Patent Application
20220041687
Immunotherapy with CAR T cells, i.e., T cells modified to express chimeric antigen receptors (CARs), is one of the most promising approaches in cancer therapy. To date, the high potential of this therapeutic strategy has been demonstrated by impressive clinical responses in patients with B cell malignancies. Further translation of this success to other tumours, however, is currently prevented by several hurdles (Lim and June, Cell. 2017; 168(4):724; Labanieh et al., Nat Biomed Eng. 2018; 2:377-391). For example, current CAR T cells are typically directed by the CAR against a single defined tumour associated antigen. This fact results in so-called on-target/off-tumour toxicity, i.e., the destruction of healthy tissue expressing this antigen, since tumour associated antigens are always expressed on healthy cells as well. Existing strategies for improving tumour specificity of CAR modified cells are based on co-expression of chimeric co-inhibitory or co-stimulatory receptors directed against a second antigen or alternatively on transcriptional regulation of CAR expression by a co-expressed chimeric Notch-based receptor directed against a second antigen (Roybal and Lim, Annu Rev Immunol. 2017; 35:229; Labanieh et al., Nat Biomed Eng. 2018; 2:377-391).
WO 2017/180993 A1 discloses Salvage Chimeric Antigen Receptor Systems,
Lanitis et al. (Cancer Immunol. Res. 1 (2013), 43-53) report that chimeric antigen receptor T cells with dissociated signaling domains exhibit focused anti-tumor activity with reduced potential for toxicity in vivo,
Kloss et al. (Nat. biotechnol. 31 (2012), 71-75) report that combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.
WO 2015/075468 A1 discloses CAR systems with CARs comprising an activating endodomain,
Wu et al. (Science 350 (2015), 293 and aab4077-1 to aab4077-10) describe remote control of therapeutic T-cells through a small molecule-gated chimeric receptor,
Ajina et al. (Mol. cancer therap. 17 (2018), 1795-1815 review strategies to address CAR tonic signaling.
It is an object of the present invention to provide a novel strategy for improving the specificity of CAR modified cells in recognizing the desired target cells. This improved specificity for target cells is achieved by specific recognition of target antigen combinations, i.e. combinatorial target antigen recognition. More specifically, the novel CARs should be applicable in vivo, especially for the treatment of human patients, without the risk of adverse reactions or at least with reduced adverse reactions. It is a further object to provide means for tumour treatment, especially immunotherapy concepts for tumour treatment.
The present invention provides a system for combinatorial target antigen recognition which is based on a group of chimeric antigen receptors (CARs) consisting of two, three or four CAR molecules,
wherein each member of the group of CARs is different in its amino acid sequence from one another, and
wherein each of the CAR molecules of the group comprise at least a transmembrane domain and an ectodomain, wherein the ectodomain comprises one or two antigen binding moieties and/or one or two binding sites to which other polypeptides each comprising at least an antigen binding moiety are able to bind, and wherein at least one CAR molecule of the group additionally comprises an endodomain, which comprises at least a signalling region which can transduce a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM), and
wherein the endodomain of each CAR molecule of the group, in case the respective CAR molecule comprises an endodomain, is located on the intracellular side of a cell membrane, if expressed in a cell, wherein the ectodomain of each CAR molecule of the group translocates to the extracellular side of a cell membrane, if expressed in a cell, and wherein the transmembrane domain of each CAR molecule of the group is located in a cell membrane, if expressed in a cell;
wherein the ectodomain of each CAR molecule of the group in its prevalent conformation is free of cysteine amino acid moieties which are able to form intermolecular disulphide bonds with other CAR molecules of the group, respectively, and
wherein the antigen binding moieties of the different CAR molecules of the group and of the different other polypeptides are specific for different target antigens which are not linked to each other covalently, and
wherein the affinity of each individual antigen binding moiety of a CAR molecule of the group to its respective target antigen is between 1 mM and 100 nM, and
wherein the affinity of each individual antigen binding moiety of another polypeptide to its respective target antigen or al-ternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 1 mM and 100 nM, and
wherein each CAR molecule of the group comprises at least one heterodimerization domain, which can mediate defined heterodimerization with other CAR molecules of the group, wherein this heterodimerization of a pair of heterodimerization domains either occurs independent of a regulating molecule, or occurs in the absence of a regulating molecule and is reduced by a regulating molecule, or is induced by a regulating molecule and optionally reduced by another regulating molecule, wherein a regulating molecule is able to bind under physiological conditions to at least one member of a pair of heterodimerization domains and by inducing or reducing heterodimerization either induces or reduces the formation of a non-covalently complexed group of CARs consisting of two, three or four CAR molecules.
The underlying principle for combinatorial antigen recognition according to the present invention is a group of CARs, in which the individual antigen binding moieties of the individual CAR molecules of the group have only a low affinity to their respective target antigens, so that a monovalent interaction only triggers weak intracellular signalling in the CAR-expressing cell, or no signalling at all. In the case of usage of other polypeptides, each of which containing at least one antigen binding moiety and additionally being able to bind to a CAR molecule of the group, either the interaction between the antigen binding moiety of the other polypeptide and its respective target antigen, or the interaction between the other polypeptide and its binding site on the respective CAR molecule of the group, must be of low affinity, so that a monovalent interaction only triggers weak intracellular signalling in the CAR-expressing cell, or no signalling at all. However, non-covalent assembly of two, three or four CAR molecules of the group with different binding specificities results in the formation of multivalent CAR complexes which are able to simultaneously interact with two, up to three or up to four different target antigens, respectively, either directly or indirectly via other polypeptides, each of which comprising at least an antigen binding moiety and being able to bind to a CAR molecule of the group. This multivalent interaction with different antigens results in synergistic amplification of the low affinities, i.e. avidity. Ultimately, this multivalent interaction with a selected combination of different target antigens triggers enhanced signalling in the cells expressing said group of CARs.
Thus, in order to ensure that only multivalent, but not monovalent interactions efficiently trigger the complexed group of CARs, the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 1 mM and 100 nM, and the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 1 mM and 100 nM. In preferred embodiments, the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM, and the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM. In other preferred embodiments, the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM, and the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM. In other preferred embodiments, the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM, and the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM. It should be noted that any affinity value given herein refers to the affinity determined by surface plasmon resonance (SPR) performed with a Biacore T200 device (GE Healthcare) at pH 7.4, 25° C., using steady state analysis, as performed, e.g., in examples 1 and 4 in the example section.
As already explained, the basic principle of generating CARs with AND gate function according to the present invention founds on defined heterodimerization, heterotrimerization or heterotetramerization of a group of CARs, of which each CAR molecule mediates low-affinity recognition of a different target antigen. The amplification of the low affinities of the different binding moieties upon target antigen recognition (i.e., the avidity effect) can then trigger efficient CAR signalling. The non-covalent interaction between the individual CAR molecules of the group according to the present invention (yielding the bi-, tri- or tetraspecific complex of CAR molecules of the group) can be either constitutive or can be dependent on the presence or absence of a regulating molecule. Importantly, dimerization or oligomerization of CAR molecules with identical target antigen specificity, as e.g. mediated by formation of intermolecular extracellular disulphide bonds, would amplify the affinity towards a single antigen and thereby prevent AND gate function of a group of CARs according to the present invention. Such dimerization or oligomerization of CAR molecules with identical target antigen specificity therefore needs to be prevented or at least minimized to the greatest extent as biologically possible.
To facilitate defined heterodimerization, heterotrimerization or heterotetramerization of a group of CARs according to the present invention, each CAR molecule of the group of CARs comprises at least one domain which can mediate heterodimerization of two CAR molecules and thereby can induce, in a constitutive or conditional manner, the formation of a defined non-covalent complex of two, three or four CAR molecules. This means that if the individual molecules of the group of CARs are co-expressed in cells and assembled into a non-covalent complex, said group of CARs (optionally dependent on the presence of one or more other polypeptides, each of which comprising at least an antigen binding moiety and being able to bind to a CAR molecule of the group) can mediate enhanced activation of those cells in response to target cells, i.e. cells expressing a selected combination of two or more target antigens, compared to the response to non-target cells, i.e. cells expressing only one target antigen or a fraction of the selected target antigens. This further means that the non-covalently complexed group of CARs can integrate several inputs, i.e. binding to different target antigens, into a defined output signal, i.e. activation of cells expressing said group of CARs. This capacity of said group of CARs represents a so-called logic AND gate function and was surprisingly efficient in discriminating target cells (i.e. cells expressing a given target combination) from non-target cells (i.e. cells expressing only one antigen).
Existing concepts for designing CARs with logic AND gate function have not exploited the synergistic amplification of individual affinities of multiple binding moieties of individual molecules directed against different target antigens, (Roybal and Lim, Annu Rev Immunol. 2017; 35:229; Chen et al., Curr Opin Immunol. 2018; 51:103-110; Labanieh et al., Nat Biomed Eng. 2018; 2:377-391). There is, however, a strategy in which binding moieties directed against two different target antigens (antigens A and B) are fused in tandem, i.e. serially linked into a single polypeptide chain, to conventional CAR backbones. Such CARs (called Tandem-CARs) were designed to mediate logic OR gate function, i.e. those CARs trigger a strong signal in cells expressing said CARs in response to target cells expressing either antigen A, OR antigen B, OR both antigens. This is due to the fact that those CARs can trigger a signal also upon monovalent interaction, because their binding moieties have high affinity (Hegde et al., J Clin Invest. 2016; 126(8):3036-3052; Grada et al., Mol Ther Nucleic Acids. 2013; 2:e105; Zah et al., Cancer Immunol Res. 2016; 4(6):498-508; De Munter et al., Int J Mol Sci. 2018 Jan. 30; 19(2) pii: E403). Examples of such CARs have been disclosed in US20170107285 A1 and US20180111992 A1. Some of the reported CARs have also AND gate function (Hedge et al., J Clin Invest. 2016; 126(8):3036-3052). The AND gate function of those reported CARs, however, is very limited due to the high affinity of the individual binding moieties and due to the fact that at least a fraction of currently used CAR backbones, when expressed in cells, form homodimers resulting from disulphide bonds between extracellular cysteine residues.
As mentioned above, the prevention or minimization of dimerization or multimerization of CAR molecules with identical target antigen specificity is an essential precondition for generating a group of CARs with AND gate function according to the present invention. Dimerization or multimerization of identical CAR molecules results in amplification of affinities of binding moieties with identical target antigen specificity and thereby prevents exploitation of the avidity effect for logic AND gate function (i.e. specific recognition of antigen combinations).
For this reason, according to the present invention the ectodomain of each CAR molecule of the group in its “prevalent conformation” (i.e. natively folded conformation) is free of cysteine amino acid moieties which are able to form intermolecular disulphide bonds with other CAR molecules of the group, respectively. In other words, the extracellular domains of the CAR molecules of the group according to the present invention must not contain any cysteines which are not involved in intramolecular disulphide bonds (i.e. formed within a given CAR molecule of the group) in the natively folded conformation of the CAR. For example, the cysteines in the hinge region of CD8α, which can form intermolecular disulphide bonds (i.e. with other CAR molecules of the group) in the native conformation, need to be excluded by, e.g., mutation or deletion. On the other hand, cysteines which are engaged in intramolecular disulphide bonds in the native conformation of the CAR molecule (and therefore not accessible for the formation of intermolecular bonds with other CAR molecules) may be present in the CARs of the group of CARs according to the present invention. As an example, cysteines within Ig domains of antibody fragments (e.g. within scFvs), which form intramolecular disulphide bonds, may be present in the CAR molecules of the group of CARs according to the present invention. Since those cysteines in, e.g., scFvs are engaged in intramolecular disulphide bonds, they are not available for intermolecular disulphide bonds (if the CAR molecule is present in its prevalent, i.e. native, conformation), thereby avoiding the formation of homodimeric or homooligomeric CAR molecules of the group.
Furthermore, antigen binding moieties of current CARs are usually based on single-chain variable fragments (scFv) which tend to oligomerize due to intermolecular heterodimerization of 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). Since this uncontrolled dimerization or oligomerization can also occur between identical CAR molecules (i.e. CAR molecules with the same antigen-specificity), this potentially precludes efficient AND gate function. Therefore, the individual molecules of a group of CARs according to the present invention preferably do not contain scFv-based antigen binding moieties or other molecular components potentially leading to unwanted and uncontrolled covalent or non-covalent complex formation of CAR molecules of the group.
Generally, any non-covalent dimerization or oligomerization of identical CAR molecules mediated by other domains of the CAR molecules would preclude efficient AND gate function according to the present invention. Therefore, such non-covalent dimerization or oligomerization needs to be prevented or at least minimized to the greatest extent as biologically possible by exclusion or engineering of such domains.
Compared to Tandem-CARs, the basic design of a group of CARs according to the present invention facilitates the adaptation of linkers and spacers of the CAR molecules for optimizing the spatial requirements for efficient interaction with each of the different target antigens. The architecture of a group of CARs according to the present invention further facilitates the optimization of the CAR molecules with respect to the geometry of pulling forces between the CAR molecules and the target antigens. This is advantageous, because it is known for T cells that mechanical forces, generated upon antigen recognition by the actomyosin cytoskeleton, play an important role in organization of the immunological synapse and improve the efficiency of T cell activation and target cell killing (Basu and Huse, Trends Cell Biol. 2017; 27(4):241-254). It is conceivable that—similarly to pulling off an adhesive strip—the geometry of force transmission via the binding moieties of the CAR molecules will affect the stability of the interaction with the target antigens and thereby the signalling efficacy of CAR molecules. That is, while in Tandem-CARs the pulling force will mostly (or exclusively) act on the binding moiety that is adjacent to the transmembrane domain, in the group of CARs according to the present invention the pulling forces are transmitted in parallel in each of the CAR molecules of the group, thereby ensuring that each interaction between a binding moiety on a CAR molecule and a target antigen can contribute to the overall pulling force. Finally, the architecture of the group of CARs optionally enables reversible regulation of the function of said group of CARs by simply making heterodimerization of individual CAR molecules conditional. Thus, compared with Tandem-CARs, which are state of the art in CAR engineering, the group of CARs according to the present invention offer several critical advantages: (i) the ability to optimize linker lengths for each binding moiety individually, (ii) improved transmission of pulling forces and (iii) the option to regulate CAR function by conditional dimerization, trimerization or tetramerization.
The basic architecture of the molecular design of the group of CARs according to the present invention can be varied at specific sites without abrogating the logic AND gate function. This enables adaptation of the system to different demands. For example, the group of CARs can consist of two CAR molecules or, alternatively, also of three or four CAR molecules in order to enhance the avidity effect and/or to generate AND gate CAR complexes that are dependent on the presence of three or four different antigens, respectively, or to integrate a logic OR gate function into the group of CARs, e.g., for mediating recognition of antigen A in combination with either antigen B OR antigen C. That is, this can be considered as an AND/OR gate, because in this example the trimeric group of CARs responds to either antigens A AND B OR to antigens A AND C, i.e. A AND (B OR C). Similarly, a tetrameric group of CARs can be designed to respond to antigens (A OR B) AND (C OR D) or to antigens A AND (B OR C OR D). The group of CARs can also easily be designed to be functionally dependent on either soluble proteins or small molecules.
In preferred embodiments for conditional regulation of CAR function, the group of CARs can consist of CAR molecules each comprising at least an extracellular binding site to which other polypeptides each comprising at least an antigen binding moiety are able to bind. Such another polypeptide thereby is defined as a soluble protein that does not belong to the group of CARs and can non-covalently bind to a binding site in a CAR molecule of the group either directly or indirectly via a covalent modification on the other polypeptide such as, for example, a covalently bound fluorescein isothiocyanate (FITC) molecule. In this preferred embodiment, the defined infusion of this other polypeptide enables the control of the function of the group of CARs. This strategy of regulating CAR function by administering a soluble antigen binding protein, which is well known in the CAR field (Cho et al., Cell. 2018; 173(6):1426-1438; Ma et al., Proc Natl Acad Sci USA. 2016; 113(4):E450-458; Urbanska et al., Cancer Res. 2012; 72(7):1844-1852) and now being tested in the clinic (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), can also be incorporated into the group of CARs according to the present invention. In this case, in principle, the low affinity-binding does not necessarily need to take place via the antigen binding moiety of the other polypeptide but can also take place at the CAR molecule via the binding site to which the other polypeptide, which comprises at least an antigen binding moiety, is able to bind.
Alternatively, the function of the group of CARs according to the present invention can also be regulated by conditional heterodimerization, heterotrimerization or heterotetramerization. Therefore, in a preferred embodiment the formation of a non-covalent complex of two, three or four CAR molecules of the group is induced by one or more regulating molecules that are able to bind under physiological conditions to at least one member of a pair of heterodimerization domains of the group of CARs. In principle, a regulating molecule can be any molecule binding to at least one heterodimerization domain and capable of inducing or reducing interaction of the members of a pair of heterodimerization domains. Those molecules are typically small molecules, however, can also be, for example, soluble proteins accumulating in the stroma of tumours, which are frequently proteins that themselves natively heterodimerize (e.g., the subunits of heterodimeric cytokines as, e.g., IL-12).
In order to exclude the possibility of fratricide by crosslinking of CAR molecules between different cells, the heterodimerization domains are preferably integrated in the endodomains and/or in the transmembrane domains of the CAR molecules of the group of CARs, more preferably in the endodomains.
In order to reduce the payload of vectors used to stably express the group of CARs in cells, the group of CARs preferably comprises three, especially two CAR molecules. In order to further reduce the complexity, each CAR molecule of the group comprises preferably only a single antigen binding moiety or optionally only a single extracellular binding site to which another polypeptide is able to bind, wherein the other polypeptide comprises at least an antigen binding moiety.
In order to integrate OR gate function in a group of CARs for example for mediating recognition of antigen A in combination with either antigen B OR antigen C, the CAR molecules of the group can also contain two antigen binding moieties or two extracellular binding sites to which another polypeptide is able to bind, wherein the other polypeptide comprises at least an antigen binding moiety.
For reducing potential immunogenicity of CAR molecules, the CAR molecules of a group of CARs preferably contain extracellular binding sites to which other polypeptides is able to bind, wherein the other polypeptide comprises at least an antigen binding moiety.
For applications, where conditional regulation of CAR function is not required, the CAR molecules of the group can contain heterodimerization domains, which do not require the presence of a regulating molecule, resulting in constitutive complex formation. Optionally, as a safety means in case of unintended adverse effects, the CAR molecules can also contain heterodimerization domains which mediate constitutive heterodimerization but can additionally bind regulating molecules that induce the dissociation of the heterodimerization domains.
As mentioned above, the basic architecture of the CAR molecules of the group, according to the present invention, can be adapted to the needs of different applications. The order of the domains in the CAR molecules of the group from the extracellular to the intracellular side preferably conforms on the surface of cell to the following basic architecture: an antigen binding moiety or a binding site to which another polypeptide comprising at least an antigen binding moiety is able to bind, optionally a linker for spatial optimization of an optional second antigen binding moiety or an optional second binding site to which another polypeptide comprising at least an antigen binding moiety is able to bind, preferably a hinge region for spatial optimization, and a transmembrane domain. The transmembrane domain is preferably followed in at least one CAR molecule by a signalling region comprising a co-stimulatory domain, wherein preferably this co-stimulatory signalling region, or optionally the transmembrane domain, is followed by at least one heterodimerization domain, and further, in at least one CAR molecule, by a signalling region comprising at least one ITAM, wherein the order of the co-stimulatory and the ITAM-containing signalling region can be inverted. CAR molecules that do not comprise an ITAM either lack a co-stimulatory signalling region, or comprise one co-stimulatory signalling region, or two co-stimulatory signalling regions, or even more co-stimulatory signalling regions, but preferably a maximum of two co-stimulatory signalling regions, or even more preferably only one co-stimulatory signalling region. Generally, the heterodimerization domains, of which at least one is mandatory for each CAR molecule of the group, can be located alternatively or additionally in the ectodomain or the transmembrane domain, however, preferably between the transmembrane domain and a signalling region, and/or especially between two signalling regions and/or especially at the intracellular end of the CAR molecules. Finally, any two adjacent components (antigen binding moieties, binding sites to which another polypeptide comprising at least an antigen binding moiety is able to bind, hinge regions, transmembrane domains, signalling regions, dimerization domains) of a CAR molecule of the group can optionally be separated by a linker.
Different groups of CARs directed against different combinations of target antigens can also be co-expressed in a cell, for example, to inhibit immune escape of tumours triggered by the loss of target antigens. A group of CARs can also be co-expressed with any other protein in a given cell.
An antigen binding moiety suitable for use in a group of CARs according to the present invention can be any antigen binding polypeptide (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), a wide variety of which are known in the art (Simeon et al., Protein Cell. 2017; Gilbreth et al., Curr Opin Struct Biol. 2012; 22(4):413-420; Koide et al., ACS Chem Biol. 2009; 4(5):325-334; Traxlmayr et al., J Biol Chem. 2016; 291(43):22496-22508). Meanwhile, many more non-antibody binding proteins have been reported (Plückthun, Alternative Scaffolds: Expanding the options of antibodies. In: Little M, ed. New York: Cambridge University Press; 2009:244-271; Chapman et al., Cell Chem Biol. 2016; 23(5):543-553; Binz et al., Nat Biotechnol. 2005; 23(10):1257-1268; Vazquez-Lombardi et al., Drug Discov Today. 2015; 20(10):1271-1283), and, in fact, synthetic library design and selection can be applied to any protein which then can potentially serve as antigen binding moiety, too (Plückthun, Alternative Scaffolds: Expanding the options of antibodies. In: Little M, ed. New York: Cambridge University Press; 2009:244-271).
In some instances, the antigen binding moiety can be a single chain Fv (scFv), other antibody based recognition domains like cAb VHH (camelid antibody variable domains) and its humanized versions, IgNAR VH (shark antibody variable domains) and its humanized versions, sdAb VH (single domain antibody variable domains) or “camelized” antibody variable domains. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCRs (scTv, single chain two-domain TCR containing VaV) can also be suitable for use. Preferably, the antigen binding moiety of each molecule of the group of CARs comprises only one protein domain, preferably a human or non-human VH or VL single domain antibody (nanobody) or an engineered antigen binding moiety based on the Z-domain of staphylococcal Protein A, lipocalins, SH3 domains, fibronectin type III (FN3) domains, knottins, Sso7d, rcSso7d, Sac7d, Gp2, DARPins or ubiquitin; or a ligand, a receptor or a co-receptor which was chosen for or engineered for low affinity binding and lack of homotypic interaction. Ligands include, for example, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; etc.); 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; 14; 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, a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.); a cellular adhesion molecule (e.g., CD11a (Park et al., Sci Rep. 2017; 7(1):14366); etc); PD-1; and the like. The antigen binding moiety of each molecule of the group of CARs preferably does not cause undesired aggregation of the CAR molecules. As discussed above, such undesired dimerization or oligomerization of CAR molecules of the group can cause multivalent interaction with single-positive non-target cells. For this reason, the antigen binding moiety is preferably not a single-chain variable fragment (scFv) derived from a monoclonal antibody. With the aim of clinical applicability of the group of CARs according to the present invention, antigen binding moieties are preferably derived from human single protein domains (e.g., fibronectin type III domain (FN3) based Monobodies).
In some embodiments the ectodomains of the CAR molecules of the group comprise a hinge region interposed between an antigen binding moiety (or a binding site to which another polypeptide comprising at least an antigen binding moiety is able to bind) and the transmembrane domain, preferably a hinge region derived from CD8 alpha (amino acid sequence position 138-182 according to UniProtKB/Swiss-Prot P01732-1), or CD28 (amino acid sequence position 114-152 according to UniProtKB/Swiss-Prot P10747), or PD-1 (amino acid sequence position 146-170 according to UniProtKB/Swiss-Prot Q15116), wherein the sequences derived from CD8 alpha, CD28 or PD-1 can be N-terminally and/or C-terminally truncated and can have any length within the borders of the said sequence region, and wherein the cysteine residues in the said hinges derived from CD8 alpha and CD28 are deleted or replaced by other amino acid residues. In principle, the flexible membrane anchors and also other parts of many more receptors are suited for use in the hinge regions and/or transmembrane domains of CAR molecules of the group (Labanieh et al., Nat Biomed Eng. 2018; 2:377-391), provided that they are modified, if necessary, for preventing dimerization according to the present invention.
Depending on the individual structural requirements for optimal binding of a selected target antigen, the hinge region of a 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, hinge regions can comprise more than 50 amino acids, for example, when structured domains are integrated (e.g. from CD34 UniProt P28906-1 aa 42-140 for facilitating enrichment of CAR modified cells, as disclosed in US2018/0094044 A1).
Preferably, also other polypeptides, preferably glycine and glycine-serine polymers can be used for the hinges since both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between the CAR components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1992; 11173-11142). Therefore, for adjusting the CAR molecules for optimal binding to their target antigens, a hinge region interposed between an antigen binding moiety (or a binding site to which another polypeptide comprising at least an antigen binding moiety is able to bind) and the transmembrane domain can comprise a glycine polymer (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 one.
Each molecule of the group of CARs comprises a transmembrane domain for insertion into a eukaryotic cell membrane. Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. For example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC of human CD8 alpha (Uniprot P01732, amino acids (aa) 183-206) can be used. Further examples of suitable TM sequences include: human CD8 beta derived: LGLLVAGVLVLLVSLGVAIHLCC (Uniprot P10966, aa 173-195); human CD4 derived: ALIVLGGVAGLLLFIGLGIFFCVRC (Uniprot P01730, aa 398-422); human CD3 zeta derived: LCYLLDGILFIYGVILTALFLRV (Uniprot P20963, aa 31-53); human CD28 derived: FWVLVVVGGVLACYSLLVTVAFIIFWV (Uniprot P10747, aa 154-179); human CD134 (OX40) derived: VAAILGLGLVLGLLGPLAILLALYLL (Uniprot P43489, aa 215-240); human CD27 derived: ILVIFSGMFLVFTLAGALFLH (Uniprot P26842, aa 192-212); human CD278 (ICOS) derived: FWLPIGCAAFVVVCILGCILI (Uniprot Q9Y6W8, aa 141-161); human CD279 (PD-1) derived: VGVVGGLLGSLVLLVWVLAVI (Uniprot Q15116, aa 171-191); human DAP12 derived: GVLAGIVMGDLVLTVLIALAV (Uniprot O43914, aa 41-61); and human CD7 derived: ALPAALAVISFLLGLGLGVACVLA (Uniprot P09564, aa 178-201).
According to the present invention at least one molecule of the group of CARs contains an endodomain that can transduce a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM). An ITAM motif is YX1X2L/I, where X1 and X2 are independently any amino acid. An ITAM-containing endodomain can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 ITAM motifs. ITAM-containing portions of signal-transducing endodomains are preferably derived from any ITAM-containing protein and do not need to contain the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM-containing polypeptides include: DAP12; FCERlG (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).
In especially preferred embodiments, at least one signalling 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 (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable ITAM-containing domain 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 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 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 aa, of either of the amino acid sequences (2 isoforms) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLQLSTATKDTYDALHMQALPPR (Uniprot P20963-3) or MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQOLSTATKDTYDALHMQALPPR (Uniprot P20963-1), where the ITAM motifs are in bold and are underlined.
Likewise, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full length CD3 zeta 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 any of the amino acid sequences RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQOLSTATKDTYDALHMQALPPR (Uniprot P20963-3 aa 52-163), NQLYNELNLGRREEYDVLDKR (Uniprot P20963-3 aa 69-89), EGLYNELQKDKMAEAYSEIGMK (Uniprot P20963-3 aa 107-128), DGLYQOLSTATKDTYDALHMQ (Uniprot P20963-3 aa 138-158) where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from T-cell surface glycoprotein CD3 delta chain (also known as 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 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 170 aa, of either of the following amino acid sequences (2 isoforms): Uniprot P04234-1; Uniprot P04234-2.
Likewise, 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 amino acid sequence DQVYQPLRDRDDAQYSHLGGN (Uniprot P04234-1 aa 146-166), where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T-cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, 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 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 205 aa, of the amino acid sequence Uniprot P07766-1.
Likewise, a suitable ITAM-containing domain can comprise an ITAM-containing portion of the full length CD3 epsilon 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 amino acid sequence NPDYEPIRKGQRDLYSGLNQR (Uniprot P07766-1 aa 185-205), where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable ITAM-containing domain 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 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 amino acid sequence MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGF LTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFV LAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (Uniprot P09693-1), where the ITAMs are in bold and are underlined.
Likewise, 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 amino acid sequence DQLYQPLKDREDDQYSHLQGN (Uniprot P09693-1 aa 157-177), where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activation 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 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 (4 isoforms); Uniprot O43914-1; Uniprot O43914-2; Uniprot O43914-3; Uniprot X6RGC9-1.
Likewise, 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 ESPYQBLQGQRSDVYSDLNTQ (Uniprot O43914-1 aa 88-108), where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-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 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 MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV YTGLSTRNQETYETLKHEKPPQ (Uniprot P30273), where the ITAMs are in bold and are underlined.
Likewise, a suitable ITAM-containing domain can comprise an ITAM motif-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 amino acid sequence DGVYTGLSTRNQETYETLKHE (Uniprot P30273 aa 62-82), where the ITAMs are in bold and are underlined.
An ITAM-containing domain can also be derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound 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 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 150 aa, from about 150 aa to about 200 aa, or from about 200 aa to about 220 aa, of either of the amino acid sequences (2 isoforms) MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANV TWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQP PPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLN LDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-1) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANV TWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFR KRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (Uniprot P11912-2), where the ITAMs are in bold and are underlined.
Likewise, 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 amino acid sequence ENLYEGLNLDDCSMYEDISRG (Uniprot P11912-1 aa 185-205), where the ITAMs are in bold and are underlined.
Therefore, according to the present invention, the endodomain of at least one CAR molecule of the group of CARs comprises at least one ITAM, said ITAM is 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).
Since the number of ITAMs correlates with the signalling efficiency of CARs (James, Sci Signal. 2018; 11(531)), the group of CARs preferably comprises altogether at last three ITAMs, wherein the ITAMs can be confined to only a single CAR molecule of the group. Alternatively, several or all CAR molecules of the group can comprise at least one ITAM. In some embodiments the ITAM containing portions in the different endodomains of the CAR molecules of the group are derived from the same receptor, whereas in other embodiments the ITAM containing portions in the different endodomains of the CAR molecules of the group are derived from different receptors. In some embodiments the group of CARs comprises only one molecule comprising an ITAM-containing portion, preferably derived from CD3 zeta. In other embodiments the group of CARs consists of two molecules, wherein both comprise parts of the cytoplasmic domain derived from CD3 zeta. With respect to vector payload, the total number of ITAMs in a group of CARs is preferably between three and six. Furthermore, the ITAM containing sequences are preferably chosen and/or engineered for minimal nucleotide sequence homology, in order minimize the risk of homologous recombination.
In preferred embodiments, the endodomain of at least one CAR molecule of the group comprises a signalling region containing a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD2, CD6, CD27, CD30, CD40, GITR, and HVEM, whereby the co-stimulatory domains comprised by a group of CARs can optionally be derived from different co-stimulatory receptors.
A co-stimulatory domain suitable for inclusion in a co-stimulatory signalling region of a CAR molecule of the group of CARs can have a length of from about 30 aa to about 70 aa, e.g., a co-stimulatory 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, the co-stimulatory 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.
In especially preferred embodiments, the co-stimulatory domain in at least one molecule of the group of CARs is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitable co-stimulatory 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.
In preferred embodiments, the co-stimulatory domain in at least one molecule of the group of CARs is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable co-stimulatory 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 177-220.
In especially preferred embodiments, the co-stimulatory domain in at least one molecule of the group of CARs is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable co-stimulatory 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.
In preferred embodiments, the co-stimulatory domain in at least one molecule of the group of CARs is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). For example, a suitable co-stimulatory 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.
In other embodiments, the co-stimulatory domain in at least one molecule of the group of CARs can be derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, a suitable co-stimulatory 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.
In other embodiments, the co-stimulatory domain in at least one molecule of the group of CARs can be derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable co-stimulatory 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.
In other embodiments, the co-stimulatory domain in at least one molecule of the group of CARs can be derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain 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.
In other embodiments, the co-stimulatory domain in at least one molecule of the group of CARs can be derived from an 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 co-stimulatory 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 Q92956 aa 224-283.
In other embodiments, the co-stimulatory domain in at least one molecule of the group of CARs can be derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatory 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 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, or from about 160 aa to about 185 aa of the amino acid sequence Uniprot P28908 aa 409-595.
The molecules of the group of CARs can include a linker between any two adjacent domains (i.e. components of the CAR molecules). For example, a linker can be disposed between the transmembrane domain and a signalling region. As another example, a linker can be disposed between a signalling region and a heterodimerization domain. As another example, a linker can be disposed between two heterodimerization domains. As another example, a linker can be disposed between two signalling regions. As another example, a linker can be disposed between a transmembrane domain and a heterodimerization domain. As another example, a linker can be disposed in the ectodomain of a CAR molecule between two antigen binding moieties. As another example, a linker can be disposed in the ectodomain of a CAR molecule between two binding sites to which other polypeptides are able to bind. As another example, a linker can be disposed in the ectodomain of a CAR molecule between an antigen binding moiety and the transmembrane domain. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a binding site to which another polypeptide is able to bind and the transmembrane domain. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a signal sequence and an antigen binding moiety. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a signal sequence and a binding site to which another polypeptide is able to bind. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a signal sequence and a heterodimerization domain. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a heterodimerization domain and an antigen binding moiety. As another example, a linker can be disposed in the ectodomain of a CAR molecule between a heterodimerization domain and a binding site to which another polypeptide is able to bind. As yet another example, a linker can be disposed in the ectodomain of a CAR molecule between an antigen binding moiety and a binding site to which another polypeptide is able to bind.
A linker can be a peptide containing about 1 to about 40 amino acids in length. The linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers preferably have a sequence that results in a generally flexible peptide. Small amino acids, such as glycine, serine and alanine, are preferably used in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. Suitable linkers can be readily selected and can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGS)n and (GGGS)n, where n is an integer of at least one, or also 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. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
The molecules of a subject group of CARs can further include one or more additional polypeptide domains, where such domains include, for example, a signal sequence; an epitope tag; and/or a polypeptide that produces a detectable signal. Signal sequences that are suitable for use in a subject group of CARs include any eukaryotic signal sequence, including a naturally occurring signal sequence, a synthetic (e.g., man-made) signal sequence, etc. Suitable epitope tags include, e.g., 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: 81)), Hexahistidine tag (6xHIS; e.g., amino acid sequence HHHHHH (SEQ ID NO: 82)), and the like. Suitable detectable signal-producing proteins include, e.g., fluorescent proteins and the like. Suitable fluorescent proteins include, e.g., green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, 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 coloured proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
Complexation of groups of CARs comprising two CAR molecules can be mediated by a single heterodimerization domain per CAR molecule. In embodiments, where the group of CARs comprises three or four CAR molecules, at least one CAR molecule of the group preferably contains more than one heterodimerization domain, in order to facilitate the formation of trimers or tetramers through heterodimerization. In this case, the heterodimerization domains of that CAR molecule are preferentially members of different pairs of heterodimerization domains in order to prevent the formation of complexes comprising two or more identical CAR molecules of the group. The prevention of such homotypic interaction of CAR molecules is important, since any homotypic interaction would generate high avidity for a single type of target antigen. As a consequence this would lead to efficient signalling by the group of CARs in response to a single type of a target antigen and thereby would abrogate the dependence of efficient signalling on multivalent interaction with different target antigens.
According to the present invention, the heterodimerization domains integrated in the CAR molecules of the group can either mediate constitutive heterodimerization, or can be optionally regulated by regulating molecules. Heterodimerization of heterodimerization domains, for example, can be induced or reduced by the presence of regulating molecules. Heterodimerization of heterodimerization domains can also be constitutive and independent of regulating molecules. Domains mediating constitutive heterodimerization are well known in the art and successfully used in different applications, such as for example, coiled-coil interaction domains (Thompson et al., ACS Synth Biol. 2012; 1(4):118-29; Cho et al., Cell. 2018; 173(6):1426-1438), however, there are many more suitable domains existent in nature. In general, any pair of polypeptides, which bind to each other, and which can be expressed in CAR molecules, is suitable for mediating constitutive heterodimerization of two CAR molecules of the group of CARs according to the present invention. Below, a lipocalin-fold molecule based system is described (chapter 8.1.2), which can be engineered for conditional but also for constitutive heterodimerization. A lipocalin-fold molecule based system, contrary to coiled-coil domains, can furthermore easily be engineered for off-switching heterodimerization by a regulating molecule.
8.1.1. Conditional Heterodimerization of CAR Molecules Based on Ligand Binding Domains from Nuclear Receptors:
In preferred embodiments at least two CAR molecules of a group of CARs according to the present invention can be heterodimerized by a pair of heterodimerization domains comprising one member which is a ligand binding domain (LBD) from a nuclear receptor and a second member which is a co-regulator peptide. LBDs derived from nuclear receptors upon binding of appropriate small molecules (i.e., regulating molecules according the present invention) can heterodimerize with respective co-regulator peptides. This system can be used for heterodimerization of proteins of interest. Suitable sequences of LBDs and co-regulator peptides together with suitable regulating molecules have been disclosed for example in US 2017/0306303 A1. Suitable LBDs can 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 Adrostrane Receptor, SF-1, LRH-1, DAX-1, SHP, TLX, PNR, NGF1-B-alpha, NGF1-B-beta, NGF1-B-gamma, ROR-alpha, ROR-beta, ROR-gamma, ERR-alpha, ERR-beta, ERR-gamma, GCNF, TR2/4, HNF-4, COUP-TF-alpha, COUP-TF-beta and COUP-TF-gamma.
Abbreviations for nuclear receptors (synonymous with nuclear hormone receptors) are as follows. ER: Estrogen Receptor; PR: Progesterone Receptor; AR: Androgen Receptor; GR: Glucocorticoid Receptor; MR: Mineralocorticoid Receptor; RAR: Retinoic Acid Receptor; TR-alpha/beta: Thyroid Receptor; VDR: Vitamin D3 Receptor; EcR: Ecdysone Receptor; RXR: Retinoic Acid X Receptor; PPAR: Peroxisome Proliferator Activated Receptor; LXR: Liver X Receptor; FXR: Farnesoid X Receptor; PXR/SXR: Pregnane X Receptor/Steroid and Xenobiotic Receptor; SF-1: Steroidogenic Factor 1; DAX-1: Dosage sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, gene 1; LRH-1: Liver Receptor Homolog 1; SHP: Small Heterodimer Partner; TLX: Tail-less Gene; PNR: Photoreceptor-Specific Nuclear Receptor; NGF1-B: Nerve Growth Factor; ROR: RAR related orphan receptor; ERR: Estrogen Related Receptor; GCNF: Germ Cell Nuclear Factor; TR2/4: Testicular Receptor; HNF-4: Hepatocyte Nuclear Factor; COUP-TF: Chicken Ovalbumin Upstream Promoter, Transcription Factor.
Mineralocorticoid Receptor:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of a mineralocorticoid receptor (MR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an MR (Uniprot P08235).
For example, the LBD of a MR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).
For example, the LBD of a MR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P08235 aa 686-984 and has a length of from about 250 amino acids to 299 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 299 amino acids).
For example, the LBD of a MR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P08235 aa 737-984 and has a length of from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).
For example, the LBD of a MR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P08235 aa 686-984 with S810L substitution, and has a length of from about 250 amino acids to 299 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 299 amino acids).
For example, the LBD of a MR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P08235 aa 737-984 with S810L substitution, and has a length of from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of an MR, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence SLTARHKILHRLLQEGSPSDI (Uniprot Q15788 aa 681-701), where the co-regulator peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Where one member of the pair of heterodimerization domains is an LBD of an MR, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence QEAEEPSLLKKLLLAPANTQL (Uniprot Q9UBK2 aa 136-156), where the co-regulator peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Where one member of the pair of heterodimerization domains is an LBD of an MR, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence SKVSQNPILTSLLQITGNGGS (Uniprot Q15648 aa 596-616), where the co-regulator peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the co-regulator 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).
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 (AR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an AR (Uniprot P10275).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 619-919 and has a length of from about 250 amino acids to 301 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 301 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 690-919 and has a length of from about 190 amino acids to 230 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, or from 210 amino acids to 230 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 619-919 with T877A substitution, and has a length of from about 250 amino acids to 301 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 301 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 690-919 with T877A substitution, and has a length of from about 190 amino acids to 230 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, or from 210 amino acids to 230 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 619-919 with F876L substitution, and has a length of from about 250 amino acids to 301 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 301 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 690-919 with F876L substitution, and has a length of from about 190 amino acids to 230 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, or from 210 amino acids to 230 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 619-919 with F876L and T877A substitution, and has a length of from about 250 amino acids to 301 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, or from 275 amino acids to 301 amino acids).
For example, the LBD of an AR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10275 aa 690-919 with F876L T877A substitution, and has a length of from about 190 amino acids to 230 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, or from 210 amino acids to 230 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of an AR, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence ESKGHKKLLQLLTCSSDDR (Uniprot Q9Y6Q9 aa 614-632) where the co-regulator peptide has a length of from about 19 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Progesterone Receptor:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of a progesterone receptor (PR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a PR (Uniprot P06401).
For example, the LBD of a PR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 9-256; and has a length of from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).
For example, the LBD of a PR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P06401 aa 678-933 and has a length of from about 200 amino acids to 256 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 256 amino acids; e.g., has a length of 256 amino acids).
For example, the LBD of a PR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P06401 aa 686-933 and has a length of from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 250 amino acids; e.g., has a length of 248 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a PR, the second member of the dimerization pair can be a co-regulator peptide comprising the amino acid sequence GHSFADPASNLGLEDIIRKALMGSF (Uniprot O75376 aa 2251-2275) where the co-regulator peptide has a length of from about 25 amino acids to about 50 amino acids (e.g., the co-regulator peptide has a length of from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids).
Thyroid Hormone Receptor-Beta:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of thyroid hormone receptor-beta (TR-beta). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a TR-beta (Uniprot P10828).
For example, the LBD of a TR-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 has a length of from about 200 amino acids to 250 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 230 amino acids, from 230 amino acids to 240 amino acids, or from 240 amino acids to 250 amino acids).
For example, the LBD of a TR-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10828 aa 202-461 and has a length of from about 200 amino acids to 260 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 260 amino acids; e.g., has a length of 260 amino acids).
For example, the LBD of a TR-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10828 aa 216-461 and has a length of from about 200 amino acids to 246 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 246 amino acids; e.g., has a length of 246 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a TR-beta, the second member of the pair can be an NCOA3/SRC3 polypeptide, for example comprising the amino acid sequence Uniprot Q9Y6Q9 aa 627-829 or Uniprot Q9Y6Q9 aa 673-750 or Uniprot Q15596 aa 721-1021.
Estrogen Receptor-Alpha:
In preferred embodiments, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of estrogen receptor-alpha (ER-alpha). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an ER-alpha (Uniprot P03372).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 has a length of from about 200 amino acids to 240 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 230 amino acids, from 230 amino acids to 235 amino acids, or from 235 amino acids to 240 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 305-533 and has a length of from about 180 amino acids to 229 amino acids (e.g., has a length of from 180 amino acids to 200 amino acids, or from 200 amino acids to 229 amino acids; e.g., has a length of 229 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 282-595 and has a length of from about 250 amino acids to 314 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, from 275 amino acids to 300 amino acids, or from 300 amino acids to 314 amino acids; e.g., has a length of 314 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 310-547 and has a length of from about 190 amino acids to 238 amino acids (e.g., has a length of from 190 amino acids to 220 amino acids, or from 220 amino acids to 238 amino acids; e.g., has a length of 238 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 305-533 with substitution D351Y; and has a length of from about 180 amino acids to 229 amino acids (e.g., has a length of from 180 amino acids to 200 amino acids, or from 200 amino acids to 229 amino acids; e.g., has a length of 229 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 282-595 with substitution D351Y; and has a length of from about 250 amino acids to 314 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, from 275 amino acids to 300 amino acids, or from 300 amino acids to 314 amino acids; e.g., has a length of 314 amino acids).
For example, the LBD of an ER-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P03372 aa 310-547 with substitution D351Y; and has a length of from about 190 amino acids to 238 amino acids (e.g., has a length of from 190 amino acids to 220 amino acids, or from 220 amino acids to 238 amino acids; e.g., has a length of 238 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of an ER-alpha, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence DAFQLRQLILRGLQDD (SEQ ID NO: 10), where the co-regulator peptide has a length of from about 16 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Where one member of the pair of heterodimerization domains is an LBD of an ER-alpha, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence SPGSREWFKDMLS (SEQ ID NO: 11), where the co-regulator peptide has a length of from about 13 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Estrogen Receptor-Beta (ER-Beta):
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of estrogen receptor-beta (ER-beta). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an ER-beta (Uniprot Q92731).
For example, the LBD of an ER-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 has a length of from about 200 amino acids to 243 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 230 amino acids, from 230 amino acids to 235 amino acids, or from 235 amino acids to 243 amino acids).
For example, the LBD of an ER-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot Q92731 aa 260-502; and has a length of from about 200 amino acids to 243 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 230 amino acids, from 230 amino acids to 235 amino acids, or from 235 amino acids to 243 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of an ER-beta, the second member of the dimerization pair can be a co-regulator peptide comprising the amino acid sequence PRQGSILYSMLTSAKQT (SEQ ID NO: 12), where the co-regulator peptide has a length of from about 17 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Peroxisome Proliferator-Activated Receptor-Gamma:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a PPAR-gamma (Uniprot P37231).
For example, the LBD of a PPAR-gamma can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences: Uniprot Q4H2X4 aa 176-417; Uniprot P37233.1 aa 129-395; 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 has a length of from about 200 amino acids to 269 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 250 amino acids, or from 250 amino acids to 269 amino acids).
For example, the LBD of a PPAR-gamma can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P37231 aa 174-475 and has a length of from about 150 amino acids to 202 amino acids (e.g., has a length of from 150 amino acids to 160 amino acids, from 160 amino acids to 170 amino acids, from 170 amino acids to 190 amino acids, or from 190 amino acids to 202 amino acids).
For example, the LBD of a PPAR-gamma can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P37231 aa 181-475 and has a length of from about 200 amino acids to 269 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 250 amino acids, or from 250 amino acids to 269 amino acids).
For example, the LBD of a PPAR-gamma can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P37231 aa 205-475 and has a length of from about 200 amino acids to 269 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 250 amino acids, or from 250 amino acids to 271 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a PPAR-gamma, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence CPSSHSSLTERHKILHRLLQEGSPS (Uniprot Q15788-1 aa 676-700), where the co-regulator peptide has a length of from about 25 amino acids to about 50 amino acids (e.g., the co-regulator peptide has a length of from 25 amino acids to 28 amino acids, from 28 amino acids to 29 amino acids, from 29 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a PPAR-gamma, the second member of the pair can be a co-regulator peptide comprising the amino acid sequence PKKENNALLRYLLDRDDPSDV (SEQ ID NO: 13) or PKKKENALLRYLLDKDDTKDI (Uniprot Q15596-1 aa 737-757), where the co-regulator peptide has a length of from about 21 amino acids to about 50 amino acids (e.g., the co-regulator 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).
Glucocorticoid Receptor:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of glucocorticoid receptor (GR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a GR having the amino acid sequence Uniprot P04150-3.
For example, the LBD of a GR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one 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 has a length of from about 200 amino acids to 247 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, from 225 amino acids to 230 amino acids, from 230 amino acids to 240 amino acids, or from 240 amino acids to 247 amino acids).
For example, the LBD of a GR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P04150-3 aa 532-778 and has a length of from about 200 amino acids to 247 amino acids (e.g., has a length of from 200 amino acids to 225 amino acids, or from 225 amino acids to 247 amino acids; e.g., has a length of 247 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a GR, the second member of the pair can be an NCOA2/SRC2 polypeptide, for example, comprising the amino acid sequence Uniprot Q15788 aa 1172-1441 or a fragment thereof, or Uniprot Q15596 aa 320-1021 or a fragment thereof.
Vitamin D Receptor:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of vitamin D receptor (VDR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a VDR (Uniprot P11473).
For example, the LBD of a VDR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one of the following amino acid sequences: Uniprot O42392.1 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 from about 250 amino acids to 310 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, from 275 amino acids to 300 amino acids, or from 300 amino acids to 310 amino acids).
For example, the LBD of a VDR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P11473 aa 124-426 and has a length of from about 250 amino acids to 303 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, from 275 amino acids to 300 amino acids, or from 300 amino acids to 303 amino acids).
Where one member of the pair of heterodimerization domains is an LBD of a VDR, the second member of the pair can be an NCOA1/SRC1 polypeptide, for example, comprising the amino acid sequence Uniprot Q15788 aa 1172-1441 or a fragment thereof, or Uniprot Q15596 aa 320-1021 or a fragment thereof.
For example, in some cases, where one member of the pair of heterodimerization domains is an 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, where the co-regulator peptide has a length of from about from about 8 amino acids to about 50 amino acids (e.g., the co-regulator peptide has a length of from about 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).
Thyroid Hormone Receptor-Alpha:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of thyroid hormone receptor-alpha (TR-alpha). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a TR-alpha (Uniprot P10827-2).
For example, the LBD of a TR-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to 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 has a length of from about 190 amino acids to about 245 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, from 210 amino acids to 230 amino acids, or from 230 amino acids to 245 amino acids).
For example, the LBD of a TR-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10827-2 aa 162-404 and has a length of from about 190 amino acids to about 243 amino acids (e.g., has a length of from 190 amino acids to 210 amino acids, from 210 amino acids to 230 amino acids, or from 230 amino acids to 243 amino acids).
A suitable co-regulator peptide for TR-alpha can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
Retinoic Acid Receptor-Beta:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of retinoic acid receptor-beta (RAR-beta). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a RAR-beta (Uniprot P10826-2).
For example, the LBD of a RAR-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to one 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 has a length of from about 180 amino acids to about 235 amino acids (e.g., has a length of from 180 amino acids to 200 amino acids, from 200 amino acids to 220 amino acids, or from 220 amino acids to 235 amino acids).
For example, the LBD of a RAR-beta can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P10826-2 aa 179-409 and has a length of from about 180 amino acids to about 231 amino acids (e.g., has a length of from 180 amino acids to 200 amino acids, from 200 amino acids to 220 amino acids, or from 220 amino acids to 231 amino acids).
A suitable co-regulator peptide for RAR-beta can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
Where one member of a pair of heterodimerization domains is an LBD of a RAR-beta, the other member of the dimerization pair can be an NCOA1/SRC1 polypeptide, for example comprising the amino acid sequence Uniprot Q15788 aa 1172-1441 or a fragment thereof.
Where one member of a pair of heterodimerization domains is an LBD of a RAR-beta, the other member of the dimerization pair can be an NCOA2/SRC2 polypeptide, for example comprising the amino acid sequence Uniprot Q15596 aa 320-1021 or a fragment thereof.
Farnesoid X Receptor:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of farnesoid X receptor (FXR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an FXR having the amino acid sequence Uniprot Q96RI1-2.
For example, the LBD of an FXR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot Q96RI1-2 aa 237-472; and has a length of from about 100 amino acids to about 136 amino acids (e.g., has a length of from 100 amino acids to 110 amino acids, from 110 amino acids to 120 amino acids, or from 120 amino acids to 136 amino acids).
A suitable co-regulator peptide for an FXR can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
LXR-Alpha:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of liver X receptor-alpha (LXR-alpha). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an LXR-alpha having the amino acid sequence Uniprot Q13133-1.
For example, the LBD of an LXR-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot Q13133-1 aa 182-447; and has a length of from about 200 amino acids to about 266 amino acids (e.g., has a length of from 200 amino acids to 220 amino acids, from 220 amino acids to 240 amino acids, or from 240 amino acids to 266 amino acids).
A suitable co-regulator peptide for an LXR-alpha can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
ROR-Gamma:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of a retinoid-related orphan receptor gamma (ROR-gamma). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an ROR-gamma having the amino acid sequence Uniprot P51449-2.
For example, the LBD of an ROR-gamma can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P51449-2 aa 237-497; and has a length of from about 200 amino acids to about 261 amino acids (e.g., has a length of from 200 amino acids to 220 amino acids, from 220 amino acids to 240 amino acids, or from 240 amino acids to 261 amino acids).
A suitable co-regulator peptide for an ROR-gamma can be an NCORNR peptide (CDPASNLGLEDIIRKALMGSFDDK, Uniprot Q7Z516-1 aa 2160-2182).
A suitable co-regulator peptide for an ROR-gamma can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
RXR-Alpha:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of a retinoid-X receptor-alpha (RXR-alpha). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of an RXR-alpha having the amino acid sequence Uniprot P19793-1.
For example, the LBD of an RXR-alpha can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot P19793-1 aa 225-462; and has a length of from about 190 amino acids to about 238 amino acids (e.g., has a length of from 190 amino acids to 200 amino acids, from 200 amino acids to 210 amino acids, or from 210 amino acids to 238 amino acids).
A suitable co-regulator peptide for an RXR-alpha can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
PXR:
In some cases, an LBD suitable for inclusion as a member of a pair of heterodimerization domains can be an LBD of a Pregnane X Receptor (PXR). For example, in some cases, the LBD can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the LBD of a PXR having the amino acid sequence Uniprot O75469-1. In some cases, the LBD comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 143-428 of the amino acid sequence Uniprot O75469-1. In some cases, the LBD comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 205-434 of the amino acid sequence depicted in Uniprot O75469-1.
For example, the LBD of a PXR can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence Uniprot 075469-1 aa 130-434; and has a length of from about 250 amino acids to about 302 amino acids (e.g., has a length of from 250 amino acids to 275 amino acids, from 275 amino acids to 290 amino acids, or from 290 amino acids to 302 amino acids).
A suitable co-regulator peptide for a PXR can be an SRC1 polypeptide or a fragment thereof (e.g., a peptide of from 8 amino acids to 50 amino acids in length, derived from an SRC1 polypeptide).
Suitable co-regulator polypeptides include full-length naturally-occurring nuclear hormone co-regulator polypeptides. Suitable co-regulator polypeptides include fragments of naturally-occurring nuclear hormone co-regulator polypeptides. Suitable co-regulator polypeptides include synthetic or recombinant nuclear hormone co-regulator polypeptides.
Suitable co-regulator polypeptides can have a length of from 8 amino acids to 2000 amino acids. Suitable co-regulator polypeptides can have a length of from 8 amino acids to 50 amino acids, e.g., 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 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. Suitable co-regulator polypeptides can have a length of from 50 amino acids to 100 amino acids, e.g., from 50 amino acids to 60 amino acids, from 60 amino acids to 70 amino acids, from 70 amino acids to 80 amino acids, from 80 amino acids to 90 amino acids, or from 90 amino acids to 100 amino acids. Suitable co-regulator polypeptides can have a length of from 100 amino acids to 200 amino acids, from 200 amino acids to 300 amino acids, from 300 amino acids to 400 amino acids, from 400 amino acids to 500 amino acids, from 500 amino acids to 600 amino acids, from 600 amino acids to 700 amino acids, from 700 amino acids to 800 amino acids, from 800 amino acids to 900 amino acids, or from 900 amino acids to 1000 amino acids. Suitable co-regulator polypeptides can have a length of from 1000 amino acids to 2000 amino acids.
Suitable co-regulator peptides include Steroid Receptor Coactivator (SRC)-1, SRC-2, SRC-3, TRAP220-1, TRAP220-2, NR0B1, NRIP1, CoRNR box, 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, RIP140-3, RIP140-4, RIP140-5, RIP140-6, RIP140-7, RIP140-8, RIP140-9, PRIC285-1, PRIC285-2, PRIC285-3, PRIC285-4, and PRIC285-5.
A co-regulator polypeptide suited for heterodimerization with a respective LBD dimerization partner preferably 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 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; and preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a stretch of from 8 to 50 contiguous amino acids of the amino acid sequences: SRC1 (Uniprot Q15788-1), SRC2 (Uniprot Q15596-1), SRC3 (Uniprot 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 Q9HOE9-2), 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);
In preferred embodiments, a suitable co-regulator peptide comprises an LXXLL motif, where X is any amino acid; where the co-regulator peptide has a length of from 8 amino acids to 50 amino acids, e.g., from 8 amino acids to 10 amino acids, from 10 amino acids to 12 amino acids, from 12 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, from 30 amino acids to 35 amino acids, from 35 amino acids to 40 amino acids, from 40 amino acids to 45 amino acids, or from 45 amino acids to 50 amino acids.
Examples of suitable co-regulator peptides are as follows:
In some cases, a given LBD can be paired with two or more different co-regulator polypeptides. For example, PPAR-gamma (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: 13; 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: 11), 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: 11), 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: 13; 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: 13; 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: 13; Uniprot Q9Y6Q9-5 aa 614-632), or TRAP220 (Uniprot Q15648-1 aa 596-616; Uniprot Q15648-1 aa 637-657).
Where one member of a pair of heterodimerization domains is an LBD of a nuclear hormone receptor, at least one type of the used regulating molecules are able to bind the LBD in a first CAR molecule of the group which then can heterodimerize with a co-regulator peptide in a second CAR molecule of the group.
Suitable regulating molecules for LBD-based heterodimerization systems are known in the art. Examples of regulating 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]phenanthren-3-one); cortisone ((8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,12,14,15,16-decahydrocyclopenta[a]phenanthrene-3,11-dione); 18-hydroxycorticosterone ((8S,9S,10R,11S,13R,14S,17S)-11-hydroxy-17-(2-hydroxyacetyl)-13-(hydroxymethyl)-10-methyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); 1alpha-hydroxycorticosterone ((1S,8S,9S,10R,11S,13S,14S,17S)-1,11-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one); aldosterone ((8S,9S,10R,11S,13R,14S,17S)-11-hydroxy-17-(2-hydroxyacetyl)-10-methyl-3-oxo-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthrene-13-carbaldehyde); androstenedione ((8R,9S,10R,13S,14S)-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione); 4-hydroxy-androstenedione ((8R,9S,10R,13S,14S)-4-hydroxy-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione); 11beta-hydroxyandrostenedione ((8S,9S,10R,11S,13S,14S)-11-hydroxy-10,13-dimethyl-2,6,7,8,9,11,12,14,15,16-decahydro-1H-cyclopenta[a]phenanthrene-3,17-dione); androstanediol ((3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,17-diol); androsterone ((3R,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecahydrocyclopenta[a]phenanthren-17-one); epiandrosterone ((3S,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecahydrocyclopenta[a]phenanthren-17-one); adrenosterone ((8S,9S,10R,13S,14S)-10,13-dimethyl-1,2,6,7,8,9,12,14,15,16-decahydrocyclopenta[a]phenanthrene-3,11,17-trione); dehydroepiandrosterone ((3S,8R,9S,10R,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-17-one); dehydroepiandrosterone sulphate ([(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 sulphate); 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); 5alpha-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); 5beta-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); 5beta-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); 11beta-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]phenanthren-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′-oxolane]-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); bexlosteride ((4aR,10bR)-8-chloro-4-methyl-1,2,4a,5,6,10b-hexahydrobenzo[f]quinolin-3-one); bicalutamide (N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenyl)sulfonyl-2-hydroxy-2-methylpropanamide); N-butylbenzene-sulfonamide (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-butylbenzene-sulfonamide (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′-methylidenespiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthrene-17,2′-oxolane]-3-one); ORG-31806 ((7S,8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-7,13-dimethylspiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthrene-17,2′-oxolane]-3-one); ZK-112993 ((8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); ORG-31376 ((8S,11R,13S,14R,17S)-11-[4-(dimethylamino)phenyl]-13-methylspiro[1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]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-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); ZK-230211 ((8S,11R,13S,14S,17S)-11-(4-acetylphenyl)-17-hydroxy-13-methyl-17-(1,1,2,2,2-pentafluoroethyl)-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); ZK-136798; ZK-98229; ZK-98734 ((8S,11R,13S,14S,17R)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-[(Z)-3-hydroxyprop-1-enyl]-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); ZK-137316; Asoprisnil ((8S,11R,13S,14S,17S)-11-[4-[(E)-hydroxyiminomethyl]phenyl]-17-methoxy-17-(methoxymethyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one); 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)-17p-hydroxy-17α-(1,1,2,2,2-penta-fluoroethyl)estra-4,9-dien-3-one; 11β-(4-Acetylphenyl)-19,24-dinor-17,23-epoxy-17alpha-chola-4,9,20-trie-n-3-one; (Z)-11beta, 19-[4-(3-Pyridinyl)-o-phenylene]-17beta-hydroxy-17α-[3-hydroxy-1-propenyl]-4-androsten-3-one; 11beta-[4-(1-methylethenyl)phenyl]-17α-hydroxy-17beta-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); bazedoxifene (1-[[4-[2-(azepan-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methylindol-5-ol); falsodex ((7R,8R,9S,13S,14S,17S)-13-methyl-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol); clomifene (2-[4-[(E)-2-chloro-1,2-diphenylethenyl]phenoxy]-N,N-diethylethanamine); femarelle ( ); ormeloxifene (1-[2-[4-[(3R,4R)-7-methoxy-2,2-dimethyl-3-phenyl-3,4-dihydrochromen-4-yl]phenoxy]ethyl]pyrrolidine); toremifiene (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 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); 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); Thiazolidinedione: (eg. Rosiglitazone (5-[[4-[2-[methyl(pyridin-2-yl)amino]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); lobeglitazone (5-[[4-[2-[[6-(4-methoxyphenoxy)pyrimidin-4-yl]-methylamino]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione); troglitazone (5-[[4-[(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydrochromen-2-yl)methoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione)), farglitazar ((2S)-2-(2-benzoylanilino)-3-[4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]phenyl]propanoic acid); aleglitazar ((2S)-2-methoxy-3-[4-[2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy]-1-benzothiophen-7-yl]propanoic acid); and fenofibric acid (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid); benzopyranoquinoline A 276575, 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]phenanthren-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), paricalitol ((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]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-nor-vitamin D3, 2-methylene-19-nor-(24R)-1α,25-dihydroxyvitamin D2, 2-methylene-(20R,25S)-19,26-dinor-1α,25-dihydroxyvitamin D3, 2-methylene-19-nor-1α-hydroxy-pregnacalciferol, 1α-hydroxy-2-methylene-19-nor-homopregnacalciferol, (20R)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol, 2-methylene-19-nor-(20S)-1α-hydroxy-trishomopregnacalciferol, 2-methylene-23,23-difluoro-1α-hydroxy-19-nor-bishomopregnacalcifero-1,2-methylene-(20S)-23,23-difluoro-1α-hydroxy-19-nor-bishomopregnan-calciferol, (2-(3′ hydroxypropyl-1′,2′-idene)-19,23,24-trinor-(20S)-1α-hydroxyvitamin D3, 2-methylene-18,19-dinor-(20S)-1α,25-dihydroxyvitamin D3, and the like. Retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), all-trans-retinoic acid ((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), 9-cis-retinoic acid ((2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), tamibarotene (4-[(5,5,8,8-tetramethyl-6,7-dihydronaphthalen-2-yl)carbamoyl]benzoic acid), 13-cis-retinoic acid ((2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid), (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexeneyl)nona-2,4,6, -8-tetraenoic acid, 9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-nona-2,4,6,8-tetraenoic acid, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-napthoic 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]phenanthren-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 7alpha,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).
Where a pair of heterodimerization domains comprises an LBD of a mineralocorticoid receptor (MR) and a corresponding co-regulator peptide, a suitable regulating molecule includes spironolactone, and eplerenone. Spironolactone can be administered at a dose ranging from 10 to 35 mg per day, e.g., 25 mg per day.
Where a pair of heterodimerization domains comprises an LBD of an androgen receptor (AR) and a corresponding co-regulator peptide, a suitable regulating molecule includes cyproterone acetate, hydroxyflutamide, enzalutamide, ARN-509, 3,3′-diindolylmethane (DIM), bexlosteride, bicalutamide, N-butylbenzene-sulfonamide (NBBS), dutasteride, epristeride, finasteride, flutamide, izonsteride, ketoconazole, N-butylbenzene-sulfonamide, nilutamide, megestrol, a steroidal antiandrogen, and turosteride.
Where a pair of heterodimerization domains comprises an LBD of a progesterone receptor (PR) and a corresponding co-regulator peptide, a suitable regulating molecule includes mifepristone (RU-486; 11beta-[4 N,N-dimethylaminophenyl]-17beta-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one); Lilopristone (11beta-(4 N,N-dimethylaminophenyl)-17beta-hydroxy-17-((Z)-3-hydroxypropenyl) estra-4, 9-dien-3-one); onapristone (11beta-(4 N,N-dimethylaminophenyl)-17alpha-hydroxy-17-(3-hydroxypropyl)-13alpha-estra-4,9-dien-3-one); asoprisnil (benzaldehyde, 4-[(11beta, 17beta)-17-methoxy-17-(methoxymethyl)-3-oxoestra-4, 9-dien-11-yl]-1-(E)-oxim; J867); J912 (4-[17beta-Hydroxy-17alpha-(methoxymethyl)-3-oxoestra-4, 9-dien-11beta-yl]benzaldehyd-(1E)-oxim); and CDB-2914 (17alpha-acetoxy-11beta-(4-N,N-dimethylaminophenyl)-19-norpregna-4, 9-dien-3, 20-dione). Other suitable dimerization agents include, e.g., JNJ-1250132, (6alpha,11beta, 17beta)-11-(4-dimethylaminophenyl)-6-methyl-4′,5′-di-hydrospiro[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); (7beta, 11beta, 17beta)-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-[17beta-Methoxy-17alpha-(methoxymethyl)-3-oxoestra-4, 9-dien-11beta-yl] benzaldehyde-1-(E)-oxime; 4-[17beta-Methoxy-17alpha-(methoxymethyl)-3-oxoestra-4, 9-dien-11beta-yl]benzaldehyde-1-(E)-[O-(ethylamino) carbonyl] oxime; 4-[17beta-Methoxy-17alpha-(methoxymethyl)-3-oxoestra-4, 9-dien-11beta-yl]benzaldehyde-1-(E)-[O-(ethylthio)carbonyl]oxime; (Z)-6′-(4-cyanophenyl)-9, 11alpha-dihydro-17beta-hydroxy-17alpha-[4-(1-oxo-3-methylbutoxy)-1-butenyl]4′H-naphtho[3′,2′,1′; 10,9,11]estr-4-en-3-one; 11beta-(4-acetylphenyl)-17beta-hydroxy-17alpha-(1,1,2,2,2-penta-fluoroethyl)estra-4,9-dien-3-one; 11beta-(4-Acetylphenyl)-19,24-dinor-17,23-epoxy-17alpha-chola-4,9,20-trien-3-one; (Z)-11beta, 19-[4-(3-Pyridinyl)-o-phenylene]-17beta-hydroxy-17alpha-[3-hydroxy-1-propenyl]-4-androsten-3-one; 11beta-[4-(1-methylethenyl) phenyl]-17alpha-hydroxy-17beta-beta-hydroxypropyl)-13alpha-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, and drospirenone.
Where a pair of heterodimerization domains comprises an LBD of thyroid receptor-beta (TR-beta) and a corresponding co-regulator peptide, a suitable regulating molecule includes 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)-phenoxy acetic 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-oxido-[1,3,2]-dioxaphosphonane); QH2; and (3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl) phenoxy) methylphosphonic acid (MB07344).
Where a pair of heterodimerization domains comprises an LBD of estrogen receptor-alpha (ER-alpha) and a corresponding co-regulator peptide, a suitable regulating molecule includes tamoxifen, 4-OH-tamoxifen, raloxifene, lasofoxifene, bazedoxifene, falsodex, clomifene, femarelle, ormeloxifene, toremifiene, ospemifene, and ethinyl estradiol.
Where a pair of heterodimerization domains comprises an LBD of estrogen receptor-beta (ER-beta) and a corresponding co-regulator peptide, a suitable regulating molecule includes estradiol (E2; or 17-beta-estradiol), and ethinyl estradiol.
Where a pair of heterodimerization domains comprises an LBD PPAR-gamma and a corresponding co-regulator peptide, a suitable regulating molecule includes a thiazolidinedione (e.g., rosiglitazone, pioglitazone, lobeglitazone, troglitazone), farglitazar, aleglitazar, and fenofibric acid.
Where a pair of heterodimerization domains comprises an LBD of a GR and a corresponding co-regulator peptide, a suitable regulating molecule can be a selective GR agonist (SEGRA) or a selective GR modulator (SEGRM).
Where a pair of heterodimerization domains comprises an LBD of a GR and a corresponding co-regulator peptide, a suitable regulating molecule includes benzopyranoquinoline A 276575, Mapracorat, ZK 216348, 55D1E1, dexamethasone, prednisolone, prednisone, methylprednisolone, fluticasone propionate, beclomethasone-17-monopropionate, betamethasone, rimexolone, paramethasone, and hydrocortisone.
Where a pair of heterodimerization domains comprises an LBD of a VDR and a corresponding co-regulator peptide, a suitable regulating molecule can be 1,25-dihydroxyvitamin D3 (calcitriol), paricalitol, doxercalciferol, 25-hydroxyvitamin D3 (calcifediol), cholecalciferol, ergocalciferol, tacalciol, 22-dihydroergocalciferol, (6Z)-Tacalciol, 2-methylene-19-nor-20(S)-1alpha-hydroxy-bishomopregnacalciferol, 19-nor-26,27-dimethylene-20(S)-2-methylene-1alpha,25-dihydroxyvitamin D3, 2-methylene-1alpha,25-dihydroxy-(17E)-17(20)-dehydro-19-nor-vitamin D3, 2-methylene-19-nor-(24R)-1alpha,25-dihydroxyvitamin D2, 2-methylene-(20R,25S)-19,26-dinor-1alpha,25-dihydroxyvitamin D3, 2-methylene-19-nor-1alpha-hydroxy-pregnacalciferol, 1alpha-hydroxy-2-methylene-19-nor-homopregnacalciferol, (20R)-1alpha-hydroxy-2-methylene-19-nor-bishomopregnacalciferol, 2-methylene-19-nor-(20S)-1alpha-hydroxy-trishomopregnacalciferol, 2-methylene-23,23-difluoro-1alpha-hydroxy-19-nor-bishomopregnacalcifero-1,2-methylene-(20S)-23,23-difluoro-1alpha-hydroxy-19-nor-bishomopregna-n-calciferol, (2-(3′ hydroxypropyl-1′,2′-idene)-19,23,24-trinor-(20S)-1alpha-hydroxyvitamin D3, 2-methylene-18,19-dinor-(20S)-1alpha,25-dihydroxyvitamin D3, and the like.
Where a pair of heterodimerization domains comprises an LBD of a RAR-beta and a corresponding co-regulator peptide, a suitable regulating molecule can be retinoic acid, all-trans-retinoic acid, 9-cis-retinoic acid, tamibarotene, 13-cis-retinoic acid, (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexeneyl)nona-2,4,6, -8-tetraenoic acid, 9-(4-methoxy-2,3,6-trimethyl-phenyl)-3,7-dimethyl-nona-2,4,6,8-tetraenoic acid, 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-napthoic 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-butyrate, retinoyl pinacol, and retinoyl cholesterol.
Where a pair of heterodimerization domains comprises an FXR and a corresponding co-regulator peptide, a suitable regulating molecule includes 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).
Where a pair of heterodimerization domains comprises an LBD of LXR-alpha and a corresponding co-regulator peptide, a suitable regulating molecule includes 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).
Where a pair of heterodimerization domains comprises an LBD of an ROR-gamma and a corresponding co-regulator peptide, a suitable regulating molecule includes 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).
Where a pair of heterodimerization domains comprises an LBD of an ROR-gamma and a corresponding co-regulator peptide, a suitable regulating molecule includes 7beta, 27-dihydroxycholesterol, and 7alpha, 27-dihydroxycholesterol.
Where a pair of heterodimerization domains comprises an LBD of an RXR-alpha and a corresponding co-regulator peptide, a suitable regulating molecule includes 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).
Where a pair of heterodimerization domains comprises an LBD of a PXR and a corresponding co-regulator peptide, a suitable regulating molecule can be rifampicin, chlotrimazole, and lovastatin.
In other preferred embodiments at least two CAR molecules of a group of CARs according to the present invention can 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 EP17208924.5 filed on 20 Dec. 2017.
According to a preferred embodiment, a lipocalin-fold based heterodimerization system comprises:
(a) a lipocalin-fold molecule
(b) a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, and
(c) a lipocalin-fold binding interaction partner, wherein the lipocalin-fold molecule can bind to the 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 which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand,
and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 μM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.
According to a further preferred embodiment, a lipocalin-fold based heterodimerization system comprises:
(a) a lipocalin-fold molecule
(b) a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, and
(c) a lipocalin-fold binding interaction partner,
wherein the lipocalin-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; and
wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand in the second conformation binds to the lipocalin-fold binding interaction partner with an affinity which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand in the first conformation,
and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 μM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.
This lipocalin-fold molecule based system for conditional heterodimerization generally relies on a substantial difference in the affinities of the lipocalin-fold molecule to the lipocalin-fold binding interaction partner depending on whether the lipocalin-fold ligand is bound or not. It is preferred that the affinity window (i.e. the affinities of the lipocalin-fold binding interaction partner to the lipocalin-fold molecule bound or not bound to the lipocalin-fold ligand, respectively) is present in a reasonable affinity range which allows for regulation of heterodimerization under physiological conditions. Therefore, it is preferred that the affinity of the lipocalin-fold binding interaction partner to the lipocalin-fold molecule in the ligand-bound state is below 10 μM, preferably below 2 μM, especially below 400 nM.
Depending on whether a lipocalin-fold binding interaction partner is engineered for binding to a lipocalin-fold molecule charged with a lipocalin-fold ligand or not charged with a lipocalin-fold ligand a lipocalin-fold molecule based system can be used for conditional heterodimerization (i.e., for on-switching) or for constitutive heterodimerization, respectively. Since a lipocalin-fold binding interaction partner can also be engineered for binding to a lipocalin-fold molecule in the absence but not in the presence of a lipocalin-fold ligand, the system can also be used for conditionally preventing heterodimerization (i.e., for off-switching). In principle, a lipocalin-fold molecule based system can optionally also be engineered for binding at least two different lipocalin-fold ligands, wherein an accordingly selected lipocalin-fold binding interaction partner can distinguish between the two differentially induced conformational states which then allows for conditional on- and off-switching by sequentially adding the two different lipocalin-fold ligands.
A lipocalin-fold molecule, which can be used as a heterodimerization domain according to the present invention, may be any protein that contains the structural motif of a lipocalin-fold to which (or in which) the lipocalin-fold ligand binds and which enables binding of the lipocalin-fold molecule to the lipocalin-fold binding interaction partner.
A lipocalin-fold molecule is defined as any naturally occurring molecule classified into the lipocalin superfamily in the SCOP database (version 1.75), or a mutant thereof. However, it is preferred to exchange only a limited number of amino acids.
According to a preferred embodiment, the lipocalin-fold molecule is a molecule identical with a naturally occurring iLBP (intracellular lipid binding protein), a naturally occurring lipocalin or an anticalin, and derivatives of any of these molecules with 1-30 amino acid exchanges and fragments thereof. In another preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring lipocalin or iLBP with at least one, two, three, four, five, six, seven, eight, nine, ten, 25 or 30 amino acid exchanges.
According to a preferred embodiment of the present invention, the lipocalin-fold molecule is engineered by one or more amino acid exchanges, insertions and/or deletions to optimize lipocalin-fold ligand binding. According to a preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring or otherwise disclosed (by its amino acid sequence) lipocalin-fold molecule with at least 70%, preferably at least 80%, especially at least 90% sequence identity in the β-barrel structure, whereby this β-barrel structure is defined as the regions preferably corresponding structurally to the regions of amino acid residues selected from
According to a preferred embodiment, the lipocalin-fold molecule is a fragment of a naturally occurring lipocalin or a derivative thereof with a length of at least 80, preferably at least 100, especially at least 120, amino acids covering at least the structurally conserved β-barrel structure of the lipocalin-fold, or wherein the lipocalin-fold molecule is a fragment of a naturally occurring iLBP or a derivative thereof with a length of at least 80, preferably at least 85, especially at least 90, amino acids covering at least the structurally conserved β-barrel structure of the lipocalin-fold, wherein the structurally conserved β-barrel structure comprises or consists of amino acid positions preferably corresponding structurally to the regions of amino acid residues selected from
According to a further preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring lipocalin or iLBP with 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 of the structurally conserved β-barrel structure, preferably corresponding structurally to the regions of amino acid residues selected from
In another preferred embodiment, the lipocalin-fold molecule is a derivative of a naturally occurring member of the lipocalin superfamily with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid exchanges.
According to a further preferred embodiment, the lipocalin-fold molecule used as heterodimerization domain according to the present invention is a lipocalin, i.e., a protein containing an eight-stranded up-and-down β-barrel arranged in a +1 topology, followed by an α-helix after the C-terminal end of the eighth β-strand.
The lipocalin-fold ligand, which can be used as a regulating molecule according to the present invention, is a “small molecule”, e.g. “small” compared to polypeptides and proteins, such as the lipocalin-fold molecule. Accordingly, the lipocalin-fold ligand has a molecular weight of 1500 Da or less, preferably 1000 Da or less, especially 750 Da or less. Preferred Mw ranges of the lipocalin-fold ligand are 50 to 1500 Da, preferably 75 to 1500 Da, especially 150 to 750 Da. Preferably, the lipocalin-fold ligand can bind in the calyx of the lipocalin-fold molecule formed by the barrel and the loop regions of the lipocalin-fold structure.
It is preferred that the lipocalin-fold ligand has an affinity to the lipocalin-fold molecule of below 1 mM, preferably of below 100 μM, especially of below 10 μM. This affinity between the lipocalin-fold ligand and the lipocalin-fold molecule is defined as a Kd (dissociation constant) value and preferably determined by isothermal titration calorimetry (ITC) using an automated MicroCal PEAQ-ITC instrument (Malvern Instruments).
Examples from which lipocalin-fold ligands can be selected are:
According to the present invention, a pair of heterodimerization domains for heterodimerization of two CAR molecules of the group of CARs, for example, can also be selected from:
a) FKBP and FKBP-rapamycin associated protein (FRB, mutant T82L)
c) FKBP and calcineurin catalytic subunit A (CnA)
d) FKBP and cyclophilin
The sequences of these heterodimerization domains as well as the regulating molecules suited for dimerization of these heterodimerization domains are well known in the art (Rutkowska et al., Angew Chem Int Ed Engl. 2012; 51(33):8166) and are disclosed, for example, in WO2014127261.
The 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 amino acids or more; e.g., the 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.
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.
As another example, a heterodimerization domain can be derived from calcineurin catalytic subunit A (also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin-dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform; etc.) and can 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).
As another example, a heterodimerization domain can be derived from cyclophilin (also known cyclophilin A, PPIA, CYPA, CYPH, PPIase A, 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 P62937-1.
As another example, a heterodimerization domain can be derived from MTOR (also known as FKBP-rapamycin associated protein; FK506 binding protein 12-rapamycin associated protein 1; FK506 binding protein 12-rapamycin associated protein 2; FK506-binding protein 12-rapamycin complex-associated protein 1; FRAP; 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).
As another example, a heterodimerization domain can be derived from a PYL protein (also known as abscisic acid receptor and as RCAR) 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 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 Q9FLB1-1); PYL6 (Uniprot Q8S8E3-1); PYL7 (Uniprot Q1ECF1-1); PYL8 (Uniprot Q9FGM1-1); PYL9 (Uniprot Q84MC7-1); PYR1 (Uniprot O49686-1).
As another example, a heterodimerization domain can be derived from an 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 2C 56, AtPP2C56, P2C56, and PP2C ABI1) and/or ABI2 (also known as P2C77, Protein phosphatase 2C 77, AtPP2C77, ABSCISIC ACID-INSENSITIVE 2, Protein phosphatase 2C ABI2, and PP2C ABI2). For example, a suitable heterodimerization 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 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).
As another example, a heterodimerization domain can be derived from the GAI Arabidopsis thaliana protein (also known as Gibberellic Acid Insensitive, and DELLA protein GAI) 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 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 the amino acid sequence Uniprot Q9LQT8-1.
As another example, a heterodimerization domain can be derived from a GID1 Arabidopsis thaliana protein (also known as Gibberellin receptor GID1) 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 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: GID1A (Uniprot Q9MAA7-1); GID1B (Uniprot Q9LYC1-1); GID1C (Uniprot Q940G6-1).
Heterodimerization of the heterodimerization domains described in 8.1.3 can be achieved by different regulating molecules (shown in the 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 the gibberellin analog GA-3M).
As noted above, rapamycin (PubChem CID 5284616) can serve as a regulating molecule. Alternatively, a rapamycin derivative or analog can be used. See, e.g., WO96/41865; WO 99/36553; WO 01/14387; and Ye et al (1999) Science 283:88-91. For example, analogs, homologs, derivatives and other compounds related structurally to rapamycin (“rapalogs”) include, among others, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, elimination or replacement of the methoxy at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxy at CI 3, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered pipecolate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional information is presented in, e.g., U.S. Pat. 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, e.g., WO 01/14387. Additional synthetic regulating molecules suitable for use as an alternative to rapamycin include those described in U.S. Patent Publication No. 2012/0130076, for example, 28-epirapamycin (PubChem CID 131668123).
Also suitable as a rapalog is a compound of the formula:
(as disclosed for example in U.S. Pat. No. 7,067,526B1) where n is 1 or 2; R28 and R43 are independently H, or a substituted or unsubstituted aliphatic or acyl moiety; one of R7a and R7b is H and the other is halo, RA, ORA, SRA, —OC(O) RA, —OC(O)NRARB, —NRARB, —NRBC(OR) RA, NRBC(O)ORA, —NRBSO2RA, or NRBSO2NRARB′; or R7a and R7b, taken together, are H in the tetraene moiety:
where RA is H or a substituted or unsubstituted aliphatic, hetero aliphatic, aryl, or heteroaryl moiety and where RB and RB′ are independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety.
The non-covalent complexation of at least two CAR molecules of a group of CARs according to the present invention can also be induced by secreted soluble factors, e.g., proteins accumulating in the tumour stroma, whereby, frequently, these proteins can itself homo- or heterodimerize. In this case, these soluble factors serve as regulating molecules according to the present invention. Dimerization domains then can be, for example, domains of native receptors (or short peptides derived therefrom; e.g., Young et al., J Biol Chem. 2004; 279(46):47633-42) to which the soluble factors are able to bind, or any antigen binding polypeptide (as already described above in chapter 1 “Antigen binding moiety”) engineered for binding to a selected soluble factor (e.g., Dotor et al., Cytokine. 2007; 39(2):106-15); Lobner et al., MAbs. 2017; 9(7):1088-1104) (VEGF binding domain used for complexation of a group of CARs in example 6)).
In preferred embodiments according to the present invention each antigen binding moiety of a group of CARs and of other polypeptides being able to bind to CAR molecules of the group binds to a target antigen present on a cell, preferably a target antigen of a cell, on a solid surface, or a lipid bilayer.
According to the present invention the specific target antigens specifically recognized by the antigen binding moieties of a group of CARs, or, alternatively, by the antigen binding moieties of the other polypeptides able to bind to the CAR molecules of the group, can be naturally occurring cellular surface antigens or polypeptides, carbohydrates or lipids bound to naturally occurring cellular surface antigens.
Examples of antigens, to which an antigen binding moiety of a group of CARs, and of another polypeptide being able to bind to a CAR molecule of the group, can specifically bind, include, e.g., 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, 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, CACNA11, 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, SLC30A-1, 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, CHRNB4, KIAA1524, NRM.3, RPRM, GRM8, KCNH4, Melanocortin 1 receptor, PTPRH, SDK1, SCN9A, SORCS1, CLSTN2, Endothelin converting enzyme like-1, Lysophosphatic acid receptor 2, LTB4R, TLR2, Neurotropic tyrosine kinase 1, MUC16, B7-H4, epidermal growth factor receptor (EGFR), ERBB2, HER3, EGFR variant III (EGFRvIII), HGFR, FOLR1, MSLN, CA-125, MUC-1, prostate-specific membrane antigen (PSMA), mesothelin, epithelial cell adhesion molecule (EpCAM), L1-CAM, CEACAM1, CEACAM5, CEACAM6, VEGFR1, VEGFR2, high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A 1, IL-13R-α2, disialogangliosides (GD2 and GD3), tumour-associated carbohydrate antigens (CA-125, CA-242, Tn and sialyl-Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), C-MET, CCR1, CCR4, FAP, fibronectin extra domain-B (ED-B), GPNMB, IGF-1 receptor, integrin α5β1, integrin αvβ, ITB5, ITGAX, embigin, PDGF-Rα, ROR1, Syndecan 1, TAG-72, tenascin C, TRAIL-R1, TRAIL-R2, NKG2D-Ligands, a major histocompatibility complex (MHC) molecule presenting a tumour-specific peptide epitope, preferably 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, MHC molecules, and the like.
Another aspect of the present invention relates to nucleic acids comprising nucleotide sequences encoding the CAR molecules of a group of CARs according to the present invention. The nucleic acid according to the present invention will in some embodiments be DNA or RNA, including, e.g., an expression vector. The nucleic acids according to the present invention may also be provided in other form, e.g. in viral vectors. The nucleic acids may be active or conditionally active in cells and be present or presents in some embodiments as RNA, e.g., in vitro synthesized RNA. Introducing RNA or DNA into a host cell can be carried out in vitro or ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding the CAR molecules of the group of CARs.
In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence encoding the CAR molecules of a group of CARs according to the present invention, consisting either of two, or three or four CAR molecules. In some cases, the nucleic acids of the present disclosure comprise one, two, three, or four separate nucleotide sequences each encoding one molecule of the group of CARs, consisting either of two, three or four CAR molecules.
In the case where the CAR molecules of the group of CARs are encoded by different nucleic acid molecules, the present invention provides a kit of at least two nucleic acids encoding one, two, three or four molecules of a group of CAR, wherein, again, the nucleic acids are preferably selected from DNA, RNA or in vitro transcribed RNA.
The present invention also provides a vector comprising the nucleic acids according to the present invention (i.e. encoding the CAR molecules of the group of CARs) and/or the kit of nucleic acids (encoding the CAR molecules of the group of CARs).
Such a vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating the 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, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia). Vectors can have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable vectors include viral vectors (e.g. viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, a retroviral vector (e.g., Murine Leukaemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like). Preferred vectors, due to the ability of efficiently integrating into the genome of the transduced cells, are retroviral vectors, especially gamma-retroviral vectors and lentiviral vectors, i.e. vectors derived from at least a portion of a retrovirus genome. An example of a preferred retroviral vector is a self-inactivating lentiviral vector (as provided in Milone et al., Mol Ther. 2009; 17(8):1453-1464). Other examples of lentivirus vectors that may be used in the clinic include, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector System from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one 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 technologies (e.g., based on Zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs)) or on CRISPR/Cas-technology (as described, e.g., by Gaj et al., Trends Biotechnol. 2013; 31(7):397-405; and Ren et al., Protein Cell 2017; 8(9):634-643). These technologies allow for integration of defined nucleotide sequences from any DNA molecule (single stranded DNA or double stranded DNA; in the form of a vector, PCR amplicon etc.) and are attractive because the gene of interest can be integrated into the genome downstream of endogenous promoters (as described, e.g., by Eyquem et al., Nature. 2017; 543(7643):113-117).
The present invention also provides a kit of at least two vectors, wherein each vector comprises nucleic acid sequences encoding one, two, three or four CAR molecules of the group of CARs according to the present invention. The vectors may be provided with the same or different regulation sequences in order to achieve expression in the same or different host systems (e.g. suitable cells where the vectors express the CAR molecules after transformation with the vector or propagation).
In the vector or kit of vectors according to the present invention, the nucleic acids encoding the CAR molecules of the group of CARs can be operably linked to a transcriptional control element, yielding an expression vector. Such a transcriptional control element can be a promoter, an enhancer, etc., wherein suitable promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include light and/or heavy chain immunoglobulin gene promoter and enhancer elements, cytomegalovirus immediate early promoter, herpes simplex virus thymidine kinase promoter, early and late SV40 promoters, promoter present in long terminal repeats from a retrovirus (e.g. the 5′-LTR of a gamma retrovirus or a promoter sequence comprising subelements R and U3 of the 5′-LTR of the Moloney murine leukaemia virus (MMLV)), promoter present in the murine stem cell virus (MSCV), mouse metallothionein-I promoter, EF1-alpha with or without intron, promoter of phosphoglycerate kinase (PGK), and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g. eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g. a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include alcohol regulated promoters (e.g. alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g. promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g. rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g. metallothionein promoter systems, 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, and the like.
In some instances, the locus or construct or transgene containing the suitable promoter can be irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination. Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating 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-specific promoter. For example, a CD4 gene promoter can be used. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of a Neri (p46) promoter. In some embodiments, e.g. for expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADHl promoter, a PGK l promoter, an ENO promoter, a PYK l promoter and the like; or a regulatable promoter such as a GALl promoter, a GALlO promoter, an ADH2 promoter, a PH05 promoter, a CUPl promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYCl promoter, a HIS3 promoter, an ADHl promoter, a PGK promoter, a GAPDH promoter, an ADCl promoter, a TRPl promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TPl promoter, and AOX l (e.g. for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g. a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter, a pagC promoter, a nirB promoter, and the like; a sigma70 promoter, e.g. a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g. a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2; an actA promoter; an rpsM promoter; a tet promoter; an SP6 promoter; and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include Trc, Tac, T5, T7, and PLambda. Examples of operators for use in bacterial host cells include a lactose promoter operator (Laci repressor protein changes conformation when contacted with lactose, thereby preventing the Laci repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator.
According to a preferred embodiment of the present invention, the vector or the kit of at least two vectors comprise a T lymphocyte-specific promoter or an NK cell-specific promoter or an EF1-alpha promoter operably linked to nucleotide sequences encoding CAR molecules of the group of CARs.
According to a further aspect, the present invention also relates to a genetically modified cell which has been modified to produce all CAR molecules of a group of CARs according to the present invention. The cells of the present invention may also be used to produce the vectors of the present invention (e.g. as virus or plasmid supernatant) from where they may then be further purified and provide these vectors in amplified and purified form.
According to a preferred embodiment, the cell is a mammalian cell which is genetically modified to produce the CAR molecules of a group of CARs according to the present invention. Preferred mammalian cells are stem cells, progenitor cells, or cells derived from a stem cell or a progenitor cell, preferably lymphocytes. Further preferred cells to be genetically modified according to the present invention are primary cells and immortalized cell lines. For pharmaceutical uses, human cells, especially lymphocytes, are specifically preferred. However, also non-human primary cells and cell lines may be suitable cell types, especially for addressing scientific questions with the system according to the present invention, e.g. non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
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. CCLlO), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATl cells, mouse L cells (ATCC No. CCLl0.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like. In some preferred instances, the cell according to the present invention is not an immortalized cell line, but is instead a cell (e.g. a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.
According to a specifically preferred embodiment, the mammalian cell according to the present invention, which is transformed with a vector or a kit of at least two vectors encoding the individual CAR molecules of a group of CARs according to the present invention, is a T cell or an NK cell.
According to a further aspect, the present invention relates to a pharmaceutical preparation which comprises a nucleic acid according to the present invention, a kit of nucleic acids according to the present invention, a vector or a kit of vectors according to the present invention, or a cell or a kit of cells according to the present invention.
The present disclosure provides a method of generating a cell capable of combinatorial antigen recognition. The method generally involves genetically modifying a mammalian cell with a vector, or a kit of vectors, or an RNA (e.g., in vitro transcribed RNA), comprising nucleotide sequences encoding the molecules of a group of CARs of the present disclosure. The genetic modification can be carried out in vivo, in vitro, or ex vivo. The cell can be an immune cell (e.g., a T lymphocyte or NK cell), a stem cell, a progenitor cell, etc.
The genetic modification is preferably carried out ex vivo. For example, a T lymphocyte (i.e., T cell), a stem cell, or an NK cell can be obtained from an individual and the cell obtained from the individual is genetically modified to express a group of CARs according to the present disclosure. In some cases, the genetically modified cell is activated ex vivo. Where the genetically modified cell is introduced into an individual (e.g., the individual from whom the cell was obtained), the genetically modified cell is activated in vivo when it comes into contact with a selected combination of target antigens present at physiological expression levels on the surface of a cell in the individual. For example, where the genetically modified cell is a T lymphocyte or NK cell, the genetically modified cell can exhibit cytotoxicity toward a cell that presents a selected combination of target antigens at physiological expression levels on its surface to which the group of CARs (and/or the antigen binding moieties of the other polypeptides) binds. In cases of a conditionally active group of CARs, the genetically modified cell comes into contact with a selected combination of target antigens present on the surface of a cell at physiological expression levels in the individual and is efficiently activated only upon administration to the individual of one or more regulating molecules and/or one or more other polypeptides that each are able to bind to a binding site of a molecule of the group of CARs and comprise at least an antigen binding moiety. In some other cases, the activation of a genetically modified cell upon contact with the target antigens present on the surface of a cell at physiological expression levels in the individual is reduced upon administration of a regulating molecule to the individual.
The present disclosure provides various treatment methods using a subject group of CARs.
A group of CARs according to the present invention, when present in a T lymphocyte or an NK cell, can mediate cytotoxicity toward a target cell. A non-covalently complexed group of CARs according to the present invention, in some cases dependent on the presence of (an)other polypeptide(s) binding to target antigen(s), can bind to a selected combination of target antigens present on a target cell, thereby mediating killing of a target cell by a T lymphocyte or an NK cell genetically modified to produce the group of CARs.
Target cells include cancer cells. Thus, the present disclosure provides methods of killing, or inhibiting the growth of, a target cancer cell, the method involving contacting a target cancer cell with a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) that is genetically modified to produce a subject group of CARs, such that the T lymphocyte or NK cell recognizes a selected combination of target antigens present on the surface of a target cancer cell, and mediates killing of the target cell.
The present disclosure provides a method of treating cancer in an individual having a cancer. In a preferred embodiment the method comprises: i) genetically modifying NK cells or preferably T lymphocytes obtained from the individual with at least one vector comprising nucleotide sequences encoding the respective CAR molecules of a group of CARs according to the present invention, wherein the antigen binding moieties of the group of CARs are specific for target antigens on a cancer cell in the individual, and wherein said genetic modification is carried out 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 regulating molecule for either inducing or reducing heterodimerization of the respective CAR molecules of the group, preferably inducing heterodimerization of the respective CAR molecules of the group, thereby either inducing or reducing non-covalent complexation of the group of CAR, preferably inducing non-covalent complexation of the group of CARs, wherein the non-covalently complexed group of CARs, upon contact with a cancer cell expressing the respective target antigen combination at physiological expression levels, mediates activation of the genetically modified cell, which leads to killing of the cancer cell and thereby enables treating the cancer. In another preferred embodiment the method comprises: i) genetically modifying NK cells or preferably T lymphocytes obtained from the individual with at least one vector comprising nucleotide sequences encoding the respective CAR molecules of a group of CARs according to the present invention, wherein the antigen binding moieties of the CAR molecules of the group, and/or the antigen binding moieties of the other polypeptide(s) being able to bind to CAR molecules of the group, are specific for target antigens on a cancer cell in the individual, and wherein heterodimerization of the respective CAR molecules of the group does not require the administration of a regulating molecule, and wherein said genetic modification is carried out 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 other polypeptide that comprises at least an antigen binding moiety and is able to bind to a binding site in a CAR molecule of the group of CARs, which, upon contact with a cancer cell expressing the respective target antigen combinations at physiological expression levels, mediates activation of the genetically modified cell, which leads to killing of the cancer cell and thereby enables treating the cancer. In yet another preferred embodiment the method comprises: i) genetically modifying NK cells or preferably T lymphocytes obtained from the individual with at least one vector comprising nucleotide sequences encoding the respective CAR molecules of a group of CARs according to the present invention, wherein the antigen binding moieties of the CAR molecules of the group are specific for target antigens on a cancer cell in the individual, and wherein heterodimerization of the respective CAR molecules of the group does not require the administration of a regulating molecule, and wherein said genetic modification is carried out in vitro or ex vivo; ii) introducing the genetically modified cells into the individual, wherein this enables killing of the cancer cell, thereby treating the cancer.
Carcinomas that can be amenable to therapy by a method disclosed herein include esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, 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, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be amenable to therapy by a method disclosed herein include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio-sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be amenable to therapy by a method disclosed herein include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Leukaemias that can be amenable to therapy by a method disclosed herein include a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukaemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukaemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukaemia, and hairy cell leukaemia; and d) acute lymphoblastic leukaemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's lymphoma, and the like. Other cancers that can be amenable to treatment according to 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 neurofibroma mediastinum.
A subject method can also be used to treat inflammatory conditions and autoimmune disease. A subject group of CARs can be expressed in a T-helper cell or a regulatory T (Treg) cell for use in an immunomodulatory method. Immunomodulatory methods include, e.g., enhancing an immune response in a mammalian subject toward a pathogen; enhancing an immune response in a subject who is immunocompromised; reducing an inflammatory response; reducing an immune response in a mammalian subject to an autoantigen, e.g., to treat an autoimmune disease; and reducing an immune response in a mammalian subject to a transplanted organ or tissue, to reduce organ or tissue rejection. Where the method involves reducing an immune response to an autoantigen, at least one of the target antigens used to activate the group of CARs preferably is an autoantigen. Where the method involves reducing an immune response to a transplanted organ or tissue, at least one of the antigens used to activate the group of CARs preferably is an antigen specific to the transplanted organ.
As discussed above, a treatment method of the present disclosure in preferred cases involves the administration to an individual in need thereof of an effective amount of one or more different regulating molecules and/or one or more different other polypeptides, wherein each of the other polypeptides comprises at least an antigen binding moiety and being able to bind to an extracellular binding site of a CAR molecule of the group of CARs.
The required effective amount of each regulating molecule, administered to an individual in need thereof having received T lymphocytes or NK cells expressing a group of CARs according to the present invention (“effector cells”), is defined by the different response of those effector cells upon contact with target cells expressing the respective antigen combination in presence vs. absence of each required regulating molecule. The response of those effector cells thereby is defined by the excretion of interferon-gamma, and/or Macrophage inflammatory protein-1 (MIP-1) alpha, and/or MIP-1 beta, and/or granzyme B, and/or IL-2, and/or TNF, and/or IL-10, and/or IL-4, and/or by effector cell degranulation, wherein cell degranulation is preferably detected by the percentage of effector cells translocating CD107a onto their surface, i.e., the percentage of CD107a-positive effector cells detected by flow cytometric analysis using a degranulation assay (for example, as described in Proff et al., Front Micro-biol. 2016; 7:844), after contact with a target cell expressing more than 100,000 molecules of each target antigen per cell, optionally in the presence of an effective concentration of each required other polypeptide comprising at least an antigen binding moiety and being able to bind to binding sites of the CAR molecules of the group of CARs. In preferred embodiments, the response of the effector cells in presence vs. absence of an effective concentration of each required regulating molecule differs by at least 20%, preferably by at least 50%, or even more preferably by at least 100%, wherein the effective concentration of each required regulating molecule is the concentration achieved by administration of an effective amount of each required regulating molecule in one or more doses to an individual in need thereof. The effective concentration of each required other polypeptide comprising at least an antigen binding moiety and being able to bind to the group of CARs is defined by the response of the fully complexed subject group of CARs (i.e., all dimerization domains comprised by the group of CARs are dimerized), after contact with a target cell expressing more than 100,000 molecules of each target antigen per cell, in presence vs. absence of each required other polypeptide comprising at least an antigen binding moiety and being able to bind to the group of CARs, wherein the response preferably differs by at least 20%, preferably by at least 50%, or even more preferably by at least 100%, and wherein the effective concentration of each required other polypeptide comprising at least an antigen binding moiety and being able to bind to the group of CARs is the concentration achieved by administration of an effective amount of each of those other polypeptides in one or more doses to an individual in need thereof having received T lymphocytes or NK cells expressing the subject group of CARs.
Both the regulating molecules and the antigen-specific other polypeptides being able to bind to a CAR molecule of a group of CARs according to the present invention are hereafter together referred to as “agents specifically binding to the group of CARs”.
In the subject methods, an “agent specifically binding to the group of CARs” can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect or diagnostic effect. Thus, the “agent(s) specifically binding to the group of CARs” can be incorporated into a variety of formulations for therapeutic administration. More particularly, an “agent specifically binding to the group of CARs” can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In pharmaceutical dosage forms, an “agent specifically binding to the group of CARs” can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary:
Suitable excipient vehicles can be, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the required “agent(s) specifically binding to the group of CARs” adequate to achieve the desired state in the subject being treated. The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. For oral preparations, an “agent specifically binding to the group of CARs” can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatines; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavouring agents.
An “agent specifically binding to the group of CARs” can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
Pharmaceutical compositions comprising an “agent specifically binding to the group of CARs” can be prepared by mixing the “agent (s) specifically binding to the group of CARs” having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).
The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is usually to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.
An “agent specifically binding to the group of CARs” can be optionally formulated also in a controlled release formulation. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the “agent(s) specifically binding to the group of CARs” in which the matrices are in the form of shaped articles, e.g. films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(−)-3-hydroxybutyric acid. Possible loss of biological activity may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.
A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular “agent(s) specifically binding to the group of CARs” to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. An “agent specifically binding to the group of CARs” may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.
Those of skill will readily appreciate that dose levels can vary as a function of the specific “agent specifically binding to the group of CARs”, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
One or more “agents specifically binding to the group of CARs” can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Conventional and 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 may be combined, if desired, or adjusted depending upon the “agent(s) specifically binding to the group of CARs” and/or the desired effect. An “agent specifically binding to the group of CARs” can be administered in a single dose or in multiple doses. In preferred embodiments with conditionally active groups of CARs, an “agent specifically binding to the group of CARs” can be administered orally or alternatively intravenously. In other embodiments, an “agent specifically binding to the group of CARs” can be administered via an inhalational route. In yet other embodiments, an “agent specifically binding to the group of CARs” can be administered intranasally, locally, or also intratumourally. In yet other embodiments, an “agent specifically binding to the group of CARs” can be administered peritumourally. In preferred embodiments with conditionally active groups of CARs for treatment of brain tumours, an “agent specifically binding to the group of CARs” can be administered intracranially.
The “agent(s) specifically binding to the group of CARs” can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include enteral, parenteral, or inhalational routes. Parenteral routes of administration other than inhalation administration include topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous routes. Parenteral administration can be carried to effect systemic or local delivery of an “agent specifically binding to the group of CARs”. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations. An “agent specifically binding to the group of CARs” can also be delivered to the subject by enteral administration. Enteral routes of administration include oral and rectal (e.g., using a suppository) delivery.
By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as cancer. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.
An “agent specifically binding to the group of CARs” can be administered by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue. An “agent specifically binding to the group of CARs” can also be administered directly to a target site e.g., by direct injection, by implantation of a drug delivery device such as an osmotic pump or slow release particle, by biolistic delivery to the target site, etc. Furthermore, an “agent specifically binding to the group of CARs” can be administered as an adjuvant therapy to a standard cancer therapy. Standard cancer therapies include surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, antibody treatment, biological response modifier treatment, and certain combinations of the foregoing.
A variety of subjects are suitable for treatment with a subject method of treating cancer. Suitable subjects include any individual, e.g., a human or non-human animal who has cancer, who has been diagnosed with cancer, who is at risk for developing cancer, who has had cancer and is at risk for recurrence of the cancer, who has been treated with other therapeutics and failed to respond to such treatment, or who relapsed after initial response to such treatment.
Subjects suitable for treatment with a subject immunomodulatory method include individuals who have an autoimmune disorder; individuals who are organ or tissue transplant recipients; and the like; individuals who are immunocompromised; and individuals who are infected with a pathogen.
The first example shows a strategy for generating an antigen binding moiety with low affinity that is suited for use as an antigen binding moiety in a group of CARs, according to the present invention. Reduced charge Sso7d (rcSso7d) is a charge-reduced version of a small (˜7 kDa) DNA-binding protein from the archaeon Sulfolobus solfataricus. Charge-reduction minimizes unspecific binding due to reduced electrostatic interactions. rcSso7d is a single-domain protein antigen binding moiety with high thermal stability and monomeric behaviour and therefore is an example of a suited binding scaffold. Starting from the well characterized antigen binding moiety rcSso7d E11.4.1, which binds to human EGFR with a Kd of 19 nM (Traxlmayr et al., J Biol Chem. 2016; 291(43):22496-22508), we generated low affinity mutants by performing an alanine scan in which we replaced all amino acids potentially involved in epitope binding by alanine, i.e. in each mutant one position involved in antigen binding was mutated to alanine. Mutants of rcSso7d E11.4.1 were fused to sfGFP and expressed as soluble proteins in a bacterial expression system. The schematics of the architecture of the fusion proteins are shown in
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 the QuikChange Primer Design software (Agilent Genomics) and oligonucleotides were synthesized by Biomers.
Expression and Purification of rcSso7d-Based Antigen Binding Moieties:
Binding scaffolds were expressed as sfGFP fusion proteins (consisting of an N-terminal hexahistidine tag followed by either rcSso7d or the Affibody and sfGFP) using the pE-SUMO vector (Life Sensors). The nucleotide sequence that encodes 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 cultivation at 37° C., cultures were 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. while shaking. When cultures reached an A600 of roughly 2, expression of the transgene was induced by addition of 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were further cultured overnight at 20° C. Cells were harvested by centrifugation (5000 g, 20 minutes, 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 seconds, duty cycle 50%, amplitude set to 5) and centrifuged again to remove cell debris. Hexahistidine-tagged fusion proteins were purified from crude cell extracts using TALON metal affinity resin (Clontech Laboratories). After addition of 10 mM imidazole, the sonicated supernatants were applied onto the resin twice, followed by washing step with equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) with increasing amounts of imidazole (5-15 mM). Binding scaffolds were eluted by applying equilibration buffer supplemented with 250 mM imidazole. After buffer exchange to PBS using Amicon Ultra-15 10K centrifugal filters (Merck Millipore), concentrations were determined by measuring the absorbance at 280 nm using the respective molar absorption coefficient and finally proteins were directly frozen at −80° C.
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 regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads (Thermo Scientific), a flow cytometer-based cell counting platform.
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit (Qiagen). Briefly, the mRNA solution was diluted with a mixture of RLT buffer (Qiagen), ethanol (Merck) and 2-mercaptoethanol (Merck). The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water (Thermo Scientific) and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Jurkat T cells were resuspended in FACS buffer (PBS (Thermo Scientific), 0.2% human albumin (CSL Behring) and 0.02% sodium azide) and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then directly processed with a BD LSRFortessa. Expression of the engineered target antigen tEGFR was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
Jurkat T cells were engineered to express high levels of the respective tumour antigen. Hence, 3 μg of mRNA coding for tEGFR were electroporated into Jurkat T cells one day prior to the co-culture with the effector cells. After washing in PBS, cells were resuspended in PBS containing 0.1% BSA (Sigma Aldrich) and incubated with varying concentrations of the binder proteins fused to sfGFP in order to determine the affinities of the antigen binding moieties towards the respective tumour antigen. After incubation for 1 hour at 4° C. while shaking, plates were centrifuged (450 g, 7 minutes, 4° C.), the supernatant was discarded and cells were acquired with a BD LSRFortessa. Cells were kept on ice to avoid endocytosis. The Kd was obtained by curve fitting using Microsoft Excel (Microsoft Corporation).
SPR experiments were performed with a Biacore T200 instrument (GE Healthcare). All experiments were conducted 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 flow rate of 10 μL/min for 60 seconds at a concentration 6.67 μg/mL. To determine the affinity of rcSso7d-based antigen binding moieties, five concentrations (depending on the expected Kd of the antigen binding moiety) of the respective protein were injected at a flow rate of 30 μL/min for 15 seconds in the single-cycle kinetic mode, 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. The Kd was obtained by curve fitting using the Biacore T200 Evaluation Software (GE Healthcare).
Extracellular disulphide-bond forming cysteines in extracellular hinge regions as e.g. CD8a can prevent the exploitation of the avidity effect according to present invention. This is demonstrated in example 2, in which the low affinity mutant of the binding moiety “E11.4.1 G32A” of example 1 was fused to CAR signalling backbones in which the two extracellular cysteine residues in the hinge region of CD8a (UniProt ID P01732, positions C164 and C181) were substituted by serine residues or not, respectively. Whereas the cysteine-containing CAR-variant (“Cys”) efficiently triggered T cell activation in response to target cells, the serine-containing variant (“Ser”) did not or only poorly trigger the T cells. This example thus illustrates the importance of preventing disulphide-bond formation for generating CAR molecules that are suited for use in a group of CARs according to the present invention. The schematics in
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos 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. CD3pos T Cells were activated with anti-CD3/CD28 beads (Thermo Scientific) according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide) and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, Genscript) as a primary antibody and a PE- or APC-conjugated secondary antibody (eBioscience). Expression of the engineered target antigen tEGFR was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the signal peptide CD33, the human CD8a hinge, human monomeric CD8a hinge (UniProt ID P01732, C164S and C181S) and CD8a transmembrane domain, the 4-1BB co-stimulatory domain and the CD3ζ ITAM signalling domain were synthesized by GenScript. Sequences encoding the extracellular and transmembrane domain of EGFR was obtained from Addgene (plasmid #11011). Insertion of a Strep II tag (NWSHPQFEK) and flexible linkers was performed by PCR. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix (New England BioLabs), according to the manufacturer's instructions. The schematics and sequences are shown in
Luciferase-expressing tumour cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1 with 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, remaining living cells were quantified by determination of the residual luciferase activity of the co-culture. After equilibration to room temperature for 10 minutes, luciferin was added to the cell suspension (150 μg/mL final concentration; Perkin Elmer) and luciferase activity was measured 20 minutes later using the ENSPIRE Multimode plate reader. The percentage of specific lysis was determined with the following formula:
% specific lysis=100−((RLU from well with effector and target cell co-culture)/(RLU from well with target cells only)×100)).
Cytokine secretion of primary CAR T cells was assessed by co-cultivation with target cells at E:T ratios of 1:1 or 2:1 in flat-bottom 96 well plates for 4 hours or 24 hours at 37° C. In some experiments, released cytokines were quantified in the supernatants from the co-culture experiments for determining cytotoxicity. The supernatants were centrifuged (1600 rpm, 7 minutes, 4° C.) to remove remaining cells and debris and were subsequently frozen at −80° C. For analysis of secreted IFN-γ, ELISA was performed using the Human IFN gamma ELISA Ready-SET-Go!® kit (eBioscience) according to the manufacturer's instructions. Measurements were conducted using the ENSPIRE Multimode plate reader.
The third example demonstrates that the integration of scFv-based binding moieties in CAR molecules can prevent the exploitation of the avidity effect for specific recognition of antigen combinations. The schematics of the CAR constructs shown in
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos 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. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide) and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs 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 a primary antibody and a PE- or APC-conjugated secondary antibody in case of the Strep II tag antibody. Expression of the engineered target antigen tHER2 was detected with a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the GM-CSF-Ra signal peptide, the anti-human CD19 scFv FMC63, the human CD8a hinge, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain and the CD3ζITAM signalling domain were synthesized by GenScript. The nucleotide sequences encoding the signal peptide IgGk, the anti-human HER2 scFv 4D5-5 and the dimerization domain FKBP F36V were synthesized by GeneArt (Thermo Scientific). Sequences encoding the extracellular and transmembrane domain of HER2 were obtained from Addgene (plasmid #16257). Insertion of a Strep II tag (NWSHPQFEK) or FLAG tag (DYKDDDDK) and flexible linkers was performed by PCR. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
Luciferase-expressing tumour cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1with 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, remaining living cells were quantified by determination of the residual luciferase activity of the co-culture. After equilibration to room temperature for 10 minutes, luciferin was added to the cell suspension (150 μg/mL final concentration) and luciferase activity was measured 20 minutes later using the ENSPIRE Multimode plate reader. The percentage of specific lysis was determined with the following formula:
% specific lysis=100−((RLU from well with effector and target cell co-culture)/(RLU from well with target cells only)×100)).
For the FACS-based cytotoxicity assay two populations of target cells were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. 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 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours at 37° C. Target cells without the addition of CAR T cells served as a control condition (“targets only”). After the incubation period, the co-cultures were centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants were collected for subsequent cytokine measurements and the remaining cells were resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
Cytokine secretion of primary CAR T cells was assessed by co-cultivation with target cells at E:T ratios of 1:1 or 2:1 in flat-bottom 96 well plates for 4 hours or 24 hours at 37° C. In some experiments, released cytokines were quantified in the supernatants from the co-culture experiments for determining cytotoxicity. The supernatants were centrifuged (1600 rpm, 7 minutes, 4° C.) to remove remaining cells and debris and were subsequently frozen at −80° C. For analysis of secreted IFN-γ, ELISA was performed using the Human IFN gamma ELISA Ready-SET-Go!® kit (eBioscience) according to the manufacturer's instructions. Measurements were conducted using the ENSPIRE Multimode plate reader.
Dimerization of transgenes was induced prior to co-cultivation experiments. Primary T cells were diluted to the final cell concentration in the respective cell culture medium. The homodimerization agent AP20187 (MedChemExpress) was diluted in cell culture medium and was added at 10 nM final concentration. Addition of the respective vehicle control DMSO at the same concentration served as control. Cells were incubated at 37° C. for 30 minutes to ensure efficient dimerization of transgenes and subsequently used for in vitro experiments.
In the fourth example, we generated and identified an affibody based binding moiety directed against HER2 which is suitable for use in a group of CARs according to the present invention. Again, we started from a well characterized existent antigen binding moiety that was engineered for high affinity binding to human HER2 (Wikman et al., Protein Eng Des Sel. 2004; 17(5):455-462). To eliminate a potential N-glycosylation site and to reduce IgG binding, two point mutations (N23A and S33K) were introduced into the framework region of the binding scaffold (Feldwisch et al., J. Mol. Biol. 2010; 398(2):232-47), which resulted in the antigen binding moiety “zHER2-WT”. Low affinity mutants were generated by performing an alanine-scan of “zHER2-WT” by mutating all amino acids involved in antigen binding consecutively to alanine, resulting in various mutants containing one alanine-mutation each. Instead of expressing the 13 mutants in E. coli and determining their affinities for selection of appropriate antigen binding moieties, we performed a functional screening by directly integrating all mutants into a CAR backbone (exemplified with binder zHER2-WT (WT) in SEQ ID NO: 52) that could be conditionally homodimerized (
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos T cells were enriched by negative selection using RosetteSep Human T cell Enrichment Cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected via the hexahistidine tag using an AF647-conjugated anti-pentahistidine tag antibody (Qiagen). Expression of the engineered target antigen tHER2 was detected with a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was done by the FlowJo software.
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
The nucleotide sequences encoding the CD33 signal peptide, the affibody zHER2-WT, the hexahistidine tag, a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain, the dimerization domain FKBP F36V and the CD3ζ ITAM signalling domain were synthesized by GeneArt. Sequences encoding the extracellular and transmembrane domain of HER2 were obtained from Addgene (plasmid #16257). Insertion of flexible linkers was performed by PCR. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
Site-directed mutagenesis of all amino acids involved in epitope binding was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit, according to the manufacturer's instructions. Primers were designed using the QuikChange Primer Design software (Agilent Genomics) and oligonucleotides were synthesized by Biomers.
Luciferase-expressing tumour cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1 with 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, remaining living cells were quantified by determination of the residual luciferase activity of the co-culture. After equilibration to room temperature for 10 minutes, luciferin was added to the cell suspension (150 μg/mL final concentration) and luciferase activity was measured 20 minutes later using the ENSPIRE Multimode plate reader. The percentage of specific lysis was calculated with the following formula:
% specific lysis=100−((RLU from well with effector and target cell co-culture)/(RLU from well with target cells only)×100)).
Dimerization of transgenes was induced prior to co-cultivation experiments. Primary T cells were diluted to the final cell concentration in the respective cell culture medium. The homodimerization agent AP20187 was diluted in cell culture medium and was added at 10 nM final concentration. Addition of the vehicle control DMSO at the same concentration served as control. Cells were incubated at 37° C. for 30 minutes to ensure efficient dimerization of transgenes and subsequently used for in vitro experiments.
Binding scaffolds were expressed as sfGFP fusion proteins (consisting of an N-terminal hexahistidine tag followed by either rcSso7d or the Affibody and sfGFP) using the pE-SUMO vector. The schematics of the architecture of the fusion proteins are shown in
SPR experiments were performed with a Biacore T200 instrument. All experiments were conducted in degassed and filtered PBS, pH 7.4, containing 0.1% BSA and 0.05% Tween-20 (Merck Millipore), at 25° C. hHER2-Fc (R&D) was immobilized on a Protein A sensor chip at a flow rate of 10 μL/min for 60 seconds at a concentration 4 μg/mL. To determine the affinity of Affibody-based antigen binding moieties, five concentrations (depending on the expected Kd of the antigen binding moiety) of the respective protein were injected at a flow rate of 30 μL/min for 15 seconds (zHER2-R10A and zHER2-R32A) or 60 seconds (zHER2-WT) in the single-cycle kinetic mode, followed by a dissociation step (60 seconds for zHER2-R10A and zHER2-R32A and 180 seconds for zHER2-WT). Regeneration was performed using 10 mM Glycine-HCl, pH 1.5 at a flow rate of 30 μL/min for 30 seconds. The Kdwas obtained by curve fitting using the Biacore T200 Evaluation Software (GE Healthcare).
In example 5 we show in a leukaemia model with immunodeficient NOD.Cg-Prkdcscid Il2rgtm1WJI/SzJ (NSG) mice that tumour growth can efficiently be inhibited by lentivirally transduced T cells expressing the low-affinity CAR “S(G32A)-8ser-BB-FKBP (36V)-3z” (SEQ ID NO: 46) in the presence but not the absence of a regulating molecule. As there is currently no system available for conditional heterodimerization that would be suitable for long-term in vivo experiments, we used an FKBP-based system for homodimerization in order to demonstrate the in vivo proof of concept of regulating the function of T cells by regulating the avidity of CAR molecules. This is based on the conclusion that the monospecific low-affinity CAR “S(G32A)-8ser-BB-FKBP (36V)-3z” represents in its complexed, i.e, dimeric state a bivalent anti-EGFR/EGFR-CAR and in principle compares to an anti-EGFR/HER2-CAR that recognizes a double positive target cell (i.e. bivalent interaction).
For the in vivo model we used the B-ALL cell line Nalm6 which was transduced with a vector for high expression of tEGFR (approx. 1×106 tEGFR molecules per cell) and firefly luciferase for in vivo quantification of tumour growth by using bioluminescence imaging. Intravenous (i.v.) injection of 0.5×106 Nalm6-tEGFR-fLuc cells into NSG mice resulted in exponential tumour growth in untreated mice.
Expression of the CARs was detected with an anti-Strep II tag antibody in case of rcSso7d-based CARs and with Protein L in case of the CD19-specific CAR (
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos 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. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
Virus production of pantropic VSV-G pseudotyped lentivirus was performed from Lenti-X 293T cells with a third-generation Puromycin-selectable pCDH transgene vector and second-generation viral packaging plasmids pMD2.G and psPAX2 (both obtained from Addgene, plasmids #12259 and #12260 respectively). Co-transfection was performed using Purefection Transfection Reagent according to the manufacturer's instructions. Supernatants were collected one and two days after transfection and were concentrated using the Lenti-X Concentrator according to the manufacturer's instructions.
Twenty-four hours prior to the 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, according to the manufacturer's instructions, to promote co-localization of lentivirus and primary T cells. Cells were exposed to concentrated lentiviral supernatants for one day, followed by removal of the virus particles. After three days, T cells were treated with 1 μg/mL Puromycin to ensure high and uniform expression of the transgene. T cells were expanded in T cell transduction medium, consisting of AIM-V supplemented with 2% Octaplas, 1% L-Glutamine, 2.5% HEPES and 200 IU/mL recombinant human IL-2.
Cell lines were split 24 hours before lentiviral transduction to ensure exponential cell growth at the time point of transduction. Cells were exposed to varying concentrations of lentiviral supernatants for one day. Puromycin selection was performed three days after transduction with concentrations varying from 1 to 8 μg/mL in order to exclude non-transduced cells.
The nucleotide sequences encoding the CD33 signal peptide, the low affinity rcSso7d variant E11.4.1-G32A, the Strep II tag (NWSHPQFEK), a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain, the dimerization domain FKBP F36V and the CD3ζ ITAM signalling domain were synthesized by GeneArt (Thermo Scientific). The nucleotide sequences encoding the GM-CSF-Ra signal peptide and the anti-human CD19 scFv FMC63 were synthesized by GenScript. The nucleotide sequence encoding the extracellular and transmembrane domain of EGFR was obtained from Addgene (plasmid #11011). Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, Genscript) or Protein L in case of the CD19-BBz CAR as primary antibodies and a PE- or APC-conjugated secondary antibody. Expression EGFR was detected with a PE-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
Luciferase-expressing tumour cells were co-cultured with CAR T cells at an E:T cell ratio of 2:1 with 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, remaining living cells were quantified by determination of the residual luciferase activity of the co-culture. After equilibration to room temperature for 10 minutes, luciferin was added to the cell suspension (150 μg/mL final concentration) and luciferase activity was measured 20 minutes later using the ENSPIRE Multimode plate reader. The percentage of specific lysis was calculated with the following formula:
% specific lysis=100−((RLU from well with effector and target cell co-culture)/(RLU from well with target cells only)×100)).
Dimerization of transgenes was induced prior to co-cultivation experiments. Primary T cells were diluted to the final cell concentration in the respective cell culture medium. The homodimerization agent AP20187 was diluted in cell culture medium and was added at 10 nM final concentration. Addition of the respective vehicle control DMSO at the same concentration served as control. Cells were incubated at 37° C. for 30 minutes to ensure efficient dimerization of transgenes and subsequently used for in vitro experiments.
NOD.Cg-Prkdcscid Il2rgtm1WJI/SzJ (NSG) mice were housed in the Anna Spiegel facility for animal breeding. For subsequent experiments, mice were transferred to the preclinical research laboratories (PIL) of the Medical University of Vienna. All procedures were performed as approved (GZ: 813267/2015/24) by the Magistratsabteilung 58, Vienna.
Primary T cells were engineered to express the CD19-specific control CAR (CD19-BBz), the EGFR-specific high and low affinity CARs (“E11.4.1-WT” and “E11.4.1-G32A”, respectively) using the protocol depicted in “Transduction of T cells and cell lines”. After transduction, the CAR T cells were expanded for over 14 days prior to in vivo experiments to generate sufficient cell numbers.
The homodimerization agent AP20187 (Clontech Laboratories) was dissolved in vehicle solution according to manufacturer's instructions. Briefly, AP20187 was initially dissolved to a concentration of 12.5 mg/mL in ethanol with rigorous vortexing. The compound was then diluted to the final working concentration of 0.5 mg/mL using an appropriate mixture of PEG-400 (Sigma Aldrich) and Tween-80 (Sigma Aldrich) in water. The resulting vehicle solution consisted of 4% ethanol, 10% PEG-400 and 1.7% Tween-80 in water for injection. The working stock of AP20187 was prepared immediately prior to injection, sterile-filtered and was used within 30 minutes.
Nalm6 cells engineered to express high levels of tEGFR-FKBP and fLuc (“Nalm6-tEGFR-fLuc”) were resuspended in PBS, filtered through a 35 μm cell strainer (Corning Falcon) and set to a final concentration of 5×106/mL. 0.5×106 cells were injected intravenously (i.v.) into the tail-vein of each NSG mouse (mixture of male and female mice, Jackson Laboratory). Three days later, mice were treated with respective CAR T cells (10×106 CAR T cells i.v. into the tail-vein), followed by injection of the homodimerization agent AP20187 (2 mg/kg dosage) or vehicle control using intraperitoneal (i.p.) injections. The dimerization agent AP20187 (2 mg/kg) or the vehicle control was administered on day 0 (right after T cell injection), day 1, day 2, day 4, day 7, day 9 and day 11 where indicated. All control conditions were treated with the respective vehicle control (4% ethanol, 10% PEG-400 and 1.7% Tween-80 in water for injection). Tumour growth and control was monitored by bioluminescence imaging (BLI). Mice were sacrificed by cervical dislocation at the end of the experiment.
BLI imaging of tumour growth was performed at the preclinical research laboratory (PIL) of the Medical University of Vienna using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer). D-Luciferin substrate (Perkin Elmer) was dissolved in PBS to a final concentration of 15 mg/mL and sterile filtered. Mice were anesthetized with isoflurane and received i.p. injections of the luciferin working stock (final dosage 150 mg/kg body weight). After 15-20 minutes, 1-3 mice were transferred to the IVIS Imaging System, bioluminescence was measured in medium binning mode at an acquisition time of 1 seconds to 2 minutes to obtain unsaturated images. Luciferase activity was analysed with the Living Image Software (Caliper) and photon flux was determined within the region of interest that encompassed the entire body of the mouse.
The sixth example demonstrates a strategy to create a group of CARs that can be complexed by an extracellular soluble factor, which in this case serves as the regulating molecule according to the present invention. In the shown example VEGF was used as a potential dimerization agent (i.e. regulating molecule) for homodimerization of the CAR S(G32A)-J.CT6-8ser-BB-3z (SEQ ID NO: 66 and SEQ ID NO: 67). For this purpose, an engineered CH2-CH3-IgG1-Fc domain was integrated into the ectodomain of a CAR molecule (SEQ ID NO: 67) and co-expressed with a soluble construct comprising the CH2-CH3-Fc domain “Janus CT6” (SEQ ID NO: 66), that was engineered for high affinity binding to VEGF (Lobner et al., MAbs. 2017; 9(7):1088-1104) and that covalently heterodimerizes via formation of disulphide bridges with the CH2-CH3-Fc domain in the ectodomain of the CAR molecule. The CH2-CH3 domains of both constructs were engineered for minimizing homodimerization (Lobner et al., MAbs. 2017; 9(7):1088-1104). In the given example E11.4.1-G32A was used as antigen binding moiety due to its dependence on cooperative binding.
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos 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 (Sigma Aldrich) and 10% DMSO until use. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected either via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, Genscript) or via the Fc domain by using a biotinylated anti-human-IgG1-antibody (clone JDC-10, Biozol) as a primary antibody and a PE-conjugated streptavidin as secondary staining reagent. Expression of the engineered target antigen tEGFR was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the CD33 signal peptide, low affinity rcSso7d variant E11.4.1-G32A, the Strep II tag (NWSHPQFEK), a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain and the CD3ζ ITAM signalling domain were synthesized by GeneArt (Thermo Scientific). Plasmids comprising the CH2-CH3-Fc domain “Janus CT6” and the mutated “WT” CH2-CH3-Fc domain were a kind gift from Elisabeth Lobner at the University of Natural Resources and Life Sciences in Vienna. Sequences encoding the extracellular and transmembrane domains of EGFR were obtained from Addgene (plasmid #11011). Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
For the FACS-based cytotoxicity assay two populations of target cells were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. 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 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours at 37° C. Target cells without the addition of CAR T cells served as a control condition (“targets only”). After the incubation period, the co-cultures were centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants were collected for subsequent cytokine measurements and the remaining cells were resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
Recombinant expression of a truncated form of human VEGF (residues 14-108) was previously described by (Lobner et al., MAbs. 2017; 9(7):1088-1104).
In example 7 we exemplified a strategy for generating a group of CARs which can be conditionally complexed by the regulating molecule AP21967 and which allows for specific recognition of the combined expression of EGFR and HER2 on target cells. For this purpose, we grafted two different single domain binding scaffolds, i.e., low affinity mutants of the rcSso7d based EGFR-specific antigen binding moiety E11.4.1 (Traxlmayr et al., J Biol Chem. 2016; 291(43):22496-22508) and the affibody-based HER2-specific antigen binding moiety (Wikman et al., Protein Eng Des Sel. 2004; 17(5):455-462), onto a second generation signalling backbone comprising either the heterodimerization domain FKBP or FRB, i.e., “S(G32A)-8ser-BB-FKBP-3z” (SEQ ID NO: 48) and “A(R10A)-ser8-BB-FRB-3z” (SEQ ID NO: 54), respectively (
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos 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. CD3pos T Cells were activated with anti-CD3/CD28 beads (Thermo Scientific) according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
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 a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells or Jurkat T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocols were used for the respective cell types: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary human T cells or tumour cell lines were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide) and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected either via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, Genscript) or via the FLAG tag using an anti-FLAG antibody (clone L5, BioLegend) as a primary antibody and a PE- or APC-conjugated secondary antibody (eBioscience). Expression of the engineered target antigens tEGFR and tHER2 was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend) or with a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the CD33 signal peptide, low affinity rcSsso7d variant E11.4.1-G32A, the low affinity affibody variant zHER2-R10A, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain, the dimerization domains FKBP F36V and FRB and the CD3ζ ITAM signalling domain were synthesized by GeneArt (Thermo Scientific). The nucleotide sequences encoding the dimerization domain FKBP was synthesized by Genscript. Sequences encoding the extracellular and transmembrane domains of EGFR and HER2 were obtained from Addgene (plasmids #11011 and #16257, respectively). Flexible linkers, the FLAG tag and the Strep II tag were inserted by using respective PCR primers. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix (New England Biolabs), according to the manufacturer's instructions. The schematics and sequences are shown in
For the FACS-based cytotoxicity assay two populations of target cells were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. 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 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours at 37° C. Target cells without the addition of CAR T cells served as a control condition (“targets only”). After the incubation period, the co-cultures were centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants were collected for subsequent cytokine measurements and the remaining cells were resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
Dimerization of transgenes was induced prior to co-cultivation experiments. Primary T cells and Jurkat T cells were diluted to the final cell concentration in the respective cell culture medium. The heterodimerization agent AP21967 (Clontech Laboratories) was diluted in cell culture medium and was added at a concentration of 500 nM. Control conditions were treated with the same concentration of ethanol (vehicle control). Cells were incubated at 37° C. for 30 minutes to ensure efficient dimerization of transgenes and subsequently used for in vitro experiments.
By using two orthogonal dimerization platforms (FKBP/FRB using AP21967; FKBP F36V/FKBP F36V using AP20187) and the low affinity antigen binding moieties E11.4.1-G32A, we exemplify a strategy to create conditionally active groups of CARs comprising three (comprising the two constructs (SEQ ID NO: 69) and (SEQ ID NO: 48)) or four CAR molecules (comprising the two constructs (SEQ ID NO: 70) and (SEQ ID NO: 48)) in the complexed state.
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos T cells were enriched by negative selection using RosetteSep Human T cell Enrichment Cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. A Jurkat T reporter cell line engineered with an NF-κB-dependent eGFP gene and a NF-AT-dependent CFP gene is a kind gift from Dr. Peter Steinberger at the Medical University of Vienna and is maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines are regularly tested for mycoplasma contamination and authentication is performed at Multiplexion, Germany. Cell densities are monitored with AccuCheck counting beads.
In Vitro Transcription and Electroporation of mRNA
In vitro transcription is performed using the mMessage mMachine T7 Ultra Kit according to the manufacturer's instructions. 50-200 ng of column purified PCR product are used as a reaction template. The resulting mRNA is purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution is diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture is loaded onto an RNeasy column and purification is performed according to the manufacturer's instructions. Elution is performed with nuclease-free water and purified mRNAs are frozen at −80° C. until electroporation. For transient transgene expression, Jurkat T cells are electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocol is used: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary T cells and Jurkat T cells are resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells are stained with the respective primary antibody for 25 minutes at 4° C. Stained cells are washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs is detected either 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 a primary antibody and a PE- or APC-conjugated secondary antibody, in the case of anti-Strep II tag antibodies. Expression of the engineered target antigen tEGFR was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the CD33 signal peptide, the low affinity rcSso7d variant E11.4.1-G32A, the Strep II tag, a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain, the dimerization domains FKBP F36V and FRB and the CD3ζ ITAM signalling domain are synthesized by GeneArt (Thermo Scientific). The nucleotide sequence encoding the dimerization domain FKBP is synthesized by Genscript. Sequences encoding the extracellular and transmembrane domain of EGFR are obtained from Addgene (plasmid #11011). Flexible linkers and the FLAG tag are inserted by using respective PCR primers. Assembly of nucleotide sequences into functional transgenes is performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
The Jurkat T reporter cell line is differentially labelled with distinct fluorescent proteins to enable efficient differentiation of Jurkat T reporter cells and Jurkat T target cells expressing the respective tumour antigen within the same well. Suitable fluorescent protein can be dKeima (Addgene #54618), mAmetrine (Addgene #54505) or similar proteins that have minimal cross-talk to the reporter proteins. Activity of the transcription factors NFAT and NFκB in Jurkat T reporter cells expressing the respective CAR is assessed by co-cultivation with target cells at an E:T ratios of 0.25:1, 0.5:1, 1:1 or 2:1 in round-bottom 96 well plates for 4 hours, 8 hours, 16 hours or 24 hours at 37° C. Cells are acquired using a BD LSRFortessa and activation of Jurkat T reporter cells is determined by measuring the geometric mean of the fluorescence intensity of the respective reporter protein or the percentage of reporter protein positive cells.
Dimerization of transgenes is induced prior to co-cultivation experiments. Primary T cells and Jurkat T cells are diluted to the final cell concentration in the respective cell culture medium. The homodimerization agent AP20187 and the heterodimerization agent AP21967 are diluted in cell culture medium and are added at 10 nM and 500 nM final concentration, respectively. Addition of the respective vehicle control DMSO or ethanol, respectively, at the same concentration served as control. Cells are incubated at 37° C. for 30 minutes to ensure efficient dimerization of transgenes and subsequently used for in vitro experiments.
For the FACS-based cytotoxicity assay two populations of target cells are generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. These two populations are mixed at a 1:1 ratio and co-cultured with CAR T cells at an E:T cell ratio of 4:1:1 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours or 24 hours at 37° C. Target cells without the addition of CAR T cells serve as a control condition (“targets only”). After the incubation period, the co-cultures are centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants are collected for subsequent cytokine measurements and the remaining cells are resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations is determined using a BD LSRFortessa flow cytometer and specific lysis is calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
By using heterodimerizing leucine zippers (Moll et al., Protein Sci. 2001; 10(3):649-655) and the low affinity antigen binding moieties E11.4.1-G32A and zHER2-R10A, we exemplify a strategy for generating a group of CARs that is formed by CAR molecules comprising domains that can efficiently heterodimerize independent of the presence of regulating molecules.
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos T cells were enriched by negative selection using RosetteSep Human T cell Enrichment Cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
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 were used as a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocol is used: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary T cells were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected either 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 a primary antibody and a PE- or APC-conjugated secondary antibody, in the case of anti-Strep II tag antibodies. Expression of the engineered target antigens tEGFR and tHER2 was detected either with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend) or with a PE-conjugated anti-HER2 antibody (clone 24D2, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the CD33 signal peptide, the low affinity rcSso7d variant E11.4.1-G32A, the Strep II tag, a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain and the CD3ζ ITAM signalling domain were synthesized by GeneArt (Thermo Scientific). The nucleotide sequence encoding the EE and RR leucine zipper was synthesized by Biocat. Sequences encoding the extracellular and transmembrane domain of EGFR were obtained from Addgene (plasmid #11011). Flexible linkers and the FLAG tag were inserted by using respective PCR primers. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
For the FACS-based cytotoxicity assay two populations of target cells were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. 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 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours or 24 hours at 37° C. Target cells without the addition of CAR T cells served as a control condition (“targets only”). After the incubation period, the co-cultures were centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants were collected for subsequent cytokine measurements and the remaining cells were resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
Over the last two decades it has been demonstrated that CAR molecules of the 2nd generation containing different co-stimulatory domains can efficiently activate T cells. Example 10 shows that CAR molecules comprising different co-stimulatory domains in their co-stimulatory signalling region also work in the context of a group of CARs of the present invention for discriminating between monovalent and multivalent interaction with target antigens.
Primary human T cells were obtained from de-identified healthy donor's blood after apheresis (Buffy coats from the Austrian Red Cross, Vienna, Austria). CD3pos T cells were enriched by negative selection using RosetteSep Human T cell Enrichment Cocktail (STEMCELL Technologies). Isolated and purified T cells were cryopreserved in RPMI-1640 medium supplemented with 20% FCS and 10% DMSO until use. CD3pos T Cells were activated with anti-CD3/CD28 beads according to the manufacturer's instructions and were 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 cultivated for at least 14 days before experiments were conducted. Jurkat T cells were a gift from Dr. Sabine Strehl at the CCRI and were maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. A Jurkat T reporter cell line engineered with an NFκB-dependent eGFP gene and a NFAT-dependent CFP gene is a kind gift from Dr. Peter Steinberger at the Medical University of Vienna and is maintained in RPMI-1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Cell lines were regularly tested for mycoplasma contamination and authentication was performed at Multiplexion, Germany. Cell densities were monitored with AccuCheck counting beads.
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 were used as a reaction template. The resulting mRNA was purified with an adapted protocol using the RNeasy column purification kit. Briefly, the mRNA solution was diluted with a mixture of RLT buffer, ethanol and 2-mercaptoethanol. The mixture was loaded onto an RNeasy column and purification was performed according to the manufacturer's instructions. Elution was performed with nuclease-free water and purified mRNAs were frozen at −80° C. until electroporation. For transient transgene expression, primary T cells were electroporated with varying amounts of the respective mRNA using the Gene Pulser (Biorad). Following protocol is used: primary T cells (square wave protocol, 500 V, 5 ms and 4 mm cuvettes), Jurkat T cells (square wave protocol, 500 V, 3 ms and 4 mm cuvettes).
Primary T cells were resuspended in FACS buffer (PBS, 0.2% human albumin and 0.02% sodium azide and treated for 10 minutes at 4° C. with 10% human serum. Cells were stained with the respective primary antibody for 25 minutes at 4° C. Stained cells were washed two times in FACS buffer and then either stained with the secondary antibody for 25 minutes at 4° C. or processed directly with a BD LSRFortessa. Expression of CAR constructs was detected via the Strep II tag using an anti-Strep II tag antibody (clone 5A9F9, Genscript) as a primary antibody and a PE- or APC-conjugated secondary antibody. Expression of the engineered target antigens tEGFR was detected with a PE- or APC-conjugated anti-EGFR antibody (clone AY13, BioLegend). Analysis was done by the FlowJo software.
The nucleotide sequences encoding the CD33 signal peptide, the low affinity rcSso7d variant E11.4.1-G32A, the Strep II tag, a flexible G4S linker, the human monomeric CD8α hinge (UniProt ID P01732, C164S and C181S) and CD8α transmembrane domain, the 4-1BB co-stimulatory domain, the dimerization domain FKBP F36V and the CD3ζ ITAM signalling domain were synthesized by GeneArt (Thermo Scientific). The nucleotide sequences encoding the intracellular domains of CD28, ICOS and OX40 were derived from cDNA clones (Sino Biological). Sequences encoding the extracellular and transmembrane domain of EGFR were obtained from Addgene (plasmid #11011). Flexible linkers were inserted by using respective PCR primers. Assembly of nucleotide sequences into functional transgenes was performed by using the Gibson Assembly Master Mix, according to the manufacturer's instructions. The schematics and sequences are shown in
The Jurkat T reporter cell line was differentially labelled with distinct fluorescent proteins to enable efficient differentiation of Jurkat T reporter cells and Jurkat T target cells expressing the respective tumour antigen within the same well. Suitable fluorescent proteins were dKeima (Addgene #54618), mAmetrine (Addgene #54505) or similar proteins that have minimal cross-talk to the reporter proteins. Activity of the transcription factors NFAT and NFκB in Jurkat T reporter cells expressing the respective CAR was assessed by co-cultivation with target cells at an E:T ratios of 0.25:1, 0.5:1, 1:1 or 2:1 in round-bottom 96 well plates for 24 hours at 37° C. Cells were acquired using a BD LSRFortessa and activation of Jurkat T reporter cells was determined by measuring the geometric mean of the fluorescence intensity of the respective reporter protein or the percentage of reporter protein positive cells.
For the FACS-based cytotoxicity assay two populations of target cells were generated: (i) Jurkat cells electroporated with mRNA encoding eGFP and RNA encoding the respective target antigen, and (ii) Jurkat cells only electroporated with mRNA encoding mCherry. 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 with 20,000 target cells/well in round-bottom 96 well plates for 4 hours or 24 hours at 37° C. Target cells without the addition of CAR T cells served as a control condition (“targets only”). After the incubation period, the co-cultures were centrifuged (5 minutes, 1600 rpm, 4° C.), supernatants were collected for subsequent cytokine measurements and the remaining cells were resuspended in 100 μL of FACS buffer, consisting of PBS, 0.2% human albumin and 0.02% sodium azide. The viability of target antigenpos and target antigenneg cell populations was determined using a BD LSRFortessa flow cytometer and specific lysis was calculated with the following formula:
% specific lysis=(1−(((% eGFPpos cells of the sample)/(% mCherrypos cells of the sample)/(% eGFPpos cells of the “targets only” control)/(% mCherrypos cells of the “targets only” control))))*100.
The present invention therefore discloses the following preferred embodiments:
1. A group of chimeric antigen receptors (CARs) consisting of two, three or four CAR molecules, wherein each member of the group of CARs is different in its amino acid sequence from one another, and wherein each of the CAR molecules of the group comprise at least a transmembrane domain and an ectodomain, wherein the ectodomain comprises one or two antigen binding moieties and/or one or two binding sites to which other polypeptides each comprising at least an antigen binding moiety are able to bind, and wherein at least one CAR molecule of the group additionally comprises an endodomain, which comprises at least a signalling region which can transduce a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM), and
wherein the endodomain of each CAR molecule of the group, in case the respective CAR molecule comprises an endodomain, is located on the intracellular side of a cell membrane, if expressed in a cell, wherein the ectodomain of each CAR molecule of the group translocates to the extracellular side of a cell membrane, if expressed in a cell, and wherein the transmembrane domain of each CAR molecule of the group is located in a cell membrane, if expressed in a cell;
wherein the ectodomain of each CAR molecule of the group in its prevalent conformation is free of cysteine amino acid moieties which are able to form intermolecular disulphide bonds with other CAR molecules of the group, respectively, and
wherein the antigen binding moieties of the different CAR molecules of the group and of the different other polypeptides are specific for different target antigens which are not linked to each other covalently, and
wherein the affinity of each individual antigen binding moiety of a CAR molecule of the group to its respective target antigen is between 1 mM and 100 nM, and
wherein the affinity of each individual antigen binding moiety of another polypeptide to its respective target antigen or al-ternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 1 mM and 100 nM, and
wherein each CAR molecule of the group comprises at least one heterodimerization domain, which can mediate defined heterodimerization with other CAR molecules of the group, wherein this heterodimerization of a pair of heterodimerization domains either occurs independent of a regulating molecule, or occurs in the absence of a regulating molecule and is reduced by a regulating molecule, or is induced by a regulating molecule and optionally reduced by another regulating molecule, wherein a regulating molecule is able to bind under physiological conditions to at least one member of a pair of heterodimerization domains and by inducing or reducing heterodimerization either induces or reduces the formation of a non-covalently complexed group of CARs consisting of two, three or four CAR molecules.
2. A group of CARs according to embodiment 1, wherein the antigen binding moiety comprises only one protein domain.
3. A group of CARs according to embodiment 1 or 2, wherein the antigen binding moiety comprises only one protein domain and does not cause dimerization or oligomerization of CAR molecules of the group when expressed on the surface of a human cell, and wherein said protein domain preferably is selected from the group consisting of a human or non-human VH or VL single domain antibody (nanobody) or an engineered antigen binding moiety based on the Z-domain of staphylococcal Protein A, lipocalins, SH3 domains, fibronectin type III (FN3) domains, knottins, Sso7d, rcSso7d, Sac7d, Gp2, DARPins, ubiquitin, a receptor, a ligand of a receptor, or a co-receptor.
4. A group of CARs according to any one of embodiments 1 to 3, wherein the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM, and
wherein the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 1 mM and 150 nM, preferably between 1 mM and 200 nM, more preferably between 1 mM and 300 nM, especially between 1 mM and 400 nM.
5. A group of CARs according to any one of embodiments 1 to 3, wherein the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM, and
wherein the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 500 μM and 100 nM, preferably between 250 μM and 100 nM, more preferably between 125 μM and 100 nM, especially between 50 μM and 100 nM.
6. A group of CARs according to any one of embodiments 1 to 3, wherein the affinity of each individual antigen binding moiety of a CAR molecule of the group to its target antigen is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM, and
wherein the affinity of each individual antigen binding moiety of another polypeptide to its target antigen or alternatively the affinity of this other polypeptide to the binding site of its respective CAR molecule is between 500 μM and 150 nM, preferably between 250 μM and 200 nM, more preferably between 125 μM and 300 nM, especially between 50 μM and 400 nM.
7. A group of CARs according to any one of embodiments 1 to 6, wherein the target antigens specifically recognized by the antigen binding moieties of the group of CARs or of other polypeptides being able to bind to CAR molecules of the group are naturally occurring cellular surface antigens or polypeptides, carbohydrates or lipids bound to naturally occurring cellular surface antigens.
8. A group of CARs according to any one of embodiments 1 to 7, wherein the antigen binding moieties of the group of CARs and of other polypeptide(s) being able to bind to CAR molecules of the group bind to at least two different target antigens present on a cell, preferably at least two different target antigens of a cell, on a solid surface, or a lipid bilayer.
9. A group of CARs according to any one of embodiments 1 to 8, wherein at least one target antigen, to which an antigen binding moiety of a group of CARs, or of another polypeptide being able to bind to a CAR molecule of the group, specifically binds, comprises a molecule preferably 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, 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, CHRNB4, KIAA1524, NRM.3, RPRM, GRM8, KCNH4, Melanocortin 1 receptor, PTPRH, SDK1, SCN9A, SORCS1, CLSTN2, Endothelin converting enzyme like-1, Lysophosphatic acid receptor 2, LTB4R, TLR2, Neurotropic tyrosine kinase 1, MUC16, B7-H4, epidermal growth factor receptor (EGFR), ERBB2, HER3, EGFR variant III (EGFRvIII), HGFR, FOLR1, MSLN, CA-125, MUC-1, prostate-specific membrane antigen (PSMA), mesothelin, epithelial cell adhesion molecule (EpCAM), L1-CAM, CEACAM1, CEACAM5, CEACAM6, VEGFR1, VEGFR2, high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A 1, IL-13R-α2, disialogangliosides (GD2 and GD3), tumour-associated carbohydrate antigens (CA-125, CA-242, Tn and sialyl-Tn), 4-1BB, 5T4, BAFF, carbonic anhydrase 9 (CA-IX), C-MET, CCR1, CCR4, FAP, fibronectin extra domain-B (ED-B), GPNMB, IGF-1 receptor, integrin α5ρ1, integrin αvβ, ITB5, ITGAX, embigin, PDGF-Rα, ROR1, Syndecan 1, TAG-72, tenascin C, TRAIL-R1, TRAIL-R2, NKG2D-Ligands, a major histocompatibility complex (MHC) molecule presenting a tumour-specific peptide epitope, preferably 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.
10. A group of CARs according to any one of embodiments 1 to 9, wherein the ectodomain of the CAR molecules of the group comprises a structurally flexible hinge region interposed between an antigen binding moiety and the transmembrane domain, preferably a hinge region derived from CD8 alpha (amino acid sequence position 138-182 according to UniProtKB/Swiss-Prot P01732-1), or CD28 (amino acid sequence position 114-152 according to UniProtKB/Swiss-Prot P10747), or PD-1 (amino acid sequence position 146-170 according to UniProtKB/Swiss-Prot Q15116), wherein the sequences derived from CD8 alpha, CD28 or PD-1 can be N-terminally and/or C-terminally truncated and can have any length within the borders of the said sequence region, and wherein the cysteine residues in the said hinges derived from CD8 alpha and CD28 are deleted or replaced by other amino acid residues.
11. A group of CARs according to any one of embodiments 1 to 10, wherein the domains of the CAR molecules are derived from different proteins, wherein at least two of these domains are connected via amino acid linker sequences, wherein the linker preferably comprises 1 to 40 amino acids in length.
12. A group of CARs according to any one of embodiments 1 to 11, wherein heterodimerization of the heterodimerization domains of at least two CAR molecules of the group, preferably of all CAR molecules of the group, is enhanced by binding of a regulating molecule.
13. A group of CARs according to any one of embodiments 1 to 12, wherein heterodimerization of the heterodimerization domains of at least two CAR molecules of the group, preferably all CAR molecules of the group, does not require binding of a regulating molecule.
14. A group of CARs according to any one of embodiments 1 to 13, wherein in the case of at least two heterodimerization domains within a CAR molecule the heterodimerization domains of that CAR molecule are separated by the cell membrane, and/or are members of different pairs of heterodimerization domains not able to bind to each other in the presence and absence of a regulating molecule in order to prevent the formation of complexes comprising two or more identical CAR molecules of the group.
15. A group of CARs according to any one of embodiments 1 to 14, wherein each CAR molecule of the group comprises at least a signalling region which can transduce a signal via at least one immunoreceptor tyrosine-based activation motif (ITAM).
16. A group of CARs according to any one of embodiments 1 to 15, wherein the heterodimerization domains of at least two CAR molecules of the group are selected from: FK506 binding protein 12 (FKBP12, FKBP), FKBP-rapamycin associated protein (FRB) mutant T82L, calcineurin catalytic subunit A (CnA), cyclophilin, GAI, GID1, PYL and ABI.
17. A group of CARs according to any one of embodiments 1 to 16, wherein heterodimerization of at least two CAR molecules of the group is mediated by a pair of heterodimerization domains comprising FKBP and FKBP-rapamycin associated protein (FRB, mutant T82L) and/or pairs of interacting coiled-coil domains.
18. A group of CARs according to any one of embodiments 1 to 17, wherein heterodimerization of at least two CAR molecules of the group is mediated by a pair of heterodimerization domains comprising a ligand binding domain from a nuclear receptor and a co-regulator peptide.
19. A group of CARs according to any one of embodiments 1 to 18, wherein heterodimerization of at least two CAR molecules of the group is mediated by a pair of heterodimerization domains comprising a lipocalin-fold molecule and a lipocalin-fold binding interaction partner.
20. A group of CARs according to any one of embodiments 1 to 19, wherein heterodimerization of at least two CAR molecules of the group is mediated by a pair of heterodimerization domains comprising a ligand binding domain from a nuclear receptor and a co-regulator peptide, and wherein the ligand binding domain from a nuclear receptor is selected from an estrogen receptor, an ecdysone receptor, a glucocorticoid receptor, an androgen receptor, a thyroid hormone receptor, a mineralocorticoid receptor, a progesterone receptor, a vitamin D receptor, a PPARγ receptor, a PPARβ receptor, a PPARα receptor, a pregnane X receptor, a liver X receptor, a farnesoid X receptor, a retinoid X receptor, a RAR-related orphan receptor, a retinoic acid receptor, and the respective compatible co-regulators of the nuclear receptors selected from SRC1, GRIP1, AIB1, PGC1a, PGC1b, PRC, TRAP220, ASC2, ASC2-1, ASC2-2, CBP, CBP-1, CBP-2, P300, CIA, ARA70, ARA70-1, ARA70-2, NSD1, SMAP, Tip60, ERAP140, Nix1, LCoR, N-CoR, SMRT, RIP140, RIP140-1, RIP140-2, RIP140-3, RIP140-4, RIP140-5, RIP140-6, RIP140-7, RIP140-8, RIP140-9, PRIC285, PRIC285-1, PRIC285-2, PRIC285-3, PRIC285-4, PRIC285-5, SRC1-1, SRC1-2, SRC1-3, SRC1-4a, SRC1-4b, SRC2, SRC3, SRC3-1, PGC1, TRAP220-1, TRAP220-2, NR0B1, NRIP1, TIF1, TIF2, CoRNR Box, CoRNR1, CoRNR2, αβV, EA2, TA1, EAB1, GRIP1-1, GRIP1-2, GRIP1-3, AIB1-1, AIB1-2, AIB1-3, PGC1a, PGC1b. 21. A group of CARs according to any one of embodiments 1 to 20, wherein heterodimerization of at least two CAR molecules of the group is mediated by a pair of heterodimerization domains comprising a lipocalin-fold molecule and a lipocalin-fold binding interaction partner, and wherein the lipocalin-fold molecule is a derivative of a naturally occurring lipocalin or iLBP with 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 of the structurally conserved β-barrel structure, preferably corresponding structurally to the regions of amino acid residues selected from
| Number | Date | Country | Kind |
|---|---|---|---|
| 18198843.7 | Oct 2018 | EP | regional |
| 19175975.2 | May 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/076916 | 10/4/2019 | WO | 00 |
