPHB1(RefSeq accession number NP-004432); EPHB2(RefSeq accession number NP-001296122); EPHB3(RefSeq accession number NP-004434); EPHB4(RefSeq accession number NP-004435); EPHB6(RefSeq accession number NP-004436); ERBB2(RefSeq accession number NP-004439); ERBB3(RefSeq accession number NP-001973); ERBB4(RefSeq accession number NP-005226); FGFR1(RefSeq accession No. NP _ 075598); FGFR3(RefSeq accession No. NP _ 000133); FGFR2(RefSeq accession No. NP _ 000132); FGFR4(RefSeq accession No. NP _ 002002); LMTK2(RefSeq accession number NP-055731); FLT1(RefSeq accession No. NP _ 002010); FLT3(RefSeq accession No. NP _ 004110); FLT4(RefSeq accession No. NP _ 891555); ALK (RefSeq accession No. NP _ 004295); EPHA10(RefSeq accession No. NP _ 001092909); EPHA6(RefSeq accession No. NP _ 001265229); IGF1R (RefSeq accession No. NP _ 000866); INSR (RefSeq accession No. NP _ 000199); INSRR (RefSeq accession No. NP _ 055030); KDR (RefSeq accession No. NP _ 002244); KIT (RefSeq accession No. NP _ 000213); LTK (RefSeq accession No. NP _ 002335); MET (RefSeq accession No. NP _ 000236); MST1R (RefSeq accession No. NP _ 002438); MUSK (RefSeq accession number NP-005583); NTRK1(RefSeq accession No. NP _ 002520); NTRK2(RefSeq accession No. NP _ 001018074); NTRK3(RefSeq accession No. NP _ 001012338); ROR1(RefSeq accession No. NP _ 005003); ROR2(RefSeq accession No. NP _ 004551); DDR2(RefSeq accession No. NP _ 001014796); PDGFRA (RefSeq accession No. NP _ 006197); PDGFRB (RefSeq accession No. NP _ 002600); AXL (RefSeq accession number NP _ 068713); PTK7(RefSeq accession No. NP _ 002812); RET (RefSeq accession No. NP _ 066124); ROS1(RefSeq accession No. NP _ 002935); RYK (RefSeq accession No. NP _ 002949); TEK (RefSeq accession No. NP _ 000450); TIE1(RefSeq accession No. NP _ 005415); TYRO3(RefSeq accession number NP-006284); DDR1(RefSeq accession No. NP _ 001284583); AATK (RefSeq accession No. NP _ 001073864); and so on. The amino acid sequences of such examples are provided in fig. 65.

Dimeric pair

As noted above, conditionally active dimerization-dependent cell surface receptors of the present disclosure will typically comprise a cell surface receptor polypeptide comprising a first member of a dimerization pair that is conditionally dimerizable with a second cell surface receptor polypeptide comprising a second member of the dimerization pair. Thus, conditionally active dimerization-dependent cell surface receptors of the present disclosure will comprise members of a dimerization pair comprising the LBD or co-regulatory peptide of the nuclear hormone receptor. Two conditionally active dimerization-dependent cell surface receptors may be used as a system, wherein one conditionally active dimerization-dependent cell surface receptor comprises a first member of a dimerization pair comprising the LBD of a nuclear hormone receptor, and the other conditionally active dimerization-dependent cell surface receptor comprises a second member of the dimerization pair comprising a co-regulatory peptide of the same nuclear hormone receptor. In the presence of a dimerizing agent (e.g., a nuclear hormone or a functional derivative or analog of a nuclear hormone; also referred to herein as a "dimerizer"), the first member and the second member of the dimerization pair will bind to each other and will effect dimerization of the two conditionally active dimerization-dependent cell surface receptor polypeptides. The first member of a dimerization pair or the second member of a dimerization pair may also be referred to as a "dimerization domain".

The ligand binding domain of a nuclear hormone receptor may be from any of a variety of nuclear hormone receptors, including but not limited to those described above. Suitable co-regulatory polypeptides include full-length naturally occurring nuclear hormone co-regulatory polypeptides. Suitable co-regulatory polypeptides include fragments of naturally occurring nuclear hormone co-regulatory polypeptides. Suitable co-regulatory polypeptides include synthetic or recombinant nuclear hormone co-regulatory polypeptides. Non-limiting examples of suitable co-modulatory polypeptides include those described above.

Nucleic acids

The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a conditionally active polypeptide of a heterodimer of the present disclosure. A single nucleic acid molecule can include multiple sequences encoding two or more portions of a conditionally active polypeptide of a heterodimer of the disclosure. In some cases, two or more portions of a conditionally active polypeptide of a heterodimer of the present disclosure can be isolated across multiple separate nucleic acid molecules (e.g., multiple nucleic acid vectors). In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a conditionally active polypeptide of a heterodimer of the present disclosure will be DNA, including for example, a recombinant expression vector. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a conditionally active polypeptide of a heterodimer of the present disclosure will be an RNA, e.g., an RNA synthesized in vitro.

In some cases, a nucleic acid of the disclosure comprises a nucleotide sequence encoding only a first portion (e.g., a first polypeptide chain) of a conditionally active polypeptide of a heterodimer of the disclosure. In some cases, a nucleic acid of the disclosure comprises a nucleotide sequence encoding only a second portion of a conditionally active polypeptide (e.g., a second polypeptide chain) of a heterodimer of the disclosure. In some cases, a nucleic acid of the disclosure comprises a nucleotide sequence of two polypeptide chains encoding a conditionally active polypeptide of a heterodimer of the disclosure.

In some cases, a single nucleic acid of the disclosure can comprise one or more nucleotide sequences encoding two or more conditionally active polypeptides of the disclosure. For example, in some cases, a nucleic acid of the disclosure can encode a first conditionally active polypeptide comprising a first member of a dimerization pair and a second conditionally active polypeptide comprising a second member of the dimerization pair. In some embodiments, a nucleic acid of the present disclosure may comprise one or more sequences encoding a first conditionally active dimerization-dependent cell surface receptor comprising a first member of a dimerization pair and a second conditionally active dimerization-dependent cell surface receptor comprising a second member of the dimerization pair. In some embodiments, the two conditionally active dimerization-dependent cell surface receptors, although each comprising half of a dimerization pair, may be encoded by sequences present on separate nucleic acids.

In some cases, the nucleic acids of the invention provide conditionally active polypeptides that produce the heterodimers of the invention, e.g., in mammalian cells. In other cases, the nucleic acids of the invention provide for amplification of nucleic acids encoding heterodimerically active polypeptides.

The nucleotide sequences encoding conditionally active polypeptides of the heterodimers of the present disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers, and the like. In some cases, the nucleic acid encoding the heterodimeric, conditionally active polypeptide is operably linked to a tissue-specific promoter for expression in a particular cell type of interest. For example, a heterodimeric, conditionally active polypeptide can be operably linked to an immune cell-specific promoter for specific expression in one or more immune cell types. In other instances, the heterodimeric, conditionally active polypeptide can be operably linked to a universal (i.e., non-immune cell-specific) promoter, which includes, for example, ubiquitous promoters (ubiquitosu promoters), constitutive promoters, heterologous promoters, regulatable promoters (e.g., inducible promoters, reversible promoters, etc.), and the like.

Universal promoters

Suitable promoter and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, λ P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to: cytomegalovirus immediate early promoter; a herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; a promoter present in a long terminal repeat from a retrovirus; mouse metallothionein-I promoter; and various promoters known in the art.

Suitable promoters for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7RNA polymerase promoter; a trp promoter; a lactose operon promoter; hybrid promoters (e.g., lac/tac hybrid promoter, tac/trc hybrid promoter, trp/lac promoter, T7/lac promoter); a trc promoter; tac promoter, and the like; the araBAD promoter; in vivo regulated promoters (such as the ssaG promoter or related promoters (see, e.g., U.S. patent publication No. 20040131637), the pagC promoter (Pulkkien and Miller, J.Bacteriol.,1991:173(1): 86-93; Alpuche-Aranda et al, PNAS, 1992; 89(21):10079-83), the nirB promoter (Harborne et al (1992) mol.Micro.6: 2805-; a sigma 70 promoter (e.g., consensus sigma 70 promoter (see, e.g., GenBank accession nos. AX798980, AX798961, and AX 798183)); stationary phase promoters (e.g., dps promoter, spv promoter, etc.); promoters derived from the pathogenic island SPI-2 (see, e.g., WO 96/17951); the actA promoter (see, e.g., Shetron-Rama et al (2002) feed. Immun.70: 1087-; the rpsM promoter (see, e.g., Valdivia and Falkow (1996). mol. Microbiol.22: 367); the tet promoter (see, e.g., Hillen, W., and Wissmann, A. (1989) In Saenger, W., and Heinemann, U. (eds.), Topics In Molecular and structural Biology, Protein-Nucleic Acid interaction, Macmillan, London, UK, Vol.10, p.143-; the SP6 promoter (see, e.g., Melton et al (1984) Nucl. acids Res.12: 7035); and so on. Suitable strong promoters for prokaryotes such as E.coli include, but are not limited to, Trc, Tac, T5, T7, and P λ. Non-limiting examples of operons for use in bacterial host cells include the lactose promoter operon (the LacI repressor protein changes conformation when contacted with lactose, thereby preventing binding of the LacI repressor protein to the operon), the tryptophan promoter operon (the TrpR repressor protein has a conformation that binds to the operon when complexed with tryptophan; the TrpR repressor protein has a conformation that does not bind to the operon in the absence of tryptophan), and the tac promoter operon (see, e.g., deBoer et al (1983) Proc. Natl. Acad. Sci. U.S.A.80: 21-25).

Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. Modifications of reversible promoters derived from a first organism for use in a second organism (e.g., first and second prokaryotes, etc.) are well known in the art. Such reversible promoters and systems based on such reversible promoters but also including additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase i (alca) gene promoters, promoters responsive to alcohol transactivator (AlcR), etc.), tetracycline regulated promoters (e.g., promoter systems including Tet-activator, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), thermoregulatory promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.)), photoregulated promoters, synthetically inducible promoters, and the like.

In some embodiments, for example for expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, and the like; or a regulatable promoter such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for pichia pastoris). Selection of appropriate vectors and promoters is within the level of ordinary skill in the art.

Immune cell promoters

In some cases, a nucleic acid of the present disclosure includes an immune cell-specific promoter expressed in one or more immune cell types, including but not limited to lymphocytes, hematopoietic stem cells, and/or progeny thereof (i.e., immune cell progenitors), and the like. Any convenient and suitable promoter for an immune cell-specific gene may be used in the nucleic acids of the present disclosure. In some cases, the immune cell-specific promoter of a nucleic acid of the present disclosure can be a T cell-specific promoter. In some cases, an immune cell-specific promoter of a nucleic acid of the present disclosure may be a light chain and/or heavy chain immunoglobulin gene promoter, and may or may not include one or more associated enhancer elements.

In some cases, the immune cell-specific promoter of a nucleic acid of the present disclosure may be a promoter of the B29 gene, a promoter of the CD14 gene, a promoter of the CD43 gene, a promoter of the CD45 gene, a promoter of the CD68 gene, a promoter of the IFN- β gene, a promoter of the WASP gene, a promoter of the β chain gene of the T cell receptor, a promoter of the V9 gamma (TRGV9) gene, a promoter of the V2 delta (TRDV2) gene, or the like.

In some cases, the immune cell-specific promoter of a nucleic acid of the present disclosure can be a viral promoter that is expressed in an immune cell. Thus, in some instances, viral promoters for use in the nucleic acids of the present disclosure include viral promoters derived from immunocytoviruses including, but not limited to, for example, lentiviral promoters (e.g., HIV, SIV, FIV, EIAV, or Visna promoters) (including, for example, LTR promoters, etc.), retroviral promoters (including, for example, HTLV-1 promoters, HTLV-II promoters, etc.), and the like.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter may be used; see, e.g., Salmonon et al (1993) Proc. Natl. Acad. Sci. USA 90: 7739; and Marodon et al (2003) Blood101: 3416. As another example, the CD8 gene promoter may be used. NK cell-specific expression can be achieved by using Ncr1(p46) promoter; see, e.g., Eckelhart et al (2011) Blood 117: 1565.

Additional nucleic acid components, constructs and uses thereof

In some cases, a locus or construct or transgene containing a suitable promoter is irreversibly switched by induction by an induction system. Suitable systems for inducing irreversible transformation are well known in the art, e.g., inducing irreversible transformation can utilize Cre-lox mediated recombination (see, e.g., Fuhrmann-Benzakein, et al, PNAS (2000)28: e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art may be used to generate the irreversibly switched promoter. The methods, mechanisms and requirements for performing site-specific recombination described elsewhere herein can be used to generate irreversibly switched promoters and are well known in the art, see, e.g., Grindeley et al (2006) Annual Review of Biochemistry, 567-.

The nucleotide sequence encoding the conditionally active polypeptide of the heterodimer may be present in an expression vector and/or a cloning vector. In the case where the conditionally active polypeptide of the heterodimer of the present invention is split between two or more separate polypeptides, the nucleotide sequences encoding the two or more polypeptides may be cloned in the same or different vectors. Expression vectors may include selectable markers, origins of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for the production of recombinant constructs of the invention. The following vectors are provided by way of example. Of the bacterium: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5(Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (pharmacia).

Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for insertion of a nucleic acid sequence encoding a heterologous protein. The selectable marker may be present efficiently in the expression host. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al, Invest Opthalmol Vis Sci 35: 25432549,1994; Borras et al, Gene Ther 6:515524,1999; Li and Davidson, PNAS 92: 77007704,1995; Sakamoto et al, H Gene Ther 5:10881097,1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated viruses (see, e.g., Ali et al, Hum Gene Ther 9: 8186,1998; Flannery et al, PNAS94: 69166921,1997; Bennett et al, Invest Opthalmol Vis Sci 38: 28572863,1997; Jomary et al, Gene Therr 4: 48; Rolling et al, 39m Hu R10: 641648,1999; Ali et al, Themhalt 5: Moval et al; Sri 3829J 3828; Srl 38J. 3828; Melson et al, virol (1988)166: 154-165; and Flotte et al, PNAS (1993)90: 10613-10617); SV 40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al, PNAS94: 1031923,1997; Takahashi et al, J Virol 73: 78127816,1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus and vectors derived from retroviruses such as rous sarcoma virus, hayweed sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and so on.

As noted above, in some embodiments, a nucleic acid comprising a nucleotide sequence encoding a conditionally active polypeptide of a heterodimer of the present disclosure will be an RNA in some embodiments, e.g., an RNA synthesized in vitro. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising the nucleotide sequences of the first and/or second polypeptides encoding conditionally active polypeptides of the heterodimers of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al (2010) Cancer Res.15: 9053. Introduction of RNA comprising the nucleotide sequences encoding the first and/or second polypeptides of the conditionally active polypeptides of the heterodimers of the disclosure into a host cell may be performed in vitro or ex vivo or in vivo. For example, a host cell (e.g., NK cell, cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a first and/or second polypeptide encoding a conditionally active polypeptide of a heterodimer of the disclosure.

Cells

The present disclosure provides a mammalian cell genetically modified to produce a conditionally active polypeptide of a heterodimer of the present disclosure.

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC number CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), Human Embryonic Kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

In some cases, suitable cells include those described in Themeli et al, Cell Stem cell.2015, 4 months and 2 days; 16(4) 357-66, the disclosure of which is incorporated herein by reference in its entirety.

In some cases, the cell is not an immortalized cell line, but a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell, an immune cell progenitor cell, or an immune stem cell obtained from an individual. As an example, the cell is a T lymphocyte or a progenitor thereof obtained from an individual. As another example, the cell is a cytotoxic cell or progenitor thereof obtained from an individual. As another example, the cell is a stem cell or a progenitor cell obtained from an individual.

Practicality of use

The conditionally active polypeptides of the heterodimers of the present disclosure are useful in a variety of research and therapeutic methods provided herein.

In some cases, where the heterodimeric, conditionally active polypeptide of the present disclosure comprises a first heterologous polypeptide and a second heterologous polypeptide, which do not individually exhibit activity, but do exhibit activity when present in the heterodimeric, conditionally active polypeptide of the present disclosure and in the presence of a dimerizer that induces LBD in the heterodimeric, conditionally active polypeptide in combination with a co-regulatory peptide, the present disclosure provides a method of activating the activity. Thus, for example, the present disclosure provides a method of activating a polypeptide (inducing polypeptide activity), the method comprising contacting a heterodimeric, conditionally active polypeptide of the present disclosure with a dimerizing agent, wherein the polypeptide is present in the heterodimeric polypeptide as a first heterologous polypeptide and a second heterologous polypeptide, wherein the first heterologous polypeptide and the second heterologous polypeptide do not individually exhibit activity, but do exhibit activity when present in the heterodimeric, conditionally active polypeptide of the present disclosure and in the presence of the dimerizing agent. The activity induced depends on the nature of the first heterologous polypeptide and the second heterologous polypeptide. For example, where the first heterologous polypeptide and the second heterologous polypeptide are a receptor and a co-receptor, respectively, the activity can be binding of the receptor to the co-receptor or a downstream activity resulting from binding of the receptor to the co-receptor. In some cases, contacting the conditionally active polypeptide of a heterodimer of the present disclosure occurs in vitro, wherein the conditionally active polypeptide of a heterodimer of the present disclosure is not in a cell. In some cases, contacting the conditionally active polypeptide of a heterodimer of the present disclosure occurs in vitro in a cell, wherein the conditionally active polypeptide of a heterodimer of the present disclosure is in a cell. In some cases, contacting of the conditionally active polypeptide of a heterodimer of the present disclosure occurs in vivo in a cell, wherein the conditionally active polypeptide of a heterodimer of the present disclosure is in a cell.

Methods for modulating cellular activity

The present disclosure provides methods for modulating (activating; repressing) cellular activity. The methods generally involve contacting a cell expressing a conditionally active polypeptide of a heterodimer of the present disclosure with a dimerizing agent. In some cases, the methods involve contacting a cell expressing a conditionally active polypeptide of a heterodimer of the present disclosure with a dimerizing agent and a second agent (e.g., an antigen).

Method for activating immune cells

The present disclosure provides methods of activating an immune cell, wherein the immune cell expresses a switch-open CAR of the present disclosure. The present disclosure provides methods of activating immune cells in vitro, in vivo, or ex vivo. The methods generally involve contacting an immune cell (in vitro, in vivo, or ex vivo) with a dimerizing agent and an antigen, wherein the immune cell is genetically modified to produce a heterodimeric, conditionally active CAR of the present disclosure. In the presence of a dimerizing agent and an antigen, the heterodimeric, conditionally active CAR dimerizes and activates an immune cell, thereby generating an activated immune cell. Immune cells include, for example, cytotoxic T lymphocytes, NK cells, CD4⁺T cells, T regulatory (Treg) cellsAnd the like.

Contacting a genetically modified immune cell (e.g., T lymphocyte, NK cell) with a dimerizing agent and a second member of a specific binding pair (e.g., antigen, ligand, receptor) can increase the cytokine produced by the immune cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the amount of cytokine produced by the immune cell in the absence of the second member of the specific binding pair and/or the dimerizing agent. Cytokines whose production can be increased include, but are not limited to, IL-2 and IFN-gamma.

Contacting a genetically modified immune cell (e.g., T lymphocyte, NK cell) with a dimerizing agent and an antigen can increase cytokine production by the immune cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the amount of cytokine produced by the immune cell in the absence of the antigen and/or dimerizing agent. Cytokines whose production can be increased include, but are not limited to, IL-2 and IFN-gamma.

Contacting a genetically modified cytotoxic cell (e.g., a cytotoxic T lymphocyte) with a dimerizing agent and a second member of a specific binding pair (e.g., an antigen, a ligand, a receptor) can increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

Contacting a genetically modified cytotoxic cell (e.g., a cytotoxic T lymphocyte) with a dimerizing agent and an antigen can increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold as compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

In other embodiments, for example, depending on the host immune cell, contacting a genetically modified host cell with a dimerizing agent and an antigen may increase or decrease cell proliferation, cell survival, cell death, and the like.

The present disclosure provides methods of producing conditionally activatable cells. The methods generally involve genetically modifying a mammalian cell with an expression vector or RNA (e.g., an in vitro transcribed RNA) comprising a nucleotide sequence encoding a conditionally active CAR of a heterodimer of the disclosure. The genetically modified cell is conditionally activated in the presence of: a) an antigen to which a first polypeptide of the CAR binds; and b) a dimerizer (dimerizer). Genetic modification may be performed in vivo, in vitro or ex vivo. The cells can be immune cells (e.g., T lymphocytes or NK cells), stem cells, progenitor cells, and the like.

In some cases, the genetic modification is performed ex vivo. For example, T lymphocytes, stem cells, or NK cells are obtained from an individual; and genetically modifying a cell obtained from the individual to express the CAR of the disclosure. The genetically modified cell is conditionally activated in the presence of: a) an antigen to which a first polypeptide of the CAR binds; and b) a dimerizing agent. In some cases, the genetically modified cells are activated ex vivo. In other cases, the genetically modified cell is introduced into an individual (e.g., an individual from which the cell was obtained); and the genetically modified cells are activated in vivo, for example, by administering a dimerizing agent to the individual. For example, where the antigen is present on the surface of a cell of an individual, administration of the antigen is not required. Contacting the genetically modified cell with an antigen present on the cell surface of the individual; and the genetically modified cells are activated upon administration of the dimerizing agent to the individual. For example, where the genetically modified cell is a T lymphocyte, the genetically modified cell can exhibit cytotoxicity against a cell presenting an antigen to which the CAR binds on its surface.

Method for suppressing immune cell activation

The present disclosure provides methods of suppressing immune cell activation, such methods being useful in vitro, in vivo, or ex vivo. The methods generally involve contacting an immune cell (in vitro, in vivo, or ex vivo) with a dimerizing agent, wherein the immune cell is genetically modified to produce a conditionally-repressed synthetic ICR of a heteromer of the disclosure. In the presence of a dimerizing agent, the heteromer conditions repress ICR dimerization and repress activation of immune cells, thereby producing repressed immune cells. Immune cells include, for example, cytotoxic T lymphocytes, NK cells, CD4+ T cells, T regulatory (Treg) cells, and the like.

Contacting a genetically modified immune cell (e.g., T lymphocyte, NK cell) with a dimerizing agent and an antigen can repress a lymphocyte surface antigen expressed by the immune cell (e.g., a cell surface antigen indicative of immune cell activation, T cell activation) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, as compared to the amount of cell surface antigen expressed by the activated immune cell in the absence of the dimerizing agent. Lymphocyte surface antigens whose production can be repressed include, but are not limited to, for example, CD 69.

Contacting a genetically modified immune cell (e.g., T lymphocyte, NK cell) with a dimerizing agent can suppress cytokine production by the immune cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, as compared to the amount of cytokine produced by an activated immune cell in the absence of the dimerizing agent. Cytokines whose production can be repressed include, but are not limited to, IL-2 and IFN-gamma.

Formulations, dosages and routes of administration

As discussed above, the therapeutic methods of the present disclosure involve administering an effective amount of a dimerizing agent to an individual in need thereof, and may also involve administering an antigen.

In some instances, an "effective amount" of a dimerizing agent is an amount that, when administered to an individual in need thereof in one or more doses, increases the level of cytotoxic activity of T lymphocytes expressing a CAR of the invention by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the cytotoxic activity of T lymphocytes in the absence of the dimerizing agent.

In some instances, an "effective amount" of a dimerizing agent is an amount that, when administered to an individual in need thereof at one or more doses, increases the level of cytotoxic activity of NK cells expressing a CAR of the invention by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the cytotoxic activity of NK cells in the absence of the dimerizing agent.

In some cases, an "effective amount" of a dimerizing agent is an amount that, when administered to an individual in need thereof in one or more doses, reduces the number of cancer cells and/or the mass of a tumor in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 75% as compared to the number of cancer cells and/or the mass of a tumor in the absence of the dimerizing agent.

In some embodiments, an effective amount of a dimerizing agent is an amount effective to reduce one or more of tumor growth rate, cancer cell number, and tumor mass by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, when administered alone (e.g., in monotherapy) or in combination (e.g., in combination therapy) with one or more additional therapeutic agents, as compared to the tumor growth rate, cancer cell number, and tumor mass when not treated with the dimerizing agent.

Preparation

In the methods of the invention, the dimerizing agent may be administered to the host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Accordingly, the dimerizing agent may be incorporated into a variety of formulations for therapeutic administration. More specifically, the dimerizing agent is formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable carrier or diluent, and may be formulated into preparations in the form of solid, semisolid, liquid, or gas, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

In pharmaceutical dosage forms, the dimerizing agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate combination and combination with other pharmaceutically active compounds. The following methods and excipients are exemplary only and are in no way limiting.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition (if desired), the vehicle may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents. The actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.See also，For exampleRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17 th edition, 1985. In any event, the composition or formulation to be administered will contain an amount sufficient to achieve the desired state in the subject being treatedA dimerizing agent.

Pharmaceutically acceptable excipients (such as vehicles, adjuvants, carriers or diluents) are readily available to the public. Furthermore, pharmaceutically acceptable auxiliary substances (such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizing agents, wetting agents, etc.) are readily available to the public.

For oral formulations, the compounds of the invention can be used alone or in combination with suitable additives to make tablets, powders, granules or capsules, for example in combination with conventional additives such as lactose, mannitol, corn starch or potato starch; in combination with a binder (such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin); in combination with a disintegrant (such as corn starch, potato starch, or sodium carboxymethyl cellulose); in combination with a lubricant (such as talc or magnesium stearate); and, if desired, in combination with diluents, buffers, wetting agents, preservatives and flavoring agents.

The dimerizing agent may be formulated for injection by: dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable oil or other similar oil, synthetic aliphatic acid glyceride, ester of higher aliphatic acid or propylene glycol; and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

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

The pharmaceutical composition may be in liquid form, lyophilized form, or reconstituted liquid form from a lyophilized form, wherein the lyophilized formulation is reconstituted with a sterile solution prior to administration. The standard procedure for reconstitution of lyophilized compositions is to add back a volume of purified water (usually equivalent to the volume removed during lyophilization); however, solutions containing antibacterial agents may be used in the manufacture of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18,1311-54.

As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of dimerizing agent, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specification for a given dimerizing agent may depend on the particular dimerizing agent used and the effect to be achieved as well as the pharmacodynamics associated with each dimerizing agent in the host.

In some embodiments, the dimerizing agent is formulated as a controlled release formulation. Sustained release formulations can be prepared using methods well known in the art. Suitable examples of sustained-release articles include semipermeable matrices of solid hydrophobic polymers containing the dimerizing agent, wherein the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D- (-) -3-hydroxybutyric acid. Possible loss of biological activity can be prevented by the use of appropriate additives, by controlling the moisture content, and by developing a specified polymer matrix composition.

Dosage form

An attending physician or other qualified medical professional can determine the appropriate dosage based on various clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the size of the patient, the body surface area, the age, the particular dimerizing agent to be administered, the sex of the patient, the time and route of administration, general health, and other drugs currently being administered. The dimerizing agent may be administered in an amount between 1ng/kg body weight and 20mg/kg body weight, such as between 0.1mg/kg body weight and 10mg/kg body weight, such as between 0.5mg/kg body weight and 5mg/kg body weight per dose; however, dosages below or above this exemplary range are contemplated, especially in view of the above-mentioned factors. If the regimen is a continuous infusion, it may also be in the range of 1 μ g to 10mg per kg body weight per minute.

The skilled artisan will readily appreciate that dosage levels may vary with the given dimerizing agent, the severity of the symptoms, and the subject's susceptibility to side effects. The preferred dosage of a given compound can be readily determined by one skilled in the art in a variety of ways.

Route of administration

The dimerizing agent is administered to the individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration.

Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, buccal and other enteral and parenteral routes of administration. The routes of administration may be combined (if desired) or adjusted according to the dimerizing agent and/or the desired effect. The dimerizing agent may be administered in a single dose or in multiple doses. In some embodiments, the dimerizing agent is administered orally. In some embodiments, the dimerizing agent is administered via the inhalation route. In some embodiments, the dimerizing agent is administered intranasally. In some embodiments, the dimerizing agent is administered topically. In some embodiments, the dimerizing agent is administered intratumorally. In some embodiments, the dimerizing agent is administered peritumorally. In some embodiments, the dimerizing agent is administered intracranially. In some embodiments, the dimerizing agent is administered intravenously.

The agents may be administered to the host using any available conventional method and route (systemic or local route) suitable for delivering conventional drugs. In general, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

In addition to inhalation administration, parenteral routes of administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration is performed to achieve systemic or local delivery of the dimerizing agent. Where systemic delivery is desired, administration typically involves local or mucosal administration of the drug formulation for invasive or systemic absorption.

The dimerizing agent may also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using suppositories) delivery.

Treatment means at least improving the symptoms associated with the pathological condition afflicting the host, where broadly improvement is used to refer to at least reducing the magnitude of a parameter (e.g., symptom) associated with the pathological condition being treated, such as cancer. Thus, treatment also includes situations in which the pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or halted (e.g., terminated) such that the host is no longer subjected to the pathological condition, or at least the symptoms characteristic of the pathological condition.

In some embodiments, the dimerizing agent is administered by injection and/or delivery, e.g., to a site in a cerebral artery or directly to brain tissue. The dimerizing agent may also be administered directly to the target site, for example, by direct injection, by implantation of a drug delivery device (such as an osmotic pump or slow release particles), by biolistic delivery to the target site, and the like.

Examples of non-limiting aspects of the disclosure

Aspects of the inventive subject matter described above, including embodiments, can be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure numbered 1-45 are provided below. It will be apparent to those skilled in the art upon reading this disclosure that each of the individually numbered aspects can be used together or in combination with any of the previously or below individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below:

1. a conditionally active polypeptide of a heterodimer, comprising: a) a first chimeric polypeptide comprising a first member of a dimerization pair and a first heterologous polypeptide; and b) a second chimeric polypeptide comprising a second member of a dimerization pair and a second heterologous polypeptide, wherein the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein the first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and the second member of the dimerization pair comprises the LBD of the nuclear hormone receptor; and wherein said first chimeric polypeptide and said second chimeric polypeptide dimerize in the presence of a dimerizing agent that induces binding of said LBD to said co-regulator.

2. The conditionally active polypeptide of a heterodimer of aspect 1, wherein a) the first heterologous polypeptide is a T Cell Receptor (TCR) α chain and b) the second heterologous polypeptide is a TCR β chain.

3. The conditionally active polypeptide of a heterodimer of aspect 1, wherein: a) the first heterologous polypeptide is a first polypeptide of a Chimeric Antigen Receptor (CAR) heterodimer; and b) the second heterologous polypeptide is a second polypeptide of a CAR heterodimer.

4. The conditionally active polypeptide of a heterodimer of aspect 1, wherein: a) the first heterologous polypeptide is the N-terminal portion of an RNA-guided endonuclease; and b) the second heterologous polypeptide is the C-terminal portion of the RNA-guided endonuclease, wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide mediated by a dimerizing agent that induces binding of LBD to a co-regulatory factor restores the enzymatic function of the RNA-guided endonuclease.

5. The conditionally active polypeptide of a heterodimer of aspect 4, wherein the RNA-guided endonuclease is a class 2 CRISPR/Cas endonuclease.

6. The conditionally active polypeptide of a heterodimer of aspect 5, wherein the class 2 CRISPR/Cas endonuclease is a type II CRISPR/Cas protein, a type V CRISPR/Cas protein, or a type VI CRISPR/Cas protein.

7. The conditionally active polypeptide of a heterodimer of aspect 1, wherein: a) the first heterologous polypeptide is an N-terminal portion of the enzyme; and b) the second heterologous polypeptide is the C-terminal portion of the enzyme, wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide restores the enzymatic activity of the enzyme.

8. The conditionally active polypeptide of a heterodimer of aspect 7, wherein the enzyme is a kinase, a protease, a phosphatase, or a caspase.

9. The conditionally active polypeptide of a heterodimer of aspect 1, wherein the first polypeptide and the second polypeptide exhibit activity when in proximity after dimerization mediated by a dimerizing agent, but do not individually exhibit activity.

10. The conditionally active polypeptide of a heterodimer of aspect 1, wherein: a) the first heterologous polypeptide is the N-terminal portion of the antigen receptor; and b) the second heterologous polypeptide is the C-terminal portion of the antigen receptor, wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide restores the signaling activity of the antigen receptor.

11. The conditionally active polypeptide of a heterodimer of aspect 1, wherein: a) the first heterologous polypeptide is the N-terminal portion of the receptor; and b) the second heterologous polypeptide is the C-terminal portion of the antigen receptor, wherein dimerization of the first and second chimeric polypeptides mediated by the dimerizing agent restores the signaling activity of the receptor.

12. The conditionally active polypeptide of a heterodimer of any one of aspects 1-11, wherein the LBD of the nuclear hormone binding member of the dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

13. The heterodimeric, conditionally active polypeptide of any one of aspects 1-12, wherein the co-modulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

14. The conditionally active polypeptide of a heterodimer of any one of aspects 1-12, wherein the co-regulator of a nuclear hormone receptor is selected from:

15. a heterodimeric, conditionally active receptor comprising: a) a first chimeric polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second chimeric polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain; or comprises: a) a first chimeric polypeptide comprising: i) a first member of a specific binding pair; ii) a regulatory domain; iii) a first member of a dimerization pair; iv) a transmembrane domain interposed between the first member of the specific binding pair and the regulatory domain; and b) a second chimeric polypeptide comprising: i) a second member of a dimerization pair; and ii) an intracellular signaling domain, wherein a first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and a second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein a first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and a second member of the dimerization pair comprises a LBD of the nuclear hormone receptor, wherein the first and second chimeric polypeptides dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

16. The conditionally active receptor of a heterodimer of aspect 15, wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain.

17. The conditionally active receptor of a heterodimer of aspect 15, wherein the first member of the specific binding pair is an antibody or antibody fragment, a ligand, a receptor, or a non-antibody based recognition scaffold.

18. The conditionally active receptor of a heterodimer of aspect 17, wherein said hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD 8.

19. The conditionally active receptor of a heterodimer of aspect 15, wherein said first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD 28.

20. The conditionally active receptor of a heterodimer of aspect 15, wherein said intracellular signaling domain is selected from ZAP70 and CD 3-zeta.

21. The conditionally active receptor of heterodimer of aspect 15, wherein said intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

22. The conditionally active receptor of a heterodimer of any one of aspects 15-21, wherein the LBD of the nuclear hormone binding member of the dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

23. The heterodimeric, conditionally active receptor of any of aspects 15-22, wherein the co-modulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

24. The conditionally active receptor of a heterodimer according to any one of aspects 15-22, wherein the co-modulator of the nuclear hormone receptor is selected from:

25. the conditionally active receptor of the heterodimer of aspect 18, wherein: i) the first regulatory domain and the second regulatory domain are derived from 4-1 BB; ii) the first member and the second member of the dimerization pair are PPAR γ and SRC 3; and ii) the signaling domain comprises ITAM.

26. The conditionally active receptor of a heterodimer of aspect 15, wherein the first member of the specific binding pair is a single chain Fv.

27. The conditionally active receptor of a heterodimer of aspect 15, wherein the first member of the specific binding pair binds to an epitope present on a cell, a solid surface, or a lipid bilayer.

28. The conditionally active receptor for a heterodimer of aspect 27, wherein said cell is a cancer cell.

29. The conditionally active receptor of a heterodimer of aspect 15, wherein said intracellular signaling domain is an intracellular inhibitory domain.

30. The conditionally active receptor of a heterodimer of aspect 29, wherein said intracellular inhibitory domain is derived from a protein selected from the group consisting of: PD-1, CTLA4, HPK1, SHP1, SHP2, Sts1 and Csk.

31. A heterodimeric, conditionally blocked synthetic Immune Cell Receptor (ICR), comprising: a synthetic stimulatory ICR comprising a first member of a dimerization pair linked to the synthetic stimulatory ICR; and a synthetic ICR repressor factor comprising a second member of a dimerization pair linked to an intracellular inhibitory domain, wherein a first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and a second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein the first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and the second member of the dimerization pair comprises an LBD of the nuclear hormone receptor, wherein the synthetic stimulatory ICR and the synthetic ICR repressor dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

32. The conditionally repressed synthetic ICR of aspect 31, wherein the synthetically stimulatory ICR comprises an intracellular co-stimulatory domain.

33. The conditionally blocked synthetic ICR of aspect 32, wherein the intracellular co-stimulatory domain is selected from the group consisting of: 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR and HVEM.

34. The conditionally-repressed synthetic ICR of any of aspects 31-33, wherein a first member of the dimerization pair is intracellularly linked to a synthetic stimulatory ICR and a second member of the dimerization pair is intracellularly linked to an intracellular inhibitory domain.

35. The conditionally blocked synthetic ICR of any of aspects 31-34, wherein the synthetic ICR repressor further comprises a transmembrane domain.

36. The conditionally-repressed synthetic ICR of aspect 35, wherein the second member of the dimerization pair is intracellularly linked to a transmembrane domain.

37. The conditionally-repressed synthetic ICR of aspect 35, wherein the second member of the dimerization pair is extracellular and linked to an intracellular inhibitory domain through a transmembrane domain.

38. The conditionally blocked synthetic ICR of any of aspects 31-37, wherein the stimulatory ICR binds to a soluble antigen.

39. The conditionally blocked synthetic ICR of any of aspects 31-38, wherein the stimulatory ICR binds to a cell surface antigen.

40. The conditionally-repressed synthetic ICR of any of aspects 31-39, wherein the intracellular inhibitory domain is an inhibitory domain derived from a protein selected from the group consisting of: PD-1, CTLA4, HPK1, SHP1, SHP2, Sts1 and Csk.

41. The conditionally blocked synthetic ICR of any of aspects 31-40, wherein the synthetic stimulatory ICR comprises an intracellular signaling domain selected from the group consisting of: a CD 3-zeta signaling domain, a ZAP70 signaling domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

42. The conditionally-repressed synthetic ICR of any one of aspects 31-41, wherein the LBD of the nuclear hormone binding member of the dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of an estrogen receptor, an ecdysone receptor, a PPAR γ receptor, a glucocorticoid receptor, an androgen receptor, a thyroid hormone receptor, a mineralocorticoid receptor, a progesterone receptor, a vitamin D receptor, a PPAR β receptor, a PPAR α receptor, a pregnane X receptor, a liver X receptor, a farnesoid X receptor, a RAR-related orphan receptor, and a retinoic acid receptor.

43. The conditionally blocked synthetic ICR of any one of aspects 41-42, wherein the co-regulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

44. The conditionally blocked synthetic ICR of any one of aspects 41-42, wherein the co-regulator of the nuclear hormone receptor is selected from the group consisting of:

45. the conditionally blocked synthetic ICR of any of aspects 31-44, wherein the synthetic stimulatory ICR is a synthetic Chimeric Antigen Receptor (CAR) or a portion thereof.

46. The conditionally blocked synthetic ICR of any of aspects 31-45, wherein the synthetic stimulatory ICR is a synthetic T Cell Receptor (TCR) or a portion thereof.

47. A heterodimeric, conditionally-repressed, synthetic Chimeric Antigen Receptor (CAR), comprising: a) a synthetic stimulatory CAR comprising: i) an extracellular recognition domain; ii) a transmembrane domain linked to an extracellular recognition domain; iii) a first member of a dimerization pair linked to a transmembrane domain; and iv) an intracellular stimulation domain; and b) a synthetic CAR repressor comprising: i) a second member of a dimerization pair; and ii) an intracellular inhibitory domain linked to a second member of a dimerization pair, wherein the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein the first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and the second member of the dimerization pair comprises a LBD of the nuclear hormone receptor; and wherein the synthetic stimulatory CAR and the synthetic CAR repressor dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

48. The heterodimeric, conditionally-repressed synthetic CAR of aspect 47, wherein the synthetic CAR repressor further comprises a transmembrane domain linked to the second member of the dimerization pair, the intracellular inhibitory domain, or both.

49. A conditionally-repressed synthetic T-cell receptor (TCR) of a heterodimer comprising a) a synthetic stimulatory TCR comprising i) a transmembrane domain, ii) a first member of a dimerization pair linked to a transmembrane domain, iii) an engineered TCR polypeptide comprising at least one TCR α or β chain, wherein the at least one TCR α or β chain is linked to the transmembrane domain or a first member of the dimerization pair, and b) a synthetic TCR repressor comprising i) a second member of the dimerization pair, and ii) an intracellular inhibitory domain linked to a second member of the dimerization pair, wherein the first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and the second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein the first member of the dimerization pair is a co-regulator of the nuclear hormone receptor and the second member of the dimerization pair comprises a LBD of the nuclear hormone receptor, and wherein the synthetic stimulatory TCR and the synthetic TCR induce binding of the co-regulator in the presence of the dimerization factor.

50. The conditionally-repressed synthetic TCR of aspect 49, wherein the synthetic TCR repressor further comprises a transmembrane domain linked to a second member of a dimerization pair, an intracellular inhibitory domain, or both.

51. The conditionally blocked synthetic TCR of aspect 49 or 50, wherein the engineered TCR polypeptide further comprises a TCR γ chain.

52. A heterodimeric, conditionally active Chimeric Antigen Receptor (CAR), comprising: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a first regulatory domain; iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and b) a second polypeptide comprising: i) a transmembrane domain; ii) a second regulatory domain; iii) a second member of a dimerization pair; and iv) an intracellular signaling domain; or comprises: a) a first polypeptide comprising: i) a first member of a specific binding pair; ii) a regulatory domain; iii) a first member of a dimerization pair; iv) a transmembrane domain interposed between the first member of the specific binding pair and the regulatory domain; and b) a second polypeptide comprising: i) a second member of a dimerization pair; and ii) an intracellular signaling domain, wherein a first member of the dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and a second member of the dimerization pair comprises a co-regulator of the nuclear hormone receptor, or wherein a first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and a second member of the dimerization pair comprises a LBD of the nuclear hormone receptor, wherein the first polypeptide and the second polypeptide dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

53. The heterodimeric, conditionally active CAR of aspect 52, wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain.

54. The heterodimeric, conditionally active CAR of aspect 52, wherein the first member of the specific binding pair is an antibody or antibody fragment, ligand, or receptor.

55. The heterodimeric, conditionally active CAR of aspect 53, wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD 8.

56. The heterodimeric, conditionally active CAR of aspect 52, wherein the first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD 28.

57. The heterodimeric, conditionally active CAR of aspect 52, wherein said intracellular signaling domain is selected from ZAP70 and CD 3-zeta.

58. The heterodimeric, conditionally active CAR of aspect 52, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

59. The heterodimeric, conditionally active CAR of any of aspects 52-58, wherein the LBD of the nuclear hormone binding member of the dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

60. The heterodimeric, conditionally active CAR of any of aspects 52-59, wherein the co-regulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

61. The heterodimeric, conditionally active CAR of any of aspects 52-59, wherein the co-regulator of the nuclear hormone receptor is selected from:

63. the heterodimeric, conditionally active CAR of any of aspects 52-62, wherein the first member of the specific binding pair is a single chain Fv.

64. The heterodimeric, conditionally active CAR of any of aspects 52-63, wherein the first member of the specific binding pair binds to an epitope present on a cell, a solid surface, or a lipid bilayer.

65. The heterodimeric, conditionally active CAR of aspect 64, wherein the cell is a cancer cell.

66. A mammalian cell genetically modified to produce a conditionally active polypeptide or receptor of the heterodimer of any of aspects 1-65.

67. The cell of aspect 66, wherein the cell is a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell.

68. The cell of aspect 66, wherein the cell is a T lymphocyte or an NK cell.

69. A nucleic acid comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of the heterodimer of any of aspects 1-65.

70. The nucleic acid of aspect 69, wherein the nucleotide sequence is operably linked to a promoter.

71. The nucleic acid of aspect 69, wherein the promoter is an inducible promoter.

72. The nucleic acid of aspect 69, wherein the promoter is a cell type-specific promoter or a tissue-specific promoter.

73. The nucleic acid of aspect 72, wherein the promoter is a T lymphocyte-specific promoter or an NK cell-specific promoter.

74. The nucleic acid of any one of aspects 69-73, wherein the nucleic acid is in vitro transcribed RNA.

75. A recombinant expression vector comprising the nucleic acid of any one of aspects 69-74.

76. A method of modulating eukaryotic cell activity, the method comprising: a) expressing a conditionally active polypeptide or receptor of the heterodimer of any one of aspects 1-65 in a eukaryotic cell; and b) contacting the cell with the ligand.

77. A method of modulating T lymphocyte activity, said method comprising contacting a T lymphocyte with a dimerizing agent and a second member of a specific binding pair, wherein said T lymphocyte is genetically modified to produce a conditionally active receptor for a heterodimer of any of aspects 15-65, and wherein in the presence of the dimerizing agent and the second member of the specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates the activity of the T lymphocyte, thereby producing a modulated T lymphocyte.

78. The method of aspect 77, wherein the second member of the specific binding pair is an antigen.

79. The method of aspect 77, wherein the contacting occurs in vivo.

80. The method of aspect 77, wherein said T lymphocytes are activated, thereby producing activated T lymphocytes.

81. The method of aspect 80, wherein the activated T lymphocytes mediate killing of target cells.

82. The method of aspect 80, wherein the activated T lymphocytes produce IL-2 and/or IFN- γ.

83. The method of aspect 81, wherein the target cell is a cancer cell.

84. A method of making the cell of any one of aspects 66-68, the method comprising genetically modifying a mammalian cell with an expression vector comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of a heterodimer of any one of aspects 1-65, or genetically modifying a mammalian cell with an RNA comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of a heterodimer of any one of aspects 1-65.

85. The method of aspect 84, wherein the genetic modification is performed ex vivo.

86. The method of aspect 84, wherein the cell is a T lymphocyte, a stem cell, an NK cell, a progenitor cell, a cell derived from a stem cell, or a cell derived from a progenitor cell.

87. A method of treating cancer in an individual, the method comprising: i) genetically modifying a T lymphocyte obtained from an individual with an expression vector comprising a nucleotide sequence encoding a heterodimeric, conditionally active Chimeric Antigen Receptor (CAR) of any of aspects 49-65, wherein the antigen-binding domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell of the individual, and wherein the genetic modification is performed ex vivo; ii) introducing genetically modified T lymphocytes into the individual; and iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of conditionally active receptors of heterodimers, wherein the dimerization provides activation of genetically modified T lymphocytes and killing of cancer cells, thereby treating the cancer.

88. The method of aspect 87, wherein the dimerizing agent is a nuclear hormone that binds to LBD of a nuclear hormone receptor and a co-regulator.

89. A method of modulating host cell activity, the method comprising contacting a host cell with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a conditionally active receptor for a heterodimer of any of aspects 49-65, and wherein in the presence of the dimerizing agent and the second member of a specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates at least one activity of the host cell.

90. The method of aspect 89, wherein the activity is proliferation, cell survival, apoptosis, gene expression, or immune activation.

91. The method of aspect 89, wherein the second member of the specific binding pair is an antigen.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pairs; kb, kilobases; pl, picoliter; s or sec, seconds; min, min; h or hr, hours; aa, an amino acid; kb, kilobases; bp, base pair; nt, nucleotide; i.m., intramuscular (intramyogenic); i.p., intraperitoneal (intraperitoneally); s.c., subcutaneous (s.c.); and the like.

Materials and methods

The following materials and methods were used for the experiments described in examples 1 and 2.

Construction of PPAR γ -based CAR with turn-on switch

Sequences encoding anti-human CD19scFv were cloned into an expression vector. The human 4-1BB costimulatory and CD3 ζ ITAM signaling chain was cloned from cDNA supplied by Open Biosystems. PPAR γ LBD and SRC3 coactivated peptide coding sequences were custom synthesized from IDT DNA. Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, homology-based DNA recombination, etc.) were applied to generate lentiviral expression plasmids for each CAR molecule that opened the switch.

Effector and target cell culture conditions

Jurkat cell line expressing green fluorescent protein after NFAT activation was used. The cell lines were maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin. K562 target cells from u.penn (CD19+/-) were cultured in IMDM supplemented with 10% FBS.

Engineering effector cells with lentiviruses

Pantropic VSV-G pseudotyped lentiviruses were produced by Lenti-X293T cells (Clontech Laboratories #632180) co-transfected with pHR' SIN: CSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies # 15338). Infection medium supernatants were collected 48 hours post transfection and used directly for transduction.

Jurkat cells were split 1-2 days prior to transduction to ensure that the cell culture was in log phase at the time of transduction. The transduced cells were cultured for at least 7 days and were sorted for abundant CAR expression as needed before performing the experiment. The expression level of the CAR molecule with the switch open was quantified by flow cytometry. Part 1 of the CAR (featuring scFv) with the switch open was stained with a fluorophore-conjugated anti-myc antibody that recognized a myc epitope upstream of the anti-CD 19 scFv. Part 2 of the CAR with the switch open (characterized by CD3 ζ ITAM) was quantified using the fluorescence of mCherry located downstream of ITAM.

Quantification of IL-2 production by Jurkat cells with switch open

Jurkat T cells expressing the CAR with the switch open were co-cultured with CD19+/-K562 target cells at a 1:2 effector to target ratio. Rosiglitazone (Sigma-Aldrich # R2408) was dissolved in DMSO and added to the sample to a final concentration of 10. mu.M. Samples without dimerizing agent contained the corresponding vehicle controls (1:3000 diluted dimethyl sulfoxide (DMSO) or 1:1000 diluted ethanol). After 18 hours of incubation, the media supernatants were collected and analyzed with the BD OptEIA human IL-2ELISA kit (BD Biosciences # 555190).

Construction of CAR based on opening switch of hormone receptor

The sequence encoding anti-human CD19scFv was cloned. The human 4-1BB costimulatory and CD3 ζ ITAM signaling chain was cloned from cDNA supplied by Open Biosystems. Estrogen receptor LBD and coactivating peptide coding sequences were custom synthesized by IDT DNA. The sequence of the coactivating peptide was obtained from Heldring et al J Biol chem.2007, 4.6; 282(14) 10449-55, PMID 17283072. Standard molecular cloning techniques (PCR, restriction digestion, ligation, homology-based DNA recombination, etc.) were applied to generate lentiviral expression plasmids for each CAR molecule that opened the switch.

Quantification of CD-69 expression by open-switch Jurkat cells

Jurkat cell line expressing green fluorescent protein after NFAT activation was used. The cell lines were maintained in RPMI-1640 medium supplemented with 10% FBS and glutamate. K562 target cells (CD19+/-) were cultured in DMEM supplemented with 10% FBS. Jurkat cells infected with lentivirus with CAR construct were co-cultured with K562 target cells expressing either the target antigen (CD19) or an unrelated antigen (mesothelin). 4-hydroxy tamoxifen (Sigma-Aldrich # H7904) was dissolved in DMSO and added to the samples to a final concentration of 0-10. mu.M. The rapamycin analogue A/C heterodimerization agent (Clontech laboratories #635055) was dissolved in ethanol and added to a final concentration of 500 nM. Cells were cultured for 24 hours, stained for CD69 expression, and analyzed on a flow cytometer.

Engineering effector cells with lentiviruses

Pantropic VSV-G pseudotyped lentiviruses were produced by Lenti-X293T cells (Clontech Laboratories #632180) that were co-transfected with pHR' SIN: CSW transgene expression vector, viral packaging plasmids pCMVdR8.91 and pMD2.G using Lipofectamine LTX (Life Technologies # 15338). Infection medium supernatants were collected 48 hours post transfection and used directly for transduction.

Example 1: PPAR gamma based CAR constructs for opening switches

CAR constructs that generate PPAR γ -based open switches. Figure 12 presents a schematic of the overall structure of a generalized nuclear hormone Ligand Binding Domain (LBD)/coactivator peptide open switch CAR. Figure 13 presents a schematic of the overall structure of the construct. The constructs are listed in table 3. Open-switch constructs with FKBP and FRB domains (bCW197, bCW206, bCW207) were used as positive and negative control CARs.

TABLE 3

Construct ID #	Encoded CAR molecules
		bCW492	Part 1 (antigen binding) Comodulatory peptide with SRC3, short form
bCW493	Portion 1 co-modulates peptide, short form, 3 tandem copies with SRC3
		bCW494	Part 1 co-regulated peptide with SRC3, elongated form
bCW495	Moiety 2 with PPAR γ LBD

The amino acid sequence of the Ligand Binding Domain (LBD) of PPAR γ and the amino acid sequence of the co-regulatory peptide used, included in the CAR construct, are shown below.

The amino acid sequence of the LBD of PPAR γ included in CAR construct bCW495 was as follows:

ESADLRALAKHLYDSYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY(SEQ ID NO:695)。

the amino acid sequence of the human SRC3 co-regulatory peptide (short form (P729 to a750)) in construct bCW492 is as follows: PKKENNALLRYLLDRDDPSDA (SEQ ID NO: 696).

Construct bCW493 contained three tandem copies of the shorter form of the human SRC3 co-regulatory peptide (P729-a750) with a linker/spacer between the copies such that the amino acid sequence of the co-regulatory peptide with linker/spacer in bCW493 is as follows: PKKENNALLRY LLDRDDPSDAGGGSGGGSPKKENNALLRYLLDRDDPSDAGGGSGGGSPKKENNALLRYLLDRDDPSDA (SEQ ID NO:697), wherein the linker/spacer is underlined.

Construct bCW494 contained a long form of the human SRC3 co-modulatory peptide (M673-a 750); bCW494 the amino acid sequence of human SRC3 is: MHGSLLQEKHRILHKLLQNGNSPAEVAKITAEATGKDTSSITSCGDGNVVKQEQLSPKKKENNALLRYLLDRDDPSDA (SEQ ID NO: 698).

The amino acid sequence of the polypeptide chain containing the co-regulatory peptide of the PPAR γ CAR construct p51 (part 1 of the p51 bCW492 myc anti-CD 19 open switch with 1x SRC3 short co-regulatory peptide and 4-1BB) is as follows:

in which the anti-CD 19 sequence is in bold, the CD8 α Transmembrane (TM) domain is double underlined, the 4-1BB sequence is in italic and bold text, and the co-regulatory peptide is underlined (single underlined).

The amino acid sequence of PPAR γ CAR construct p52 (part 1 of the p52 bCW494 myc a cd19 open switch with 1x SRC3 long co-regulatory peptide and 4-1BB) is as follows:in which the anti-CD 19 sequence is in bold, the CD8 α Transmembrane (TM) domain is double underlined, the 4-1BB sequence is in italic and bold text, and the co-regulatory peptide is underlined (single underlined).

The amino acid sequence of PPAR γ CAR construct p55(p 55 bCW493 dimeric myc aCD19 CD8a hinge TM 41BB linker 3x short SRC3 peptide in pHR) is as follows:

The amino acid sequence of the PPAR γ CAR construct P56 (P56 bCW495Kozak dDAP10CD8a TM-41BB-GSx8-PPARg LBD-GSx 4-zeta-GSx 4-mCherry in pHR) is as follows:

LBD underlined (single underlined), CD8 α TM domain underlined, 4-1BB in bold text, and ζ in bold and italics.

Results

Figure 14 depicts the production of IL-2 cytokines by CAR + Jurkat cells (transduced with the lentiviral constructs shown) with the switch open after 18 hours of co-culture with K562 target cells (+/-CD19Ag, as shown) in the presence or absence of rosiglitazone dimerizer (10 micromolar).

Example 2CAR constructs based on the opening switch of ER- α

Constructs of CARs based on the opening switch of ER α figure 15 presents a schematic of the overall structure of the constructs are listed in table 4 constructs with opening switches of FKBP and FRB domains (bCW197, bCW206, bCW207) were used as positive and negative control CARs.

TABLE 4

The amino acid sequences of the various components of the CAR based on the opening switch of ER α are as follows.

a. LBD of human estrogen receptor α in construct bCW 503:

DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTS(SEQ ID NO:703)；

b. LBD of mutated human estrogen receptor α in construct bCW 504:

DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTS(SEQ ID NO:704)；

c. LBD of mutated human estrogen receptor α in construct bCW 505:

DRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMKCKNVVPLFDLLLEMLDAHRLHAPTS(SEQ ID NO:705)；

3x αβ V co-regulatory peptide in bcw501:

SGSGPGSREWFKDMLGGGSGGGSSGSGPGSREWFKDMLGGGSGGGSSGSGPGSREWFKDM(SEQ IDNO:706)；

3x CoRNR co-regulatory peptides in bCBW 502:

DAFQLRQLILRGLQDDGGGSGGGSDAFQLRQLILRGLQDDGGGSGGGSDAFQLRQLILRGLQDD(SEQID NO:707)。

the amino acid sequence of the bCW501 construct (p25bCW501myc aCD19 open switch part 1 with 3x αβ V peptide and 4-1BB) is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSSGSGPGSREWFKDMLGGGSGGGSSGSGPGSREWFKDMLGGGSGGGSSGSGPG SREWFKDMLGS (SEQ ID NO:708), in which the co-regulatory peptide αβ V is underlined.

The amino acid sequence of the bCW502 construct (p26bCW502myc aCD19 open switch part 1 with 3x CoRNR peptide and 4-1BB) is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSDAFQLRQLILRGLQDDGGGSGGGSDAFQLRQLILRGLQDDGGGSGGGSDAFQ LRQLILRGLQDDGS (SEQ ID NO:709) with the 3 x CoRNR co-regulatory peptide underlined.

The amino acid sequence of the bCW503 construct (CAR part 2 with human ER α LBD with p27bCW503 open switch) was as follows:

MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGSGSGSTSDRRGGRMLKHKR QRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGL LTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTL QQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSGSGSGSGSSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD (SEQ ID NO:710), wherein ER LBD is underlined.

The amino acid sequence of the bCW504 construct (CAR part 2 with p28bCW504 open switch and human ER α LBD w Y537F) was as follows:

where ER α LBD is underlined and the Y537F substitution is in bold.

bCW505 the amino acid sequence of the construct (CAR moiety 2 with p29bCW505 open switch and human ER α LBD w 537F G521R) was as follows:

where ER α LBD is underlined and the Y536F and G521R substitutions are in bold.

The amino acid sequence of the bCW506 construct (CAR part 2 with p30bCW506 open switch and human ER α LBD without helix 12) is as follows:

MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGSGSGSTSDRRGGRMLKHKR QRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGL LTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTL QQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVAGSGSGSGSSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD (SEQ ID NO:713), wherein ER LBD (without helix 12) is underlined.

The amino acid sequence of the bCW507 construct (CAR part 2 with p31bCW506 open switch and human ER α LBD without helix 12w G521R) is as follows:

where ER α LBD (without helix 12) is underlined and the G521R substitution is in bold.

The amino acid sequence of the construct termed "p 40 dimeric myc a CD19 CD8a hinge TM 41BB linker 3 x NR0B1 peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSPRQGSILYSMLTSAKQTGGGSGGGSPRQGSILYSMLTSAKQTGGGSGGGSPR QGSILYSMLTSAKQTGS (SEQ ID NO:715), in which the 3 x NR0B1 co-regulatory peptide is underlined.

The amino acid sequence of the construct termed "p 41 dimeric myc a CD19 CD8a hinge TM 41BB linker 3 x NCOA1 peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSLTERHKILHRLLQEGSPSDGGGSGGGSLTERHKILHRLLQEGSPSDGGGSGG GSLTERHKILHRLLQEGSPSDGS (SEQ ID NO:716) in which the 3 x NCOA1 peptide is underlined.

The amino acid sequence of the construct termed "p 42 dimeric myc a CD19 CD8a hinge TM 41BB linker 3 x NCOA2 peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSSKGQTKLLQLLTTKSDQGGGSGGGSSKGQTKLLQLLTTKSDQGGGSGGGSSK GQTKLLQLLTTKSDQGS (SEQ ID NO:717), in which the 3 x NCOA2 peptide is underlined.

The amino acid sequence of the construct termed "p 43 dimeric myc a CD19 CD8a hinge TM 41BB linker 3 x PGC-1 peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSAEEPSLLKKLLLAPANTGGGSGGGSAEEPSLLKKLLLAPANTGGGSGGGSAE EPSLLKKLLLAPANTGS (SEQ ID NO:718), wherein the 3 XPGC-1 peptide is underlined.

The amino acid sequence of the construct termed "p 44 dimeric myc a CD19 CD8a hinge TM 41BB linker 3 x NRIP1 peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSAANNSLLLHLLKSQTIPGGGSGGGSAANNSLLLHLLKSQTIPGGGSGGGSAA NNSLLLHLLKSQTIPGS (SEQ ID NO:719), wherein the 3 x NRIP1 peptide is underlined.

The amino acid sequence designated "p 45Kozak dDAP10 CD8a TM-41BB-GSx8-ERbLBD-GSx 4-zeta-GSx 4-mCherry in pHR" is as follows:

MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGSGSGSTSVKCGSRRERCGY RLVRRQRSADEQLHCAGKAKRSGGHAPRVRELLLDALSPEQLVLTLLEAEPPHVLISRPSAPFTEASMMMSLTKLA DKELVHMISWAKKIPGFVELSLFDQVRLLESCWMEVLMMGLMWRSIDHPGKLIFAPDLVLDRDEGKCVEGILEIFD MLLATTSRFRELKLQHKEYLCVKAMILLNSSMYPLVTATQDADSSRKLAHLLNAVTDALVWVIAKSGISSQQQSMR LANLLMLLSHVRHASNKGMEHLLNMKCKNVVPVYDLLLEMLNAHVLRGCKSSITGSECSPAEDSKSKEGSQNPQSQGSGSGSGSSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD (SEQ ID NO:720), wherein ER β LBD is underlined.

The amino acid sequence of the construct termed "p 179 dimeric myc aCD19 CD8a hinge TM 41BB linker 3 x SRC-1 NRIV peptide in pHR" is as follows:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSQAQQKSLLQQLLTEGGGSGGGSQAQQKSLLQQLLTEGGGSGGGSQAQQKSLL QQLLTEGS (SEQ ID NO:721), in which the 3 xNRIV 1 peptide is underlined

Results

The data are depicted in fig. 16A and 16B. Figure 16a CD69 upregulation by Jurkat cells expressing ER CAR + (bCW502 and bCW505) after 24 hours of co-culture with K562 target cells expressing CD19 or an unrelated antigen mesothelin ("meso") in the presence or absence of a small molecule dimerizer (4-hydroxytamoxifen or rapamycin analog AP 21967). CD69 expression in ER CAR + Jurkat cells was strongly induced by 4-hydroxytamoxifen (EC 50-100 nM) only in the presence of K562 cells expressing CD 19. Rapamycin CARs and dead rapamycin CARs (with mutations in ITAMs that abrogate signaling) were tested as positive and negative controls, respectively. Figure 16b FACS data from the experiment described in figure 16A, showing up-regulation of CD69 in ER CAR + Jurkat cells depending on the manner of CD19 antigen and 4-hydroxytamoxifen treatment.

Example 3: dimerization agent controlled activation of primary human T cells

The control of cellular function by induced heterodimerization of the ligand binding domain of human estrogen receptor β with a small peptide derived from a transcriptional co-repressor in the presence of the drug 4-hydroxytamoxifen was evaluated the present method does not involve DNA binding and/or transcriptional activation or any DNA binding or transcriptional activation function of human estrogen receptor β from which the polypeptide component was derived.

Construction of a CD 19-specific open switch CAR comprising a human estrogen receptor β ligand binding domain and a co-repressor peptide (cockr.) a schematic of the general constructs within the T cell membrane and the associated CD19 antigen expressed on the surface of the target cell is provided in fig. 17 a stable expression was observed after introduction into primary CD8+ T cells and the transduced cells showed increased T cell activation upon addition of the drug 4-hydroxytamoxifen.

Nucleic acid sequences encoding the following constructs were used: part 1 of the "p 26 bCW502 myc aCD19 open switch with 3 x CoRNR peptide and 4-1 BB" having the following sequence:

MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRGSGSGSTSDAFQLRQLILRGLQDDGGGSGGGSDAFQLRQLILRGLQDDGGGSGGGSDAFQ LRQLILRGLQDDGS (SEQ ID NO:722), in which the 3 x CoRNR co-regulatory peptide is underlined, and "p 45Kozak dDAP10 CD8a TM-41BB-GSx8-ERb LBD-GSx4- ζ -GSx 4-mCherry" in pHR has the following sequence:

MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPIYIWAPLAGTCGVLLLSLVITLYCSLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGSGSGSGSGSGSGSTSVKCGSRRERCGY RLVRRQRSADEQLHCAGKAKRSGGHAPRVRELLLDALSPEQLVLTLLEAEPPHVLISRPSAPFTEASMMMSLTKLA DKELVHMISWAKKIPGFVELSLFDQVRLLESCWMEVLMMGLMWRSIDHPGKLIFAPDLVLDRDEGKCVEGILEIFD MLLATTSRFRELKLQHKEYLCVKAMILLNSSMYPLVTATQDADSSRKLAHLLNAVTDALVWVIAKSGISSQQQSMR LANLLMLLSHVRHASNKGMEHLLNMKCKNVVPVYDLLLEMLNAHVLRGCKSSITGSECSPAEDSKSKEGSQNPQSQGSGSGSGSSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRGSGSGSGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKCVTD (SEQ ID NO:723), wherein ER β LBD is underlined.

Specifically, the up-regulation of CD69 of primary human CD8+ T cells constructed as CARs expressing a CD19 specific open switch including a human estrogen receptor β ligand binding domain (ER- β) and a co-repressor peptide (cockr) was measured after 24 hours of co-culture with CD19 or an unrelated antigen mesothelin ("meso") in the presence or absence of a small molecule dimerizing agent (4-hydroxytamoxifen or rapamycin analog "rapa". as shown in fig. 18A, activation of primary human CD8+ T cells of a CD19 antigen-induced CAR construct expressing an ER- β/cockr open switch was dependent on the dose of small molecule dimerizing agent provided to the culture fig. 18B provides a histogram from flow cytometry data showing the correlation between increased 4-hydroxytamoxifen dose and increased number of primary human CD8+ T cells positive to CD 69.

The percentage of K562 target cells expressing CD19 remaining after 22 hours of co-culture with primary human CD8+ T cells expressing a CD 19-specific ER- β/crnr on-off CAR (ER-CAR) in the presence of various concentrations of drug (4-hydroxytamoxifen (4HT) or rapamycin analogue) was measured (figure 52) the cells expressing CD 19-specific single-chain second generation CAR ("second generation CAR") and cells expressing CD 19-specific rapamycin analogue on-off switch and administered rapamycin analogue dimerisation agent was used as a positive control negative control including cells expressing ER-CAR T cells in the absence of 4HT (ethanol vehicle only, "ER-EtOH") and cells expressing only part 2 of the primary CD 19-on-off construct (CAR without part 1) demonstrating that these cells express the drug on-off CAR (CAR) induces cytotoxic activity.

Increased T cell activation was also observed in primary CD8+ T cells expressing a split open-switch CAR construct, in which the heterodimerization module consists of a portion of the human vitamin D receptor ligand binding domain and the SRC2-3 coactivating peptide. In the present system, T cells show increased activation in the presence of the vitamin D analogue calcipotriol.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A conditionally active polypeptide of a heterodimer, comprising:

a) a first chimeric polypeptide comprising a first member of a dimerization pair and a first heterologous polypeptide; and

b) a second chimeric polypeptide comprising a second member of a dimerization pair and a second heterologous polypeptide,

wherein a first member of said dimerization pair comprises a Ligand Binding Domain (LBD) of a nuclear hormone receptor and a second member of said dimerization pair comprises a co-regulator of said nuclear hormone receptor, or

Wherein a first member of the dimerization pair is a co-regulator of a nuclear hormone receptor and a second member of the dimerization pair comprises the LBD of the nuclear hormone receptor; and is

Wherein said first chimeric polypeptide and said second chimeric polypeptide dimerize in the presence of a dimerizing agent that induces binding of said LBD to said co-regulator.

2. The heterodimeric, conditionally active polypeptide of claim 1, wherein:

a) the first heterologous polypeptide is a T Cell Receptor (TCR) α chain, and

b) the second heterologous polypeptide is a TCR β chain.

3. The heterodimeric, conditionally active polypeptide of claim 1, wherein:

a) the first heterologous polypeptide is a first polypeptide of a Chimeric Antigen Receptor (CAR) heterodimer; and is

b) The second heterologous polypeptide is a second polypeptide of a CAR heterodimer.

4. The heterodimeric, conditionally active polypeptide of claim 1, wherein:

a) the first heterologous polypeptide is the N-terminal portion of an RNA-guided endonuclease; and is

b) The second heterologous polypeptide is the C-terminal portion of the RNA-guided endonuclease,

wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide mediated by a dimerizing agent that induces binding of the LBD to the co-regulator restores the enzymatic function of the RNA-guided endonuclease.

5. The conditionally active polypeptide of a heterodimer of claim 4, wherein the RNA-guided endonuclease is a class 2 CRISPR/Cas endonuclease.

6. The heterodimeric, conditionally active polypeptide of claim 5, wherein the class 2 CRISPR/Cas endonuclease is a type II CRISPR/Cas protein, a type V CRISPR/Cas protein, or a type VI CRISPR/Cas protein.

7. The heterodimeric, conditionally active polypeptide of claim 1, wherein:

a) the first heterologous polypeptide is an N-terminal portion of an enzyme; and is

b) Said second heterologous polypeptide is the C-terminal part of said enzyme,

wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide restores enzymatic activity of the enzyme.

8. The conditionally active polypeptide of a heterodimer of claim 7, wherein the enzyme is a kinase, a protease, a phosphatase, or a caspase.

9. The conditionally active polypeptide of a heterodimer of claim 1, wherein the first polypeptide and the second polypeptide exhibit activity when in proximity after dimerization mediated by the dimerizing agent, but do not individually exhibit activity.

10. The conditionally active polypeptide of claim 1, wherein

a) The first heterologous polypeptide is the N-terminal portion of an antigen receptor; and is

b) Said second heterologous polypeptide is the C-terminal part of said antigen receptor,

wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide restores the signaling activity of the antigen receptor.

11. The heterodimeric, conditionally active polypeptide of claim 1, wherein:

a) the first heterologous polypeptide is the N-terminal portion of the receptor; and is

wherein dimerization of the first chimeric polypeptide and the second chimeric polypeptide mediated by the dimerizing agent restores signaling activity of the receptor.

12. A conditionally active polypeptide of a heterodimer according to any one of claims 1-11, wherein the LBD of the nuclear hormone binding member of the dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

13. The heterodimeric, conditionally active polypeptide of any of claims 1-12, wherein the co-regulator of nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

14. The conditionally active polypeptide of a heterodimer of any one of claims 1-12, wherein the co-regulator of a nuclear hormone receptor is selected from:

15. a heterodimeric, conditionally active receptor comprising:

a) a first chimeric polypeptide comprising:

i) a first member of a specific binding pair;

ii) a first regulatory domain;

iii) a first member of a dimerization pair; and

iv) a transmembrane domain interposed between the first member of the specific binding pair and the first regulatory domain; and

b) a second chimeric polypeptide comprising:

i) a transmembrane domain;

ii) a second regulatory domain;

iii) a second member of said dimerized pair; and

iv) an intracellular signaling domain;

or comprises:

a) a first chimeric polypeptide comprising:

i) a first member of a specific binding pair;

ii) a regulatory domain;

iii) a first member of a dimerization pair;

iv) a transmembrane domain interposed between the first member of the specific binding pair and the regulatory domain; and

b) a second chimeric polypeptide comprising:

i) a second member of said dimerized pair; and

ii) an intracellular signaling domain.

Wherein a first member of said dimerization pair is a co-regulator of a nuclear hormone receptor and a second member of said dimerization pair comprises the LBD of said nuclear hormone receptor,

16. The conditionally active receptor of a heterodimer of claim 15, wherein said first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain.

17. The conditionally active receptor of a heterodimer of claim 15, wherein the first member of the specific binding pair is an antibody or antibody fragment, a ligand, a receptor, or a non-antibody based recognition scaffold.

18. The conditionally active receptor of heterodimer of claim 17, wherein said hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD 8.

19. The conditionally active receptor of a heterodimer of claim 15, wherein said first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD 28.

20. The conditionally active receptor of a heterodimer of claim 15, wherein said intracellular signaling domain is selected from ZAP70 and CD3- ζ.

21. The conditionally active receptor of heterodimer of claim 15, wherein said intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

22. The conditionally active receptor of a heterodimer of any one of claims 15-21, wherein the LBD of the nuclear hormone binding member of the dimerization pair is the LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

23. The heterodimeric, conditionally active receptor of any of claims 15-22, wherein the coregulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

24. The heterodimeric, conditionally active receptor of any of claims 15-22, wherein the co-modulator of the nuclear hormone receptor is selected from:

25. the conditionally active multi-receptor of heterodimer of claim 18, wherein:

i) the first regulatory domain and the second regulatory domain are derived from 4-1 BB;

ii) the first member and the second member of the dimerization pair are PPAR γ and SRC 3; and is

ii) the signaling domain comprises ITAM.

26. The conditionally active receptor of claim 15, wherein the first member of the specific binding pair is a single chain Fv.

27. The conditionally active receptor of a heterodimer of claim 15, wherein the first member of the specific binding pair binds to an epitope present on a cell, a solid surface, or a lipid bilayer.

28. The conditionally active receptor for a heterodimer of claim 27, wherein said cell is a cancer cell.

29. The conditionally active receptor of a heterodimer of claim 15, wherein said intracellular signaling domain is an intracellular inhibitory domain.

30. The conditionally active receptor of a heterodimer of claim 29, wherein said intracellular inhibitory domain is derived from a protein selected from the group consisting of: PD-1, CTLA4, HPK1, SHP1, SHP2, Sts1 and Csk.

31. A heterodimeric, conditionally blocked synthetic Immune Cell Receptor (ICR), comprising:

a synthetic stimulatory ICR comprising a first member of a dimerization pair linked to the synthetic stimulatory ICR; and

a synthetic ICR repressor factor comprising a second member of the dimerization pair linked to an intracellular inhibitory domain,

wherein said synthetic stimulatory ICR and said synthetic ICR repressor dimerize in the presence of a dimerizing agent that induces binding of said LBD to said co-regulator.

32. The conditionally repressed synthetic ICR of claim 31, wherein said synthetically stimulatory ICR comprises an intracellular co-stimulatory domain.

33. The conditionally blocked synthetic ICR of claim 32, wherein said intracellular co-stimulatory domain is selected from the group consisting of: 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR and HVEM.

34. The conditionally-repressed synthetic ICR of any one of claims 31-33, wherein a first member of said dimerization pair is intracellularly linked to said synthetic stimulatory ICR and a second member of said dimerization pair is intracellularly linked to said intracellular inhibitory domain.

35. The conditionally blocked synthetic ICR of any one of claims 31-34, wherein the synthetic ICR repressor further comprises a transmembrane domain.

36. The conditionally-repressed synthetic ICR of claim 35, wherein a second member of the dimerization pair is intracellularly linked to the transmembrane domain.

37. The conditionally-repressed synthetic ICR of claim 35, wherein a second member of the dimerization pair is extracellular and linked to the intracellular inhibitory domain through the transmembrane domain.

38. The conditionally blocked synthetic ICR of any one of claims 31-37, wherein the stimulatory ICR binds to a soluble antigen.

39. The conditionally blocked synthetic ICR of any one of claims 31-38, wherein the stimulatory ICR binds to a cell surface antigen.

40. The conditionally blocked synthetic ICR of any one of claims 31-39, wherein the intracellular inhibitory domain is an inhibitory domain derived from a protein selected from the group consisting of: PD-1, CTLA4, HPK1, SHP1, SHP2, Sts1 and Csk.

41. The conditionally blocked synthetic ICR of any one of claims 31-40, wherein the synthetic stimulatory ICR comprises an intracellular signaling domain selected from the group consisting of: a CD 3-zeta signaling domain, a ZAP70 signaling domain, and an immunoreceptor tyrosine-based activation motif (ITAM).

42. The conditionally-repressed synthetic ICR of any one of claims 31-41, wherein the LBD of the nuclear hormone binding member of said dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of an estrogen receptor, an ecdysone receptor, a PPAR γ receptor, a glucocorticoid receptor, an androgen receptor, a thyroid hormone receptor, a mineralocorticoid receptor, a progesterone receptor, a vitamin D receptor, a PPAR β receptor, a PPAR α receptor, a pregnane X receptor, a liver X receptor, a farnesoid X receptor, a RAR-related orphan receptor, and a retinoic acid receptor.

43. The conditionally blocked synthetic ICR of any one of claims 41-42, wherein the co-regulator of the nuclear hormone receptor is selected from the group consisting of SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

44. The conditionally blocked synthetic ICR of any one of claims 41-42, wherein the co-regulator of the nuclear hormone receptor is selected from the group consisting of:

45. the conditionally blocked synthetic ICR of any one of claims 31-44, wherein the synthetic stimulatory ICR is a synthetic Chimeric Antigen Receptor (CAR) or a portion thereof.

46. The conditionally blocked synthetic ICR of any one of claims 31-45, wherein the synthetic stimulatory ICR is a synthetic T Cell Receptor (TCR) or portion thereof.

47. A heterodimeric, conditionally-repressed, synthetic Chimeric Antigen Receptor (CAR), comprising:

a) a synthetic stimulatory CAR comprising:

i) an extracellular recognition domain;

ii) a transmembrane domain linked to the extracellular recognition domain;

iii) a first member of a dimerization pair linked to the transmembrane domain; and

iv) an intracellular stimulation domain; and

b) a synthetic CAR repressor comprising:

i) a second member of said dimerized pair; and

ii) an intracellular inhibitory domain attached to the second member of the dimerization pair,

Wherein the synthetic stimulatory CAR and the synthetic CAR repressor factor dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

48. The heterodimeric, conditionally-repressed synthetic CAR of claim 47, wherein the synthetic CAR repressor further comprises a transmembrane domain linked to the second member of the dimerization pair, the intracellular inhibitory domain, or both.

49. A heterodimerically conditionally blocked synthetic T Cell Receptor (TCR) comprising:

a) a synthetic stimulatory TCR comprising:

i) a transmembrane domain;

ii) a first member of a dimerization pair linked to the transmembrane domain;

iii) an engineered TCR polypeptide comprising at least one TCR α or β chain, wherein the at least one TCR α or β chain is linked to the transmembrane domain or the first member of the dimerization pair, and

b) a synthetic TCR repressor, comprising:

i) a second member of said dimerized pair; and

Wherein the synthetic stimulatory TCR and the synthetic TCR repressor dimerize in the presence of a dimerizing agent that induces binding of the LBD to the co-regulator.

50. The conditionally-repressed synthetic TCR of claim 49, wherein the synthetic TCR repressor further comprises a transmembrane domain linked to the second member of the dimerization pair, the intracellular inhibitory domain, or both.

51. The conditionally blocked synthetic TCR of claim 49 or 50, wherein the engineered TCR polypeptide further comprises a TCR γ chain.

52. A heterodimeric, conditionally active Chimeric Antigen Receptor (CAR), comprising:

a) a first polypeptide comprising:

i) a first member of a specific binding pair;

ii) a first regulatory domain;

iii) a first member of a dimerization pair; and

b) a second polypeptide comprising:

i) a transmembrane domain;

ii) a second regulatory domain;

iii) a second member of said dimerized pair; and

iv) an intracellular signaling domain;

or comprises:

a) a first polypeptide comprising:

i) a first member of a specific binding pair;

ii) a regulatory domain;

iii) a first member of a dimerization pair;

b) a second polypeptide comprising:

i) a second member of said dimerized pair; and

ii) an intracellular signaling domain,

Wherein said first polypeptide and said second polypeptide dimerize in the presence of a dimerizing agent that induces binding of said LBD to said co-regulator.

53. The heterodimeric, conditionally active CAR of claim 52, wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain.

54. The heterodimeric, conditionally active CAR of claim 52, wherein the first member of the specific binding pair is an antibody or antibody fragment, ligand, or receptor.

55. The heterodimeric, conditionally active CAR of claim 53, wherein said hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD 8.

56. The heterodimeric, conditionally active CAR of claim 52, wherein the first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD 28.

57. The heterodimeric, conditionally active CAR of claim 52, wherein said intracellular signaling domain is selected from ZAP70 and CD3- ζ.

58. The heterodimeric, conditionally active CAR of claim 52, wherein said intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

59. The conditionally active CAR of a heterodimer of any one of claims 52-58, wherein the LBD of the nuclear hormone binding member of said dimerization pair is an LBD of a nuclear hormone receptor selected from the group consisting of estrogen receptor, ecdysone receptor, PPAR γ receptor, glucocorticoid receptor, androgen receptor, thyroid hormone receptor, mineralocorticoid receptor, progesterone receptor, vitamin D receptor, PPAR β receptor, PPAR α receptor, pregnane X receptor, liver X receptor, farnesoid X receptor, RAR-related orphan receptor, and retinoic acid receptor.

60. The heterodimeric, conditionally active CAR of any of claims 52-59, wherein the co-regulator of the nuclear hormone receptor is selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, CBP, P300, CIA, ARA70, TIF1, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, and PRIC 285.

61. The heterodimeric, conditionally active CAR of any of claims 52-59, wherein said coregulator of the nuclear hormone receptor is selected from:

62. the heterodimeric, conditionally active CAR of any of claims 52-62, wherein the first member of said specific binding pair is a single chain Fv.

63. The heterodimeric, conditionally active CAR of any of claims 52-63, wherein the first member of the specific binding pair binds to an epitope present on a cell, a solid surface, or a lipid bilayer.

64. The heterodimeric, conditionally active CAR of claim 64, wherein said cell is a cancer cell.

65. A mammalian cell genetically modified to produce a conditionally active polypeptide or receptor of the heterodimer of any one of claims 1-65.

66. The cell of claim 66, wherein the cell is a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell.

67. The cell of claim 66, wherein the cell is a T lymphocyte or an NK cell.

68. A nucleic acid comprising a nucleotide sequence encoding a conditionally active receptor or polypeptide of the heterodimer of any one of claims 1-65.

69. The nucleic acid of claim 69, wherein the nucleotide sequence is operably linked to a promoter.

70. The nucleic acid of claim 69, wherein the promoter is an inducible promoter.

71. The nucleic acid of claim 69, wherein the promoter is a cell type-specific promoter or a tissue-specific promoter.

72. The nucleic acid of claim 72, wherein the promoter is a T lymphocyte-specific promoter or an NK cell-specific promoter.

73. The nucleic acid of any one of claims 69-73, wherein the nucleic acid is in vitro transcribed RNA.

74. A recombinant expression vector comprising the nucleic acid of any one of claims 69-74.

75. A method of modulating eukaryotic cell activity, the method comprising:

a) expressing in the eukaryotic cell the conditionally active polypeptide or receptor of the heterodimer of any one of claims 1-65; and

b) contacting the cell with the ligand.

76. A method of modulating the activity of a T lymphocyte, comprising contacting the T lymphocyte with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a conditionally active receptor for a heterodimer of any of claims 15-65, and wherein in the presence of the dimerizing agent and the second member of the specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates the activity of the T lymphocyte, thereby producing a modulated T lymphocyte.

77. The method of claim 77, wherein the second member of the specific binding pair is an antigen.

78. The method of claim 77, wherein the contacting occurs in vivo.

79. The method of claim 77, wherein the T lymphocytes are activated, thereby producing activated T lymphocytes.

80. The method of claim 80, wherein the activated T lymphocytes mediate killing of target cells.

81. The method of claim 80, wherein the activated T lymphocytes produce IL-2 and/or IFN- γ.

82. The method of claim 81, wherein the target cell is a cancer cell.

83. A method of making the cell of any one of claims 66-68, comprising genetically modifying a mammalian cell with an expression vector comprising a nucleotide sequence encoding the conditionally active receptor or polypeptide of the heterodimer of any one of claims 1-65, or genetically modifying a mammalian cell with RNA comprising a nucleotide sequence encoding the conditionally active receptor or polypeptide of the heterodimer of any one of claims 1-65.

84. The method of claim 84, wherein said genetic modification is performed ex vivo.

85. The method of claim 84, wherein the cell is a T lymphocyte, a stem cell, an NK cell, a progenitor cell, a cell derived from a stem cell, or a cell derived from a progenitor cell.

86. A method of treating cancer in an individual, the method comprising:

i) genetically modifying T lymphocytes obtained from the individual with an expression vector comprising a nucleotide sequence encoding a heterodimeric, conditionally active Chimeric Antigen Receptor (CAR) of any of claims 49-65, wherein the antigen-binding domain of the heterodimeric, conditionally active CAR is specific for an epitope on a cancer cell in the individual, and wherein the genetic modification is performed ex vivo;

ii) introducing the genetically modified T lymphocyte into the individual; and

iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of conditionally active receptors of the heterodimers, wherein the dimerization provides for activation of the genetically modified T lymphocytes and killing of the cancer cells, thereby treating the cancer.

87. The method of claim 87, wherein said dimerizing agent is a nuclear hormone that binds to the LBD of said nuclear hormone receptor and said co-regulator.

88. A method of modulating the activity of a host cell, the method comprising contacting the host cell with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a conditionally active receptor for the heterodimer of any one of claims 49-65, and wherein in the presence of the dimerizing agent and the second member of the specific binding pair, the conditionally active receptor for the heterodimer dimerizes and modulates at least one activity of the host cell.

89. The method of claim 89, wherein the activity is proliferation, cell survival, apoptosis, gene expression, or immune activation.

90. The method of claim 89, wherein the second member of the specific binding pair is an antigen.