Group of chimeric antigen receptors (CARS)

Abstract

To provide novel strategy to improve the specificity of CAR modified cells in recognition of desired target cells.SOLUTION: A group of chimeric antigen receptors (CARs) comprising two, three or four CAR molecules are disclosed, where each member of the group of CARs is different in its amino acid sequence from one another, and where each of the CAR molecules of the group includes at least a transmembrane domain and an ectodomain, where the ectodomain has one or two antigen binding moieties and / or one or two binding sites to which other polypeptides each including at least an antigen binding moiety are able to bind; where the ectodomain of each CAR molecule of the group in its prevalent conformation is free of cysteine amino acid moieties which are able to form intermolecular disulfide bonds with other CAR molecules of the group, respectively, and where each CAR molecule of the group includes at least one heterodimerization domain.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a family of chimeric antigen receptors (CARs) consisting of two, three or four CAR molecules. [Background technology]

[0002] Immunotherapy using CAR T cells, i.e. T cells modified to express a chimeric antigen receptor (CAR), is one of the most promising approaches in cancer treatment. To date, the high potential of this therapeutic strategy has been proven by excellent clinical responses in patients with B-cell malignancies. However, the further application of this success to other tumors is currently hindered by several hurdles (Lim and June, Cell. 2017;168(4):724; Labanieh et al., Nat Biomed Eng. 2018;2:377-391). For example, current CAR T cells are typically directed against a single defined tumor-associated antigen by the CAR. As tumor-associated antigens are always expressed also on healthy cells, this fact results in the so-called on-target / off-tumor toxicity, i.e. the destruction of healthy tissues expressing this antigen. Existing strategies to improve the tumor specificity of CAR-modified cells are based on the co-expression of a chimeric co-inhibitory or co-stimulatory receptor against a second antigen, or the transcriptional regulation of CAR expression by a co-expressed chimeric Notch-based receptor against a second antigen (Roybal and Lim, Annu Rev Immunol. 2017;35:229; Labanieh et al., Nat Biomed Eng. 2018; 2:377-391).

[0003] WO 2017 / 180993A1 discloses a salve-dimeric chimeric antigen receptor system, Lanitis et al. (Cancer Immunol. Res. 1 (2013), 43-53) reported that chimeric antigen receptor T cells with dissociated signaling domains exhibit focused antitumor activity with low potential for toxicity in vivo. Kloss et al. (Nat. biotechnol. 31 (2012), 71-75) report that combinatorial antigen recognition accompanied by balanced signaling promotes selective tumor eradication by engineered T cells.

[0004] WO 2015 / 075468A1 discloses a CAR system comprising a CAR that includes an activation internal domain, Wu et al. (Science 350 (2015), 293 and aab4077-1 to aab4077-10) describe remote control of therapeutic T cells via small molecule gated chimeric receptors. Ajina et al. (Mol. cancer therap. 17 (2018), 1795-1815) outline strategies to address CAR-enhanced signaling. Summary of the Invention

[0005] The object of the present invention is to provide a new strategy for improving the specificity of CAR modified cells in recognizing desired target cells. This improved specificity for target cells is achieved by specific recognition of a combination of target antigens, i.e., combinatorial target antigen recognition. More specifically, the new CAR should be applicable in vivo, especially for the treatment of human patients, without the risk of side effects or at least with reduced side effects. A further object is to provide a means for tumor therapy, especially the concept of immunotherapy for tumor therapy.

[0006] Summary of the Invention The present invention provides a system for combinatorial target antigen recognition based on a group of chimeric antigen receptor (CAR) molecules consisting of two, three or four CAR molecules, wherein each member of the CAR family differs from the others in its amino acid sequence; wherein each CAR molecule of the group comprises at least a transmembrane domain and an ectodomain, the ectodomain comprising one or two antigen-binding moieties and / or one or two binding sites to which other polypeptides, each comprising at least an antigen-binding moiety, can bind, and at least one CAR molecule of the group further comprises an endodomain, which comprises at least a signaling region capable of transmitting a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM); wherein the endodomain of each CAR molecule of the group, if expressed intracellularly, is located on the intracellular side of the cell membrane, if each CAR molecule comprises an endodomain, and the ectodomain of each CAR molecule of the group, if expressed intracellularly, is transferred to the extracellular side of the cell membrane, and the transmembrane domain of each CAR molecule of the group, if expressed intracellularly, is located within the cell membrane; wherein the ectodomain of each CAR molecule of the group does not, in its general conformation, contain a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with each of the other CAR molecules of the group; wherein the antigen-binding portions of the different CAR molecules of the group and of the different other polypeptides are specific for different target antigens that are not covalently linked to each other; wherein the affinity of each antigen-binding portion of the group of CAR molecules for each target antigen is between 1 mM and 100 nM; wherein the affinity of the individual antigen-binding portion of the other polypeptide for each target antigen, or the affinity of the other polypeptide for the binding site of each CAR molecule, is between 1 mM and 100 nM; wherein each CAR molecule of the group comprises at least one heterodimerization domain capable of mediating a defined heterodimerization with other CAR molecules of the group, and wherein this heterodimerization of pairs of heterodimerization domains occurs independently of a regulatory molecule, or occurs in the absence of a regulatory molecule and is reduced by a regulatory molecule, or is induced by a regulatory molecule and optionally reduced by another regulatory molecule, wherein a regulatory molecule is capable of binding to at least one member of the pair of heterodimerization domains under physiological conditions, and wherein inducing or reducing heterodimerization induces or reduces the formation of a non-covalently complexed CAR group consisting of two, three, or four CAR molecules. [Brief description of the drawings]

[0007] [Figure 1-1] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Figure 1-2] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Figure 1-3] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Figure 1-4] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Figure 1-5] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Figure 1-6] 1 shows a schematic representation of an exemplary configuration of CAR groups. [Diagram 2] 1 shows the Kd values ​​of various rcSso7d-based antigen-binding moieties against human EGFR. [Figure 3-1] We show that extracellular disulfide bond forming cysteines can prevent the use of avidity effects on the AND gate function of CARs. [Figure 3-2] We show that extracellular disulfide bond forming cysteines can prevent the use of avidity effects on the AND gate function of CARs. [Figure 4-1] We show that dimerization / oligomerization of scFv-containing CAR molecules prevents the use of avidity effects to generate CAR populations with AND gate functionality. [Figure 4-2]We show that dimerization / oligomerization of scFv-containing CAR molecules prevents the use of avidity effects to generate CAR populations with AND gate functionality. [Figure 4-3] We show that dimerization / oligomerization of scFv-containing CAR molecules prevents the use of avidity effects to generate CAR populations with AND gate functionality. [Figure 5-1] 1 shows the generation and function of affibody-based CARs against HER2. [Figure 5-2] 1 shows the generation and function of affibody-based CARs against HER2. [Figure 5-3] 1 shows the generation and function of affibody-based CARs against HER2. [Figure 6] 1 shows regulation of function of CARs expressed in stably transduced T cells in vivo. [Figure 7-1] 1 shows functional in vitro characterization of CAR-modified T cells used for in vivo experiments. [Figure 7-2] 1 shows functional in vitro characterization of CAR-modified T cells used for in vivo experiments. [Figure 7-3] 1 shows functional in vitro characterization of CAR-modified T cells used for in vivo experiments. [Figure 8-1] VEGF regulation of the binding activity of CARs. [Figure 8-2] VEGF regulation of the binding activity of CARs. [Figure 9-1] The function of CARs directed against EGFR and HER2 and that can be controlled by regulatory molecules is shown. [Figure 9-2] The function of CARs directed against EGFR and HER2 and that can be controlled by regulatory molecules is shown. [Figure 9-3] The function of CARs directed against EGFR and HER2 and that can be controlled by regulatory molecules is shown. [Figure 10-1] CAR groups consisting of three or four CAR molecules are shown. [Figure 10-2] CAR groups consisting of three or four CAR molecules are shown. [Figure 11-1] FIG. 1 shows the function of CARs containing heterodimerization domains for constitutive complex formation. [Figure 11-2] FIG. 1 shows the function of CARs containing heterodimerization domains for constitutive complex formation. [Figure 12-1] CARs containing different costimulatory domains are shown. [Figure 12-2] CARs containing different costimulatory domains are shown. [Figure 13-1] 1 shows the expression of CAR molecules containing different rcSso7d and affibody-based binding moieties fused to different CAR signaling scaffolds. [Figure 13-2] 1 shows the expression of CAR molecules containing different rcSso7d and affibody-based binding moieties fused to different CAR signaling scaffolds. [Figure 14-1] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-2] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-3] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-4] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-5] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-6] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-7] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-8] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-9] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-10] Schematic diagrams of various CAR molecule designs are shown. [Figure 14-11] Schematic diagrams of various CAR molecule designs are shown. [Figure 15-1] The amino acid sequences of various CAR molecules are shown. [Figure 15-2] The amino acid sequences of various CAR molecules are shown. [Figure 15-3] The amino acid sequences of various CAR molecules are shown. [Figure 15-4] The amino acid sequences of various CAR molecules are shown. [Figure 15-5] The amino acid sequences of various CAR molecules are shown. [Figure 15-6] The amino acid sequences of various CAR molecules are shown. [Figure 15-7] The amino acid sequences of various CAR molecules are shown. [Figure 15-8] The amino acid sequences of various CAR molecules are shown. [Figure 15-9] The amino acid sequences of various CAR molecules are shown. [Figure 15-10] The amino acid sequences of various CAR molecules are shown. [Figure 15-11] The amino acid sequences of various CAR molecules are shown. [Figure 15-12] The amino acid sequences of various CAR molecules are shown. [Figure 15-13] The amino acid sequences of various CAR molecules are shown. [Figure 15-14] The amino acid sequences of various CAR molecules are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Detailed Description of the Preferred Embodiments The principle underlying the combinatorial antigen recognition of the present invention is a group of CARs, where the individual antigen-binding portions of the individual CAR molecules of the group have only low affinity for their respective target antigens, so that monovalent interactions induce only weak intracellular signaling in CAR-expressing cells or no signaling at all. When using other polypeptides, each of which contains at least one antigen-binding portion and can further bind to the CAR molecules of the group, the interaction between the antigen-binding portion of the other polypeptide and its respective target antigen, or between the other polypeptide and its binding site on each CAR molecule of the group, must have low affinity, so that monovalent interactions induce only weak intracellular signaling in CAR-expressing cells or no signaling at all. However, non-covalent assembly of two, three, or four CAR molecules of the group with different binding specificities results in the formation of a multivalent CAR complex that can simultaneously interact with two, up to three, or up to four different target antigens, respectively, directly or indirectly via other polypeptides, where each of the other polypeptides contains at least an antigen-binding portion and can bind to the CAR molecules of the group. This multivalent interaction with different antigens results in synergistic amplification of low affinity, i.e. avidity. Ultimately, this multivalent interaction with selected combinations of different target antigens induces enhanced signaling in cells expressing the CARs.

[0009] Thus, to ensure that only polyvalent and not monovalent interactions are efficiently elicited in the complexed CAR population, the affinity of each antigen-binding portion of the CAR molecules of the population to its target antigen is between 1 mM and 100 nM, and the affinity of each antigen-binding portion of the other polypeptide to its target antigen or to the binding site of this other polypeptide to its respective CAR molecule is between 1 mM and 100 nM. In a preferred embodiment, the affinity of each antigen-binding portion of the CAR molecules of the population to its target antigen is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, in particular between 1 mM and 400 nM, and the affinity of each antigen-binding portion of the other polypeptide to its target antigen or to the binding site of this other polypeptide to its respective CAR molecule is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, in particular between 1 mM and 400 nM. In another preferred embodiment, the affinity of the individual antigen-binding portions of the CAR molecules of the group to each target antigen is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, in particular 50 μM to 100 nM, and the affinity of the individual antigen-binding portion of the other polypeptide to each target antigen, or the affinity of this other polypeptide to the binding site of each CAR molecule, is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, in particular 50 μM to 100 nM. In another preferred embodiment, the respective affinity of the individual antigen-binding portions of the CAR molecules of the group to their target antigen is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, and in particular between 50 μM and 400 nM, and the affinity of the individual antigen-binding portion of the other polypeptide to its target antigen, or the affinity of this other polypeptide to the binding site of its respective CAR molecule, is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, and in particular between 50 μM and 400 nM.It should be noted that any affinity values ​​presented herein refer to affinities determined by surface plasmon resonance (SPR) performed at pH 7.4 and 25°C using steady-state analysis, e.g., as performed in Examples 1 and 4 of the Examples section.

[0010] As already explained, the basic principle of generating CARs with the AND gate function of the present invention is found in the defined heterodimerization, heterotrimerization, or heterotetramerization of the CAR group, where each CAR molecule mediates low affinity recognition of a different target antigen. Then, the amplification of the low affinity of the different binding moieties upon target antigen recognition (i.e., avidity effect) can induce efficient CAR signaling. The non-covalent interactions between the individual CAR molecules of the group of the present invention (resulting in a bi-, tri-, or tetra-specific complex of the CAR molecules of the group) can be constitutive or dependent on the presence or absence of a regulatory molecule. Importantly, dimerization or oligomerization of CAR molecules with the same target antigen specificity, for example mediated by the formation of intermolecular extracellular disulfide bonds, would amplify the affinity for a single antigen, thereby interfering with the AND gate function of the CAR group of the present invention. Therefore, such dimerization or oligomerization of CAR molecules with the same target antigen specificity needs to be prevented as much as possible, or at least minimized as much as biologically possible.

[0011] To facilitate the defined heterodimerization, heterotrimerization, or heterotetramerization of the CARs of the present invention, each CAR molecule of the CARs comprises at least one domain capable of mediating the heterodimerization of two CAR molecules, thereby inducing the formation of a defined non-covalent complex of two, three, or four CAR molecules in a constitutive or conditional manner. This means that if the individual molecules of the CARs are co-expressed in a cell and assembled into a non-covalent complex, the CARs (optionally depending on the presence of one or more other polypeptides, each of which comprises at least an antigen-binding portion and can bind to the CAR molecules of the group) can mediate enhanced activation of those cells in response to target cells, i.e. cells expressing a selected combination of two or more target antigens, compared to non-target cells, i.e. cells expressing only the target antigen or only a part of the selected target antigen. This means that the non-covalently complexed CARs can integrate several inputs, i.e. binding to different target antigens, into a defined output signal, i.e. activation of the cells expressing the CARs. This ability of the CARs represents a so-called logical AND gate function, and they were surprisingly efficient at distinguishing target cells (i.e., cells expressing a given combination of targets) from non-target cells (i.e., cells expressing only one antigen).

[0012] Existing concepts for designing CARs with logic AND gate functionality do not use synergistic amplification of the individual affinities of multiple binding moieties of individual molecules for different target antigens (Roybal and Lim, Annu Rev Immunol. 2017;35:229; Chen et al., Curr Opin Immunol. 2018;51:103-110; Labanieh et al., Nat Biomed Eng. 2018;2:377-391). However, strategies exist in which binding moieties for two different target antigens (antigens A and B) are fused in tandem, i.e., linked sequentially into a single polypeptide chain, into a conventional CAR scaffold. Such CARs (called tandem CARs) are designed to mediate a logic OR gate functionality, i.e., these CARs induce a strong signal in cells expressing the CAR in response to target cells expressing either antigen A, or antigen B, or both antigens. This is due to the fact that these CARs can induce signals even with monovalent interactions due to the high affinity of their binding moieties (Hegde et al., J Clin Invest. 2016;126(8):3036-3052; Grada et al., Mol Ther Nucleic Acids. 2013;2:e105; Zah et al., Cancer Immunol Res. 2016;4(6):498-508; De Munter et al., Int J Mol Sci. 2018 Jan 30;19(2) pii: E403). Examples of such CARs are disclosed in US Patent Application Publication No. 20170107285A1 and US Patent Application Publication No. 20180111992A1. Some of the reported CARs also have AND gate functionality (Hedge et al., J Clin Invest. 2016;126(8):3036-3052).However, the AND-gating function of these reported CARs is severely limited due to the high affinity of the individual binding moieties and because at least some of the currently used CAR scaffolds form homodimers when expressed intracellularly due to disulfide bonds between the bonds between extracellular cysteine ​​residues.

[0013] As mentioned above, the prevention or minimization of dimerization or multimerization of CAR molecules with the same target antigen specificity is an essential prerequisite for generating a CAR population with the AND gate function of the present invention. Dimerization or multimerization of identical CAR molecules leads to affinity amplification of binding moieties with the same target antigen specificity, thereby preventing the use of avidity effects (i.e., specific recognition of antigen combinations) for the logical AND gate function.

[0014] For this reason, according to the present invention, the ectodomain of each CAR molecule of the group in its "common conformation" (i.e., in its naturally folded conformation) does not contain a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with each of the other CAR molecules of the group. In other words, the extracellular domain of a CAR molecule of the group of the present invention must not contain a cysteine ​​in the naturally folded conformation of the CAR that is not involved in an intramolecular disulfide bond (i.e., formed within a given CAR molecule of the group). For example, cysteines in the hinge region of CD8α that can form an intermolecular disulfide bond (i.e., with other CAR molecules of the group) in the native conformation must be excluded, for example by mutation or deletion. On the other hand, cysteines that are involved in an intramolecular disulfide bond in the native conformation of the CAR molecule (and therefore are not accessible for the formation of intermolecular bonds with other CAR molecules) can be present in the CAR of the CAR group of the present invention. As an example, cysteines within the Ig domain of an antibody fragment (e.g., within an scFv) that form intramolecular disulfide bonds can be present in a CAR molecule of a CAR population of the invention, such that cysteines within an scFv are involved in intramolecular disulfide bonds and therefore unavailable for intermolecular disulfide bonds (when the CAR molecule is present in its common, or native, conformation), thereby preventing the formation of homodimeric or homooligomeric CAR molecules of the population.

[0015] In addition, the antigen-binding portion of current CARs typically consists of variable light (VL) and variable heavy (VH) domains between individual molecules. HCARs are based on single chain variable fragments (scFvs) that tend to oligomerize due to intermolecular heterodimerization of the scFv domains (Hudson et al., J Immunol Methods. 1999; 231(1-2):177-89; Long et al., Nat Med. 2015;21(6):581-90). This uncontrolled dimerization or oligomerization can occur even between identical CAR molecules (i.e., CAR molecules with the same antigen specificity), which can prevent efficient AND gate function. Thus, the individual molecules of the CAR group of the present invention preferably do not contain scFv-based antigen-binding moieties or other molecular components that can lead to undesired and uncontrolled covalent or non-covalent complexation of the CAR molecules of the group.

[0016] In general, non-covalent dimerization or oligomerization of the same CAR molecule mediated by other domains of the CAR molecule would prevent efficient AND gate function of the present invention. Therefore, such non-covalent dimerization or oligomerization should be prevented or at least minimized as far as biologically possible by elimination or engineering of such domains.

[0017] Compared to tandem CARs, the basic design of the CARs of the present invention facilitates the adaptation of the linkers and spacers of the CAR molecules to optimize the spatial requirements for efficient interaction with each of the different target antigens. The configuration of the CARs of the present invention further facilitates the optimization of the CAR molecules with respect to the shape of the pulling force between the CAR molecule and the target antigen. This is advantageous because it is known for T cells that the mechanical force generated upon antigen recognition by the actomyosin cytoskeleton plays a key role in the organization of the immune synapse, improving the efficiency of T cell activation and target cell killing (Basu and Huse, Trends Cell Biol. 2017;27(4):241-254). Similar to pulling off an adhesive strip, it is believed that the shape of the force transmission through the binding portion of the CAR molecule affects the stability of the interaction with the target antigen, thereby affecting the signaling efficiency of the CAR molecule. That is, in tandem-CARs, the pulling force acts mostly (or exclusively) on the binding moiety adjacent to the transmembrane domain, whereas in the CAR group of the present invention, the pulling force is transmitted in parallel in each CAR molecule of the group, thereby ensuring that each interaction between the binding moiety on the CAR molecule and the target antigen contributes to the overall pulling force. Finally, the configuration of the CAR group allows for optional reversible regulation of the function of the CAR group by simply making the heterodimerization of individual CAR molecules conditional. Thus, compared to the tandem-CAR, which is the state of the art in CAR engineering, the CAR group of the present invention offers several important advantages: (i) the ability to individually optimize the linker length of each binding moiety, (ii) improved transmission of pulling force, and (iii) the option to tune CAR function by conditional dimerization, trimerization, or tetramerization.

[0018] The basic structure of the molecular design of the CAR group of the present invention can be varied at certain sites without invalidating the logical AND gate function. This allows the system to be adapted to various demands. For example, the CAR group can consist of two CAR molecules, or three or four CAR molecules, to enhance the avidity effect and / or to generate an AND gate CAR complex that depends on the presence of three or four different antigens, respectively, or to incorporate a logical OR gate function into the CAR group, for example to mediate the recognition of antigen A in combination with either antigen B or antigen C. That is, this can be considered as an AND / OR gate, because in this example, the trimeric CAR group responds to either antigen A and (AND) B or (OR) antigen A and (AND) C, i.e., A and (AND) B or (OR) C). Similarly, the tetrameric group of CARs can be designed to respond to antigen (A or (OR) B) and (AND) (C or (OR) D), or to antigen A and (AND) (B or (AND) C or (AND) D). CARs can also be easily engineered to be functionally dependent on either soluble proteins or small molecules.

[0019] In a preferred embodiment for the conditional regulation of CAR function, the CAR group may consist of CAR molecules, each of which comprises at least an extracellular binding site to which another polypeptide, each of which comprises at least an antigen-binding portion, can bind. Such other polypeptides are thereby defined as soluble proteins that do not belong to the CAR group and can bind non-covalently directly to the binding site of the CAR molecules of the group, or indirectly through the covalent modification of another polypeptide, such as, for example, a covalently bound fluorescein isothiocyanate (FITC) molecule. In this preferred embodiment, the defined injection of this other polypeptide allows the control of the function of the CAR group. This strategy of modulating CAR function by administering soluble antigen-binding proteins, which is well known in the CAR field (Cho et al., Cell. 2018;173(6):1426-1438; Ma et al., Proc Natl Acad Sci US A. 2016;113(4):E450-458; Urbanska et al., Cancer Res. 2012;72(7):1844-1852) and is currently being tested in the clinical setting (Labanieh et al., Nat Biomed Eng. 2018;2:377-391), can also be incorporated into the CARs of the present invention. In this case, in principle, low affinity binding does not necessarily have to occur via the antigen-binding portion of another polypeptide, but can also occur in the CAR molecule via a binding site to which another polypeptide can bind that contains at least an antigen binding.

[0020] Alternatively, the function of the CAR group of the present invention can also be regulated by conditional heterodimerization, heterotrimerization, or heterotetramerization.Thus, in a preferred embodiment, the formation of a non-covalent complex of two, three, or four CAR molecules of the group is induced by one or more regulatory molecules that can bind to at least one member of the pair of heterodimerization domains of the CAR group under physiological conditions.In principle, the regulatory molecule can be any molecule that can bind to at least one heterodimerization domain and induce or reduce the interaction of the members of the pair of heterodimerization domains.These molecules are usually small molecules, but can also be, for example, soluble proteins that accumulate in the stroma of tumors, which are often proteins that themselves naturally heterodimerize (e.g., subunits of heterodimeric cytokines such as IL-12).

[0021] To eliminate the possibility of fratricide due to cross-linking of CAR molecules between different cells, the heterodimerization domain is preferably incorporated into the internal domain and / or transmembrane domain, more preferably the internal domain, of the CAR molecules of the CAR group.

[0022] To reduce the burden of vectors used to stably express the CARs in cells, the CAR panel preferably comprises three, in particular two, CAR molecules. To further reduce the complexity, each CAR molecule of the panel preferably comprises only a single antigen-binding moiety, or optionally only a single extracellular binding site to which another polypeptide can bind, where the other polypeptide comprises at least the antigen-binding moiety.

[0023] To incorporate an OR gate function into the CAR panel, e.g., to mediate recognition of antigen A in combination with either antigen B or (OR) antigen C, the CAR molecules of the panel can also comprise two antigen-binding moieties, or two extracellular binding sites to which other polypeptides can bind, where the other polypeptide comprises at least an antigen-binding moiety.

[0024] To reduce the potential immunogenicity of the CAR molecules, the CAR molecules of the CAR family preferably comprise an extracellular binding site to which another polypeptide can bind, where the other polypeptide comprises at least an antigen-binding portion.

[0025] For applications in which conditional regulation of CAR function is not required, the CAR molecules of the group can contain a heterodimerization domain that does not require the presence of a regulatory molecule, resulting in constitutive complex formation. Optionally, as a safety measure in the event of unintended adverse effects, the CAR molecule can also contain a heterodimerization domain that mediates constitutive heterodimerization, but can further bind to a regulatory molecule that induces dissociation of the heterodimerization domain.

[0026] As mentioned above, the basic structure of the CAR molecules of the present invention can be adapted to the needs of different applications. The order of the domains of the CAR molecules of the present invention from outside the cell to inside the cell preferably corresponds to the following basic structure on the surface of the cell: a binding site to which an antigen-binding portion or other polypeptides comprising at least an antigen-binding portion can bind, optionally a linker for spatial optimization of any second antigen-binding portion or any second binding site to which other polypeptides comprising at least an antigen-binding portion can bind, preferably a hinge region for spatial optimization, and a transmembrane domain. The transmembrane domain is preferably followed in at least one CAR molecule by a signaling region comprising a costimulatory domain, and preferably this costimulatory signaling region or optionally the transmembrane domain is followed by at least one heterodimerization domain, and further, in at least one CAR molecule, by a signaling region comprising at least one ITAM, where the order of the costimulatory signaling region and the ITAM-containing signaling region can be reversed. An ITAM-free CAR molecule lacks a costimulatory signaling region, or contains one costimulatory signaling region, or contains two costimulatory signaling regions, or contains even more costimulatory signaling regions, but preferably contains up to two costimulatory signaling regions, or more preferably contains only one costimulatory signaling region. In general, the heterodimerization domain, at least one of which is essential for each CAR molecule of the group, can alternatively or additionally be located in the ectodomain or the transmembrane domain, but preferably between the transmembrane domain and the signaling region, and / or in particular between the two signaling regions, and / or in particular at the intracellular end of the CAR molecule. Finally, any two adjacent components of the CAR molecules of the group (antigen-binding portion, a binding site to which another polypeptide containing at least an antigen-binding portion can bind, hinge region, transmembrane domain, signaling region, dimerization domain) can be optionally separated by a linker.

[0027] Different CARs against different combinations of target antigens can also be co-stimulated in cells, for example, to suppress tumor immune evasion caused by loss of target antigens. CARs can also be co-expressed with other proteins in a particular cell.

[0028] 1. Antigen binding moiety: Antigen-binding moieties suitable for use in the CARs of the invention can be any antigen-binding polypeptide (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), a wide variety of which are known in the art (Simeon et al., Protein Cell. 2017; Gilbreth et al., Curr Opin Struct Biol. 2012;22(4):413-420; Koide et al., ACS Chem Biol. 2009;4(5):325-334; Traxlmayr et al., J Biol Chem. 2016;291(43):22496-22508). Meanwhile, many more non-antibody binding proteins have been reported (Plückthun, Alternative Scaffolds: Expanding the options of antibodies. In: Little M, ed. New York: Cambridge University Press; 2009:244-271; Chapman et al., Cell Chem Biol. 2016;23(5):543-553; Binz et al., Nat Biotechnol. 2005;23(10):1257-1268; Vazquez-Lombardi et al., Drug Discov Today. 2015;20(10):1271-1283), and in fact the design and selection of synthetic libraries can be applied to any protein that may also function as an antigen-binding moiety (Plückthun, Alternative Scaffolds: Expanding the options of antibodies. In: Little M, ed. New York: Cambridge University Press; 2009:244-271).

[0029] In some cases, the antigen-binding portion may be a single chain Fv (scFv), other antibody-based recognition domain, e.g., a cAb V H (Camelidae Antibody Variable Domain) and its humanized version, IgNAR V H(Shark antibody variable domain) and its humanized version, sdAb V H (single domain antibody variable domain), or "camelized" antibody variable domain. In some cases, T cell receptor (TCR) based recognition domains such as single chain TCRs (single chain two domain TCRs including scTv, VaV) may also be suitable for use. Preferably, the antigen binding portion of each molecule of the CAR family is composed of only one protein domain, preferably a human or non-human V H or VL single domain antibodies (nanobodies), or engineered antigen binding moieties based on the Z domain of Staphylococcus aureus protein A, lipocalin, SH3 domain, fibronectin type III (FN3) domain, knottin, Sso7d, rcSso7d, Sac7d, Gp2, DARPin, or ubiquitin; or ligands, receptors, or co-receptors selected or engineered for low affinity binding and lack of homotypic interactions. Ligands include, for example, cytokines (e.g., IL-13); growth factors (e.g., heregulin); and the like. The ligand may be a receptor-binding fragment of a ligand (e.g., a peptide of HGF (Thayaparan et al., Oncoimmunology. 2014;14;6(12):e1363137); an integrin-binding peptide (e.g., a peptide containing the sequence Arg-Gly-Asp)). Similarly, the receptor may be a ligand-binding fragment of a receptor. Suitable receptors include, for example, cytokine receptors (e.g., IL-13 receptor; IL-2 receptor); cell adhesion molecules (e.g., CD11a (Park et al., Sci Rep. 2017;7(1):14366)); PD-1. The antigen-binding portion of each molecule of the CAR series preferably does not cause undesired aggregation of the CAR molecules. As mentioned above, such undesired dimerization or oligomerization of the CAR molecules of the series may cause multivalent interactions with single positive non-target cells. For this reason, it is preferable that the antigen-binding portion is not a single chain variable fragment (scFv) derived from a monoclonal antibody. For the purpose of clinical applicability of the CAR series of the present invention, the antigen-binding portion is preferably derived from a human single protein domain (e.g., a fibronectin type III domain (FN3)-based monobody).

[0030] 2. Hinge area: In some embodiments, the ectodomain of the CAR molecule of the group comprises a hinge region inserted between the antigen-binding portion (or at least a binding site to which another polypeptide comprising an antigen-binding portion can bind) and the transmembrane domain, preferably a hinge region derived from CD8 alpha (amino acid sequence positions 138-182 according to UniProtKB / Swiss-Prot P01732-1), or CD28 (amino acid sequence positions 114-152 according to UniProtKB / Swiss-Prot P10747), or PD-1 (amino acid sequence positions 146-170 according to UniProtKB / Swiss-Prot Q15116), where the sequence derived from CD8 alpha, CD28, or PD-1 can be N-terminally and / or C-terminally truncated and have any length within the boundaries of said sequence region, and where the cysteine ​​residues of the hinge derived from CD8 alpha and CD28 have been deleted or replaced by other amino acid residues. In principle, the flexible membrane anchors and other parts of many receptors are suitable for use in the hinge regions and / or transmembrane domains of the CAR molecules of the group (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), provided that these are modified, if necessary, to prevent dimerization in the present invention.

[0031] Depending on the individual structural requirements for optimal binding of the selected target antigen, the hinge region of the CAR molecule can have a length of about 2 amino acids to about 50 amino acids, e.g., about 4 amino acids (aa) to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. Optionally, the hinge region can comprise more than 50 amino acids, e.g., when a structured domain is incorporated (e.g., from CD34 UniProt P28906-1 aa 42 to 140 to facilitate enrichment of CAR modified cells, as disclosed in U.S. Patent Application Publication No. 2018 / 0094044A1).

[0032] Preferably, other polypeptides, preferably glycine and glycine-serine polymers, can also be used in the hinge, since both Gly and Ser are relatively unstructured and therefore can function as neutral tethers between CAR components. Glycine has much greater access to the phi-psi space than alanine and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1992; 11173-11142). Thus, to tailor CAR molecules for optimal binding to their target antigens, the hinge region inserted between the antigen-binding portion (or at least the binding site to which other polypeptides containing antigen-binding portions can bind) and the transmembrane domain comprises glycine polymers (G)n and / or glycine-serine polymers (GS)n, (GSGGS)n, (GGS)n, (GGGS)n, (GGGGS)n, where n is an integer of at least 1.

[0033] 3. Transmembrane domain: Each molecule of the CAR family contains a transmembrane domain for insertion into the eukaryotic cell membrane. Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. For example, the TM sequence of human CD8 alpha IYIWAPLAGTCGVLLLSLVITLYC (Uniprot P01732, amino acids (aa) 183-206) can be used. Further examples of suitable TM sequences include: from human CD8 beta: LGLVAAGVLVLLVSLGVAIHLCC (Uniprot P10966, aa 173-195); from human CD4: ALIVLGGVAGLLLFIGLGIFFCVRC (Uniprot P01730, aa 398-422); from human CD3 zeta: LCYLLDGILFIYGVILTALFLRV (Uniprot P20963, aa 31-53); from human CD28: FWVLVVVGGVLACYSLLVTVAFIIFWV (Uniprot P10747, aa 154-179); from human CD134 (OX40): VAAILGLGLVLGLLGPLAILLALYLL (Uniprot P43489, aa 215-240); from human CD27: ILVIFSGMFLVFTLAGALFLH (Uniprot P43489, aa 215-240); P26842, aa192-212); from human CD278 (ICOS): FWLPIGCAAFVVVCILGCILI (Uniprot Q9Y6W8, aa141-161); from human CD279 (PD-1): VGVVGGLLGSLVLLVWVLAVI (Uniprot Q15116, aa171-191); from human DAP12: GVLAGIVMGDLVLTVLIALAV (Uniprot O43914, aa41-61); and from human CD7: ALPAALAVISFLLGLGLGVACVLA (Uniprot P09564, aa178-201).

[0034] 4. Immunoreceptor tyrosine-based activation motifs (ITAMs): According to the present invention, at least one molecule of the CAR family contains an internal domain capable of signaling through at least one immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1 X 2 L / I, where X 1 and X 2 are independently any amino acid. An ITAM-containing endodomain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ITAM motifs. The ITAM-containing portions of a signaling endodomain are preferably derived from any ITAM-containing protein and need not comprise the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM-containing polypeptides include: DAP12; FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex associated protein alpha chain).

[0035] In particularly preferred embodiments, at least one signaling domain in at least one CAR molecule of the CAR population is derived from the cytoplasmic domain of 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 ITAM-containing domain has the amino acid sequence (two isoforms): MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-3) or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPQRRKNPQEGL YNELQKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-1) (wherein the ITAM motif is bold and underlined), about 50 amino acids to about 60 amino acids (aa), about 60 aa to about 70 aa, about 70 aa to about 80 aa, about 80 aa to about 90 aa, about 90 aa to about 100 aa, about 100 aa to about 110 aa, about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 12 It 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 with a contiguous stretch of 0 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa.

[0036] Similarly, a suitable ITAM-containing domain can include an ITAM-containing portion of the full-length CD3 zeta amino acid sequence. Amino acid sequence RVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-3 aa52~163), NQL YNEL NLGRREE YDVL DKR (Uniprot P20963-3 aa69~89), EGL YNEL QKDKMAEA YSEI GMK(Uniprot P20963-3 aa107~128), DGL YQGL STATKDT YDALHMQ (Uniprot P20963-3 aa138-158) (wherein the ITAM is bold and underlined) (wherein the ITAM is bold and underlined) (wherein the ITAM is underlined).

[0037] The ITAM-containing domain may also be derived from the T cell surface glycoprotein CD3 delta chain (also called CD3D; CD3-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 ITAM-containing domain 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%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 170 aa of any of the following amino acid sequences (two isoforms) (Uniprot P04234-1; Uniprot P04234-2).

[0038] Similarly, a suitable ITAM-containing domain may comprise an ITAM-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable ITAM-containing domain may comprise the amino acid sequence DQV YQPL RDRDDAQ YSHL The amino acid sequence may include 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 GGN (Uniprot P04234-1 aa146-166) (wherein the ITAM is bolded and underlined).

[0039] The ITAM-containing domain may be derived from the T cell surface glycoprotein CD3 epsilon chain (also called 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 ITAM-containing domain can include 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 205 aa of the amino acid sequence Uniprot P07766-1.

[0040] Similarly, a suitable ITAM-containing domain may comprise an ITAM-containing portion of the full-length CD3 epsilon amino acid sequence. YEPI RKGQRDL YSGL The amino acid sequence may include 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 with NQR (Uniprot P07766-1 aa185-205) (wherein the ITAM is bolded and underlined).

[0041] An ITAM-containing domain may be derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable ITAM-containing domain may have the amino acid sequence MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL YQPL KDREDDQ YSHL The amino acid sequence may include 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 180 aa of QGNQLRRN (Uniprot P09693-1) (where the ITAMs are bolded and underlined).

[0042] Similarly, a suitable ITAM-containing domain may comprise an ITAM-containing portion of the full-length CD3 gamma amino acid sequence. YQPL KDREDDQ YSHL The amino acid sequence may include 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 with QGN (Uniprot P09693-1 aa157-177) (wherein the ITAMs are bolded and underlined).

[0043] The ITAM-containing domain may be derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; Kira-activated receptor associated protein; Kira-activated receptor associated protein, etc.). For example, a suitable ITAM-containing domain may include 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 of the following amino acid sequences (four isoforms): Uniprot O43914-1; Uniprot O43914-2; Uniprot O43914-3; Uniprot X6RGC9-1.

[0044] Similarly, a suitable ITAM-containing domain can include an ITAM-containing portion of the full-length DAP12 amino acid sequence. YQEL QGQRSDV YSDL The amino acid sequence may include 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 with NTQ (Uniprot O43914-1 aa88-108) (wherein the ITAM is bolded and underlined).

[0045] The ITAM-containing domain may be derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon R1-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain, etc.). For example, a suitable ITAM-containing domain may be derived from the amino acid sequence MIPAVVLLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV YTGL STRNQET YETL The amino acid sequence can include 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 with KHEKPPQ (Uniprot P30273) (wherein the ITAMs are bolded and underlined).

[0046] Similarly, a suitable ITAM-containing domain can include an ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Thus, a suitable ITAM-containing domain can include the amino acid sequence DGV YTGL STRNQET YETL The polypeptide may include 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 KHE (Uniprot P30273 aa62-82) (wherein the ITAM is bold and underlined).

[0047] An ITAM-containing domain may be derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein, etc.). For example, a suitable ITAM-containing domain may be derived from the amino acid sequence (two isoforms): MPGGPGVLQALPATIFLLFLLSAVYLPGGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL YEGL NLDDCSM YEDI SRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-1) or MPGGP YEGL NLDDCSM YEDI The amino acid sequence may include 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 150 aa, about 150 aa to about 200 aa, or about 200 aa to about 220 aa of any of the following: SRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-2) (where the ITAM is bold and underlined).

[0048] Similarly, a suitable ITAM-containing domain can include an ITAM-containing portion of the full-length CD79A amino acid sequence. YEGL NLDDCSM YEDI The amino acid sequence may include 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 SRG (Uniprot P11912-1 aa185-205) (wherein the ITAM is bolded and underlined).

[0049] Thus, according to the present invention, the internal domain of at least one CAR molecule of the CAR group comprises at least one ITAM, said ITAM preferably being selected from CD3 zeta, DAP12, Fc-epsilon receptor 1 gamma chain, CD3 delta, CD3 epsilon, CD3 gamma, and CD79A (antigen receptor complex associated protein alpha chain).

[0050] Since the number of ITAMs correlates with the signaling efficiency of the CAR (James, Sci Signal. 2018;11(531)), it is preferred that the CAR population contains a total of three ITAMs, where the ITAM can be limited to only a single CAR molecule of the population. Alternatively, some or all of the CAR molecules of the population can contain at least one ITAM. In some embodiments, the ITAM-containing moieties in different internal domains of the CAR molecules of the population are derived from the same receptor, while in other embodiments, the ITAM-containing moieties in different internal domains of the CAR molecules of the population are derived from different receptors. In some embodiments, the CAR population contains only one molecule, preferably containing an ITAM-containing moiety derived from CD3 zeta. In other embodiments, the CAR population consists of two molecules, both containing a portion of the cytoplasmic domain derived from CD3 zeta. For vector loading, it is preferred that the total number of ITAMs in the CAR population is between 3 and 6. Furthermore, the ITAM-containing sequences are preferably selected and / or engineered to minimize nucleotide sequence homology, to minimize the risk of homologous recombination.

[0051] 5. Co-stimulatory domain: In a preferred embodiment, the internal domain of at least one CAR molecule of the panel comprises a signaling region that includes costimulatory domains derived from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD2, CD6, CD27, CD30, CD40, GITR, and HVEM, such that the costimulatory domains included in the CAR panel can, optionally, be derived from different costimulatory receptors.

[0052] A costimulatory domain suitable for inclusion in the costimulatory signaling region of a CAR molecule of the CAR group can have a length of about 30 aa to about 70 aa, for example, the costimulatory domain can have a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa. Optionally, the costimulatory domain can have a length of about 70 aa to about 100 aa, about 100 aa to about 200 aa, or more than 200 aa.

[0053] In particularly preferred embodiments, the costimulatory domain in at least one molecule of the CARs is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9, CD137, 4-1BB, CDw137, ILA, etc.). For example, a suitable costimulatory domain 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%, or 100% amino acid sequence identity to the amino acid sequence Uniprot Q07011 aa214-255.

[0054] In a preferred embodiment, the costimulatory domain in at least one molecule of the CARs is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory domain can include 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 with the amino acid sequence Uniprot P10747 aa177-220.

[0055] In particularly preferred embodiments, the costimulatory domain in at least one molecule of the CAR family is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable costimulatory domain 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%, or 100% amino acid sequence identity to the amino acid sequence Uniprot Q9Y6W8 aa165-199.

[0056] In a preferred embodiment, the costimulatory domain in at least one molecule of the CARs is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable costimulatory domain 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%, or 100% amino acid sequence identity to the amino acid sequence Uniprot P26842 aa212-260.

[0057] In other embodiments, the costimulatory domain in at least one molecule of the CARs can be derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, a suitable costimulatory domain can include 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 Uniprot P43489 aa241-277.

[0058] In other embodiments, the costimulatory domain in at least one molecule of the CARs can be derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable costimulatory domain can include 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 Uniprot Q7Z6A9 aa176-289.

[0059] In other embodiments, the costimulatory domain in at least one molecule of the CARs can be 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 costimulatory domain can include 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 Uniprot Q9Y5U5 aa188-241.

[0060] In other embodiments, the costimulatory domain in at least one molecule of the CARs can be derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable costimulatory domain can include 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 Uniprot Q92956 aa224-283.

[0061] In other embodiments, the costimulatory domain in at least one molecule of the CARs may be derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable costimulatory domain can include 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa of the amino acid sequence Uniprot P28908 aa409-595.

[0062] 6. Linker: The molecules of the CAR group can include a linker between any two adjacent domains (i.e., components of the CAR molecule). For example, the linker can be placed between the transmembrane domain and the signaling region. As another example, the linker can be placed between the signaling region and the heterodimerization domain. As another example, the linker can be placed between two heterodimerization domains. As another example, the linker can be placed between two signaling regions. As another example, the linker can be placed between the transmembrane domain and the heterodimerization domain. As another example, the linker can be placed in the ectodomain of the CAR molecule between two antigen binding moieties. As another example, the linker can be placed in the ectodomain of the CAR molecule between two binding sites to which other polypeptides can bind. As another example, the linker can be placed in the ectodomain of the CAR molecule between the antigen binding moiety and the transmembrane domain. As another example, the linker can be placed in the ectodomain of the CAR molecule between the binding site to which other polypeptides can bind and the transmembrane domain. As another example, the linker can be placed in the ectodomain of the CAR molecule between the signal sequence and the antigen binding moiety. As another example, the linker can be placed in the external domain of the CAR molecule between the signal sequence and the binding site to which other polypeptides can bind. As another example, the linker can be placed in the external domain of the CAR molecule between the signal sequence and the heterodimerization domain. As another example, the linker can be placed in the external domain of the CAR molecule between the heterodimerization domain and the antigen binding portion. As another example, the linker can be placed in the external domain of the CAR molecule between the heterodimerization domain and the binding site to which other polypeptides can bind. As yet another example, the linker can be placed in the external domain of the CAR molecule between the antigen binding portion and the binding site to which other polypeptides can bind.

[0063] The linker may be a peptide comprising a length of about 1 to about 40 amino acids. The linking peptide may have virtually any amino acid sequence, taking into consideration that a suitable linker preferably has a sequence that generally results in a flexible peptide. In creating a flexible peptide, preferably small amino acids such as glycine, serine, alanine, etc. are used. Creating such sequences is routine for those skilled in the art. Suitable linkers can be easily selected and may be of different lengths, such as, for example, 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 may be 1, 2, 3, 4, 5, 6, or 7 amino acids. Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS), (GSGGS), (GGS), and (GGGS), where n is an integer of at least 1), or glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary flexible linkers include GGSG (SEQ ID NO: 1), GGSGG (SEQ ID NO: 2), GSGSG (SEQ ID NO: 3), GSGGG (SEQ ID NO: 4), GGGSG (SEQ ID NO: 5), GSSSG (SEQ ID NO: 6), and the like. One of ordinary skill in the art will recognize that the design of a peptide attached to any of the above elements can include a linker that is fully or partially flexible, and thus the linker can include a flexible linker as well as one or more moieties that provide a less flexible structure.

[0064] 7. Additional Domains: The subject CAR molecules can further comprise one or more additional polypeptide domains, such as signal sequences, epitope tags, and / or polypeptides that generate detectable signals. Signal sequences suitable for use in the subject CARs include any eukaryotic signal sequence, such as naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, and the like. Suitable epitope tags include, for example, hemagglutinin (HA, e.g., amino acid sequence YPYDVPDYA (SEQ ID NO: 7)), FLAG (e.g., amino acid sequence DYKDDDDK (SEQ ID NO: 8)), c-myc (e.g., amino acid sequence EQKLISEEDL (SEQ ID NO: 9)), StrepII (e.g., amino acid sequence NWSHPQFEK (SEQ ID NO: 81)), hexahistidine tag (6xHIS, e.g., amino acid sequence HHHHHH (SEQ ID NO: 82)), and the like. Suitable detectable signal-generating proteins include, for example, fluorescent proteins, and the like. Suitable fluorescent proteins include, for example, green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), extended CFP (ECFP), extended YFP (EYFP), GFPS65T, emerald, topaz (TYFP), venus, citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCF These include Pm, cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster-GFP, paGFP, Kaede proteins and kindling proteins, phycobiliproteins and phycobiliprotein conjugates, such as B-phycoerythrin, R-phycoerythrin, and allophycocyanin.Further examples of fluorescent proteins include mhoneydew, mbanana, morange, dtomato, tdtomato, mtangerine, mstrawberry, mcherry, mgrape, mraspberry, mgrape2, mplum (Shaner et al. (2005) Nat. Methods 2:905-909). A variety of fluorescent and colored proteins from anthozoans are suitable for use, for example, as described in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0065] 8. Heterodimerization domain: The complexation of a CAR group comprising two CAR molecules can be mediated by one heterodimerization domain per CAR molecule. In an embodiment in which a CAR group comprises three or four CAR molecules, at least one CAR molecule of the group preferably comprises two or more heterodimerization domains to promote the formation of trimers or tetramers by heterodimerization. In this case, the heterodimerization domains of the CAR molecule are preferentially members of different pairs of heterodimerization domains to prevent the formation of complexes comprising two or more identical CAR molecules of the group. Preventing such homotypic interactions of CAR molecules is important because homotypic interactions generate high avidity for a single type of target antigen. As a result, this results in efficient signaling by the CAR group in response to a single type of target antigen, thereby negating the dependency of efficient signaling on multivalent interactions with different target antigens.

[0066] According to the present invention, the heterodimerization domain incorporated in the CAR molecules of the group can mediate constitutive heterodimerization or can be optionally regulated by a regulatory molecule. For example, the heterodimerization of the heterodimerization domain can be induced or reduced by the presence of a regulatory molecule. The heterodimerization of the heterodimerization domain can also be constitutive and independent of a regulatory molecule. Domains that mediate constitutive heterodimerization are well known in the art and have been successfully used in different applications, such as, for example, coiled-coil interaction domains (Thompson et al., ACS Synth Biol. 2012; 1(4):118-29; Cho et al., Cell. 2018;173(6):1426-1438), but there are many more suitable domains in nature. In general, any pair of polypeptides that can bind to each other and be expressed in a CAR molecule is suitable for mediating constitutive heterodimerization of two CAR molecules of the CAR group of the present invention. Below, a lipocalin-fold molecule-based system is described (Chapter 8.1.2), which can be engineered for conditional as well as constitutive heterodimerization. In contrast to coiled-coil domains, lipocalin-fold molecule-based systems can be more easily engineered to switch off heterodimerization by regulatory molecules.

[0067] 8.1. Domains for conditional heterodimerization: 8.1.1. Conditional heterodimerization of CAR molecules based on ligand-binding domains from nuclear receptors: In a preferred embodiment, at least two CAR molecules of the CAR group of the present invention can be heterodimerized by a pair of heterodimerization domains, one member of which is a ligand binding domain (LBD) from a nuclear receptor and the second member of which is a coregulatory peptide. Upon binding of an appropriate small molecule (i.e., a regulatory molecule of the present invention), the LBD from the nuclear receptor can heterodimerize with each coregulatory peptide. This system can be used to heterodimerize proteins of interest. Suitable sequences of LBDs and coregulatory peptides, along with suitable regulatory molecules, are disclosed, for example, in US Patent Application Publication No. 2017 / 0306303A1. Suitable LBDs can be selected from any of a variety of nuclear receptors, such as ER-alpha, ER-beta, PR, AR, GR, MR, RAR-alpha, RAR-beta, RAR-gamma, TR-alpha, TR-beta, VDR, EcR, RXR-alpha, RXR-beta, RXR-gamma, PPAR-alpha, PPAR-beta, PPAR-gamma, LXR-alpha, LXR-beta, FXR, PXR, SXR, constitutive Adrostrane receptor, SF-1, LRH-1, DAX-1, SHP, TLX, PNR, NGF1-B-alpha, NGF1-B-beta, NGF1-B-gamma, ROR-alpha, ROR-beta, ROR-gamma, ERR-alpha, ERR-beta, ERR-gamma, GCNF, TR2 / 4, HNF-4, COUP-TF-alpha, COUP-TF-beta, and COUP-TF-cancer markers.

[0068] The abbreviations for nuclear receptors (synonymous with nuclear hormone receptors) are as follows: ER: estrogen receptor; PR: progesterone receptor; AR: androgen receptor; GR: glucocorticoid receptor; MR: mineralocorticoid receptor; RAR: retinoic acid receptor; TR-alpha / beta: thyroid receptor; VDR: vitamin D3 receptor; EcR: ecdysone receptor; RXR: retinoic acid X receptor; PPAR: peroxisome proliferator-activated receptor; LXR: liver X receptor; FXR: farnesoid X receptor; PXR / SXR: pregnane X receptor / steroid and xenobiotic receptor; SF-1: steroidogenic factor 1; DAX-1: dosage-sensitive sex reversal-adrenal dysplasia congenital critical region on the X chromosome, gene 1; LRH-1: liver receptor homolog 1; SHP: small heterodimer partner; TLX: tailless gene; PNR: photoreceptor-specific nuclear receptor; NGF1-B: nerve growth factor; ROR: RAR-related orphan receptor; ERR: estrogen-related receptor; GCNF: germline nuclear factor; TR2 / 4: testis receptor; HNF-4: hepatocyte nuclear factor; COUP-TF: chicken ovalbumin upstream promoter, transcription factor.

[0069] LBD: Mineralocorticoid Receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of the mineralocorticoid receptor (MR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of MR (Uniprot P08235).

[0070] For example, the LBD of MR is identified by the amino acid sequence (Uniprot Q9IAC6.1 aa112~359; Uniprot Q91573.1 aa365~612; Uniprot Q157N1 aa734~981; GenBank CAG11072.1 aa173~501; PDB 2AA6_A aa28~275; PDB 2AA2_A aa28~275; PDB 2A3I_A aa6~253; PDB 2OAX_A aa9~256; PDB 1Y9R_A aa8~255; PDB 2ABI_A aa1~256 ... The amino acid sequence may have 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 any of the following amino acids: aa9-256, and may have a length of about 200 amino acids to 250 amino acids (e.g., a length of 200 amino acids to 225 amino acids, or a length of 225 amino acids to 250 amino acids; e.g., a length of 248 amino acids).

[0071] For example, the LBD of MR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P08235 aa686-984, and has a length of about 250 amino acids to 299 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or has a length of 275 amino acids to 299 amino acids).

[0072] For example, the LBD of MR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P08235 aa737-984, and has a length of about 200 amino acids to 250 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or has a length of 225 amino acids to 250 amino acids, e.g., has a length of 248 amino acids).

[0073] For example, the LBD of MR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P08235 aa686-984 having an S810L substitution, and has a length of about 250 amino acids to 299 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or has a length of 275 amino acids to 299 amino acids).

[0074] For example, the LBD of MR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P08235 aa737-984 having an S810L substitution, and has a length of about 200 amino acids to 250 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or has a length of 225 amino acids to 250 amino acids, e.g., has a length of 248 amino acids).

[0075] Where one member of a pair of heterodimerization domains is the LBD of MR, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence SLTARHKILHRLLQEGSPSDI (Uniprot Q15788 aa681-701), where the coregulatory peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0076] Where one member of a pair of heterodimerization domains is the LBD of MR, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence QEAEEPSLLKKLLLAPANTQL (Uniprot Q9UBK2 aa136-156), where the coregulatory peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0077] Where one member of a pair of heterodimerization domains is the LBD of MR, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence SKVSQNPILTSLLQITGNGGS (Uniprot Q15648 aa596-616), where the coregulatory peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0078] Androgen Receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of the androgen receptor (AR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of AR (Uniprot P10275).

[0079] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa619-919, and has a length of about 250 amino acids to 301 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or 275 amino acids to 301 amino acids).

[0080] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa690-919, and has a length of about 190 amino acids to 230 amino acids (e.g., has a length of 190 amino acids to 210 amino acids, or 210 amino acids to 230 amino acids).

[0081] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa619-919 having a T877A substitution, and has a length of about 250 amino acids to 301 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or 275 amino acids to 301 amino acids).

[0082] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa690-919 having a T877A substitution, and has a length of about 190 amino acids to 230 amino acids (e.g., has a length of 190 amino acids to 210 amino acids, or 210 amino acids to 230 amino acids).

[0083] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa619-919 having an F876L substitution, and has a length of about 250 amino acids to 301 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or 275 amino acids to 301 amino acids).

[0084] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa690-919 having an F876L substitution, and has a length of about 190 amino acids to 230 amino acids (e.g., has a length of 190 amino acids to 210 amino acids, or 210 amino acids to 230 amino acids).

[0085] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa619-919 having F876L and T877A substitutions, and has a length of about 250 amino acids to 301 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, or 275 amino acids to 301 amino acids).

[0086] For example, the LBD of AR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10275 aa690-919 having F876L and T877A substitutions, and has a length of about 190 amino acids to 230 amino acids (e.g., has a length of 190 amino acids to 210 amino acids, or 210 amino acids to 230 amino acids).

[0087] Where one member of a pair of heterodimerization domains is the LBD of AR, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence ESKGHKKLLQLLTCSSDDR (Uniprot Q9Y6Q9 aa 614-632), where the coregulatory peptide has a length of from about 19 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0088] Progesterone Receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of a progesterone receptor (PR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of PR (Uniprot P06401).

[0089] For example, the LBD of a PR 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 any of the following amino acid sequences: (Uniprot Q8UVY3 aa456-703; Uniprot P07812.1 aa539-786; GenBank CAQ14518.1 aa306-553; PDB 1SR7_A aa12-259; PDB 1SQN_A aa14-261; PDB 1E3K aa11-258; PDB 1A28_A aa9-256) and has a length of about 200 amino acids to 250 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or 225 amino acids to 250 amino acids, e.g., 248 amino acids).

[0090] For example, the LBD of a PR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P06401 aa678-933, and has a length of about 200 amino acids to 256 amino acids (e.g., a length of 200 amino acids to 225 amino acids, or a length of 225 amino acids to 256 amino acids; e.g., a length of 256 amino acids).

[0091] For example, the LBD of a PR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P06401 aa686-933, and has a length of about 200 amino acids to 250 amino acids (e.g., a length of 200 amino acids to 225 amino acids, or a length of 225 amino acids to 250 amino acids; e.g., a length of 248 amino acids).

[0092] Where one member of a pair of heterodimerization domains is the LBD of a PR, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence GHSFADPASNLGLEDIIRKALMGSF (Uniprot O75376 aa2251-2275), where the coregulatory peptide has a length of from about 25 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0093] Thyroid hormone receptor beta: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of thyroid hormone receptor beta (TR-beta). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of TR-beta (Uniprot P10828).

[0094] For example, the LBD of TR-beta has the following amino acid sequence (Uniprot Q4T8V6 aa223~502; Uniprot Q90382.1 aa159~401; Uniprot P18115.2 aa170~412; Uniprot Q9PVE4.2 aa141~392; Uniprot P10828.2 aa216~458; GenBank ABS11249.1 aa179~419; NCBI REF SEQ XP_001185977.1 aa186~416; PDB 1NAV_A aa17~259; PDB 2PIN_A aa8~250; PDB 3D57_A aa22~264; PDB 1N46_A aa13~255; PDB 1BSX_A aa13~256; PDB 1N46_A aa13~255; PDB 1N46_A aa13~256 ... The amino acid sequence may have 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 any of the sequences (aa15 to 257) and may have a length of about 200 to 250 amino acids (e.g., a length of 200 to 225 amino acids, 225 to 230 amino acids, 230 to 240 amino acids, or 240 to 250 amino acids).

[0095] For example, the LBD of TR-beta can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10828 aa202-461, and has a length of about 200 amino acids to 260 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or 225 amino acids to 260 amino acids, e.g., 260 amino acids).

[0096] For example, the LBD of TR-beta can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10828 aa216-461, and has a length of about 200 amino acids to 246 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or 225 amino acids to 246 amino acids, e.g., 246 amino acids).

[0097] Where one member of the pair of heterodimerization domains is the LBD of TR-beta, the second member of the pair may be, for example, an NCOA3 / SRC3 polypeptide comprising the amino acid sequence Uniprot Q9Y6Q9 aa627-829, or Uniprot Q9Y6Q9 aa673-750, or Uniprot Q15596 aa721-1021.

[0098] Estrogen receptor-alpha: In a preferred embodiment, an LBD suitable for inclusion as a member of a pair of heterodimerization domains may be the LBD of estrogen receptor alpha (ERalpha). For example, in some cases, the LBD can comprise an amino acid sequence having 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 the LBD of ER-alpha (Uniprot P03372).

[0099] For example, the LBD of ER-alpha has the following amino acid sequence (Uniprot P06212.1 aa304-541; Uniprot P81559.1 aa302-539; Uniprot Q7ZU32 aa280-517; GenBank ACB10649.1 aa303-529; GenBank ABQ42696.1 aa226-468; GenBank ACC85903.1 aa141-375; PDB 1XP9_A aa4-241; PDB 1YY4_A aa1-241). aa1-236), and has a length of about 200 amino acids to 240 amino acids (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).

[0100] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa305-533, and has a length of about 180 amino acids to 229 amino acids (e.g., has a length of 180 amino acids to 200 amino acids, or 200 amino acids to 229 amino acids, e.g., 229 amino acids).

[0101] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa282-595, and has a length of about 250 amino acids to 314 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 314 amino acids, e.g., 314 amino acids).

[0102] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa310-547, and has a length of about 190 amino acids to 238 amino acids (e.g., has a length of 190 amino acids to 220 amino acids, or 220 amino acids to 238 amino acids, e.g., 238 amino acids).

[0103] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa305-533 having the substitution D351Y, and has a length of about 180 amino acids to 229 amino acids (e.g., has a length of 180 amino acids to 200 amino acids, or 200 amino acids to 229 amino acids, e.g., 229 amino acids).

[0104] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa282-595 having the substitution D351Y, and has a length of about 250 amino acids to 314 amino acids (e.g., 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 314 amino acids, e.g., a length of 314 amino acids).

[0105] For example, the LBD of ER-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P03372 aa310-547 having the substitution D351Y, and has a length of about 190 amino acids to 238 amino acids (e.g., has a length of 190 amino acids to 220 amino acids, or 220 amino acids to 238 amino acids, e.g., 238 amino acids).

[0106] Where one member of the pair of heterodimerization domains is the LBD of ER-alpha, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence DAFQLRQLILRGLQDD (SEQ ID NO: 10), where the coregulatory peptide has a length of from about 16 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of from 16 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).

[0107] Where one member of the pair of heterodimerization domains is the LBD of ER-alpha, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence SPGSREWFKDMLS (SEQ ID NO:11), where the coregulatory peptide has a length of from about 13 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of from 13 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).

[0108] Estrogen Receptor-Beta (ER-Beta): In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of estrogen receptor-beta (ER-beta). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of ER-beta (Uniprot Q92731).

[0109] For example, the LBD of ER-beta has the following amino acid sequence (Uniprot P06212.1 aa304-541; Uniprot P81559.1 aa302-539; Uniprot Q7ZU32 aa280-517; GenBank ACB10649.1 aa303-529; GenBank ABQ42696.1 aa226-468; GenBank ACC85903.1 aa141-375; PDB 1XP9_A aa4-241; PDB 1YY4_A aa1-241). aa1-236), and has a length of about 200 amino acids to 243 amino acids (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).

[0110] For example, the LBD of ER-beta can comprise an amino acid sequence having 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 the amino acid sequence Uniprot Q92731 aa260-502, and has a length of about 200 amino acids to 243 amino acids (e.g., has a length of 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).

[0111] Where one member of the pair of heterodimerization domains is the LBD of ER-beta, the second member of the dimerization pair can be a coregulatory peptide comprising the amino acid sequence PRQGSILYSMLTSAKQT (SEQ ID NO: 12), where the coregulatory peptide has a length of from about 17 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of from 17 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).

[0112] Peroxisome proliferator-activated receptor-gamma: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of PPAR-gamma (Uniprot P37231).

[0113] For example, the LBD of PPAR-gamma has the following amino acid sequence (Uniprot Q4H2X4 aa176-417; Uniprot P37233.1 aa129-395; Uniprot Q7T029 aa95-435; GenBank AAL26245.1 aa95-435; NCBI REF SEQ XP_781750.1 aa137-378; NCBI REF SEQ XP 784429.2 aa219-478; NCBI REF SEQ NP_001001460.1 aa207-474; PDB 2J14_A aa17-284; PDB 1FM6_D The amino acid sequence can include an amino acid sequence having 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 one of the sequences (aa4-271) and has a length of about 200 amino acids to 269 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 269 amino acids).

[0114] For example, the LBD of PPAR-gamma can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P37231 aa174-475, and has a length of about 150 amino acids to 202 amino acids (e.g., has a length of 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).

[0115] For example, the LBD of PPAR-gamma can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P37231 aa181-475, and has a length of about 200 amino acids to 269 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 269 amino acids).

[0116] For example, the LBD of PPAR-gamma can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P37231 aa205-475, and has a length of about 200 amino acids to 269 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, 225 amino acids to 250 amino acids, or 250 amino acids to 271 amino acids).

[0117] Where one member of the pair of heterodimerization domains is the LBD of PPAR-gamma, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence CCPSSHSSLTERHKILHRLLQEGSPS (Uniprot Q15788-1 aa676-700), where the coregulatory peptide has a length of from about 25 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of from 25 amino acids to 28 amino acids, from 28 amino acids to 29 amino acids, from 29 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).

[0118] Where one member of the pair of heterodimerization domains is the LBD of PPAR-gamma, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence PKKENNALLRYLLDRDDPSDV (SEQ ID NO: 13) or PKKKENALLRYLLDKDDTKDI (Uniprot Q15596-1 aa 737-757), where the coregulatory peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0119] Glucocorticoid receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of the glucocorticoid receptor (GR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of GR having the amino acid sequence Uniprot P04150-3.

[0120] For example, the LBD of GR has the following amino acid sequence (Uniprot Q4RIR9 aa110-356; Uniprot P49844.1 aa530-776; NCBI REF SEQ NP_001032915.1 aa526-772; PDB 1NHZ_A 34-280; PDB 1M2Z_A aa11-257; PDB 3BQD_A aa9-255; PDB 3CLD_A The amino acid sequence may include an amino acid sequence having 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 one of the sequences (aa13-259) and having a length of about 200 amino acids to 247 amino acids (e.g., having a length of 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).

[0121] For example, the LBD of GR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P04150-3 aa532-778, and has a length of about 200 amino acids to 247 amino acids (e.g., has a length of 200 amino acids to 225 amino acids, or 225 amino acids to 247 amino acids, e.g., 247 amino acids).

[0122] Where one member of a pair of heterodimerization domains is the LBD of GR, the second member of the pair may be, for example, an NCOA2 / SRC2 polypeptide comprising the amino acid sequence Uniprot Q15788 aa1172-1441, or a fragment thereof, or Uniprot Q15596 aa320-1021, or a fragment thereof.

[0123] Vitamin D Receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of the Vitamin D Receptor (VDR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of the VDR (Uniprot P11473).

[0124] For example, the LBD of a VDR can comprise an amino acid sequence having 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 one of the following amino acid sequences (Uniprot O42392.1 aa147-450; NCBI REF SEQ NP_001079288.1 aa125-421; PDB 2HBH_A aa5-301; PDB 1S0Z_A aa11-262) and has a length of about 250 amino acids to 310 amino acids (e.g., has a length of 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 310 amino acids).

[0125] For example, the LBD of a VDR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P11473 aa124-426, and has a length of about 250 amino acids to 303 amino acids (e.g., 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 303 amino acids).

[0126] When one member of a pair of heterodimerization domains is the LBD of a VDR, the second member of the pair can be, for example, an NCOA1 / SRC1 polypeptide comprising the amino acid sequence Uniprot Q15788 aa1172-1441 or a fragment thereof, or Uniprot Q15596 aa320-1021 or a fragment thereof.

[0127] For example, if one member of a pair of heterodimerization domains is the LBD of a VDR, the other member of the pair can be an NCOA2 / SRC2 polypeptide comprising the amino acid sequence Uniprot Q15596 aa744-751, where the coregulatory peptide has a length of about 8 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0128] Thyroid hormone receptor-alpha: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of thyroid hormone receptor-alpha (TR-alpha). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of TR-alpha (Uniprot P10827-2).

[0129] For example, the LBD of TR-alpha has the following amino acid sequence (Uniprot Q4T8V6 aa223~502; Uniprot Q90382.1 aa159~401; Uniprot P18115.2 aa170~412; Uniprot Q9PVE4.2 aa141~392; Uniprot P10828.2 aa216~458; GenBank ABS11249.1 aa179~419; NCBI REF SEQ XP_001185977.1 aa186~416; PDB 1NAV_A aa17~259; PDB 2PIN_A aa8~250; PDB 3D57_A aa22~264; PDB 1N46_A aa13~255; PDB 1BSX_A aa13~256; PDB 1N46_A aa13~25 ... The amino acid sequence may have 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 with a sequence selected from the group consisting of aa 15-257, and may have a length of about 190 amino acids to about 245 amino acids (e.g., a length of 190 amino acids to 210 amino acids, 210 amino acids to 230 amino acids, or 230 amino acids to 245 amino acids).

[0130] For example, the LBD of TR-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10827-2 aa162-404, and has a length of about 190 amino acids to about 243 amino acids (e.g., a length of 190 amino acids to 210 amino acids, 210 amino acids to 230 amino acids, or 230 amino acids to 243 amino acids).

[0131] A suitable coregulatory peptide for TR-alpha may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 to 50 amino acids in length derived from an SRC1 polypeptide).

[0132] Retinoic acid receptor-beta: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of retinoic acid receptor-beta (RAR-beta). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of RAR-beta (Uniprot P10826-2).

[0133] For example, the LBD of RAR-beta has the following amino acid sequence (Uniprot Q4H2W2 aa400-634; Uniprot P22448.2 aa186-416; Uniprot P28699.2 aa209-439; Uniprot Q91392.2 aa176-406; NCBI REF SEQ XP_779976.2 aa134-362; NCBI REF SEQ XP_002204386.1 aa179-409; PDB 1XAP_A aa32-262; PDB 1XDK_B aa34-264; PDB 1DKF_B aa36-264). aa5-235), and has a length of about 180 amino acids to about 235 amino acids (e.g., 180 amino acids to 200 amino acids, 200 amino acids to 220 amino acids, or 220 amino acids to 235 amino acids).

[0134] For example, the LBD of RAR-beta can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P10826-2 aa179-409, and has a length of about 180 amino acids to about 231 amino acids (e.g., a length of 180 amino acids to 200 amino acids, 200 amino acids to 220 amino acids, or 220 amino acids to 231 amino acids).

[0135] A suitable coregulatory peptide of RAR-beta may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 to 50 amino acids in length derived from the SRC1 polypeptide).

[0136] When one member of the pair of heterodimerization domains is the LBD of RAR-beta, the other member of the dimerization pair may be, for example, an NCOA1 / SRC1 polypeptide comprising the amino acid sequence Uniprot Q15788 aa1172-1441 or a fragment thereof.

[0137] When one member of the pair of heterodimerization domains is the LBD of RAR-beta, the other member of the dimerization pair may be, for example, an NCOA2 / SRC2 polypeptide comprising the amino acid sequence Uniprot Q15596 aa320-1021 or a fragment thereof.

[0138] Farnesoid X Receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of farnesoid X receptor (FXR). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of FXR having the amino acid sequence Uniprot Q96RI1-2.

[0139] For example, the LBD of FXR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot Q96RT1-2 aa237-472, and has a length of about 100 amino acids to about 136 amino acids (e.g., a length of 100 amino acids to 110 amino acids, 110 amino acids to 120 amino acids, or 120 amino acids to 136 amino acids).

[0140] A suitable coregulatory peptide for FXR may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 amino acids to 50 amino acids in length derived from an SRC1 polypeptide).

[0141] LXR-Alpha: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of Liver X Receptor-alpha (LXR-alpha). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of LXR-alpha having the amino acid sequence Uniprot Q13133-1.

[0142] For example, the LBD of LXR-alpha can include an amino acid sequence having 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 the amino acid sequence Uniprot Q13133-1 aa182-447, and can have an amino acid sequence having a length of about 200 amino acids to about 266 amino acids (e.g., having a length of 200 amino acids to 220 amino acids, 220 amino acids to 240 amino acids, or 240 amino acids to 266 amino acids).

[0143] A suitable coregulatory peptide for LXR-alpha can be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 amino acids to 50 amino acids in length derived from an SRC1 polypeptide).

[0144] ROR-Gamma: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of retinoid-related orphan receptor gamma (ROR-gamma). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of ROR-gamma having the amino acid sequence Uniprot P51449-2.

[0145] For example, the LBD of ROR-gamma can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P51449-2 aa 237-497, and has a length of about 200 amino acids to about 261 amino acids (e.g., has a length of 200 amino acids to 220 amino acids, 220 amino acids to 240 amino acids, or 240 amino acids to 261 amino acids).

[0146] A suitable coregulatory peptide for ROR-gamma may be the NCORNR peptide (CDPASNLGLEDIIRKALMGSFDDK, Uniprot Q7Z516-1 aa2160-2182).

[0147] A suitable coregulatory peptide for ROR-gamma may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 to 50 amino acids in length derived from an SRC1 polypeptide).

[0148] RXR-Alpha: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of retinoid-X receptor-alpha (RXR-alpha). For example, in some cases, the LBD can include an amino acid sequence having 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 the LBD of RXR-alpha having the amino acid sequence Uniprot P19793-1.

[0149] For example, the LBD of RXR-alpha can comprise an amino acid sequence having 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 the amino acid sequence Uniprot P19793-1 aa225-462, and has a length of about 190 amino acids to about 238 amino acids (e.g., a length of 190 amino acids to 200 amino acids, 200 amino acids to 210 amino acids, or 210 amino acids to 238 amino acids).

[0150] A suitable coregulatory peptide for RXR-alpha may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 to 50 amino acids in length derived from an SRC1 polypeptide).

[0151] PXR: In some cases, the LBD suitable for inclusion as a member of the pair of heterodimerization domains may be the LBD of pregnane X receptor (PXR). For example, in some cases, the LBD may comprise an amino acid sequence having 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 with the LBD of PXR having the amino acid sequence Uniprot O75469-1. In some cases, the LBD may comprise an amino acid sequence having 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 with amino acids 143-428 of the amino acid sequence of Uniprot O75469-1. In some cases, the LBD comprises an amino acid sequence having 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 amino acids 205 to 434 of the amino acid sequence set forth in Uniprot O75469-1.

[0152] For example, the LBD of PXR can comprise an amino acid sequence having 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 the amino acid sequence Uniprot O75469-1 aa130-434, and has a length of about 250 amino acids to about 302 amino acids (e.g., a length of 250 amino acids to 275 amino acids, 275 amino acids to 290 amino acids, or 290 amino acids to 302 amino acids).

[0153] A suitable coregulatory peptide for PXR may be an SRC1 polypeptide or a fragment thereof (eg, a peptide of 8 amino acids to 50 amino acids in length derived from an SRC1 polypeptide).

[0154] 8.1.1.2. Coregulatory Polypeptides: Suitable coregulatory polypeptides include full-length naturally occurring nuclear hormone coregulatory polypeptides. Suitable coregulatory polypeptides include fragments of naturally occurring nuclear hormone coregulatory polypeptides. Suitable coregulatory polypeptides include synthetic or recombinant nuclear hormone coregulatory polypeptides.

[0155] A suitable coregulatory polypeptide may have a length of 8 to 2000 amino acids. A suitable coregulatory polypeptide may have a length of 8 to 50 amino acids, such as 8 to 10 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, 20 to 25 amino acids, 25 to 30 amino acids, 30 to 35 amino acids, 35 to 40 amino acids, 40 to 45 amino acids, or 45 to 50 amino acids. A suitable coregulatory polypeptide may have a length of 50 to 100 amino acids, such as 50 to 60 amino acids, 60 to 70 amino acids, 70 to 80 amino acids, 80 to 90 amino acids, or 90 to 100 amino acids. Suitable coregulatory polypeptides may have a length of 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids. Suitable coregulatory polypeptides may have a length of 1000 to 2000 amino acids.

[0156] Suitable coregulatory peptides include steroid receptor coactivator-(SRC)-1, SRC-2, SRC-3, TRAP220-1, TRAP220-2, NR0B1, NRIP1, CoRNR box, alpha-beta V, 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, PGC1a, PGC1b, PRC, ASC2-1, ASC2-2, CBP- 1, CBP-2, P300, CIA, ARA70-1, ARA70-2, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, CoRNR1 (N-CoR), CoRNR2, SMRT, RIP140-C, RIP140-1, RIP140-2, RIP140-3, RIP140-4, RIP140-5, RIP140-6, RIP140-7, RIP140-8, RIP140-9, PRIC285-1, PRIC285-2, PRIC285-3, PRIC285-4, and PRIC285-5.

[0157] Coregulatory polypeptides suitable for heterodimerization with the respective LBD dimerization partners preferably have a length of 8 to 10 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, 20 to 25 amino acids, 25 to 30 amino acids, 30 to 35 amino acids, 35 to 40 amino acids, 40 to 45 amino acids, or 45 to 50 amino acids; preferably have 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 with a stretch of 8 to 50 consecutive amino acids of the following amino acid sequences: SRC1 (Uniprot Q15788-1), SRC2 (Uniprot Q15596-1), SRC3 (Uniprot Q9Y6Q9-5), PGC1a (Uniprot Q9UBK2-1), PGC1b (Uniprot Q15788-1), SRC4 (Uniprot Q15788-1), SRC5 (Uniprot Q15788-2), SRC6 (Uniprot Q15788-3), SRC7 (Uniprot Q15788-4), SRC8 (Uniprot Q15788-5), SRC9 (Uniprot Q15788-6), SRC1b (Uniprot Q15788-7), SRC1c (Uniprot Q15788-8), SRC1d (Uniprot Q15788-9), SRC1e (Uniprot Q15788-1), SRC2f (Uniprot Q15788-1), SRC3f (Uniprot Q15788-1), SRC4f (Uniprot Q15788-1), SRC5f (Uniprot Q15788-2), SRC6f (Uniprot Q86YN6-1), PPRC-1(Uniprot Q5VV67-1), TRAP220(Uniprot Q15648-1), NCOA6(Uniprot Q14686-1), CREBBP(Uniprot Q92793-1), EP300(Uniprot Q09472-1), NCOA5(Uniprot Q9HCD5-1), NCOA4(Uniprot Q13772-1), TRIM24(Uniprot O15164-2), NSD1(Uniprot Q96L73-1), BRD8(Uniprot Q9H0E9-2), KAT5(Uniprot Q92993-1), NCOA7(Uniprot Q9BQI9-1), LCoR(Uniprot Q96JN0-1), N-CoR (Uniprot O75376-1), NCOR2 (Uniprot Q9Y618-1), RIP140 (Uniprot P48552-1), PRIC285 (Uniprot Q9BYK8-2).

[0158] In a preferred embodiment, a suitable coregulatory peptide comprises an LXXLL motif, where X is any amino acid, where the coregulatory peptide has a length of 8 to 50 amino acids, e.g., 8 to 10 amino acids, 10 to 12 amino acids, 12 to 15 amino acids, 15 to 20 amino acids, 20 to 25 amino acids, 25 to 30 amino acids, 30 to 35 amino acids, 35 to 40 amino acids, 40 to 45 amino acids, or 45 to 50 amino acids.

[0159] Examples of suitable coregulatory peptides are as follows: SRC1: (Uniprot Q15788-1 aa676-700) CPSSHSSLTERHKILHRLLQEGSPS; SRC1-2: (Uniprot Q15788-1 aa682-702; SNP rs1049021 E685A) SLTARHKILHRLLQEGSPSDI; SRC3-1: (Uniprot Q9Y6Q9-5 aa614-632) ESKGHKKLLQLLTCSSDDR; SRC3: (SEQ ID NO: 13) PKKENNALLRYLLDRDDPSDV; PGC-1: (Uniprot Q9UBK2-1 aa138-154) AEEPSLLKKLLLAPANT; PGC1a: (Uniprot Q9UBK2-1 aa136~156)QEAEEPSLLKKLLLAPANTQL;TRAP220-1:(Uniprot Q15648-1 aa596~616)SKVSQNPILTSLLQITGNGGS;NCoR:(Uniprot O75376-1 aa2251~2275)GHSFADPASNLGLEDIIRKALMGSF;NR0B1:(Uniprot P51843-1 aa74~90)PRQGSILYSMLTSAKQT;NRIP1:(Uniprot P48552-1 aa374~390)AANNSLLLHLLKSQTIP;TIF2:(Uniprot Q15596-1 aa737~757)PKKKENALLRYLLDKDDTKDI;CoRNR Box: (SEQ ID NO: 14) DAFQLRQLILRGLQDD; abV: (SEQ ID NO: 11) SPGSREWFKDMLS; TRAP220-2: (Uniprot Q15648-1 aa 637-657) GNTKNHPMLMNLLKDNPAQDF; EA2: (SEQ ID NO: 15) SSKGVLWRMLAEPVSR; TA1: (SEQ ID NO: 16) SRTLQLDWGTLYWSR; EAB1: (SEQ ID NO: 17) SSNHQSSRLIELLSR; SRC2: (Uniprot Q15596-1 aa 683-701) LKEKHKILHRLLQDSSSPV; SRC1-3: (Uniprot Q15788-1 aa 1428-1441) QAQQKSLLQQLLTE;SRC1-1:(Uniprot Q15788-1 aa625~645)KYSQTSHKLVQLLTTTAEQQL;SRC1-2:(Uniprot Q15788-1 aa682~702;SNP rs1049021 E685A)SLTARHKILHRLLQEGSPSDI;SRC1-3:(Uniprot Q15788-1 aa741~761)KESKDHQLLRYLLDKDEKDLR;SRC1-4a:(Uniprot Q15788-1 aa1427~1441)PQAQQKSLLQQLLTE;SRC1-4b:(Uniprot Q15788-1 aa1427~1441 L1435R)PQAQQKSLRQQLLTE;GRIP1-1:(Uniprot Q15596-1 aa633~653)HDSKGQTKLLQLLTTKSDQME;GRIP1-2:(Uniprot Q15596-1 aa682~702)SLKEKHKILHRLLQDSSSPVD;GRIP1-3:(Uniprot Q15596-1 aa737~757)PKKKENALLRYLLDKDDTKDI;AIB1-1:(Uniprot Q9Y6Q9-5 aa613~633)LESKGHKKLLQLLTCSSDDRG;AIB1-2:(Uniprot Q9Y6Q9-5 aa677~697)LLQEKHRILHKLLQNGNSPAE;AIB1-3:(Uniprot Q9Y6Q9-5 aa730~750)KKKENNALLRYLLDRDDPSDA;PGC1a:(Uniprot Q9UBK2-1 aa136~156)QEAEEPSLLKKLLLAPANTQL;PGC1b:(Uniprot Q86YN6-1 aa148~168)PEVDELSLLQKLLLATSYPTS;PRC:(Uniprot Q5VV67-1 aa156~176)VSPREGSSLHKLLTLSRTPPE;TRAP220-1:(Uniprot Q15648-1 aa596~616)SKVSQNPILTSLLQITGNGGS;TRAP220-2:(Uniprot Q15648-1 aa637~657)GNTKNHPMLMNLLKDNPAQDF;ASC2-1:(Uniprot Q14686-1 aa879~899)DVTLTSPLLVNLLQSDISAGH;ASC2-2:(Uniprot Q14686-1 aa1483~1503)AMREAPTSLSQLLDNSGAPNV;CBP-1:(Uniprot Q92793-1 aa62~82)DAASKHKQLSELLRGGSGSSI;CBP-2:(Uniprot Q92793-1 aa350~370)KRKLIQQQLVLLLHAHKCQRR;P300:(Uniprot Q09472-1 aa73~93)DAASKHKQLSELLRSGSSPNL;CIA:(Uniprot Q9HCD5-1 aa337~357)GHPPAIQSLINLLADNRYLTA;ARA70-1:(Uniprot Q13772-1 aa84~104)TLQQQAQQLYSLLGQFNCLTH;ARA70-2:(Uniprot Q13772-1 aa320~340)GSRETSEKFKLLFQSYNVNDW;TIF1:(Uniprot O15164-2 aa718~738)NANYPRSILTSLLLNSSQSST;NSD1:(Uniprot Q96L73-1 aa899~919)IPIEPDYKFSTLLMMLKDMHD;SMAP:(Uniprot Q9H0E9-2 aa263~283)ATPPPSPLLSELLKKGSLLPT;Tip60:(Uniprot Q92993-1 aa481~501)VDGHERAMLKRLLRIDSKCLH;ERAP140:(Uniprot Q8NI08-1 aa514~534)HEDLDKVKLIEYYLTKNKEGP;Nix1:(Uniprot Q9BQI9-1 aa236~256)ESPEFCLGLQTLLSLKCCIDL;LCoR:(Uniprot Q96JN0-1 aa45~65)AATTQNPVLSKLLMADQDSPL;CoRNR1(N-CoR):(Uniprot O75376-1 aa239~268)MGQVPRTHRLITLADHICQIITQDFARNQV;CoRNR2(N-CoR):(Uniprot O75376-1 aa2260~2273)NLGLEDIIRKALMG;CoRNR1(SMRT):(Uniprot Q9Y618-1 aa2131~2170)APGVKGHQRVVTLAQHISEVITQDTYRHHPQQLSAPLPAP;CoRNR2(SMRT):(Uniprot Q9Y618-1 aa2347~2360)NMGLEAIIRKALMG;RIP140-C:(SEQ ID NO: 18)RLTKTNPILYYMLQKGGNSVA;RIP140-1:(Uniprot P48552-1 aa13~33)QDSIVLTYLEGLLMHQAAGGS;RIP140-2:(Uniprot P48552-1 aa125~145)KGKQDSTLLASLLQSFSSRLQ;RIP140-3:(Uniprot P48552-1 aa177~197)CYGVASSHLKTLLKKSKVKDQ;RIP140-4:(Uniprot P48552-1 RIP140-5: (Uniprot P48552-1 aa372-392) KQAANNSLLLHLLKSQTIPKP; RIP140-6: (Uniprot P48552-1 aa493-513) NSHQKVTLLQLLLGHKNEENV; RIP140-7: (SEQ ID NO: 19) NLLERRTVLQLLLGNPTKGRV; RIP140-8: (Uniprot P48552-1 aa811-831) FSFSKNGLLSRLLRQNQDSYL; RIP140-9: (Uniprot P48552-1 aa928-948) RESKSFNVLKQLLLSENCVRD; PRIC285-1: (Uniprot Q9BYK8-2 aa458~518)ELNADDAILRELLDESQKVMV;PRIC285-2:(Uniprot Q9BYK8-2 aa541~561)YENLPPAALRKLLRAEPERYR;PRIC285-3:(Uniprot Q9BYK8-2 aa596~616) MAFAGDEVLVQLLSGDKAPEG; PRIC285-4: (Uniprot Q9BYK8-2 aa1435~1455) SCCYLCIRLEGLLAPTASPRP; and PRIC285-5: (Uniprot Q9BYK8-2 aa1652~1672) PSNKSVDVLAGLLLRRMELKP. ;

[0160] In some cases, a given LBD can be paired with two or more different coregulatory polypeptides. For example, PPAR-gamma (Uniprot P37231) is a nucleotide sequence encoding SRC1 (Uniprot Q15788-1 aa625-645; Uniprot Q15788-1 aa676-700; Uniprot Q15788-1 aa682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa741-761; Uniprot Q15788-1 aa1428-1441; Uniprot Q15788-1 aa1427-1441; Uniprot Q15788-1 aa1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa683-701), SRC3 (SEQ ID NO: 13; Uniprot Q9Y6Q9-5 As another example, ER-alpha (Uniprot P03372) can be paired with CoRNR (Uniprot O75376-1 aa239-268; Uniprot O75376-1 aa2260-2273; Uniprot Q9Y618-1 aa2131-2170; Uniprot Q9Y618-1 aa2347-2360), alpha-beta V (SEQ ID NO: 11), or TA1 (SEQ ID NO: 16). As another example, ER-beta (Uniprot Q92731) can pair with CoRNR (Uniprot O75376-1 aa239-268; Uniprot O75376-1 aa2260-2273; Uniprot Q9Y618-1 aa2131-2170; Uniprot Q9Y618-1 aa2347-2360), alpha-beta V (SEQ ID NO: 11), or TA1 (SEQ ID NO: 16).As another example, AR (Uniprot P10275) is a nucleotide sequence encoding SRC1 (Uniprot Q15788-1 aa625-645; Uniprot Q15788-1 aa676-700; Uniprot Q15788-1 aa682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa741-761; Uniprot Q15788-1 aa1428-1441; Uniprot Q15788-1 aa1427-1441; Uniprot Q15788-1 aa1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa683-701), SRC3 (SEQ ID NO: 13; Uniprot Q9Y6Q9-5 It can pair with TRAP220 (Uniprot Q15648-1 aa596-616; Uniprot Q15648-1 aa637-657), or TRAP220 (Uniprot Q15648-1 aa596-616; Uniprot Q15648-1 aa637-657). As another example, PR (Uniprot P06401) is a nucleotide sequence encoding SRC1 (Uniprot Q15788-1 aa625-645; Uniprot Q15788-1 aa676-700; Uniprot Q15788-1 aa682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa741-761; Uniprot Q15788-1 aa1428-1441; Uniprot Q15788-1 aa1427-1441; Uniprot Q15788-1 aa1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa683-701), SRC3 (SEQ ID NO: 13; Uniprot Q9Y6Q9-5 It can pair with TRAP220 (Uniprot Q15648-1 aa596-616; Uniprot Q15648-1 aa637-657), NR0B1 (Uniprot P51843-1 aa74-90), PGC1B (Uniprot Q86YN6-1 aa148-168), NRIP1 (Uniprot P48552-1 aa374-390), EA2 (SEQ ID NO: 15), or EAB1 (SEQ ID NO: 17).As another example, TR-beta (Uniprot P10828) is a nucleotide sequence encoding SRC1 (Uniprot Q15788-1 aa625-645; Uniprot Q15788-1 aa676-700; Uniprot Q15788-1 aa682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa741-761; Uniprot Q15788-1 aa1428-1441; Uniprot Q15788-1 aa1427-1441; Uniprot Q15788-1 aa1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa683-701), SRC3 (SEQ ID NO: 13; Uniprot Q9Y6Q9-5 It can pair with TRAP220 (Uniprot Q15648-1 aa596-616; Uniprot Q15648-1 aa637-657), or TRAP220 (Uniprot Q15648-1 aa596-616; Uniprot Q15648-1 aa637-657).

[0161] 8.1.1.3. Regulatory molecules for LBD-based heterodimerization: When one member of the pair of heterodimerization domains is the LBD of a nuclear hormone receptor, at least one type of regulatory molecule used can bind to the LBD of a first CAR molecule of the group, which can then heterodimerize with a co-regulatory peptide in a second CAR molecule of the group.

[0162] Regulatory molecules suitable for LBD-based heterodimerization systems are known in the art. Examples of regulatory molecules for LBD-based heterodimerization systems include: corticosterone ((8S,9S,10R,11S,13S,14S,17S)-11-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); deoxycorticosterone ((8S,9S,10R,13S,14S,17S)-17 -(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one; Cortisol ((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-3-one); Tren-3-one; 11-deoxycortisol ((8R,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-3-one); Cortisone ((8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl -1,2,6,7,8,9,12,14,15,16-Decahydrocyclopenta[a]phenanthrene-3,11-dione;18-hydroxycorticosterone ((8S,9S,10R,11S,13R,14S,17S)-11-hydroxy-17-(2-hydroxyacetyl)-13-(hydroxymethyl)-10-methyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthrene-3-one);1α-hydroxycorticosterone ((1S,8S,9S,10R,11S,13S,14S,17S)-1,11-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); aldosterone ((8S,9S,10R,11S,13R,14S,17S)-11-hydroxy-17-(2-hydroxyacetyl)-10-methyl-3-oxo-1,2,6,7,8,9,11,12,14,15,16, 17-Dodecahydrocyclopenta[a]phenanthrene-13-carbaldehyde;Androstenedione ((8R,9S,10R,13S,14S)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione);4-Hydroxy-androstenedione ((8R,9S,10R,13S,14S)-4-hydroxy-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione) -3,17-dione; 11β-hydroxyandrostenedione ((8S,9S,10R,11S,13S,14S)-11-hydroxy-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione); androstanediol ((3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3 ,17-diol;Androsterone ((3R,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecahydrocyclopenta[a]phenanthren-17-one);Epiandrosterone ((3S,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecahydrocyclopenta[a]phenanthren-17-one);Adrenosterone ((8S,9S,10R,13S,14S)-10,13-dimethyl-1,2,6,7,8,9,12,14,15,16-decahydrocyclopenta[a]phenanthrene-3,11,17-trione); Dehydroepiandrosterone ((3S,8R,9S,10R,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthrene-17-one); Dehydroepiandrosterone sulfate ([( 3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-oxo-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-3-yl]hydrogensulfate; Testosterone ((8R,9S,10R,13S,14S,17S)-17-hydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); Epitestosterone ((8R,9S ,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one; 5α-dihydrotestosterone ((5S,8R,9S,10S,13S,14S,17S)-17-hydroxy-10,13-dimethyl-1,2,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-3-one); 5β-dihydrotestosterone ( (5R,8R,9S,10S,13S,14S,17S)-17-hydroxy-10,13-dimethyl-1,2,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-3-one; 5β-dihydrotestosterone ((5R,8R,9S,10S,13S,14S,17S)-17-hydroxy-10,13-dimethyl-1,2,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydrocyclopenta[a]phenanthren-3-one);11β-hydroxytestosterone ((8S,9S,10R,11S,13S,14S,17S)-11,17-dihydroxy-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); 11-ketotestosterone ((8S,9S,10R,13S,14S,17S)-17-hydroxy-10,13-dimethyl-2,6,7,8,9,12,14,15,16,17-decahydro-1H-cyclopenta[ a]phenanthrene-3,11-dione; estrone ((8R,9S,13S,14S)-3-hydroxy-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-17-one); estradiol ((8R,9S,13S,14S,17S)-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol); estriol ((8R,9S,13S, 14S,16R,17R)-13-Methyl-6,7,8,9,11,12,14,15,16,17-Decahydrocyclopenta[a]phenanthrene-3,16,17-triol; Pregnenolone (1-[(3S,8S,9S,10R,13S,14S,17S)-3-Hydroxy-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-17-yl]ethanone); 17-Hydroxypregnenolone (1 -[(3S,8R,9S,10R,13S,14S,17R)-3,17-dihydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-17-yl]ethanone;Progesterone ((8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one);17-Hydroxyprogesterone ((8R,9S,10R,13S,14S,17R)-17-acetyl-17-hydroxy-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-3-one); T3 ((2S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoic acid); T4 ((2S)-2-amino-3-[ 4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoic acid;Spironolactone (S-[(7R,8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3,5'-dioxospiro[2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-17,2'-oxolan]-7-yl]ethanethioate);Eplerenone (PubChem CID 443872);

[0163] Cyproterone acetate (PubChem CID 9880); Hydroxyflutamide (2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide); Eanzalutamide (4-[3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-4-oxo-2-sulfanylideneimidazolidin-1-yl]-2-fluoro-N-methylbenzamide); ARN-509 (4-[7-[6-cyano-5-(trifluoromethyl)pyridin-3-yl]-8-oxo-6-sulfanylidene-5,7-diazaspiro[3 .4]octan-5-yl]-2-fluoro-N-methylbenzamide;3,3'-diindolylmethane (DIM) (3-(1H-indol-3-ylmethyl)-1H-indole);bexlosteride ((4aR,10bR)-8-chloro-4-methyl-1,2,4a,5,6,10b-hexahydrobenzo[f]quinolin-3-one);bicalutamide (N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenyl)sulfonyl-2-hydroxy-2-methylpropanamide);N-butylbenzene -Sulfonamides (NBBS) (N-butylbenzenesulfonamides); Dutasteride ((1S,3aS,3bS,5aR,9aR,9bS,11aS)-N-[2,5-bis(trifluoromethyl)phenyl]-9a,11a-dimethyl-7-oxo-1,2,3,3a,3b,4,5,5a,6,9b,10,11-dodecahydroindeno[5,4-f]quinoline-1-carboxamide); Epristeride ((8S,9S,10R,13S,14S,17S)-17-(tert-butylcarbamoyl)-10,13-dimethyl -2,7,8,9,11,12,14,15,16,17-Decahydro-1H-cyclopenta[a]phenanthrene-3-carboxylic acid;Finasteride ((1S,3aS,3bS,5aR,9aR,9bS,11aS)-N-tert-butyl-9a,11a-dimethyl-7-oxo-1,2,3,3a,3b,4,5,5a,6,9b,10,11-dodecahydroindeno[5,4-f]quinoline-1-carboxamide);Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide);Izonsteride ((4aR,10bR)-8-[(4-ethyl-1,3-benzothiazol-2-yl)sulfanyl]-4,10b-dimethyl-2,4a,5,6-tetrahydro-1H-benzo[f]quinolin-3-one); Ketoconazole (1-[4-[4-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]ethanone); N-Butylbenzene-sulfonamide (N-butylbenzenesulfonamide); Nilutamide (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl)phenyl]imidazo Lysine-2,4-dione; Megestrol ((8R,9S,10R,13S,14S,17R)-17-acetyl-17-hydroxy-6,10,13-trimethyl-2,8,9,11,12,14,15,16-octahydro-1H-cyclopenta[a]phenanthren-3-one); Turosteride ((1S,3aS,3bS,5aR,9aR,9bS,11aS)-6,9a,11a-trimethyl-7-oxo-N-propan-2-yl-N-(propan-2-ylcarbamoyl)-2,3,3a,3b,4,5,5a,8,9,9b,10,11-dodecahydro-1H-indeno[5,4-f]quinoline-1-carboxamide);

[0164] Mifepristone ((8S,11R,13S,14S,17S)-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); Lilopristone ((8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-[(Z)-3-hydroxyprop-1-enyl]-13-methyl-1,2,6,7,8,11 ,12,14,15,16-Decahydrocyclopenta[a]phenanthren-3-one; Onapristone ((8S,11R,13R,14S,17S)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(3-hydroxypropyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-Decahydrocyclopenta[a]phenanthren-3-one); Asoprisnil ((8S,11R,13S,14S,17S)-11-[4-[(E)-hydroxyiminomethyl]phenyl]-17- Methoxy-17-(methoxymethyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one; J912 ((8S,11R,13S,14S,17S)-17-hydroxy-11-[4-[(Z)-hydroxyiminomethyl]phenyl]-17-(methoxymethyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); CDB-2914 ((8S,13S,14S,17 R)-17-acetyl-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one; JNJ-1250132 ([(8S,11R,13S,14S,17R)-17-acetyl-13-methyl-3-oxo-11-(4-piperidin-1-ylphenyl)-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17-yl]acetate);ORG-31710 ((6R,8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-6,13-dimethylspiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthrene-17,2'-oxolane]-3-one); ORG-33628 ((8S,11R,13S,14S,17R)-11-(4-acetylphenyl)-13-methyl-3'-methylidenespiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthrene-17, 2'-oxolane]-3-one; ORG-31806 ((7S,8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-7,13-dimethylspiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthrene-17,2'-oxolane]-3-one); ZK-112993 ((8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16 -Decahydrocyclopenta[a]phenanthren-3-one; ORG-31376 ((8S,11R,13S,14R,17S)-11-[4-(dimethylamino)phenyl]-13-methylspiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17,2'-oxolane]-3-one); ORG-33245 ((8S,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-13-methyl-3'-methylidenespiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-17,2'-oxolane]-3-one); carbhydrocyclopenta[a]phenanthrene-17,2'-oxolan]-3-one;ORG-31167;ORG-31343;RU-2992;RU-1479;RU-25056;RU-49295;RU-46556;RU-26819;LG1127;LG120753(3-(2,2,4-trimethyl-1H-quinolin-6-yl)benzonitrile);LG120830(3-fluoro-5-(2,2,4-trimethyl-1H-quinolin-6-yl)benzonitrile);LG1447;LG121046;CGP-19984A(sodium;Methyl [(2Z)-3-methyl-2-[(Z)-[5-methyl-3-(2-methylprop-2-enyl)-4-oxo-1,3-thiazolidin-2-ylidene]hydrazinylidene]-4-oxo-1,3-thiazolidin-5-yl]phosphate;RTI-3021-012 (8S,11R,13S,14S,17R)-17-acetyl-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a] Phenanthren-3-one;RTI-3021-022((8S,11R,13S,14S,17R)-17-acetyl-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one);RTI-3021-020;RWJ-25333((3,4-dichlorophenyl)-(6-phenyl-4,5-dihydro-3H-pyridazin-2-yl)methanone);ZK-136796 ;ZK-114043 ((8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-17-[(E)-3-hydroxyprop-1-enyl]-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one);ZK-230211 ((8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-13-methyl-17-(1,1,2,2,2-pentafluoroethyl) )-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one;ZK-136798;ZK-98229;ZK-98734((8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-[(Z)-3-hydroxyprop-1-enyl]-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one);ZK-137316;Asoprisnil ((8S,11R,13S,14S,17S)-11-[4-[(E)-hydroxyiminomethyl]phenyl]-17-methoxy-17-(methoxymethyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one);

[0165] 4-[17β-Methoxy-17α-(methoxymethyl)-3-oxoestra-4,9-dien-11β-yl]benzaldehyde-1-(E)-[O-(ethylamino)carbonyl]oxime;(Z)-6′-(4-cyanophenyl)-9,11α-dihydro-17β-hydroxy-17α-[4-(1-oxo-3-methylbutoxy)-1-butenyl]4′H-naphtho[3′,2′,1′;10,9,11]estra-4-en-3-one;11β-(4-acetylphenyl)-17β-hydroxy-17α-(1,1,2,2,2-pentafluoroethyl)estra-4,9-dien-3-one;11β-(4-acetylphenyl)-19 ,24-Dinor-17,23-epoxy-17α-chola-4,9,20-trien-3-one;(Z)-11β,19-[4-(3-pyridinyl)-o-phenylene]-17β-hydroxy-17α-[3-hydroxy-1-propenyl]-4-androsten-3-one;11β-[4-(1-methylethenyl)phenyl]-17α-hydroxy-17β-β-hydroxypropyl)-13α-estra-4,9-dien-3-one;4',5'-Dihydro-11β-[4-(dimethylamino)phenyl]-6β-methylspiro[estra-4,-9-dien-17β,2'(3'H)-furan]-3-one;Drospirenone (PubChem CID 68873);

[0166] T3 ((2S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoic acid); KB-141 (2-[3,5-dichloro-4-(4-hydroxy-3-propan-2-ylphenoxy)phenyl]acetic acid); Sobetirome (2-[4-[(4-hydroxy-3-propan-2-ylphenyl)methyl]-3,5-dimethylphenoxy]acetic acid); GC-2 4(2-[4-[(3-benzyl-4-hydroxyphenyl)methyl]-3,5-dimethylphenoxy]acetic acid);4-OH-PCB106(2-chloro-4-(2,3,4,5-tetrachlorophenyl)phenol);Eprotirom(3-[3,5-dibromo-4-(4-hydroxy-3-propan-2-ylphenoxy)anilino]-3-oxopropanoic acid);MB07811(PubChem CID 15942005;QH2(2-[(2E)-3,7-dimethylocta-2,6-dienyl]-5,6-dimethoxy-3-methylbenzene-1,4-diol);MB07344([4-[(4-hydroxy-3-propan-2-ylphenyl)methyl]-3,5-dimethylphenoxy]methylphosphonic acid);Tamoxifen(2-[4-[(Z)-1,2-diphenylbut-1-enyl]phenoxy]-N,N-dimethylethanamine);4-OH-Tamoxifen(4-[(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-phenylbutanyl) tert-1-enyl]phenol;raloxifene ([6-hydroxy-2-(4-hydroxyphenyl)-1-benzothiophen-3-yl]-[4-(2-piperidin-1-ylethoxy)phenyl]methanone;lasofoxifene ((5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol);bazedoxifene (1-[[4-[2-(azepan-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methylindol-5-ol);Falsodex ((7R,8R,9S,13S,14S,17S)-13-methyl-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol); Clomiphene (2-[4-[(E)-2-chloro-1,2-diphenylethenyl]phenoxy] -N,N-Diethylethanamine;Femalele();Ormeloxifene(1-[2-[4-[(3R,4R)-7-Methoxy-2,2-dimethyl-3-phenyl-3,4-dihydrochromen-4-yl]phenoxy]ethyl]pyrrolidine;Toremifien(2-[4-[(Z)-4-Chloro-1,2-diphenylbut-1-enyl]phenoxy]-N,N-Dimethylethanamine);Ospemif and ethinylestradiol ((8R,9S,13S,14S,17R)-17-ethynyl-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3,17-diol); estradiol ((8R,9S,13S,1 4S,17S)-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol; Ethynylestradiol ((8R,9S,13S,14S,17R)-17-ethynyl-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3,17-diol);

[0167] Thiazolidinediones: (e.g. rosiglitazone (5-[[4-[2-[methyl(pyridin-2-yl)amino]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); lobeglitazone (5-[[4-[2-[[6-(4-methoxyphenoxy)pyrimidin-4-yl]-methylamino]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); troglitazone (5-[[4-[(6-hydroxy-2,5,7,8-tetramethyl-3 ,4-dihydrochromen-2-yl)methoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); farglitazar ((2S)-2-(2-benzoylanilino)-3-[4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]phenyl]propanoic acid); aleglitazar ((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]-1-benzothiophen-7-yl]propanoic acid); and fenofibric acid (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid); benzopyranoquinoline A 276575, mapracolate ((2R)-1,1,1-trifluoro-4-(5-fluoro-2,3-dihydro-1-benzofuran-7-yl)-4-methyl-2-[[(2-methylquinolin-5-yl)amino]methyl]pentan-2-ol); ZK216348 (4-(2,3-dihydro-1-benzofuran-7-yl)-2-hydroxy-4-methyl-N-(4-methyl-1-oxo-2,3-benzyl) benzoxazin-6-yl)-2-(trifluoromethyl)pentanamide;55D1E1;Dexamethasone ((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one);Prednisolone ((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one); Prednisone ((8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,12,14,15,16 -octahydrocyclopenta[a]phenanthrene-3,11-dione; methylprednisolone ((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-6,10,13-trimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3-one); fluticasone propionate ([(6S,8S,9R,10S,11S,13S,14S,16R ,17R)-6,9-difluoro-17-(fluoromethylsulfanylcarbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-17-yl]propanoate; beclomethasone-17-monopropionate ([(8S,9R,10S,11S,13S,14S,16S,17R)-9-chloro-11-hydroxy-17-(2-hydroxyacetyl)-10, 13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-17-yl]propanoate; Betamethasone ((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one);Rimexolone ((8S,9S,10R,11S,13S,14S,16R,17S)-11-hydroxy-10,13,16,17-tetramethyl-17-propanoyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one); Paramethasone ((6S,8S,9S,10R,11S,13S,14S,16R,17R)-6-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl) )-10,13,16-trimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one; and hydrocortisone ((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthren-3-one);

[0168] 1,25-Dihydroxyvitamin D3 (calcitriol) ((1R,3S,5Z)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol), paricalitol ((1R,3R)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(E,2R,5S)-6-hydroxy-5,6-dimethylhept-3-yl]- doxercalciferol ((1R,3S,5Z)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(E,2R,5R)-5,6-dimethylhept-3-en-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol), 25-hydroxyvitamin D3 (calciferol) ((1S,3Z)-3-[(2E)-2-[(1R,3aS,7aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol), cholecalciferol ((1S,3Z)-3-[(2E)-2-[(1R,3aS,7aR)-7a-methyl-1-[(2R)-6-methylheptan-2-yl]-2,3,3a,5,6,7-hexahydro- 1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol), ergocalciferol ((1S,3Z)-3-[(2E)-2-[(1R,3aS,7aR)-1-[(E,2R,5R)-5,6-dimethylhept-3-en-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol), tacalcitol ((1R,3S,5Z)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(2R,5R)-5-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol), 22-dihydroergocalciferol ((1S,3Z)-3-[(2E)-2-[(1R,3aS,7aR)-1-[(2R,5S)-5,6-dimethylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane xan-1-ol), (6Z)-tacalciol ((1S)-3-[(Z)-2-[(1R,7aR)-7a-methyl-1-[(2R)-6-methylheptan-2-yl]-1,2,3,3a,6,7-hexahydroinden-4-yl]ethenyl]-4-methylcyclohex-3-en-1-ol), 2-methylene-19-nor-20(S)-1α-hydroxy-bishomopregnacalciferol ((1R,3R)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(2S)-but-2-yl]-7a-methyl-2,3, 3a,5,6,7-Hexahydro-1H-inden-4-ylidene]ethylidene]-2-methylidenecyclohexane-1,3-diol), 19-nor-26,27-dimethylene-20(S)-2-methylene-1alpha,25-dihydroxyvitamin D3, 2-methylene-1α,25-dihydroxy-(17E)-17(20)-dehydro-19-nor-vitamin D3, 2-methylene-19-nor-(24R)-1alpha,25-dihydroxyvitamin D2, 2-methylene-(20R,25S)-19,26-dinor-1alpha,25 -Dihydroxyvitamin D3, 2-methylene-19-nor-1α-hydroxy-pregnacalciferol, 1α-hydroxy-2-methylene-19-nor-homopregnacalciferol, (20R)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol, 2-methylene-19-nor-(20S)-1α-hydroxy-trishomopregnacalciferol, 2-methylene-23,23-difluoro-1α-hydroxy-19-nor-bishomopregnacalciferol, 2-methylene-(20S)-23,23-difluoro-1α-hydroxy-19-nor-bishomopregnane-calciferol, (2-(3′hydroxypropyl-1′,2′-idene)-19,23,24-trinor-(20S)-1α-hydroxyvitamin D3, 2-methylene-18,19-dinor-(20S)-1alpha,25-dihydroxyvitamin D3, etc.

[0169] Retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), all-trans-retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), 9-cis-retinoic acid ((2E,4E,6Z, 8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), Tamibarotene (4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid), 13-cis-retinoic acid ((2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona -2,4,6,8-tetraenoic acid), (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexeneyl)nona-2,4,6,-8-tetraenoic acid, 9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-nona-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)ethenyl]benzoic acid, retinobenzoic acid (4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid), ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)ethynyl]pyridine-3-carboxylate, retinoyl t-butylate, retinoyl pinacol and retinoyl cholesterol,

[0170] Obeticholic acid ((4R)-4-[(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid), LY2562175 (6-(4-( (5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazolyl-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]ethenyl]benzoic acid); T0901 317 (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]benzeneacetic acid hydrochloride), and LXR-623 (2-[(2-chloro-4-fluorophenyl)methyl]-3-(4-fluorophenyl)-7-(trifluoromethyl)indazole); GNE-3500 (27,1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazin-2-yl-methyl)-phenyl]-piperazin-1-yl}-ethanone);7β,27-dihydroxycholesterol ((3S,7R,8S,9S,10R,13R,14S,17R)-17-[(2R)-7-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol) and 7α,27-dihydroxycholesterol ((3S,7S,8S,9S,10R,13R,14S,17R)-17-[(2R)-7-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17 -dodecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol; 9-cis retinoic acid ((2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), LGD100268 (6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)cyclopropyl]pyridine-3-carboxylic acid), CD3254 (3-[4-hydroxy-3-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-phenyl]-2-propenoic acid), and CD2915 (Sorensen et al. (1997) Skin Pharmacol. 10:144).

[0171] Where the heterodimerization domain pair comprises the LBD of the mineralocorticoid receptor (MR) and a corresponding coregulatory peptide, suitable regulatory molecules include spironolactone and eplerenone. Spironolactone can be administered at a dose ranging from 10 to 35 mg / day, e.g., 25 mg / day.

[0172] When the heterodimerization domain pair comprises the LBD of the androgen receptor (AR) and a corresponding coregulatory peptide, suitable regulatory molecules include cyproterone acetate, hydroxyflutamide, enzalutamide, ARN-509, 3,3'-diindolylmethane (DIM), bexlosteride, bicalutamide, N-butylbenzene-sulfonamide (NBBS), dutasteride, epristeride, finasteride, flutamide, izonsteride, ketoconazole, N-butylbenzene-sulfonamide, nilutamide, megestrol, steroidal antiandrogens, and turosteride.

[0173] When the heterodimerization domain pair comprises the LBD of the progesterone receptor (PR) and a corresponding coregulatory peptide, suitable regulatory molecules include mifepristone (RU-486; 11β-[4N,N-dimethylaminophenyl]-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one); lilopristone (11β-(4N,N-dimethylaminophenyl)-17β-hydroxy-17-((Z)-3-hydroxypropenyl)-estra-4,9-dien-3-one); onapristone (11β-(4N,N-dimethylaminophenyl)-17α-hydroxy-17-(3-hydroxypropenyl)-estra-4,9-dien-3-one); asoprisnil (benzaldehyde, 4-[(11β,17β)-17-methoxy-17-(methoxymethyl)-3-oxoestr-4,9-dien-11-yl]-1-(E)-oxime; J867); J912 (4-[17β-hydroxy-17α-(methoxymethyl)-3-oxoestr-4,9-dien-11β-yl]benzaldehyde-(1E)-oxime); and CDB-2914 (17α-acetoxy-11β-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione). Other suitable dimerizing agents include, for example, JNJ-1250132, (6α,11β,17β)-11-(4-dimethylaminophenyl)-6-methyl-4',5'-dihydrospiro[estra-4,9-diene-17,2'(3'H)-furan]-3-one (ORG-31710); (11β,17α)-11-(4-acetylphenyl)-17,23-epoxy-19,24-dinorchol-4,9-,20-trien-3-one (ORG-33628); (7β,11β,17β)-11-(4 -Dimethylaminophenyl-7-methyl]-4',5'-dihydrospiro[estra-4,9-diene-17,2'(3'H)-furan]-3-one (ORG-31806);ZK-112993;ORG-31376;ORG-33245;ORG-31167;ORG-31343;RU-2992;RU-1479;RU-25056;RU-49295;RU-46556;RU-26819;LG1127;LG120753;LG120830;LG1447;LG121046;CGP-19984A;RTI-3021-012;RTI-3021-022;RTI-3021-020;RWJ-25333;ZK-136796;ZK-114043;ZK-230211;ZK-136798;ZK-98229;ZK-98734;ZK-137316;4-[17β-methoxy-17α-(methoxymethyl)-3-oxoestra-4,9-dien-11β-yl]benzaldehyde-1-(E)-oxime;4-[17β-methoxy-17α-( 4-[17β-Methoxy-17α-(methoxymethyl)-3-oxoestra-4,9-dien-11β-yl]benzaldehyde-1-(E)-[O-(ethylthio)carbonyl]oxime;(Z)-6'-(4-cyanophenyl)-9,11α-dihydro-17β-hydroxy-17α-[4-(1-oxo-3-methyl) 11β-(4-acetylphenyl)-17β-hydroxy-17α-(1,1,2,2,2-pentafluoroethyl)estra-4,9-dien-3-one;11β-(4-acetylphenyl)-19,24-dinor-17,23-epoxy-17α-cholate-4,9,20-trien-3-one;(Z)-11β,19-[4-(3-pyridinyl)-o-phenyl 11β-[4-(1-methylethenyl)phenyl]-17α-hydroxy-17β-β-hydroxypropyl)-13α-estra-4,9-dien-3-one; 4',5'-dihydro-11β-[4-(dimethylamino)phenyl]-6β-methylspiro[estra-4,9-dien-17β,2'(3'H)-furan]-3-one, and drospirenone.

[0174] Where the heterodimerization domain pair comprises the LBD of thyroid receptor beta (TR-beta) and a corresponding coregulatory peptide, suitable regulatory molecules include T3 (3,5,3'-triiodo-L-thyronine); KB-141 (3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid); sobetirome (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)phenylacetate); benzylphenoxyacetic acid); 4-OH-PCB106 (4-OH-2',3,3',4',5'-pentachlorobiphenyl); eprotirom; MB07811 ((2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methyl]-2-oxide-[1,3,2]-dioxaphosphonane); QH2; and (3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344).

[0175] When the heterodimerization domain pair comprises the LBD of estrogen receptor alpha (ERalpha) and a corresponding coregulatory peptide, suitable regulatory molecules include tamoxifen, 4-OH-tamoxifen, raloxifene, lasofoxifene, bazetoxifene, farsodex, clomiphene, femarele, ormeloxifene, toremifene, ospemifene, and ethinyl estradiol.

[0176] When the heterodimerization domain pair comprises the LBD of estrogen receptor beta (ER-beta) and a corresponding coregulatory peptide, suitable regulatory molecules include estradiol (E2; or 17-β-estradiol) and ethinyl estradiol.

[0177] When the heterodimerization domain pair comprises an LBD PPAR-gamma and a corresponding coregulatory peptide, suitable regulatory molecules include thiazolidinediones (e.g., rosiglitazone, pioglitazone, lobeglitazone, troglitazone), farglitazar, aleglitazar, and fenofibric acid.

[0178] Where the pair of heterodimerization domains comprises the LBD of GR and a corresponding co-regulatory peptide, suitable regulatory molecules may be selective GR agonists (SEGRAs) or selective GR modulators (SEGRMs).

[0179] When the heterodimerization domain pair comprises the LBD of GR and a corresponding coregulatory peptide, suitable regulatory molecules include benzopyranoquinoline A276575, maplacorat, ZK216348, 55D1E1, dexamethasone, prednisolone, prednisone, methylprednisolone, fluticasone propionate, betamethasone-17-monopropionate, betamethasone, rimexolone, paramethasone, and hydrocortisone.

[0180] When the heterodimerization domain pair comprises the LBD of the VDR and a corresponding coregulatory peptide, suitable regulatory molecules include 1,25-dihydroxyvitamin D3 (calcitriol), paricalitol, doxercalciferol, 25-hydroxyvitamin D3 (calcifediol), cholecalciferol, ergocalciferol, tacalciol, 22-dihydroergocalciferol, (6Z)-tacalciol, 2-methylene-19-nor- 20(S)-1α-hydroxy-bishomopregnacalciferol, 19-nor-26,27-dimethylene-20(S)-2-methylene-1α,25-dihydroxyvitamin D3, 2-methylene-1α,25-dihydroxy-(17E)-17(20)-dehydro-19-norvitamin D3, 2-methylene-19-nor-(24R)-1α,25-dihydroxyvitamin D2, 2-methylene-(20R,25S)-19,26-dinor-1α, 25-Dihydroxyvitamin D3, 2-methylene-19-nor-1α-hydroxy-pregnacalciferol, 1α-hydroxy-2-methylene-19-nor-homopregnacalciferol, (20R)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol, 2-methylene-19-nor-(20S)-1α-hydroxy-trishomopregnacalciferol, 2-methylene-23,23-difluoro-1α -hydroxy-19-nor-bishomopregnacalcifero-1,2-methylene-(20S)-23,23-difluoro-1α-hydroxy-19-nor-bishomopregna-n-calciferol, (2-(3'hydroxypropyl-1',2'-idene)-19,23,24-trinor-(20S)-1α-hydroxyvitamin D3, 2-methylene-18,19-dinor-(20S)-1α,25-dihydroxyvitamin D3, and the like.

[0181] When the pair of heterodimerization domains comprises the LBD of RAR-beta and a corresponding coregulatory peptide, suitable regulatory molecules include 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-nona-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)ethenyl]benzoic acid, retinobenzoic acid, ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)ethynyl]pyridine-3-carboxylate, retinoyl t-butylate, retinoyl pinacol, and retinoyl cholesterol.

[0182] When the heterodimerization domain pair comprises FXR and a corresponding coregulatory peptide, suitable regulatory molecules include 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]ethenyl]benzoic acid).

[0183] When the heterodimerization domain pair comprises the LBD of LXR-alpha and a corresponding coregulatory peptide, suitable regulatory molecules include 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]benzeneacetic acid hydrochloride), and LXR-623 (2-[(2-chloro-4-fluorophenyl)methyl]-3-(4-fluorophenyl)-7-(trifluoromethyl)indazole).

[0184] When the heterodimerization domain pair comprises the LBD of ROR-gamma and a corresponding coregulatory peptide, a suitable regulatory molecule includes GNE-3500 (27,1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazin-2-yl-methyl)-phenyl]-piperazin-1-yl}-ethanone).

[0185] When the heterodimerization domain pair comprises the LBD of ROR-gamma and a corresponding coregulatory peptide, suitable regulatory molecules include 7β,27-dihydroxycholesterol and 7α,27-dihydroxycholesterol.

[0186] When the heterodimerization domain pair comprises the LBD of RXR-alpha and a corresponding coregulatory peptide, suitable regulatory molecules include 9-cis retinoic acid, LGD100268, CD3254 (3-[4-hydroxy-3-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-phenyl]-2-propenoic acid), and CD2915 (Sorensen et al. (1997) Skin Pharmacol. 10:144).

[0187] When the heterodimerization domain pair comprises the LBD of PXR and a corresponding coregulatory peptide, suitable regulatory molecules can be rifampicin, clotrimazole, and lovastatin.

[0188] 8.1.2. Conditional heterodimerization of CAR molecules based on lipocalin-fold molecules: In another preferred embodiment, at least two CAR molecules of the CAR population of the invention may be heterodimerized by a pair of heterodimerization domains, comprising one member that is a lipocalin fold molecule and a second member that is a lipocalin fold binding interaction partner (as disclosed in EP17208924.5, filed December 20, 2017). According to a preferred embodiment, the lipocalin fold based heterodimerization system comprises: (a) Lipocalin-fold molecule (b) low molecular weight lipocalin-fold ligands of 1500 Da or less; and (c) a lipocalin-fold binding interaction partner, wherein the lipocalin-fold molecule is capable of binding to a lipocalin-fold ligand, and wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand binds to the lipocalin-fold binding interaction partner with an affinity that is at least 10-fold greater than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand; Here, the lipocalin-fold binding interaction partner is not a naturally occurring protein that has an affinity of <10 μM for a naturally occurring lipocalin-fold molecule in the presence of a lipocalin-fold ligand.

[0189] According to a further preferred embodiment, the lipocalin fold based heterodimerization system comprises: (a) Lipocalin-fold molecule (b) low molecular weight lipocalin-fold ligands of 1500 Da or less; and (c) a lipocalin-fold binding interaction partner, wherein the lipocalin-fold molecule has at least a first conformation when a lipocalin-fold ligand is not bound to the lipocalin-fold molecule and has at least a second conformation when a lipocalin-fold ligand is bound to the lipocalin-fold molecule; wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand in the second conformation binds to the lipocalin-fold binding interaction partner with an affinity that is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand in the first conformation; and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein that has an affinity of <10 μM for a naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.

[0190] This lipocalin-fold molecule-based system for conditional heterodimerization generally relies on substantial differences in the affinity of lipocalin-fold molecules to lipocalin-fold binding interaction partners depending on whether a lipocalin-fold ligand is bound or not. It is preferred that the affinity window (i.e. the affinity of the lipocalin-fold binding interaction partner to the lipocalin-fold molecule bound or not to the lipocalin-fold ligand) is in a reasonable affinity range that allows heterodimerization under physiological conditions. Thus, it is preferred that the affinity of the lipocalin-fold binding interaction partner to the lipocalin-fold molecule in the ligand-bound state is less than 10 μM, preferably less than 2 μM, in particular less than 400 nM.

[0191] Depending on whether the lipocalin fold binding interaction partner is engineered for binding to lipocalin fold molecules loaded with lipocalin fold ligands or not loaded with lipocalin fold ligands, the lipocalin fold molecule-based system can be used for conditional heterodimerization (i.e. for on-switching) or for constitutive heterodimerization, respectively. The lipocalin fold binding interaction partner can also be engineered to bind to lipocalin fold molecules in the absence but not in the presence of lipocalin fold ligands, so that the system can also be used to conditionally prevent heterodimerization (i.e. for off-switching). In principle, the lipocalin fold molecule-based system can also be arbitrarily engineered to bind at least two different lipocalin fold ligands, and the lipocalin fold binding interaction partner selected accordingly can distinguish between two differently induced conformational states, which allows for conditional on-switching and off-switching by sequential addition of two different lipocalin fold ligands.

[0192] A lipocalin-fold molecule that can be used as a heterodimerization domain of the present invention can be any protein that contains a lipocalin-fold structural motif to (or within) which a lipocalin-fold ligand can bind, allowing binding of the lipocalin-fold molecule to a lipocalin-fold binding interaction partner.

[0193] Lipocalin-fold molecules are defined as naturally occurring molecules classified in the lipocalin spars family in the SCOP database (version 1.75), or variants thereof, however, preferably with only a limited number of amino acid exchanges.

[0194] According to a preferred embodiment, the lipocalin-fold molecule is a molecule identical to naturally occurring iLBP (intracellular lipid-binding protein), naturally occurring lipocalin or anticalin, and any derivative of these molecules having 1-30 amino acid exchanges, or a fragment thereof. In another preferred embodiment, the lipocalin-fold molecule is a derivative of naturally occurring lipocalin or iLBP having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, or 30 amino acid exchanges.

[0195] According to a preferred embodiment of the invention, the lipocalin fold molecule is engineered by one or more amino acid exchanges, insertions and / or deletions to optimize lipocalin fold ligand binding. According to a preferred embodiment, the lipocalin fold molecule is a derivative of a naturally occurring or otherwise disclosed lipocalin fold molecule (by its amino acid sequence) having at least 70%, preferably at least 80%, in particular at least 90% sequence identity in the β-barrel structure, whereby this β-barrel structure is preferably defined as a region structurally corresponding to a region of amino acid residues selected from the following: - amino acid residues 21-30, 41-47, 52-58, 71-78, 85-88, 102-109, 114-120, and 132-138 of human RBP4 (according to the amino acid residue numbering scheme of PDB entry 1RBP), which define the structurally conserved β-strand of human RBP4; - amino acid residues 14-23, 37-43, 48-54, 62-69, 76-79, 84-91, 96-102, and 111-117 of human tear lipocalin (TLC; defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand of human TLC; - amino acid residues 44-53, 69-75, 81-87, 96-103, 110-113, 119-126, 131-137, and 142-148 of human apolipoprotein M (ApoM; defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand of human ApoM; - amino acid residues 5-12, 41-45, 50-54, 61-65, 71-73, 81-87, 93-96, 108-112, 119-124, and 129-135 of human cellular retinoic acid binding protein II (CRABPII; according to the amino acid residue numbering scheme of PDB entry 2FS6), which define the structurally conserved β-strand of human CRABP1I; - Amino acid residues 5-12, 39-43, 48-52, 59-63, 69-71, 79-85, 91-94, 99-103, 109-114, and 119-125 of human fatty acid binding protein 1 (FABP1; according to the amino acid residue numbering scheme of PDB entry 2F73), which define the structurally conserved β-strand of human FABP1.

[0196] According to a preferred embodiment, the lipocalin fold molecule is a fragment of a naturally occurring lipocalin or a derivative thereof having a length of at least 80, preferably at least 100, in particular at least 120 amino acids, covering at least the structurally conserved β-barrel structure of the lipocalin fold, or wherein the lipocalin fold molecule is a fragment of a naturally occurring iLPB or a derivative thereof having a length of at least 80, preferably at least 85, in particular at least 90 amino acids, covering at least the structurally conserved β-barrel structure of the lipocalin fold, wherein the structurally conserved β-barrel structure preferably comprises or consists of amino acid positions structurally corresponding to a region of amino acid residues selected from the following: - amino acid residues 21-30, 41-47, 52-58, 71-78, 85-88, 102-109, 114-120, and 132-138 of human RBP4 (according to the amino acid residue numbering scheme of PDB entry 1RBP), which define the structurally conserved β-strand of human RBP4; - amino acid residues 14-23, 37-43, 48-54, 62-69, 76-79, 84-91, 96-102, and 111-117 of human tear lipocalin (TLC; defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand of human TLC; - amino acid residues 44-53, 69-75, 81-87, 96-103, 110-113, 119-126, 131-137, and 142-148 of human apolipoprotein M (ApoM; defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand of human ApoM; - amino acid residues 5-12, 41-45, 50-54, 61-65, 71-73, 81-87, 93-96, 108-112, 119-124, and 129-135 of human cellular retinoic acid binding protein II (CRABPII; according to the amino acid residue numbering scheme of PDB entry 2FS6), which define the structurally conserved β-strand of human CRABP1I; - Amino acid residues 5-12, 39-43, 48-52, 59-63, 69-71, 79-85, 91-94, 99-103, 109-114, and 119-125 of human fatty acid binding protein 1 (FABP1; according to the amino acid residue numbering scheme of PDB entry 2F73), which define the structurally conserved β-strand of human FABP1.

[0197] According to a further preferred embodiment, the lipocalin fold molecule is a derivative of a naturally occurring lipocalin or iLBP, which, apart from the structurally conserved β-barrel structure, has a deletion of up to 15, up to 30, or up to 50 amino acids and / or an insertion of up to 15, up to 30, or up to 50 amino acids, preferably corresponding structurally to a region of amino acid residues selected from the following: - amino acid residues 1-20, 31-40, 48-51, 59-70, 79-84, 89-101, 110-113, 121-131, and 139-183 of human RBP4, which define the regions adjacent to the structurally conserved β-strand of human RBP4 according to the amino acid residue numbering scheme of PDB entry 1RBP; - amino acid residues 1-13, 24-36, 44-47, 55-61, 70-75, 80-83, 92-95, 103-110, and 118-158 of human TLC (according to the amino acid residue numbering scheme of Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand-adjacent regions in human TLC; - amino acid residues 1-43, 54-68, 76-80, 88-95, 104-109, 114-118, 127-130, 138-141, and 149-188 of human ApoM (according to the amino acid residue numbering scheme of Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand-adjacent regions of human ApoM; - amino acid residues 1-4, 13-40, 46-49, 55-60, 66-70, 74-80, 88-92, 97-107, 113-118, 125-128, and 136-137 of human CRABPII (according to the amino acid residue numbering scheme of PDB entry 2FS6), which define the structurally conserved β-strand-adjacent region of human CRABPII; - amino acid residues 1-4, 13-38, 44-47, 53-58, 64-68, 72-78, 86-90, 95-98, 104-108, 115-118, and 126-127 of human FABP1 (according to the amino acid residue numbering scheme of PDB entry 2F73), which define a region adjacent to the structurally conserved β-strand of human FABP1.

[0198] In other preferred embodiments, the lipocalin-fold molecule is a derivative of a naturally occurring member of the lipocalin sparing family having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid exchanges.

[0199] According to a further preferred embodiment, the lipocalin fold molecule used as heterodimerization domain of the present invention is a lipocalin, i.e. a protein consisting of eight strands of upper and lower β-barrels arranged in a +1 topology, followed by an α-helix following the C-terminus of the eighth β-strand.

[0200] The lipocalin fold ligands that can be used as regulatory molecules of the present invention are "small" in comparison to "small molecules", such as polypeptides and proteins, such as lipocalin fold molecules. Thus, the molecular weight of the lipocalin fold ligand is 1500 Da or less, preferably 1000 Da or less, in particular 750 Da or less. The preferred Mw range of the lipocalin fold ligand is 50-1500 Da, preferably 75-1500 Da, in particular 150-750 Da. Preferably, the lipocalin fold ligand is capable of binding to the jaw of the lipocalin fold molecule, which is formed by the barrel and loop regions of the lipocalin fold structure.

[0201] The lipocalin-fold ligand preferably has an affinity for the lipocalin-fold molecule of less than 1 mM, preferably less than 100 μM, in particular less than 10 μM. This affinity between the lipocalin-fold ligand and the lipocalin-fold molecule is known as the K d(dissociation constant) value, which is preferably determined by isothermal titration calorimetry (ITC) using an automated MicroCal PEAQ-ITC instrument (Malvern Instruments).

[0202] Examples of lipocalin fold ligands that can be selected are: Nr Database HMDB 1. Nafcillin 2. Gerberinol 3. Montelukast 4. Flurazepam 5 Quinidine barbiturates 6. Glyceollin I 7 Glyceollin II 8 (-)-Sympterocarpine 9 Canzonol W 10 6α-hydroxyphaseolin 11 (1a,5b,6a)-7-Protoyl-1,5,6,14-tetrol 14-(2,4-dihydroxy-6-methylbenzoic acid) 12 4'-O-Methylcanzophenol W 13 Cyclokieviton 14 Lycoflanone 15 Aluminum Lattin 16 2-(4-Methyl-3-pentenyl)anthraquinone 17 Sorafenib beta-D-glucuronide 18 Heterophilin 192'-O-Methylphaseolin isoflavan 20 Chiapride 21 Gluten Exorphin C 22 Murbero Franc M 23 Dalxanthon G 24 Sclareo-R 25 Corpus docurus a 26 Canzonol F 27 Mangostinone 28 Gankaonin X 29 Rubraflavone D 30 Cycloquivitone Hydrate 31 Griseolidine II 32 Cyclandreit 33 Dalxanthon E 34 Morsin 35 (E)-2',4,4'-Trihydroxy-3-prenylchalcone 36 Dalxanthon H 37 Judeol 38 Artonin E 39 Canzonol T 40 Fragransole A 41 Dalxanthon F 42 Murbero Fran T 43 Garcimangoson A 44 Artonin B 45 Asturtoxin Database KEGG 46 Oxyfedrine 47 Profluthrin 48 Monfluorothrin 49 Xyloylsulfamine 50 Cetotiamine hydrochloride hydrate 51 Dicethiamine hydrochloride hydrate 52 Diacetamine 53 Oxolamin 54 Oxalamin 55 Chrysositol mesylate 56 Iofurbenzamide I 57 Ceritinib 58 Zikadia 59 Imiprothrin 60 Triclabendazole 61 Fascinex 62 Brivanivualaninet 63 Transfluthrin 64 Enolicum sodium 65 Enolicum sodium monohydrate 66 Zivcain 67 Cincocaine 68 Nupercain 69 Trametinib Database WDI 70 Flucloxacillin 71 S-Farnesylthiosalicylic acid 72 2-Fluorotropapride 73 Butampicillin 74 Dicloxacillin sulfate 75 Floxacillin 76 Ibcillin 77 Prazocillin 78 Saretamide 79 Tetrachlorosalicylanilide 80 Carbenicillin 81 Dichloromethide 82 Methyl-sulfometuron 83 Trichlorosalicylanilide 84 Chlometocillin 85 Cystoditin-F 86 Data Nosaru 87 Diaballone 88 Diclofop 89 Epipheneticillin 90 Phthalyl-Mediol 91 Saretamide hydrochloride 92 Tiapride Hydrochloride 93 Tranqulin-A 94 Deoxypentalenyl glucuronide 95 Lafurnimus 96 Melazolam 97 Dibsador 98 Phenethicillin Curium 99 Plano monkey 100 Dareformis 101 Diphenicillin 102 Phenoterol hydrochloride 103 Giant acid 104 Halothraline-B 105 Isopropicillin 106 Proguanil hydrochloride 107 Piranotkunton-B 108 Tuberosine 109 Zopfieramide-B 110 Cloxacillin Sodium 111 Retimide Hydrochloride 112 Bygen-B 113 Bidwilon-B 114 Carbenicillin disodium 115 Hydroxyprocaine 116 Ochratoxin-A 117 Terox 118 Marker Rangaflavanone-B 119 Menoximycin-B 120 Penicillin-S 121 Psoralizin 122 Rubidinone-C1 123 Secopseudolopterosin-E 124 Asadithium sulfide 125 Balangcadoic acid-A 126 Bephedon 127 Meledonal-B 128 Nelaminol 129 Phomopsolido-A 130 Fast Acid 131 Zopfieramide-A 132 Cystodactylid-B 133 Dicloxacillin 134 Fluoropropranolol 135 Iriosicollin-B 136 Indicanin-B 137 Zhakaryubin 138 Cottamid-C 139 Mexolamine 140 Mycaperoxide-H 141 Otogirin 142 Oxolamine hydrochloride 143 Oxopropaline-A 144 Purvalanol-A 145 Rubiginone-C2 146 Terakrylshikonin 147 Clodinafop-propargyl-ester 148 Mazaticol 149 Sethoxydim 150 Sulfaguanol 151 Balaperidone 152 Flucloxacillin Sodium 153 Geodiamolid-TA 154 Lucanthone sulfoxide 155 Meleoride-D 156 Nadoxol hydrochloride 157 Becloprid Acid Glucuronide 158 Cloxacillin 159 Hypergynon-B 160 Oligosporol-A 161 Propoxycaine hydrochloride 162 Ronifibrate 163 Su-Dumble-GN 164 Trichodermamide-B 165 Botryllamide-A 166 Carfecillin 167 Kresodim 168 Dutadolpin 169 Epicoclioquinone-B-14 170 Flurazepam 171 Hydroxyflucloxacillin 172 Rinacanthin-C 173 Teflubenzuron 174 Zenisrate 175 Antimycin-A8A 176 Alto Indonesian-U 177 Bronco Cain 178 Enolicum 179 Ipadildo 180 Modern Brown-1 181 Propranolol Phenobarbital 182 Pugiinin-A 183 Amphibin-H 184 Almirallic Acid 185 Kalindacillin 186 Chlorobiochate 187 Chloropropylguanil 188 Synlindrol-B 189 Ecliptal Vine 190 Galcigerin-A 191 O-Demethylchlorothricin 192 Sangenon-C 193 Tetraptero-G 194 Chlorsulfuron 195 Dexamethasone diethylaminoacetate 196 Diethixim 197 Pyridovericin 198 Subendazol 199 Thiocaine 200 Trapezifollixanthon 201 Trikrazan 202 3',4'-Dichlorobenzamil 203 Chaetoviridin-C 204 Cycloglutin 205 Flurazepam monohydrochloride 206 Fusidilactone-B 207 Griseochelin methyl ester 208 Isobutylshikonin 209 Misiniketan 210 Ajudazole B 211 Cystogytin-E 212 Demethylprecanthone-A 213 Destruxin-A 214 Diphenidol embonate 215 Discochioride-B 216 Irumanolide-2 217 Lactoquinomycin 218 Neobornirbal 219 Saroslalen-D 220 Abyssinon-V 221 Akichirome 222 Chlorfluazuron 223 Chondrilidien-18,20 224 Euglobal-G2 225 Idarubicin Hydrochloride 226 Whiplash Maranon-A 227 3-O-Methylcarpocarpine 228 Allochloramide hydrochloride 229 Anopterin 230 Dioxathione 231 Euglobal-IIC 232 Irisicoline-C 233 Mycinolide-II 234 Suirin 235 Tocotrienol-gamma 236 Indomicinone-beta 237 Isothiol 238 Mebeverine 239 Whiplash Maranon-D 240 Nympheal-C 241 Pursokine 242 Jimeto-20-84 243 2,4-D-Butoxypropyl 244 Abyssinon-IV 245 Bygen-A 246 Chrysocratic Acid 247 Epolon-B 248 Ethoxycaine 249 Fluorenamil 250 Mefenamic acid guaiacol 251 Maxima-Isoflavone-C 252 Sarcodictyin-B 253 Triclabendazole 254 Akiyofran 255 Asteriquinone 256 Diethylglycolate trihydrazide 257 Maroto Philippenes-D 258 Naphthoxate 259 Patydictyol-A-epoxide 260 Ratjadon 261 Salverine Hydrochloride 262 4-Menahydroquinone 263 Botryllamide-C 264 Elsa Sewing Machine 265 Fenfluthrin 266 Mutisifenone-A 267 Sangenon-A 268 Schweinfurtchin-A 269 ​​Vanillin Roll 270 4-O-Methylmerleolide 271 Acetyl Bismuth-F 272 Alfentanil hydrochloride 273 Asperteronin-A 274 Beta-Hydroxyisovalerylshikonin 275 Carbisocaine 276 Chlorguanil hydrochloride 277 Cryptophycin-52 278 Didemethyltocotrienol 279 Fluosol-DA 280 Pyridoxiphene 281 Tiazesim Hydrochloride 282 Butoctamide 283 Deoxyshikonin 284 Gambieric acid-A 285 Lycoflanone 286 Prednylidene-diethylaminoacetate 287 Pseudopterosin-G 288 Trikendiol 289 Zoapatanol 290 Ergokinin-C 291 Pentamoxane hydrochloride 292 Scandinavian 293 Actinopyrone-C 294 Amiton 295 Kratoxyarborenone-C 296 Simopol 297 Doxycycline-Hyclate 298 Flavidurol-C 299 Franc-12 300 Miriaporon-3 301 Orinosinolide 302 Tonabelsat 303 Vismion-B 304 Amykelin 305 Butamoxane hydrochloride 306 Chlorobiocin 307 Cyclocomunin 308 Fenazaflor 309 Vismion-D 310 3-Trichlororemethaphos 311 Aminopropyl phenylbutazone 312 Geocrenol 313 Grifolin 314 Hyper-Jovinol-A 315 Tamorarijin 316 Tomentol 317 Trans-delta-tocotrienol acid 318 Diacetol hydrochloride 319 Dutomycin 320 Liglobutoside-O 321 Lespyrolle-B 322 Electokion-A 323 Salotralen-A 324 Shitamakin 325 Trichoporin-II 326 3-Hydroxytrufazepam 327 Dimethopramide 328 Phenoterol hydrobromide 329 Fencapton 330 Kratoxyarborenone-B 331 Etomoxan hydrochloride 332 Isocentratelin 333 Kalimantasin-A 334 Hispaglabridin-A 335 Lankacyclinol 336 Maclulaxanthone 337 Pivampicillin hydrochloride 338 Pluraflavin-A 339 Sigmoidin-I 340 Phenalamide 341 Hydroxyclasinomycin-M,2 342 Soforadin 343 Ascochlorin 344 Beta-Hydroxysanshur 345 Bistramide-K 346 Chlortetracycline borate 347 Dehydroascochlorin-8+,9+ 348 Dimethium chloride 349 Marker Rangin 350 Marcelomycin 351 Benzoyl gomisin-H 352 Clindamycin hydrochloride 353 Hydroxyascochlorin-8+ 354 Neocardilin-A 355 Chaetoviridin-B 356 Chlortetracycline bisulfate 357 Napyradiomycin-C1 358 Saloaspidin-B 359 Sarasin 360 Electon-B 361 Homohardian Acid 362 Asteriquinone-B1 363 Datamid 364 Rubraxanthone 365 Dolastatin-19 366 Edetol 367 Phaseolign 368 Gedkarnir 369 Manumycin-B 370 Santarol-beta-salicylate 371 Kowanin 372 Injibrin 373 Cowanol 374 Karatabishijin 375 Picloxydine 376 Proxazole 377 Strobilurin-E 378 Electroquinone-B 379 Sri Kainid 380 Diethylaminomethylrutin 381 Tropesin 382 Picloxydine hydrochloride 383 HEX-1 384 Decrobaniloviocin Database MDDR 385 Rubidinone C2 386 Frobfen 387 Tetronothiodin 388 Lafurnimus 389 Micaperoxide A 390 Flurazepam hydrochloride 391 Rubidinone C1 392 Chrysositol mesylate 393 Dolastatin D 394 Epolactaen 395 Lexiparphant Database CHEMBL 396 Carbenicillin 397 Diclofop 398 Epiphenicillin 399 Flobufen 400 Giant Acid 401 Phenethicillin Curium 402 Diphenicillin Database Pubchem 403 Acotiamide 404 Acosiborole 405 Akuma Pimod 406 Apalutamide 407 ASP3026 408 AZD1480 409 BIIB021 410 Bran Plum 411 Brekinar 412 Chlorproguanil 413 Emrikasan 414 Enasidenib 415 Enolicum 416 Flurazepam 417 ILX295501 418 Injibrin 419 Metoclopramide 420 Mevastatin 421 MK0686 422 Navarixin 423 Nefazodone 424 Pantoprazole 425 Pavinetant 426 SCYX-7158 427 Shikanin 428 Sulfoguanol 429 Sunitinib 430 Suvorexant 431 Chiapride 432 Tonabelsat 433 Urimorelin 434 Xypammid 435 MGGBYMDAPCCKCT-UHFFFAOYSA-N 436 VNBRGSXVFBYQNN-UHFFFAOYSA-N 437 YUHNXUAATAMVKD-PZJWPPBQSA-N

[0203] 8.1.3. Further Systems for Conditional Heterodimerization: According to the present invention, a pair of heterodimerization domains for heterodimerization of, for example, two CAR molecules of a CAR population may also be selected from: a) FKBP and FKBP-rapamycin-related protein (FRB, mutated T82L), b) GAI and GID1, c) FKBP and calcineurin catalytic subunit A (CnA); d) FKBP and cyclophilin, e) PYL and ABI.

[0204] The sequences of these heterodimerization domains, as well as the sequences of regulatory molecules suitable for dimerization of these heterodimerization domains, are known in the art (Rutkowska et al., Angew Chem Int Ed Engl. 2012;51(33):8166) and are disclosed, for example, in WO2014127261.

[0205] A member of a pair of heterodimerization domains selected from GAI, GID1, FKBP, CnA, cyclophilin, PYL, and ABI can have a length of about 50 amino acids to about 300 amino acids or more, for example, a member of a pair of heterodimerization domains can have a length of about 50 aa to about 100 aa, about 100 aa to about 150 aa, about 150 aa to about 200 aa, about 200 aa to about 250 aa, about 250 aa to about 300 aa, or 300 aa or more.

[0206] For example, a preferred heterodimerization domain can be derived from FKBP and can include 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 Uniprot P62942-1.

[0207] As another example, the heterodimerization domain can be derived from calcineurin catalytic subunit A (also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin-dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform, etc.) and can include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence Uniprot Q08209-1 amino acids (aa) 56-347 (PP2Ac domain).

[0208] As another example, the heterodimerization domain can be derived from cyclophilin (also known as cyclophilin A, PPIA, CYPA, CYPH, PPIase A, etc.) and can include 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 Uniprot P62937-1.

[0209] As another example, the heterodimerization domain can be derived from MTOR (also known as FKBP-rapamycin related protein, FK506 binding protein 12-rapamycin related protein 1; FK506 binding protein 12-rapamycin related protein 2; FK506 binding protein 12-rapamycin complex associated protein 1; FRAP; FRAP1; FRAP2; RAFT1; and RAPT1) and can include 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 Uniprot P42345-1 aa2021-2113 (also known as "Frb": Fkbp-rapamycin binding domain).

[0210] As another example, the heterodimerization domain can be derived from the PYL proteins (also known as abscisic acid receptors and RCARs) and can be represented by the following amino acid sequences: PYL10 (Uniprot Q8H1R0-1); PYL11 (Uniprot Q9FJ50); PYL12 (Uniprot Q9FJ49-1); PYL13 (Uniprot Q9SN51-1); PYL1 (Uniprot Q8VZS8-1); PYL2 (Uniprot O80992-1); PYL3 (Uniprot Q9SSM7-1); PYL4 (Uniprot O80920-1); PYL5 (Uniprot Q9FLB1-1); PYL6 (Uniprot Q8S8E3-1); PYL7 (Uniprot Q1ECF1-1); PYL8 (Uniprot Q9FGM1-1); PYL9 (Uniprot Q9FGM1-1); ...9FGM1-1); PYL11 (Uniprot Q9FGM Q84MC7-1; PYR1 (Uniprot O49686-1); PYR1 (Uniprot O49686-1)).

[0211] As another example, the heterodimerization domain can be derived from the ABI protein (also known as abscisic acid insensitive) and can be derived from the Arabidopsis thaliana proteins ABI1 (also known as abscisic acid insensitive 1, protein phosphatase 2C 56, AtPP2C56, P2C56, and PP2C ABI1) and / or ABI2 (also known as P2C77, protein phosphatase 2C 77, AtPP2C77, abscisic acid insensitive 2, protein phosphatase 2C ABI2, and PP2C ABI2). For example, suitable heterodimerization domains have the following amino acid sequences: ABI1 (Uniprot P49597-1); ABI2 (Uniprot P49597-2); The present invention can include 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, about 160 aa to about 170 aa, about 170 aa to about 180 aa, about 180 aa to about 190 aa, or about 190 aa to about 200 aa of any of the following:

[0212] As another example, the heterodimerization domain can be derived from the GAI Arabidopsis thaliana protein (also known as gibberellic acid insensitive, and DELLA protein GAI), which has the amino acid sequence Uniprot The amino acid sequence may have 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 stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, about 160aa to about 170aa, about 170aa to about 180aa, about 180aa to about 190aa, or about 190aa to about 200aa of Q9LQT8-1.

[0213] As another example, the heterodimerization domain can be derived from the GID1 Arabidopsis protein (also known as the gibberellin receptor GID1) and has the following amino acid sequences: GID1A (Uniprot Q9MAA7-1); GID1B (Uniprot Q9LYC1-1); GID1C (Uniprot Q9LYC1-1); GID1D (Uniprot Q9LYC1-1); GID1E (Uniprot Q9LYC1-1); GID1F (Uniprot Q9LYC1-1); GID1G (Uniprot Q9LYC1-1); GID1H (Uniprot Q9LYC1-1); GID1I (Uniprot Q9LYC1-1); GID1J (Uniprot Q9LYC1-1); GID1K (Uniprot Q9LYC1-1); GID1L (Uniprot Q9LYC1-1); GID1M (Uniprot Q9LYC1-1 Q940G6-1)) or a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, about 160 aa to about 170 aa, about 170 aa to about 180 aa, about 180 aa to about 190 aa, or about 190 aa to about 200 aa.

[0214] Heterodimerization of the heterodimerization domains described in 8.1.3 can be achieved by coupling different regulatory molecules (shown in brackets following the pair of heterodimerization domains): b) FKBP and CnA (rapamycin); c) FKBP and cyclophilin (rapamycin); d) FKBP and FRG (rapamycin); h) PYL and ABI (abscisic acid); j) This can be achieved by GAI and GID1 (gibberellin or the gibberellin analog GA-3M).

[0215] As mentioned above, rapamycin (PubChem CID 5284616) functions as a regulatory molecule. Alternatively, rapamycin derivatives or analogs can be used. See, for example, WO 96 / 41865; WO 99 / 36553; WO 01 / 14387; and Ye et al (1999) Science 283:88-91. For example, analogs, congeners, derivatives, and other compounds structurally related to rapamycin ("rapalogs") include variants of rapamycin that have one or more of the following modifications, particularly relative to rapamycin: demethylation, removal, or replacement of methoxy at C7, C42, and / or C29. removal, derivatization, or replacement of hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of ketone at C14, C24, and / or C30; replacement of the 6-membered pipecolic acid ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional information is provided, for example, in U.S. Patent Nos. 5,525,610; 5,310,903; 5,362,718; and 5,527,907. Selective epimerization of the C-28 hydroxyl group has been reported; see, for example, WO 01 / 14387. Additional synthetic regulatory molecules suitable for use as alternatives to rapamycin include those described in U.S. Patent Publication No. 2012 / 0130076, such as 28-epilapamycin (PubChem CID 131668123).

[0216] Also suitable as rapalogs are compounds of the formula:

[0217] [ka]

[0218] (e.g., as disclosed in U.S. Patent Application Publication No. 7,067,526 B1), where n is 1 or 2; 28 and R 43 are independently H or a substituted or unsubstituted aliphatic or acyl moiety; R 7a and R 7b One of them is H, the other is halo, R A , OR A , S.R. A , -OC(O)R A , -OC(O)NR A R B , -NR A R B , -NR B C(OR)R A , N.R. B C(O)OR A , -NR B SO 2 R A , or NR B SO 2 NR A R B' or R 7a and R 7b together are the H of the tetraene part:

[0219] [ka]

[0220] In the formula, R A is H or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety; R B and R B' is independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety.

[0221] 8.1.4. Extracellular dimerization by secreted soluble factors: Non-covalent complexation of at least two CAR molecules of the CAR group of the present invention can also be induced by secreted soluble factors, such as proteins that accumulate in the tumor stroma, whereby these proteins can often homo- or heterodimerize themselves. In this case, these soluble factors serve as the regulatory molecules of the present invention. The dimerization domain can then be, for example, a domain of a natural receptor (or a short peptide derived therefrom; e.g., Young et al., J Biol Chem. 2004;279(46):47633-42) to which the soluble factor can bind, or any antigen-binding polypeptide (as already described in Chapter 1 "Antigen-binding Moieties") engineered to bind to the selected soluble factor (e.g., Dotor et al., Cytokine. 2007;39(2):106-15; Lobner et al., MAbs. 2017;9(7):1088-1104) (VEGF-binding domain used for complexing the CAR group of Example 6).

[0222] 9. Target antigen: In a preferred embodiment of the invention, each antigen-binding portion of the CAR family, and each antigen-binding portion of another polypeptide capable of binding to a CAR molecule of said family, binds to a target antigen present on a cell, preferably a target antigen on a cell, on a solid surface, or on a lipid bilayer.

[0223] According to the present invention, the particular target antigen specifically recognized by the antigen-binding portion of the CAR family, or by the antigen-binding portion of another polypeptide capable of binding to a CAR molecule of said family, may be a naturally occurring cell surface antigen, or a polypeptide, carbohydrate, or lipid bound to a naturally occurring cell surface antigen.

[0224] Examples of antigens that can be bound by antigen-binding portions of the CAR family, as well as antigen-binding portions of other polypeptides that can bind to the CAR molecules of the family, include, for example, CD19, CD20, CD22, CD23, CD28, CD30, CD33, CD35, CD38, CD40, CD42c, CD43, CD44, CD44v6, CD47, CD49D, CD52, CD53, CD56, CD70, CD72, CD73, CD74, CD79A, CD79B, CD80, CD82, CD85A, CD85B, CD85D, CD85H, CD85K, CD96, CD107a , CD112, CD115, CD117, CD120b, CD123, CD146, CD148, CD155, CD185, CD200, CD204, CD221, CD271, CD276, CD279, CD280, CD281, CD301, CD312, CD353, CD362, BCMA, CD16V, CLL-1, Igkappa, TRBC1, TRBC2, CKLF, CLEC2D, EMC10, EphA2, FR-a, FLT3LG, FLT3, Lewis-Y, HLA-G, ICAM5, IGHA1 / IgA1, IL-1RAP, IL-17RE, I L-27RA, MILR1, MR1, PSCA, PTCRA, PODXL2, PTPRCAP, ULBP2, AJAP1, ASGR1, CADM1, CADM4, CDH15, CDH23, CDHR5, CELSR3, CSPG4, FAT4, GJA3, GJB2, GPC2, G PC3, IGSF9, LRFN4, LRRN6A / LINGO1, LRRC15, LRRC8E, LRIG1, LGR4, LYPD1, MARVELD2, MEGF10, MPZLI1, MTDH, PANX3, PCDHB6, PCDHB10, PCDHB12, PCDHB13 , PCDHB18, PCDHGA3, PEP, SGCB, Bezatin, DAGLB, SYT11, WFDC10A, ACVR2A, ACVR2B, Anaplastic Lymphoma Kinase, Cadherin 24, DLK1, GFRA2, GFRA3, EPHB2, EPHB3, EPHB4, EFNB1, EPOR, FGFR2, FGFR4, GALR2, GLG1, GLP1R, HBEGF, IGF2R, UNC5C, VASN, DLL3, FZD10, KREMEN2, TMEM169, TMEM198, NRG1, TMEFF1, ADRA2C, CHRNA1, CHRNB4,<h2 style=";text-align:left;direction:ltr">CHRNA3、CHRNG、DRD4、GABRB3、GRIN3A、GRIN2C、GRIK4、HTR7、APT8B2、NKAIN 1、NKAIN4、CACNA1A、CACNA1B、CACNA1I、CACNG8、CACNG4、CLCN7、KCNA4、KCN G2, KCNN3, KCNQ2, KCNU1, PKD1L2, PKD2L1, SLC5A8, SLC6A2, SLC6A6, SLC6A11, SLC6A15, SLC7A1, SLC7A5P1, SLC7A6, SLC9A1, SLC10A3, SLC10A4, SLC13A5 SLC16A8, SLC18A1, SLC18A3, SLC19A1, SLC26A10, SLC29A4, SLC30A1, SLC30A5, SLC35E2, SLC38A6, SLC38A9, SLC39A7, SLC39A8, SLC43A3, TRPM4, TRPV 4、TMEM16J、TMEM142B、ADORA2B、BAI1、EDG6、GPR1、GPR26、GPR34、GPR44、GP R56、GPR68、GPR173、GPR175、LGR4、MMD、NTSR2、OPN3、OR2L2、OSTM1、P2RX3、P 2RY8、P2RY11、P2RY13、PTGE3、SSTR5、TBXA2R、ADAM22、ADAMTS7、CST11、MMP 14、LPPR1、LPPR3、LPPR5、SEMA4A、SEMA6B、ALS2CR4、LEPROTL1、MS4A4A、ROM 1、TM4SF5、VANGL1、VANGL2、C18orf1、GSGL1、ITM2A、KIAA1715、LDLRAD3、OZ D3、STEAP1、MCAM、CHRNA1、CHRNA3、CHRNA5、CHRNA7、CHRNB4、KIAA1524、NRM. 3、RPRM、GRM8、KCNH4、DLK1、GFRA2、GFRA3、EPHB2、EPHB3、EPHB4、EFNB1、EPO R、FGFR2、FGFR4、GLP1R、HBEGF、IGF2R、UNC5C、VASN、DLL3、FZD10、KREMEN2、 TMEM169、TMEM198、NRG1、TMEFF1、ADRA2C、CHRNA1、CHRNB4、CHRNA3、CHRNG、 DRD4、GABRB3、GRIN3A、GRIN2C、GRIK4、NKAIN4、CACNA1A、CACNA1B、CACNA1I、CACNG8, CACNG4, CLCN7, KCNA4, KCNG2, KCNN3, KCNQ2, KCNU1, PKD1L2, PKD2L1, SLC5A8, SLC6A2, SLC6A6, SLCA10, SLC6A2, SLC6A6, SLC6A SLC13A5, SLC16A8, SLC18A1, SLC18A3, SLC19A1, SLC26A10, SLC29A4, SLC30A1, SLC30A5, SLC35E2, SLC38A6, SLC38A9, SLC39A7, SLC39A8, SLC43A3, TRPM4, TRPV4, TMEM GPR44, GPR56, GPR68, GPR173, GPR175, LGR4, MMD, NTSR2, OPN3, OR2L2, OSTM1, P2RX3, P2RY8, P2RY11, P2RY13, PTGE3, SSTR5, TBXA2R, ADAM22, ADAMTS7, CST11, MMP14, LP1 SEMA4A, SEMA6B, ALS2CR4, LEPRO TL1, MS4A4A, ROM1, TM4SF5, VANGL1, VANGL2, C18orf1, GSGL1, ITM2A, KIAA1715, LDLRAD3, OZD3, STEAP1, MCAM, CHRNA1, CHRNA3, CHRNA5, CHRNA7, CHRNB4, KIAA1524, NRM.3, RPC, GRM8, KCNH4, melanocortin 1 receptor, PTPRH, SDK1, SCN9A, SORCS1, CLSTN2, endothelin-converting enzyme-like-1, lysophosphatase receptor 2, L-lysozyme ... TB4R, TLR2, neurotropic tyrosine kinase 1, MUC16, B7-H4, epidermal growth factor receptor (EGFR), ERBB2, HER3, EGFR variant III (EGFRvIII), HGFR, FOLR1, MSLN, CA-125, MUC-1, prostate-specific membrane antigen (PSMA), mesothelin, epithelial cell adhesion molecule (EpCAM), L1-CAM, CEACAM1, CEACAM5, CEACAM6, VEGFR1, VEGFR2, high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A 1, IL-13R-α2, disialogangliosides (GD2 and GD3), tumor-associated carbohydrate antigens (CA-125, CA-242, Tn, and sialylated Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), C-MET, CCR1, CCR4, FAP,Fibronectin extra domain B (ED-B), GPNMB, IGF-1 receptor, integrin α5β1, integrin αvβ3, ITB5, ITGAX, embigin, PDGF-Rα, ROR1, syndecan 1, TAG-72, tenascin C, TRAIL-R1, TRAIL-R2, NKG2D-ligand, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, preferably PR1 / HLA-A2, lineage-specific or tissue-specific tissue antigens, preferably CD3, CD4, CD5, CD7, CD8, CD24, CD25, CD34, CD80, CD86, CD133, CD138, CD152, CD319, endoglin, MHC molecules, etc.

[0225] 10. Nucleic Acids, Cell Preparations and Therapeutic Uses: Another aspect of the present invention relates to a nucleic acid comprising a nucleotide sequence encoding a CAR molecule of the CAR group of the present invention. The nucleic acid of the present invention is, in some embodiments, a DNA or RNA, including, for example, an expression vector. The nucleic acid of the present invention can also be provided in other forms, such as a viral vector. The nucleic acid can be active or conditionally active in a cell, and in some embodiments, can be present as RNA, for example RNA synthesized in vitro. The introduction of RNA or DNA into a host cell can be performed in vitro or ex vivo or in vivo. For example, a host cell (e.g., NK cell, cytotoxic T lymphocyte) can be electroporated in vitro or ex vivo with an RNA comprising a nucleotide sequence encoding a CAR molecule of the CAR group.

[0226] In some cases, the nucleic acid of the disclosure comprises a nucleotide sequence encoding a CAR molecule of a CAR group of the invention consisting of either two, three, or four CAR molecules. In some cases, the nucleic acid of the disclosure comprises one, two, three, or four separate nucleotide sequences, each encoding one molecule of a CAR group consisting of either two, three, or four CAR molecules.

[0227] In the case where the CAR molecules of the CAR group are encoded by different nucleic acid molecules, the invention provides a kit of at least two nucleic acids encoding one, two, three or four molecules of the CAR group, where again the nucleic acids are preferably selected from DNA, RNA, or in vitro transcribed RNA.

[0228] The present invention also provides vectors comprising the nucleic acids (i.e. encoding a CAR molecule of the CAR family) of the invention, and / or kits of nucleic acids (encoding a CAR molecule of the CAR family).

[0229] Such vectors may include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector. Suitable vectors include, for example, plasmids, viral vectors, and the like. A large number of suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating recombinant constructs of the present invention. The following vectors are provided by way of example: Bacterial: pBs, phagescript, PsiX 174, 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). The vector may have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker functional in the expression host may be present. Suitable vectors include viral vectors (e.g., vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Due to their ability to efficiently integrate into the genome of transduced cells, preferred vectors are retroviral vectors, particularly gamma retroviral vectors, and lentiviral vectors, i.e., vectors derived from at least a portion of a retroviral genome. An example of a preferred retroviral vector is a self-inactivating lentiviral vector (provided in Milone et al., Mol Ther. 2009;17(8):1453-1464).Other examples of lentiviral vectors that can be used clinically include, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, LENTIMAX™ vector system from Lentigen, etc. Non-clinical lentiviral vectors are also available and will be known to those skilled in the art. Other types of preferred vectors that can efficiently integrate into the genome of transfected cells are transposon vectors, preferably PiggyBAC-based vectors and Sleeping beauty-based vectors. Further important non-viral strategies for integrating a gene of interest into the genome of a cell are based on site-specific nuclease technology (e.g., based on zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN)), or CRISPR / Cas technology (e.g., Gaj et al., Trends Biotechnol. 2013;31(7):397-405; and Ren et al., Protein Cell 2017;8(9):634-643). These techniques are attractive because they allow the incorporation of defined nucleotide sequences from any DNA molecule (single- or double-stranded DNA; in the form of vectors, PCR amplicons, etc.) and allow the integration of a gene of interest into the genome downstream of an endogenous promoter (reviewed, for example, in Eyquem et al., Nature. 2017;543(7643):113-117).

[0230] The invention also provides a kit of at least two vectors, each vector comprising a nucleic acid sequence encoding one, two, three or four CAR molecules of the CAR group of the invention. The vectors may comprise the same or different regulatory sequences to achieve expression in the same or different host systems (e.g., suitable cells in which the vector expresses the CAR molecule after transformation or propagation with the vector).

[0231] In the vector or vector kit of the present invention, the nucleic acid encoding the CAR molecule of the CAR group can be operably linked to a transcriptional control element to produce an expression vector. Such a transcriptional control element can be a promoter, an enhancer, etc., and suitable promoter and enhancer elements are known in the art. Suitable promoters for expression in bacterial cells include lac1, lacZ, T3, T7, gpt, lambda P, and trc. Suitable promoters for expression in eukaryotic cells include the light and / or heavy chain immunoglobulin gene promoter and enhancer elements, the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the early and late SV40 promoters, promoters present in long terminal repeats from retroviruses (e.g., promoter sequences comprising the 5'-LTR of gammaretroviruses or subelements R and U3 of the 5'-LTR of Moloney Murine Leukemia Virus (MMLV)), promoters present in mouse stem cell virus (MSCV), the mouse metallothionein-I promoter, EF1-alpha with or without an intron, the promoter of phosphoglycerate kinase (PGK), and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. The modification of a reversible promoter from a first organism for use in a second organism, e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc., is known in the art.Such reversible promoters, and systems based on such reversible promoters and also including additional regulatory proteins, include alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, 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.), temperature-regulated promoters, such as heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0232] In some cases, a locus or construct or transgene containing a suitable promoter can be irreversibly switched through induction of an inducible system. Suitable systems for induction of an irreversible switch are known in the art, for example, induction of an irreversible switch can use Cre-lox mediated recombination. Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art can be used to create an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, are used in creating irreversibly switched promoters and are well known in the art. In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK-specific promoter. For example, the CD4 gene promoter can be used. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the Neri (p46) promoter. In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter; or regulatable promoters, such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYCl 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 use in Pichia). Selection of appropriate vectors and promoters is within the level of ordinary skill in the art.Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters, the pagC promoter, the nirB promoter, etc.; the sigma70 promoter, such as the consensus sigma70 promoter (see, for example, GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, such as the dps promoter, the spv promoter; promoters from the pathogenicity island SPI-2; the actA promoter; the rpsM promoter; the tet promoter; the SP6 promoter; etc. Suitable strong promoters for use in prokaryotes, such as E. coli, include Trc, Tac, T5, T7, and PLambda. Examples of operators for use in bacterial host cells include the lactose promoter operator (the Laci repressor protein changes structure when contacted with lactose, thereby preventing the Laci repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator.

[0233] According to a preferred embodiment of the present invention, the vector or the kit of at least two vectors comprises a T lymphocyte-specific promoter, or a NK cell-specific promoter, or an EF1-alpha promoter, operably linked to a nucleotide sequence encoding a CAR molecule of the CAR group.

[0234] According to a further aspect, the present invention also relates to genetically modified cells modified to produce all CAR molecules of the CAR group of the present invention. The cells of the present invention can also be used to produce the vectors of the present invention (e.g., as viral or plasmid supernatants), which can be further purified therefrom, providing these vectors in amplified and purified form.

[0235] According to a preferred embodiment, the cell is a mammalian cell genetically modified to produce a CAR molecule of the CAR group of the present invention.Preferred mammalian cells are stem cells, progenitor cells, or cells derived from stem cells or progenitor cells, preferably lymphocytes.More preferred cells to be genetically modified according to the present invention are primary cells and immortalized cell lines.For pharmaceutical applications, human cells, especially lymphocytes, are particularly preferred.However, non-human primary cells and cell lines can also be suitable cell types, such as non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, especially for addressing scientific questions with the system of the present invention.

[0236] Further preferred cells of the present invention include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH The cells may be 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like. In some preferred examples, the cells of the present invention are not immortalized cell lines, but are cells (e.g., primary cells) obtained from an individual. For example, in some cases, the cells are immune cells obtained from an individual. In one example, the cells are T lymphocytes obtained from an individual. In another example, the cells are cytotoxic cells obtained from an individual. In another example, the cells are stem or progenitor cells obtained from an individual.

[0237] According to a particularly preferred embodiment, the mammalian cell of the invention transformed with a vector or a kit of at least two vectors encoding an individual CAR molecule of the CAR group of the invention is a T cell or a NK cell.

[0238] According to a further aspect, the present invention relates to a pharmaceutical preparation comprising a nucleic acid according to the invention, a kit of nucleic acids according to the invention, a vector or kit of vectors according to the invention, or a cell or kit of cells according to the invention.

[0239] The present disclosure provides a method for generating cells capable of combinatorial antigen recognition. The method generally involves genetically modifying a mammalian cell with a vector or kit of vectors, or RNA (e.g., in vitro transcribed RNA) that includes a nucleotide sequence encoding a molecule of the CAR family of the present disclosure. The genetic modification can be performed in vivo, in vitro, or ex vivo. The cell can be an immune cell (e.g., T lymphocyte or NK cell), stem cell, progenitor cell, etc.

[0240] Genetic modification is preferably performed ex vivo. For example, T lymphocytes (i.e., T cells), stem cells, or NK cells can be obtained from an individual, and the cells obtained from the individual are genetically modified to express the CARs of the present disclosure. In some cases, the genetically modified cells are activated ex vivo. When the genetically modified cells are introduced into an individual (e.g., the individual from whom the cells were obtained), the genetically modified cells are activated in vivo upon contact with a selected combination of target antigens present at physiological expression levels on the surface of the individual's cells. For example, when the genetically modified cells are T lymphocytes or NK cells, the genetically modified cells can exhibit cytotoxicity against cells that present a selected combination of target antigens at physiological expression levels on their surface to which the CARs (and / or antigen-binding portion of other polypeptides) bind. In the case of conditionally active CARs, the genetically modified cells are only effectively activated when contacted with a selected combination of target antigens present on the surface of cells at physiological expression levels in an individual, and when administered to the individual one or more regulatory molecules and / or one or more other polypeptides that are each capable of binding to a binding site of a molecule of the CARs and that include at least an antigen-binding portion. In some other cases, activation of the genetically modified cells when contacted with target antigens present on the surface of cells at physiological expression levels in an individual is reduced when a regulatory molecule is administered to the individual.

[0241] The present disclosure provides various therapeutic methods using the subject CARs.

[0242] The CARs of the invention, when present in a T lymphocyte or NK cell, can mediate cytotoxicity against a target cell. The non-covalently complexed CARs of the invention can, in some cases, bind to selected combinations of target antigens present on a target cell, depending on the presence of other polypeptides that bind to the target antigens, thereby mediating the killing of the target cell by the T lymphocyte or NK cell genetically engineered to generate the CARs.

[0243] Target cells include cancer cells. Thus, the present disclosure provides a method of killing or inhibiting the proliferation of target cancer cells, comprising contacting the target cancer cells with cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) genetically engineered to generate a set of subject CARs, such that the T lymphocytes or NK cells recognize a selected combination of target antigens present on the surface of the target cancer cells and mediate the killing of the target cells.

[0244] The present disclosure provides a method for treating cancer in an individual with cancer. In a preferred embodiment, the method includes: i) genetically modifying NK cells or preferably T lymphocytes obtained from an individual with at least one vector comprising a nucleotide sequence encoding each CAR molecule of the CAR group of the present invention, wherein the antigen-binding portion of the CAR group is specific for a target antigen on the cancer cells of the individual, and the genetic modification is performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual; iii) administering to the individual an effective amount of at least one regulatory molecule for inducing or reducing the heterodimerization of each CAR molecule of the group, preferably for inducing the heterodimerization of each CAR molecule of the group, thereby inducing or reducing the non-covalent complexation of the CAR group, preferably inducing the non-covalent complexation of the CAR group, wherein the non-covalently complexed CAR group, when contacted with a cancer cell expressing a combination of each target antigen at a physiological expression level, mediates the activation of the genetically modified cells, which leads to the death of the cancer cell, thereby allowing the treatment of cancer. In another preferred embodiment, the method comprises: i) genetically modifying NK cells or preferably T lymphocytes obtained from the individual with at least one vector comprising a nucleotide sequence encoding each CAR molecule of the CAR group of the invention, wherein the antigen-binding portion of the CAR molecule of said group and / or the antigen-binding portion of another polypeptide capable of binding to the CAR molecule of said group is specific for a target antigen on cancer cells of the individual, wherein the heterodimerization of each CAR molecule of said group does not require the administration of a regulatory molecule, and wherein said genetic modification is performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual; iii) administering to said individual an effective amount of at least one regulatory molecule comprising at least one antigen-binding portion and capable of binding to a binding site of a CAR molecule of said CAR group, wherein, upon contact with cancer cells expressing the combination of each target antigen at a physiological expression level, it mediates the activation of the genetically modified cells, which leads to the killing of the cancer cells, thereby allowing the treatment of the cancer.In yet another preferred embodiment, the method comprises: i) genetically modifying NK cells or preferably T lymphocytes obtained from an individual with at least one vector comprising a nucleotide sequence encoding each CAR molecule of a CAR panel of the invention, wherein the binding moieties of the CAR molecules of the panel are specific for a target antigen on cancer cells of the individual, and wherein heterodimerization of each CAR molecule of the panel does not require administration of a regulatory molecule, and wherein said genetic modification is performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual, which allows for the killing of cancer cells, thereby treating cancer. .

[0245] Cancers that may be treatable by the methods disclosed herein include esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma including small cell carcinoma and non-small cell carcinoma of the lung, adrenal cortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that may be treated by the methods disclosed herein include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that may be treated by the methods disclosed herein include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Leukemias that may be treated by the methods disclosed herein include: a) chronic myeloproliferative syndromes (neoplastic disorders of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells with restricted lineage potential); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and functionally dysfunctional small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; d) acute lymphoblastic leukemia (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using the subject methods include B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like. Other cancers that may be treatable by the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and mediastinal neurofibroma.

[0246] The subject method can also be used to treat inflammatory conditions and autoimmune diseases. The subject CARs can be expressed in T helper cells or regulatory T (Treg) cells for use in immunomodulatory methods. Immunomodulatory methods include, for example, enhancing the immune response of a mammalian subject to a pathogen; enhancing the immune response of an immunocompromised subject; reducing inflammatory responses; reducing the immune response of a mammalian subject exposed to an autoantigen, for example to treat an autoimmune disease; reducing the immune response of a mammalian subject exposed to a transplanted organ or tissue, for reducing organ or tissue rejection. When the method includes reducing the immune response to an autoantigen, at least one of the target antigens used to activate the CARs is preferably an autoantigen. When the method includes reducing the immune response to a transplanted organ or tissue, at least one of the antigens used to activate the CARs is preferably an antigen specific to the transplanted organ.

[0247] As noted above, the therapeutic methods of the present disclosure in preferred cases comprise the administration to an individual in need thereof of an effective amount of one or more distinct regulatory molecules and / or one or more distinct other polypeptides, each of which comprises at least an antigen-binding portion and is capable of binding to the extracellular binding site of a CAR molecule of the CAR group.

[0248] The required effective amount of each regulatory molecule to be administered to a required individual receiving T lymphocytes or NK cells ("effector cells") expressing CARs in accordance with the present invention is defined by the differential response of these effector cells upon contact with target cells expressing each antigen combination in the presence and absence of each required regulatory molecule. The response of these effector cells is thereby defined by secretion of interferon gamma, and / or macrophage inflammatory protein-1 (MIP-1) alpha, and / or MIP-1 beta, and / or granzyme B, and / or IL-2, and / or TNF, and / or IL-10, and / or IL-4, and / or degranulation of the effector cells, whereby cellular degranulation is preferably detected by the proportion of effector cells translocating CD107a on their surface, i.e., the proportion of CD107a positive effector cells detected by flow cytometric analysis using a degranulation assay (e.g., as described in Proff et al., Front Micro-biol. 2016 ;7:844), optionally following contact with target cells expressing more than 100,000 molecules of each target antigen per cell, optionally in the presence of an effective concentration of each other required polypeptide that comprises at least an antigen binding portion and is capable of binding to a binding site of a CAR molecule of the CAR group. In a preferred embodiment, the response of effector cells in the presence versus absence of an effective concentration of each necessary regulatory molecule differs by at least 20%, preferably at least 50%, or even more preferably at least 100%, where an effective concentration of each necessary regulatory molecule is a concentration achieved by administering an effective amount of each necessary regulatory molecule in one or more administrations to an individual in need thereof.The effective concentration of each of the other required polypeptides that comprise at least an antigen binding portion and are capable of binding to the CARs is defined by the response of the fully complexed subject CARs (i.e., all dimerization domains contained in the CARs) following contact with target cells expressing more than 100,000 molecules of each target antigen per cell in the presence versus absence of each of the other required polypeptides that comprise at least an antigen binding portion and are capable of binding to the CARs, wherein the response preferably differs by at least 20%, preferably at least 50%, or even more preferably at least 100%, and wherein the effective concentration of each of the other required polypeptides that comprise at least an antigen binding portion and are capable of binding to the CARs is the concentration achieved by administering an effective amount of each of these other polypeptides in one or more administrations to an individual in need thereof that has been administered T lymphocytes or NK cells expressing the subject CARs.

[0249] Both regulatory molecules and other antigen-specific polypeptides capable of binding to the CAR molecules of the CAR group of the present invention are hereinafter collectively referred to as "agents that specifically bind to the CAR group."

[0250] In the subject method, the "agent that specifically binds to the CAR group" can be administered to the host using any convenient means that can produce the desired therapeutic or diagnostic effect. That is, the "agent that specifically binds to the CAR group" can be incorporated into various formulations for therapeutic administration. More specifically, the "agent that specifically binds to the CAR group" can be formulated into a pharmaceutical composition by combining with a suitable pharma- ceutically acceptable carrier or diluent, and can be formulated into a preparation in the form of a solid, semi-solid, liquid, or gas, such as a tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, aerosol, etc. In the pharmaceutical dosage form, the "agent that specifically binds to the CAR group" can be administered in the form of their pharma- ceutically acceptable salts, or they can also be used alone or in appropriate combination, as well as in combination with other pharma- ceutically active compounds. The following methods and excipients are merely exemplary:

[0251] Suitable excipient vehicles may be, 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. Actual methods for preparing such dosage forms are known or will become apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. In any event, the composition or formulation to be administered will contain the necessary "agent that specifically binds to the CAR group" in a sufficient amount to achieve the desired state of the subject being treated. Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. In addition, pharma- ceutical acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public. For oral formulations, the "agent that specifically binds to the CAR group" can be used alone or in combination with suitable excipients to produce tablets, powders, granules, or capsules using conventional excipients such as lactose, mannitol, corn starch, potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc and magnesium stearate; and, if desired, diluents, buffers, humectants, preservatives, and flavors.

[0252] The "agents that specifically bind to the CAR group" can be formulated into injectable preparations 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 fatty acid or ester of propylene glycol, if desired, using conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0253] Pharmaceutical compositions comprising an "agent that specifically binds to a CAR group" can be prepared by mixing an "agent that specifically binds to a CAR group" having a desired purity with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer, and / or tonicity agent. Acceptable carriers, excipients, and / or stabilizers are preferably non-toxic to recipients at the dosages and concentrations used, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants such as 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, phenylalan ... monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin and serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants, such as Tween, Brizipluronic, Triton X, or polyethylene glycol (PEG).

[0254] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, where the lyophilized preparation is reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is usually to add a certain amount of pure water (usually equal to the amount removed during lyophilization), but solutions containing antimicrobial agents can be used to prepare pharmaceutical compositions for parenteral administration. See also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0255] The "agent that specifically binds to the CAR group" can also be optionally formulated in a controlled release formulation. The sustained release preparation can be prepared using methods known in the art. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the "agent that specifically binds to the CAR group", the matrices being in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. The potential loss of biological activity can be prevented by using appropriate additives, controlling the water content, and developing specific polymer matrix compositions.

[0256] The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical arts, the dosage for a single patient depends on many factors, such as the patient's size, body surface area, age, the particular "agent that specifically binds to a CAR group" administered, the patient's sex, the time and route of administration, general health, and other agents being administered simultaneously. The "agent that specifically binds to a CAR group" may be administered in an amount of 1 ng / kg body weight to 20 mg / kg body weight per administration, such as 0.1 mg / kg body weight to 10 mg / kg body weight, such as 0.5 mg / kg body weight to 5 mg / kg body weight, although doses less than or greater than this exemplary range are contemplated, particularly considering the aforementioned factors. If the formulation is a continuous infusion, it may also range from 1 μg to 10 mg per kilogram of body weight per minute.

[0257] The skilled artisan will readily appreciate that dosage levels can vary as a function of the particular "agent that specifically binds to a CAR group," the severity of the symptoms, and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0258] One or more "agents that specifically bind to CARs" can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes. Conventional pharma- ceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, intranasal, oral, and other enteral and parenteral routes of administration. Routes of administration can be combined as needed or tailored depending on the "agents that specifically bind to CARs" and / or the desired effect. The "agents that specifically bind to CARs" can be administered in a single dose or multiple doses. In a preferred embodiment of a conditionally active CAR, the "agents that specifically bind to CARs" can be administered orally or alternatively intravenously. In other embodiments, the "agents that specifically bind to CARs" can be administered via an inhalation route. In yet other embodiments, the "agents that specifically bind to CARs" can also be administered intranasally, topically, or intratumorally. In yet another embodiment, the "agent that specifically binds to the CARs" can be administered peritumorally. In a preferred embodiment having conditionally active CARs for the treatment of brain tumors, the "agent that specifically binds to the CARs" can be administered intracranially.

[0259] The "agent that specifically binds to the CAR group" can be administered to a host using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. In general, routes of administration contemplated by the present invention include enteral, parenteral, or inhalation routes. Parenteral routes of administration other than inhalation administration include topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes. Parenteral administration can be performed to provide systemic or local delivery of the "agent that specifically binds to the CAR group". When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of a pharmaceutical formulation. The "agent that specifically binds to the CAR group" can also be delivered to a subject by enteral administration. Enteral routes of administration include oral and rectal (e.g., using a suppository) delivery.

[0260] Treatment means at least the amelioration of symptoms associated with the pathological condition afflicting the host, where amelioration is used broadly to refer to at least a decrease in a parameter associated with the pathological condition being treated, e.g., cancer, e.g., the magnitude of the symptoms. Treatment thus includes situations in which the pathological condition, or at least the symptoms associated therewith, are completely suppressed, e.g., prevented from occurring or halted, e.g., terminated, such that the host does not suffer from the pathological condition, or at least the symptoms characterizing the pathological condition.

[0261] The "agent that specifically binds to the CARs" can be administered by injection and / or delivery, for example, to the site of a cerebral artery or directly to brain tissue. The "agent that specifically binds to the CARs" can 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 sustained release particles, by biolistic delivery to the target site. Additionally, the "agent that specifically binds to the CARs" can be administered as an adjunct therapy to standard cancer treatments. Standard cancer treatments include surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy treatment, antibody treatment, biological response modifier treatment, and certain combinations of the above.

[0262] A variety of subjects are suitable for treatment with a subject method of treating cancer. Suitable subjects include any individual, such as a human or non-human animal, that has cancer, has been diagnosed with cancer, is at risk for developing cancer, has cancer and is at risk for recurrence of cancer, has been treated with other therapies but has failed to respond to such treatment, or has recurred after an initial response to such treatment.

[0263] Subjects suitable for treatment with a subject immunomodulatory method include individuals with autoimmune disorders, immunocompromised individuals, such as those who are organ or tissue transplant recipients, and individuals infected with a pathogen. EXAMPLES

[0264] Figure 1: Schematic representation of exemplary configurations of a CAR group. Figure 1A shows the basic structure of a CAR molecule of a CAR group in a version in which an antigen-binding moiety is incorporated into the CAR molecule (left) and in a version in which the CAR molecule comprises a binding site that binds to a binding site of another polypeptide, illustratively comprising one antigen-binding moiety or two antigen-binding moieties (directed against different target antigens). The low affinity interaction occurs either between the antigen-binding moiety and the target antigen, or between the binding site of the CAR molecule and the binding site of another polypeptide that binds to the binding site of the CAR molecule. At least one of the CAR molecules of a CAR group must comprise at least one signaling region that comprises at least one ITAM. In the example of the CAR molecule shown, the internal domain illustratively comprises a single signaling region. The lines between each component of the CAR molecule shown indicate optional linkers. The heterodimerization domains (at least one of which is mandatory for each CAR molecule of the group) and optional additional domains or components are not shown.

[0265] FIG. 1B shows a schematic of a version of a CAR molecule that includes two antigen-binding moieties, or two binding sites to which other polypeptides containing at least an antigen-binding moiety can bind, or a combination of an antigen-binding moiety and a binding site to which other polypeptides containing at least an antigen-binding moiety can bind. In the example CAR molecule shown, the internal domain illustratively includes a single signaling region. The lines between each component of the CAR molecule shown indicate optional linkers. The heterodimerization domain and optional additional domains or components are not shown.

[0266] FIG. 1C shows a schematic of how many CAR molecules in a CAR group of 2, 3, or 4 CAR molecules can contain at least one signaling region (the entire signaling region of a given CAR molecule is represented by a white square symbol). Of the CAR molecules that contain at least one signaling region, all or some, but at least one, CAR molecule contains at least one ITAM. For simplicity, the ectodomain, heterodimerization domain, and optional additional domains or components are not shown. The lines between each component of the depicted CAR molecules indicate optional linkers.

[0267] FIG. 1D shows the schematic arrangement of signaling regions using the example of a CAR group consisting of two CAR molecules. The depicted example shows only some of the possible combinations of different arrangements. For example, a CAR molecule can contain two or more ITAM-containing signaling regions, or two or more costimulatory signaling regions. For simplicity, the ectodomain, heterodimerization domain, and optional additional domains or components are not shown. The lines between each component of the depicted CAR molecule indicate optional linkers.

[0268] 1E-1K show schematic diagrams of how heterodimerization domains can be used for non-covalent complexation of CARs. The depicted examples show only some of the possible arrangements. In the depicted examples, different pairs of heterodimerization domains are shown in different locations in the CAR molecule, each of which illustratively includes one or two signaling regions. For simplicity, the figures show only the signaling region, the transmembrane domain, and the heterodimerization domain. The lines between each component of the depicted CAR molecule indicate optional linkers. Similar arrangements of heterodimerization domains can also be incorporated extracellularly.

[0269] FIG. 1L illustrates the non-covalent complexation of CARs with extracellular soluble factors acting as regulatory molecules. The regulatory molecules are illustrated diagrammatically using naturally heterodimerizing proteins. The illustrated CAR molecules illustratively contain only one signaling region and one antigen-binding moiety or one binding site to which other polypeptides can bind. Optional additional heterodimerization domains and optional additional domains or components are not shown. The order of the antigen-binding domain (or binding site) and the dimerization domain can also be reversed. That is, the antigen-binding domain (or binding site) can also be closer to the plasma membrane than the heterodimerization domain. The lines between the components of the illustrated CAR molecules indicate optional linkers.

[0270] Figure 2 shows the K values ​​of different rcSso7d-based antigen-binding moieties against human EGFR fused to superfolder GFP (sfGFP) as determined by three complementary methods. d Values ​​are shown: (a) Quantification by flow cytometry of the amount of different sfGFP fusion proteins bound to Jurkat T cells expressing high levels of truncated EGFR (tEGFR), (b) and (c) Surface Plasmon Resonance (SPR) analysis by using matrices coated with a chimeric EGFR protein containing the extracellular domain of EGFR fused to the Fc domain of IgG1. Affinity was determined either in kinetic mode (b) or in steady-state analysis mode (c).

[0271] Figure 3: Cysteine ​​forming extracellular disulfide bonds prevent the use of avidity effects of AND gate function. (A) Schematic representation of the configuration of the CAR signaling scaffolds "Cys" of S-8cys-BB-3z ("Cys") and "Ser" of S-8ser-BB-3z ("Ser"), which allow or do not allow disulfide bonds, respectively. These signaling scaffolds were fused to different rcSso7d-based antigen-binding moieties and expressed for functional testing in primary human T cells. (B) Typical CAR expression (shown for rcSso7d variant "E11.4.1-WT" fused to either "Cys" or "Ser" CAR scaffolds) 20 hours after electroporation of 5 μg of mRNA in primary human T cells. T cells not expressing a transgene ("no CAR") served as negative controls. (C) Expression of tEGFR in Jurkat cells used as target cells 20 hours after electroporation with 3 μg of mRNA encoding tEGFR. Jurkat T cells without construct ("No construct") and the respective isotype control ("Isotype control") served as negative controls. CAR functionality was tested by determining the ability of primary human T cells modified with different CARs for target cell killing (D) and IFN-γ production (E). T cells from four different donors (indicated by different symbols) were electroporated with 5 μg of mRNA of the indicated CAR constructs and the next day co-cultured with Jurkat T cells (electroporated with 3 μg of tEGFR-mRNA) at an effector:target (E:T) ratio of 2:1 at 37°C for 4 hours. T cells without CAR ("No CAR") served as negative controls.

[0272] Figure 4: Dimerization / multimerization of scFv-containing CAR molecules prevents the use of avidity effects of AND gate function. (A) Schematic representation of the CAR used in the experiment. (B, C, D) Expression of CAR constructs in human primary T cells 20 hours after electroporation with 5 μg of mRNA each. T cells without CAR ("No CAR") served as negative control. (E) Expression of tHER2 in Jurkat cells used as target cells 20 hours after electroporation with 5 μg of mRNA encoding tEGFR. Jurkat T cells without construct ("No Construct") served as negative control. CAR T cells and Jurkat-tHER2 cells were co-cultured at an E:T ratio of 2:1 for 4 hours at 37°C. Figures 4F and 4G show the ability of CAR, where scFv 4D5-5 was fused to two different CAR signaling scaffolds (with or without disulfide bonds available, i.e., "Cys" in 4D5-5-8cys-BB-3z (SEQ ID NO: 61) and "Ser" in 4D5-5-8ser-BB-3z (SEQ ID NO: 62)), to induce cytotoxicity (G) and IFN-gamma production (F). H and V L Two separate polypeptides ("V H and V L "; the function of CAR fused to 4D5-5(split)-8ser-BB-FKBP(36V)-3z (SEQ ID NO:58 and SEQ ID NO:59) is shown in (H). Primary T cells expressing CAR 4D5-5-8ser-BB-3z (SEQ ID NO:62) in which a monomeric signaling scaffold is fused to 4D5-5-scFv served as a positive control. CAR T cells from three different donors (indicated with different symbols) were co-cultured with a mixture of tHER2-transfected and non-transfected Jurkat T cells at an E:T ratio of 4:1:1 for 4 hours at 37°C (T cells:tHER2 pos Jurkat cells: tHER2 neg Jurkat cells), survival-active tHER2 pos and tHER2 negThe proportion of target cells was determined by flow cytometry. To induce dimerization, T cells were pretreated with the dimerizer AP20187 (10 nM, 30 min, 37°C). Treatment with the same concentration of DMSO served as the control condition. T cells without CAR ("no CAR") and V H T cells expressing only the 4D5-5(V H )-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 59)) was used as a negative control.

[0273] Figure 5: Screening of affibody-based binding moieties suitable for use in the CAR series of the invention. The affinity of the affibody zHER2-WT was reduced by replacing all amino acids potentially involved in epitope binding with alanine. Different variants of zHER2-WT were fused to the CAR signaling scaffold 8ser-BB-FKBP(36V)-3z, which contains the intracellular homodimerization domain FKBP(F36V) for conditional dimerization. The configurations of these CARs are shown in (A). (B and C) Expression of the target antigen tHER2 in Jurkat T cells and expression of affibody-based CARs in primary T cells, respectively. Primary and Jurkat T cells were electroporated with 5 μg of mRNA encoding each construct, and expression was measured 20 hours after electroporation. Primary and Jurkat T cells not expressing the construct ("no CAR" and "no construct", respectively) were used as negative controls. (D) Expression of 13 different affibody-based CARs in primary T cells ("L9A", "R10A", "Q11A", "Y13A", "W14A", "Q17A", "W24A", "T25A", "S27A", "R28A", "R32A", "Y35A", and "zHER2-WT", respectively). (Sequences of the affibody-based antigen-binding moieties fused to the CAR signaling scaffold are shown in SEQ ID NO:26-SEQ ID NO:38. Primary T cells were electroporated with 5 μg of mRNA encoding each construct and expression was measured 20 hours after electroporation. T cells not expressing the construct ("no CAR") were used as negative control. (E) Dimerization-induced activation of affibody-based CARs. Primary T cells from two donors (indicated with different symbols) were electroporated with 5 μg of each construct. Specific lysis of target cells was observed in the presence of different CARs. This was determined in a luciferase-based cytotoxicity assay after co-incubation of T cells with tHER2 transfection (4 h at 37°C, E:T ratio 2:1). CAR dimerization was induced by pre-treatment of T cells with AP20187 (10 nM, 30 min, 37°C). Treatment with the same concentration of DMSO served as the control condition. T cells without CAR ("no CAR") served as a negative control.FIG. 5F shows the K of each affibody-based binding moiety for human HER2 (fused to superfolder-GFP (sfGFP)) determined by SPR analysis using matrices coated with a chimeric HER2 protein containing the extracellular domain of HER2 fused to the Fc domain of IgG1. d Values ​​are shown. Affinities were determined in kinetic mode.

[0274] Figure 6: Modulation of function of stably transduced CAR T cells in vivo in the NSG mouse model. (A) Efficacy of dimerization-induced activation of CAR in the in vivo NSG mouse model. 9-16 week old NSG mice were stably transduced with vectors encoding luciferase and tEGFR. 0.5x10 6 Three days later, NSG mice were intravenously injected with 10x10 Nalm6 cells ("Nalm6-tEGFR-fLuc"). 6 The mice were treated by intravenous administration of CAR T cells (14 days after activation with anti-CD3 / CD28 antibody-coated beads, i.e., 13 days after stable transduction). Injection of phosphate-buffered saline (PBS) served as the control condition. Five NSG mice were used for each treatment group, except for the group treated with S(WT)-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 49), which consisted of four mice. Dimerization was induced by intraperitoneal administration of 2 mg / kg of the homodimerizer AP20187 for 11 days (days of injection are indicated by arrows). Groups that did not receive dimerizers received the respective vehicle solution intraperitoneally as control conditions. Tumor sizes (average for each treatment group) are shown as total photon flux determined within the region of interest that included the whole body of NSG mice. (B) shows that extended administration of the dimerizer in mice treated with S(G32A)-8ser-BB-FKBP(36V)-3z (sequence number 46) CAR T cells but not administered the dimerizer AP20187 until day 11 results in efficient CAR activation and control of tumor growth.

[0275] Figure 7: Modulation of the function of stably transduced CAR T cells in vitro. (A) Schematic representation of the homodimerized CAR molecules used in the in vivo experiments of Example 5. (B) Expression of CAR molecules containing antigen-binding moieties with high or low affinity for EGFR (i.e., S(WT)-8ser-BB-FKBP(36V)-3z "E11.4.1-WT" (SEQ ID NO: 49) and S(G32A)-8ser-BB-FKBP(36V)-3z "E11.4.1-G32A" (SEQ ID NO: 46), respectively) in primary human T cells 14 days after activation with anti-CD3 / CD28 antibody-coated beads (i.e., 13 days after stable transduction with each CAR construct). T cells without a CAR ("No CAR") served as a negative control. (C) Expression of anti-CD19 CAR CD19-8cys-BB-3z "CD19-BBz" (SEQ ID NO: 60) in primary human T cells 14 days after activation with anti-CD3 / CD28 antibody-coated beads (i.e., 13 days after stable transduction). T cells without a CAR ("No CAR") served as a negative control. (D and E) Expression of tEGFR in Nalm6-fLuc and Nalm6-tEGFR-fLuc cells, respectively. Unstained cells and the respective isotype controls served as negative controls. (F and G) Lysis of Nalm6-fLuc and Nalm6-tEGFR-fLuc cells by primary human T cells expressing different CAR molecules. Primary T cells from three donors (indicated by different symbols) were stably transduced with vectors encoding an rcSso7d-based CAR (S(WT)-8ser-BB-FKBP(36V)-3z "E11.4.1-WT" and S(G32A)-8ser-BB-FKBP(36V)-3z "E11.4.1-G32A"), or an scFv-based CAR against CD19 (CD19-8cys-BB-3z "CD19-BBz"), which served as a positive control. T cells without a CAR ("No CAR") served as a negative control. AP20187 was used as a modulator of non-covalent dimerization of "E11.4.1-WT" (S(WT)-8ser-BB-FKBP(36V)-3z) and "E11.4.1-G32A" (S(G32A)-8ser-BB-FKBP(36V)-3z). Dimerization was induced by pre-treating T cells with 10 nM AP20187 for 30 min at 37°C.Treatment with the same concentration of DMSO served as the control condition. Cytotoxicity of modified T cells was determined by quantification of viable, luciferase-expressing target cells after co-culture at an E:T ratio of 10:1 for 4 h at 37 °C.

[0276] Figure 8: Generation of CARs that can be regulated by VEGF as an example of an extracellular factor that accumulates in the tumor microenvironment. In the example shown, the soluble factor VEGF was used as the regulatory molecule and the EGFR-specific antigen-binding moiety E11.4.1-G32A was used as the antigen-binding moiety. Schematic diagrams of the respective CAR constructions are shown in (A) and (B). Expression of the target antigen tEGFR in Jurkat T cells 20 hours after electroporation of 5 μg of mRNA is shown in (C). Expression of the two polypeptides in primary human T cells was detected using an anti-IgG1 antibody (D). For detection of CAR molecules containing a VEGF-binding site (Janus-CT6-Fc domain without a transmembrane domain) and a control CAR without an IgG-Fc domain, an anti-StrepII-tag antibody was additionally used. Cytotoxicity caused by the different CARs in primary T cells was determined in a FACS-based cytotoxicity assay (E). CAR T cells from three different donors (indicated by different symbols) were co-transfected with tEGFR-transfected Jurkat cells at an E:T ratio of 4:1:1 (T cells:tEGFR pos Jurkat cells: tEGFR neg Jurkat cells) for 4 h at 37° C. CAR dimerization was induced by pre-treating T cells with VEGF (indicated concentrations, 30 min, 37° C.). T cells without CAR ("No CAR") served as a negative control, and T cells with CAR S(WT)-8ser-BB-FKBP(36V)-3z served as a positive control.

[0277] Figure 9: Functional characterization of CAR T cells able to specifically recognize target cells co-expressing EGFR and HER2. (A) Schematic representation of the CAR family "S(G32A)-8ser-BB-FKBP-3z+A(R10A)-8ser-BB-FRB-3z" (SEQ ID NO: 48 and SEQ ID NO: 54), which contains the low affinity binding moieties E11.4.1-G32A (targeting EGFR) and zHER2-R10A (targeting HER2) fused to a signaling scaffold in which the extracellular cysteine ​​residues are replaced by serine residues. The use of two unrelated epitope tags (FLAG and StrepII tags) allows efficient detection of the expression of both chains. The heterodimerization domains FKBP and FRB mediate the specific heterodimerization of the CAR molecules via the regulatory molecule AP21967. (B) Two different low affinity binding moieties (E11.4.1-G32A and zHER2-R10A) were attached to the ectodomain of a single CAR molecule (flexible 2xG 4 Schematic diagram of the tandem-CAR "A(R10A)-S(G32A)-8ser-BB-FKBP(36V)-3z" (SEQ ID NO: 71), which is sequentially integrated (through a 3S linker). (C and D) Expression of the CAR construct in human primary T cells 20 hours after electroporation with 5 μg of mRNA each. Primary T cells without CAR ("No CAR") served as negative control. (E and F) Expression of tEGFR and / or tHER2 in Jurkat T cells 20 hours after electroporation with 5 μg of mRNA each. Jurkat T cells not expressing a transgene ("No Construct") served as negative control. The function of the CARs in the presence and absence of the regulatory molecule AP21967 is shown in (G). T cells (indicated with different symbols) from three different donors expressing the CAR group "S(G32A)-8ser-BB-FKBP-3z+A(R10A)-8ser-BB-FRB-3z" or the tandem-CAR "A(R10A)-S(G32A)-8ser-BB-FKBP(36V)-3z" or no CAR (as indicated) were co-cultured with Jurkat T cells expressing tEGFR ("EGFR") or tHER ("HER2") or both ("EGFR / HER2"). Co-culture with T cells (E:T ratio 4:1:1 (T cells:transgene pos Jurkat cells: transgeneneg Lysis of target antigen-expressing Jurkat cells after 4 h at 37° C. (Jurkat cells) was quantified by flow cytometry. Dimerization of the CAR molecules of the population was induced by pre-treating T cells with 500 nM AP21967 for 30 min at 37° C. Treatment with the same concentration of ethanol served as the control condition. T cells lacking a CAR ("no CAR") served as a negative control.

[0278] Figure 10: Functional characterization of CAR panels consisting of three and four CAR molecules. (A and B) Schematic representation of CAR panels consisting of three or four CAR molecules, respectively. (C and D) Expression of trimeric and tetrameric CAR panels in Jurkat T cells 20 hours after electroporation with 5 μg of mRNA of each construct. Jurkat T cells expressing no construct ("No CAR") were used as negative control.

[0279] FIG. 11: Functional characterization of CARs containing heterodimerization domains for constitutive complex formation. (A) Schematic representation of CARs consisting of two CAR molecules, each containing a leucine zipper-based heterodimerization domain ("EE" and "RR"). (B and C) Expression of CARs in Jurkat T cells 20 hours after electroporation of 5 μg of mRNA of each construct. Jurkat T cells not expressing the construct ("no CAR") were used as negative controls. (D and E) Expression of CARs in primary human T cells 20 hours after electroporation of 5 μg of mRNA of each construct. Primary human T cells not expressing the construct ("no CAR") were used as negative controls. Function of CARs in primary human T cells (indicated by different symbols) from four different donors is shown in (F). Cytotoxicity of T cells responding to target cells expressing both EGFR and HER2, or only EGFR or HER2, was determined using a FACS-based cytotoxicity assay. T cells not expressing the constructs ("No CAR") and CAR T cells expressing the indicated constructs were incubated with the respective Jurkat T cells ("tEGFR", "tHER2", or "tEGFR / tHER2") for 4 hours at 37°C in an E:T ratio of 4:1:1 (T cells:transgene pos Jurkat cells: transgene negJurkat cells). Statistical significance was calculated using one-way ANOVA (using GraphPad PRISM software) (*=p<0.05; ns=p>0.05).

[0280] Figure 12: Expression and function of CARs containing different costimulatory molecules. (A) Schematic representation of a CAR panel consisting of two CAR molecules, each containing either the CD28 or ICOS or OX40 costimulatory domain in its costimulatory signaling region. (B and C) Expression of CAR molecules 20 hours after electroporation of 5 μg of mRNA in Jurkat T cells or primary human T cells, respectively (red histograms). Jurkat T cells or primary human T cells expressing no construct ("no CAR") were used as negative controls, respectively (filled blue histograms). (D) Induction of NF-κB and NF-AT promoters in Jurkat T cells electroporated with 5 μg of mRNA encoding S(G32A)-8ser-OX40-FKBP(36V)-3z. These cells were or were not cocultured for an additional 20 hours with Jurkat T cells electroporated with 5 μg of mRNA encoding tEGFR, in the presence or absence of AP20187. Induction of NF-κB and NF-AT promoters in reporter cells expressing CAR was detected by flow cytometric analysis of enhanced green fluorescent protein (eGFP) and cyan fluorescent variant of GFP (CFP) expression, respectively. Cytotoxicity of primary human T cells expressing the indicated CAR molecules is shown in (E). Twenty hours after electroporation of 5 μg of mRNA encoding each CAR molecule, T cells were mixed with target cells at an E:T ratio of 4:1:1 (T cells:tEGFR pos Jurkat cells: tEGFR neg Jurkat cells) and co-cultured for an additional 4 or 20 hours at 37° C. Dimerization of the CAR molecules in the groups was induced by pre-treatment of Jurkat cells (D) and primary human T cells (E) with 10 nM AP20187 for 30 min at 37° C. Treatment with DMSO at the same concentration served as the control condition.

[0281] Figure 13: Expression of CAR molecules containing rcSso7d and affibody-based binding moieties fused to different CAR signaling scaffolds. (A) Expression of CAR constructs "Myc-S(18.4.2)-8cys-BB-3z" (SEQ ID NO: 39), "S(18.4.2)-8cys-BB-3z" (SEQ ID NO: 40), and "S(18.4.2)-G4S-8cys-BB-3z" (SEQ ID NO: 41) in primary human T cells 20 hours after electroporation of 5 μg of mRNA each. Primary T cells without CAR served as negative control (filled histogram). Expression was detected using a fusion protein consisting of the extracellular domain of human EGFR, the Fc domain of IgG1, and an anti-human IgG1 antibody. (B) Expression of CAR constructs "S(WT)-G4S-myc-8cys-BB-3z" (SEQ ID NO: 42), "S(WT)-G4S-StrepII-8cys-BB-3z" (SEQ ID NO: 43), and "S(WT)-G4S-his-8cys-BB-3z" (SEQ ID NO: 45) in primary human T cells 20 hours after electroporation with 5 μg of mRNA each. Primary T cells without CAR served as negative control (filled histograms). CAR expression was detected using either anti-c-myc, anti-StrepII, or anti-hexahistidine antibodies, respectively. (C) Expression of CAR constructs "S(WT)-8cys-BB-3z" (SEQ ID NO: 79), "S(G25A)-8cys-BB-3z" (SEQ ID NO: 77), "S(G32A)-8cys-BB-3z" (SEQ ID NO: 43), "S(WT)-8ser-BB-3z" (SEQ ID NO: 80), "S(G25A)-8ser-BB-3z" (SEQ ID NO: 78), "S(G32A)-8ser-BB-3z" (SEQ ID NO: 44) in primary human T cells 20 hours after electroporation with 5 μg of each mRNA. Primary T cells without CAR served as negative control (filled histogram). Expression was detected using an anti-StrepII antibody.(D) Expression of CAR constructs "S(G32A)-8ser-BB-FKBP(36V)-3z" (SEQ ID NO: 46), "S(G32A)-8ser-BB-FRB-3z" (SEQ ID NO: 47), "S(G32A)-8ser-BB-FKBP-3z" (SEQ ID NO: 48), "A(WT)-8ser-BB-FKBP(36V)-3z" (SEQ ID NO: 52), "A(WT)-8ser-BB-FRB-3z" (SEQ ID NO: 53), "A(R10A)-8ser-BB-FRB-3z" (SEQ ID NO: 54) in primary human T cells 20 hours after electroporation with 5 μg of mRNA each. Primary T cells without CAR served as negative control (filled histograms). Expression was detected using either anti-FLAG, anti-StrepII, or anti-hexahistidine antibodies, as indicated.

[0282] Figure 14 shows a schematic diagram of the design of various CAR molecules. The corresponding amino acid sequences are shown in Figure 15.

[0283] FIG. 15 shows the amino acid sequences of various CAR molecules.

[0284] Example 1 Generation of low affinity single domain antigen-binding moieties based on rcSso7d for use in the CARs of the invention The first example shows a strategy for creating low affinity antigen-binding moieties suitable for use as antigen-binding moieties in the CARs of the invention. Charge-reduced Sso7d (rcSso7d) is a charge-reduced version of a small (~7 kDa) DNA-binding protein from the archaeon Sulfolobus solfataricus. Charge reduction minimizes non-specific binding due to reduced electrostatic interactions. rcSso7d is an example of a suitable binding scaffold, as it is a single domain protein antigen-binding moiety that exhibits high thermal stability and monomeric behavior. It has a K of 19 nM. dStarting from the well-characterized antigen-binding moiety rcSso7d E11.4.1 (Traxlmayr et al., J Biol Chem. 2016;291(43):22496-22508) that binds to human EGFR at 1 kDa, we created low-affinity mutants by performing an alanine scan in which all amino acids potentially involved in epitope binding were replaced by alanine, i.e., one position involved in antigen binding was mutated to alanine in each variant. Mutants of rcSso7d E11.4.1 were fused to sfGFP and expressed as soluble proteins in a bacterial expression system. A schematic representation of the fusion protein construction is shown in Figure 14G. Binding affinities were determined (i) by performing titration experiments of soluble fusion proteins of the binding moieties with sfGFP on Jurkat T cells engineered by mRNA electroporation to express high levels of the respective target antigen EGFR, and (ii) by performing SPR experiments on Protein A chips loaded with the extracellular domain of EGFR fused to IgG-Fc. The results of the alanine scan and the obtained affinities of the antigen-binding moieties are shown in Figure 2.

[0285] Alanine scanning of protein antigen-binding sites: Site-directed mutagenesis of all amino acids involved in epitope binding was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Genomics) according to the manufacturer's instructions. Primers were engineered using QuikChange Primer Design software (Agilent Genomics) and oligonucleotides were synthesized by Biomers.

[0286] Expression and purification of rcSso7d-based antigen-binding moieties: The binding scaffolds were expressed as sfGFP fusion proteins (consisting of an N-terminal hexahistidine tag followed by rcSso7d or affibody and sfGFP) using the pE-SUMO vector (Life Sensors). The nucleotide sequence encoding the sfGFP reporter protein was obtained from Addgene (plasmid #54737). Briefly, E. coli cells (Tuner DE3) were transformed using heat shock transformation with sequence verified plasmids. After overnight growth at 37 °C, the cultures were transferred to Terrific Broth (TB) medium (12 g / L tryptone, 24 g / L yeast extract, 4% glycerol, 2.31 g / L KH2O2) supplemented with kanamycin (50 μg / mL) and incubated for 3 h at 4 °C. 2 PO 4 , and 16.43 g / LK 2 HPO 4 3H 2 O) and incubated with shaking at 37°C. The cultures reached an A of approximately 2 600When the chromatin concentration reached 100 mM, transgene expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were further grown overnight at 20°C. Cells were harvested by centrifugation (5000g, 20 min, 4°C), resuspended in sonication buffer (50 mM sodium phosphate, 300 mM NaCl, 3% glycerol, 1% Triton X-100, pH 8.0), sonicated (2x90 sec, duty cycle 50%, amplitude set to 5), and centrifuged again to remove cell debris. Hexahistidine-tagged fusion proteins were purified from crude cell extracts using TALON metal affinity resin (Clontech Laboratories). After adding 10 mM imidazole, the sonicated supernatant was applied twice to the resin, followed by washing steps with equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) with increasing amounts of imidazole (5-15 mM). The bound scaffold was eluted by applying equilibration buffer supplemented with 250 mM imidazole. After buffer exchange into PBS using Amicon Ultra-15 10K centrifugal filters (Merck Millipore), the concentration was determined by measuring the absorbance at 280 nm using the respective molar extinction coefficient, and finally the protein was directly frozen at -80 °C.

[0287] Maintenance of human cell lines: Jurkat T cells were kindly provided by Dr. Sabine Strehl at the Children's Cancer Research Institute (CCRI) and maintained in RPMI-1640 (Thermo Scientific) supplemented with 10% FCS (Sigma Aldrich) and 1% penicillin-streptomycin (Thermo Scientific). Cell lines were routinely tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell density was monitored using AccuCheck counting beads (Thermo Scientific), a flow cytometer-based cell counting platform.

[0288] In vitro transcription and electroporation of mRNA: In vitro transcription was performed using the mMessage mMachine T7 Ultra kit (Ambion) according to the manufacturer's instructions. 50–200 ng of column-purified PCR product was used as reaction template. The resulting mRNA was purified using an RNeasy column purification kit (Qiagen) using an adapted protocol. Briefly, the mRNA solution was diluted with a mixture of RLT buffer (Qiagen), ethanol (Merck), and 2-mercaptoethanol (Merck). The mixture was loaded onto an RNeasy column and purified according to the manufacturer's instructions. Elution was performed with nuclease-free water (Thermo Scientific), and the purified mRNA was frozen at −80°C until the time of electroporation. For transient transgene expression, Jurkat T cells were electroporated with various amounts of each mRNA using a Gene Pulser (Biorad). The following protocol was used for each cell type: Jurkat T cells (square wave protocol, 500 V, 3 ms, and 4 mm cuvettes).

[0289] Antibodies and flow cytometry: Jurkat T cells were resuspended in FACS buffer (PBS (Thermo Scientific), 0.2% human albumin (CSL Behring), 0.02% sodium azide) and treated with 10% human serum for 10 min at 4°C. Cells were stained with each primary antibody for 25 min at 4°C. Stained cells were washed twice with FACS buffer and then directly processed with BD LSRFortessa. Expression of the engineered target antigen tEGFR was detected with either PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was performed by FlowJo software.

[0290] Determination of binding affinity on cell membrane: Jurkat T cells were engineered to express high levels of each tumor antigen. Thus, Jurkat T cells were electroporated with 3 μg of mRNA encoding tEGFR and co-cultured with effector cells one day later. After washing with PBS, cells were resuspended in PBS containing 0.1% BSA (Sigma Aldrich) and incubated with various concentrations of binder proteins fused to sfGFP to determine the affinity of the antigen-binding moiety for each tumor antigen. After 1 h of incubation at 4°C with shaking, plates were centrifuged (450 g, 7 min, 4°C), the supernatant was discarded, and cells were retrieved using a BD LSRFortessa. To avoid endocytosis, cells were placed on ice. K d was obtained by curve fitting using Microsoft Excel (Microsoft Corporation).

[0291] Determining binding affinity using surface plasmon resonance (SPR): SPR experiments were performed using a Biacore T200 instrument (GE Healthcare). All experiments were performed at 25°C in degassed and filtered PBS, pH 7.4, containing 0.1% BSA and 0.05% Tween-20 (Merck Millipore). hEGFR-Fc (R&D) was immobilized at a concentration of 6.67 μg / mL on a Protein A sensor chip (GE Healthcare) at a flow rate of 10 μL / min for 60 s. To determine the affinity of the rcSso7d-based antigen-binding moieties, five concentrations of each protein (expected K of the antigen-binding moiety) were used. d (depending on K) was injected in single cycle kinetic mode at a flow rate of 30 μL / min for 15 s, followed by a dissociation step (30 s). Regeneration was performed with 10 mM glycine-HCl, pH 1.7, at a flow rate of 30 μL / min for 30 s. d was obtained by curve fitting using Biacore T200 evaluation software (GE Healthcare).

[0292] Example 2 Extracellular disulfide bond-forming cysteines prevent full utilization of the avidity effect on the AND gating function of CARs Extracellular disulfide bond forming cysteines in the extracellular hinge region, such as CD8α, can prevent the use of the avidity effect of the present invention. This is demonstrated in Example 2, where a low affinity mutant of the binding moiety "E11.4.1 G32A" of Example 1 was fused to a CAR signaling scaffold, where two extracellular cysteine ​​residues in the hinge region of CD8α (UniProt ID P01732, positions C164 and C181) were either substituted or not substituted with serine residues, respectively. The cysteine-containing CAR variant ("Cys") efficiently induced T cell activation in response to target cells, whereas the serine-containing variant ("Ser") did not or only weakly induced T cells. This example therefore shows the importance of preventing disulfide bond formation to create CAR molecules suitable for use in the CAR group of the present invention. The diagram in Figure 3A shows the engineering of the constructs tested. Figures 3B and 3C show the expression of the CAR and the target antigen. Primary human T cells were electroporated with 5 μg of mRNA of each construct and CAR expression was detected via the StrepII tag 20 hours after electroporation. Jurkat T cells were electroporated with 3 μg of mRNA encoding a truncated version of EGFR (tEGFR). Full-length EGFR was truncated at both the N- and C-termini to create a functionally inactive human polypeptide, which has reduced dimerization properties due to its inability to bind its natural ligand EGF and the absence of a kinase domain (Wang et al., Blood. 2011;118(5):1255-1263). The resulting transgene consisted of amino acids 334-675 of human EGFR (Uniprot P00533), including the leader sequence of the granulocyte-macrophage colony-stimulating factor 2 receptor alpha subunit (GM-CSF-Ra) and the two extracellular membrane-proximal and transmembrane domains. Transgene expression was detected 20 hours after electroporation via antibodies against EGFR. Twenty hours after electroporation of T cells, CAR function was determined using a luciferase-based cytotoxicity assay (Figure 3D) and by quantifying cytokines released from T cells using ELISA (Figure 3E).Figures 3D and 3E show that the antigen-binding moiety with the lowest affinity tested ("E11.4.1-G32A") can only induce T cells in the case of bivalent interaction with target cells, i.e., when fused to a CAR containing cysteines in the CD8α hinge ("Cys"), but not or only weakly when fused to a CAR in which these cysteines are replaced by serine ("Ser"). Conversely, antigen-binding moieties with increased affinity (E11.4.1-WT and E11.4.1-G25A) induced strong cytotoxicity even in monovalent interactions when fused to the "Ser" CAR scaffold. Taken together, this example shows that CAR molecules containing cysteines can homodimerize, resulting in activation of the CAR by single-positive target cells (i.e., target cells expressing only one antigen). However, the aim of the present invention is to construct a panel of CARs that specifically recognize target cells expressing a given combination of antigens (i.e., AND-gated CAR function). Thus, the ectodomain of each CAR molecule of the CAR population of the invention does not contain a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with each of the other CAR molecules of the population.

[0293] Maintenance of human cell lines: Primary human T cells were obtained from anonymous healthy donor blood after apheresis (buffy coat from the Austrian Red Cross, Vienna, Austria). pos T cells were enriched by negative selection using RosetteSep Human T Cell Enrichment Cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO (Sigma Aldrich) until use. CD3 posT cells were activated with anti-CD3 / CD28 beads (Thermo Scientific) according to the manufacturer's instructions and expanded in human T cell medium consisting of RPMI-1640 supplemented with 10% FCS, 1% penicillin-streptomycin, and 200 IU / mL recombinant human IL-2 (Peprotech). Primary T cells were cultured for at least 14 days before experiments were performed. Jurkat T cells were kindly provided by Dr. Sabine Strehl at CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell density was monitored using AccuCheck counting beads.

[0294] In vitro transcription and electroporation of mRNA: In vitro transcription was performed using the mMessage mMachine T7 Ultra kit according to the manufacturer's instructions. 50–200 ng of column-purified PCR product was used as reaction template. The resulting mRNA was purified using the RNeasy column purification kit using an adapted protocol. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol, and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purified according to the manufacturer's instructions. Elution was performed with nuclease-free water, and the purified mRNA was frozen at −80°C until the time of electroporation. For transient transgene expression, primary or Jurkat T cells were electroporated with various amounts of each mRNA using a Gene Pulser (Biorad). The following protocol was used for each cell type: primary T cells (square wave protocol, 500 V, 5 ms, and 4 mm cuvette), Jurkat T cells (square wave protocol, 500 V, 3 ms, and 4 mm cuvette).

[0295] Antibodies and flow cytometry: Primary human T cells or tumor cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin, and 0.02% sodium azide) and treated with 10% human serum for 10 min at 4°C. Cells were stained with each primary antibody for 25 min at 4°C. Stained cells were washed twice with FACS buffer and then stained with secondary antibodies for 25 min at 4°C or directly treated with BD LSRFortessa. Expression of CAR constructs was detected via the StrepII tag using an anti-StrepII tag antibody (clone 5A9F9, Genscript) as the primary antibody and a PE- or APC-conjugated secondary antibody (eBioscience). Expression of the engineered target antigen tEGFR was detected using either a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was performed by FlowJo software.

[0296] Construction of transgene constructs: Nucleotide sequences encoding the signal peptide CD33, human CD8α hinge, human monomeric CD8α hinge (UniProt ID P01732, C164S, and C181S), and CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ ITAM signaling domain were synthesized by GenScript. Sequences encoding the extracellular and transmembrane domains of EGFR were obtained from Addgene (plasmid #11011). Insertion of the StrepII tag (NWSHPQFEK) and flexible linker was performed by PCR. Assembly of the nucleotide sequences into a functional transgene was performed using Gibson Assembly Master Mix (New England BioLabs) according to the manufacturer's instructions. Diagrams and sequences are shown in Figure 14 and Figure 15, respectively. The resulting construct was amplified by PCR and then used for in vitro transcription.

[0297] Luciferase-based cytotoxicity assay: Luciferase-expressing tumor cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1 in white round-bottom 96-well plates (Sigma Aldrich) with 10,000 target cells / well in cytotoxicity assay medium consisting of phenol-free RPMI (Thermo Scientific), 10% FCS, 1% L-glutamine (Thermo Scientific), and 1% penicillin-streptomycin for 4 h at 37 °C. Finally, the remaining viable cells were quantified by measuring the residual luciferase activity of the co-cultures. After equilibration at room temperature for 10 min, luciferin was added to the cell suspension (150 μg / mL final concentration; Perkin Elmer) and luciferase activity was measured after 20 min using an ENSPIRE multimode plate reader. The specific percentage of lysis was determined by the following formula: % specific lysis=100-((RLU from wells containing co-cultures of effector and target cells) / (RLU from wells containing target cells only)×100).

[0298] Cytokine release by CAR T cells: Cytokine secretion of primary CAR T cells was assessed by co-culturing with target cells at E:T ratios of 1:1 or 2:1 in flat-bottom 96-well plates for 4 or 24 hours at 37°C. In some experiments, released cytokines were quantified in supernatants from co-culture experiments to determine cytotoxicity. Supernatants were centrifuged (1600 rpm, 7 min, 4°C) to remove remaining cells and debris, and subsequently frozen at -80°C. For analysis of secreted IFN-γ, ELISA was performed using the Human IFN Gamma ELISA Ready-SET-Go!® Kit (eBioscience) according to the manufacturer's instructions. Measurements were performed using an ENSPIRE multimode plate reader.

[0299] Example 3 Single-chain variable fragments (scFv) can induce clustering of CARs at the cell membrane, thereby preventing the use of avidity effects to specifically recognize antigen combinations The third example shows that the incorporation of scFv-based binding moieties into car molecules can prevent the use of avidity effects for specific recognition of antigen combinations. The schematic diagram of the CAR construct shown in Figure 4A shows the design of the tested CAR variants (4D5-5-8cys-BB-3z, 4D5-5-8ser-BB-3z, 4D5-5(split)-8ser-BB-FKBP(36V)-3z). In the example shown, the scFv 4D5-5 against HER2 was used as the antigen binding moiety and was incorporated into either a monomeric ("Ser") or dimeric ("Cys") CAR signaling scaffold. Figure 4B shows the expression of the CAR in primary T cells. The effective binding affinity of scFv 4D5-5 was reported to be 1.1 μM (Liu et al., Cancer Res. 2015;75(17):3596-3607), which is comparable to that of E11.4.1-G32A. Jurkat T cells expressing truncated HER2 (tHER2) served as the target cell line (Figure 4E). IFN-gamma secretion by CAR T cells (Figure 4F) and target cell lysis (Figure 4G) induced by the serine-containing CAR "4D5-5-8ser-BB-3z" were slightly reduced compared to the cysteine-containing CAR "4D5-5-8cys-BB-3z" despite being a low-affinity scFv, which is in stark contrast to the observations with the low-affinity rcSso7d-based antigen-binding portion of CAR S(G32A)-8ser-BB-3z (SEQ ID NO: 44) in Example 2 (Figure 3). V bound together in scFv on the surface of T cells, as observed in diabody formation. H and V Lcan not only dimerize within the same single chain molecule (i.e., within the same CAR molecule), but also form intermolecular links (i.e., between different CAR molecules). This would mediate dimerization or even oligomerization as reported for purified scFv proteins (Atwell et al., Protein Eng. 1999;12(7):597-604) and explain the previously observed CAR clustering (Long et al., Nat Med. 2015;21(6):581-590). Finally, this dimerization or oligomerization of scFvs would result in the formation of bivalent or multivalent CARs, even when based on a monomeric CAR scaffold. Thus, V H and V L Cleavage of the linker between V prevents oligomerization and thus activation of low affinity CARs based on monomeric CAR scaffolds. H and V L The V domain also heterodimerizes, at least in part, on the surface of T cells to form functional V H / V L We hypothesized that heterodimers (i.e., Fvs) could be formed. In the absence of nonspecific adhesion between the Fvs, these Fvs would be able to form heterodimers (i.e., Fvs) by controlling the dimerization of two Fvs (V H Thus, T cell activation should only be induced by the FKBP F36V domain fused intracellularly to the carrier chain. Indeed, Figure 4H shows that the V domain of the low affinity scFv 4D5-5 H and V L This is shown to be the case, as shown by the separation of tHER2 on two separate membrane-anchored molecules (SEQ ID NO: 58 and SEQ ID NO: 59). As expected, CAR T cells expressing these constructs (Figures 4C and 4D) express tHER2 pos was not activated by target cells. HWhen the constructs were homodimerized by AP20187, T cells were activated by these target cells (Figure 4H). This activation of T cells in the presence of dimers confirmed that the two separate constructs indeed formed functional Fvs on the T cell surface and that the lack of activation in the absence of dimers was due to the monovalent nature of the Fvs. This is consistent with the low affinity of 4D5-5 and with the observations obtained with the rcSso7d-based antigen-binding moiety of Example 2. For comparison, we also tested the scFv version (i.e., V connected by the linker "218"). H and V L ) expression, V H The expression levels obtained with the construct (Figure 4C) were adjusted to the large marker. This resulted in strong activation of CAR T cells despite the low expression (Figure 4H). Taken together, the data in Figure 4 suggest that at least certain versions of scFvs can mediate V-interactions between adjacent molecules on the T cell surface. H and V L This strongly suggests that the CAR molecules dimerize (or oligomerize) partially through intermolecular heterodimerization of the scFvs. Similar to the dimerization or oligomerization caused by cysteine ​​amino acid residues (described above), uncontrolled dimerization or oligomerization of CAR molecules mediated by at least certain scFvs variants may cause homodimerization of the same CAR molecules. This in turn may result in avidity effects when encountering a single positive cell, thus preventing the desired specific recognition of the antigen combination on the target cell by the CAR group of the present invention. Therefore, in a preferred embodiment, neither the antigen-binding portion of the CAR molecule of the CAR group nor the antigen-binding portion of the other polypeptide that binds to the CAR molecule of the group of the present invention is an scFv.

[0300] Maintenance of human cell lines: Primary human T cells were obtained from anonymous healthy donor blood after apheresis (buffy coat from the Austrian Red Cross, Vienna, Austria). posT cells were enriched by negative selection using RosetteSep human T cell enrichment cocktail. Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3 pos T cells were activated with anti-CD3 / CD28 beads according to the manufacturer's instructions and expanded in human T cell medium consisting of RPMI-1640 supplemented with 10% FCS, 1% penicillin-streptomycin, and 200 IU / mL recombinant human IL-2. Primary T cells were cultured for at least 14 days before experiments were performed. Jurkat T cells were kindly provided by Dr. Sabine Strehl at CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell density was monitored using AccuCheck counting beads.

[0301] In vitro transcription and electroporation of mRNA: In vitro transcription was performed using the mMessage mMachine T7 Ultra kit according to the manufacturer's instructions. 50–200 ng of column-purified PCR product was used as reaction template. The resulting mRNA was purified using the RNeasy column purification kit using an adapted protocol. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol, and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purified according to the manufacturer's instructions. Elution was performed with nuclease-free water, and the purified mRNA was frozen at −80°C until the time of electroporation. For transient transgene expression, primary or Jurkat T cells were electroporated with various amounts of each mRNA using a Gene Pulser (Biorad). The following protocol was used for each cell type: primary T cells (square wave protocol, 500 V, 5 ms, and 4 mm cuvette), Jurkat T cells (square wave protocol, 500 V, 3 ms, and 4 mm cuvette).

[0302] Antibodies and flow cytometry: Primary human T cells or tumor cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin, and 0.02% sodium azide) and treated with 10% human serum for 10 min at 4°C. Cells were stained with each primary antibody for 25 min at 4°C. Stained cells were washed twice with FACS buffer and then stained with secondary antibodies for 25 min at 4°C or directly treated with BD LSRFortessa. Expression of CAR constructs was detected via StrepII tag using anti-StrepII tag antibody (clone 5A9F9, Genscript) or via FLAG tag using anti-FLAG tag antibody (clone L5, BioLegend) as primary antibody, and in the case of StrepII tag antibody, using PE- or APC-conjugated secondary antibody. Expression of engineered target antigen tHER2 was detected using PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was performed by FlowJo software.

[0303] Construction of transgene constructs: Nucleotide sequences encoding the GM-CSF-Ra signal peptide, anti-human CD19 scFv FMC63, human CD8α hinge, human monomeric CD8α hinge (UniProt ID P01732, C164S, and C181S), and CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ ITAM signaling domain were synthesized by GenScript. Nucleotide sequences encoding the signal peptide IgGk, anti-human HER2 scFv 4D5-5, and dimerization domain FKBP F36V were synthesized by GeneArt (Thermo Scientific). Sequences encoding the extracellular and transmembrane domains of HER2 were obtained from Addgene (plasmid #16257). Insertion of the StrepII tag (NWSHPQFEK) or FLAG tag (DYKDDDDK) and flexible linker was performed by PCR. Assembly of the nucleotide sequences into functional transgenes was performed using Gibson assembly master mix according to the manufacturer's instructions. The diagram and sequence are shown in Figures 14 and 15, respectively. The resulting construct was amplified by PCR and then used for in vitro transcription.

[0304] Luciferase-based cytotoxicity assay: Luciferase-expressing tumor cells were co-cultured with CAR T cells at a 2:1 E:T cell ratio, and 10,000 target cells / well in white round-bottom 96-well plates were cultured in cytotoxicity assay medium consisting of phenol-free RPMI, 10% FCS, 1% L-glutamine, and 1% penicillin-streptomycin for 4 h at 37°C. Finally, the remaining viable cells were quantified by measuring the residual luciferase activity of the co-cultures. After equilibration at room temperature for 10 min, luciferin was added to the cell suspension (150 μg / mL final concentration), and luciferase activity was measured after 20 min using an ENSPIRE multimode plate reader. The specific percentage of lysis was determined by the following formula: % specific lysis=100-((RLU from wells containing co-cultures of effector and target cells) / (RLU from wells containing target cells only)×100).

[0305] FACS-based cytotoxicity assay: For the FACS-based cytotoxicity assay, two target cell populations were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding each target antigen, and (ii) Jurkat cells electroporated with mRNA encoding mCherry alone. These two populations were mixed in a 1:1 ratio and co-cultured with CAR T cells at an E:T cell ratio of 4:1:1 at 20,000 target cells / well in round-bottom 96-well plates for 4 h at 37°C. Target cells without added CAR T cells served as the control condition ("target only"). After the incubation period, the co-cultures were centrifuged (5 min, 1600 rpm, 4°C), the supernatant was collected for subsequent cytokine measurements, and the remaining cells were resuspended in 100 μL of FACS buffer consisting of PBS, 0.2% human albumin, and 0.02% sodium azide. Target antigens pos Cells and target antigens neg The viability of the cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated by the following formula: % specific lysis = (1-((eGFP in sample pos % cells / (mCherry in sample pos Cells%) / (eGFP in "target only" control pos % cells / (mCherry in "target only" control pos Cells%))))×100.

[0306] Cytokine release by CAR T cells: Cytokine secretion of primary CAR T cells was assessed by co-culturing with target cells at E:T ratios of 1:1 or 2:1 in flat-bottom 96-well plates for 4 or 24 hours at 37°C. In some experiments, released cytokines were quantified in supernatants from co-culture experiments to determine cytotoxicity. Supernatants were centrifuged (1600 rpm, 7 min, 4°C) to remove remaining cells and debris, and subsequently frozen at -80°C. For analysis of secreted IFN-γ, ELISA was performed using the Human IFN Gamma ELISA Ready-SET-Go!® Kit (eBioscience) according to the manufacturer's instructions. Measurements were performed using an ENSPIRE multimode plate reader.

[0307] In vitro dimerization of transgenes: Transgene dimerization was induced prior to co-culture experiments. Primary T cells were diluted to the final cell concentration in the respective cell culture medium. The homodimerizer AP20187 (MedChemExpress) was diluted in cell culture medium and added at a final concentration of 10 nM. Addition of the same concentration of each vehicle control DMSO served as a control. To ensure efficient dimerization of the transgene and subsequent in vitro experiments, cells were incubated at 37 °C for 30 min and then used in the in vitro experiments.

[0308] Example 4 Generation and function of affibody-based CARs against HER2 In a fourth example, we created and identified an affibody-based binding moiety for HER2 suitable for use in the CARs of the invention. Here again, we started with a well-characterized existing antigen-binding moiety engineered for high affinity binding to human HER2 (Wikman et al., Protein Eng Des Sel. 2004;17(5):455-462). To eliminate potential N-glycosylation sites and reduce IgG binding, two point mutations (N23A and S33K) were introduced into the framework region of the binding scaffold (Feldwisch et al., J. Mol. Biol. 2010;398(2):232-47), which resulted in the antigen-binding moiety "zHER2-WT". An alanine scan of "zHER2-WT" was performed to create low affinity variants by sequentially mutating all amino acids involved in antigen binding to alanine, resulting in a variety of variants each containing a single alanine mutation. Instead of expressing the 13 variants in E. coli and determining their affinity to select the appropriate antigen-binding moiety, we performed a functional screen by directly incorporating all variants that can conditionally homodimerize (Figure 5A) into a CAR scaffold (exemplified by binder-zHER2-WT (WT) in SEQ ID NO: 52). In this way, T cells expressing different CARs can be directly screened for activation in the presence and absence of homodimers in co-culture with Jurkat T cells electroporated with 5 μg of mRNA encoding tHER2. Figure 5B shows the expression of tHER2 in Jurkat cells. Primary human T cells were electroporated with 5 μg of mRNA for each CAR construct and expression was detected via the hexahistidine tag 20 hours after electroporation (Figure 5C). Expression of all 13 different affibody-based CARs was comparable (Figure 5D). Primary T cells not expressing the constructs were used as a negative control. For functional screening, dimerization of the CAR molecule was induced by treating CAR T cells with 10 nM AP20187 for 30 min at 37° C. and then co-culturing with Jurkat T cells. Vehicle control DMSO served as a control.After 4 hours of co-culture at 37°C with an E:T ratio of 2:1, the ability of the CARs to induce cytotoxicity was determined by performing a luciferase-based cytotoxicity assay. The ability of various CARs to induce T cell cytotoxicity in the presence or absence of 10 nM AP20187 is shown in Figure 5E. The high affinity affibody antigen binding moiety zHER2-WT induced efficient target cell lysis that was independent of the presence of AP20187. Similarly, CARs containing the mutants Q11A, Q17A, W24A, T25A, S27A, and R28A showed no significant dependency on the presence of dimerizers. No cytotoxicity was induced by CARs containing affibody antigen binding moieties with substitutions Y13A and W14A, while Y35A induced cytotoxicity at low levels. Dimerization-induced activation was observed with mutants L9A-, R10A-, and R32A, which are therefore suitable binding moieties for incorporation into the CAR molecules of the present invention.

[0309] Maintenance of human cell lines: Primary human T cells were obtained from anonymous healthy donor blood after apheresis (buffy coat from the Austrian Red Cross, Vienna, Austria). pos T cells were enriched by negative selection using RosetteSep human T cell enrichment cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3 posT cells were activated with anti-CD3 / CD28 beads according to the manufacturer's instructions and expanded in human T cell medium consisting of RPMI-1640 supplemented with 10% FCS, 1% penicillin-streptomycin, and 200 IU / mL recombinant human IL-2. Primary T cells were cultured for at least 14 days before experiments were performed. Jurkat T cells were kindly provided by Dr. Sabine Strehl at CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell density was monitored using AccuCheck counting beads.

[0310] Antibodies and flow cytometry: Primary human T cells or tumor cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin, and 0.02% sodium azide) and treated with 10% human serum for 10 min at 4°C. Cells were stained with each primary antibody for 25 min at 4°C. Stained cells were washed twice with FACS buffer and then stained with secondary antibodies for 25 min at 4°C or directly treated with BD LSRFortessa. Expression of CAR constructs was detected via the hexahistidine tag using an AF647-conjugated anti-pentahistidine tag antibody (Qiagen). Expression of the engineered target antigen tHER2 was detected using a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was performed by FlowJo software.

[0311] In vitro transcription and electroporation of mRNA: In vitro transcription was performed using the mMessage mMachine T7 Ultra kit according to the manufacturer's instructions. 50–200 ng of column-purified PCR product was used as reaction template. The resulting mRNA was purified using the RNeasy column purification kit using an adapted protocol. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol, and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purified according to the manufacturer's instructions. Elution was performed with nuclease-free water, and the purified mRNA was frozen at −80°C until the time of electroporation. For transient transgene expression, primary or Jurkat T cells were electroporated with various amounts of each mRNA using a Gene Pulser (Biorad). The following protocol was used for each cell type: primary T cells (square wave protocol, 500 V, 5 ms, and 4 mm cuvette), Jurkat T cells (square wave protocol, 500 V, 3 ms, and 4 mm cuvette).

[0312] Construction of transgene constructs: CD33 signal peptide, affibody zHER2-WT, hexahistidine tag, flexible G 4 Nucleotide sequences encoding the S linker, human monomeric CD8α hinge (UniProt ID P01732, C164S, and C181S), and CD8α transmembrane domain, 4-1BB costimulatory domain, dimerization domain FKBP F36V, and CD3ζ ITAM signaling domain were synthesized by GenArt. Sequences encoding the extracellular and transmembrane domains of HER2 were obtained from Addgene (plasmid #16257). Insertion of the flexible linker was performed by PCR. Assembly of the nucleotide sequences into a functional transgene was performed using Gibson Assembly Master Mix according to the manufacturer's instructions. Diagram and sequences are shown in Figures 14 and 15, respectively. The resulting construct was amplified by PCR and then used for in vitro transcription.

[0313] Alanine scanning of protein antigen-binding sites: Site-directed mutagenesis of all amino acids involved in epitope binding was performed using the QuikChange Lightning site-directed mutagenesis kit according to the manufacturer's instructions. Primers were engineered using QuikChange Primer Design software (Agilent Genomics) and oligonucleotides were synthesized by Biomers.

[0314] Luciferase-based cytotoxicity assay: Luciferase-expressing tumor cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1 in white round-bottom 96-well plates (Sigma Aldrich) with 10,000 target cells / well of cytotoxicity assay medium consisting of phenol-free RPMI (Thermo Scientific), 10% FCS, 1% L-glutamine (Thermo Scientific), and 1% penicillin-streptomycin for 4 h at 37 °C. Finally, the remaining viable cells were quantified by measuring the residual luciferase activity of the co-culture. After equilibration at room temperature for 10 min, luciferin was added to the cell suspension (150 μg / mL final concentration) and luciferase activity was measured after 20 min using an ENSPIRE multimode plate reader. The percentage of specific lysis was determined by the following formula: % specific lysis=100-((RLU from wells containing co-cultures of effector and target cells) / (RLU from wells containing target cells only)×100).

[0315] In vitro dimerization of transgenes: Transgene dimerization was induced prior to co-culture experiments. Primary T cells were diluted to the final cell concentration in the respective cell culture medium. The homodimerizer AP20187 was diluted in cell culture medium and added at a final concentration of 10 nM. Addition of the same concentration of the respective vehicle control DMSO served as a control. To ensure efficient dimerization of the transgene and subsequent in vitro experiments, cells were incubated at 37°C for 30 min and then used in the in vitro experiments.

[0316] Expression and purification of affibody-based antigen-binding moieties: The binding scaffolds were expressed as sfGFP fusion proteins (consisting of an N-terminal hexahistidine tag followed by either rcSso7d or affibody and sfGFP) using the pE-SUMO vector. A schematic representation of the fusion protein construction is shown in Figure 14G. Different variants of the affibody-based binder zHER2 were fused to sfGFP in the same manner as shown in Figure 14G. The nucleotide sequence encoding the sfGFP reporter protein was obtained from Addgene (plasmid #54737). Briefly, E. coli cells (Tuner DE3) were transformed with sequence-verified plasmids using heat shock transformation. After overnight growth at 37 °C, the cultures were transferred to TB medium (12 g / L tryptone, 24 g / L yeast extract, 4% glycerol, 2.31 g / L KH2O2) supplemented with kanamycin (50 μg / mL) and incubated for 1 h at 37 °C for 2 h. 2 PO 4 , and 16.43 g / LK 2 HPO 4 3H 2 O) and incubated with shaking at 37°C. The cultures reached an A of approximately 2 600When the β-terminal tail reached 100 μg, transgene expression was induced by addition of 1 mM IPTG and cells were further cultivated overnight at 20 °C. Cells were harvested by centrifugation (5000 g, 20 min, 4 °C), resuspended in sonication buffer (50 mM sodium phosphate, 300 mM NaCl, 3% glycerol, 1% Triton X-100, pH 8.0), sonicated (2x90 s, duty cycle 50%, amplitude set to 5) and centrifuged again to remove cell debris. Hexahistidine-tagged fusion proteins were purified from crude cell extracts using TALON metal affinity resin. After addition of 10 mM imidazole, the sonicated supernatant was applied twice to the resin, followed by washing steps with equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) with increasing amounts of imidazole (5-15 mM). The bound scaffold was eluted by applying equilibration buffer supplemented with 250 mM imidazole. After buffer exchange into PBS using Amicon Ultra-15 10K centrifugal filters, the concentration was determined by measuring the absorbance at 280 nm using the respective molar extinction coefficients, and finally the protein was directly frozen at -80°C.

[0317] Determining binding affinity using surface plasmon resonance (SPR): SPR experiments were performed using a Biacore T200 instrument (GE Healthcare). All experiments were performed at 25° C. in degassed and filtered PBS, pH 7.4, containing 0.1% BSA and 0.05% Tween-20 (Merck Millipore). hHER2-Fc (R&D) was immobilized at a concentration of 4 μg / mL on a Protein A sensor chip for 60 s at a flow rate of 10 μL / min. To determine the affinity of the affibody-based antigen-binding moieties, five concentrations of each protein (depending on the expected Kd of the antigen-binding moiety) were injected in single cycle kinetics mode at a flow rate of 30 μL / min for 15 s (zHER2-R10A and zHER2-R32A) or 60 s (zHER2-WT), followed by a dissociation step (60 s for zHER2-R10...

Claims

1. A chimeric antigen receptor (CAR) group consisting of two, three, or four CAR molecules, wherein each member of the CAR group differs from the other in amino acid sequence; wherein each CAR molecule of the group comprises at least a transmembrane domain and an ectodomain, the ectodomain comprising one or two antigen binding moieties and / or one or two binding sites to which other polypeptides, each comprising at least an antigen binding moiety, can bind, and at least one CAR molecule of the group additionally comprises an endodomain comprising at least a signaling region capable of transmitting a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM); wherein the endodomain of each CAR molecule of the group, when each CAR molecule comprises an endodomain, is located on the intracellular side of the cell membrane when expressed in a cell, the ectodomain of each CAR molecule of the group, when expressed in a cell, is moved to the extracellular side of the cell membrane, and the transmembrane domain of each CAR molecule of the group, when expressed in a cell, is located on the cell membrane; wherein the ectodomain of each CAR molecule of the group does not, in its general conformation, comprise a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with each of the other CAR molecules of the group; wherein the antigen-binding portions of the different CAR molecules of the group and of the different other polypeptides are specific for different target antigens that are not covalently linked to each other; wherein the affinity of each antigen-binding portion of the CAR molecules of the group for its respective target antigen is between 1 mM and 100 nM; wherein the affinity of each antigen-binding portion of the other polypeptide for its respective target antigen, or the affinity of the other polypeptide for the binding site of its respective CAR molecule, is between 1 mM and 100 nM; a CAR group, wherein each CAR molecule of the group comprises at least one heterodimerization domain capable of mediating a defined heterodimerization with other CAR molecules of the group, and wherein this heterodimerization of a pair of heterodimerization domains occurs independently of or occurs in the absence of a regulatory molecule, is reduced by a regulatory molecule, or is induced by a regulatory molecule, and optionally is reduced by another regulatory molecule, wherein a regulatory molecule is capable of binding under physiological conditions to at least one member of the pair of heterodimerization domains, and by inducing or reducing heterodimerization, induces or reduces the formation of a non-covalently complexed CAR group consisting of 2, 3 or 4 CAR molecules.

2. 2. The group of CARs according to claim 1, wherein the affinity of each antigen-binding portion of the CAR molecules of the group for its target antigen is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM; and wherein the affinity of the individual antigen-binding portion of the other polypeptide for its target antigen, or the affinity of this other polypeptide for the binding site of its respective CAR molecule, is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM.

3. 2. The group of CARs according to claim 1, wherein the affinity of each antigen-binding portion of the CAR molecules of the group for its target antigen is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM; and wherein the affinity of each antigen-binding portion of the other polypeptide for its target antigen, or the affinity of this other polypeptide for the binding site of each CAR molecule, is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM.

4. 2. The group of CARs according to claim 1, wherein the affinity of each antigen-binding portion of the CAR molecules of the group for its target antigen is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM; and wherein the affinity of the individual antigen-binding portion of the other polypeptide for its target antigen, or the affinity of this other polypeptide for the binding site of each CAR molecule, is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM.

5. 5. The CAR group of any one of claims 1 to 4, wherein the target antigen specifically recognised by the antigen-binding portion of said CAR group or of another polypeptide capable of binding to a CAR molecule of said group is a naturally occurring cell surface antigen, or a polypeptide, carbohydrate, or lipid bound to a naturally occurring cell surface antigen.

6. 6. The CAR group according to any one of claims 1 to 5, wherein the antigen binding portion of said CAR group or of another polypeptide capable of binding to a CAR molecule of said group preferably specifically binds to or comprises a molecule selected from the following CAR group: CD19, CD20, CD22, CD23, CD28, CD30, CD33, CD35, CD38, CD40, CD42c, CD43, CD44, CD44v6, CD47, CD49D, CD52, CD53, CD56, CD70, CD72, CD73, CD74, CD79A, CD79B, CD80, CD90, CD100, CD110, CD120, CD130, CD140, CD150, CD160, CD170, CD180, CD190, CD200, CD22, CD23, CD28, CD30, CD33, CD35, CD38, CD40, CD42c, CD43, CD44, CD44v6, CD47, CD49D, CD52, CD53, CD56, CD70, CD72, CD73, CD74, CD79A, CD79B, CD80, CD180, CD190, CD200, CD220, CD230, CD280, CD280, CD300, CD330, CD35, CD38, CD40, CD42c, CD43, CD44, CD44v6, CD47, CD49D, CD52, CD53, CD56, CD70, CD72, CD73, CD74, CD79A, CD79B, CD80, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CD1 D82, CD85A, CD85B, CD85D, CD85H, CD85K, CD96, CD107a, CD112, CD115, CD11 7, CD120b, CD123, CD146, CD148, CD155, CD185, CD200, CD204, CD221, CD271 , CD276, CD279, CD280, CD281, CD301, CD312, CD353, CD362, BCMA, CD16V, CL L-1, Ig kappa, TRBC1, TRBC2, CKLF, CLEC2D, EMC10, EphA2, FR-a, FLT3LG, FLT3, Lewis-Y, HLA-G, ICAM5, IGHA1 / IgA1, IL-1RAP, IL-17RE, IL-27RA, MILR1, MR1 , PSCA, PTCRA, PODXL2, PTPRCAP, ULBP2, AJAP1, ASGR1, CADM1, CADM4, CDH15 , CDH23, CDHR5, CELSR3, CSPG4, FAT4, GJA3, GJB2, GPC2, GPC3, IGSF9, LRFN4 , LRRN6A / LINGO1, LRRC15, LRRC8E, LRIG1, LGR4, LYPD1, MARVELD2, MEGF10, M PZLI1, MTDH, PANX3, PCDHB6, PCDHB10, PCDHB12, PCDHB13, PCDHB18, PCDHGA3, PEP, SGCB, Bezatin, DAGLB, SYT11, WFDC10A, ACVR2A, ACVR2B, anaplastic lymphoma kinase, cadherin 24, DLK1, GFRA2, GFRA3, EPHB2, EPHB3, EPHB4, EFNB1, EPOR, FGFR2, FGFR4, GALR2, GLG1, GLP1R, HBEGF, IGF2R, UNC5C, VASN, DLL3, FZD10, KREME N2,TMEM169, TMEM198, NRG1, TMEFF1, ADRA2C, CHRNA1, CHRNB4, CHRNA3, CHRNG, DRD4, GABRB3, GRIN3A, GRIN2C, GRIK4, HTR7, APT8B2, NKAIN1, NKAIN4, CACNA1A, CACNA1B, CACNA1I, CACNG8, CACNG4, CLCN7, KCNA4, KCNG2, KCNN3, KCNQ2, KCNU1, PKD1L 2, PKD2L1, SLC5A8, SLC6A2, SLC6A6, SLC6A11, SLC6A15, SLC7A1, SLC7A5P1, SLC7A6, SLC9A1, SLC10A3, SLC10A4, SLC13A5 , SLC16A8, SLC18A1, SLC18A3, SLC19A1, SLC26A10, SLC29A4, SLC30A1, SLC30A5, SLC35E2, SLC38A6, SLC38A9, SLC39A7, SL C39A8, SLC43A3, TRPM4, TRPV4, TMEM16J, TMEM142B, ADORA2B, BAI1, EDG6, GPR1, GPR26, GPR34, GPR44, GPR56, GPR68, GPR 173, GPR175, LGR4, MMD, NTSR2, OPN3, OR2L2, OSTM1, P2RX3, P2RY8, P2RY11, P2RY13, PTGE3, SSTR5, TBXA2R, ADAM22, ADMT S7, CST11, MMP14, LPPR1, LPPR3, LPPR5, SEMA4A, SEMA6B, ALS2CR4, LEPROTL1, MS4A4A, ROM1, TM4SF5, VANGL1, VANGL2, C1 8orf1, GSGL1, ITM2A, KIAA1715, LDLRAD3, OZD3, STEAP1, MCAM, CHRNA1, CHRNA3, CHRNA5, CHRNA7, CHRNB4, KIAA1524, NRM. 3, RPRM, GRM8, KCNH4, melanocortin 1 receptor, PTPRH, SDK1, SCN9A, SORCS1, CLSTN2, endothelin-converting enzyme-like-1, lysophosphatidic acid receptor 2, LTB4R, TLR2, neurotropic tyrosine kinase 1, MUC16, B7-H4, epidermal growth factor receptor (EGFR), ERBB2, HER3, EGFR variant III (EGFRvIII), HGFR, FOLR1, MSLN, CA-125, MUC-1,Prostate-specific membrane antigen (PSMA), mesothelin, epithelial cell adhesion molecule (EpCAM), L1-CAM, CEACAM1, CEACAM5, CEACAM6, VEGFR1, VEGFR2, high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A l, IL-13R-α2, disialogangliosides (GD2 and GD3), tumor-associated carbohydrate antigens (CA-125, CA-242, Tn, and cialyl Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), C-MET, CCR1, CCR4, FAP, fibronectin extra domain B (ED-B), GPNMB, IGF-1 receptor, integrin α5β1, integrin αvβ3, ITB5, ITGAX, embigin, PDGF-Rα, R OR1, syndecan 1, TAG-72, tenascin C, TRAIL-R1, TRAIL-R2, NKG2D-ligand, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, preferably PR1 / HLA-A2, lineage- or tissue-specific antigens, preferably CD3, CD4, CD5, CD7, CD8, CD24, CD25, CD34, CD80, CD86, CD133, CD138, CD152, CD319, endoglin, and MHC molecules.

7. 7. A nucleic acid molecule comprising a nucleotide sequence encoding an individual CAR molecule of the CAR group of any one of claims 1 to 6, wherein the nucleic acid is selected from DNA, RNA, or in vitro transcribed RNA.

8. 7. A kit of nucleic acid molecules comprising a nucleotide sequence encoding an individual CAR molecule of the CAR group of any one of claims 1 to 6, wherein the nucleic acid is selected from DNA, RNA, or in vitro transcribed RNA.

9. A vector or a kit of vectors comprising a nucleotide sequence encoding an individual CAR molecule of the CAR group according to any one of claims 1 to 6, wherein the nucleic acid is DNA or RNA.

10. A cell modified in vitro or ex vivo using the nucleic acid molecule or kit of nucleic acid molecules according to claim 7 or 8, or using the vector or kit of vectors according to claim 9, to generate an individual CAR molecule of the CAR group according to any one of claims 1 to 6, or a kit comprising two or more of said modified cells.

11. 11. A pharmaceutical formulation comprising a nucleic acid or a kit of nucleic acids according to claim 7 or 8, a vector or a kit of vectors according to claim 9, or a cell or a kit of cells according to claim 10.

12. 7. A CAR according to any one of claims 1 to 6 for use in a method of treating cancer in an individual, said method comprising: i) genetically modifying NK cells or preferably T lymphocytes obtained from the individual with at least one vector comprising sequences encoding each CAR molecule of a CAR panel, wherein the antigen-binding portion of said CAR panel and / or the antigen-binding portion of another polypeptide capable of binding to a CAR molecule of said panel is specific for a target antigen on cancer cells of the individual, said genetic modification being performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual; and optionally iii) administering to the individual an effective amount of at least one other polypeptide, which comprises at least one antigen binding moiety and is capable of binding to a binding site in a CAR molecule of said CAR group, and / or an effective amount of at least one regulatory molecule, to induce or reduce heterodimerization of each CAR molecule of the group, preferably to induce heterodimerization of each CAR molecule of the group, wherein the non-covalently complexed CARs, upon contact with a cancer cell expressing the combination of each target antigen at physiological expression levels, mediate activation of the genetically modified cell, which leads to the death of the cancer cell, thereby allowing the treatment of cancer.

13. 11. The cell of claim 10 for use in a method of treating cancer in an individual, wherein the antigen binding portion of the CAR group and / or the antigen binding portion of another polypeptide capable of binding to a CAR molecule of said group is specific for a target antigen on cancer cells of the individual, the method comprising: i) introducing the cells into an individual; and ii) administering to the individual an effective amount of at least one other polypeptide capable of binding to a binding site of a CAR molecule of the CAR group, and / or an effective amount of at least one regulatory molecule, which comprises at least an antigen binding portion, to induce or reduce heterodimerization of each CAR molecule of the group, preferably to induce heterodimerization of each CAR molecule of the group, wherein the non-covalently complexed CARs, upon contact with cancer cells expressing the respective target antigen, mediate activation of the genetically modified cells, which leads to the death of the cancer cells, thereby allowing the treatment of cancer.

14. Kit includes: - a CAR according to any one of claims 1 to 6, a vector or a kit of vectors according to claim 9, or a cell or a kit of cells according to claim 10, and at least one other polypeptide comprising at least an antigen-binding portion and capable of binding to the binding site of a CAR molecule of the CAR population and / or to at least one regulatory molecule.

15. for use in the treatment of diseases characterized by the need for binding of T lymphocytes or NK cells to a target antigen on a cell, preferably for use in the treatment of tumor patients, in particular Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, osteogenic sarcoma, mesothelioma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, leiomyosarcoma, melanoma, glioma, astrocytoma, myeloma, Blastoblastoma, neuroblastoma, retinoblastoma, oligodendroglioma, meningioma, craniopharyngioma, ependymoma, pinealoma, hemangioma, acoustic neuroma, chronic myeloproliferative disorders, acute myeloid leukemia, chronic lymphocytic leukemia (CLL) including B-cell CLL, T-cell CLL, prolymphocytic leukemia and hairy cell leukemia, acute lymphoblastic leukemia, B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma, tonsilloma, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma, tonsillar cancer, leukemia ... Squamous cell carcinoma, bladder cancer, transitional cell carcinoma, bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell carcinoma and non-small cell carcinoma of the lung), adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer Cancer, seborrheic adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma CARs according to any one of claims 1 to 6, a vector or a kit of vectors according to claim 9, a cell or a kit of cells according to claim 10, in particular a T lymphocyte or NK cell, or a kit according to claim 8, 9 or 14, for use in the treatment of a tumor patient having a tumor selected from: atypical meningioma, islet cell carcinoma, medullary carcinoma, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, clear cell carcinoma, and mediastinal neurofibroma.

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