Group of chimeric antigen receptor (CAR)

Abstract

To provide a group of CAR formed of two, three or four chimeric antigen receptor (CAR) molecules.SOLUTION: There is provided a group of chimeric antigen receptor (CAR) formed of two, three or four CAR molecules, where the members of the group of the CAR may be different from each other or equal, in amino acid sequences of the members, and each CAR molecule of the group include an external domain including at least any of a membrane domain, an antigen-binding part and a binding portion to which another polypeptide having an antigen-binding part can bind, each CAR molecule in the group includes at least one dimerization domain, the dimerization of a pair of dimerization domains is induced by a regulatory molecule, and arbitrarily, reduced by another regulatory molecule, or may occur in absence of a regulatory molecule and may be reduced by a regulatory molecule.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

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

[0002] Immunotherapy using CAR T cells, i.e., T cells modified to express chimeric antigen receptors (CARs), is one of the most promising approaches in cancer treatment. To date, the high potential of this therapeutic strategy has been demonstrated by impressive clinical responses in patients with B-cell malignancies. However, further translation of this success to other tumors is currently hindered by several hurdles (Lim and June, Cell. 2017;168(4):724). For example, CAR T cells are pharmaceutical agents that replicate after administration, and their activity cannot be adequately controlled reversibly. To date, several strategies for conditional CARs have been developed (Lim and June, Cell. 2017;168(4):724). These strategies allow for reversible modulation by administration of small molecule drugs or injection of bispecific proteins that bind to the target antigen and the CAR (Wu et al. Science. 2015;350(6258):aab4077; Juillerat et al. Sci Rep. 2016;6:18950; Ma et al. Proc Natl Acad Sci US A. 2016;113(4):E450; Urbanska et al. J Transl Med. 2014;12:347; Urbanska et al. Cancer Res. 2012;72(7):1844; Cartellieri et al. Blood Cancer J. 2016;6(8):e458). The present invention provides a novel strategy for modulating CAR function based on modulating avidity, i.e., synergistic amplification of the affinity of the individual antigen-binding moieties of a CAR panel and / or of the individual antigen-binding moieties of other polypeptides that can bind to the CAR molecules of the panel. Specifically, the novel CARs 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. It is a further objective to provide a means for tumor therapy, especially an immunotherapy concept for the treatment of tumors.

[0003] International Publication No. WO2017 / 180993A1 discloses a salvage chimeric antigen receptor system.

[0004] 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 in vivo toxicity.

[0005] 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.

[0006] International Publication WO 2015 / 075468 A1 discloses a CAR system comprising a CAR that includes an activation endodomain.

[0007] 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.

[0008] Ajina et al. (Mol. cancer therap. 17 (2018), 1795-1815) review strategies to address CAR tonic signaling. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a system based on a group of chimeric antigen receptors (CARs) consisting of two, three or four CAR molecules, wherein the members of the CAR family may be different or identical to each other in their amino acid sequence, and each CAR molecule of the family comprises at least a membrane domain and an ectodomain that comprises either an antigen-binding portion or a binding site to which another polypeptide comprising an antigen-binding portion can bind; and wherein at least one CAR molecule of the group further comprises an endodomain comprising at least a signaling region capable of signaling via at least one immunoreceptor tyrosine-based activation motif (ITAM) or at least one immunoreceptor tyrosine-based inhibition motif (ITIM); and wherein the endodomain of each CAR molecule of the group, when expressed in a cell, if each CAR molecule comprises an endodomain, is located on the intracellular side of the cell membrane; the ectodomain of each CAR molecule of the group, when expressed in a cell, is located on 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 plasma membrane; each CAR molecule of the group comprises at least one dimerization domain capable of mediating homo- or heterodimerization with other CAR molecules of the group, wherein this dimerization of a pair of dimerization domains is induced by a regulatory molecule, and optionally reduced by another regulatory molecule, or occurs in the absence of a regulatory molecule and is reduced by a regulatory molecule, wherein the regulatory molecule is capable of binding to at least one member of the pair of dimerization domains under physiological conditions and induces or reduces dimerization, thereby inducing or reducing the formation of a non-covalently complexed group of CARs consisting of 2, 3, or 4 CAR molecules; and The ectodomain of each CAR molecule of the group in its general conformation does not have a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with another CAR molecule of the group, and The CAR molecules of the group and the antigen-binding portions of other polypeptides to which the CAR molecules of the group can bind are specific for one target antigen or for a non-covalent complex of different target molecules; and the affinity of each individual antigen-binding portion of the group of CAR molecules for its target antigen is between 1 mM and 100 nM; and The affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or on the other hand, the affinity of this other polypeptide to the binding site of its respective CAR molecule, is between 1 mM and 100 nM. [Means for solving the problem]

[0010] To date, several strategies for conditionally active CARs have been developed. Examples of such CARs are disclosed in European Patent No. 2,956,175 B1, U.S. Patent No. 20170081411A1, and International Publication No. WO2017032777 A1. However, the strategy of the present invention represents a fundamentally different principle for modulating CAR function. The underlying principle for modulating CAR function according to the present invention is a group of CARs, where the individual antigen-binding portions of the individual CAR molecules in the group have low affinity for their respective target antigens, resulting in only monovalent interactions and weak or no intracellular signaling in CAR-expressing cells. In the case of the use of other polypeptides each containing an antigen-binding portion and capable of further binding 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 be of low affinity, resulting in a monovalent interaction that induces only weak or no intracellular signaling in CAR-expressing cells. However, noncovalent assembly of two, three, or four CAR molecules of the group results in the formation of a multivalent CAR complex that can interact with their respective target antigens (or noncovalent or coexisting complexes of different target antigens) in a bivalent or tetravalent manner, directly or indirectly through other polypeptides. This multivalent interaction results in a synergistic amplification of low affinity, i.e., avidity. Importantly, such multivalent interaction relies on the noncovalent complex formation of the CAR molecules of the group. This noncovalent complexation can be regulated, ultimately facilitating modulation of CAR function. [Effects of the Invention]

[0011] Therefore, to ensure efficient induction of complex populations of CARs, not only monovalent but also polyvalent interactions, the affinity of each individual antigen-binding portion of the CAR molecules of the group to its target antigen is 1 mM to 100 nM, and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or, on the other hand, the affinity of this other polypeptide to the binding site of its respective CAR molecule, is 1 mM to 100 nM. According to a preferred embodiment, the affinity of each individual antigen-binding portion of the CAR molecules of the group to its target antigen is 1 mM to 150 nM, preferably 1 mM to 200 nM, more preferably 1 mM to 300 nM, and particularly 1 mM to 400 nM, and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or, on the other hand, the affinity of this other polypeptide to the binding site of its respective CAR molecule, is 1 mM to 150 nM, preferably 1 mM to 200 nM, more preferably 1 mM to 300 nM, and particularly 1 mM to 400 nM. According to another preferred embodiment, the affinity of each individual antigen-binding portion of the group of CAR molecules to its target antigen is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, and in particular 50 μM to 100 nM, and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or on the other hand, the affinity of this other polypeptide to its respective binding site of the CAR molecule, is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, and in particular 50 μM to 100 nM. According to another preferred embodiment, the affinity of each individual antigen-binding portion of the group of CAR molecules to its target antigen is 500 μM to 150 nM, preferably 250 μM to 200 nM, more preferably 125 μM to 300 nM, and in particular 50 μM to 400 nM, and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or on the other hand, the affinity of this other polypeptide to its respective binding site of the CAR molecule, is 500 μM to 150 nM, preferably 250 μM to 200 nM, more preferably 125 μM to 300 nM, and in particular 50 μM to 400 nM.It should be noted that any affinity values ​​presented herein refer to affinities determined by surface plasmon resonance (SPR) using steady-state analysis, e.g., as performed in Examples 1 and 9 in the Examples section, at pH 7.4 and 25°C, performed on a Biacore T200 instrument (GE Healthcare).

[0012] To promote the defined dimerization, trimerization, or tetramerization of the CAR group according to the present invention, each CAR molecule of the CAR group contains at least one dimerization domain. According to the present invention, the dimerization of a pair of these dimerization domains is regulated by a regulatory molecule capable of binding to at least one member of the pair of dimerization domains under physiological conditions, thereby inducing or preventing the formation of a defined noncovalent complex of two, three, or four CAR molecules of the group. Depending on whether the CAR group contains an activating ITAM or an inhibitory ITIM, the noncovalently complexed group of CARs can efficiently activate or inhibit cells modified to express the group of CARs in response to target cells expressing the respective target antigens, or respective noncovalent or covalent complexes of different target antigens.

[0013] The avidity regulation facilitated by the design of the CAR group according to the present invention would not be possible with the currently used CAR molecule formats. First, this is due to the fact that at least some of the currently used CAR molecules form dimers (or oligomers) when expressed in cells due to disulfide bonds between extracellular system residues. However, such uncontrolled dimerization or oligomerization of CAR molecules prevents efficient regulation of CAR complexation and therefore prevents the realization of the resulting avidity effect based on the recognition of target antigens or covalently or non-covalently complexed target antigens.

[0014] For this reason, according to the present invention, the ectodomain of each CAR molecule of a group in its "general conformation" (i.e., its naturally folded conformation) does not contain a cysteine ​​amino acid moiety that can form intermolecular disulfide bonds with other CAR molecules of the group. In other words, the extracellular domain of a CAR molecule of a group according to the present invention should not contain any cysteines that are not involved in intramolecular disulfide bonds in the naturally folded conformation of the CAR (i.e., formed within a particular CAR molecule of the group). For example, cysteines in the hinge region of CD8α that can form intermolecular disulfide bonds in the native conformation (i.e., with other CAR molecules of the group) must be excluded, for example, by mutation or deletion. On the other hand, cysteines that are involved in intramolecular disulfide bonds 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 a CAR of a group of CARs according to the present invention. As one 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 group of CARs according to the invention, such that, because those cysteines in, for example, an scFv, are involved in intramolecular disulfide bonds, they are unavailable for intermolecular disulfide bonds (when the CAR molecule is present in its normal, i.e., native, conformation), thereby avoiding undesired covalent dimerization or oligomerization of the group of CAR molecules.

[0015] In general, non-covalent dimerization or oligomerization of CAR molecules mediated by domains other than those intended to mediate complexation of the CAR group according to the present invention will also prevent efficient modulation of CAR complexation and, therefore, modulation of the function of the CAR group according to the present invention.

[0016] Therefore, such non-covalent dimerization or oligomerization of CAR molecules needs to be prevented, or at least minimized, as far as biologically possible by elimination or engineering of such domains.

[0017] For example, the antigen-binding portions of current CARs are typically based on single-chain variable fragments (csFvs), which tend to oligomerize due to intermolecular heterodimerization of the variable light (VL) and variable heavy (VH) domains between individual molecules (Hudson et al., J Immunol Methods. 1999; 231(1-2):177-89; Long et al., Nat Med. 2015; 21(6):581-90). Thus, the individual molecules of the CAR population of the present invention preferably do not contain scFv-based antigen-binding portions or other molecular components that potentially lead to undesired and uncontrolled covalent or non-covalent complex formation of the CAR molecule.

[0018] The basic structure of the molecular design of the CAR group of the present invention can be varied at specific sites without destroying the non-covalent complexation of the CAR group and thereby the possibility of conditional regulation of the avidity of the CAR group based on target antibody recognition.

[0019] For example, a group of CARs can consist of two CAR molecules, or alternatively, three or four CAR molecules to further enhance the efficiency of binding based on target antigen recognition and the ability to also recognize low density target antigen molecules on target cells.

[0020] According to the present invention, the CAR group is designed to be functionally dependent on the presence or absence of a regulatory molecule, which can be any molecule that can bind to at least one dimerization domain and induce or reduce the interaction of a pair of dimerization domains. These molecules are usually small molecules, but can also be soluble proteins that accumulate in the tumor stroma, which are often proteins that naturally heterodimerize (e.g., subunits of heterodimerizing cytokines such as IL-12) or naturally homodimerize (e.g., VEGF (as used in Example 7), TGF-β1, etc.). Under physiological conditions, the regulatory molecule can bind to at least one member of a pair of dimerization domains located in the endodomain, transmembrane domain, and / or ectodomain of the CAR molecule. However, to eliminate the possibility of fratricide due to cross-linking of CAR molecules between different cells, the dimerization domain is incorporated into the endodomain and / or transmembrane domain of the CAR molecule of the CAR group, more preferably the endodomain.

[0021] Individual CAR molecules of a group of CARs can bind directly to a target antigen or to a non-covalent or covalent complex of a different target antigen via an integrated antigen-binding moiety, or indirectly via another polypeptide that can bind to the CAR molecule and contains an antigen-binding moiety. Such another polypeptide is thereby defined as a soluble protein that does not belong to the group of CARs and that can non-covalently bind to a binding site on a CAR molecule of said group, directly or indirectly, via covalent modification of another polypeptide, such as, for example, a covalently attached fluorescein isothiocyanate (FITC) molecule. This principle of indirect CAR binding to antigens, 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 clinic (Labanieh et al., Nat Biomed Eng. 2018;2:377-391), can be incorporated into the CAR family 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 containing an antigen-binding portion can bind.

[0022] However, to avoid the need to administer a separate polypeptide that contains the antigen-binding portion and is capable of binding to the CAR molecule, it is preferred that the ectodomain of each CAR molecule of the CAR population itself contains the antigen-binding portion.

[0023] As described above, the basic structure of the CAR molecules of the present invention can be adapted to the needs of different applications. The order of domains in the CAR molecules of the present invention, from the outside to the inside of the cell, preferably corresponds to the following basic structure on the surface of the cell: an antigen-binding portion or a binding site to which another polypeptide containing an antigen-binding portion can bind, a hinge region, preferably for spatial optimization, and a transmembrane domain. According to a preferred embodiment of the ITAM-containing CAR molecule, the transmembrane domain is preferably followed in at least one CAR molecule by a signaling region containing a costimulatory domain, where preferably this costimulatory signaling region, or optionally the transmembrane domain, is followed by at least one dimerization domain and, further, in at least one CAR molecule, a signaling region containing at least one ITAM, where the order of the costimulatory and ITAM-containing signaling regions can be reversed. CAR molecules that do not contain an ITAM lack a costimulatory signaling region or contain one costimulatory signaling region, or two costimulatory signaling regions, or even more costimulatory signaling regions, but preferably contain no more than two costimulatory signaling regions, or even more preferably only one costimulatory signaling region. In the case of ITIM-containing CARs, the transmembrane domain is followed, preferentially in at least one CAR molecule, by an inhibitory signaling region comprising an ITIM. This inhibitory signaling region, or optionally the transmembrane domain, is followed by at least one dimerization domain and, optionally, a second inhibitory signaling region. Generally, in ITAM- and ITIM-containing CARs, the dimerization 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 is preferably located between the transmembrane domain and the signaling domain, and / or, in particular, between the two signaling regions and / or at the intracellular end of the CAR molecule. Finally, any two adjacent components of the CAR molecules of the group (e.g., antigen-binding portion, binding site to which another polypeptide comprising an antigen-binding portion can bind, hinge region, transmembrane domain, signaling region, dimerization domain) can optionally be separated by a linker.

[0024] Different groups of CARs directed against different target antigens, or different non-covalent or covalent complexes of different target antigens, can also be co-expressed in cells to inhibit tumor immune escape induced by loss of the target antigen. Groups of CARs can also be co-expressed with other proteins in a given cell.

[0025] 1. Antigen binding part: The antigen-binding portion of each CAR molecule in a group of CARs according to the present invention can be directed against a single selected epitope of a target antigen molecule. In this case, efficient signaling by the complexed group of CARs requires either a high density of the target antigen or the ability of the target antigen to dimerize / oligomerize. To enable efficient and sensitive targeting of monomeric target antigen molecules, each CAR molecule in a group of CARs according to the present invention can also be directed against different (i.e., non-overlapping) epitopes of the selected target antigen, or against different epitopes located on different molecules that naturally form covalent complexes (e.g., TCRα / TCRβ chains) or non-covalent complexes (e.g., MMC IIα / β chains or ErbB-1 / ErbB-2). In these cases, i.e., when targeting non-overlapping epitopes on the same target antigen or different epitopes on complex target antigens, the binding efficiency is greatly increased because the target epitopes of the CAR group are located on a single molecule or on a complex molecule corresponding to a very high local concentration of epitopes.

[0026] Antigen-binding moieties suitable for use in the population of CARs according to 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 (Pluckthun, 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 Discovery Today. 2015;20(10):1271-1283), and in fact, synthetic library design and selection can be applied to any protein that can potentially also function as an antigen-binding moiety (Pluckthun, Alternative Scaffolds: Expanding the options of antibodies. In: Little M, ed. New York: Cambridge University Press; 2009:244-271).

[0027] In some cases, the antigen-binding moiety may be a single-chain Fv (scFv), other antibody-based recognition domains such as cAb VHH (camelized antibody variable domain) or human versions thereof, IgNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (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 in the CAR group comprises: only one protein domain, preferably a human or non-human VH or VL single-domain antibody (nanobody), or an engineered antigen-binding portion based on the Z domain of Staphylococcal protein A, lipocalin, SH3 domain, fibronectin type III (FN3) domain, knottin, Sso7d, rcSso7d, Sac7d, Gp2, DARPins, or ubiquitin; a ligand, receptor, or co-receptor selected or engineered for low affinity binding and lack of homotypic interactions. Ligands include, for example, cytokines (such as IL-13); growth factors (such as heregulin); and the like. The ligand can be a receptor-binding fragment of a ligand (e.g., a peptide of HGF (Thayaparan et al., Oncoimmunology. 2014;6(12):e1363137); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp); etc. Similarly, the receptor can be a ligand-binding fragment of a receptor. Suitable receptors include, for example, cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); cell adhesion molecules (e.g., CD11a (Park et al., Sci Rep. 2017;7(1):14366); etc.); PD-1; and the like. The antigen-binding portion of each molecule of the CAR group preferably does not cause undesired aggregation of the CAR molecules. As noted above, such undesired dimerization or oligomerization of the CAR molecules of the group prevents efficient control of the dimerization, trimerization, or tetramerization state of the CAR molecules of the group.For this reason, it is preferred that the antigen-binding portion is not a single-chain variable fragment (scFv) derived from a monoclonal antibody. For the clinical applicability of the CAR group according to 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).

[0028] 2. Hinge area: According to some embodiments, the ectodomain of the CAR molecules 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 can bind) and the transmembrane domain, preferably from CD8α (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), wherein the sequence from CD8α, CD28, or PD-1 can be N- and / or C-terminally truncated and can have any length within the boundaries of said sequences, and wherein cysteine ​​residues in the hinge from CD8α and CD28 are deleted or substituted with other amino acid residues. In principle, flexible membrane anchors, and also other parts of many receptors, are suitable for use in the hinge region and / or transmembrane domain of the CAR molecules of this group (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), provided that, if necessary, they are modified to prevent dimerization according to the present invention.

[0029] 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 from about 2 amino acids to about 50 amino acids, e.g., from about 4 amino acids (aa) to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. Optionally, the hinge region can comprise more than 50 amino acids, for example, if a structural domain is incorporated (e.g., from CD34 UniProt P28906-1 aa 42-140 to facilitate enrichment of CAR-modified cells, as disclosed in U.S. Patent No. 2018 / 0094044A1).

[0030] Preferably, other polypeptides, preferably glycine and glycine-serine polymers, can be used for the hinge, since both Gly and Ser are relatively unstructured and can therefore function as neutral dethers between CAR components. Glycine has access to much more phi-phi space than alanine and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1992; 11173-11142). Therefore, 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 another polypeptide containing the antigen-binding portion 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.

[0031] 3.Transmembrane domain: Each molecule of the CAR group contains a transmembrane domain for insertion into the eukaryotic cell membrane. Any transmembrane (TM) domain that allows the polypeptide to be inserted into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. For example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC of human CD8α (Uniprot P01732, amino acid (aa) 183-206) can be used. Further examples of suitable TM sequences include: from human CD8β: LGLVAGVLVLLVSLGVAIHLCC (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 P26842, aa 192-212); from human CD278 (ICOS): FWLPIGCAAFVVVCILGCILI (Uniprot Q9Y6W8, aa 141-161); from human CD279 (PD-1): VGVVGGLLGSLVLLVWVLAVI (Uniprot Q15116, aa 171-191); from human DAP12: GVLAGIVMGDLVLTVLIALAV (Uniprot O43914, aa 41-61); and from human CD7: ALPAALAVISFLLGLGLGVACVLA (Uniprot P09564, aa 178-201).

[0032] 4. Immunoreceptor tyrosine-based activation motifs (ITAMs): According to the present invention, at least one molecule in the CAR group contains an endodomain capable of signaling via at least one immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. The ITAM-containing endodomain contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ITAM motifs. The ITAM-containing portion of the signaling endodomain is preferably derived from any ITAM-containing protein and need not comprise the entire sequence of the entire protein from which it is 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).

[0033] According to particularly preferred embodiments, at least one signaling domain in at least one CAR molecule of the group of CARs is derived from the cytoplasmic domain of the T cell surface glycoprotein CD3 zeta chain (CD3Z, also known as T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, suitable ITAM-containing domains include any of the following amino acid sequences (two isoforms): from about 50 amino acids to about 60 amino acids (aa): from about 60 aa to about 70 aa: from about 70 aa to about 80 aa: from about 80 aa to about 90 aa: from about 90 aa to about 100 aa: from about 100 aa to about 110 aa: from about 110 aa to about 115 aa: from about 115 aa to about 115 aa: 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 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-3 ) or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPQRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-1), where the ITAM motif is bold and underlined.

[0034] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length CD3 zeta amino acid sequence. Accordingly, 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 any of the following amino acid sequences: RVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR (Uniprot P20963-3 aa 52-163), NQL YNEL NLGRREE YDVLDKR (Uniprot P20963-3 aa 69-89), EGL YNEL QKDKMAEA YSEI GMK (Uniprot P20963-3 AA 107-128), DGL YQGL STATKDT YDAL HMQ (Uniprot P20963-3 aa 138-158), where the ITAM is bold and underlined.

[0035] 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.

[0036] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length CD3 delta amino acid sequence. Thus, 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 the following amino acid sequence: DQV YQPL RDRDDAQ YSHLGGN (Uniprot P04234-1 aa 146-166), where the ITAM is bold and underlined.

[0037] The ITAM-containing domain may also be derived from the T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T-cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3c chain, AI504783, CD3, CD3 epsilon, T3e, 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 205 aa of the following amino acid sequence: Uniprot P07766-1.

[0038] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length CD3 epsilon amino acid sequence. Accordingly, 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 the following amino acid sequence: NPD YEPI RKGQRDL YSGL NQR (Uniprot P07766-1 aa 185-205), where the ITAM is bold and underlined.

[0039] ITAM-containing domains can also be derived from the T-cell surface glycoprotein CD3 gamma chain (also known as D3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex, etc.)). For example, suitable ITAM-containing domains 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 from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 180 aa of the following amino acid sequences: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL YQPL KDREDDQ YSHL QGNQLRRN (Uniprot P09693-1), where the ITAM is in bold and underlined.

[0040] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length CD3 gamma amino acid sequence. Thus, 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 the following amino acid sequence: DQL YQPL KDREDDQ YSHL QGN (Uniprot P09693-1 aa 157-177), where the ITAM is bold and underlined.

[0041] The ITAM-containing domain may also 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; killer activating receptor-associated protein; killer activating receptor-associated protein; etc.). For example, a suitable ITAM-containing domain may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences (four isoforms): Uniprot O43914-1; Uniprot O43914-2; Uniprot O43914-3; Uniprot X6RGC9-1.

[0042] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length DAP12 amino acid sequence. Thus, 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 the following amino acid sequence: ESP YQEL QGQRSDV YSDL NTQ (Uniprot O43914-1 aa 88-108), where the ITAM is bold and underlined.

[0043] An ITAM-containing domain can also be derived from FCER1G (also referred to as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilonRI-gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). For example, a suitable ITAM-containing domain comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: MIPAVVLLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV YTGL STRNQET YETL KHEKPPQ (Uniprot P30273), where the ITAM is bold and underlined.

[0044] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length FCER1G amino acid sequence. Thus, 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 the following amino acid sequence: DGV YTGL STRNQET YETL KHE (Uniprot P30273 aa 62-82), where the ITAM is bold and underlined.

[0045] The ITAM-containing domain may also be derived from CD79A (B-cell antigen receptor complex-associated protein alpha chain; also known as 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 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 150 aa, about 150 aa to about 200 aa, or about 200 aa to about 220 aa of any of the following amino acid sequences (two isoforms): MPGGPGVLQALPATIFLLFLLSAVYLPGGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL YEGL NLDDCSM YEDI SRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-1) or MPGGP YEGL NLDDCSM YEDI SRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-2), where the ITAM motif is bold and underlined.

[0046] Similarly, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full-length CD79A amino acid sequence. Thus, 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 the following amino acid sequence: ENL YEGL NLDDCSM YEDI SRG (Uniprot P11912-1 aa 185-205), where the ITAM is bold and underlined.

[0047] Thus, according to the present invention, the endodomain of at least one CAR molecule of the group of CARs preferentially comprises at least one ITAM, said ITAM being preferably 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).

[0048] Because the number of ITAMs correlates with the signaling efficiency of a CAR (James, Sci Signal. 2018;11(531)), a group of CARs preferably contains all three ITAMs, although an ITAM may be limited to only a single CAR molecule in the group. Alternatively, some or all CAR molecules in the group may contain at least one ITAM. According to some embodiments, the ITAM-containing moieties in different endodomains of the CAR molecules in the group are derived from the same receptor, while according to other embodiments, the ITAM-containing moieties in different endodomains of the CAR molecules in the group are derived from different receptors. According to some embodiments, a group of CARs contains only one molecule containing an ITAM-containing moiety, preferably derived from CD3 zeta. According to other embodiments, a group of CARs consists of two molecules, both of which contain a portion of the cytoplasmic domain derived from CD3 zeta. With respect to vector payloads, the total number of ITAMs in a group of CARs is preferably 3 to 6. Additionally, the ITAM-containing sequences are selected and / or engineered to minimize nucleotide sequence homology in order to minimize the risk of homologous recombination.

[0049] 5.Co-stimulatory domain: According to a preferred embodiment, the endodomain of at least one CAR molecule of the group comprises a signaling region comprising costimulatory domains from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD2, CD6, CD27, CD30, CD40, GITR, and HVEM, whereby the costimulatory domains comprised in the group of CARs can optionally be derived from different costimulatory receptors.

[0050] Costimulatory domains suitable for inclusion in the costimulatory signaling region of a CAR molecule of the CAR group can have a length of from about 30 aa to about 70 aa, for example, a costimulatory domain can have a length of from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. Optionally, a costimulatory domain can have a length of from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

[0051] According to particularly preferred embodiments, the costimulatory domain in at least one molecule of the group of 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 aa 214-255.

[0052] According to a preferred embodiment, the costimulatory domain in at least one molecule of the group of CARs is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). 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 P10747 aa 1778-220.

[0053] According to particularly preferred embodiments, the costimulatory domain in at least one molecule of the group of CARs 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 aa 165-199.

[0054] According to a preferred embodiment, the costimulatory domain in at least one molecule of the CAR group 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 aa 212-260.

[0055] According to a preferred embodiment, the costimulatory domain in at least one molecule of the group of CARs is 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 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 P43489 aa 241-277.

[0056] According to other embodiments, the costimulatory domain in at least one molecule of the group of CARs is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). 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 Q7Z6A9 aa 176-289.

[0057] According to other embodiments, the costimulatory domain in at least one molecule of the group of CARs is 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 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 Q9Y5U5 aa 188-241.

[0058] According to other embodiments, the costimulatory domain in at least one molecule of the group of CARs is 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 may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the amino acid sequence Uniprot Q92956 aa 224-283.

[0059] According to other embodiments, the costimulatory domain in at least one molecule of the group of CARs is 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 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, about 150 aa to about 160 aa, or about 160 aa to about 185 aa of the amino acid sequence Uniprot P28908 aa 409-595.

[0060] 6. Inhibitory domain: If the group of CARs is also intended to induce an inhibitory signal, the endodomain of at least one CAR molecule of said group comprises a signaling region comprising the ITIM-containing cytoplasmic portion (or part thereof) of an inhibitory receptor preferentially selected from PD-1, CD85A, CD85C, CD85D, CD85J, CD85K, LAIR1, TIGIT, CEACAM1, CD96, KIR2DL, KIR3DL, SLAM family members, CD300 / LMIR family members, CD22 and other Siglec family members, whereby the inhibitory signaling regions comprised by the group of CARs can optionally be derived from different inhibitory receptors. Inhibitory ITIMs are well known in the art (Ravetch and Lanier, Science. 2000;290(5489):84; Barrow and Trowsdale, Eur J Immunol. 2006;36(7):1646), and the sequences of the respective inhibitory domains are disclosed, for example, in US 2018 / 0044399 A1 and US 2017 / 0260268 A1. In principle, the cytoplasmic domains of other inhibitory receptors that mediate their inhibitory function independently of ITIMs, such as CTLA-4, LAG3, TIM3, or CD5, are also suitable for inclusion in the inhibitory signaling region of a CAR molecule.

[0061] Inhibitory domains suitable for inclusion in the inhibitory signaling region of a CAR molecule of the CAR group can have a length of from about 30 aa to about 100 aa, e.g., an inhibitory domain has a length of from about 30 aa to about 50 aa, from about 50 aa to about 70 aa, or from about 70 aa to about 100 aa. In other cases, an inhibitory domain can have a length of from about 100 aa to about 100 aa to about 200 aa, or greater than 200 aa.

[0062] For example, a suitable inhibitory 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 any of the following amino acid sequences: PD-1 (Uniprot Q15116 aa 192-288), CD85A (Uniprot O75022 aa 465-631), CD85C (Uniprot O75023 aa 480-590), CD85D (Uniprot Q8N423 aa 483-598), CD85K (Uniprot Q8NHJ6 aa 281-448), KIR3DL3 (Uniprot Q8N743 aa 344-410), KIR3DL1 (Uniprot P43629 aa 361-444), KIR3DL2(Uniprot P43630 aa 361-455), CTLA4(Uniprot P16410 aa 183-223), LAG3(Uniprot P18627 aa 472-525), TIM3(Uniprot Q8TDQ0 aa 224-301), LAIR1(Uniprot Q6GTX8 aa 187-287), KIR2DL2(Uniprot P43627 aa 265-348), CD85J(Uniprot Q8NHL6 aa 483-650), TIGIT(Uniprot Q495A1 aa 163-244), CEACAM1(Uniprot P13688 aa 453-526), ​​CD5(Uniprot P06127 aa 403-495), CD96 (Uniprot P40200 aa 541-585), CD22 (Uniprot P20273 aa 707-847), CSF1R (Uniprot P07333 aa 539-972).

[0063] 7. 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 dimerization domain. As another example, the linker can be placed between two dimerization 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 dimerization domain. As another example, the linker can be placed in the ectodomain of the CAR molecule between the antigen-binding portion 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 another polypeptide 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 portion. As another example, the linker can be placed in the ectodomain of the CAR molecule between the signal sequence and the binding site to which another polypeptide can bind. As another example, the linker can be placed in the ectodomain of the CAR molecule between the signal sequence and the dimerization domain. As another example, a linker can be placed in the ectodomain of a CAR molecule between the dimerization domain and the antigen-binding portion. As another example, a linker can be placed in the ectodomain of a CAR molecule between the dimerization domain and a binding site to which another polypeptide can bind.

[0064] The linker can be a peptide comprising about 1 to about 40 amino acids in length. The linking peptide can have virtually any amino acid sequence, keeping in mind that suitable linkers preferably have sequences that generally result in flexible peptides. Small amino acids such as glycine, serine, and alanine are preferably used in creating flexible peptides. Creating such sequences is routine for those of skill in the art. Suitable linkers can be readily selected and are of different lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids in length. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGS)n, and (GGGS)n, 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 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 consequently, the linker can include one or more moieties that provide a flexible linker and a non-flexible structure.

[0065] 8. Additional Domains: The CAR target group molecules can further comprise one or more additional polypeptides, where such domains include, for example, a signal sequence; an epitope tag; and / or a polypeptide that generates a detectable signal. Signal sequences suitable for use in the CAR target group include any eukaryotic signal sequence, including 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)), Strep II (e.g., amino acid sequence NWSHPQFEK (SEQ ID NO: 76)), hexahistidine tag (6xHIS; e.g., amino acid sequence HHHHHH (SEQ ID NO: 77)), 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), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP ( dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, 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 including B-phycoerythrin, R-phycoerythrin, and allophycocyanin.Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from flower buds are suitable for use, for example, as described in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0066] 9. Dimerization domain: Complexation of a group of CARs comprising two CAR molecules can be mediated by a single dimerization domain per CAR molecule, whereby this domain can be for homodimerization or heterodimerization. According to embodiments in which a group of CARs comprises three or four CAR molecules, at least one CAR molecule of the group preferably comprises two or more dimerization domains to promote the formation of trimers or tetramers through dimerization.

[0067] 9.1 Dimerization domain for conditional homodimerization: According to a preferred embodiment, examples of suitable dimerization domains for homodimerization include FK506 binding protein (FKBP), FKPB mutant F36V (dmrB), gyrase B (GyrB), and dihydrofolate reductase (DHFR). The sequences of these homodimerization domains and suitable regulatory molecules for their homodimerization are known in the art (Rutkowska et al., Angew Chem Int Ed Engl. 2012;51(33):8166) and are disclosed, for example, in International Publication No. WO201427261.

[0068] For example, the dimerization domain can be derived from FKBP and 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 P62942-1.

[0069] As another example, the dimerization domain is derived from GyrB (also known as DNA gyrase subunit B) and 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 200 aa to about 300 aa, about 300 aa to about 400 aa, about 400 aa to about 500 aa, about 500 aa to about 600 aa, about 600 aa to about 700 aa, or about 700 aa to about 800 aa of the GyrB amino acid sequence of E. coli Uniprot P0AES6-1 (or the DNA gyrase subunit B sequence of any organism). In some cases, the dimerization domain comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to amino acids 1-220 of the GyrB amino acid sequence from E. coli.

[0070] As another example, the dimerization domain is derived from DHFR (also known as dihydrofolate reductase, DHFRP1, and DYR) and comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence Uniprot P00374-1.

[0071] In another example, the dimerization domain is derived from a DmrB binding domain (i.e., a DmrB homodimerization domain) and comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to SEQ ID NO:10.

[0072] Dimerization can be mediated by different regulatory molecules, such as FK1012 and AP1510 for the homodimerization of FKBP (Amara et al. PNAS 1997;94(20):10618); coumermycin or a coumermycin analog (PubChem CID 54675768) for the homodimerization of GyrB (Farrar et al. Nature. 1996;383:178; and U.S. Pat. No. 6,916,846); a homobifunctional dimer of methotrexate (PubChem CID 126941) for the homodimerization of DHFR (e.g., as disclosed in U.S. Pat. No. 8,236,925); and, according to a preferred embodiment, AP20187 (PubChem CID 78357784) and AP1903 (PubChem CID 78357784) for the homodimerization of DmrB. This can be achieved by the use of a method such as the one described in CID 16135625.

[0073] 9.2. Dimerization Domains for Conditional Heterodimerization: 2.2.1. Conditional Heterodimerization of CAR Molecules Based on Ligand Binding Domains from Nuclear Receptors:

[0074] According to a preferred embodiment, at least two CAR molecules of a group of CARs according to the present invention can be heterodimerized by a pair of heterodimerization domains, one of which is a ligand binding domain (LBD) from a nuclear receptor and the second of which is a coregulatory peptide. Upon binding of an appropriate small molecule (i.e., a regulatory molecule according to the present invention), the LBD from the nuclear receptor can heterodimerize with the respective coregulatory peptide. This system can be used for heterodimerization of proteins of interest. Suitable sequences for LBDs and coregulatory peptides, along with suitable regulatory molecules, are disclosed, for example, in U.S. Patent Publication No. 2017 / 0306303 A1. Suitable LBDs may be selected from any of a variety of nuclear receptors, including: 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 adrenoceptor, 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-gamma.

[0075] The abbreviations for nuclear receptors (synonymous with nuclear hormone receptors) are as follows: R: 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-X-chromosome adrenal dysplasia congenital critical region, 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; R2 / 4: testis receptor; HNF-4: hepatocyte nuclear factor; COUP-TF: chicken ovalbumin upstream promoter, transcription factor.

[0076] 9.2.1.1 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 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 LBD of the MR (Uniprot P08235).

[0077] For example, the LBD of the MR 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 any of the following amino acid sequences: Uniprot Q9IAC6.1 aa 112-359; Uniprot Q91573.1 aa 365-612; Uniprot Q157N1 aa 734-981; GenBank CAG11072.1 aa 173-501; PDB 2AA6_A aa 28-275; PDB 2AA2_A aa 28-275; PDB 2A3I_A aa 6-253; PDB 2OAX_A aa 9-256; PDB 1Y9R_A aa 8-255; PDB 2ABI_A aa 9-256 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 has a length of 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).

[0078] For example, the LBD of MR 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 P08235 aa 686-984, 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 275 amino acids to 299 amino acids).

[0079] For example, the LBD of MR 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 P08235 aa 737-984, 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 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).

[0080] For example, the LBD of MR 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 P08235 aa 686-984 (with 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 275 amino acids to 299 amino acids).

[0081] For example, the LBD of MR 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 P08235 aa 737-984 (with 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 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).

[0082] When 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 aa 681-701), 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).

[0083] When 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 aa 136-156), where the coregulatory peptide has a length of about 21 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 21 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).

[0084] When 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 aa 596-616), 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).

[0085] Androgen receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of an androgen receptor. For example, in some cases, the LBD 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% sequence identity to the LBD of AR (Uniprot P10275).

[0086] For example, the LBD of AR 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 P10275 aa 619-919, 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).

[0087] For example, the LBD of AR 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 P10275 aa 690-919, 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).

[0088] For example, the LBD of AR 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 P10275 aa 619-919 (with 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).

[0089] For example, the LBD of AR 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 P10275 aa 690-919 (with 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).

[0090] For example, the LBD of AR 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 P10275 aa 619-919 (with 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).

[0091] For example, the LBD of AR 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 P10275 aa 690-919 (with 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).

[0092] For example, the LBD of AR 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 P10275 aa 619-919 (with 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).

[0093] For example, the LBD of AR 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 P10275 aa 690-919 (with 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).

[0094] When 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).

[0095] Progesterone receptor: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of the progesterone (PR) receptor. For example, in some cases, the LBD 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% sequence identity to the LBD of the PR (Uniprot P06401).

[0096] For example, the LBD of a PR 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 any of the following amino acid sequences: Uniprot Q8UVY3 aa 456-703; Uniprot P07812.1 aa 539-786; GenBank CAQ14518.1 aa 306-553; PDB 1SR7_A aa 12-259; PDB 1SQN_A aa 14-261; PDB 1E3K aa 11-258; PDB 1A28_A aa and having a length of about 200 amino acids to 250 amino acids (e.g., having a length of 200 amino acids to 225 amino acids, or having a length of 225 amino acids to 250 amino acids; e.g., having a length of 248 amino acids).

[0097] For example, the LBD of a PR 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 any of the following amino acid sequences: Uniprot P06401 aa 678-933; and having a length of about 200 amino acids to 256 amino acids (e.g., having a length of 200 amino acids to 225 amino acids, or 225 amino acids to 256 amino acids; e.g., having a length of 256 amino acids).

[0098] For example, the LBD of a PR 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 any of the following amino acid sequences: Uniprot P06401 aa 686-933; and having a length of about 200 amino acids to 250 amino acids (e.g., having a length of 200 amino acids to 225 amino acids, or 225 amino acids to 250 amino acids; e.g., having a length of 248 amino acids).

[0099] When one member of a pair of heterodimerization domains is the LBD of a PR, the second member of the dimerization pair can be a coregulatory peptide comprising the amino acid sequence GHSFADPASNLGLEDIIRKALMGSF (Uniprot O75376 aa 2251-2275), where the coregulatory peptide has a length of about 25 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 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).

[0100] Thyroid hormone receptor-β: 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-β (TR-β). For example, in some cases, the LBD 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% sequence identity to the LBD of TR-β (Uniprot P10828).

[0101] For example, the LBD of TR-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to one of the following amino acid sequences: Uniprot Q4T8V6 aa 223-502; Uniprot Q90382.1 aa 159-401; Uniprot P18115.2 aa 170-412; Uniprot Q9PVE4.2 aa 141-392; Uniprot P10828.2 aa 216-458; GenBank ABS11249.1 aa 179-419; NCBI REF SEQ XP_001185977.1 aa 186-416; PDB 1NAV_A aa 17-259; PDB 2PIN_A aa 8-250; PDB 3D57_A aa 22-264; PDB 1N46_A aa 13-255; PDB 1BSX_A aa 15-257; and having a length of about 200 amino acids to 250 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 250 amino acids).

[0102] For example, the LBD of TR-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P10828 aa 202-461; and having a length of about 200 amino acids to 260 amino acids (e.g., having 200 amino acids to 225 amino acids, or having 225 amino acids to 260 amino acids; e.g., having a length of 260 amino acids).

[0103] For example, the LBD of TR-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P10828 aa 216-461; and having a length of about 200 amino acids to 246 amino acids (e.g., having 200 amino acids to 225 amino acids, or having 225 amino acids to 246 amino acids; e.g., having a length of 246 amino acids).

[0104] When one member of a pair of heterodimerization domains is the LBD of TR-β, the second member of the pair can be, for example, an NCOA3 / SRC3 polypeptide comprising the amino acid sequence Uniprot Q9Y6Q9 aa 627-829 or Uniprot Q9Y6Q9 aa 673-750 or Uniprot Q15596 aa 721-1021.

[0105] Estrogen receptor-α: According to a preferred embodiment, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of estrogen receptor-α (ER-α). For example, in some cases, the LBD 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% sequence identity to the LBD of ER-α (Uniprot P03372).

[0106] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot P06212.1 aa 304-541; Uniprot P81559.1 aa 302-539; Uniprot Q7ZU32 aa 280-517; GenBank ACB10649.1 aa 303-529; GenBank ABQ42696.1 aa 226-468; GenBank ACC85903.1 aa 141-375; PDB 1XP9_A aa 4-241; PDB 1YY4_A aa 1-236; and having a length of about 200 amino acids to 240 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 235 amino acids, or 235 amino acids to 240 amino acids).

[0107] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 305-533; and having a length of about 180 amino acids to 229 amino acids (e.g., having a length of 180 amino acids to 200 amino acids, or 200 amino acids to 229 amino acids; e.g., having a length of 229 amino acids).

[0108] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 282-595; and having a length of about 250 amino acids to 314 amino acids (e.g., having 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., having a length of 314 amino acids).

[0109] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 310-547; and having a length of about 190 amino acids to 238 amino acids (e.g., having a length of 190 amino acids to 220 amino acids, or 220 amino acids to 238 amino acids; e.g., having a length of 238 amino acids).

[0110] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 305-533 (with 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., has a length of 229 amino acids).

[0111] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 282-595 (with substitution D351Y); and has a length of about 250 amino acids to 314 amino acids (e.g., having 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., having a length of 314 amino acids).

[0112] For example, the LBD of ER-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: Uniprot P03372 aa 310-547 (with 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., has a length of 238 amino acids).

[0113] When one member of a pair of heterodimerization domains is the LBD of ER-α, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence DAFQLRQLILRGLQDD (SEQ ID NO: 11), 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).

[0114] When one member of a pair of heterodimerization domains is the LBD of ER-α, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence SPGSREWFKDMLS (SEQ ID NO: 12), 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).

[0115] Estrogen receptor-β (ER-β): In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be the LBD of estrogen receptor-β (ER-β). For example, in some cases, the LBD 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% sequence identity to the LBD of ER-β (Uniprot Q92731).

[0116] For example, the LBD of ER-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot P06212.1 aa 304-541; Uniprot P81559.1 aa 302-539; Uniprot Q7ZU32 aa 280-517; GenBank ACB10649.1 aa 303-529; GenBank ABQ42696.1 aa 226-468; GenBank ACC85903.1 aa 141-375; PDB 1XP9_A aa 4-241; PDB 1YY4_A aa 1-236; and having a length of about 200 amino acids to 243 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 235 amino acids, or 235 amino acids to 243 amino acids).

[0117] For example, the LBD of ER-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot Q92731 aa 260-502; and having a length of about 200 amino acids to 243 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 235 amino acids, or 235 amino acids to 243 amino acids).

[0118] When one member of a pair of heterodimerization domains is the LBD of ER-β, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence PRQGSILYSMLTSAKQT (SEQ ID NO: 13), 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).

[0119] Peroxisome proliferator-activated receptor-γ: 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-γ (PPAR-γ). For example, in some cases, the LBD 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% sequence identity to the LBD of PPAR-γ (Uniprot P37231).

[0120] For example, the LBD of PPAR-γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot Q7T029 aa 95-435; GenBank AAL26245.1 aa 95-435; NCBI REF SEQ XP_781750.1 aa 137-378; NCBI REF SEQ XP 784429.2 aa 219-478; NCBI REF SEQ NP_001001460.1 aa 207-474; PDB 2J14_A aa 17-284; PDB 1FM6_D aa 4-271; and having a length of about 200 amino acids to 269 amino acids (e.g., having 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).

[0121] For example, the LBD of PPAR-γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P37231 aa 174-475; and having a length of about 150 amino acids to 202 amino acids (e.g., having 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).

[0122] For example, the LBD of PPAR-γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P37231 aa 181-475; and having a length of about 200 amino acids to 269 amino acids (e.g., having 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).

[0123] For example, the LBD of PPAR-γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P37231 aa 205-475; and having a length of about 200 amino acids to 269 amino acids (e.g., having 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).

[0124] When one member of a pair of heterodimerization domains is the LBD of PPAR-γ, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence CPSSHSSLTERHKILHRLLQEGSPS (Uniprot Q15788-1 aa 676-700), where the coregulatory peptide has a length of about 25 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of 25 amino acids to 28 amino acids, 28 amino acids to 29 amino acids, 29 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids).

[0125] When one member of a pair of heterodimerization domains is the LBD of PPAR-γ, the second member of the pair can be a coregulatory peptide comprising the amino acid sequence PKKENNALLRYLLDRDDPSDV (SEQ ID NO: 14) 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).

[0126] 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 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% sequence identity to the LBD of GR having the amino acid sequence Uniprot P04150-3.

[0127] For example, the LBD of GR 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 any of the following amino acid sequences: Uniprot Q4RIR9 aa 110-356; Uniprot P49844.1 aa 530-776; NCBI REF SEQ NP_001032915.1 aa 526-772; PDB 1NHZ_A 34-280; PDB 1M2Z_A aa 11-257; PDB 3BQD_A aa 9-255; PDB 3CLD_A aa 13-259; 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).

[0128] For example, the LBD of GR 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 following amino acid sequence: amino acid sequence Uniprot P04150-3 aa 532-778; 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, or 225 amino acids to 247 amino acids; e.g., having a length of 247 amino acids).

[0129] When one member of a pair of heterodimerization domains is the LBD of GR, the second member of the pair can be, for example, an NCOA2 / SRC2 polypeptide comprising the amino acid sequence Uniprot Q15788 aa 1172-1441 or a fragment thereof, or Uniprot Q15596 aa 320-1021 or a fragment thereof.

[0130] 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 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% sequence identity to the LBD of the VDR (Uniprot P11473).

[0131] For example, the LBD of a VDR can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot O42392.1 aa 147-450; NCBI REF SEQ NP_001079288.1 aa 125-421; PDB 2HBH_A aa 5-301; PDB 1S0Z_A aa 11-262; 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).

[0132] For example, the LBD of a VDR can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P11473 aa 124-426; and having a length of about 250 amino acids to 30 amino acids (e.g., having a length of 250 amino acids to 275 amino acids, 275 amino acids to 300 amino acids, or 300 amino acids to 303 amino acids).

[0133] 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 aa 1172-1441 or a fragment thereof, or Uniprot Q15596 aa 320-1021 or a fragment thereof.

[0134] For example, in some cases, when 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 aa 744-751, wherein the coregulatory peptide has a length of from about 8 amino acids to about 50 amino acids (e.g., the coregulatory peptide has a length of from 8 amino acids to 10 amino acids, from 10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 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).

[0135] Thyroid hormone receptor-α: 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-α (TR-α). For example, in some cases, the LBD 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 TR-α (Uniprot P10827-2).

[0136] For example, the LBD of TR-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot P18115.2 aa 170-412; Uniprot Q9PVE4.2 aa 141-392; Uniprot P10828.2 aa 216-458; GenBank ABS11249.1 aa 179-419; NCBI REF SEQ XP_001185977.1 aa 186-416; PDB 1NAV_A aa 17-259; PDB 2PIN_A aa 8-250; PDB 3D57_A aa 22-264; PDB 1N46_A aa 13-255; PDB 1BSX_A aa 15-257; and having a length of about 190 amino acids to 245 amino acids (e.g., having 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).

[0137] For example, the LBD of TR-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P10827 aa 162-404; and having a length of about 190 amino acids to 243 amino acids (e.g., having 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).

[0138] A suitable coregulatory peptide for TR-α can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0139] Retinoic acid receptor-β: 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-β (RAR-β). For example, in some cases, the LBD 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 RAR-β (Uniprot P10826-2).

[0140] For example, the LBD of RAR-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: Uniprot Q4H2W2 aa 400-634; Uniprot P22448.2 aa 186-416; UNIPROT P28699.2 aa 209-439; Uniprot Q91392.2 aa 176-406; NCBI REF SEQ XP_779976.2 aa 134-362; NCBI REF SEQ XP_002204386.1 aa 179-409; PDB 1XAP_A aa 32-262; PDB 1XDK_B aa 34-264; PDB 1DKF_B aa 5-235; and having a length of about 180 amino acids to 235 amino acids (e.g., having a length of 180 amino acids to 200 amino acids, 200 amino acids to 220 amino acids, or 220 amino acids to 235 amino acids).

[0141] For example, the LBD of RAR-β can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P10826 aa 179-409; and having a length of about 180 amino acids to 231 amino acids (e.g., having 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).

[0142] A suitable coregulatory peptide for RAR-β can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0143] When one member of a pair of heterodimerization domains is the LBD of RAR-β, the other member of the pair can be, for example, an NCOA1 / SRC1 polypeptide comprising the amino acid sequence Uniprot Q15788 aa 1172-1441 or a fragment thereof.

[0144] When one member of a pair of heterodimerization domains is the LBD of RAR-β, the other member of the heterodimer pair can be, for example, an NCOA2 / SRC2 polypeptide comprising the amino acid sequence Uniprot Q155596 aa 320-1021 or a fragment thereof.

[0145] 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 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 FXR having the amino acid sequence Uniprot Q96RI1-2.

[0146] For example, the LBD of FXR 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 following amino acid sequence: amino acid sequence Uniprot Q96RI1-2 aa 237-472; and having a length of about 100 amino acids to 136 amino acids (e.g., having 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).

[0147] A suitable coregulatory peptide for FXR can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0148] LXR-α: 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-α (LXR-α). For example, in some cases, the LBD 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 LBD of LXR-α, which has the amino acid sequence Uniprot Q13133-1.

[0149] For example, the LBD of LXR-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot Q13133-1 aa 182-447; and having a length of about 200 amino acids to 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).

[0150] A suitable coregulatory peptide for LXR-α can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0151] ROR-γ: 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-γ (ROR-γ). For example, in some cases, the LBD 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 LBD of ROR-γ having the amino acid sequence Uniprot P51449-2.

[0152] For example, the LBD of ROR-γ can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P51449-2 aa 237-497; and having a length of about 200 amino acids to 261 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 261 amino acids).

[0153] A suitable coregulatory peptide for ROR-γ may be the NCORNR peptide (CDPASNLGLEDIIRKALMGSFDDK, Uniprot Q7Z516-1 aa 2160-2182).

[0154] A suitable coregulatory peptide for ROR-γ can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0155] RXR-α: 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-α (RXR-α). For example, in some cases, the LBD 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 LBD of RXR-α having the amino acid sequence Uniprot P19793-1.

[0156] For example, the LBD of RXR-α can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid sequence: amino acid sequence Uniprot P19793-1 aa 225-462; and having a length of about 190 amino acids to 238 amino acids (e.g., having 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).

[0157] A suitable coregulatory peptide for RXR-α can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0158] PXR: In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains may be the LBD of pregnane X receptor (PXR-α). For example, in some cases, the LBD may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the 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 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 amino acids 143-428 of the amino acid sequence Uniprot O75469-1. In some cases, the LBD 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 set forth in the amino acid sequence Uniprot O75469-1.

[0159] For example, the LBD of PXR 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 following amino acid sequence: amino acid sequence Uniprot O75469-1 aa 130-434; and having a length of about 250 amino acids to 302 amino acids (e.g., having 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).

[0160] A suitable coregulatory peptide for PXR can be an SRC1 polypeptide or a fragment thereof (eg, a peptide derived from an SRC1 polypeptide, between 8 amino acids and 50 amino acids in length).

[0161] 9.2.1.2. 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. Suitable coregulatory polypeptides can be 8 amino acids to 2000 amino acids in length. Suitable coregulatory polypeptides can be 8 amino acids to 50 amino acids, e.g., 8 amino acids to 10 amino acids, 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids in length. Suitable coregulatory polypeptides can have a length of 50 to 100 amino acids, e.g., 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 can 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 can have a length of 1000 to 2000 amino acids.

[0162] Suitable co-regulators include: steroid receptor coactivator (SRC)-1, SRC-2, SRC-3, TRAP220-1, TRAP220-2, NR0B1, NRIP1, CoRNRbox, alpha-betaV, 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, RIP14 0-3, RIP140-4, RIP140-5, RIP140-6, RIP140-7, RIP140-8, RIP140-9, PRIC285-1, PRIC285-2, PRIC285-3, PRIC285-4 and PRIC285-5.

[0163] Coregulatory polypeptides suitable for heterodimerization with their 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; and preferably 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% sequence identity to a stretch of 8 to 50 contiguous amino acids of the following amino acid sequences: SRC1 (Uniprot Q15788-1), SRC2 (Uniprot Q15596-1), SRC3 (Uniprot Q15601-1), SRC4 (Uniprot Q15602-1), SRC5 (Uniprot Q15603-1), SRC6 (Uniprot Q15604-1), SRC7 (Uniprot Q15605-1), SRC8 (Uniprot Q15606-1), SRC9 (Uniprot Q15607-1), SRC10 (Uniprot Q15608-1), SRC11 (Uniprot Q15609-1), SRC12 (Uniprot Q156109-1), SRC13 (Uniprot Q15611-1), SRC14 (Uniprot Q15612-1), SRC15 (Uniprot Q15613-1), SRC16 (Uniprot Q15614-1), SRC17 (Uniprot Q15615-1), SRC18 (Uniprot Q15616-1), SRC19 (Uniprot Q15617-1), SRC20 Q9Y6Q9-5), PGC1a (Uniprot Q9UBK2-1), PGC1b (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 Q8NI08-1), Nix1 (Uniprot Q9BQI9-1), LCoR (Uniprot Q96JN0-1), N-CoR (Uniprot O75376-1), NCOR2 (Uniprot Q9Y618-1), RIP140 (Uniprot P48552-1), PRIC285 (Uniprot Q9BYK8-2).

[0164] According to preferred embodiments, a suitable coregulatory peptide comprises an LXXLL motif, where X is any amino acid; wherein the coregulatory peptide has a length of 8 amino acids to 50 amino acids, e.g., 8 amino acids to 10 amino acids, 10 amino acids to 12 amino acids, 12 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 25 amino acids, 25 amino acids to 30 amino acids, 30 amino acids to 35 amino acids, 35 amino acids to 40 amino acids, 40 amino acids to 45 amino acids, or 45 amino acids to 50 amino acids.

[0165] Examples of suitable coregulatory peptides are as follows: SRC1: (Uniprot Q15788-1 aa 676-700) CPSSHSSLTERHKILHRLLQEGSPS; SRC1-2: (Uniprot Q15788-1 aa 682-702; SNP rs1049021 E685A) SLTARHKILHRLLQEGSPSDI; SRC3-1: (Uniprot Q9Y6Q9-5 aa 614-632) ESKGHKKLLQLLTCSSDDR; SRC3: (SEQ ID NO: 14) PKKENNALLRYLLDRDDPSDV; PGC-1: (Uniprot Q9UBK2-1 aa 138-154) AEEPSLLKKLLLAPANT; PGC1a: (Uniprot Q9UBK2-1 aa 136-156) QEAEEPSLLKKLLLAPANTQL; TRAP220-1: (Uniprot Q15648-1 aa 596 - 616) SKVSQNPILTSLLQITGNGGS; NCoR: (Uniprot O75376-1 aa 2251 - 2275) GHSFADPASNLGLEDIIRKALMGSF; NR0B1: (Uniprot P51843-1 aa 74 - 90) PRQGSILYSMLTSAKQT; NRIP1: (Uniprot P48552-1 aa 374 - 390) AANNSLLLHLLKSQTIP; TIF2: (Uniprot Q15596-1 aa 737 - 757) PKKKENALLRYLLDKDDTKDI; CoRNR Box: (SEQ ID NO: 11) DAFQLRQLILRGLQDD; abV: (SEQ ID NO: 12) 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 aa 625 - 645) KYSQTSHKLVQLLTTTAEQQL; SRC1-2: (Uniprot Q15788-1 aa 682 - 702; SNP rs1049021 E685A) SLTARHKILHRLLQEGSPSDI; SRC1-3: (Uniprot Q15788-1 aa 741 - 761) KESKDHQLLRYLLDKDEKDLR; SRC1-4a: (Uniprot Q15788-1 aa 1427 - 1441) PQAQQKSLLQQLLTE; SRC1-4b: (Uniprot Q15788-1 aa 1427 - 1441 L1435R) PQAQQKSLRQQLLTE; GRIP1-1: (Uniprot Q15596-1 aa 633 - 653) HDSKGQTKLLQLLTTKSDQME; GRIP1-2: (Uniprot Q15596-1 aa 682 - 702) SLKEKHKILHRLLQDSSSPVD; GRIP1-3: (Uniprot Q15596-1 aa 737 - 757) PKKKENALLRYLLDKDDTKDI; AIB1-1: (Uniprot Q9Y6Q9-5 aa 613 - 633) LESKGHKKLLQLLTCSSDDRG; AIB1-2: (Uniprot Q9Y6Q9-5 aa 677 - 697) LLQEKHRILHKLLQNGNSPAE; AIB1-3: (Uniprot Q9Y6Q9-5 aa 730 - 750) KKKENNALLRYLLRDDPSDA; PGC1a: (Uniprot Q9UBK2-1 aa 136 - 156) QEAEEPSLLKKLLLAPANTQL; PGC1b: (Uniprot Q86YN6-1 aa 148 - 168) PEVDELSLLQKLLLATSYPTS; PRC: (Uniprot Q5VV67-1 aa 156 - 176) VSPREGSSLHKLLTLSRTPPE;TRAP220-1: (Uniprot Q15648-1 aa 596 - 616) SKVSQNPILTSLLQITGNGGS; TRAP220-2: (Uniprot Q15648-1 aa 637 - 657) GNTKNHPMLMNLLKDNPAQDF; ASC2-1: (Uniprot Q14686-1 aa 879 - 899) DVTLTSPLLVNLLQSDISAGH; ASC2-2: (Uniprot Q14686-1 aa 1483 - 1503) AMREAPTSLSQLLDNSGAPNV; CBP-1: (Uniprot Q92793-1 aa 62 - 82) DAASKHKQLSELLRGGSGSSI; CBP-2: (Uniprot Q92793-1 aa 350 - 370) KRKLIQQQLVLLLHAHKCQRR; P300: (Uniprot Q09472-1 aa 73 - 93) DAASKHKQLSELLRSGSSPNL; CIA: (Uniprot Q9HCD5-1 aa 337 - 357) GHPPAIQSLINLLADNRYLTA; ARA70-1: (Uniprot Q13772-1 aa 84 - 104) TLQQQAQQLYSLLGQFNCLTH; ARA70-2: (Uniprot Q13772-1 aa 320 - 340) GSRETSEKFKLLFQSYNVNDW; TIF1: (Uniprot O15164-2 aa 718 - 738) NANYPRSILTSLLLNSSQSST; NSD1: (Uniprot Q96L73-1 aa 899 - 919) IPIEPDYKFSTLLMMLKDMHD; SMAP: (Uniprot Q9H0E9-2 aa 263 - 283) ATPPPSPLLSELLKKGSLLPT; Tip60: (Uniprot Q92993-1 aa 481 - 501) VDGHERAMLKRLLRIDSKCLH; ERAP140: (Uniprot Q8NI08-1 aa 514 - 534) HEDLDKVKLIEYYLTKNKEGP; Nix1: (Uniprot Q9BQI9-1 aa 236 - 256) ESPEFCLGLQTLLSLKCCIDL;LCoR: (Uniprot Q96JN0-1, amino acids 45 - 65) AATTQNPVLSKLLMADQDSPL; CoRNR1 (N-CoR): (Uniprot O75376-1, amino acids 239 - 268) MGQVPRTHRLITLADHICQIITQDFARNQV; CoRNR2 (N-CoR): (Uniprot O75376-1, amino acids 2260 - 2273) NLGLEDIIRKALMG; CoRNR1 (SMRT): (Uniprot Q9Y618-1, amino acids 2131 - 2170) APGVKGHQRVVTLAQHISEVITQDTYRHHPQQLSAPLPAP; CoRNR2 (SMRT): (Uniprot Q9Y618-1, amino acids 2347 - 2360) NMGLEAIIRKALMG; RIP140-C: (SEQ ID NO: 18) RLTKTNPILYYMLQKGGNSVA; RIP140-1: (Uniprot P48552-1, amino acids 13 - 33) QDSIVLTYLEGLLMHQAAGGS; RIP140-2: (Uniprot P48552-1, amino acids 125 - 145) KGKQDSTLLASLLQSFSSRLQ; RIP140-3: (Uniprot P48552-1, amino acids 177 - 197) CYGVASSHLKTLLKKSKVKDQ; RIP140-4: (Uniprot P48552-1, amino acids 258 - 278) KPSVACSQLALLLSSEAHLQQ; RIP140-5: (Uniprot P48552-1, amino acids 372 - 392) KQAANNSLLLHLLKSQTIPKP; RIP140-6: (Uniprot P48552-1, amino acids 493 - 513) NSHQKVTLLQLLLGHKNEENV; RIP140-7: (SEQ ID NO: 19) NLLERRTVLQLLLGNPTKGRV; RIP140-8: (Uniprot P48552-1, amino acids 811 - 831) FSFSKNGLLSRLLRQNQDSYL; RIP140-9: (Uniprot P48552-1, amino acids 928 - 948) RESKSFNVLKQLLLSENCVRD;PRIC285-1: (Uniprot Q9BYK8-2 aa 458 - 518) ELNADDAILRELLDESQKVMV; PRIC285-2: (Uniprot Q9BYK8-2 aa 541 - 561) YENLPPAALRKLLRAEPERYR; PRIC285-3: (Uniprot Q9BYK8-2 aa 596 - 616) MAFAGDEVLVQLLSGDKAPEG; PRIC285-4: (Uniprot Q9BYK8-2 aa 1435 - 1455) SCCYLCIRLEGLLAPTASPRP; and PRIC285-5: (Uniprot Q9BYK8-2 aa 1652 - 1672) PSNKSVDVLAGLLLRRMELKP。;

[0166] In some cases, a given LBD may be paired with two or more different co-regulatory polypeptides. For example, PPAR-γ (Uniprot P37231) can be paired with: SRC1 (Uniprot Q15788-1 aa 625-645; Uniprot Q15788-1 aa 676-700; Uniprot Q15788-1 aa 682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa 741-761; Uniprot Q15788-1 aa 1428-1441; Uniprot Q15788-1 aa 1427-1441; Uniprot Q15788-1 aa 1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa 683-701), SRC3 (SEQ ID NO: 15596-1 aa 684-701), SRC4 (Uniprot Q15596-1 aa 685-701), SRC5 (Uniprot Q15596-1 aa 685-701), SRC6 (Uniprot Q15596-1 aa 686-701), SRC7 (Uniprot Q15596-1 aa 687-701), SRC8 (Uniprot Q15596-1 aa 688-701), SRC9 (Uniprot Q15596-1 aa 689-710), SRC10 (Uniprot Q15596-1 aa 690-710), SRC11 (Uniprot Q15596-1 aa 691-710), SRC12 (Uniprot Q15596-1 aa 692-712), SRC13 (Uniprot Q 14; Uniprot Q9Y6Q9-5 aa 614-632) or TRAP220 (Uniprot Q15648-1 aa 596-616; Uniprot Q15648-1 aa 637-657). As another example, ER-ALPHA (Uniprot P03372) can be paired with CoRNR (Uniprot O75376-1 aa 239-268; Uniprot O75376-1 aa 2260-2273; Uniprot Q9Y618-1 aa 2131-2170; Uniprot Q9Y618-1 aa 2347-2360), alpha-betaV (SEQ ID NO: 12) or TA1 (SEQ ID NO: 16). As another example, ER-beta (Uniprot Q92731) can be paired with: CoRNR (Uniprot O75376-1 aa 239-268; Uniprot O75376-1 aa 2260-2273; Uniprot Q9Y618-1 aa 2131-2170; Uniprot Q9Y618-1 aa 2347-2360), alpha-betaV (SEQ ID NO: 12) or TA1 (SEQ ID NO: 16).As another example, AR (Uniprot P10275) can be paired with: SRC1 (Uniprot Q15788-1 aa 625-645; Uniprot Q15788-1 aa 676-700; Uniprot Q15788-1 aa 682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa 741-761; Uniprot Q15788-1 aa 1428-1441; Uniprot Q15788-1 aa 1427-1441; Uniprot Q15788-1 aa 1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa 683-701), SRC3 (SEQ ID NO: 14; Uniprot Q9Y6Q9-5 aa 614-632) or TRAP220 (Uniprot Q15648-1 aa 596-616; Uniprot Q15648-1 aa 637-657). As another example, PR (Uniprot P06401) can be paired with: SRC1 (Uniprot Q15788-1 aa 625-645; Uniprot Q15788-1 aa 676-700; Uniprot Q15788-1 aa 682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa 741-761; Uniprot Q15788-1 aa 1428-1441; Uniprot Q15788-1 aa 1427-1441; Uniprot Q15788-1 aa 1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa 683-701), SRC3 (SEQ ID NO: 14; Uniprot Q9Y6Q9-5 aa 614-632), TRAP220 (Uniprot Q15648-1 aa 596-616; Uniprot Q15648-1 aa 637-657), NR0B1 (Uniprot P51843-1 aa 74-90), PGC1B (Uniprot Q86YN6-1 aa 148-168), NRIP1 (Uniprot P48552-1 aa 374-390), EA2 (SEQ ID NO: 15) or EAB1 (SEQ ID NO: 17).As another example, TR-beta (Uniprot P10828) can be paired with: SRC1 (Uniprot Q15788-1 aa 625-645; Uniprot Q15788-1 aa 676-700; Uniprot Q15788-1 aa 682-702, SNP rs1049021 E685A; Uniprot Q15788-1 aa 741-761; Uniprot Q15788-1 aa 1428-1441; Uniprot Q15788-1 aa 1427-1441; Uniprot Q15788-1 aa 1427-1441 L1435R), SRC2 (Uniprot Q15596-1 aa 683-701), SRC3 (SEQ ID NO: 14; Uniprot Q9Y6Q9-5 aa 614-632), or TRAP220 (Uniprot Q15648-1 aa 596-616; Uniprot Q15648-1 aa 637-657).

[0167] 9.2.1.3. Regulatory molecules for LBD-based heterodimerization: When one member of a heterodimerization domain pair 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 and then dimerize with a co-regulatory peptide in a second CAR molecule of the group.

[0168] Suitable regulatory molecules 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); 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]phenanthrene-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]phenanthrene-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); 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]phenanthrene-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]phenanthren-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] hydrogen sulfate); 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]phenanthren-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); cyproterone acetate (PubChem CID 9880); hydroxyflutamide (2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide); Enzalutamide (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); bequilosteride ((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-butylbenzenesulfonamide (NBBS) (N-butylbenzenesulfonamide); 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-butylbenzenesulfonamide (N-butylbenzenesulfonamide); Nilutamide (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl)phenyl]imidazolidine-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); 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,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); 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'-methylidenediamine; pyro[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]phenanthrene-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]phenanthrene-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]phenanthrene-17,2'-oxolane]-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-12,6,7,8,11,12,14,15,16-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);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]estr-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-dinol-17,23-epoxy-17alpha-chola-4,9,20-tri-n-3-one; (Z)-11beta,19-[4-(3-pyridinyl)-o-phenylene]-17beta-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-11beta-[4-(dimethylamino)phenyl]-6beta-methylspiro[estra-4,-9-dien-17beta,2'(3'H)-furan]-3-one; drospirenone (PubChem CID 68873); 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-24 (2-[4-[(3-benzyl-4-hydroxyphenyl)methyl]-3,5-dimethylphenoxy]acetic acid); 4-OH-PCB106 (2-chloro-4-(2,3,4,5-tetrachlorophenyl)phenol); Eprotirome (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-phenylbut-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); azedoxifene (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); Femarel (); 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); Ospemifene (2-[4-[(Z)-4-chloro-1,2-diphenylbut-1-enyl]phenoxy]ethanol);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,14S,17S)-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol); Ethinyl estradiol ((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); 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 A276575, Mapracorat ((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); ZK 216348 (4-(2,3-dihydro-1-benzofuran-7-yl)-2-hydroxy-4-methyl-N-(4-methyl-1-oxo-2,3-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); 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);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) and paricaritol ((1R,3R)-5-[(2E)-2-[(1R,3aS,7aR)-1-[(E,2R,5S)-6-hydroxy-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); hexahydro-1H-inden-4-ylidene]ethylidene]cyclohexane-1,3-diol); 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 (calcifediol) ((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-methylidenecyclohexan-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)-butan-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-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-dinol-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-trichomopregnacalciferol, 2-methylene-23,23-difluoro-1α-hydroxy-19-nor-bishomopregnacalcifero-1,2-methylene-(20S)-23, 23-difluoro-1α-hydroxy-19-nor-bishomopregnan-calciferol, (2-(3'hydroxypropyl-1',2'-idene)-19,23,24-triol-(20S)-1α-hydroxyvitamin D3, 2-methylene-18,19-dinol-(20S)-1α, 25-dihydroxyvitamin D3, and the like; 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-cyclohexenyl)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-butyrate, retinoyl pinacol, and retinoyl cholesterol; 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]phenanthrene-17-yl]pentanoic 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); 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); GNE-3500 (27,1-{4- [3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-yl-methyl)-phenyl]-piperazin-1-yl}-ethanone; 7beta,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).

[0169] When the heterodimerization domain pair comprises the LBD of the mineralocorticoid receptor (MR) and the corresponding co-receptor peptide, suitable modulatory molecules include spironolactone and eplerenone. Spironolactone may be administered at doses ranging from 10 to 35 mg / day, e.g., 25 mg / day.

[0170] When the heterodimerization domain pair comprises the LBD of the androgen receptor (AR) and the corresponding co-receptor peptide, suitable regulatory molecules include: cyproterone acetate, hydroxyflutamide, enzalutamide, ARN-509, 3,3'-diindolylmethane (DIM), bequilosteride, bicalutamide, N-butylbenzene-sulfonamide (NBBS), dutasteride, epristeride, finasteride, flutamide, zonsteride, ketoconazole, N-butylbenzene-sulfonamide, nilutamide, megestrol, steroidal antiandrogens, and turosteride.

[0171] When the heterodimerization domain pair comprises the LBD of the progesterone receptor (PR) and the corresponding co-receptor peptide, suitable regulatory molecules include: mifepristone (RU-486; 11beta-[4N,N-dimethylaminophenyl]-17beta-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one); lilopristone (11beta-(4N,N-dimethylaminophenyl)-17beta-hydroxy-17-((Z)-3-hydroxypropenyl)estra-4,9-dien-3-one); onapristone (11β-(4N,N-dimethylaminophenyl)-17α-hydroxy-17-(3-hydroxypropyl)-13α-estra-4,9-dien-3-one); Asoprisnil (benzaldehyde, 4-[(11beta,17beta)-17-methoxy-17-(methoxymethyl)-3-oxoestra-4,9-dien-11-yl]-1-(E)-oxime; J867); J912 (4-[17beta-hydroxy-17alpha-(methoxymethyl)-3-oxoestra-4,9-dien-11beta-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, (6alpha, 11beta, 17beta)-11-(4-dimethylaminophenyl)-6-methyl-4',5'-dihydrospiro[estra-4,9-diene-17,2'(3'H)-furan]-3-one (ORG-31710); (11beta, 17alpha)-11-(4-acetylphenyl)-17,23-epoxy-19,24-dinorchola-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-(methoxymethyl)-3-oxoestra-4,9-dien-11β-yl]benzaldehyde-1-(E)-[O-(ethylamino)carbonyl]oxime; 4-[17beta-Methoxy-17α-(methoxymethyl)-3-oxoestra-4,9-dien-11β-yl]benzaldehyde-1-(E)-[O-(ethylthio)carbonyl]oxime; (Z)-6'-(4-cyanophenyl)-9,11alpha-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-dinol-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, and drospirenone;

[0172] When the heterodimerization domain pair comprises the LBD of thyroid receptor-β (TR-β) and the corresponding co-receptor 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)benzylphenoxyacetic acid); 4-OH-PCB106 (4-OH-2',3,3',4',5'-pentachlorobiphenyl); eprotirome; 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).

[0173] When the heterodimerization domain pair comprises the LBD of estrogen receptor-α (ER-α) and the corresponding co-receptor peptide, suitable regulatory molecules include: tamoxifen, 4-OH-tamoxifen, raloxifene, lasofoxifene, bazedoxifene, falsodex, clomiphene, femarel, ormeloxifene, toremifene, ospemifene, and ethinyl estradiol.

[0174] When the heterodimerization domain pair comprises the LBD of estrogen receptor-β (ER-β) and the corresponding co-receptor peptide, suitable regulatory molecules include: estradiol (E2; or 17-beta-estradiol), and ethinyl estradiol.

[0175] When the heterodimerization domain pair comprises the LBD of PPAR-γ and the corresponding co-receptor peptide, suitable regulatory molecules include: thiazolidinediones (e.g., rosiglitazone, pioglitazone, lobeglitazone, troglitazone), farglitazar, aleglitazar, and fenofibric acid.

[0176] When the heterodimerization domain pair comprises the LBD of GR and the corresponding co-receptor peptide, suitable regulatory molecules can be: selective GR agonists (SEGRAs) or selective GR modulators (SEGRMs).

[0177] When the heterodimerization domain pair comprises the LBD of GR and the corresponding co-receptor peptide, suitable regulatory molecules include: benzopyranoquinoline A276575, mapracorat, ZK 216348, 55D1E1, dexamethasone, prednisolone, prednisone, methylprednisolone, fluticasone propionate, beclomethasone-17-monopropionate, betamethasone, rimexolone, paramethasone, and hydrocortisone.

[0178] When the heterodimerization domain pair comprises the LBD of a VDR and a corresponding co-receptor peptide, suitable regulatory molecules can be: 1,25-dihydroxyvitamin D3 (calcitriol), paricaritol, 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)-dihydro-19-nor -Vitamin D3, 2-methylene-19-nor-(24R)-1α, 25-dihydroxyvitamin D2, 2-methylene-(20R,25S)-19,26-dinol-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-trichomopregnacalciferol, 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-triol-(20S)-1α-hydroxyvitamin D3, 2-methylene-18,19-dinol-(20S)-1α, 25-dihydroxyvitamin D3, etc.

[0179] When the heterodimerization domain pair comprises the LBD of RAR-β and the corresponding coreceptor peptide, suitable regulatory molecules may be the following: 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)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, ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)ethynyl]pyridine-3-carboxylate, retinoyl t-butyric acid, retinoyl pinacol, and retinoyl cholesterol.

[0180] When the heterodimerization domain pair comprises FXR and the corresponding co-receptor 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).

[0181] When the heterodimerization domain pair comprises the LBD of LXR-α (PR) and the corresponding co-receptor peptide, suitable modulatory 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).

[0182] When the heterodimerization domain pair comprises the LBD of ROR-γ and the corresponding co-receptor peptide, suitable regulatory molecules include: GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-yl-methyl)-phenyl]-piperazin-1-yl}-ethanone).

[0183] When the heterodimerization domain pair comprises the LBD of ROR-γ and the corresponding co-receptor peptide, suitable regulatory molecules include: 7β, 27-dihydroxycholesterol and 7α, 27-dihydroxycholesterol.

[0184] When the heterodimerization domain pair comprises the LBD of RXR-α and the corresponding coreceptor 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).

[0185] When the heterodimerization domain pair comprises the LBD of PXR and the corresponding co-receptor peptide, suitable regulatory molecules include: rifampicin, clotrimazole, and lovastatin.

[0186] 9.2.2. Conditional Heterodimerization of CAR Molecules Based on Lipocalin-Fold Molecules: According to another preferred embodiment, at least two CAR molecules of the group of CARs of the present invention may be heterodimerized by a pair of heterodimerization domains comprising one member which is a lipocalin fold molecule and a second member which is a lipocalin fold binding interaction partner, as disclosed in European Patent No. 17208924.5 filed December 20, 2017. According to a preferred embodiment, the lipocalin fold-based heterodimerization system comprises: (a) Lipocalin fold molecule, (b) lipocalin fold ligands having a low molecular weight of 1500 Da or less, and (c) lipocalin fold binding interaction partners; wherein said 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 times higher than the affinity of the lipocalin fold molecule not bound to the lipocalin fold ligand; and Here, a lipocalin fold binding interaction partner is not a naturally occurring protein that has an affinity of 10 μM or less for any naturally occurring lipocalin fold molecule in the presence of any lipocalin fold ligand.

[0187] According to a further preferred embodiment, the lipocalin fold based heterodimerization system comprises: (a) Lipocalin fold molecule, (b) lipocalin fold ligands having a low molecular weight of 1500 Da or less, and (c) lipocalin fold binding interaction partners; wherein the ligand-fold molecule has at least a first conformation when the lipocalin-fold ligand is not bound to the lipocalin-fold molecule, and at least a second conformation when the lipocalin-fold ligand is bound to the lipocalin-fold molecule; wherein the lipocalin fold molecule that is bound to the lipocalin fold in the second conformation binds to the lipocalin fold binding interaction partner with an affinity that is at least 10 times higher than the affinity of the lipocalin fold molecule that is not bound to the lipocalin fold ligand in the first conformation; and Here, a lipocalin fold binding interaction partner is not a naturally occurring protein that has an affinity of 10 μM or less for any naturally occurring lipocalin fold molecule in the presence of any lipocalin fold ligand.

[0188] This lipocalin fold molecule-based system for conditional heterodimerization generally relies on a substantial difference in the affinity of the lipocalin fold molecule for its lipocalin fold binding interaction partner depending on whether or not a lipocalin fold ligand is bound. Preferably, the affinity window (i.e., the affinity of the lipocalin fold binding interaction partner for the lipocalin fold molecule bound to or unbound to a lipocalin fold ligand, respectively) lies within a reasonable affinity range that allows for modulation of heterodimerization under physiological conditions. Thus, it is preferred that the affinity of the lipocalin fold binding interaction partner for the lipocalin fold molecule in the liganded state is less than 10 μM, preferably less than 2 μM, and particularly less than 400 nM.

[0189] Depending on whether the lipocalin fold binding partner is designed to bind to lipocalin fold molecules charged or uncharged with a lipocalin fold ligand, lipocalin fold molecule-based systems can be used for conditional heterodimerization (i.e., switching) or constitutive heterodimerization, respectively. Because lipocalin fold binding interaction partners can also be designed to bind to lipocalin fold molecules independently of, but not in the presence of, a lipocalin fold ligand, the system can also be used to conditionally prevent heterodimerization (i.e., switching). In principle, lipocalin fold molecule-based systems can optionally be designed to bind at least two different lipocalin fold ligands, where the correspondingly selected lipocalin fold binding interaction partners can distinguish between two differentially induced conformational states, which can then be conditionally switched on and off by sequential addition of two different lipocalin fold ligands.

[0190] A lipocalin fold molecule that can be used as a heterodimerization domain according to the present invention can be any protein that contains the structural motif of the lipocalin fold to which (or in which) a lipocalin fold ligand binds and that allows binding of the lipocalin fold molecule to a lipocalin fold interaction partner.

[0191] A lipocalin-fold molecule is defined as any naturally occurring molecule classified in the lipocalin superfamily in the SCOP database (version 1.75), or a variant thereof, although it is preferred to exchange only a limited number of amino acids.

[0192] According to a preferred embodiment, the lipocalin-fold molecule is a molecule identical to a naturally occurring iLBP (intracellular lipid-binding protein), a naturally occurring lipocalin or anticalin, and any derivatives and fragments thereof of these molecules with 1 to 30 amino acid exchanges. According to another preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring lipocalin or iLBP with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or 30 amino acid exchanges.

[0193] According to a preferred embodiment of the present invention, the lipocalin fold molecule is designed by one or more amino acid exchanges, insertions and / or deletions in order 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 (by its amino acid sequence) lipocalin fold molecule having at least 70%, preferably at least 80%, in particular at least 90% sequence identity in its β-barrel structure, whereby this β-barrel structure is defined as a region preferably 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 in human RBP4 (according to the amino acid residue numbering scheme of PDB entry 1RBP), which define the structurally conserved β-strand in human RBP4; - amino acid residues 14-23, 37-43, 48-54, 62-69, 76-79, 84-91, 96-102 and 111-117 in human tear lipocalin (TLC: as defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand in human TLC; - amino acid residues 44-53, 69-75, 81-87, 96-103, 110-113, 119-126, 131-137 and 142-148 in human apolipoprotein M (ApoM; as defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand in 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 in human cellular retinoic acid binding protein II (CRABPII; according to the amino acid residue numbering scheme in PDB entry 2FS6), which define the structurally conserved β-strand in human CRABPII; - Amino acid residues 5-12, 39-43, 48-52, 59-63, 69-71, 79-85, 91-94, 99-103, 109-114, and 119-125 in human fatty acid binding protein 1 (FABP1; according to the amino acid numbering scheme in PDB entry 2F73), which define the structurally conserved β-strand in human FABP1.

[0194] According to a preferred embodiment, the lipocalin fold molecule is a fragment of a naturally occurring lipocalin or a derivative thereof at least 80, preferably at least 100, in particular at least 120 amino acids in length, 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 iLBP or a derivative thereof at least 80, preferably at least 85, in particular at least 90 amino acids in length, covering at least the structurally conserved β-barrel structure of the lipocalin fold, wherein said structurally conserved β-barrel structure comprises or consists of amino acid positions which preferably structurally correspond 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 in human RBP4 (according to the amino acid residue numbering scheme of PDB entry 1RBP), which define the structurally conserved β-strand in human RBP4; - amino acid residues 14-23, 37-43, 48-54, 62-69, 76-79, 84-91, 96-102 and 111-117 in human tear lipocalin (TLC: as defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand in human TLC; - amino acid residues 44-53, 69-75, 81-87, 96-103, 110-113, 119-126, 131-137 and 142-148 in human apolipoprotein M (ApoM; as defined by Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the structurally conserved β-strand in 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 in human cellular retinoic acid binding protein II (CRABPII; according to the amino acid residue numbering scheme in PDB entry 2FS6), which define the structurally conserved β-strand in human CRABPII; according to the amino acid residue numbering scheme in PDB entry 2F3; - Amino acid residues 5-12, 39-43, 48-52, 59-63, 69-71, 79-85, 91-94, 99-103, 109-114, and 119-125 in human fatty acid binding protein 1 (FABP1; according to the amino acid numbering scheme in PDB entry 2F73), which define the structurally conserved β-strand in human FABP1.

[0195] According to a further preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring lipocalin or iLBP, preferably having up to 15, up to 30 or up to 50 amino acid deletions and / or up to 15, up to 30 or up to 50 amino acid insertions outside the structurally conserved β-barrel structure corresponding to the amino acid residues selected from: - Amino acid residues 1-20, 31-40, 48-51, 59-70, 79-84, 89-101, 110-113, 121-131, and 139-183 in human RBP4, which define the regions adjacent to the structurally conserved β-strand in human RBP4 according to the amino acid residue numbering in 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 in human TLC, which define the region adjacent to the structurally conserved β-strand in human TLC (according to the amino acid residue numbering in Schiefner et al., Acc Chem Res. 2015;48(4):976-985); - amino acid residues 1-43, 54-68, 76-80, 88-95, 104-109, 114-118, 127-130, 138-141 and 149-188 in human ApoM (according to the amino acid residue numbering as defined by ApoM; Schiefner et al., Acc Chem Res. 2015;48(4):976-985), which define the region adjacent to the structurally conserved β-strand in 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 in human CRABPII (according to the amino acid residue numbering scheme in PDB entry 2FS6), which define the region adjacent to the structurally conserved β-strand in 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 in human FABP1 (according to the amino acid residue numbering scheme in PDB entry 2F73), which define the region adjacent to the structurally conserved β-strand in human FABP1.

[0196] According to another preferred embodiment, the lipocalin fold molecule is a derivative of a naturally occurring member of the lipocalin superfamily with at least 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.

[0197] According to a further preferred embodiment, the lipocalin-fold molecule used as a heterodimerization domain according to the present invention is a lipocalin, i.e., a protein comprising an eight-stranded upper and lower β-barrel arranged in a +1 topology, with the C-terminus of the eighth β-strand followed by an α-strand.

[0198] Lipocalin fold ligands that can be used as regulatory molecules according to the present invention are "small" compared to "small molecules," e.g., polypeptides and proteins, such as lipocalin fold molecules. Thus, lipocalin fold ligands have a molecular weight of 1500 Da or less, preferably 1000 Da or less, and in particular 750 Da or less. A preferred Mw range for lipocalin fold ligands is 50-1500 Da, preferably 75-1500 Da, and in particular 150-750 Da. Preferably, the lipocalin fold ligand can bind to the tail of a lipocalin fold molecule, which is formed by the barrel and loop regions of the lipocalin fold structure.

[0199] The lipocalin fold ligand preferably has an affinity for the lipocalin fold molecule of less than 1 mM, preferably less than 100 μM, and especially on the order of 10 μM. This affinity between the lipocalin fold ligand and the lipocalin fold molecule is defined as the Kd (dissociation constant) value and is preferably determined by isothermal titration calorimetry (ITC) using an automated MicroCal PEAQ-ITC instrument (Malvern Instruments).

[0200] Examples of lipocalin fold ligands from which may be selected are: Nr Database HMDB 1. Nafcillin 2. Gerberinol 3. Montelukast 4. Flurazepam 5. Quinidine barbiturate 6 Glyceollin I 7. Glyceollin II 8 (-)-Simpterocarpine 9 Canzonor W 10 6α-hydroxyphaseolin 11 (1a,5b,6a)-7-Protoiludene-1,5,6,14-tetrol 14-(2,4-dihydroxy-6-methylbenzoic acid) 12 4'-O-Methylcanzolinol W 13 Szkrokiewitone 14 Lycoflanone 15 Almiratin 16 2-(4-methyl-3-pentenyl)anthraquinone 17 Sorafenib beta-D-glucuronide 18 Heterophilin 19 2'-O-Methyl Phaseolin Isoflavan 20 Chia Prides 21 Gluten Exorphin C 22 Marbello Franc M 23 Dalxanthon G 24 Sclareol 25 Corpse Dogs 26 Canzonol F 27 Mangostinone 28 Gankaonin X 29 Rubraflavone D 30 Cycloquivitone Hydrate 31 Griseolidine II 32 Cyclandrelate 33 Dalxanthon E 34 Morcin 35 (E)-2',4,4'-Trihydroxy-3-prenylchalcone 36 Dalxanthon H 37 Judeol 38 Artonin E 39 Canzonol T 40 Fragransol A 41 Dalxanthon F 42 Marbello Fran T 43 Garcimangozon A 44 Artonin B 45 Asteltoxin Database KEGG database 46 Oxyfedrine 47 Profluthrin 48 Monflorotrin 49 Xyloylsulfamine 50 Cetotiamine hydrochloride hydrate 51 Daisethiamine hydrochloride hydrate 52 Diacetamine 53 Oxolamine 54 Oxalamin 55 Clinathol mesylate 56 Iofurbenzamide I 57 Ceritinib 58 Zykadia 59 Imiprothrin 60 Triclabendazole 61 Fascinex 62 Brivanib alaninate 63 Transfluthrin 64 Enolicum sodium 65 Enolicum sodium monohydrate 66 Zivcain 67 Shinchocaine 68 Nupercain 69Trametinib Database WDI 70 Flucloxacillin 71 S-Farnesylthiosalicylic acid 72 2-Fluorotropapride 73 Butampicillin 74 Dicloxacillin sulfate 75 Floxacillin 76 Ibcillin 77 Prazocillin 78 Saletamide 79 Tetrachlorosalisilanilide 80 Carbenicillin 81 Dichloromethide 82 Methylsulfometron 83 Trichlorosalisilanilide 84 Clomethocillin 85 Cystodactylin-F 86 Detanozaru 87 Diaballone 88 Diclofop 89 Epipheneticillin 90 FTALIL-MEDEYOL 91 Salutamide hydrochloride 92 Tiapride Hydrochloride 93 Tranculin-A 94 Deoxyfentalenyl glucuronide 95 Lafurnimas 96 Melazolam 97 Dibsaddle 98 Feneticillin potassium 99 Plano Sal 100 Dareformis 101 Diphenicillin 102 Fenoterol hydrochloride 103 Gigantic Acid 104 Halothraline-B 105 Isopropylidin 106 Proguanil Hydrochloride 107 Piranokhunthong-B 108 Tuberosin 109 Zofielamide-B 110 Cloxacillin sodium 111 Retimide hydrochloride 112 Bygene-B 113 Bidwilon-B 114 Carbenicillin disodium 115 Hydroxyprocaine 116 Ochratoxin-A 117 Celox 118 Macaragaflavanone-B 119 Menoximycin-B 120 Penicillin-S 121 Psoralidine 122 Rubiginone-C1 123 Secopseudocopterosin-E 124 Asadisulfide 125 Balancadic acid-A 126 Bephedon 127 Meldnar-B 128 Neraminol 129 Phomopsolid-A 130 Strong acid 131 Zofielamide-A 132 Cystodactylin-B 133 Dicloxacillin 134 Fluoropropranolol 135 Iriosicollin-B 136 Indicanin-B 137 Jacarevin 138 Cottamide C 139 Mexolamine 140 Mica peroxide-H 141 Otogirin 142 Oxolamine Hydrochloride 143 Oxopropaline-A 144 Purvalanol-A 145 Rubiginone-C2 146 Teracrilshikonin 147 Clodinafop-propargyl-ester 148 Mazaticol 149 Sehokdim 150 Sulfaguanol 151 Balaperidone 152 Flucloxacillin Sodium 153 Geodiamolidota 154 Lucantone sulfoxide 155 Meleoride-D 156 Nadoxolol Hydrochloride 157 Becloric Acid Glucuronide 158 Cloxacillin 159 Hypergynon-B 160 Oligosporol-A 161 Propoxycaine Hydrochloride 162 Ronifibrate 163 Sudan Blue GN 164 Trichodermamid-B 165 Botryllamide-A 166 Carfecillin 167 Chresodim 168 Dutadolpin 169 Epicoclioquinone-B-14 170 Flurazepam 171 Hydroxyflucloxacillin 172 Rinacanthin-C 173 Teflubenzuron 174 Zenisarat 175 Antimycin-A8A 176 ARTOINDONESIANIN-U 177 Bronco Cain 178 Enolicum 179 Ipadilade 180 Mordant Brown 1 181 Propranolol Phenobarbital 182 Puginin A 183 Amphibin-H 184 Aluminum Acid 185 Kalindasilin 186 Chlorobiocate 187 Chlorproguanil 188 Cylindrol-B 189 Ecliptalvine 190 Garsigarin-A 191O-demethylchlorothricin 192 Sanguenon-C 193 Tetraperol-G 194 Chlorsulfuron 195 Dexamethasone diethylaminoacetate 196 Diet 197 Pyridovericin 198 Subendazole 199 Thiocaine 200 Trapezifolixanthone 201 Trikrazan 202 3',4'-Dichlorobenzamil 203 Chaetoviridin-C 204 Cycloregatin 205 Flurazepam monohydrochloride 206 Fusidilactone-B 207 Griseochelin methyl ester 208 Isobutylshikonin 209 Misinicate 210 Ajudazole-B 211 Cystodactylin-E 212 Demethylpraecanthon-A 213 Destruxin-A 214 Diphenidol Embonate 215 Discochioride-B 216 Irumano Ride-2 217 Lactoquinomycin 218 Neobornival 219 Salotralen-D 220 Abyssinon-V 221 Acrylonitrile 222 Chlorfluazuron 223 Chondrijien-18,20 224 EUGLOBAL-G2 225 Idarubicin hydrochloride 226 Muticicmaranon-A 227 3-O-Methylcalpocarpine 228 Alloclamide hydrochloride 229 Anopterin 230 Deoxidation 231 EUGLOBAL-IIC 232 Ilicicolin-C 233 Mysinolide II 234 Suirin 235 Tocotrienol Gamma 236 Indochinanonbeta 237 Isothiobamine 238 Mebeverine 239 Muticicmaranon-D 240 Nympheaol-C 241 Pursocine 242 ZIMET-20-84 243 2,4-D-Butoxypropyl 244 Abyssinon IV 245 Bygene-A 246 Chrysocratic Acid 247 Epolon-B 248 Ethoxycaine 249 Florenamin 250 Guaiacol-Mefenamate 251 Maxima-Isoflavone-C 252 Sarcodictin-B 253 Triclabendazole 254 Akio Furan 255 Asteriquinone 256 Diethylglycolate trihydrazide 257 Marot Philippens-D 258 Naphthoxate 259 PACHYDICTYOL-A-EPOXIDE 260 Ratjadong 261 Salverine Hydrochloride 262 4-Menahydroquinone 263 Botryllamide-C 264 Elsa Sewing Machine 265 Fenfluthrin 266 Muticifenone-A 267 Sanguenon-A 268 Schweinfachin-A 269 ​​Vanilligirole 270 4-O-Methylmelleolide 271 Acetyl Bismuth-F 272 Alfentanil hydrochloride 273 Aspertetronin-A 274 Beta-hydroxyisovalerylshikonin 275 Carbisocaine 276 Chlorproguanil hydrochloride 277 Cryptophycin-52 278 Didemethyltocotrienol 279 Furusol-DA 280 Pyridoxifene 281 Tiazesim Hydrochloride 282 Butoctamide 283 Deoxyshikonin 284 Cambieric acid-A 285 Lycoflanone 286 Prednylidene diethylaminoacetic acid 287 Pseudoopterosin-G 288 Trikendior 289 Zoapatanol 290 Ergoquine-C 291 Pentamoxane Hydrochloride 292 Scandenin 293 Actinopyrone-C 294 Amiton 295 Kratokiyaborenone-C 296 Simopol 297 Doxycycline Hydrate 298 Flavidurol-C 299 francs - 12 300 Miriaporon-3 301 Orinosinolide 302 Tonabelsat 303 Vismione-B 304 Amykelin 305 Butamoxan hydrochloride 306 Chlorobiosin 307 Cyclocom Unit 308 Fenazaflor 309 Bismione D 310 3-Trichlorometaphos 311 Aminopropyl phenylbutazone 312 Diocrenor 313 Grifolin 314 Hyperjovinol-A 315 Tamora Rin 316 Tomentol 317 Trans-delta tocotrienol 318 Diacetol hydrochloride 319 Dutomycin 320 Liglobastosid-O 321 Rice Parole-B 322 Electione-A 323 Salotralen-A 324 Shitamakin 3 325 Trichoporin II 326 3-Hydroxytorfazepam 327 Dimetapramide 328 Fenoterol hydrochloride 329 Fencapton 330 Kratokiyaborenone-B 331 Etomoxan hydrochloride 332 Isocentratelin 333 Kalimantan-A 334 Hispaglabridin-A 335 Lancacyclinol 336 Macroxanthone 337 Picillin Hydrochloride 338 Purflavin-A 339 Sigmoidine I 340 Fenaraamide 341 Hydroxyaclasinomycin-M,2 342 Soforadin 343 Ascochlorin 344 Beta-Hydroxysanshur 345 Bistromide-K 346 Chlortetracycline borate 347 Dehydroascochlorin-8+,9+ 348 Dimethylium Chloride 349 Macarangin 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 Sarashiine 360 Electone B 361 Homodinic Acid 62 Asteriquinone-B1 363 Detamid 364 Rubraxanthon 365 Dolastatin-19 366 Edetol 367 Phaseolyn 368 Gedkarnir 369 Man Sewing Machine-B 3 70 Santalol-beta-salicylic acid 371 Kowanin 372 Indibrin 373 Coinole 374 Karatabishi 375 Picroxidyne 376 Proxazole 377 Strobilurin-E 378 Elekkion-B 379 Sri Kainid 380 Diethylaminomethylrutin 381 Tropesin 382 Picloxidin hydrochloride 383 HEX-1 384 Decrobaniloubiocin Database MDDR 385 Rubizinone C2 386 Phlobaphen 387 Tetronothiodin 388 Lafurnimas 389 Mica peroxide A 390 Flurazepam hydrochloride 391 Rubiginone C1 392 Clinathol mesylate 393 Dolastatin D 394 Epolactine 395 Lexipaphant CHEMBL database 396 Carbenicillin 397 Diclofop 398 Epiphenicillin 399 Phlobaphene 400 Gigantic Acid 401 Phenethicillin potassium 402 Diphenicillin Database Pubchem 403 Acotiamide 404 Acodiborol 405 Akuma Pimod 406 Apalutamide 407ASP3026 408AZD1480 409 BIIB021 410 Bran Plum 411 Brekinar 412 Chlorproguanil 413 Emrikasan 414 Enasidenib 415 Enolicum 416 Flurazepam 417 ILX295501 418 Indibrin 419 Metoclopramide 420 Mevastatin 421 MK0686 422 Navarixin 423 Nefazodone 424 Pantoprazole 425 Pavinetant 426 SCYX-7158 427 Siccanin 428 Sulfoguanol 429 Sunitinib 430 Suvorexant 431 Chiapride 432 Tonabarsat 433 Urimorelin 434 Sipamid 435 MGGBYMDAPCCKCT-UHFFFAOYSA-N 436 VNBRGSXVFBYQNN-UHFFFAOYSA-N 437 YUHNXUAATAMVKD-PZJWPPBQSA-N

[0201] 9.2.3. Additional Systems for Conditional Heterodimerization: According to the present invention, for example, a pair of dimerization domains for heterodimerization of two CAR molecules of a group of CARs can also be selected from: a) FKBP and FKBP-rapamycin-related protein (FRB, mutant T82L) b) GAI and GID1 c) FKBP and calcineurin catalytic subunit A (CnA) d) FKBP and cyclophilin e) PYL and ABI.

[0202] The sequences of these heterodimerization domains and suitable regulatory molecules for dimerization of these heterodimerization domains are well known to those skilled in the art (Rutkowska et al., Angew Chem Int Ed Engl. 2012;51(33):8166) and are disclosed, for example, in International Publication No. WO2014127261.

[0203] Members of a pair of heterodimerization domains selected from GAI, GID1, FKBP, CnA, cyclophilin, PYL, and ABI can have a length of from about 50 amino acids to about 300 or more amino acids; for example, members of a pair of heterodimerization domains can have a length of from about 50 aa to about 100 aa, from about 100 aa to about 150 aa, from about 150 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to about 300 aa, or more than 300 aa.

[0204] For example, a preferred heterodimerization domain can be derived from FKBP and 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 P62942-1.

[0205] As another example, the heterodimerization domain can be derived from calcineurin catalytic subunit A (also known as PPP3CA; CALN; calla; calla1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin Aα; calmodulin-dependent calcineurin A subunit α isoform; protein phosphatase 2B, catalytic subunit, α isoform, etc.) and 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 Q08209-1 amino acids (aa) 56-347 (PP2Ac domain).

[0206] As another example, the heterodimerization domain can be derived from a 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.

[0207] 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; FLAP; FRAP1; FRAP2; RAFT1; and RAPT1) and 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 P42345-1 aa 2021-2113 (also known as "Frb": Fkbp-Rapamycin Binding Domain).

[0208] As another example, the heterodimerization domain can be derived from a PYL protein (also known as abscisic acid receptor and RCAR) 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 any of 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 Q8VZS8-1); Q9FLB1-1);PYL6(Uniprot Q8S8E3-1);PYL7(Uniprot Q1ECF1-1);PYL8(Uniprot Q9FGM1-1); PYL9(Uniprot Q84MC7-1);PYR1(Uniprot O49686-1).

[0209] As another example, the heterodimerization domain can be derived from the ABI protein (also known as abscisic acid insensitive) and can be derived from proteins such as those of Arabidopsis thaliana: ABI1 (also known as abscisic acid insensitive 1, protein phosphatase 2C56, AtPP2C56, P2C56, and PP2C ABI1) and / or ABI2 (also known as P2C77, protein phosphatase 2C77, AtPP2C77, abscisic acid insensitive 2, protein phosphatase 2C ABI2, PP2C ABI2). For example, suitable heterodimerization domains include those having from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the following amino acid sequences: ABI1 (Uniprot P49597-1); ABI2 (Uniprot O04719-1). The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of aa of

[0210] 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) and has the amino acid sequence: Uniprot Q9LQT8-1 from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 190 aa. It 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 over a contiguous stretch of about 200 aa.

[0211] As another example, the heterodimerization domain can be derived from the GID1 Arabidopsis thaliana protein (also known as the gibberellin receptor GID1) and can be from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, or from about 170 aa to about 180 aa, of any of the amino acid sequences: GID1A (Uniprot Q9MAA7-1); GID1B (Uniprot Q9LYC1-1); GID1C (Uniprot Q940G6-1). It 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 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa.

[0212] Heterodimerization of the heterodimerization domains described in 9.2.3 can be achieved with different regulatory molecules (shown in parentheses 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) GAI and GID1 (gibberellin or gibberellin analog GA-3M).

[0213] As mentioned above, rapamycin (PubChem CID 5284616) can act as a regulatory molecule. Alternatively, rapamycin derivatives or analogs can also be used. See, for example, International Publication Nos. WO96 / 41865; WO99 / 36553; WO01 / 14387; and Ye et al. (1999) Science 283:88-91. For example, analogs, homologs, derivatives, and other compounds structurally related to rapamycin, known as "rapalogs," include, among others, variants of rapamycin having one of the following modifications relative to rapamycin: demethylation, removal, or substitution of methoxy at C7, C42, and / or C29; removal, derivatization, or substitution 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. Further 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 described; see International Publication No. 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).

[0214] Compounds of the following formula are also suitable as rapalogs: [ka] (e.g., as disclosed in U.S. Pat. No. US7067526B1), where n is 1 or 2; R 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, and 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 SO2R A or NR B SO2NR A R B‘ or R 7a and R 7b together are the H's of the tetraene part: [ka] where R A is H or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, and R B and R B’ is independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety.

[0215] 9.3. Extracellular dimerization by secreted soluble factors: Non-covalent complexation of at least two CAR molecules of the CAR group according to the invention can also be induced by secreted soluble factors, such as proteins that accumulate in the tumor stroma, and which are often capable of homo- or heterodimerization themselves, in which case these soluble factors act as regulatory molecules according to the invention. The dimerization domain can then be, for example, a domain of the native 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 (already described above in Chapter 1 "Antigen-binding moieties") engineered to bind to a secreted soluble factor (e.g., a TGF-β1-binding peptide (Dotor et al., Cytokine. 2007;39(2):106-15), or a VEGF-binding CH2-CH3-Fc domain (Lobner et al., MAbs. 2017;9(7):1088-1104), as used as a dimerization domain in Example 7).

[0216] 10.Target antigen: According to a preferred embodiment of the present invention, each antigen-binding portion of the group of CARs and other polypeptides capable of binding to a CAR molecule of said group binds to a target antigen present on a cell, preferably a target antigen present on a cell, a solid surface, or a lipid bilayer.

[0217] According to the present invention, the particular target antigen specifically recognized by the antigen-binding portion of a group of CARs, or alternatively by the antigen-binding portion of another polypeptide capable of binding to a CAR molecule of said group, can be a naturally occurring cell surface antigen or polypeptide, a carbohydrate or lipid bound to a naturally occurring cell surface antigen.

[0218] To maximize binding efficiency, the antigen-binding portions of the CAR group, or alternatively, the antigen-binding portions of other polypeptides capable of binding to the CAR molecules of the group, are preferably directed to different, non-overlapping epitopes on the same target antigen, or to target antigens that naturally form covalent or non-covalent complexes. The advantages of targeting such co-localized epitopes or antigens are demonstrated in Example 6.

[0219] Examples of antigens to which the antigen-binding portion of the CAR group, and the antigen-binding portion of another polypeptide capable of binding to a CAR molecule of the group, can specifically bind 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, 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, 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、 CHRNA3、 CHRNG、 DRD4、 GABRB3、 GRIN3A、 GRIN2C、 GRIK4、 HTR7、 APT8B2、 NKAIN1、 NKAIN4、 CACNA1A、CACNA1B、 CACNA1I、 CACNG8、 CACNG4、 CLCN7、 ​​KCNA4、 KCNG2、 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、TRPV4、TMEM16J、TMEM142B、ADORA2B、BAI1、 EDG6、GPR1、GPR26、GPR34、GPR44、GPR56、GPR68、GPR173、GPR175、LGR4、MMD、NTSR2、OPN3、OR2L2、OSTM1、P2RX3、P2RY8、 P2RY11、 P2RY13, PTGE3, SSTR5, TBXA2R, ADAM22, ADAMTS7, CST11, MMP14, LPPR1, LPPR3, LPPR5, SEMA4A, SEMA6B, ALS2CR4, LEPROTL1, MS4A4A, ROM1, TM4SF5, VANGL1, VANGL2, C18orf1, GSGL1, ITM2A, KIAA1715, LDLRAD3, OZD3, STEAP1, MCAM, CHRNA1, CHRNA3, CHRNA5, CHRNA7, CHRNB4KIAA1524, NRM.3, RPRM, GRM8, KCNH4, melanocortin 1 receptor, PTPRH, SDK1, SCN9A, SORCS1, CLSTN2, endothelin-converting enzyme-like-1, lysophosphoryl 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 1, IL-13R-α2, disialoganglioside (GD2 and GD3), tumor-associated carbohydrate antigens (CA-125, CA-242, Tn and sialyl-Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), -MET, CCR1, CCR4, FAP, fibronectin extra domain B (ED-B), GPNMB, IGF-1 receptor, integrin α5β1, integrin αvβ3, ITB5, ITGAX, enbigin, PDGF-Rα, ROR1, syndecan-1, TAG-72, tenascin-C, TRAIL-R1, TRAIL-R2, and NKG2D-ligands, preferably PR1 / PR2, which are major histocompatibility complex (MHC) molecules that present tumor-specific peptide epitopes. 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.

[0220] 11. Nucleic Acids, Cell Preparation, and Therapeutic Applications: Another aspect of the invention relates to nucleic acids comprising a nucleotide sequence encoding a CAR molecule of the CAR group of the present invention. In some embodiments, the nucleic acids of the present invention are DNA or RNA, including, for example, an expression vector. The nucleic acids of the present invention can also be provided in other forms, such as in the form of a viral vector. The nucleic acids are active or conditionally active in cells and, in some embodiments, can be present as RNA, for example, RNA synthesized in vitro. Introduction of RNA or DNA into host cells can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a CAR molecule of the CAR group.

[0221] In some cases, the nucleic acid of the disclosure comprises a nucleotide sequence encoding a CAR molecule of a group of CARs according to 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 group of CARs consisting of either two, three, or four CAR molecules.

[0222] When the CAR molecules of the group of CARs are encoded by different nucleic acid molecules, the invention provides a kit of at least two nucleic acids encoding 1, 2, 3, or 4 molecules of the group of CARs, where again the nucleic acids are preferably selected from DNA, RNA, or in vitro transcribed RNA.

[0223] The present invention also provides a vector comprising a nucleic acid (i.e. encoding a CAR molecule of the CAR group) and / or a kit of nucleic acids (encoding a CAR molecule of the CAR group) according to the present invention.

[0224] 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. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs according to 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, CA, 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, polyvirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, viral vectors based on retrovirus vectors (e.g., murine leukemia virus, spleen necrosis virus, retrovirus-derived vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, mammary tumor virus), and the like). Due to their ability to efficiently integrate into the genome of transduced cells, preferred vectors are retroviral vectors, particularly gammaretroviral 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 in clinics include, for example, Oxford BioMedica's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX® vector system. Non-clinical lentiviral vectors are also available and 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 (ZFNs) or transcription activator-like effector nucleases (TALENs)) or CRISPR / Cas technology (e.g., as described by 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 integration of defined nucleotide sequences from any DNA molecule (single- or double-stranded DNA; in the form of vectors, PCR amplicons, etc.), and genes of interest can be integrated into the genome downstream of their endogenous promoters (e.g., Eyquem et al., Nature. 2017;543(7643):113-117).

[0225] The present 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 group of CARs according to the present invention. The vectors can be provided with 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).

[0226] 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 generate 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. For expression in bacterial cells, suitable promoters include lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters 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 retroviral long terminal repeats (e.g., promoter sequences containing the R and U3 subelements of the 5'-LTR of gammaretroviruses or the 5'-LTR of Moloney murine leukemia virus (MMLV)), promoters present in murine stem cell virus (MSCV), the mouse metallothionein-I promoter, EF1-α with or without an intron, the phosphoglycerate kinase (PGK) promoter, 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. 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 well known in the art.Such reversible promoters, and systems based on such reversible promoters but which also include 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 (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90), soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0227] In some cases, a locus, construct, or transgene containing an appropriate promoter can be irreversibly switched via induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art; for example, induction of irreversible switches can utilize Cre-lox-mediated recombination. Any suitable combination of recombinases, endonucleases, ligases, recombination sites, and the like known in the art can be used to generate irreversibly switchable promoters. Methods, mechanisms, and requirements for site-specific recombination are described elsewhere herein and are used to generate 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 an NK cell-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 through the use of the Neri (p46) promoter. In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, AD1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of an appropriate vector and promoter is well within the level of ordinary skill in the art.Promoters suitable 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, and the like; the araBAD promoter; in vivo-regulated promoters, such as the ssaG promoter or related promoters, the pagC promoter, the nirB promoter, and the like; sigma70 promoters, such as the consensus sigma70 promoter (see, for example, Genbank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters, such as the dps promoter, the spv promoter, and the like; promoters from the pathogenicity island SPI-2; the actA promoter; the rpsM promoter; the tet promoter; the SP6 promoter; and the like. Strong promoters suitable 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 (when contacted with lactose, the Laci repressor protein changes structure, 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.

[0228] According to a preferred embodiment of the present invention, the vector or kit of at least two vectors comprises a T lymphocyte-specific promoter or an NK cell-specific promoter or an EF1-α promoter operably linked to a nucleotide sequence encoding a CAR molecule of the CAR group.

[0229] According to a further aspect, the present invention also relates to genetically modified cells that have been modified to produce all CAR molecules of the group of CARs according to the present invention. The cells of the present invention can also be used to produce the vectors of the present invention (e.g., as virus or plasmid supernatants), from which they can be further purified to provide these vectors in amplified and purified form.

[0230] According to a preferred embodiment, the cells are mammalian cells genetically modified to produce CAR molecules of the CAR group according to the present invention. Preferred mammalian cells are stem cells, progenitor cells, or cells derived from stem cells or progenitor cells, preferably lymphocytes. Further 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 may be suitable cell types, such as non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc., particularly for addressing scientific questions using the system according to the present invention.

[0231] Further preferred cells according to the present invention may be: 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 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, such as Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, YTS), etc. In some preferred embodiments, the cells according to the present invention are not immortalized cell lines, but rather are cells (e.g., primary cells) obtained from an individual. For example, in some cases, the cells are immune cells obtained from an individual. Illustratively, 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.

[0232] According to a particularly preferred embodiment, the mammalian cells according to the invention transformed with a vector or kit of at least two vectors encoding individual CAR molecules of the group of CARs according to the invention are T cells or NK cells.

[0233] In a further aspect, the present invention relates to 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.

[0234] The present disclosure provides methods for generating conditionally activatable cells. The methods generally involve genetically modifying mammalian cells with a vector or kit of vectors, or RNA (e.g., in vitro-transcribed RNA) containing a nucleotide sequence encoding a conditionally active group of CARs according to the present disclosure. In a preferred embodiment, the genetically modified cells are conditionally activated in the presence of a) a target antigen bound by the antigen-binding portion of the CAR group or a naturally complexed target antigen, and b) at least one regulatory molecule. Optionally, the genetically modified cells are activatable in the presence of a target antigen bound by the antigen-binding portion of the CAR group or a naturally complexed target antigen, and are difficult to activate in the additional presence of at least one regulatory molecule. Genetic modification can be performed in vivo, in vitro, or ex vivo. The cells can be immune cells (e.g., T lymphocytes or NK cells), stem cells, progenitor cells, etc.

[0235] Preferably, genetic modification is 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 a group of CARs according to the present disclosure. According to a preferred embodiment, the genetically modified cells are conditionally activatable in the presence of: a) a target antigen or a naturally complexed target antigen to which the antigen-binding portion of the CARs or the antigen-binding portion of another polypeptide capable of binding to the CARs binds; or b) at least one regulatory molecule. In some cases, the genetically modified cells are activated ex vivo, for example, during the cell expansion process prior to administration. When the genetically modified cells are introduced into an individual (e.g., the individual from whom the cells were obtained), the genetically modified cells can be activated in vivo, for example, by administering at least one regulatory molecule to the individual, provided that the target antigen of each of the antigen-binding portions is present at a physiological expression level on the cell surface of the individual. The genetically modified cells are contacted with a target antigen that is present at a physiological expression level on the cell surface of an individual; and according to a preferred embodiment, administering at least one regulatory molecule to the individual activates the genetically modified cells. Optionally, in embodiments in which the complex formation of the CAR group can be inhibited by the presence of a regulatory molecule, the genetically modified cells can be less likely to be activated after administering at least one regulatory molecule to the individual.

[0236] For example, if the genetically modified cells are T lymphocytes or NK cells, upon activation, the CAR group (or antigen-binding portion of other polypeptides) can bind on their surface.

[0237] The present disclosure provides various therapeutic methods using subject populations of CARs.

[0238] Non-covalently conjugated CARs according to the invention, when present on T lymphocytes or NK cells, can mediate cytotoxicity against target cells. Non-covalently conjugated CARs according to the invention can bind to a selected target antigen or a selected conjugate of a naturally conjugated target antigen present on a target cell, optionally depending on the presence of other polypeptides that comprise at least an antigen-binding portion and can bind to the CAR molecule, thereby mediating target cell killing by T lymphocytes or NK cells genetically engineered to generate the CARs, according to preferred embodiments.

[0239] Target cells include cancer cells. Accordingly, the present disclosure provides a method of killing or inhibiting the growth of target cancer cells, the method comprising contacting the target cancer cells with cytotoxic immune effector cells (e.g., cytotoxic T cells or NK cells) that have been genetically engineered to generate a targeted population of CARs such that the T lymphocytes or NK cells recognize the target, such that the T lymphocytes or NK cells recognize a target antigen or target antigen complex present on the surface of the target cancer cells and mediate killing of the target cells.

[0240] The present disclosure provides a method for treating cancer in an individual having cancer. According to a preferred embodiment, the method includes: 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 a group of CARs according to the present invention, wherein each antigen-binding portion of the group of CARs is specific for a target antigen on the individual's cancer cells, and said genetic modification is performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual; and iii) introducing at least one CAR molecule to induce or reduce dimerization of each CAR molecule of the group, preferably to induce dimerization of each CAR molecule of the group. The method includes administering to an individual an effective amount of one regulatory molecule, thereby inducing or reducing non-covalent complex formation of the group of CARs, preferably inducing non-covalent complex formation of the group of CARs, whereby upon contact with cancer cells expressing the respective target antigens or respective covalent or non-covalent complexes of different target antigens, the non-covalent complexes of CARs mediate activation of the genetically modified cells and kill the cancer cells, allowing for the treatment of cancer.

[0241] Carcinomas amenable to treatment by the methods disclosed herein include: esophageal carcinoma, hepatocellular carcinoma, bladder carcinoma including basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), transitional cell carcinoma (a malignant neoplasm of the bladder), bronchial carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical 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, ductal 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 amenable to treatment with the methods disclosed herein include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial sarcoma, mesothelial sarcoma, Ewing's sarcoma, soft tissue sarcoma, and other sarcomas. Other solid tumors amenable to treatment with the methods disclosed herein include: glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Leukemias amenable to treatment with 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 limited lineage potential); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using the present methods include B-cell lymphomas (e.g., Burkitt lymphoma), Hodgkin lymphoma, non-Hodgkin lymphoma, and the like. Other cancers amenable to treatment 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.

[0242] The method can also be used to treat inflammatory conditions and autoimmune diseases. The target group of CARs can be expressed in helper T cells or regulatory T (Treg) cells for use in immunomodulatory methods. Immunomodulatory methods include, for example, enhancing the immune response in a mammalian subject to pathogens; enhancing the immune response in an immunocompromised subject; reducing inflammatory responses; reducing the immune response of a mammalian subject to autoantigens, for example, to treat autoimmune diseases; and reducing the immune response of a mammalian subject to transplanted organs or tissues, for reducing organ or tissue rejection. When the method involves reducing the immune response to autoantigens, it is preferred that at least one of the target antigens used to activate the group of CARs is an autoantigen. When the method involves reducing the immune response to a transplanted organ or tissue, it is preferred that at least one of the antigens used to activate the group of CARs is an antigen specific to the transplanted organ.

[0243] As noted above, the therapeutic methods of the present disclosure comprise administering to an individual in need thereof an effective amount of one or more different regulatory molecules and optionally one or more different other polypeptides, each of which comprises an antigen-binding portion, wherein each of the other polypeptides comprises an antigen-binding portion and is capable of binding to the extracellular binding site of a CAR molecule of a group of CARs.

[0244] The required effective amount of each regulatory molecule to be administered to an individual in need thereof who has received T lymphocytes or NK cells ("effector cells") expressing a group of CARs according to the invention is defined by the differential response of those effector cells upon contact with antigen-expressing target cells in the presence versus absence of each required regulatory molecule. Thus, the response of these effector cells is defined by interferon-gamma, and / or macrophage inflammatory protein-1 (MIP-1)α, and / or MIP-1β, and / or granzyme B, and / or IL-2, and / or TNF, and / or IL-10, and / or IL-4, and / or effector cell degranulation, where cellular degranulation is preferably detected by the percentage of effector cells that translocate CD107a to their surface after contact with target cells expressing more than 100,000 target antigen molecules or target antigen complexes per cell, optionally in the presence of an effective concentration of each of the necessary other polypeptides that contain at least an antigen-binding portion and are capable of binding to the binding site of a CAR molecule of the CAR group (i.e., the percentage of CD107a-positive effector cells detected by flow cytometry analysis using a degranulation assay) (e.g., Proff et al., Front Micro-Biol. 2016;7:844). According to preferred embodiments, the response of effector cells in the presence versus absence of an effective concentration of each required regulatory molecule differs by at least 20%, preferably at least 50%, or even more preferably at least 100%, wherein the effective concentration of each required regulatory molecule is the concentration achieved by administering an effective amount of each required regulatory molecule one or more times to an individual in need thereof.The effective concentration of each of the required other polypeptides that comprise at least an antigen-binding portion and are capable of binding to the population of CARs is defined by the response of the fully complexed target population of CARs (i.e., all dimerization domains included in the population of CARs) after contact with target cells expressing greater than 100,000 target antigen molecules or target antigen complexes per cell, in the presence and absence of the required other polypeptides that comprise at least one antigen-binding portion and are capable of binding to the population of CARs, where the response preferably differs by at least 20%, preferably at least 50%, or even more preferably at least 100%, and the effective concentration of each of the required other polypeptides that comprise at least an antigen-binding portion and are capable of binding to the population of CARs is the concentration achieved by administering an effective amount of each of those other polypeptides in one or more doses to an individual in need thereof that is receiving T lymphocytes or NK cells expressing the target population of CARs.

[0245] Both regulatory molecules and antigen-specific other polypeptides capable of binding to CAR molecules of the CAR group according to the present invention are hereinafter collectively referred to as "agents that specifically bind to the CAR group."

[0246] In this method, the "agent that specifically binds to the CAR group" can be administered to a host using any convenient means capable of producing the desired therapeutic or diagnostic effect. Accordingly, 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 it with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into a solid, semi-solid, liquid, or gaseous formulation. It can be formulated into a solid, semi-solid, liquid, or gaseous formulation, such as a tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, or aerosol. In pharmaceutical dosage forms, the "agent that specifically binds to the CAR group" can be administered in the form of a pharmaceutically acceptable salt, or they can be used alone or in appropriate combinations, or in combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary.

[0247] Suitable excipient vehicles can be, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, if necessary, the vehicle can contain minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, or pH buffering agents. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. In any case, the composition or formulation to be administered will contain the necessary "agent that specifically binds to the CAR group" in an amount sufficient to achieve the desired state in the treated subject. Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc., are readily available to the public. In the case of oral preparations, the "agent that specifically binds to the CAR group" can be used alone or in combination with the following suitable additives to prepare tablets, powders, granules, or capsules, for example, conventional additives such as lactose, mannitol, corn starch, or 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 or magnesium stearate; and, if necessary, diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0248] The "agent that specifically binds to the CAR group" can be made into an injectable preparation by dissolving, suspending, or emulsifying it in the following aqueous or non-aqueous solvents: vegetable oil or other similar oil, synthetic fatty acid glyceride, ester of higher fatty acid, or propylene glycol; and, if necessary, conventional additives such as solubilizers, isotonicity adjusting agents, suspending agents, emulsifiers, stabilizers, and preservatives can be added.

[0249] Pharmaceutical compositions containing "agents that specifically bind to the CAR group" can be prepared by mixing "agents that specifically bind to the CAR group" having the desired purity with any physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, excipients, and / or stabilizers are preferably non-toxic to recipients at the dosages and concentrations used, and 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, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants, such as Tween®, Brij Pluronics®, Triton®-X, or polyethylene glycol (PEG).

[0250] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, where the lyophilized formulation must be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is usually to return it to a volume of pure water (usually equivalent to the volume removed during lyophilization); however, solutions containing antibacterial agents can be used to prepare pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0251] The "agent that specifically binds to a group of CAR" can also be optionally formulated in a sustained-release formulation. Sustained-release formulations can be prepared using methods well known in the art. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the "agent that specifically binds to a group of CAR," where the matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity can be prevented by using appropriate additives, controlling the water content, and developing specific polymer matrix compositions.

[0252] 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 field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the particular "agent that specifically binds to a group of CARs" administered, the patient's gender, the time and route of administration, general health, and other medications being administered concomitantly. The "agent that specifically binds to a group of CARs" may be administered in an amount of 1 ng / kg body weight to 20 mg / kg body weight, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight per dose; however, doses lower or higher than this exemplary range are contemplated, particularly considering the aforementioned factors. If the administration regimen is a continuous infusion, the range may be 1 μg to 10 mg per kg body weight per minute.

[0253] Those skilled in the art will readily appreciate that dosage levels can vary as a function of the particular "agent that specifically binds to a group of CARs," the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound can be readily determined by those of skill in the art by a variety of means.

[0254] One or more "agents that specifically bind to CAR groups" 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 routes of administration. Conventional pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration can be combined or tailored as needed, depending on the "agents that specifically bind to CAR groups" and / or the desired effect. "Agents that specifically bind to CAR groups" can be administered in a single dose or multiple doses. According to preferred embodiments, "agents that specifically bind to CAR groups" can be administered orally or intravenously. According to other embodiments, "agents that specifically bind to CAR groups" can be administered via the inhalation route. According to still other embodiments, "agents that specifically bind to CAR groups" can also be administered intranasally, topically, or intratumorally. According to yet other embodiments, the "agent that specifically binds to a group of CARs" can be administered peritumorally. According to other embodiments for the treatment of brain tumors, the "agent that specifically binds to a group of CARs" can be administered intracranially.

[0255] The "agent that specifically binds to the CAR group" can be administered to a host using available conventional methods and routes suitable for conventional drug delivery, including systemic or local routes. Generally, administration routes contemplated by the present invention include enteral, parenteral, or inhalation routes. Parenteral administration routes other than inhalation administration include topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes. Parenteral administration can be used to achieve systemic or local delivery of the "agent that specifically binds to the CAR group." When systemic delivery is desired, administration typically involves the administration of invasive or systemically absorbed topical or mucosal formulations. The "agent that specifically binds to the CAR group" can also be delivered to a subject by enteral administration. Enteral administration routes include oral and rectal (e.g., using a suppository) delivery.

[0256] Treatment refers to at least an amelioration of symptoms associated with a pathological condition afflicting a host, where amelioration is used in a broad sense to refer to a decrease in at least the parameters, e.g., the magnitude of symptoms, associated with the pathological condition being treated, e.g., cancer, etc. Treatment therefore also includes situations in which the pathological condition, or at least the symptoms associated therewith, are completely inhibited, e.g., prevented from occurring, or arrested, e.g., terminated, such that the host is no longer afflicted with the pathological condition, or at least the symptoms that characterize the pathological condition.

[0257] "Agents that specifically bind to a group of CARs" can be administered by injection and / or delivery, for example, to a site within a cerebral artery or directly to brain tissue. "Agents that specifically bind to a group of CARs" can also be administered directly to a 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, etc. Additionally, "agents that specifically bind to a group of CARs" can be administered as adjunctive 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 modification treatment, and certain combinations thereof.

[0258] A variety of subjects are suitable for treatment with a subject method of treating cancer. Suitable subjects include, for example, individuals, e.g., human or non-human animals, who have cancer, have been diagnosed with cancer, are at risk for developing cancer, have cancer and are at risk for cancer recurrence, have been treated with other therapies and have failed to respond to such treatment, or have relapsed after an initial response to such treatment.

[0259] Subjects suitable for treatment with the present immunomodulatory methods include individuals with autoimmune diseases; individuals who are organ or tissue transplant recipients; immunocompromised individuals; and individuals infected with a pathogen. [Brief explanation of the drawings]

[0260] [Figure 1-1] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0261] [Figure 1-2] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0262] [Figure 1-3] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0263] [Figure 1-4] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0264] [Figure 1-5] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0265] [Figure 1-6] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0266] [Figure 1-7] FIG. 1 shows a schematic diagram of an exemplary architecture of a group of CARs.

[0267] [Figure 2] FIG. 2 shows the Kd values ​​of various rcSso7d-based antigen-binding moieties against human EGFR.

[0268] [Figure 3-1] FIG. 3 shows that extracellular disulfide bond forming cysteines can prevent the utilization of avidity effects for the modulation of CAR function.

[0269] [Figure 3-2] FIG. 3 shows that extracellular disulfide bond forming cysteines can prevent the utilization of avidity effects for the modulation of CAR function.

[0270] [Figure 4-1]Figure 4 shows that dimerization / oligomerization of scFv-containing CAR molecules prevents the exploitation of avidity effects for modulation of CAR function.

[0271] [Figure 4-2] Figure 4 shows that dimerization / oligomerization of scFv-containing CAR molecules prevents the exploitation of avidity effects for modulation of CAR function.

[0272] [Figure 4-3] Figure 4 shows that dimerization / oligomerization of scFv-containing CAR molecules prevents the exploitation of avidity effects for modulation of CAR function.

[0273] [Figure 5-1] FIG. 5 shows modulation of CAR function by modulating the avidity of a group of CARs through conditional homodimerization.

[0274] [Figure 5-2] FIG. 5 shows modulation of CAR function by modulating the avidity of a group of CARs through conditional homodimerization.

[0275] [Figure 6] FIG. 6 shows the modulation of function of stably transduced T cells expressing a panel of CARs in vivo.

[0276] [Figure 7-1] Figure 7 shows the functional in vitro characteristics of CAR-modified T cells used in in vivo experiments.

[0277] [Figure 7-2] Figure 7 shows the functional in vitro characteristics of CAR-modified T cells used in in vivo experiments.

[0278] [Figure 7-3] Figure 7 shows the functional in vitro characteristics of CAR-modified T cells used in in vivo experiments.

[0279] [Figure 8-1] Figure 8 shows the avidity of CAR groups through heterodimerization, modulating the sensitivity of CAR T cells depending on whether the target antigen is a monomer or dimer.

[0280] [Figure 8-2] Figure 8 shows the avidity of CAR groups through heterodimerization, modulating the sensitivity of CAR T cells depending on whether the target antigen is a monomer or dimer.

[0281] [Figure 8-3] Figure 8 shows the avidity of CAR groups through heterodimerization, modulating the sensitivity of CAR T cells depending on whether the target antigen is a monomer or dimer.

[0282] [Figure 8-4] Figure 8 shows the avidity of CAR groups through heterodimerization, modulating the sensitivity of CAR T cells depending on whether the target antigen is a monomer or dimer.

[0283] [Figure 9-1] FIG. 9 shows the regulation of avidity of a group of CARs by VEGF.

[0284] [Figure 9-2] FIG. 9 shows the regulation of avidity of a group of CARs by VEGF.

[0285] [Figure 10-1] FIG. 10 shows the generation and function of affibody-based CARs against HER2.

[0286] [Figure 10-2] FIG. 10 shows the generation and function of affibody-based CARs against HER2.

[0287] [Figure 10-3] FIG. 10 shows the generation and function of affibody-based CARs against HER2.

[0288] [Figure 11-1] FIG. 11 shows a group of CARs consisting of 3 or 4 CAR molecules.

[0289] [Figure 11-2] FIG. 11 shows a group of CARs consisting of 3 or 4 CAR molecules.

[0290] [Figure 12-1] Figure 12 shows a group of CARs containing different costimulatory domains.

[0291] [Figure 12-2] Figure 12 shows a group of CARs containing different costimulatory domains.

[0292] [Figure 13-1] FIG. 13 shows the expression of CAR molecules containing different rcSso7d and affibody-based binding moieties fused to different CAR signaling backbones.

[0293] [Figure 13-2] FIG. 13 shows the expression of CAR molecules containing different rcSso7d and affibody-based binding moieties fused to different CAR signaling backbones.

[0294] [Figure 14-1] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0295] [Figure 14-2] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0296] [Figure 14-3] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0297] [Figure 14-4] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0298] [Figure 14-5] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0299] [Figure 14-6] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0300] [Figure 14-7] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0301] [Figure 14-8] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0302] [Figure 14-9] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0303] [Figure 14-10] FIG. 14 shows a schematic diagram of the design of different CAR molecules.

[0304] [Figure 15-1] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0305] [Figure 15-2] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0306] [Figure 15-3] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0307] [Figure 15-4] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0308] [Figure 15-5] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0309] [Figure 15-6] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0310] [Figure 15-7] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0311] [Figure 15-8] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0312] [Figure 15-9] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0313] [Figure 15-10] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0314] [Figure 15-11] FIG. 15 shows the amino acid sequences of the different CAR molecules.

[0315] [Figure 15-12] FIG. 15 shows the amino acid sequences of the different CAR molecules. [Example]

[0316] Figure 1: Schematic representation of a typical architecture of a CAR group. Figure 1A is a schematic representation of the basic structure of a CAR molecule in the CAR group herein, in which an antigen-binding moiety is incorporated into the CAR molecule (left), and the CAR molecule contains a binding site that binds to the binding site of another polypeptide containing the antigen-binding moiety (right). A low-affinity interaction occurs 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 CAR molecule in the group must contain at least one signaling region containing either at least one ITAM or at least one ITIM. In the example CAR molecule shown, the endodomain illustratively contains a single signaling region. Lines between components of the illustrated CAR molecule indicate optional linkers. Dimerization domains (at least one required for each CAR molecule in the group) and optional additional domains or components are not shown. Figure IB is a schematic diagram showing the number of CAR molecules that may contain at least one signaling region in a group of 2, 3, or 4 CAR molecules (the entire signaling region of a given CAR molecule is represented by a white box). Of the CAR molecules that contain at least one signaling region, at least one, but only some, CAR molecule contains at least one ITAM or at least one ITIM. For brevity, the ectodomain and dimerization domain, as well as optional additional domains or components, are not shown. Lines between components of the illustrated CAR molecules indicate optional linkers. Figure 1C is a schematic diagram showing the arrangement of signaling regions using a group of CARs consisting of two CAR molecules as an example. The illustrated example shows only some of the possible combinations of various arrangements. For example, a CAR molecule may contain two or more ITAM-containing signaling regions or two or more costimulatory signaling regions. In an inhibitory group of CARs (not shown), the ITAM-containing signaling region is replaced by an ITIM-containing signaling region, and the costimulatory signaling region is omitted or replaced by another inhibitory domain. For simplicity, the ectodomain and dimerization domain, as well as optional additional domains or components, are not shown. Lines between components of the illustrated CAR molecules indicate optional linkers. Figures 1D-1N are schematic diagrams showing how dimerization domains can be used for non-covalent complex formation of CARs. The illustrated examples show only some of the possible configurations. In the illustrated examples, different pairs of homodimerization and heterodimerization domains are shown at different locations on the CAR molecule, each illustratively containing one or two signaling domains. In a preferred example (Figure 1E, left), a group of three CAR molecules contains only two members of a single pair of heterodimerization domains and can accordingly be controlled by a single type of regulatory molecule. For simplicity, only the signaling domain, transmembrane domain, and dimerization domain are illustrated. Lines between some components of the illustrated CAR molecules indicate optional linkers. Similar configurations of dimerization domains can also be incorporated extracellularly. Figure 10 illustrates the non-covalent complex formation of CARs with extracellular soluble factors that act as regulatory molecules. The regulatory molecules are illustrated schematically using monomeric proteins and proteins that naturally homodimerize or heterodimerize. The illustrated CAR molecule illustratively contains only one signaling region. Optional additional dimerization domains and optional additional domains or components are not shown. The order of the antigen-binding domain (or binding site) and dimerization domain may be reversed. That is, the antigen-binding domain (or binding site) may be closer to the plasma membrane than the dimerization domain. The lines between the components of the illustrated CAR molecule indicate optional linkers.

[0317] Figure 2 shows the K of various rcSso7d-based antigen-binding moieties (fused to superfolder GFP (sfGFP)) against human EGFR, determined by three complementary methods. d Figure 1 shows values ​​for (a) flow cytometric quantification of the amount of various sfGFP fusion proteins bound to Jurkat T cells expressing high levels of truncated EGFR (tEGFR). (b) and (c) surface plasmon resonance (SPR) analysis using matrices coated with a chimeric EGFR protein containing the extracellular domain of EGFR fused to the Fc domain of IgG1. Affinity was determined by either (b) kinetic analysis or (c) steady-state analysis.

[0318] Figure 3: Cysteine-forming extracellular disulfide bonds prevent the use of avidity effects to control CAR function. (A) Schematic showing whether the architectures of the CAR signaling scaffolds S-8cys-BB-3z ("Cys") and S-8ser-BB-3z ("Ser") can form disulfide bonds. These signaling scaffolds were fused to various rcSso7d-based antigen-binding moieties and expressed for functional testing in primary human T cells. (B) Typical CAR expression 20 hours after electroporation of 5 μg of mRNA in primary human T cells (shown for the rcSso7d variant "E11.4.1-WT" fused to either the "Cys" or "Ser" CAR scaffold). T cells not expressing the transgene ("No CAR") were used as a negative control. (C) Expression of tEGFR in Jurkat cells used as target cells 20 hours after electroporation with 3 μg of tEGFR-encoding mRNA. Jurkat T cells without the construct ("No Construct") and the respective isotype control ("Isotype Control") were used as negative controls. CAR function was tested by measuring the ability of primary human T cells modified with various 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 for the indicated CAR constructs and the following day cultured with Jurkat T cells (electroporated with 3 μg of tEGFR-mRNA) at a 2:1 effector:target (E:T) ratio for 4 hours at 37°C. T cells without the CAR ("No CAR") were used as a negative control.

[0319] Figure 4: Dimerization / multimerization of scFv-containing CAR molecules prevents the use of avidity effects to control CAR function. (A) Schematic of the CAR used in the experiment. (B, C, and D) Expression of CAR constructs in human primary T cells 20 hours after electroporation of 5 μg of each mRNA. T cells without a CAR ("No CAR") were used as a negative control. (E) Expression of tHER2 in Jurkat cells used as target cells 20 hours after electroporation of 5 μg of tEGFR-encoding mRNA. Jurkat T cells without the construct ("No Construct") were used as a negative control. CAR T cells were co-cultured with Jurkat-tHER2 cells at an E:T ratio of 2:1 for 4 hours at 37°C. Figures 4F and 4G show the ability of CAR, scFv 4D5-5, to induce cytotoxicity (G) and IFN-γ production (F) when fused to two different CAR signaling scaffolds (disulfide bond formation capable or incapable, i.e., "Cys" for 4D5-5-8cys-BB-3z (SEQ ID NO: 59) and "Ser" for 4D5-5-8ser-BB-3z (SEQ ID NO: 60)). 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: 56 and SEQ ID NO: 57) is shown in (H). Primary T cells expressing CAR 4D5-5-8ser-BB-3z (SEQ ID NO: 60), in which a monomeric signaling scaffold is fused to 4D5-5-scFv, were used as a positive control. CAR T cells from three different donors (indicated by different symbols) were incubated with a mixture of tHER2-transfected and non-transfected Jurkat T cells at an E:T ratio of 4:1:1 (T cells:tHER2) for 4 hours at 37°C. pos Jurkat cells: tHER2 neg Jurkat cells) and viable HER2 pos and tHER2 negThe proportion of target cells was determined by flow cytometry. To induce dimerization, the T cells were pretreated with the dimerizer AP20187 (10 nM, 30 min, 37°C). Treatment with the same concentration of DMSO served as a control condition. T cells without CAR ("No CAR") and V H T cells expressing only the 4D5-5 chain (V H )-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 57)) was used as a negative control.

[0320] Figure 5: Control of CAR binding activity by homodimerization of CAR molecules. (A) Schematic diagram of CARs conjugated with the regulatory molecule AP20187. In the illustrated example, an rcSso7d-based antigen-binding moiety against EGFR was used as the antigen-binding moiety. (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") were used as a negative control. (C) Expression of EGFR in the target cell line "A431-fLuc." Unstained cells ("Unstained") and the respective isotype control ("Isotype Control") were used as negative controls. (D) Ability of primary human T cells transduced with different CAR constructs to kill the luciferase-expressing target cell line A431-fLuc. Primary T cells from three individual donors (indicated by different symbols) were stably transduced with vectors encoding either the rcSso7d-based CARs S(WT)-8ser-BB-FKBP(36V)-3z ("E11.4.1-WT") and S(32)-8ser-BB-FKBP(36V)-3z ("E11.4.1-G32A") against CD19 (CD19-8cys-BB-3z "CD19-BBz" (SEQ ID NO: 58)) or scFv-based CARs, which were used as negative controls. T cells without CAR ("No CAR") were used as an additional negative control. AP20187 was used as a control molecule for noncovalent 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 pretreatment of T cells with 10 nM AP20187 for 30 minutes at 37°C. Treatment with the same concentration of DMSO was used as a control condition.The cytotoxicity of modified T cells was determined by quantifying viable luciferase expressing A431-fLuc target cells after 4 h of co-culture at 37°C at an E:T ratio of 10:1.

[0321] Figure 6: Regulation of the function of stably transduced CAR T cells in vivo in the NSG mouse model. (A) Efficacy of dimerization induced CAR activation in the in vivo NSG mouse model. Nine to 16-week-old NSG mice were stably transduced with vectors encoding luciferase and tEGFR, 0.5 x 10 6 Three days later, the NSG mice were intravenously injected with 10 x 10 Nalm6 cells ("Nalm6-tEGFR-fLuc"). 6 NSG mice were treated with intravenous administration of CAR T cells (14 days after activation with anti-CD3 / CD28 antibody-coated beads, i.e., 13 days after stable transduction). Phosphate-buffered saline (PBS) injection was used as a 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 (injection days indicated by arrows). Groups that did not receive the dimerizer received the respective vehicle solution intraperitoneally as a control condition. Tumor size (average for each treatment group) is shown as total photon flux measured within the region of interest encompassing the entire body of NSG mice. (B) Delaying administration of dimerizer AP20187 to mice treated with S(32)-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 46) CAR T cells but not administered until day 11 results in efficient CAR activation and modulation of tumor growth.

[0322] Figure 7: Regulation of in vitro function of stably transduced CAR T cells. (A) 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") were used as a negative control. (B) Expression of anti-CD19 CAR CD19-8cys-BB-3z "CD19-BBz" (SEQ ID NO: 58) 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") were used as a negative control. (C and D) Expression of tEGFR in Nalm6-fLuc and Nalm6-tEGFR-fLuc cells, respectively. Unstained cells and the respective isotype controls were used as negative controls. (E and F) Lysis of Nalm6-fLuc and Nalm6-tEGFR-fLuc cells by primary human T cells expressing different CAR molecules. Primary T cells from three individual donors (indicated by different symbols) were stably transduced with vectors encoding either the rcSso7d-based CARs S(WT)-8ser-BB-FKBP(36V)-3z (E11.4.1-WT) and S(G32A)-8ser-BB-FKBP(36V)-3z (E11.4.1-G32A) or scFv-based CARs directed against CD19 (CD19-8cys-BB-3z "CD19-BBz") and were used as negative controls. T cells without a CAR ("No CAR") were used as negative controls. AP20187 was used as a control molecule for noncovalent 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 pretreatment of T cells with 10 nM AP20187 for 30 min at 37°C.Treatment with DMSO at the same concentration was used as a control condition. The cytotoxicity of modified T cells was determined by quantifying viable luciferase expressing target cells after 4 h of co-culture at 37°C at an E:T ratio of 10:1.

[0323] Figure 8: Control of CAR binding activity by heterodimerization of CAR molecules and the effect of colocalization of target antigens on CAR T cell sensitivity. (A) Illustrates the expression of tEGFR fusion protein ("FKBP F36V", conditional homodimerization) in Jurkat cells used as target cells 20 hours after electroporation of 5 μg of each mRNA. Unstained cells ("mock") and the respective isotype control ("isotype") were used as negative controls. (B and C) Correlation of tEGFR expression levels in Jurkat T cells electroporated with different amounts of each mRNA. (D and E) Expression of different CARs in primary human T cells 20 hours after electroporation of 5 μg of each mRNA. For conditional heterodimerization with the regulatory molecule AP21967, high-affinity and low-affinity rcSso7d variants ("E11.4.1-WT" and "E11.4.1.-G32A," respectively) were fused to both the 8ser-BB-FKBP-3z and 8ser-BB-FRB-3z backbones. Expression of the resulting constructs (S(G32A)-8ser-BB-FKBP-3z (SEQ ID NO: 48), S(G32A)-8ser-BB-FRB-3z (SEQ ID NO: 47), S(WT)-8ser-BB-FKBP-3z (SEQ ID NO: 50), and S(WT)-8ser-FRB-3z (SEQ ID NO: 51)) was detected via their incorporated tags (Strep II for 8ser-BB-FRB-3z and FLAG for 8ser-BB-FKBP-3z). (F) Molecular structure of the experimental system used to measure the sensitivity of dimerization-induced activation of CAR T cells in response to target cells expressing monomeric or dimeric target antigen tEGFR. In this system, CAR molecules were conditionally heterodimerized with the control molecule AP21967, and the target antigen tEGFR was conditionally homodimerized with AP20187. The ability of CARs to induce cytotoxicity and IFN-γ production in primary human T cells, respectively, depending on both the number of tEGFR molecules expressed on the target cells and the presence or absence of AP21967 and AP20187 (mean ± standard deviation, n = 3, three different donors) is shown in (G) and (H).Primary T cells from three individual donors were electroporated with 5 μg of mRNA for each CAR molecule (i.e., the low-affinity construct S(G32A)-8ser-BB-FKBP-3z plus S(G32A)-ser8-BB-FRB-3z or the high-affinity construct S(WT)-8ser-BB-FKBP-3z plus S(WT)-8ser-BB-FRB-3z, as shown) and cocultured with Jurkat T cells expressing various amounts of tEGFR. Cocultures were performed at an E:T ratio of 4:1:1 (T cells:tEGFR). pos Jurkat cells: tEGFR neg The cytotoxicity of CAR T cells was measured using 1000-kDa tEGFR-1000 cells (Jurkat cells) at 37°C for 4 hours. pos Target cells and tEGFR neg The proportion of target cells was determined by quantification by flow cytometry. Primary human T cells were pretreated with AP21967 (500 nM, 30 min, 37°C) to induce CAR dimerization, and Jurkat T cells were pretreated with AP20187 (10 nM, 30 min, 37°C) to induce EGFR dimerization. Treatment with the same concentration of ethanol (for AP21967) or DMSO (for AP20187) was used as a control condition.

[0324] Figure 9: Generation of CARs that can be controlled by VEGF, an example of an extracellular factor that accumulates in the tumor microenvironment. In the illustrated example, the soluble factor VEGF was used as the control molecule, and the EGFR-specific rcSso7d-based binder E11.4.1-G32A was used as the antigen-binding moiety. Schematic diagrams of the architecture of each CAR 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). An anti-Strep II tag antibody was additionally used to detect CAR molecules containing a VEGF-binding site (Janus-CT6-Fc domain without a transmembrane domain) and a control CAR without an IgG-Fc domain. Cytotoxicity induced by the various CARs in primary T cells was measured using a FACS-based cytotoxicity assay (E). CAR T cells from three different donors (indicated by different symbols) were incubated with tEGFR-transfected Jurkat cells at an E:T ratio of 4:1:1 (T cells:tEGFR) for 4 hours at 37°C. pos Jurkat cells: tEGFR neg T cells were co-cultured with VEGF-positive Jurkat cells. CAR dimerization was induced by pre-treating T cells with VEGF (concentrations as indicated, 30 min, 37°C). T cells without CAR ("No CAR") were used as a negative control, and T cells containing CAR S(WT)-8ser-BB-FKBP(36V)-3z were used as a positive control.

[0325] Figure 10: Screening of affibody-based binding moieties suitable for use in a panel of CARs according to the present invention. The affinity of the affibody zHER2-WT was reduced by substituting all amino acids potentially involved in epitope binding with alanine. Various variants of zHER2-WT were fused to 8ser-BB-FKBP(36V)-3z, a CAR signaling scaffold containing the intracellular homodimerization domain FKBP(F36V) for conditional dimerization. The architecture of these CARs is shown in (A). (B and C) Expression of the target antigen tHER2 in Jurkat T cells and expression of the affibody-based CAR in primary T cells. Primary and Jurkat T cells were electroporated with 5 μg of mRNA encoding each construct, and expression was measured 20 hours after electroporation. Primary T cells and Jurkat T cells not expressing the construct ("No CAR" and "No Construct," respectively) were used as negative controls. (D) Expression of 13 affibody-based CARs ("L9A," "R10A," "Q11A," "Y13A," "W14A," "Q17A," "W24A," "T25A," "S27A," "R28A," "R32A," "Y35A," and "zHER2-WT") in primary T cells (the sequences of the affibody-based antigen-binding moieties fused to the CAR signaling backbone are shown in SEQ ID NOs: 26 to 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 a negative control. (E) Activation of affibody-based CARs by dimerization. Primary T cells from two donors (shown with different symbols) were electroporated with 5 μg of each construct. Specific lysis of target cells was measured in a luciferase-based cytotoxicity assay after co-incubation of different tHER2-transfected CAR T cells (E:T ratio 2:1, 4 h at 37°C). 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 a control condition. T cells without CAR ("no CAR") were used as a negative control. Figure 10F shows the K of each affibody-based binding moiety (fused to superfolder GFP (sfGFP)) for human HER2, as determined by SPR analysis using a matrix coated with a chimeric HER2 protein containing the extracellular domain of HER2 fused to the Fc domain of IgG1. d The values ​​are shown. The affinity was determined by kinetic methods.

[0326] Figure 11: Functional characterization of CAR panels consisting of three and four CAR molecules. (A and B) Schematic diagram of CAR panels consisting of three or four CAR molecules, respectively. (C and D) Expression of CAR trimer and tetramer panels in Jurkat T cells 20 hours after electroporation of 5 μg of mRNA for each construct. Jurkat T cells not expressing the construct ("No CAR") were used as a negative control.

[0327] Figure 12: Expression and function of CARs containing different costimulatory molecules. (A) Schematic diagram of a panel of CARs consisting of two CAR molecules, each containing the costimulatory domain of CD28, ICOS, or OX40 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 (red histograms). Jurkat T cells or primary human T cells expressing no construct ("no CAR") were used as negative controls, respectively (blue filled 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 co-cultured with or without Jurkat T cells electroporated with 5 μg of tEGFR-encoding mRNA for an additional 20 hours in the presence or absence of AP20187. Induction of the NF-κB and NF-AT promoters in the CAR-expressing reporter cells was detected by flow cytometry analysis of the expression of enhanced green fluorescent protein (eGFP) and the cyan fluorescent variant of GFP (CFP), 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 the respective CAR molecules, T cells were co-cultured with target cells at an E:T ratio of 4:1:1 (T cells:tEGFR) for an additional 4 or 20 hours at 37°C. pos Jurkat cells: tEGFR neg Jurkat cells). Dimerization of CAR molecules was induced by pretreatment of Jurkat cells (D) and primary human T cells (E) with 10 nM AP20187 for 30 min at 37° C. Treatment with the same concentration of DMSO served as a control condition.

[0328] 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 the respective mRNA. Primary T cells without a CAR were used as a 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(G32A)-G4S-myc-8cys-BB-3z" (SEQ ID NO: 42), "S(G32A)-G4S-StrepII-8cys-BB-3z" (SEQ ID NO: 43), and "S(G32A)-G4S-his-8cys-BB-3z" (SEQ ID NO: 45) in primary human T cells 20 hours after electroporation of 5 μg of each mRNA. Primary T cells without CAR were used as a negative control (filled histogram). CAR expression was detected using anti-c-myc, anti-Strep II, or anti-hexahistidine antibodies, respectively. (C) Expression of CAR constructs "S(WT)-8cys-BB-3z" (SEQ ID NO: 74), "S(G25A)-8cys-BB-3z" (SEQ ID NO: 72), "S(G32A)-8cys-BB-3z" (SEQ ID NO: 43), "S(WT)-8ser-BB-3z" (SEQ ID NO: 75), "S(G25A)-8ser-BB-3z" (SEQ ID NO: 73), and "S(G32A)-8ser-BB-3z" (SEQ ID NO: 44) in primary human T cells 20 hours after electroporation of 5 μg of the respective mRNA. Primary T cells without CAR were used as a negative control (filled histogram). Expression was detected using an anti-Strep II 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), and "A(WT)-8ser-BB-FKBP(36V)-3z" (SEQ ID NO: 52) in primary human T cells 20 hours after electroporation of 5 μg of the respective mRNA. Primary T cells without CAR were used as a negative control (filled histogram). Expression was detected using either anti-FLAG, anti-Strep II, or anti-hexahistidine antibodies, as indicated.

[0329] Figure 14 shows a schematic representation of the design of various CAR molecules, and the corresponding amino acid sequences are shown in Figure 15.

[0330] FIG. 15 shows the amino acid sequences of different CAR molecules.

[0331] Example 1: Generation of low affinity single domain binding moieties based on rcSso7d for use in the CARs of the present invention The first example demonstrates a strategy for generating low-affinity antigen-binding moieties suitable for use as antigen-binding moieties in the CARs of the present invention. Reduced-charge Sso7d (rcSso7d) is a reduced-charge form of a small (approximately 7 kDa) DNA-binding protein from the archaeon Sulfolobus solfataricus. Charge reduction minimizes nonspecific binding by reducing electrostatic interactions. rcSso7d is a single-domain protein antigen-binding moiety with high thermal stability and monomeric behavior, and therefore represents an example of a suitable binding scaffold. It has a K of 19 nM. dLow-affinity mutants were generated from the well-characterized antigen-binding fragment rcSso7d E11.4.1, which binds to human EGFR at 100 kJ / s (Traxlmayr et al., J Biol Chem., 2016;291(43):22496-22508). Low-affinity mutants were generated by alanine scanning, substituting all amino acids potentially involved in epitope binding with alanine. In each mutant, one position involved in antigen binding was mutated to alanine. The rcSso7d E11.4.1 mutants were fused to sfGFP and expressed as soluble proteins in a bacterial expression system. The schematic structure of the fusion protein is shown in Figure 14G. We determined the binding affinities of the target antigens (EGFR) by (i) titration experiments of soluble fusion proteins bearing sfGFP as the binding moiety to Jurkat T cells engineered by mRNA electroporation to express high levels of the antigen, and (ii) by SPR experiments on a Protein A chip loaded with the extracellular domain of EGFR fused to IgG-Fc. The alanine scan results and the resulting affinities of the antigen-binding moieties are shown in Figure 2.

[0332] 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 designed using QuikChange Primer Design software (Agilent Genomics), and oligonucleotides were synthesized by Biomers.

[0333] Expression and purification of rcSso7d-based antigen-binding moieties The binding scaffold was expressed as an sfGFP fusion protein (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, Escherichia coli cells (Tuner DE3) were transformed with sequence-verified plasmids using heat shock transformation. After overnight incubation at 37°C, the culture was diluted 1:100 in Terrific Broth (TB) medium (12 g / L tryptone, 24 g / L yeast extract, 4% glycerol, 2.31 g / L KH2PO4, and 16.43 g / L K2HPO4*3H2O) supplemented with kanamycin (50 μg / mL) and incubated at 37°C with shaking. 600When the RI reached approximately 2, transgene expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cells were further cultured 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 (2 × 90 s, 50% duty cycle, 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. Washing steps were then performed with equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) containing increasing amounts of imidazole (5–15 mM). The binding scaffold was eluted by applying equilibration buffer containing 250 mM imidazole. After buffer exchange into PBS using an Amicon Ultra-15 10K centrifugal filter (Merck Millipore), the concentration was determined by measuring the absorbance at 280 nm using the respective molar extinction coefficients. Finally, the protein was directly frozen at -80°C.

[0334] Maintenance of human cell lines Jurkat T cells were a gift from Dr. Sabine Strehl at the Children's Cancer Research Institute (CCRI) and were 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 were authenticated by Multiplexion, Germany. Cell density was monitored using AccuCheck Counting Beads (Thermo Scientific), a flow cytometer-based cell counting platform.

[0335] 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 the reaction template. The resulting mRNA was purified using an adapted RNeasy column purification kit (Qiagen). Briefly, the mRNA solution was diluted with a mixture of RLT buffer (Qiagen), ethanol (Merck), and 2-mercaptoethanol (Merck). This mixture was loaded onto an RNeasy column, and purification was performed 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 electroporation. For transient transgene expression, Jurkat T cells were electroporated with different amounts of various mRNAs using a Gene Pulser (Biorad). The following protocols were used for the various cell types: Jurkat T cells (square wave protocol, 500V, 3ms, 4mm cuvette).

[0336] Antibodies and flow cytometry Jurkat T cells were resuspended in FACS buffer (PBS (Thermo Scientific), 0.2% human albumin (CSL Behring), and 0.02% sodium azide) and treated with 10% human serum for 10 minutes at 4°C. Cells were stained with various primary antibodies for 25 minutes at 4°C. The stained cells were washed twice with FACS buffer and then directly processed on a BD LSRFortessa. Expression of the modified target antigen tEGFR was detected using a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was performed using FlowJo software.

[0337] Measurement of binding affinity on cell membranes Jurkat T cells were engineered to express various tumor antigens at high levels. To achieve this, 3 μg of tEGFR-encoding mRNA was electroporated into Jurkat T cells, and one day later, they were co-cultured with effector cells. After washing with PBS, the cells were resuspended in PBS containing 0.1% BSA (Sigma-Aldrich) and incubated with different concentrations of sfGFP-fused binder proteins to determine the affinity of the antigen-binding moiety for each tumor antigen. After 1 hour of incubation at 4°C with shaking, the plate was centrifuged (450 g, 7 minutes, 4°C), the supernatant was discarded, and the cells were harvested using a BD LSR Fortessa. The cells were kept on ice to avoid endocytosis. K was calculated by curve fitting using Microsoft Excel (Microsoft Corporation). d obtained.

[0338] Measuring binding affinity using surface plasmon resonance (SPR) SPR experiments were performed on a Biacore T200 instrument (GE Healthcare). All experiments were performed in degassed and filtered PBS (pH 7.4) containing 0.1% BSA and 0.05% Tween®-20 (Merck Millipore) at 25°C. hEGFR-Fc (R&D) was immobilized on a Protein A sensor chip (GE Healthcare) at a concentration of 6.67 μg / mL for 60 seconds at a flow rate of 10 μL / min. To determine the affinity of the rcSso7d-based antigen-binding moiety, five concentrations (expected K of the antigen-binding moiety) were used. d Various proteins (based on the K value) were injected in single-cycle kinetics mode at a flow rate of 30 μL / min for 15 seconds, followed by a dissociation step (30 seconds). Regeneration was performed using 10 mM glycine-HCl (pH 1.7) at a flow rate of 30 μL / min for 30 seconds. K values ​​were calculated by curve fitting using Biacore T200 Evaluation Software (GE Healthcare). d obtained.

[0339] Example 2: Extracellular disulfide bond-forming cysteines reversibly regulate CAR function by preventing full utilization of the avidity effect For example, extracellular disulfide bond-forming cysteines in the extracellular hinge region of CD8α can prevent the avidity effect of the present invention from being exploited. In Example 2, we demonstrated this by fusing a low-affinity variant of the binding moiety of Example 1, "E11.4.1 G32A," to a CAR signaling scaffold in which two extracellular cysteine ​​residues in the hinge region of CD8α (UniProt ID P01732, positions C164 and C181) were replaced with serine residues or not. While the cysteine-containing CAR variant ("Cys") efficiently induced T cell activation in response to target cells, the serine-containing variant ("Ser") did not or only poorly induced T cell activation. This example thus demonstrates the importance of preventing disulfide bond formation when engineering CAR molecules suitable for use in the CARs of the present invention. The schematic diagram in Figure 3A illustrates the design of the test construct. Figures 3B and 3C show the expression of CAR and target antigen. Primary human T cells were electroporated with 5 μg of mRNA for each construct, and CAR expression was detected via the Strep II tag 20 hours after electroporation. Jurkat T cells were electroporated with 3 μg of mRNA encoding a truncated form of EGFR (tEGFR). Full-length EGFR was truncated at both the N- and C-termini to generate a functionally inactive human polypeptide that cannot bind to its natural ligand, EGF, and has diminished dimerization properties due to the absence of a kinase domain (Wang et al., Blood., 2011;118(5):1255-1263). The resulting transgene consisted of the leader sequence of the granulocyte-macrophage colony-stimulating factor 2 receptor α subunit (GM-CSF-Ra) and amino acids 334-675 of human EGFR (Uniprot P00533), including the two extracellular membrane-proximal and transmembrane domains. Transgene expression was detected 20 hours after electroporation using an antibody against EGFR.Twenty hours after T cell electroporation, CAR function was measured using a luciferase-based cytotoxicity assay (Figure 3D) and ELISA to quantify cytokine release from the T cells (Figure 3E). Figures 3D and 3E show that the antigen-binding moiety with the lowest affinity tested ("E11.4.1-G32A") was able to elicit T cells only upon bivalent interaction with target cells, i.e., when fused to a CAR containing cysteines in the CD8α hinge ("Cys"), but did not or only poorly elicit T cells when fused to a CAR in which these cysteines had been replaced by serines ("Ser"). Conversely, antigen-binding moieties with increased affinity (E11.4.1-WT and E11.4.1-G25A) elicited potent cytotoxicity even upon monovalent interaction, i.e., when fused to a "Ser" CAR backbone.

[0340] Maintenance of human cell lines Primary human T cells were obtained from anonymized healthy donor blood (buffy coats obtained from the Austrian Red Cross, Vienna, Austria) after apheresis. CD3+ 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+ T cells were activated using 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. Jurkat T cells were a gift from Dr. Sabine Strehl at CCRI and maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were routinely tested for mycoplasma contamination and were authenticated by Multiplexon GmbH, Germany. Cell density was monitored using AccuCheck Counting Beads.

[0341] 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 the reaction template. The resulting mRNA was purified using an adapted RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol, and 2-mercaptoethanol. This mixture was loaded onto an RNeasy column, and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water, and the purified mRNA was frozen at -80°C until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with different amounts of various mRNAs using a Gene Pulser (BioRad). The following protocols were used for each cell type: primary T cells (square wave protocol, 500 V, 5 ms, 4 mm cuvette) and Jurkat T cells (square wave protocol, 500 V, 3 ms, 4 mm cuvette).

[0342] 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 minutes at 4°C. Cells were stained with various primary antibodies for 25 minutes at 4°C. After washing twice with FACS buffer, cells were either stained with secondary antibodies for 25 minutes at 4°C or directly processed on a BD LSRFortessa. CAR construct expression was detected via the Strep II tag using an anti-Strep II 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 a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was performed using FlowJo software.

[0343] Construction of transgene constructs Nucleotide sequences encoding the signal peptide of CD33, human CD8α hinge, human monomeric CD8α hinge (UniProt ID P01732, C164S, and C181S), 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 Strep II tag (NWSHPQFEK) and flexible linker was performed by PCR. Assembly of the nucleotide sequences into functional transgenes was performed using Gibson Assembly Master Mix (New England BioLabs) according to the manufacturer's instructions. Schematic diagrams and sequences are shown in Figures 14 and 15, respectively. The resulting constructs were amplified by PCR and then used for in vitro transcription.

[0344] Luciferase-based cytotoxicity assays Luciferase-expressing tumor cells were co-cultured with CAR T cells at a 2:1 E:T cell ratio (10,000 target cells / well) in white round-bottom 96-well plates (Sigma-Aldrich) for 4 hours at 37°C in cytotoxicity assay medium consisting of phenol-free RPMI (Thermo Scientific), 10% FCS, 1% L-glutamine (Thermo Scientific), and 1% penicillin-streptomycin. Finally, the remaining viable cells were quantified by determining the residual luciferase activity of the co-culture. After 10 minutes at room temperature, luciferin was added to the cell suspension (150 μg / mL final concentration; PerkinElmer), and luciferase activity was measured 20 minutes later using an ENSPIRE multimode plate reader. The specific lysis rate was calculated using the following formula: Percent specific lysis = 100 - ((RLU obtained from wells containing co-cultures of effector and target cells) / (RLU obtained from wells containing target cells only) x 100)).

[0345] Cytokine release by CAR T cells Cytokine secretion of primary CAR T cells was assessed by co-culturing them with target cells at an E:T ratio 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 the supernatants obtained from the co-culture experiments to determine cytotoxicity. Supernatants were centrifuged (1600 rpm, 7 minutes, 4°C) to remove remaining cells and debris and then frozen at -80°C. Analysis of secreted IFN-γ was performed using an ELISA with the Human IFN-γ ELISA Ready-SET-Go!® Kit (eBioscience) according to the manufacturer's instructions. Measurements were performed using an ENSPIRE multimode plate reader.

[0346] Example 3: Single-chain variable fragments (scFv) can induce CAR clustering at the cell membrane, preventing the exploitation of the avidity effect and allowing reversible modulation of CAR function In a third example, we demonstrate that incorporating an scFv-based binding moiety into a CAR molecule can prevent the exploitation of avidity effects and allow specific recognition of antigen combinations. The schematic diagram of the CAR construct shown in Figure 4A illustrates the design of the tested CAR variants (4D5-5-8cys-BB-3z, 4D5-5-8ser-BB-3z, and 4D5-5(split)-8ser-BB-FKBP(36V)-3z). In the illustrated example, the scFv 4D5-5 against HER2 was used as the antigen-binding moiety and 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 for scFv 4D5-5 was reported to be 1.1 μM (Liu et al., Cancer Res., 2015;75(17):3596-3607), which is comparable to the affinity of E11.4.1-G32A. Jurkat T cells expressing a truncated form of HER2 (tHER2) were used as the target cell line (Figure 3E). IFN-γ secretion by CAR T cells (Figure 3F) and target cell lysis (Figure 3G) elicited by the serine-containing CAR "4D5-5-8ser-BB-3z" were only slightly reduced compared to the cysteine-containing CAR "4D5-5-8cys-BB-3z," despite the low-affinity scFv. This is in stark contrast to what was observed using the low-affinity rcSso7d-based antigen-binding moiety for CAR S(G32A)-8ser-BB-3z (SEQ ID NO: 44) in Example 2 (Figure 3). V linked together by scFv on the T cell surface as confirmed by diabody formation H and V LNot only can V dimerize within the same single-chain molecule (i.e., within the same CAR molecule), but also form intermolecular linkages (i.e., linkages between different CAR molecules). This mediates dimerization, as reported for purified scFv proteins, or even oligomerization (Atwell et al., Protein Eng., 1999; 12(7): 597-604), and explains the previously observed CAR clustering (Long et al., Nat Med., 2015; 21(6): 581-590). Finally, this dimerization or oligomerization of scFvs leads to 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 therefore activation of low-affinity CARs based on monomeric CAR scaffolds. H Domain and V L The domains at least partially heterodimerize on the surface of T cells to form functional V H / V L We hypothesized that heterodimers (i.e., Fvs) could be formed. If there is no nonspecific adhesion between Fvs, these Fvs would have low affinity (Kd 1.1 μM in the case of 4D5-5) and therefore (V H Only after controlled dimerization of the two Fvs (via the FKBP F36V domain fused intracellularly to the carrier chain) should T cell activation be initiated. Indeed, Figure 4H shows that this is the case, with the V domain of the low affinity scFv 4D5-5 H and V L On two separate membrane-anchored molecules (SEQ ID NO: 56 and SEQ ID NO: 57), we were able to demonstrate that CAR T cells expressing these constructs (Figures 4C and 4D) were not activated by tHER2-positive target cells. However, V HWhen the constructs were homodimerized with AP20187, the T cells were activated by the target cells (Figure 4H). T cell activation in the presence of this dimerizer 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 the dimerizer was due to the monovalency of the Fvs. This is consistent with the low affinity of 4D5-5 and the findings obtained with the rcSso7d-based antigen-binding moiety in Example 2. For comparison, the scFv version (i.e., V linked by linker "218") was used. H and V L ) expression resulted in potent activation of CAR T cells despite low expression (Figure 4H), V H The expression levels were roughly adjusted to those obtained with the constructs (Figure 4C). Taken together, the data in Figure 4 demonstrate that at least certain scFv types are able to differentiate V between adjacent molecules on the T cell surface. H and V L This strongly suggests that dimerization (or oligomerization) occurs in part due to intermolecular heterodimerization of the CAR molecules. Similar to the dimerization or oligomerization caused by cysteine ​​amino acid residues (discussed above), uncontrolled dimerization or oligomerization of CAR molecules mediated by at least certain scFv variants can lead to homodimerization or homooligomerization independent of regulatory molecules. Thus, in a preferred embodiment of the present invention, neither the antigen-binding portion of a CAR molecule in a CAR group nor the antigen-binding portion of another polypeptide bound to a CAR molecule in the group is an scFv.

[0347] Maintenance of human cell lines Primary human T cells were obtained from anonymized healthy donor blood (buffy coat obtained from the Austrian Red Cross, Vienna, Austria) after apheresis. CD3+ T 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+ T cells were activated using 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. Jurkat T cells were a gift from Dr. Sabine Strehl at CCRI and maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were routinely tested for mycoplasma contamination and were authenticated by Multiplexon GmbH, Germany. Cell density was monitored using AccuCheck Counting Beads.

[0348] 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 the reaction template. The resulting mRNA was purified using an adapted RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol, and 2-mercaptoethanol. This mixture was loaded onto an RNeasy column, and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water, and the purified mRNA was frozen at -80°C until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with different amounts of various mRNAs using a Gene Pulser (BioRad). The following protocols were used for each cell type: primary T cells (square wave protocol, 500 V, 5 ms, 4 mm cuvette) and Jurkat T cells (square wave protocol, 500 V, 3 ms, 4 mm cuvette).

[0349] 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 minutes at 4°C. Cells were stained with various primary antibodies for 25 minutes at 4°C. After washing twice with FACS buffer, cells were either stained with secondary antibodies for 25 minutes at 4°C or directly processed on a BD LSRFortessa. CAR construct expression was detected via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, GenScript) or via the FLAG tag using an anti-FLAG tag antibody (clone L5, BioLegend) as the primary antibody. For the Strep II tag antibody, PE- or APC-conjugated secondary antibodies were used. Expression of the engineered target antigen, tHER2, was detected using a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was performed using FlowJo software.

[0350] 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), 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 HER2 extracellular and transmembrane domains were obtained from Addgene (plasmid #16257). Insertion of a Strep II tag (NWSHPQFEK) or FLAG tag (DYKDDDDK) and flexible linker was performed by PCR. Assembly of the nucleotide sequences into functional transgenes was performed using the Gibson Assembly Master Mix according to the manufacturer's instructions. Schematic diagrams and sequences are shown in Figures 14 and 15, respectively. The resulting constructs were amplified by PCR and then used for in vitro transcription.

[0351] Luciferase-based cytotoxicity assays Luciferase-expressing tumor cells were co-cultured with CAR T cells at a 2:1 E:T cell ratio (10,000 target cells / well) in white round-bottom 96-well plates for 4 hours at 37°C in cytotoxicity assay medium consisting of phenol-free RPMI, 10% FCS, 1% L-glutamine, and 1% penicillin-streptomycin. Finally, the remaining viable cells were quantified by determining the residual luciferase activity of the co-cultures. After 10 minutes at room temperature, luciferin was added to the cell suspension (150 μg / mL final concentration), and luciferase activity was measured 20 minutes later using an ENSPIRE multimode plate reader. Specific lysis was calculated using the following formula: specific lysis = 100 - ((RLU obtained from wells containing effector and target cell co-cultures) / (RLU obtained from wells containing target cells alone) × 100)).

[0352] 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 each target antigen, and (ii) Jurkat cells electroporated with mRNA encoding mCherry alone. These two populations were mixed at a 1:1 ratio and co-cultured with CAR T cells at an E:T cell ratio of 4:1:1 (20,000 target cells / well) in a round-bottom 96-well plate at 37°C for 4 hours. Target cells without CAR T cells served as the control condition ("target only"). After the incubation period, the co-culture was centrifuged (5 minutes, 1600 rpm, 4°C), and the supernatant was collected for subsequent cytokine measurement. The remaining cells were resuspended in 100 μL of FACS buffer consisting of PBS, 0.2% human albumin, and 0.02% sodium azide. The viability of the target antigen-positive cell population and the target antigen-negative cell population was determined using a BD LSRFortessa flow cytometer, and specific lysis was calculated using the following formula. Percent specific lysis = (1-(((% of eGFP-positive cells in sample / (% of mCherry-positive cells in sample / (% of eGFP-positive cells in "target only" control / (% of mCherry-positive cells in "target only" control)))*100.

[0353] Cytokine release by CAR T cells Cytokine secretion of primary CAR T cells was assessed by co-culturing them with target cells at an E:T ratio 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 the supernatants obtained from the co-culture experiments to determine cytotoxicity. Supernatants were centrifuged (1600 rpm, 7 minutes, 4°C) to remove remaining cells and debris and then frozen at -80°C. Analysis of secreted IFN-γ was performed using an ELISA with the Human IFN-γ ELISA Ready-SET-Go!® Kit (eBioscience) according to the manufacturer's instructions. Measurements were performed using an ENSPIRE multimode plate reader.

[0354] In vitro transgene dimerization Prior to coculture experiments, transgene dimerization was induced. Primary T cells were diluted to the final cell concentration in the appropriate cell culture medium. The homodimerizer AP20187 (MedChemExpress) was diluted in the cell culture medium and added to a final concentration of 10 nM. The same concentration of DMSO was added as a control. To ensure efficient transgene dimerization, the cells were incubated at 37°C for 30 minutes before use in in vitro experiments.

[0355] Example 4: Generation of switchable CARs by controlling avidity through homodimerization of the domain FKBP F36V In the fourth example, we demonstrate the control of CAR function by controlling the avidity of CARs through conditional homodimerization. To this end, a homodimerization domain based on the FKBP F36V variant was incorporated into a monomeric CAR scaffold (illustrated in FIG. 5A), and this scaffold was fused to a binding moiety 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). Primary human T cells were transduced with a lentiviral vector encoding the resulting CAR molecule. CAR expression was detected via a Strep II tag and is shown in FIG. 5B. For functional analysis, CAR T cells were co-cultured with the target cell line A431-fLuc, which expresses high levels of EGFR (Figure 5C), at an E:T ratio of 10:1 for 4 hours at 37°C. Target cell cytotoxicity was determined using a luciferase-based cytotoxicity assay. T cells expressing the CD19-specific standard CAR CD19-8cys-BB-3z ("CD19-BBz" (SEQ ID NO: 58)) and T cells without a CAR ("no CAR") served as control conditions. CAR dimerization was induced by adding 10 nM AP20187 to T cells prior to co-culture with target cells. The addition of the solvent control DMSO served as a control. Figure 5D shows that the function of the CAR bearing the low-affinity binding moiety E11.4.1-G32A (S(G32A)-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 46)) but not the high-affinity binding moiety E11.4.1-WT (S(WT)-8ser-BB-FKBP(36V)-3z (SEQ ID NO: 49)) was strongly dependent on the presence of the dimerizer AP20187. As seen in the control condition using T cells without CAR expression, the dimerizer itself had no effect on target cell lysis.

[0356] Maintenance of human cell lines Primary human T cells were obtained from anonymized healthy donor blood (buffy coat obtained from the Austrian Red Cross, Vienna, Austria) after apheresis. CD3+ T 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+ T cells were activated using 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. A431 epidermoid carcinoma cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FCS and 1% penicillin-streptomycin. The cell lines were routinely tested for mycoplasma contamination and were authenticated by Multiplexon GmbH, Germany. Cell density was monitored using AccuCheck Counting Beads.

[0357] Transduction of T cells and cell lines Pantropic VSV-G pseudotyped lentivirus was produced in Lenti-X 293T cells (Clontech Laboratories) using the third-generation puromycin-selectable pCDH transgene vector (System Biosciences) and the second-generation viral packaging plasmids pMD2.G and psPAX2 (both from Addgene, plasmids #12259 and #12260, respectively). Cotransfection was performed using Purefection Transfection Reagent (System Biosciences) according to the manufacturer's instructions. Supernatants were collected 1 and 2 days after transfection and concentrated using a Lenti-X Concentrator (Clontech Laboratories) according to the manufacturer's instructions. Twenty-four hours before lentiviral transduction, primary T cells were activated using anti-CD3 / 28 beads according to the manufacturer's instructions. Cell culture plates were coated with RetroNectin (Clontech Laboratories) according to the manufacturer's instructions to promote coexistence of lentivirus and primary T cells. Cells were exposed to concentrated lentiviral supernatant for 1 day, after which viral particles were removed. After 3 days, T cells were treated with 1 μg / mL puromycin (Sigma-Aldrich) to ensure high-level and uniform transgene expression. T cells were grown in T cell transduction medium consisting of AIM-V (Life Technologies) supplemented with 2% Octaplas (Octapharma), 1% L-glutamine, 2.5% HEPES (Thermo Scientific), and 200 IU / mL recombinant human IL-2. Cell lines were split 24 hours prior to lentiviral transduction to ensure exponential cell growth at the time of transduction. Cells were exposed to different concentrations of lentiviral supernatant for 1 day. Three days after transduction, non-transduced cells were selected with puromycin at concentrations ranging from 1 to 8 μg / mL to eliminate non-transduced cells.

[0358] 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 minutes at 4°C. Cells were stained with various primary antibodies for 25 minutes at 4°C. After washing twice with FACS buffer, cells were stained with secondary antibodies for 25 minutes at 4°C or directly processed with BD LSRFortessa. Expression of CAR constructs was detected via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, GenScript) or, in the case of CD19-BBz CAR, Protein L as the primary antibody, followed by a PE- or APC-conjugated secondary antibody. EGFR expression was detected with a PE-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was performed using FlowJo software.

[0359] Construction of transgene constructs Nucleotide sequences encoding the CD33 signal peptide, low-affinity rcSso7d variant E11.4.1-G32A, Strep II tag (NWSHPQFEK), flexible G4S linker, human monomeric CD8α hinge (UniProt ID P01732, C164S, and C181S), CD8α transmembrane domain, 4-1BB costimulatory domain, dimerization domain FKBP F36V, and CD3ζ ITAM signaling domain were synthesized by GeneArt (Thermo Scientific). Assembly of the nucleotide sequences into functional transgenes was performed using Gibson Assembly Master Mix according to the manufacturer's instructions. Schematic diagrams and sequences are shown in Figures 14 and 15, respectively. The resulting constructs were amplified by PCR and then used for in vitro transcription.

[0360] Luciferase-based cytotoxicity assays Luciferase-expressing tumor cells were co-cultured with CAR T cells at a 2:1 E:T cell ratio (10,000 target cells / well) in white round-bottom 96-well plates for 4 hours at 37°C in cytotoxicity assay medium consisting of phenol-free RPMI, 10% FCS, 1% L-glutamine, and 1% penicillin-streptomycin. Finally, the remaining viable cells were quantified by determining the residual luciferase activity of the co-culture. After 10 minutes at room temperature, luciferin was added to the cell suspension (150 μg / mL final concentration), and luciferase activity was measured 20 minutes later using an ENSPIRE multimode plate reader. Specific lysis was calculated using the following formula: Percent specific lysis = 100 - ((RLU obtained from wells containing co-cultures of effector and target cells) / (RLU obtained from wells containing target cells only) x 100)).

[0361] In vitro transgene dimerization Prior to co-culture experiments, transgene dimerization was induced. Primary T cells were diluted to the final cell concentration in the appropriate cell culture medium. The homodimerizer AP20187 was diluted in cell culture medium and added to a final concentration of 10 nM. The same concentration of DMSO was added as a control. To ensure efficient transgene dimerization, the cells were incubated at 37°C for 30 minutes before use in in vitro experiments.

[0362] Example 5: Treatment of tumor-bearing mice with stably transfected T cells expressing CARs with drug-controllable avidity In Example 5, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1WJI In a leukemia model in / SzJ(NSG) mice, lentivirally transduced T cells expressing the low-affinity CAR "S(G32A)-8ser-BB-FKBP(36V)-3z" (sequence number 46) were shown to efficiently inhibit tumor growth in the presence of regulatory molecules, but not in the absence of regulatory molecules. For the in vivo model described above, highly expressed tEGFR (approximately 1 × 10 6 The B-ALL cell line Nalm6 was used, transfected with vectors for tEGFR (tEGFR molecules / cell) and firefly luciferase for in vivo quantification of tumor growth by using bioluminescence imaging. 6 Intravenous (iv) injection of 10x10 Nalm6-tEGFR-fLuc cells into NSG mice resulted in exponential tumor growth in untreated mice. However, Figure 6A shows that 3 days after tumor cell injection, 10x10 Nalm6-tEGFR-fLuc cells expressing either the anti-CD19 CAR CD19-8cys-BB-3z (SEQ ID NO: 58) or the high-affinity anti-EGFR CAR "S(WT)-8ser-BB-FKBP(36V)-3z" (SEQ ID NO: 49) 6We show that intravenous injection of 100 T cells efficiently inhibited the proliferation of this cell line in NSG mice. Importantly, T cells carrying th...

Claims

1. A group of chimeric antigen receptors (CARs) consisting of two, three, or four CAR molecules, wherein the members of the CAR group may be different or identical in their amino acid sequence, and each CAR molecule of the group comprises at least a membrane domain and an ectodomain that comprises either an antigen-binding moiety or a binding site to which another polypeptide comprising an antigen-binding moiety can bind; and wherein at least one CAR molecule of the group further comprises an endodomain comprising at least a signaling region capable of signaling via at least one immunoreceptor tyrosine-based activation motif (ITAM) or at least one immunoreceptor tyrosine-based inhibition motif (ITIM); and 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 located on 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; each CAR molecule of the group comprises at least one dimerization domain capable of mediating homo- or heterodimerization with other CAR molecules of the group, wherein this dimerization of a pair of dimerization domains is induced by a regulatory molecule, and optionally reduced by another regulatory molecule, or occurs in the absence of a regulatory molecule and is reduced by a regulatory molecule, wherein the regulatory molecule is capable of binding to at least one member of the pair of dimerization domains under physiological conditions and inducing or reducing dimerization, thereby inducing or reducing the formation of a non-covalently complexed group of CARs consisting of two, three, or four CAR molecules; and the ectodomain of each CAR molecule of the group in its general conformation does not each have a cysteine ​​amino acid moiety capable of forming an intermolecular disulfide bond with another CAR molecule of the group; and the CAR molecules of the group and the antigen-binding portions of other polypeptides to which the CAR molecules of the group can bind are specific for one target antigen or for a non-covalent complex of different target molecules; and the affinity of each individual antigen-binding portion of the group of CAR molecules for its target antigen is between 1 mM and 100 nM; and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or, on the other hand, the affinity of this other polypeptide to the binding site of its respective CAR molecule, is between 1 mM and 100 nM; Group of CARs.

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

3. 2. The group of CARs according to claim 1, wherein the affinity of each individual antigen-binding portion of the CAR molecules of the group to its target antigen is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, and in particular 50 μM to 100 nM, and the affinity of each individual antigen-binding portion of another polypeptide to its target antigen, or on the other hand, this other polypeptide to the binding site of its respective CAR molecule, is 500 μM to 100 nM, preferably 250 μM to 100 nM, more preferably 125 μM to 100 nM, and in particular 50 μM to 100 nM.

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

5. The group of CARs according to any one of claims 1 to 4, wherein each target antigen specifically recognized by an antigen-binding portion of the group of CARs or of another polypeptide capable of binding to a CAR molecule of the group is a naturally occurring cell surface antigen, or a polypeptide, sugar, or lipid bound to a naturally occurring cell surface antigen.

6. The antigen-binding portion of the group of CARs or other polypeptides capable of binding to a CAR molecule of the group binds to one or more specific antigens present on a cell, solid surface, or lipid bilayer, preferably one or more target antigens on a cell, and in particular the one or more target antigens are selected from the group consisting of 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, 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, MPZLI1, MTDH, PANX3, PCDHB6, PCDHB10, PCDHB12, PCDHB13, PCDHB18, PCDHGA3, PEP, SGCB, vezatin, DAGLB, SYT11, WFDC10A, ACVR2A, ACVR2B,Undifferentiated 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, CHRNA3, CHRNG, DRD4, GABRB3, GRIN3A, GRIN2C, GRIK4, HTR7, APT8B2, NKAIN1, NKAIN4, CACNA1A, CACNA1B, CACNA1I, CACNG8, CACNG4, CLCN7, KCN§A4, KCNG2, KCNN3, KCNQ2, KCNU1, PKD1L2, PKD2L1, SLC5A8, SLC6A2, SLC6A6, SLC6A11, SLC6A15, SLC7A1, SLC7A5P1, SLC7A6, SLC9A1, SLCz0A3, SLC10A4, SLC13A5, SLC16A8, SLC18A1, SLC18A3, SLC19A1, SLC26A10, SLC29A4, SLC30A1, SLC30A5, SLC35E2, SLC38A6, SLC38A9, SLC39A7, SLC39A8, SLC43A3, TRPM4, TRPV4, TMEM16J, TMEM142B, ADORA2B, BAI1, EDG6, GPR1, GPR26, GPR34, GPR44, GPR56, GPR68, GPR173, GPR175, LGR4, MMD, NTSR2, OPN3, OR2L2, OSTM1, P2RX3, P2RY8, P2RY11, P2RY13, PTGE3, SSTR5, TBXA2R, ADAMz2, ADAMTS7, CST11, MMP14, LPPR1, LPPR3, LPPR5, SEMA4A, SEMA6B, ALS2CR4, LEPROTL1, MS4A4A, ROM1, TM4SF5, VANGL1, VANGL2, C18orf1, GSGL1, ITM2A, KIAA1715, LDLRAD3, It should be noted that there seems to be an unclear "§" in "KCN§A4" and "z" in "SLCz0A3" and "ADAMz2" in the original text. These might be errors that need to be corrected in the source for a more accurate translation.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, neurotrophic 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), mesotensin, epithelial cell adhesion molecule (EpCAM), L1-CAM, CEACAM1, CEACAM5, CEACAM6, VEGFR1, VEGFR2, high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A, IL-13R-α2, disialogangliosides (GD2 and GD3), tumor-associated glycoantigens (CA-125, CA-242, Tn, and sialyl-Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), c-MET, CCR1, CCR4, FAP, fibronectin ectodomain-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, The group of CARs according to any one of claims 1 to 5, comprising a molecule preferably selected from the group consisting of PR1 / HLA-A2, a lineage-specific or tissue-specific tissue antigen, preferably CD3, CD4, CD5, CD7, CD8, CD24, CD25, CD34, CD80, CD86, CD133, CD138, CD152, CD319, endoglin and an MHC molecule.

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

8. A kit of nucleic acid molecules comprising nucleotide sequences encoding individual CAR molecules of the group of CARs according to any one of claims 1 to 6, selected from DNA, RNA or in vitro transcribed RNA.

9. A vector or kit comprising a nucleic acid molecule comprising a nucleotide sequence encoding an individual CAR molecule of the group of CARs of 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 of claim 7 or 8, or using the vector or kit of vectors of claim 9, to produce an individual CAR molecule of the group of CARs of any one of claims 1 to 6, or a kit comprising two or more such modified cells.

11. A pharmaceutical preparation comprising a nucleic acid or a kit of nucleic acids according to any of claims 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. The group of CARs according to any one of claims 1 to 6, for use in a method for treating cancer in an individual having cancer, the method comprising the steps of: 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 a group of CARs according to the present invention, wherein each antigen-binding portion of the group of CARs is specific for a target antigen on cancer cells of the individual, and said genetic modification is performed in vitro or ex vivo; ii) introducing the genetically modified cells into the individual; and iii) administering to the individual an effective amount of at least one regulatory molecule to induce or reduce dimerization of each CAR molecule of the group, preferably to induce dimerization of each CAR molecule of the group, thereby inducing or reducing, preferably inducing non-covalent complex formation of the group of CARs, whereby inducing or reducing, preferably inducing non-covalent complex formation of the group of CARs, whereby inducing or reducing, preferably inducing non-covalent complex formation of the group of CARs, whereby upon contact with cancer cells expressing the respective target antigen or respective covalent or non-covalent complexes of different target antigens, the non-covalent complexes of CARs mediate activation of the genetically modified cells and kill the cancer cells, allowing for the treatment of cancer; Group of CARs.

13. 11. The cell of claim 10 for use in a method of treating cancer in an individual, wherein each antigen-binding portion of the group of CARs is specific for a target antigen on a cancer cell of the individual, the method comprising the steps of: i) introducing the cells into an individual; and ii) administering to the individual an effective amount of one or more regulatory molecules that induce or reduce, preferably induce, the formation of a non-covalent complex comprising two, three or four CAR molecules of said group, preferably three CAR molecules of said group, even more preferably four CAR molecules of said group, wherein the non-covalent complex group of CARs in contact with cancer cells expressing each target antigen or each covalent or non-covalent complex of different target antigens induces activation of the genetically modified cells that induces the death of the cancer cells, thereby allowing the treatment of cancer; including, cells.

14. 1. A kit comprising: - a group of CARs 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 - 1, 2 or 3 regulatory molecules, preferably 2, even more preferably 1 regulatory molecule; Includes a kit.

15. It is used in the treatment of diseases characterized by the need for the binding of T lymphocytes or NK cells to target antigens on cells, preferably in the treatment of tumor patients, in particular in the treatment of: Ewing's sarcoma, rhabdomyosarcoma, osteosarcoma, osteoblastic sarcoma, mesothelioma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, leiomyosarcoma, melanoma, glioma, astrocytoma, medulloblastoma, neuroblastoma, retinoblastoma, oligodendroglioma, meningioma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, chronic bone marrow hyperplasia proliferation tumors, acute myeloid leukemia, chronic lymphocytic leukemia (CLL) including B-cell CLL and 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, squamous cell carcinoma, transitional cell carcinoma, bladder cancer, bronchial cancer, colon cancer, colorectal cancer, 15. The group of CARs according to any one of claims 1 to 6, the vector or kit of vectors according to any one of claims 9, the cell or kit of cells according to any one of claims 10, in particular T lymphocytes or NK cells, or the kit of any one of claims 8, 9 or 14, for use in the treatment of tumor patients having a tumor selected from gastric cancer, small cell and non-small cell carcinoma of the lung, lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic cell carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma, atypical meningioma, islet cell carcinoma, medullary carcinoma, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, clear cell carcinoma and mediastinal neurofibroma.

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