Trimolecular system for protein dimerization and methods of use

Abstract

The present disclosure provides compositions and methods utilizing a target protein capable of binding to a small molecule to form a complex and a binding member that specifically binds to the complex, where the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein. The non-human protein may be derived from a viral, bacterial, fungal, or protozoan protein. These compositions and methods allow for controlled interactions between polypeptides fused individually to the target protein and binding member, and can be used to control the activity of dimer-inducing proteins, such as split transcription factors and split chimeric antigen receptors, by adding small molecules. The present disclosure provides expression vectors, binding members, dimer-inducing proteins, nucleic acids, cells, viral particles, kits, systems, and methods involving these components.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 62 / 874,025, filed July 15, 2019, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to compositions and methods that enable controlled interaction of a target protein and a polypeptide fused to a binding member. The compositions and methods utilize a target protein that binds to a small molecule to form a complex and a binding member that specifically binds to the complex, where the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein. The non-human protein may be derived from a bacterial, viral, fungal, or protozoan protein. The non-human protein may be derived from a viral protease and the small molecule is a viral protease inhibitor. The disclosure further relates to dimer-derived proteins, such as split transcription factors and split chimeric antigen receptors, that comprise the target protein and the binding member. The methods and compositions described herein find use, for example, in cell therapy and gene therapy involving controlled protein expression and / or protein activation. [Background technology]

[0003] Protein-protein interactions (PPIs) represent a common regulatory mechanism that controls multiple biological functions. For example, gene transcription, protein folding, protein localization, protein degradation, and signal transduction all depend on the interaction or proximity of one protein to another, or indeed several other proteins. By controlling protein-protein interactions over time, researchers can dissect complex biological mechanisms and easily track the functional consequences of PPIs. Furthermore, this ability to control biological function can be exploited in cell and gene therapy to control therapeutic activity, enabling safer, more personalized therapies.

[0004] A commonly used technique for controlling protein-protein interactions is so-called chemical induction of dimerization (CID), which uses small molecules to bind two proteins that would not otherwise interact, forming a three-molecule ternary complex (Non-Patent Document 1). The most widely used CID is rapamycin (an immunosuppressant derived from Streptomyces hygroscopicus) and its analogs, which form heterodimeric complexes with the proteins FKBP12 (a 12-kDa FK506-binding protein) and FRB (a domain derived from mTOR (mammalian target of rapamycin)) (Non-Patent Document 2). Along with other naturally occurring CIDs, such as the plant hormone S-(+)-abscisic acid (ABA) and gibberellin (GA3-AM), an attractive feature of rapamycin is its cooperative binding mechanism, which allows protein 2 to bind only to protein 1:CID complexes (Non-Patent Document 3). Novel CIDs have also been generated by chemically linking two small molecules that bind the same or different proteins (these proteins form a dimerized protein pair) (Non-Patent Document 4; Non-Patent Document 5). However, in these systems, at high concentrations of bifunctional CID, the non-productive complex between one protein partner and the CID outweighs the formation of the trimolecular complex, meaning that a linear dose response cannot be obtained.

[0005] Therefore, there is an urgent need for novel cooperatively binding CID systems that can be used to regulate cellular functions and expand into many orthogonal systems, including those for complex genetic circuits. Furthermore, very few CIDs have been approved for long-term human use. Recently, a method for generating a novel CID system (AbCID) using an antibody-based phage display selection method has been described (Non-Patent Document 6). The CID used in that study was ABT-737, a Bcl-2 and Bcl-xL inhibitor, with Bcl-xL itself used as one of the protein partners. Subsequently, a second protein was selected from a phage display library of single-chain Fab (scFab) molecules to be selective for the Bcl-xL:ABT-737 complex over Bcl-xL alone.

[0006] Although the approaches described in Non-Patent Document 6 and Patent Document 1, which identify complex-specific molecules by utilizing existing small molecules and their targets, are attractive, overexpressing specific human proteins (e.g., the anti-apoptotic Bcl-xL protein) and using small molecules that bind to human targets in vivo are not without risks. For example, overexpressing a functional human protein can affect the expressing cell, which may affect the health and survival of the cell. Furthermore, using small molecules whose targets are expressed in vivo can result in increased dosage requirements due to competition between the small molecule and the endogenous target for binding with the overexpressed target. Furthermore, binding of a small molecule to an endogenous target can affect the function of the protein, which may be detrimental to the cells expressing the target. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 213848A1 [Non-patent literature]

[0008] [Non-Patent Document 1] Stanton, Chory, and Crabtree 2018 [Non-patent document 2] Sabers et al. 1995 [Non-patent document 3] Banaszynski, Liu, and Wandless 2005 [Non-patent document 4] Belshaw, Ho, et al. 1996 [Non-patent document 5] Belshaw, Spencer, et al. 1996 [Non-patent document 6] Hill et al. 2018 Summary of the Invention [Means for solving the problem]

[0009] Disclosed herein is an approach aimed at overcoming the limitations of AbCID systems such as those described by Hill et al. First, the small molecules described herein are already approved for human use, facilitating a smoother path to regulatory approval. Second, and more importantly, the inventors have identified advantages in selecting small molecules that bind to non-human proteins, particularly viral proteins, rather than identifying small molecules by their human targets. For example, using small molecules without human targets is expected to improve safety when used in humans. It has also been concluded that using viral, bacterial, fungal, or protozoan target proteins eliminates the risk of endogenous small molecules "sinking" when used in humans if the small molecule binds to the target protein and also binds to an endogenous human target. Furthermore, expressing viral, bacterial, fungal, or protozoan proteins in human cells is less likely to affect the cellular physiology of the cells than human proteins with endogenous functions.

[0010] Antiviral agents have been approved that bind to and inhibit various viral proteins, including viral polymerases, integrases, transcriptases, and proteases. The present inventors have recognized that targeting proteins derived from viral proteases in particular would be beneficial because these proteases are located in the cytoplasm, are small, and consist of discrete domains.

[0011] Thus, the present disclosure: i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that binds to the T-SM complex with higher affinity than a binding member that binds both the target protein alone and the small molecule alone; One or more expression vectors are provided, wherein the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein. In one embodiment, the non-human protein is derived from a viral protein and the small molecule is an inhibitor of the viral protein. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In one embodiment, the non-human protein is derived from a bacterial protein and the small molecule is an inhibitor of the bacterial protein. In one embodiment, the non-human protein is derived from a fungal protein and the small molecule is an inhibitor of the fungal protein. In one embodiment, the non-human protein is derived from a protozoan protein and the small molecule is an inhibitor of the protozoan protein.

[0012] As shown herein, binding of a binding member to a T-SM complex results in the formation of a trimolecular complex comprised of the binding member, the target protein, and the small molecule, and the formation of this trimolecular complex can be controlled by the presence of the small molecule. Controlling the formation of the trimolecular complex is advantageous because it allows, for example, control of the interaction of the target protein and the polypeptide to which the binding member is fused.

[0013] The present disclosure also provides i) a target protein capable of binding to a small molecule such that a complex between the target protein and the small molecule (T-SM complex) is formed; ii) a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; The system provides a target protein derived from a non-human protein and a small molecule that is an inhibitor of the non-human protein. In one embodiment, the non-human protein is derived from a viral protein and the small molecule is an inhibitor of the viral protein. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In one embodiment, the non-human protein is derived from a bacterial protein and the small molecule is an inhibitor of the bacterial protein. In one embodiment, the non-human protein is derived from a fungal protein and the small molecule is an inhibitor of the fungal protein. In one embodiment, the non-human protein is derived from a protozoan protein and the small molecule is an inhibitor of the protozoan protein.

[0014] In some embodiments, the viral protease is HCV NS3 / 4A protease or HIV protease. These proteases are known to be targeted by several approved small molecules that are known to be generally well tolerated in humans and suitable for long-term administration, thus representing suitable target proteins for use herein.

[0015] In some embodiments, the viral protease is an HCV NS3 / 4A protease, such as the protease having the amino acid sequence of SEQ ID NO: 1. The HCV NS3 / 4A protease is a small, monomeric protein that can be expressed in the cytoplasm and has a limited number of endogenous human targets, making it an ideal target protein.

[0016] In some embodiments, the small molecule is selected from the group consisting of simeprevir, asunaprevir, vaniprevir, boceprevir, naraprevir, and telaprevir. All of these small molecules are approved for human treatment. In some embodiments, the small molecule is selected from the group consisting of simeprevir, boceprevir, and telaprevir. These small molecules are approved for human treatment and are generally well tolerated in humans.

[0017] In some embodiments, the small molecule is simeprevir. Simeprevir (Olysio®) is an orally administered small molecule that is cell membrane permeable and has a pharmacokinetic (PK) profile that supports once-daily dosing. Simeprevir has been used long-term (up to 39 months) in combination with ribavirin and pegylated interferon to treat HCV infection, is listed on the WHO Essential Medicines List, and has been shown to be a well-tolerated, widely administered agent.

[0018] The present inventors have determined that any potential non-specific activity caused by overexpression of a viral protease can be reduced by using a target protein with attenuated viral activity compared to the viral protease from which it is derived. Thus, in some embodiments, the target protein has attenuated viral activity compared to the viral protease from which it is derived.

[0019] For example, the target protein may contain one or more amino acid mutations compared to the viral protease from which it is derived. In certain embodiments where the viral protease is HCV NS3 / 4A protease, the target protein may have an amino acid mutation at one or more amino acids selected from positions 72, 96, 112, 114, 154, 160, and 164, where the amino acid numbering corresponds to SEQ ID NO: 1. For example, the target protein may have an amino acid mutation, e.g., to alanine, at position 154, where the amino acid numbering corresponds to SEQ ID NO: 1. As described below, positions 72, 96, 112, 114, 154, 160, and 164 of SEQ ID NO: 1 correspond to positions 57, 81, 97, 99, 139, 145, and 149, respectively, of the full-length NS3 protein set forth in SEQ ID NO: 199. The Examples refer to amino acid positions according to the amino acid numbering of the full-length NS3 protein. For example, reference in the examples to a "S139A" mutation corresponds to a "S154A" mutation, where the amino acid numbering corresponds to SEQ ID NO:1.

[0020] In some cases, when the second small molecule is different from the small molecule in the T-SM complex, it may be desirable for the competing small molecule to be able to bind to the target protein in the T-SM complex so that it can displace the small molecule in the T-SM complex. In this way, the second small molecule can decrease the half-life of the trimolecular complex formed between the binding member, the target protein, and the small molecule. This may be desirable, for example, in situations where it would be useful to use a second small molecule to hasten dissociation of the trimolecular complex, e.g., to rapidly inhibit the activity of a dimer-inducing protein that is activated by the formation of the trimolecular complex.

[0021] As shown herein, simeprevir binds to the target protein HCV NS3 / 4A protease (S139A) (SEQ ID NO: 2) with such high affinity that other small molecules that bind to the target protein cannot displace simeprevir from the T-SM complex. The inventors have discovered that certain affinity-reducing mutations can be introduced into the target protein, which reduces the affinity of simeprevir for HCV NS3 / 4A protease and allows other small molecules to "compete" with simeprevir and disrupt the formed trimolecular complex. Thus, in certain embodiments where the viral protease is HCV NS3 / 4A protease and the small molecule is simeprevir, the target protein may contain affinity-reducing amino acid substitutions at one or more amino acids selected from positions 151 and 183, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the affinity-reducing amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine (e.g., aspartic acid or asparagine), and the affinity-reducing mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine (e.g., glutamic acid). The target protein may include affinity-reducing amino acid mutations, such as amino acid mutations at one or more amino acids selected from positions 72, 96, 112, 114, 154, 160, and 164, in addition to other mutations described herein.

[0022] In some embodiments, the binding member is an antibody molecule such as a single-chain variable fragment (scFv) or an antibody mimetic, e.g., a Tn3 protein. In certain embodiments, the binding member is a Tn3 protein or scFv, e.g., a Tn3 protein and scFv as defined herein. Compared to the single-chain Fab (scFab) used in the system described by Hill et al., both the Tn3 protein and scFv are smaller in size. This can be advantageous, for example, when delivering an expression cassette in an expression vector with limited coding capacity, such as a viral vector. The development and use of specific Tn3 proteins and scFvs that bind to the complex between HCV NS3 / 4A protease and simeprevir are described herein and are shown to function as binding members in the context of the present disclosure. These Tn3 proteins and scFvs are referred to as HCV NS3 / 4A PR:simeprevir complex-specific binding (PRSIM) molecules.

[0023] It has been determined that the approach described herein can be used when the target protein and binding members are individually fused to polypeptides (referred to as "constituent polypeptides"). In particular, it has been determined that this approach can be implemented to control the activity of proteins that require dimerization or clustering to promote protein activity. Such proteins are referred to herein as "dimer-inducing proteins," including "split proteins," "dimerization-deficient proteins," and "split complexes." Split proteins are separated or divided into two or more domains, and contain a single protein whose constituent parts are nonfunctional or minimally capable of activation. However, bringing the separated constituent polypeptides into close proximity can initiate or restore function or activity. Examples include split fluorescent proteins (e.g., split GFP), split luciferases (e.g., NanoBiT), and split kinases. A further example is described: split transcription factors, in which distinct DNA-binding domains (DBDs) and activation domains (ADs) are separated such that each transcription factor domain cannot initiate transcription on its own. Only when the two domains are brought into close proximity (i.e., the two domains form a functional "transcription factor") can transcriptional activation of the associated gene be reconstituted. Dimerization-deficient proteins are proteins that require dimerization for activation, but whose intrinsic dimerization ability has been abolished, for example, by mutation or removal of the dimerization domain. One such example is the iCasp9 molecule, a caspase-9 protein with its dimerization (CARD) domain removed. A split complex refers to either a single protein or two or more distinct proteins that do not function optimally or function differently until they are brought into close proximity, i.e., "clustered." One such example is the split chimeric antigen receptor (CAR), where the specific intracellular domains of the CAR involved in activating cell signaling are physically separated, preventing full cellular activation.Bringing the domains into close proximity activates cell signaling (i.e., the domains form a fully functional CAR).

[0024] Thus, in some embodiments, the target protein is fused to a first constituent polypeptide and the binding member is fused to a second constituent polypeptide. In preferred embodiments, one or more expression vectors encode a dimer-inducing protein, such as a split transcription factor or a split CAR.

[0025] In one embodiment, (1) a first constituent polypeptide comprises a DNA-binding domain and is fused to a target protein to form a DBD-T (DBD-target protein) fusion protein, and a second constituent polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM (transcriptional regulatory domain-binding molecule) fusion protein, or (2) a first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein, and a second constituent polypeptide comprises a DNA-binding domain and is fused to a binding member to form a DBD-BM fusion protein, and the first and second constituent polypeptides dimerize to form a transcription factor.

[0026] In another embodiment, a first constituent polypeptide comprises a first costimulatory domain and is fused to a target protein, and a second constituent polypeptide comprises an intracellular signaling domain and is fused to a binding member. The first constituent polypeptide may further comprise an antigen-specific recognition domain and a transmembrane domain, and the second constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain, and the first and second constituent polypeptides form a chimeric antigen receptor (CAR) upon dimerization.

[0027] Alternatively, a first constituent polypeptide comprises an intracellular signaling domain and is fused to a target protein, a second constituent polypeptide comprises a first costimulatory domain and is fused to a binding member, the first constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain, and the second constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain, and the first and second constituent polypeptides form a chimeric antigen receptor (CAR) upon dimerization.

[0028] In another embodiment, the first constituent polypeptide comprises a first caspase component and the second constituent polypeptide comprises a second caspase component, and the first and second constituent polypeptides form a caspase upon dimerization.

[0029] In some embodiments, one or more expression vectors are viral vectors, such as AAV vectors.

[0030] The present disclosure also provides a method for producing viral particles in vitro, comprising transfecting a host cell with a viral vector as defined herein, expressing viral proteins required for forming viral particles within the host cell, and culturing the transfected cell in a culture medium so that the cell produces viral particles.

[0031] The present disclosure also provides i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; One or more viral particles are provided, wherein the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein, and the first and second expression cassettes form part of a viral genome within the one or more viral particles. In one embodiment, the non-human protein is derived from a viral protein and the small molecule is an inhibitor of the viral protein. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In another embodiment, the non-human protein is derived from a bacterial, fungal, or protozoan protein.

[0032] The expression cassettes, target proteins, small molecules, and binding members within the one or more viral particles may be as further described herein. The target proteins and binding members may be fused to first and second constituent polypeptides, respectively (e.g., to encode a dimer-inducing protein), as further described herein.

[0033] The viral particle may be an AAV particle.

[0034] In one aspect, the disclosure provides a binding member that specifically binds to a complex between i) a target protein derived from a non-human protein and ii) a small molecule that is an inhibitor of the non-human protein, wherein the binding member binds to the complex with higher affinity than it binds to both the target protein and the small molecule alone. In one embodiment, the non-human protein is derived from a viral protein and the small molecule is an inhibitor of the viral protein. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In another embodiment, the non-human protein is derived from a bacterial, fungal, or protozoan protein. As described herein, binding members specific for such complexes are useful as a method for controlling the formation of a trimolecular complex between a binding member, a target protein, and a small molecule in a manner that overcomes the shortcomings of the binding molecules described in Hill et al.

[0035] In another aspect, the present disclosure provides a dimer-inducing protein comprising a target protein and a binding member as defined herein. The dimer-inducing protein can be, for example, a split transcription factor, a split CAR, or a split caspase protein.

[0036] In one aspect, the present disclosure provides cells, such as allogeneic or autologous cells including stem cells, induced pluripotent stem (iPS) cells, or immune cells, comprising one or more of the expression cassettes, expression vectors, binding members, target proteins, or dimer-inducing proteins defined herein. The cells are capable of expressing the binding members, target proteins, or dimer-inducing proteins described herein. The present disclosure also provides methods for genetically modifying cells to generate cells that express the binding members or dimer-inducing proteins described herein, the methods comprising administering an expression vector to the cells. The methods may be performed in vitro or ex vivo.

[0037] It has further been determined that the techniques described herein for fusing target proteins and binding members to the constituent polypeptides of a split transcription factor can be used in gene therapy involving the regulation of expression of a desired expression product (e.g., a desired polypeptide) in a cell.

[0038] Thus, in one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: i) expressing a dimer-inducing protein as defined herein in a cell, wherein the dimerization of the first and second constituent polypeptides forms a transcription factor, and the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating expression of a desired expression product in the cell; ii) administering a small molecule to the cell to modulate expression of the desired expression product.

[0039] In some embodiments of the method, the target sequence of the DNA binding domain is located in a promoter operably linked to the coding sequence of the desired expression product.

[0040] The method may involve the delivery of an expression cassette encoding a dimer-inducing protein to control the expression of a desired expression product that is exogenously delivered to the cell.

[0041] Thus, in some embodiments, the method comprises administering to the cell a third expression cassette that encodes a desired expression product and that includes a target sequence for the DNA binding domain.

[0042] Alternatively, the method may involve the delivery of an expression cassette encoding a dimer-inducing protein to control the expression of a desired expression product that is already present as part of the genome of the cell (i.e., an endogenous desired expression product).

[0043] Thus, in another embodiment of the method, the target sequence is located within the genome of the cell.

[0044] It has further been determined that the techniques described herein can be used in methods of treating cells, which typically involve taking cells (autologous cells) from an individual, modifying the cells ex vivo to express a particular protein, e.g., a dimer-inducing protein, and readministering them back to the individual.

[0045] Thus, in another aspect, the present disclosure provides a method for producing a medicament for a method of manufacturing a medicament for a medical device, comprising: i) administering to an individual in need thereof a cell comprising an expression cassette encoding a dimer-inducing protein as defined herein; ii) administering the small molecule to the individual.

[0046] In one aspect, the disclosure provides nucleic acids encoding binding members, target proteins, and dimer-inducing proteins as defined herein.

[0047] In one aspect, the present disclosure provides a kit as defined herein.

[0048] Furthermore, it has been determined that additional small molecules (referred to herein as "competitor small molecules") can be utilized to induce disassembly of the trimolecular complex formed between the binding member, target protein, and small molecule. This can be useful when it is desirable to rapidly inactivate the chemical induction of dimerization (CID) disclosed herein, for example, to block transgene expression or therapeutic activity associated with the activity of the dimer-inducing protein.

[0049] In another aspect, the disclosure provides a method of inducing degradation of a trimolecular complex, comprising administering a competitor small molecule to a cell containing the trimolecular complex; a trimolecular complex is formed between the binding member and a complex formed by the target protein and the small molecule (T-SM complex), and the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein and the small molecule alone; Methods are provided in which a competing small molecule can bind to a target protein in a T-SM complex and displace the small molecule from the T-SM complex.

[0050] Methods for determining whether a competitor small molecule can bind to and displace a target protein from a T-SM complex include assays (e.g., homogeneous time-resolved fluorescence (HTFR) binding assays) in which a preformed trimolecular complex is generated and the ability of the binding member to bind to the T-SM complex is measured in the presence of increasing concentrations of the competitor small molecule. Displacement of the competitor small molecule from the T-SM complex may be possible if the binding member is able to inhibit binding to the T-SM complex by at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as measured using an HTFR binding assay. In some embodiments, the competitor small molecule is asunaprevir, paritaprevir, vaniprevir, grazoprevir, danoprevir, or glecaprevir. The binding members, target proteins, and small molecules used in this method may be as further defined herein in connection with other aspects of the disclosure.

[0051] In certain embodiments, the target protein may be derived from HCV NS3 / 4A protease, the small molecule in the T-SM complex may be simeprevir, and optionally, the binding member may be PRSIM_23. For example, the target protein may have an amino acid sequence having at least 90% identity to SEQ ID NO: 1. As shown herein, simeprevir binds to the target protein HCV NS3 / 4A protease (S139A) (SEQ ID NO: 2) with such high affinity that other small molecules that bind to the target protein cannot displace simeprevir from the T-SM complex. As further shown herein, mutations can be introduced into HCV NS3 / 4A protease that reduce the affinity of simeprevir for HCV NS3 / 4A protease and allow competing small molecules to disrupt the trimolecular complex formed between HCV NS3 / 4A protease, simeprevir, and the binding member PRSIM_23.

[0052] Thus, in embodiments where the target protein is derived from HCV NS3 / 4A protease and the small molecule is simeprevir, the target protein may have affinity-reducing amino acid mutations (e.g., substitutions) at one or more amino acids selected from positions 151 and 183, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the affinity-reducing amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine, and the affinity-reducing mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine. In some embodiments, the affinity-reducing amino acid mutation at position 151 is a mutation to aspartic acid or asparagine, and the affinity-reducing mutation at position 183 is a mutation to glutamic acid. The target protein may include affinity-reducing amino acid mutations in addition to another amino acid mutation described herein (e.g., in addition to the amino acid mutation at position 154 to alanine, etc.).

[0053] The present disclosure includes combinations of the described aspects and preferred features unless such combinations are clearly contraindicated or explicitly avoided.

[0054] Reference will now be made to the accompanying drawings to describe embodiments and experiments illustrating the principles of the present disclosure. [Brief explanation of the drawings]

[0055] [Figure 1] 1 shows a schematic diagram of the three components of an exemplary PRSIM-based chemical induction of dimerization (CID): A represents a target protein (e.g., typically an HCV NS3 / 4A PR(S139A) mutant), B represents a small molecule (e.g., typically simeprevir), and C represents a binding member (e.g., an scFv or Tn3 specific for the complex of simeprevir and HCV NS3 / 4A PR(S139A)). [Figure 2]The three-dimensional structure of simeprevir in complex with HCV NS3 / 4A PR (PBD code: 3KEE; 2.4 Å) is shown, illustrating the shallow binding site of HCV NS3 / 4A PR and the large surface exposure of simeprevir. [Figure 3A] An SDS-PAGE gel of recombinant WT and S139A HCV NS3 / 4A PR is shown. S139A HCV NS3 / 4A PR contains a serine to alanine mutation at a position corresponding to amino acid 139 of the full-length NS3 protein (SEQ ID NO: 199). This serine to alanine mutation corresponds to position 154 of the HCV NS3 / 4A protease, designated herein as SEQ ID NO: 1. [Figure 3B] 1 illustrates the marginal activity of the S139A mutant of HCV NS3 / 4A PR compared to its WT counterpart in a peptide cleavage assay. [Figure 3C] 1 shows isothermal calorimetry data demonstrating similar affinity of simeprevir for the WT and S139A versions of HCV NS3 / 4A PR. [Figure 4A] 1 shows the selection strategy employed to isolate HCV NS3 / 4A PR(S139A):simeprevir selective binding molecules (PRSIMs). [Figure 4B] Shown is the output from different rounds of selection for three different libraries, expressed as fold change in ELISA signal in the presence of simeprevir compared to the binding signal obtained in the presence of HCV NS3 / 4A PR(S139A) alone. [Figure 5] 1 shows a schematic diagram of the homogeneous time-resolved fluorescence (HTRF) assay used to measure binding of PRSIM molecules to HCV NS3 / 4A PR(S139A) alone or in complex with simeprevir. [Figure 6] HTRF data obtained with a panel of PRSIM molecules showing HCV NS3 / 4A PR(S139A):simeprevir selective binding are shown. The top row is data in the presence of simeprevir, and the bottom row is data in the absence of simeprevir. [Figure 7A-B]Figure 7A shows affinity data derived from BIAcore for binding of HCV NS3 / 4A PR(S139A) to PRSIM_57 in Figure 7A and PRSIM_23 in Figure 7B, with no significant binding in the presence of simeprevir (left) and without simeprevir (center). BSA in the presence of simeprevir was used as a control (right). The gray curve represents the measured data points, and the dashed black line represents the global fit used for the analysis. [Figure 7C] Titration curves for induction of heterodimerization by simeprevir of HCV NS3 / 4A PR(S139A) / PRSIM_57 (left; EC50 = 4.57 nM) or HCV NS3 / 4A PR(S139A) / PRSIM_23 (right; EC50 = 4.03 nM). ◇ = 40 nM HCV NS3 / 4A PR(S139A) + 0 nM simeprevir. [Figure 8] A schematic diagram (left) of the nanoBiT system (Promega) used to identify PRSIM molecules capable of reconstituting nanoLuc function by bringing the LgBiT and SmBiT domains into close proximity is shown. Different orientations of the generated and validated LgBiT and SmBiT fusion proteins are also shown (right). [Figure 9] 1 shows data from a nanoBiT screen demonstrating the fold change in luminescence signal in the presence of simeprevir relative to the signal in the absence of simeprevir, demonstrating that several PRSIM binding molecules are capable of reconstituting nanoLuc activity. [Figure 10] Figure 1 shows the components of two plasmids used in transient transfections to measure the ability of simeprevir to reconstitute split transcription factors and activate transcription of a luciferase reporter gene when the components are fused to HCV NS3 / 4A PR(S139A) and different PRSIM molecules. [Figure 11]Dose-response data from split transcription factor assays are shown for Tn3-based PRSIM molecules (Figure 11A) and scFv-based PRSIM molecules (Figure 11B). Several tested PRSIM molecules are able to activate transcription of a luciferase reporter gene in a dose-dependent manner. [Figure 12A] FIG. 1 shows dose-response data from split transcription factor assays for PRSIM_23 and PRSIM_57 compared to a rapamycin-induced FRB:FKBP12 positive control, which yielded superior fold-change and EC50 values. [Figure 12B] Figure 1 shows data from split transcription factor assays for PRSIM_23 and PRSIM_57 in the absence of simeprevir or rapamycin, respectively, compared to the rapamycin-induced FRB:FKBP12 positive control, indicating that PRSIM-based CIDs have lower basal expression levels and are therefore more tightly regulated. [Figure 13] Using three copies of the DBD-fused molecule shows increased predicted gene expression compared to a single copy by recruiting more AD domains and associated regulatory molecules. [Figure 14A] 1 shows data obtained from plasmids encoding three copies of PRSIM_23 or FKBP12 fused to the DBD compared to a single copy, demonstrating that increasing copy number has a synergistic effect on the fold change of expression. [Figure 14B] 1 shows data obtained from plasmids encoding various copy numbers of PRSIM_23 and null-Tn3 fused to the DBD, demonstrating that increasing copy number has a synergistic effect on the fold change of expression. [Figure 15A] The plasmid used to express the PRSIM-based split chimeric antigen receptor and the protein expressed from this plasmid are shown. [Figure 15B] The effect of adding simeprevir on the binding of PRSIM-based split CAR components is shown, demonstrating that the resulting cell activation was achieved. [Figure 16] We show a dose-dependent increase in IL-2 release, as a marker of T cell activation, from cells expressing a PRSIM-based split CAR in the presence of simeprevir compared to a comparable FRB:FKBP12-based CAR. [Figure 17] Reconstitution of a split transcription factor assay using CID with PRSIM_23 shows the dose response of simeprevir in inducing MEDI8852 expression. [Figure 18A] The vectors used to generate individual AAV particles encoding either an inducible luciferase transgene or the PRSIM_23 / HCV NS3 / 4A PR(S139A)-based split transcription factor components are shown. Proteins expressed after transduction with both AAV particles and luciferase expression after treatment with simeprevir are also shown. [Figure 18B] We show that when the PRSIM_23 switch and an inducible luciferase transgene are delivered to cells in separate AAV particles, the PRSIM_23 switch can activate luciferase expression in a dose-dependent manner in the presence of simeprevir. [Figure 18C] The vector used to generate AAV particles encoding both an inducible IL-2 transgene and a PRSIM_23 / HCV NS3 / 4A PR(S139A)-based split transcription factor component is shown. The proteins expressed after transduction with these AAV particles and IL-2 expression after treatment with simeprevir are also shown. [Figure 18D] We show that when the PRSIM_23 switch and an inducible IL-2 transgene are delivered to cells using the same AAV particle, the PRSIM_23 switch can activate IL-2 expression in a dose-dependent manner in the presence of simeprevir. [Figure 18E] We show that when the PRSIM_23 switch and an inducible IL-2 transgene are delivered to cells using the same AAV particle, the IL-2 expression level induced by the PRSIM_23 switch is similar to that achieved by delivering an AAV that constitutively expresses IL-2 from the CAG promoter. [Figure 19A] 1 shows the components of both the PRSIM-based activation plasmid and the IL-2-targeting gRNA plasmid used to measure the ability of simeprevir to regulate endogenous gene expression in the CRISPRa approach. [Figure 19B] This shows that IL-2 expression is induced from cells expressing both a PRSIM-based activation plasmid and an IL-2-targeting gRNA plasmid only in the presence of simeprevir. [Figure 20] We show that complex formation with a panel of small molecule HCV protease inhibitors is induced in a dose-dependent manner. [Figure 21] 1 shows a two-dimensional interaction diagram of the binding site of simeprevir on HCV NS3 / NS4A. [Figure 22] 1 shows the ability of a panel of mutant HCV proteases to form complexes with PRSIM_23 and simeprevir. [Figure 23] Octet-derived affinity data for simeprevir binding to HCV NS3 / NS4A "WT" (S139A) PR (Figure 23A), HCV NS3 / NS4A K136D PR (Figure 23B), HCV NS3 / NS4A K136N PR (Figure 23C), and HCV NS3 / NS4A D168E PR (Figure 23D) are shown. Data are representative of two to three independent experiments. [Figure 24A] Titration curves for induction of heterodimerization by simeprevir of mutant HCV NS3 / 4A PR / PRSIM_23 binding molecules are shown; HCV NS3 / 4A PR "WT" (S139A) (●), HCV PR NS3 / 4A K136D (■), HCV PR NS3 / 4A K136N (▲), and HCV PR NS3 / 4A D168E (◇). [Figure 24B-E]Figure 24B shows affinity data derived from BIAcore for binding of HCV NS3 / 4A PR "WT" (S139A) (Figure 24B), HCV PR NS3 / 4A K136D (Figure 24C), HCV PR NS3 / 4A K136N (Figure 24D), and HCV PR NS3 / 4A D168E (Figure 24E) to PRSIM_23 in the presence of simeprevir (20, 800, 40, and 20 nM simeprevir, respectively) (left), and no significant binding in the absence of simeprevir (right). The gray curve represents the measured data points, and the dashed black line represents the global fit used for analysis. Data are representative of three independent experiments. [Figure 25A] The inhibition of switch complex formation by the addition of a small molecule inhibitor of HCV NS3 / 4A PR was compared between the presence and absence of preincubation of simeprevir / HCV NS3 / 4A PR. [Figure 25B] Small molecule inhibitors of HCV NS3 / 4A PR can disrupt the switch complex by competing with simeprevir when bound to HCV NS3 / 4A PR mutants with amino acid mutations at positions 168 and 136. [Figure 26A] 1 shows data obtained from a split transcription factor assay for the PRSIM_23 HCV NS3 / 4A PR mutant compared to wild type. [Figure 26B] PRSIM_23: Vectors used to generate monoclonal cell lines expressing GFP-PEST under the control of HCV NS3 / 4 PR WT and mutants obtained by CRISPR-mediated knock-in of the AAVS1 transgene. The expressed proteins and the effect of adding simeprevir on cell activation are also shown. [Figure 26C] Representative histograms showing GFP fluorescence intensity measured by flow cytometry of cell lines expressing GFP-PEST under the control of the split transcription factor PRSIM_23 HCV NS3 / 4 PR wt and mutant. Monoclonal cell lines were induced with simeprevir for 24 hours. [Figure 26D]Data are shown from GFP fluorescence in cell lines expressing GFP-PEST under the control of the split transcription factor PRSIM_23 HCV NS3 / 4 PR WT and mutants. To induce expression, cells were treated with simeprevir. Simeprevir was removed, and GFP fluorescence was measured at various time points after removal using flow cytometry. [Figure 27A] The overall structure of the HCV NS3 / 4A(S193A)PR:PRSIM_57:simeprevir ternary complex is shown. Top image: HCV NS3 / 4A(S193A)PR (light gray) and PRSIM_57 (dark gray) are shown in surface representation, with the simeprevir molecule shown in ball-and-stick format (black) sandwiched between the interface of the two proteins. Bottom image: HCV NS3 / 4A(S193A)PR (light gray) and PRSIM_57 (dark gray) are shown in schematic format. Simeprevir is shown in ball-and-stick format (black) with 2mFo-DFc electron density contoured at 2σ. [Figure 27B] Details of the molecular interactions between HCV NS3 / 4A(S193A)PR, PRSIM_57, and simeprevir are shown. Top panel: Details of the interactions between HCV NS3 / 4A(S193A)PR and simeprevir formed by PRSIM_57. HCV NS3 / 4A(S193A)PR residues that interact with simeprevir (ball-and-stick, black) have been previously determined (PDB 3KEE) and are shown in ball-and-stick format with their side chains (carbon - light gray, oxygen / nitrogen - black). Hydrophobic residues of PRSIM_57 (Phe77, Ile74, Ile125, and Trp249), which form a hydrophobic cavity around simeprevir, are shown in ball-and-stick format (carbon - dark gray, oxygen / nitrogen - black). A direct interaction occurs between the side chain of Phe77 and the quinoline of simeprevir. Bottom panel: Details of the interaction between HCV NS3 / 4A(S193A)PR and PRSIM_57, colored as in the left panel, with interacting residues shown in ball-and-stick format. [Figure 28]The kill switch design is shown. Figure 28A: Homodimerization of caspase 9 (Casp9) via its CARD dimerization domain is crucial for inducing apoptotic cell death. Figure 28B: The CARD domain is replaced with a PRSIM switch component. Figure 28C: Addition of simeprevir induces the formation of PRSIM23-HCV PR heterodimers, which leads to dimerization of the Casp9 activation domain and subsequent induction of apoptosis. [Figure 29] Function of the kill switch is shown upon addition of simeprevir. Figure 29A: Phase-contrast images of HEK293 cells stably transduced with the wt kill switch, which undergo rapid cell death upon treatment with simeprevir. Figure 29B: Phase-contrast images of human tumor cell lines HCT116 and HT29 stably transduced with the wt kill switch, which undergo rapid cell death upon treatment with simeprevir. Figure 29C: Schematic outline of the caspase-3 assay. Figure 29D: Caspase-3 activity in wt kill-switch-transduced HEK293 + / - 10 nM simeprevir compared to treated, non-transduced HEK293 cells. Figure 29E: Caspase-3 activity in three single-cell clones of kill-switch-transduced HCT116 and HT29 compared to non-transduced HCT116 and HT29 in the presence of 10 nM simeprevir. ****p<0.0001; ns=not significant. [Figure 30] 1 shows the confluency over time of the untransduced ES cell line Sa121 and the same cell line transduced with a simeprevir-inducible wt kill switch upon the addition of increasing concentrations of simeprevir. [Figure 31]Coordinated knock-in of the B2M kill switch gene in induced pluripotent stem cells (iPSCs) enhances simeprevir-induced cell death. Figure 31A: Schematic diagram of the kill switch knock-in method. The kill switch (iCasp9) was knocked into the iPSC locus. An adeno-associated virus (AAV) vector was used to deliver a donor template containing an iCasp9 expression cassette flanked by B2M homology arms. Luminescent symbols indicate CRISPR target sites. LHA, left homology arm; RHA, right homology arm; EF1a promt, EF1α promoter; P2A, porcine teschovirus type 1-derived 2A self-cleaving peptide; Puro, puromycin resistance gene; blast, blasticidin resistance gene; bGH pA, bovine growth hormone polyadenylation signal; PrimerF, forward primer for genotyping; PrimerR, reverse primer for genotyping. Figure 31B: Genotyping of iPSC single-cell clones containing the kill switch. After gene knock-in, five single-cell iPSC clones (1B7, 1D6, 1D12, 1G8, and 2D8) were isolated. Genomic DNA was extracted from these clones. The targeted locus was amplified using the primers shown in A). Amplicons were loaded onto a 1.2% agarose gel for electrophoresis. Genotyping data showed that single-cell clones 1B7, 1D12, 1G8, and 2D8 had biallelic B2M-targeted kill switch knock-in, while clone 1D6 had a monoallelic kill switch knock-in. PSC-WT, wild-type (unmodified) iPSC; KI, amplicon of the knock-in allele; WT, amplicon of the wild-type allele. Figure 31C: Cell proliferation index quantified by the xCELLigence Real-Time Cell Analysis (RTCA) assay. iPSC single-cell clones were cultured for 1 day before simeprevir induction, and cell indices were monitored for 3 days before and after induction. [Figure 32]Figure 32A shows the function of the kill switch S196A mutant upon addition of simeprevir. Figure 32A: Phase contrast image of HEK293 cells stably transduced with the kill switch S196A mutant, showing rapid cell death upon treatment with simeprevir. Figure 32B: Caspase 3 activity in wt and S196A mutant kill switch transduced HEK293 + / - 10 nM simeprevir compared to treated, non-transduced HEK293 cells. ***p<0.0005; ns=not significant. DETAILED DESCRIPTION OF THE INVENTION

[0056] Aspects and embodiments of the present disclosure will now be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0057] Expression Vectors and Expression Cassettes As used herein, an "expression vector" refers to a DNA molecule used to express foreign genetic material in a cell. Any suitable vector known in the art can be used. Suitable vectors include DNA plasmids, binary vectors, viral vectors, and artificial chromosomes (e.g., yeast artificial chromosomes). In some embodiments, the expression vector is a viral vector, as described in more detail below. In some embodiments, the expression vector is a DNA plasmid.

[0058] An "expression cassette," as used herein, refers to a polynucleotide sequence capable of resulting in the transcription of an expression product, which may be a protein. A "coding sequence" is intended to mean a portion of the polynucleotide sequence of a gene that encodes the expression product. If the expression product is a protein, the sequence may also be referred to as a "protein-coding sequence." A protein-coding sequence typically begins at the 5' end with an initiation codon and ends at the 3' end with a stop codon. An expression cassette may be part of an expression vector, or, as described in more detail below, may be part of the viral genome within a viral particle.

[0059] An expression cassette typically contains a promoter operably linked to a protein-coding sequence. The term "operably linked" includes situations in which a selected coding sequence and a promoter are covalently linked in such a way that expression of the protein-coding sequence is under the influence or control of the promoter. Thus, a promoter is operably linked to a protein-coding sequence if it is capable of effecting transcription of the protein-coding sequence. The resulting transcript can then be translated into the desired protein, as desired.

[0060] Any suitable promoter known in the art may be used in the expression cassette, as long as it functions in the cell type used. For example, if the cell is a mammalian cell, the promoter may be a cytomegalovirus (CMV) promoter. When multiple expression cassettes are used, each coding sequence may be independently operably linked to its own promoter. Alternatively, one or more coding sequences of the expression cassette may be operably linked to the same promoter.

[0061] Where multiple expression cassettes are described, e.g., a first and a second expression cassette, they may be part of the same or different expression vectors. Thus, in some embodiments, the first and second expression cassettes may be located on the same expression vector. In other embodiments, the first expression cassette is located on a first expression vector and the second expression cassette is located on a second expression vector.

[0062] When multiple expression cassettes are located on the same expression vector, the individual expression cassettes (e.g., the first and second expression cassettes) may be separated by an internal ribosome entry site (IRES) or a 2A element. The use of an IRES or a 2A element allows the same promoter to be used to express multiple expression products. In other words, when the first and second expression cassettes are separated by an IRES or a 2A element, both the first and second expression cassettes can be operably linked to the same promoter.

[0063] Target proteins and small molecules Aspects and embodiments of the present disclosure relate to target proteins derived from non-human proteins, i.e., proteins that are not endogenous to humans. In one embodiment, the non-human protein is derived from a viral, bacterial, fungal, or protozoan protein. In one embodiment, the non-human protein is derived from a viral protein and the small molecule is an inhibitor of the viral protein. In one embodiment, the non-human protein is derived from a bacterial protein and the small molecule is an inhibitor of the bacterial protein. In one embodiment, the non-human protein is derived from a fungal protein and the small molecule is an inhibitor of the fungal protein. In one embodiment, the non-human protein is derived from a protozoan protein and the small molecule is an inhibitor of the protozoan protein. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is an inhibitor of the viral protease.

[0064] The term "derived from" in reference to a target protein is intended to mean that the target protein has an amino acid sequence similar, but not necessarily identical, to the protein from which it is derived, and that the target protein is still capable of binding to a small molecule. A target protein derived from a protein may contain an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the protein from which it is derived. A target protein derived from a protein may contain fewer than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 sequence mutations compared to the protein from which it is derived. For example, a target protein having the amino acid sequence set forth in SEQ ID NO:2 is derived from a viral protease having the sequence set forth in SEQ ID NO:1. Furthermore, a target protein may have fewer amino acids (i.e., a shorter protein) than the protein from which it is derived.

[0065] Viral proteases are enzymes encoded by the genetic material of viral pathogens. The normal function of these enzymes is to catalyze the cleavage of specific peptide bonds in viral polyprotein precursors or cellular proteins. Examples of viral proteases include those encoded by the hepatitis C virus (HCV), human immunodeficiency virus (HIV), herpesviruses, retroviruses, and human rhinovirus (HRV) families. Specific viral proteases, along with examples of small molecule inhibitors of these proteases, are described, for example, in Patick and Potts, 1998.

[0066] Small molecules are organic compounds that typically have a molecular weight below 2000 Daltons. Small molecules can be synthetic or naturally occurring.

[0067] The selection of a viral protease inhibitor as a small molecule is not particularly limited, as long as the viral protease inhibitor a) can bind to the target protein and b) has been evaluated for clinical purposes in humans. Viral protease inhibitors that have been evaluated for clinical purposes in humans include those approved for clinical use in humans by regulatory authorities, such as inhibitors approved for treatment by the Food and Drug Administration (FDA) and / or the European Medicines Agency (EMA). Viral protease inhibitors that have been evaluated for clinical purposes also include those currently being tested / have been tested in human clinical trials, preferably those that have previously progressed to Phase I clinical trials. Preferably, the viral protease inhibitor is approved for clinical use in humans. Preferably, the viral protease inhibitor is suitable for long-term administration (daily for six months or more), is cell-permeable, is orally administered, and / or is not used as a first-line treatment.

[0068] The viral protease used may be monomeric or multimeric (e.g., dimeric, trimeric, tetrameric, etc.). The use of a monomeric viral protease is preferred, e.g., when the target protein fusion protein and the binding member fusion protein are in a strictly 1:1 ratio, the desired functional activity is elicited. There may be alternative situations in which a multimeric viral protease is preferred, e.g., when the target protein is fused to a transcriptional regulatory domain within a split transcription factor, the use of a multimeric viral protease can increase the number of transcriptional regulatory domains that recruit the target gene.

[0069] In some embodiments, the viral protease is HCV NS3 / 4A protease or HIV protease. Both of these proteases are known to be targeted by several approved small molecule inhibitors, which are known to be generally well tolerated in humans and suitable for long-term administration. Examples of small molecule inhibitors targeting HCV NS3 / 4A protease are described in De Clercq. 2014. Examples of small molecule inhibitors targeting HIV protease are described in Lv et al. 2015.

[0070] In some embodiments, the viral protease is HCV NS3 / 4A protease. HCV NS3 / 4A protease is monomeric, has a relatively small size (21 kDa), can be expressed in the cytoplasm, and does not associate with DNA, making it an ideal candidate for use as a viral protease in the present disclosure. HCV NS3 / 4A protease may have the amino acid sequence from amino acids 1030 to 1206 of the amino acid sequence set forth in UniProt Accession No. A8DG50-1 (sequence version 2; sequence updated April 29, 2008). In some embodiments, HCV NS3 / 4A protease may comprise the amino acid sequence set forth in SEQ ID NO: 1. A target protein derived from HCV NS3 / 4A protease may comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0071] There are several small molecule inhibitors that are known to bind to the HCV NS3 / 4A protease and that have been approved for human use, some of which are listed in the table below.

[0072] [Table 1]

[0073] The structure of the target protein complexed with each small molecule is provided as a PBD accession number and corresponds to the crystal structure available from the Protein Data Bank (PBD). The structure and chemical name of the small molecule are also provided as a PBD accession number.

[0074] The small molecule may be a peptidomimetic. The terms "peptide mimetic," "peptidomimetic," and "peptide analog" are used interchangeably and refer to compounds that are not composed of amino acids but have substantially the same properties as peptide compounds composed entirely of amino acids.

[0075] Other small molecule inhibitors currently being tested / have been tested in human clinical trials include faldaprevir, sovaprevir, and vedroprevir.

[0076] In some embodiments, the small molecule is selected from the group consisting of simeprevir, boceprevir, telaprevir, asunaprevir, vaniprevir, voxilaprevir, glecaprevir, paritaprevir, naraprevir, danoprevir, faldaprevir, grazoprevir, sovaprevir, bedroprevir, or a pharmacologically acceptable analog or derivative thereof. All of these small molecules have been approved for use in humans and / or have been tested in human clinical trials. In some embodiments, the small molecule is selected from the group consisting of simeprevir, boceprevir, telaprevir, asunaprevir, vaniprevir, voxilaprevir, glecaprevir, paritaprevir, grazoprevir, danoprevir, and naraprevir, or a pharmacologically acceptable analog or derivative thereof. These small molecules have been approved for use in humans.

[0077] In certain embodiments, the small molecule is selected from the group consisting of simeprevir, boceprevir, and telaprevir, or a pharmacologically acceptable analog or derivative thereof. These small molecules (simeprevir, boceprevir, and telaprevir) are well tolerated in humans and approved for long-term use in humans. In certain embodiments, the small molecule may be simeprevir or a pharmacologically acceptable analog or derivative thereof. Simeprevir (Olysio®) is an orally administered small molecule that is cell membrane permeable and has a pharmacokinetic (PK) profile that supports once-daily dosing. Simeprevir has been used long-term (up to 39 months) in combination with ribavirin and pegylated interferon to treat HCV infection. It is listed on the WHO Essential Medicines List and has been shown to be a well-tolerated, widely administered agent.

[0078] Pharmaceutically acceptable analogs and derivatives of small molecules include compounds that differ from the "parent" small molecule but contain similar antiviral activity as the parent small molecule, including tautomers, positional isomers, geometric isomers, and, where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers), and, where applicable, related pharmaceutically acceptable salts and derivatives thereof (including prodrug forms). For example, analogs of simeprevir include those compounds encompassed by formula (I) as defined in WO 2007014926 A1.

[0079] Simeprevir may have the following chemical structure: [ka]

[0080] In some embodiments, the viral protease is HIV protease. HIV protease exists as a 22 kDa homodimer, with each subunit consisting of 99 amino acids. HIV protease may have the amino acid sequence of amino acids 501 to 599 of the amino acid sequence set forth in UniProt Accession No. P03366-1 (sequence version 3; sequence updated January 23, 2007). A target protein derived from HIV protease may comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of amino acids 501 to 599 of the amino acid sequence set forth in UniProt Accession No. P03366-1. A target protein derived from HIV protease may be a monomeric protein. For example, the target protein may contain one or more amino acid mutations that reduce the likelihood of forming a homodimeric protein.

[0081] There are several small molecule inhibitors known to bind to HIV protease that have been approved for human use, some of which are listed in the table below.

[0082] [Table 2]

[0083] Fosamprenavir is a prodrug form of amprenavir that has better solubility and bioavailability than amprenavir.

[0084] In some embodiments, the small molecule is selected from the group consisting of atazanavir, darunavir and fosamprenavir, amprenavir, indinavir, lopinavir / ritonavir, nelfinavir, ritonavir, saquinavir and tipranavir, or a pharmacologically acceptable analog or derivative thereof.

[0085] In certain embodiments, the small molecule is selected from the group consisting of atazanavir, darunavir, and fosamprenavir, or a pharmacologically acceptable analog or derivative thereof. These small molecules are well tolerated in humans and have good bioavailability. Furthermore, HIV protease inhibitors are typically administered to patients for long periods of time, and these small molecule inhibitors are expected to be well tolerated over long periods of time.

[0086] In some embodiments, the target protein has attenuated viral activity compared to the viral protease from which it is derived. Attenuated viral activity, as used herein, is intended to mean that the target protein has reduced enzymatic activity, e.g., reduced protease activity, compared to the viral protease from which it is derived. Enzymatic activity can be verified using, for example, a fluorogenic peptide cleavage assay, such as that described in the Examples or in Sabariegos et al. 2009. Briefly, the fluorogenic peptide cleavage assay involves incubating the target protein / viral protease with a fluorogenic protease FRET substrate containing a donor-quencher pair, whereby cleavage of the peptide separates the donor from the quencher and releases energy detectable at a specific wavelength, e.g., 490 nm.

[0087] In some embodiments, a target protein is considered to have attenuated viral activity compared to the viral protease from which it is derived if the target protein has less than 10% of the activity of the viral protease when measured in an enzymatic activity assay, such as a fluorogenic peptide cleavage assay. In some embodiments, the target protein does not exhibit any detectable viral activity when the target protein is at a concentration of less than 1 nM, less than 10 nM, less than 100 nM, or less than 1 μM when measured in an enzymatic activity assay, such as a fluorogenic peptide cleavage assay.

[0088] The target protein may contain one or more amino acid mutations (e.g., substitutions / insertions / deletions) relative to the viral protease from which it is derived (e.g., relative to SEQ ID NO: 1). A target protein containing one or more amino acid mutations should retain the ability to form a trimolecular complex with a small molecule and a binding member, which can be determined, for example, using a homogeneous time-resolved fluorescence (HTRF) assay as described in the Examples.

[0089] In some embodiments, the target protein comprises one or more amino acid mutations compared to the viral protease from which it is derived, wherein the one or more amino acid mutations attenuate the viral activity of the target protein. The one or more amino acid mutations may be present in the active site of the viral protease.

[0090] For example, HCV NS3 / 4A protease contains a catalytic triad comprising amino acid residues H57, D81, and S139 of HCV NS3 / 4A protease. See, e.g., Grakoui et al. 1993; Eckart et al. 1993; and Bartenschlager et al. 1993. These amino acid residues correspond to positions H72, D96, and S154 of the amino acid sequence of SEQ ID NO: 1. Thus, the target protein may contain amino acid mutations at one or more amino acids selected from positions 72, 96, and 154 of HCV NS3 / 4A protease, where the amino acid numbering corresponds to SEQ ID NO: 1. Other HCV NS3 / 4A protease residues known to be involved in viral activity include C97, C99, C145, and H149 of HCV NS3 / 4A protease (corresponding to positions C112, C114, C160, and H164 of SEQ ID NO: 1). See, e.g., Hikikata et al. 1993; and Stempniak et al. 1997. In some embodiments, the target protein contains an amino acid mutation (e.g., substitution) at one or more amino acids selected from positions 72, 96, 112, 114, 154, 160, and 164 of HCV NS3 / 4A protease, where the amino acid numbering corresponds to SEQ ID NO: 1.

[0091] In certain embodiments, the target protein comprises an amino acid mutation at position 154 of HCV NS3 / 4A protease, e.g., a mutation to alanine, where the amino acid numbering corresponds to SEQ ID NO: 1. In certain embodiments, the target protein has the amino acid sequence of SEQ ID NO: 2.

[0092] The full-length sequence of the NS3 protein is shown in SEQ ID NO: 199. The amino acid mutation at position 154 of SEQ ID NO: 1 described herein corresponds to position 139 of SEQ ID NO:199.

[0093] Provided below is a table highlighting the potential amino acid mutations described above and their corresponding positions in the NS3 / 4A protease set forth in SEQ ID NO:1, numbered according to the full-length NS3 protein (SEQ ID NO:199).

[0094] [Table 3]

[0095] As a further example, the HIV protease contains a catalytic triad including amino acid residues D25, T26, and G27, where the amino acid numbering is according to the HIV protease having the amino acid sequence of amino acids 501 to 599 of the amino acid sequence set forth in UniProt Accession No. P03366-1 (sequence version 3; sequence updated January 23, 2007). Thus, the target protein may contain amino acid mutations at one or more amino acids selected from positions 25, 26, and 27 of the HIV protease, where the amino acid numbering is according to the HIV protease having the amino acid sequence of amino acids 501 to 599 of the amino acid sequence set forth in UniProt Accession No. P03366-1 (sequence version 3; sequence updated January 23, 2007).

[0096] A target protein and a small molecule interact to form a complex (referred to herein as a T-SM complex) between the target protein and the small molecule. This interaction may be a covalent or non-covalent interaction. In some embodiments, the small molecule binds to the target protein with a kD of less than 1 mM, preferably less than 500 nM, more preferably less than 200 nM, even more preferably less than 100 nM, or even more preferably less than 50 nM, as measured, for example, using surface plasmon resonance or biolayer interferometry. In some embodiments, the small molecule binds to the target protein with a kD of 25 nM to 200 nM, 25 nM to 100 nM, or 25 to 75 nM, as measured, for example, using surface plasmon resonance or biolayer interferometry.

[0097] It may be desirable to introduce amino acid mutations (e.g., substitutions) into a target protein to reduce the affinity of a small molecule for the target protein and allow a second small molecule to displace the small molecule from the T-SM complex. For example, as shown herein, simeprevir binds to the target protein HCV NS3 / 4A protease (S139A) (SEQ ID NO: 2) with such high affinity that other small molecules that bind to the target protein cannot displace simeprevir from the T-SM complex. Reducing the binding affinity of simeprevir to HCV NS3 / 4A protease by introducing amino acid mutations into the target protein allows for the use of a different small molecule inhibitor of HCV NS3 / 4A protease to disrupt the trimolecular complex formed between HCV NS3 / 4A protease (S139A), simeprevir, and PRSIM_23. Thus, in some embodiments, the target protein contains one or more affinity-reducing amino acid mutations (e.g., substitutions) compared to the viral protease (SEQ ID NO: 1) from which it is derived, such that a small molecule binds to the target molecule with lower affinity than the small molecule binds to the parent target protein. As used herein, a "parent target protein" is one that lacks one or more affinity-reducing amino acid mutations but is otherwise identical to the target protein. The parent target protein may be the viral protease from which the target protein is derived (e.g., the parent target protein may have the amino acid sequence set forth in SEQ ID NO: 1), or the parent target protein may itself be derived from a viral protease (e.g., the parent target protein may have the amino acid sequence set forth in SEQ ID NO: 2).

[0098] One or more affinity-reducing amino acid mutations can result in a small molecule that binds to a target protein with an affinity that is at least 1.5-fold lower than the small molecule that binds to the parent target protein. One or more affinity-reducing amino acid mutations can result in a small molecule that binds to a target protein with an affinity that is 1.5-fold to 10-fold lower than the small molecule that binds to the parent target protein, or 1.5-fold to 5-fold lower than the small molecule that binds to the parent target protein. One or more affinity-reducing amino acid mutations can optionally result in a small molecule that binds to a target protein with a KD of 25 nM to 200 nM, 25 to 100 nM, or 25 to 75 nM when affinity is measured using biolayer interferometry, e.g., using Octet RED384.

[0099] As shown herein, amino acid substitutions at positions 151 and 183 of HCV NS3 / 4A protease, whose amino acid numbering corresponds to SEQ ID NO: 1, have been shown to reduce the affinity of simeprevir for HCV NS3 / 4A protease and enable a second small molecule to disrupt the trimolecular complex formed between HCV NS3 / 4A protease, simeprevir, and the binding member PRSIM_23. Furthermore, target proteins containing these affinity-reducing mutations have been shown to retain the functionality of dimer-inducing proteins, such as split transcription factors. Amino acids 151 and 183 of SEQ ID NO: 1 correspond to amino acids 136 and 168, respectively, of the full-length NS3 protein set forth in SEQ ID NO: 99.

[0100] Thus, in some embodiments where the target protein is derived from a viral protease, i.e., HCV NS3 / 4A protease, the target protein may have affinity-reducing amino acid mutations (e.g., substitutions) at one or more amino acids selected from positions 151 and 183, where the amino acid numbering corresponds to SEQ ID NO: 1. In some embodiments, the affinity-reducing amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine, and the affinity-reducing mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine. In some embodiments, the affinity-reducing amino acid mutation at position 151 is a mutation to aspartic acid or asparagine, and the affinity-reducing mutation at position 183 is a mutation to glutamic acid. The target protein may include affinity-reducing amino acid mutations in addition to other amino acid mutations described herein (e.g., in addition to an amino acid mutation at position 154, such as to alanine).

[0101] In one embodiment, the target protein has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1, and includes an alanine at position 154 and an aspartic acid, asparagine, or histidine (e.g., aspartic acid or asparagine) at position 151, where the amino acid numbering corresponds to SEQ ID NO:1. In one embodiment, the target protein is derived from a viral protease having the amino acid sequence set forth in SEQ ID NO: 1, and differs from this viral protease in that the target protein comprises an alanine at position 154, an aspartic acid, asparagine, or histidine (e.g., aspartic acid or asparagine) at position 151, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional sequence variations (e.g., functional conservative substitutions), where the amino acid numbering corresponds to SEQ ID NO: 1. In one embodiment, the target protein comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 211 and 215.

[0102] In one embodiment, the target protein has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1, and includes an alanine at position 154 and a glutamic acid, glutamine, or alanine (e.g., glutamic acid) at position 183, where the amino acid numbering corresponds to SEQ ID NO:1. In one embodiment, the target protein is derived from a viral protease having the amino acid sequence set forth in SEQ ID NO: 1, and differs from this viral protease in that the target protein comprises an alanine at position 154 and an aspartic acid, asparagine, or histidine (e.g., aspartic acid) at position 151, and optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional sequence variations (e.g., functional conservative substitutions), where the amino acid numbering corresponds to SEQ ID NO: 1. In one embodiment, the target protein comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 213.

[0103] Joined Members As used herein, "binding member" refers to a polypeptide or protein that specifically binds to a T-SM complex. The term "specific" can refer to the state in which the binding member does not exhibit any significant binding to molecules other than the T-SM complex. Such molecules are referred to as "non-target molecules" and include target proteins alone and small molecules alone, i.e., target proteins or small molecules when not part of a T-SM complex.

[0104] In some embodiments, a binding member is considered not to exhibit any significant binding to a non-target molecule if the extent of binding to the non-target molecule is less than about 10% of the binding of the binding member to T-SM, as measured, for example, by isothermal calorimetry, ELISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), homogeneous time-resolved fluorescence (HTRF), microscale thermophoresis (MST), or by radioimmunoassay (RIA). In some embodiments, the extent of binding to the non-target molecule is less than about 5% or less than about 1% of the binding of the binding member to T-SM. Methods used to measure the extent of binding, involving SPR (Biacore) and HTRF, are described in the Examples. In some embodiments, a binding member described herein binds to a T-SM complex with an affinity that is at least two-fold higher than the affinity to another non-target molecule, e.g., the target protein alone or a small molecule alone, when the extent of binding is measured by HTRF. In some embodiments, a binding member binds to its target molecule with an affinity that is at least 3-fold, 5-fold, 10-fold, or 20-fold higher than its affinity for another non-target molecule. Alternatively, binding specificity may be reflected in terms of binding affinity, and the binding members described herein bind to the T-SM complex with an affinity that is at least 10-fold higher than its affinity for another non-target molecule, e.g., the target protein alone or a small molecule alone. Binding affinity may be measured by surface plasmon resonance, e.g., Biacore. In some embodiments, a binding member binds to its target molecule with an affinity that is at least 50-fold, 100-fold, 1000-fold, or 10,000-fold higher than its affinity for another non-target molecule.

[0105] Binding affinity is typically measured by Kd (the equilibrium dissociation constant between a binding member and its target). As is well understood, the lower the Kd value, the higher the binding affinity of the binding member. For example, a binding member that binds to a T-SM complex with a Kd of 1 nM is considered to bind to the T-SM complex with higher affinity than a binding member that binds to a non-target molecule with a Kd of 100 nM.

[0106] The binding member may bind to the T-SM complex with an affinity having a Kd of 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM or less. The binding member may bind to the target protein alone or the small molecule alone with an affinity having a Kd of 500 nM, 1 μM, 10 μM, 100 μM, or 1 mM or more. Binding affinity may be measured by SPR, e.g., Biacore. SPR measurements may show minimal or no binding of the binding member to the target protein alone and / or the small molecule alone.

[0107] In some embodiments, a binding member specifically binds to a T-SM complex at an epitope that is present only on the T-SM complex and not on the target protein alone or the small molecule alone. For example, a binding member may bind to a site on the T-SM complex that consists of at least a portion of the small molecule and a portion of the target protein. Alternatively, formation of the T-SM complex may induce a conformational change in the target protein, resulting in the formation of a new epitope to which the binding member specifically binds. Methods for determining whether a binding member binds to a specific epitope include X-ray crystallography, peptide scanning, site-directed mutagenesis mapping, and mass spectrometry.

[0108] In embodiments in which the T-SM complex comprises a target protein derived from HCV NS3 / 4A protease (e.g., SEQ ID NO: 2) and the small molecule simeprevir, the binding member can specifically bind to the T-SM by forming an interaction with at least one of the following target protein residues, whose amino acid numbering corresponds to SEQ ID NO: 1: Tyr71, Gly75, Thr76, Val93, and Asp94. The binding member can form interactions with one, two, three, four, or most preferably all five of these residues. The binding member can also specifically bind to the T-SM complex by forming interactions with the quinoline moiety of simeprevir. At least some of these interactions may be hydrophobic and / or water-mediated. The interactions can be determined, for example, using X-ray crystallography as described in the Examples.

[0109] The binding member may be an antibody molecule such as a single chain variable fragment or an antibody mimetic, for example a Tn3 protein.

[0110] antibody molecule Aspects and embodiments of the present disclosure relate to binding members that are antibody molecules, such as single chain variable fragments (scFv).

[0111] The term "antibody molecule" describes an immunoglobulin, whether naturally produced or partially or wholly synthetically produced. An antibody molecule may be a human or humanized antibody molecule. An antibody molecule may be a monoclonal antibody molecule. Examples of antibodies include immunoglobulin isotypes, such as immunoglobulin G (IgG), their isotypic subclasses, such as IgG1, IgG2, IgG3, and IgG4, and fragments thereof.

[0112] Antibody molecules generally consist of six complementarity-determining regions (CDRs): three in the variable heavy (VH) region (HCDR1, HCDR2, and HCDR3) and three in the variable light (VL) region (LCDR1, LCDR2, and LCDR3). Together, the six CDRs define the paratope of the antibody molecule, the portion of the antibody molecule that binds to the T-SM complex. The VH and VL regions contain framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From the N-terminus to the C-terminus, the VH region is composed of the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region is composed of the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0113] There are several different conventional methods for defining the CDRs and FRs of antibodies, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), IMGT numbering as described in LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue); D413-22 as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1); D671-D674, and VBASE2. The CDRs and FRs of the VH and VL regions of the antibody molecules described herein were defined according to Kabat (Kabat, EA et al. (1991)).

[0114] The term "antibody molecule," as used herein, includes antibody fragments, provided that they are shown to bind to the relevant target molecule. Examples of antibody fragments include Fv, scFv, Fab, scFab, F(ab'), Fab, diabody, triabody, scFv-Fc, minibody, and single domain antibodies (e.g., VhH). Unless the context requires otherwise, the term "antibody molecule," as used herein, is therefore equivalent to "antibody molecule or antigen-binding fragment thereof." In certain exemplary embodiments, the antibody molecule is a single-chain variable fragment (scFv).

[0115] Antibody molecules and methods for their construction and use are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). It is possible to take monoclonal and other antibody molecules and use techniques of recombinant DNA technology to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can involve transferring the CDRs or variable regions of one antibody molecule into a different antibody molecule (see EP-A-184187, GB2188638A, and EP-A-239400).

[0116] In view of current technology related to monoclonal antibody technology, antibody molecules can be prepared for most antigens. The antigen-binding domain may be a portion of an antibody (e.g., an Fab fragment) or a synthetic antibody fragment (e.g., an scFv). Suitable monoclonal antibodies for a selected antigen may be prepared by known techniques, such as those described in "Monoclonal Antibodies; A Manual of Techniques" by H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies; Techniques and Applications" by J.G.R. Hurrell (CRC Press, 1982). Chimeric antibodies are described in Neuberger et al. (1988, 8th International Biotechnology Symposium Part 2, 792-799).

[0117] The sequence identifiers (SEQ ID NOs) for the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, variable heavy (VH) chain, variable light (VL) chain, and scFv amino acid sequences of PRSIM_57, PRSIM_01, PRSIM_04, PRSIM_67, PRSIM_72, and PRSIM_75 are listed in the table below.

[0118] [Table 4]

[0119] In some embodiments, the antibody molecule comprises the following heavy chain complementarity determining regions (HCDRs) 1 to 3 and / or light chain complementarity determining regions (LCDRs): i) PRSIM_57, as set forth in SEQ ID NOs: 151, 152, 153, 154, 155 and 156, respectively; ii) PRSIM_01, as set forth in SEQ ID NOs: 151, 152, 198, 154, 155 and 156, respectively; iii) PRSIM_04, as set forth in SEQ ID NOs: 151, 152, 163, 154, 155 and 164, respectively; iv) PRSIM_67, as set forth in SEQ ID NOs: 165, 166, 167, 168, 169 and 170, respectively; v) PRSIM_72, as set forth in SEQ ID NOs: 171, 172, 173, 174, 175 and 176, respectively; or vi) PRSIM_75 as set forth in SEQ ID NOs: 177, 178, 179, 180, 181 and 182, respectively; CDR sequences are defined according to the Kabat numbering scheme.

[0120] In some embodiments, the binding member comprises multiple sequence variations in any one or more of the CDRs defined above, for example 1, 2, 3, 4 or 5 sequence variations.

[0121] In some embodiments, the antibody molecule comprises the following variable heavy (VH) and / or variable light (VL) chains: i) PRSIM_57, as set forth in SEQ ID NOs: 186 and 187, respectively; ii) PRSIM_01, as set forth in SEQ ID NOs: 188 and 189, respectively; iii) PRSIM_04, as set forth in SEQ ID NOs: 190 and 191, respectively; iv) PRSIM_67, as set forth in SEQ ID NOs: 192 and 193, respectively; v) PRSIM_72, as set forth in SEQ ID NOs: 194 and 195, respectively; or vi) PRSIM_75, as set forth in SEQ ID NOs: 196 and 197, respectively.

[0122] In certain embodiments, the antibody molecule is a single-chain variable fragment (scFv). Typically, an scFv comprises a VH chain and a VL chain separated by a peptide linker. The peptide linker may be as defined herein. In some embodiments, the peptide linker separating the VH chain and the VL chain may comprise the amino acid sequence of SEQ ID NO: 204.

[0123] In some embodiments, the scFv comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the following amino acid sequence: i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) PRSIM_75 as set forth in SEQ ID NO: 15.

[0124] In certain embodiments, the scFv comprises the following amino acid sequence: i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) PRSIM_75 as set forth in SEQ ID NO: 15.

[0125] antibody mimics The binding member may be an antibody mimetic. An antibody mimetic is an organic compound that can specifically bind to an antigen but is structurally distinct from an antibody molecule. Examples of antibody mimics include scaffolding proteins such as Tn3 protein, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, flynomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.

[0126] In particular aspects and embodiments, the binding member is a Tn3 protein.

[0127] The Tn3 protein is based on the structure of the fibronectin type III module (FnIII) ​​and is a member of human tenascin-C. Third The production and use of Tn3 proteins is described, for example, in WO 2009 / 058379, WO 2011 / 130324, WO 2011130328, and Gilbreth et al. 2014.

[0128] The native FnIII domains from Tn3 protein and tenascin-C are characterized by the same three-dimensional structure: a β-sandwich structure with three β-strands (A, B, and E) on one side and four β-strands (C, D, F, and G) on the other side, connected by six loop regions. These loop regions are named according to the β-strands connected to the N- and C-termini of each loop. Thus, the AB loop is located between β-strands A and B, the BC loop is located between strands B and C, the CD loop is located between β-strands C and D, the DE loop is located between β-strands D and E, the EF loop is located between β-strands E and F, and the FG loop is located between β-strands F and G. FnIII domains possess solvent-exposed, randomization-permitting loops that facilitate the generation of a diverse pool of protein scaffolds capable of binding specific targets with high affinity.

[0129] The wild-type Tn3 protein may comprise the sequence of SEQ ID NO: 134. In the wild-type Tn3 protein, the BC, DE, and FG loops are located at positions 23-31, 51-56, and 75-80, with amino acid numbering corresponding to SEQ ID NO: 134. The Tn3 protein may comprise one, preferably two, more preferably three, and even more preferably four stabilizing mutations selected from the list consisting of I32F, D49K, E86I, and T89K, with amino acid numbering corresponding to SEQ ID NO: 134. The amino acid sequence of a wild-type Tn3 protein comprising all four stabilizing mutations is set forth in SEQ ID NO: 135. The Tn3 protein may further comprise one or more of the stabilizing mutations described in Gilbreth et al. 2014 (see in particular Table 1 of Gilbreth et al. 2014).

[0130] The Tn3 protein can be subjected to directed evolution designed to randomize one or more of its loops, analogous to the complementarity-determining regions (CDRs) of antibody variable regions. Such directed evolution approaches result in the generation of antibody-like binding members with high affinity for a target of interest, such as the T-SM complex described herein.

[0131] Thus, a Tn3 protein that specifically binds to a T-SM complex described herein may comprise the BC, DE, and FG loops of PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, or PRSIM_47. For example, the Tn3 protein may comprise the sequence of SEQ ID NO: 134 or SEQ ID NO: 135, in which the BC, DE, and FG loops located at positions 23-31, 51-56, and 75-80, respectively, are replaced with the BC, DE, and FG loops of PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, or PRSIM_47, where the amino acid numbering corresponds to SEQ ID NO: 134.

[0132] One of skill in the art could readily determine the amino acid sequence of the BC, DE, and FG loops of the PRSIM clones described herein, for example, by comparing the amino acid sequence of the PRSIM clone to that of a wild-type Tn3 protein, e.g., the amino acid sequence set forth in SEQ ID NO: 134 or 135.

[0133] The Tn3 sequence, amino acid positions, and sequences of the BC, DE, and FG loops of PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, or PRSIM_47 are as set forth in the table below.

[0134] [Table 5]

[0135] In some embodiments, the Tn3 protein comprises the following BC, DE, and FG loops. i) PRSIM_23, as set forth in SEQ ID NOs: 136, 137, and 138, respectively; ii) PRSIM_32, as set forth in SEQ ID NOs: 139, 140, and 141, respectively; iii) PRSIM_33, as set forth in SEQ ID NOs: 142, 143, and 144, respectively; iv) PRSIM_36, as set forth in SEQ ID NOs: 145, 146, and 147, respectively; or v) PRSIM_47, as set forth in SEQ ID NOs: 148, 149, and 150, respectively.

[0136] In some embodiments, the Tn3 protein comprises the following BC, DE, and FG loops. i) PRSIM_23, in which the BC loop comprises the amino acids at positions 23 to 32 of SEQ ID NO: 5, the DE loop comprises the amino acids at positions 52 to 57 of SEQ ID NO: 5, and the FG loop comprises the amino acids at positions 76 to 85 of SEQ ID NO: 5; ii) PRSIM_32, in which the BC loop comprises the amino acids at positions 23 to 34 of SEQ ID NO: 6, the DE loop comprises the amino acids at positions 54 to 59 of SEQ ID NO: 6, and the FG loop comprises the amino acids at positions 78 to 87 of SEQ ID NO: 6; iii) PRSIM_33, in which the BC loop comprises the amino acids at positions 23 to 34 of SEQ ID NO:7, the DE loop comprises the amino acids at positions 54 to 59 of SEQ ID NO:7, and the FG loop comprises the amino acids at positions 78 to 87 of SEQ ID NO:7; iv) PRSIM_36, in which the BC loop comprises the amino acids at positions 23 to 34 of SEQ ID NO:8, the DE loop comprises the amino acids at positions 54 to 59 of SEQ ID NO:8, and the FG loop comprises the amino acids at positions 78 to 87 of SEQ ID NO:8; v) PRSIM_47, in which the BC loop comprises the amino acids at positions 23 to 31 of SEQ ID NO:9, the DE loop comprises the amino acids at positions 51 to 56 of SEQ ID NO:9, and the FG loop comprises the amino acids at positions 75 to 84 of SEQ ID NO:9.

[0137] In some embodiments, the Tn3 protein comprises multiple sequence variations, e.g., 1, 2, 3, 4, or 5, in any one or more of the BC, DE, and EF loops defined above, hi some embodiments, the Tn3 protein comprises multiple sequence variations, e.g., 1, 2, 3, 4, or 5, outside the BC, DE, and EF loops defined above.

[0138] In some embodiments, the Tn3 protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the following amino acid sequence: i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) PRSIM_47 as set forth in SEQ ID NO:9.

[0139] In certain embodiments, the Tn3 protein comprises the following amino acid sequence: i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) PRSIM_47 as set forth in SEQ ID NO:9.

[0140] Dimer-inducing proteins In some embodiments, the target protein is fused to a first constituent polypeptide and the binding member is fused to a second constituent polypeptide, hi certain embodiments, the first and second constituent polypeptides form part of a dimer-inducing protein.

[0141] As used herein, a "dimer-inducing protein" refers to a protein or complex comprising a first and a second constituent polypeptide, wherein the first and second polypeptides dimerize to form a functional protein. The term "dimer-inducing protein" includes "split proteins," "dimerization-deficient proteins," and "split complexes." The term "constituent polypeptides" is intended to encompass both single-chain and multi-chain polypeptides. The first and second constituent polypeptides in a dimer-inducing protein typically have no or poor activity when separated, but when they are brought into close proximity by dimerization, they become active or have increased activity. As described in the Examples, the dimerization of a dimer-inducing protein can be modulated by combining specific binding members, target proteins, and small molecules described herein, such that a significant increase in activity is observed when the binding members bind to a T-SM complex compared to the separate components of the dimer-inducing protein alone.

[0142] Examples of dimer-inducing proteins include split chimeric antigen receptors (split CARs; e.g., as described in Wu et al. 2015), split kinases (e.g., as described in Camacho-Soto et al. 2014), split transcription factors (e.g., as described in Taylor et al. 2010), split apoptotic proteins (e.g., split caspases as described in Chelur et al. 2007), and split reporter systems (e.g., as described in Dixon et al. 2016).

[0143] The dimer-inducing protein exhibits increased activity when the binding member binds to the T-SM complex. The increased activity can be compared to the activity observed when the binding member is not bound to the T-SM complex (e.g., due to the absence of one or more of the target protein, small molecule, or binding member). In some embodiments, the increase in activity observed when the binding member binds to the T-SM complex is at least 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 105-fold, 110-fold, 115-fold, or 120-fold increase in activity compared to the activity observed when the binding member is not bound to the T-SM complex.

[0144] Methods for measuring the activity of a dimer-inducing protein vary depending on the particular dimer-inducing protein being tested. When a first constituent polypeptide and a second constituent polypeptide dimerize to form a chimeric antigen receptor (CAR), the activity of the CAR can be determined by measuring immune cell activity and / or proliferation. As described in the Examples, the activity of the CAR can be measured by the production of interleukin-2 (IL-2) after stimulating the CAR with an antigen, e.g., by ELISA. When a first constituent polypeptide and a second constituent polypeptide dimerize to form a kinase, the activity of the kinase can be measured by phosphate, e.g., radioactive, as described in Camacho-Soto et al. 2014. 32 P can be measured by incorporating P into a peptide substrate. If the first and second constituent polypeptides dimerize to form a transcription factor, transcriptional activity can be determined by measuring expression of a downstream desired expression cassette regulated by the split transcription factor, as described in the Examples. If the first and second constituent polypeptides dimerize to form a therapeutic protein, this activity can be measured using an assay suitable for determining the functional activity of the protein. If the first and second constituent polypeptides dimerize to form a caspase, caspase activity can be measured using a caspase activity assay or by measuring apoptotic cell death. If the first and second constituent polypeptides dimerize to form a reporter system, reporter activity can be determined by measuring expression of a reporter, such as luciferase.

[0145] The first constituent polypeptide may be fused to the C-terminus or N-terminus of the target protein or binding member. The second constituent polypeptide may be fused to the C-terminus or N-terminus of the target protein or binding member. The constituent polypeptides may be fused to the target protein or binding member via a peptide linker. Suitable peptide linkers include [G]n, [s]n, [A]n, [GS]n, [GGS]n, [GGGS]n (SEQ ID NO: 239), [GGGGS)n (SEQ ID NO: 240), [GGSG]n (SEQ ID NO: 241), [GSGG]n (SEQ ID NO: 242), [SGGG]n (SEQ ID NO: 243), [SSGG]n (SEQ ID NO: 244), [SSSG]n (SEQ ID NO: 245), [GG]n, [GGG]n, [SA]n, [TGGGGSGGGGS]n (SEQ ID NO: 185), and combinations thereof, where n is an integer from 1 to 30. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any number up to 30. The constituent polypeptides may be fused directly to the target protein or binding member, for example, in the configuration first constituent polypeptide-peptide linker-target protein. Alternatively, the constituent polypeptides may be fused indirectly to the target protein or binding member by one or more additional polypeptides separating the first constituent polypeptide from the target protein or binding member, for example, first constituent polypeptide-additional polypeptide-peptide linker-target protein.

[0146] In some embodiments, a first constituent polypeptide is fused to two or more target proteins or binding members. In some embodiments, a second constituent polypeptide is fused to two or more target proteins or binding members, or a combination of both. For example, a first or second constituent polypeptide may be fused to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding members. In some embodiments, a first or second constituent polypeptide is fused to 2-10 or 2-5 binding members. In certain embodiments, a first or second constituent polypeptide is fused to three binding members. For example, a first or second constituent polypeptide may be fused to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target proteins. In some embodiments, a first or second constituent polypeptide is fused to 2-10 or 2-5 target proteins. In certain embodiments, a first or second constituent polypeptide is fused to three target proteins. Where multiple binding members or target proteins are present, they may be fused to one another by peptide linkers, such as the peptide linkers described above.

[0147] Split transcription factors The dimer-inducing protein can be a split transcription factor. In some embodiments, a first constituent polypeptide comprises a DNA-binding domain and a second constituent polypeptide comprises a transcriptional regulatory domain, and dimerization of the first and second constituent polypeptides forms a transcription factor. "Forming a transcription factor" refers to bringing the first and second constituent polypeptides into sufficient proximity to reconstitute transcriptional regulatory activity of a desired expression product. The dimer-inducing protein has increased transcriptional regulatory activity when the binding member binds to the T-SM complex, compared to the transcriptional regulatory activity observed when the binding member is not bound to the T-SM complex.

[0148] The transcriptional regulatory domain can be a transcriptional activation domain capable of upregulating the transcription of a gene bound by the split transcription factor. Suitable transcriptional activation domains include the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)); replication and transcription activator (RTA; Lukac et al., J. Virol. 73:9348-61 (1999)), HSV VP16 activation domain (see, e.g., Hagmann et al., J. Virol. 71:5952-5962 (1997)), nuclear hormone receptors (e.g., Torchia et al. al., Curr. Opin. Cell. Biol. 10:373-383 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degrons (Molinari et al., (1999) EMBO J. 18, 6439-6447). Further exemplary activation domains include Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipel et al., EMBO J. 11, 4961-4968 (1992)), as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A, and ERF-2.For example, Robyr et al. (2000) Mol. Endocrinol.14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol. 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim.Pol.46:77-89;McKenna et al.(1999)J.Steroid Biochem.Mol.Biol.69:3-12;Malik et al.(2000)Trends Biochem.Sci.25:277-283;and Lemon et al. al. (1999) Curr. Opin. Genet. Dev. 9:499-504. Further exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7 and ARF-8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1, and modified Cas9 transactivator proteins. For example, Ogawa et al.(2000)Gene 245:21-29;Okanami et al.(1996)Genes Cells 1:87-99;Goff et al.(1991)Genes Dev.5:298-309;Cho et al.(1999)Plant Mol.Biol.40:419-429;Ulmason et al. al.(1999)Proc.Natl.Acad.Sci.USA 96:5844-5849;Sprenger-Haussels et al.(2000)Plant J.22:1-8;Gong et al.(1999)Plant Mol.Biol.41:33-44;Hobo et al.(1999)Proc.Natl.Acad.Sci.USA 96:15,348-15,353; and Perez-Pinera et al. (2013) Nature Methods 10:973-976. The transcription activation domain may comprise any combination of the exemplary activation domains listed above.In some embodiments, multiple transcription activation domains may be used, for example, tandem reports of the same domain or fusions of different domains. In some embodiments, the transcription activation domain is VPR, a trimolecular activation domain composed of VP64, p65, and Rta domains. An example of a TRD-T fusion protein containing VPR is set forth in SEQ ID NO: 225 (NS4A / 3 PR S139A-VPR). The production and use of VPR as a transcription activator is described, for example, in Chavez et al. 2015. In some embodiments, the transcription activation domain is HSF-1, optionally combined with p65.

[0149] Alternatively, the transcriptional regulatory domain may be a transcriptional repression domain capable of down-regulating the transcription of a gene bound by a split transcription factor. Examples of transcriptional repression domains include, but are not limited to, KRAB A / B, KOX, TGF-β-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2. See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; and Robertson et al. (2000) Nature Genet. 25:338-342. Further exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, e.g., Chem et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

[0150] The DNA-binding domain can be any protein that binds to a target sequence in a sequence-specific manner. For example, the DNA-binding domain can be or include a transcription factor or DNA-binding fragment thereof that binds to a target sequence in a sequence-specific manner. It is anticipated that any transcription factor or DNA-binding fragment thereof capable of binding to a target sequence in a specific manner can be used with the split transcription factors disclosed herein. The DNA-binding domain can be or include a naturally occurring DNA-binding domain, such as a binding domain from a human transcription factor. For example, the DNA-binding protein can be any of the human transcription factors described in Vaquerizas et al. (2009) (e.g., any of those listed in Supplementary Information S3), or DNA-binding fragments thereof. For example, the DNA-binding protein can be a member of the C2H2 zinc finger family, the homeodomain family, or the helix-loop-helix family, or a DNA-binding fragment thereof. In certain embodiments, the DNA-binding domain can be zinc finger homeodomain transcription factor 1 (ZFHD1). ZFHD1 contains zinc fingers 1 and 2 derived from the Zif268 transcription factor and the Oct-1 homeodomain. The design and construction of ZFHD1 is described, for example, in Pomerantz et al. 1995.

[0151] The DNA-binding domain may be or may include a DNA-binding domain such as a zinc finger DNA-binding domain, a TALE DNA-binding domain, a DNA-binding domain derived from a meganuclease (e.g., based on IsceI), or a DNA-binding domain derived from a CRISPR / Cas system. These binding domains can be engineered to bind to a selected target sequence, for example, a target sequence of a naturally occurring (endogenous) target gene in a cell, or a target sequence provided in trans (e.g., as part of a third expression cassette). Engineering zinc finger DNA-binding domains to bind to specific target sequences is described, for example, in U.S. Pat. No. 6,453,242 B1. In one embodiment, the DNA-binding domain is a TALE DNA-binding domain. Engineering TALE DNA-binding domains to bind to specific target sequences is described, for example, in WO2010079430A1. In one embodiment, the DNA-binding domain is an engineered DNA-binding domain derived from a meganuclease. Engineering meganucleases to bind to specific target sequences is described, for example, in International Publication No. WO 2007047859A1. Meganucleases can be engineered to not cleave DNA. In one embodiment, the DNA-binding domain is an engineered DNA-binding domain derived from a CRISPR / Cas system. Engineering DNA-binding domains derived from CRISPR / Cas systems to bind to specific sequences is described, for example, in International Publication No. WO 2013176772A1. CRISPR / Cas systems generally require an RNA-guided endonuclease (e.g., Cas9) that guides a specific DNA sequence through complementarity between an associated guide RNA (gRNA) and its target sequence. Thus, engineered DNA-binding domains derived from CRISPR / Cas systems typically comprise a complex of an RNA-guided endonuclease (e.g., Cas9 or a mutant thereof) and a guide RNA. Mutants of Cas9 have been generated that lack endonuclease activity but retain the ability to interact with DNA.See, for example, Chavez et al. 2015, which describes the use of nuclease null (dCas9) mutants in methods of transcriptional regulation. Thus, the DNA-binding domain can comprise a nuclease null Cas9 mutant that binds to a target sequence upon addition of a specific gRNA specific for that sequence. An example of a DBD-BM fusion protein containing dCas9 as the DNA-binding domain is set forth in SEQ ID NO: 227 (spdCas9-PRSIM_23x3). An example of a guide RNA targeting DBD-BM to human IL-2 is set forth in SEQ ID NO: 229. The use of dCas9 mutants as part of a split transcription factor is described in Hill et al. 2018 and WO 2018 / 213848 A1.

[0152] The binding member may be fused to a transcriptional regulatory domain or a DNA binding domain.

[0153] In some embodiments, (1) a first constituent polypeptide comprises a DNA-binding domain and is fused to a target protein to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein; a second component polypeptide comprising a DNA binding domain and fused to a binding member to form a DBD-BM fusion protein; The DNA binding domain, target protein, transcriptional regulatory domain, and binding member are as further defined herein.

[0154] In one embodiment, (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to a target protein to form a DBD-T fusion protein, the target protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1; the second component polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein, the target protein having an amino acid sequence having at least 90% identity to SEQ ID NO:1; a second component polypeptide comprising a DNA binding domain and fused to a binding member to form a DBD-BM fusion protein; In either (1) or (2), a) the binding member comprises the BC, DE and FG loops of PRSIM_23, or a Tn3 sequence; b) the binding member comprises the BC, DE and FG loops of PRSIM_32, or a Tn3 sequence; c) the binding member comprises the BC, DE and FG loops of PRSIM_33, or a Tn3 sequence; d) the binding member comprises the BC, DE and FG loops of PRSIM_36, or a Tn3 sequence; e) the binding member comprises the BC, DE and FG loops of PRSIM_47, or a Tn3 sequence; f) the binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_57; g) the binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_01; h) the binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_04; i) the binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_67; j) the binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_72; or k) The binding member comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_75.

[0155] The DBD-T fusion protein may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45. In a specific embodiment, the TRD-BM fusion protein defined in (1) above may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 67.

[0156] The TRD-T fusion protein may comprise an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 44. In a specific embodiment, the DBD-BM fusion protein defined in (2) above may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46 to 56.

[0157] As described in the Examples, some exemplary binding members have been observed to exhibit a preference for fusion with either a DNA-binding domain or a transcriptional regulatory domain, thereby resulting in increased transcriptional regulatory activity depending on whether a particular binding member is bound to a DNA-binding domain or fused to a transcriptional regulatory domain. Thus, in some embodiments: (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to a target protein to form a DBD-T fusion protein, the target protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1; a second component polypeptide comprising a transcriptional regulatory domain and fused to a binding member to form a TRD-BM fusion protein; where: a) the binding member of the TRD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_23, or a Tn3 sequence; b) the binding member of the TRD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_47, or a Tn3 sequence; or c) the binding member of the TRD-BM fusion protein comprises the HCDR and / or LCDR, or the VH and / or VL sequences of PRSIM_04; d) the binding member of the TRD-BM fusion protein comprises the HCDR and / or LCDR, or the VH and / or VL sequences of PRSIM_72; e) the binding member of the TRD-BM fusion protein comprises the HCDR and / or LCDR, or the VH and / or VL sequences of PRSIM_67; or f) the binding member of the TRD-BM fusion protein comprises the HCDR and / or LCDR, or the VH and / or VL sequences of PRSIM_75; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein, the target protein having an amino acid sequence having at least 90% identity to SEQ ID NO:1; a second component polypeptide comprising a DNA binding domain and fused to a binding member to form a DBD-BM fusion protein; where: g) the binding member of the DBD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_23, or a Tn3 sequence; h) the binding member of the DBD-BM fusion protein comprises the HCDR and / or LCDR, or the VH and / or VL sequences of PRSIM_01; i) the binding member of the DBD-BM fusion protein comprises the HCDRs and / or LCDRs, or the VH and / or VL sequences of PRSIM_57; j) the binding member of the DBD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_32, or a Tn3 sequence; k) the binding member of the DBD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_33, or a Tn3 sequence; or l) The binding member of the DBD-BM fusion protein comprises the BC, DE and FG loops of PRSIM_36, or the Tn3 sequence.

[0158] In some embodiments, the binding member or target protein is fused to the C-terminus of the DNA-binding domain. In other embodiments, the binding member or target protein is fused to the N-terminus of the transcriptional regulatory domain. The binding member or target protein may be fused to the DNA-binding domain or transcriptional regulatory domain via a peptide linker, such as one or more of the peptide linkers described above. In certain embodiments, the linker has the amino acid sequence TGGGGSGGGGS (SEQ ID NO: 185) or SA.

[0159] As described in the Examples, PRSIM_23 was found to provide potent gene expression regulation in both configurations. Thus, in some embodiments, (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to a target protein to form a DBD-T fusion protein, the target protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1; the second component polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein, the target protein having an amino acid sequence having at least 90% identity to SEQ ID NO:1; a second component polypeptide comprising a DNA binding domain and fused to a binding member to form a DBD-BM fusion protein; In either (1) or (2), the binding member comprises the BC, DE and FG loops of PRSIM_23 or a Tn3 sequence.

[0160] In certain embodiments, (1) the DBD-T fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45, and the TRD-BM fusion protein has an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 57; or (2) The DBD-BM fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 46, and the TRD-T fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 44.

[0161] As shown in the Examples, PRSIM-based CID can also be applied to activated CRISPR (CRISPRa) systems, which can be used, for example, to promote endogenous gene regulation.

[0162] Thus, in some embodiments, the DBD-BM fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 227, and the TRD-T fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 225. The DBD-BM fusion protein can be directed to the target sequence by using a specific guide RNA specific to the target sequence.

[0163] As shown in the Examples, split transcription factors containing a DNA-binding domain fused to multiple copies of a target protein or binding member have been shown to have increased expression compared to split transcription factors containing a DNA-binding domain fused to a single copy of a target protein or binding member.

[0164] Thus, in some embodiments, The DBD-T fusion protein comprises a DNA-binding domain fused to multiple copies of a target protein (e.g., two, three, four, five or more target proteins); or The DBD-BM fusion protein comprises a DNA binding domain fused to multiple copies of a target protein (eg, 2, 3, 4, 5 or more binding members).

[0165] The multiple binding members or multiple target proteins may be separated by a linker, e.g., one or more peptide linkers as described above. In certain exemplary embodiments, a DBD-T fusion protein comprises a DNA-binding domain fused to three target proteins, or a DBD-BM fusion protein comprises a DNA-binding domain fused to three binding members.

[0166] The first and / or second constituent polypeptide may further comprise a nuclear localization signal (such as, for example, from the SV40 middle T antigen).

[0167] The split transcription factor may also comprise a third expression cassette, the third expression cassette encoding a desired expression product, and the DNA-binding domain of the split transcription factor binds to a target sequence within the third expression cassette such that the transcription factor can regulate expression of the desired expression product. "Capable of regulating expression" is intended to mean that the DNA-binding domain can bind to a target sequence, and upon forming a transcription factor with the transcription regulatory domain (i.e., upon dimerization of the dimer-inducing protein), the DNA-binding domain has transcription regulatory activity that regulates (increases or decreases) expression of the desired expression product. The desired expression product may be RNA or may be peptidic (peptide, polypeptide, or protein). Preferably, the desired expression product is peptidic. The desired expression product may be a therapeutic protein, i.e., a protein that exerts a therapeutic effect in a subject.

[0168] The target sequence may be located in a promoter operably linked to the coding sequence of the desired expression product, or may be located proximal to the promoter. By "proximal," we mean that the target sequence is within 500 bp, 250 bp, 100 bp, 50 bp, or 25 bp of the sequence corresponding to the promoter.

[0169] Split Chimeric Antigen Receptor The dimer-inducing protein may be a split chimeric antigen receptor (split CAR).

[0170] CARs combine both antibody-like recognition functions and T cell activation functions. CARs typically consist of an antigen-specific recognition domain, such as that derived from an antibody, a transmembrane domain that anchors the CAR to T cells, a costimulatory domain, and one or more intracellular signaling domains that induce persistence and traffic the effector functions of transduced T cells. The design and use of CARs are known in the art and are described, for example, in Sadelain et al. 2013.

[0171] Split CARs have been engineered to require an exogenous, user-provided signal to activate the CAR, as described, for example, in Wu et al. (2015). These split receptor, antigen-binding, and intracellular signaling components assemble only in the presence of heterodimerizing small molecules, allowing the user to precisely control the timing, location, and dosage of T cell activation. Such split CARs are expected to mitigate toxicity, for example, by inducing fewer off-target effects.

[0172] In one embodiment, the dimer-inducing protein is a first constituent polypeptide comprising a costimulatory domain and fused to a target protein as defined herein; and a second component polypeptide comprising an intracellular signalling domain, fused to a binding member as defined herein.

[0173] The first constituent polypeptide described above may further comprise an antigen-specific recognition domain and a transmembrane domain, and the second constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain, and when the first and second constituent polypeptides dimerize, they form a chimeric antigen receptor (CAR). "Forming a CAR" means bringing the first and second constituent polypeptides into sufficient proximity to allow the reconstitution of a fully functional CAR.

[0174] In another embodiment, the dimer-inducing protein is a first constituent polypeptide comprising an intracellular signaling domain and fused to a target protein as defined herein; and a second constituent polypeptide comprising a first costimulatory domain and fused to a binding member as defined herein.

[0175] The first constituent polypeptide described above may further comprise a transmembrane domain and a second costimulatory domain, and the second constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain, and when the first constituent polypeptide and the second constituent polypeptide dimerize, they form a chimeric antigen receptor (CAR).

[0176] The split CAR has increased activity when the binding member binds to the T-SM complex, which activity is increased compared to the activity observed when the binding member is not bound to the T-SM complex.

[0177] In one embodiment, the first constituent polypeptide comprises, from N-terminus to C-terminus: i) an antigen-specific recognition domain; ii) a transmembrane domain; and ii) a first costimulatory domain; The second constituent polypeptide is, from the N-terminus to the C-terminus: i) a transmembrane domain; ii) a second costimulatory domain; and iii) an intracellular signaling domain; Dimerization of the first and second constituent polypeptides forms a CAR.

[0178] In some embodiments, the target protein and binding member are fused at a position from the C-terminus of the first and second constituent polypeptides toward the respective transmembrane domains. For example, the target protein or binding member may be fused to the N-terminus or C-terminus of the costimulatory domain of each of the first and second constituent polypeptides. In certain embodiments, one of the target protein and binding member is fused to the C-terminus of the first costimulatory domain, and the other is fused to the C-terminus of the second costimulatory domain.

[0179] For example, in one embodiment, the first constituent polypeptide comprises, from N-terminus to C-terminus: i) an antigen-specific recognition domain; ii) a transmembrane domain; and iii) a first costimulatory domain; The second constituent polypeptide is, from the N-terminus to the C-terminus: i) a transmembrane domain; ii) a second costimulatory domain; and iii) an intracellular signaling domain; The target protein is fused to the C-terminus of the first costimulatory domain and the binding member is fused to the C-terminus of the second costimulatory domain.

[0180] For example, in another embodiment, the first constituent polypeptide comprises, from N-terminus to C-terminus: i) an antigen-specific recognition domain; ii) a transmembrane domain; and iii) a first costimulatory domain; The second constituent polypeptide is, from the N-terminus to the C-terminus: i) a transmembrane domain; ii) a second costimulatory domain; and iii) an intracellular signaling domain; The binding member is fused to the C-terminus of the first costimulatory domain and the target protein is fused to the C-terminus of the second costimulatory domain.

[0181] The target protein and / or binding member may be fused to the respective costimulatory domain. More preferably, the target protein and binding member may be separated from the respective costimulatory domain by a peptide linker. The peptide linker may be as further defined herein. In some embodiments, the target protein and binding member are separated from the respective costimulatory domain by a linker comprising the amino acid sequence set forth in SEQ ID NO: 204. Similarly, various domains of the first and second constituent polypeptides may be separated by peptide linkers. For example, the transmembrane domain may be separated from the second costimulatory domain by a peptide linker, e.g., a peptide linker comprising the amino acid sequence GS, and / or the second costimulatory domain may be separated from the intracellular signaling domain by a peptide linker, e.g., a peptide linker comprising the amino acid sequence set forth in SEQ ID NO: 204.

[0182] Non-limiting examples of suitable costimulatory domains include, but are not limited to, activation domains from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM. In one embodiment, the first and second costimulatory domains are 4-1BB activation domains.

[0183] Non-limiting examples of suitable intracellular signaling domains include, but are not limited to, the cytoplasmic sequences of T cell receptors (TCRs) and coreceptors that coordinately initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities. Particular intracellular signaling domains include those containing signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAMs containing signaling domains include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the intracellular signaling domain is derived from CD3 zeta.

[0184] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region may be derived from the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4 (i.e., comprising at least the transmembrane region thereof). Alternatively, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Synthetic transmembrane domains may contain triplets of phenylalanine, tryptophan, and valine at each end. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular signaling domain of the CAR. A particularly suitable linker is provided by a glycine-serine doublet. In certain embodiments, the transmembrane domain is derived from CD28.

[0185] The first and second polypeptides may further comprise a hinge domain, such as an IgG4 or CD8a hinge domain, from the N-terminus of the first and / or second polypeptide to the transmembrane domain. Examples of hinge domains are described, for example, in Qin et al. 2017. In certain embodiments, the hinge domain is a human IgG4 hinge domain.

[0186] The antigen-specific recognition domain suitable for use in the dimer-inducing proteins of the present disclosure can be any antigen-binding polypeptide, and a wide variety of antigen-binding polypeptides are known in the art. In some cases, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains, such as cAb VHH (camelid antibody variable domain) and its humanized versions, IgNAR VH (shark antibody variable domain) and its humanized versions, sdAb HV (single-domain antibody variable domain), and "camelized" antibody variable domains, are also suitable for use. In some cases, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTv, single-chain two-domain TCRs including vvβ), are also suitable for use.

[0187] In certain embodiments, the antigen-specific recognition domain is a single-chain Fv (scFv). As described elsewhere, scFvs typically comprise a VH chain separated from a VL chain by a peptide linker, e.g., a peptide linker comprising the amino acid sequence set forth in SEQ ID NO: 204.

[0188] Antigen-specific recognition domains suitable for use in the dimer-inducing proteins of the present disclosure can have a variety of antigen-binding specificities. In some cases, the antigen-binding domain is specific for an epitope present in an antigen expressed (synthesized) by a cancer cell, i.e., a cancer cell-associated antigen. Cancer cell-associated antigens can be, for example, antigens associated with breast cancer cells, B-cell lymphoma cells, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's B-lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma cells, glioblastoma cells, medulloblastoma cells, colorectal cancer cells, etc. Cancer cell-associated antigens can also be expressed by non-cancerous cells.

[0189] In certain exemplary embodiments, the target protein used in the split CAR is derived from HCV NS3 / 4A protease, the small molecule is simeprevir, and the binding member is based on PRSIM_23 (e.g., comprising the BC, DE, and FG loops or Tn3 sequence of PRSIM_23, optionally with sequence identity and / or mutations described herein).

[0190] In some embodiments, the first constituent polypeptide comprises, from N-terminus to C-terminus: i) an antigen-specific recognition domain; ii) a transmembrane domain; and iii) a first costimulatory domain; The second constituent polypeptide is, from the N-terminus to the C-terminus: i) a transmembrane domain; ii) a second costimulatory domain; and iii) an intracellular signaling domain; The target protein is fused to the C-terminus of the first costimulatory domain, the binding member is fused to the C-terminus of the second costimulatory domain, the first constituent polypeptide fused to the target protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70, the second constituent polypeptide fused to the binding member comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 200, and optionally, an antigen-specific recognition domain (e.g., scFv) is located from the N-terminus to the amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70.

[0191] In some embodiments, the first constituent polypeptide comprises a first signal peptide located N-terminally to the antigen-specific recognition domain. The first signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 201 or SEQ ID NO: 202. In exemplary embodiments, the first signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 201.

[0192] In some embodiments, the second constituent polypeptide comprises a second signal peptide located N-terminally towards the transmembrane domain. The second signal peptide may comprise the amino acid sequence set forth in SEQ ID NO:201 or SEQ ID NO:202. In exemplary embodiments, the second signal peptide comprises the amino acid sequence set forth in SEQ ID NO:202. In one embodiment, the second constituent polypeptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:203.

[0193] Also provided are modified immune cells comprising the split CAR disclosed herein. In one embodiment, the immune cells are T cells. Also provided are methods for genetically modifying immune cells to express the split CAR disclosed herein. This method can be carried out ex vivo. The method can include administering one or more expression vectors described herein to immune cells so that the split CAR is expressed on the surface of the immune cells.

[0194] Split Reporter System The dimer-inducing protein may be a split reporter system. The split reporter system may be an enzyme or a fluorescent protein that provides an observable phenotype upon dimerization of a first constituent polypeptide and a second constituent polypeptide. The observable phenotype may be a colorimetric signal, a luminescent signal, or a fluorescent signal. A specific example of a split reporter system is provided in Dixon et al. 2017.

[0195] In some embodiments, the first constituent polypeptide comprises a first reporter moiety and the second constituent polypeptide comprises a second reporter moiety, and when the first and second constituent polypeptides dimerize they form a reporter system, and optionally the reporter system provides an increase in a colorimetric, luminescent, or fluorescent signal when the binding member binds to the T-SM complex.

[0196] Split Apoptosis Protein The dimer-inducing protein may be a split apoptosis protein. A split apoptosis protein is any protein that can induce apoptosis upon dimerization of a first and second constituent polypeptides of the split apoptosis protein. An example of a split apoptosis protein is a split caspase (e.g., split caspase 9 or split caspase 3) that can induce apoptosis upon dimerization and can therefore be used to kill specific cells containing the split apoptosis protein (e.g., diseased cells or therapeutic cells administered for cell therapy). Examples of split caspases are provided in Chelur et al. 2007. The use of an inducible caspase 9 suicide gene system is described, for example, in Gargett et al. 2014.

[0197] In some embodiments, the first constituent polypeptide comprises a first caspase component and the second constituent polypeptide comprises a second caspase component, and the first and second constituent polypeptides dimerize to form a caspase. The split caspase may be capable of inducing cell death when the binding member binds to the T-SM complex.

[0198] In some embodiments, the first and second caspase components are identical, e.g., both caspase components contain a caspase-9 activation domain. An exemplary caspase-9 activation domain is provided as amino acid residues 152-414 of the human caspase-9 amino acid sequence, available under NCBI Accession No. AAO21133.1 (Version 1, last updated December 1, 2009). When the first and second caspase components are identical, they may be encoded by the same expression cassette. For example, a split apoptosis protein may be encoded by one or more expression cassettes encoding a target protein, a binding member, and a caspase-9 activation domain, with both the target protein and the binding member fused to the caspase-9 activation domain. Upon expression, multiple proteins containing the target protein, the binding member, and the caspase-9 activation domain are produced, and dimerization of the caspase-9 activation domain (i.e., at least the first and second caspase-9 activation domains) can be regulated by the addition of a small molecule.

[0199] In certain exemplary embodiments, the split apoptotic protein comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:223.

[0200] Other dimer-inducing proteins Other dimerizing proteins contemplated for use in the present disclosure include split therapeutic proteins, split TEV proteases, and split Cas9. A split therapeutic protein is any protein that can exert a therapeutic effect upon dimerization of a first constituent polypeptide and a second constituent polypeptide of the split therapeutic protein.

[0201] Viral vectors and viral particles In one embodiment, the expression vector is a viral vector. Suitable viral vectors for use include adeno-associated viral vectors, adenoviral vectors, herpes simplex viral vectors, retroviral vectors, lentiviral vectors, alphaviral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviral vectors, and picornaviral vectors.

[0202] As used herein, a viral vector refers to a DNA expression vector that contains first and second expression cassettes such that, when expressed intracellularly, the expression cassettes, along with components necessary for viral particle assembly, are converted into a viral genome packaged within the viral particle. Additionally, in one embodiment, the viral vector contains a third expression cassette encoding a desired expression product.

[0203] In certain embodiments, the expression vector is an adeno-associated virus (AAV) vector. AAV is one of the most extensively studied gene therapy vehicles, characterized by an excellent safety profile and highly efficient transduction in a wide range of target tissues. The use of AAV as a gene therapy vector is described, for example, in Naso et al. 2017 and Colella et al. 2018.

[0204] Various AAV serotypes can also be used in accordance with the present disclosure, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6.2FF, AAV8, AAV8.2, AAV9, and AAV rh10, as well as pseudotyped AAVs such as AAV2 / 8, AAV2 / 5, and AAV2 / 6. Further examples of serotypes and their isolation are described in Srivastava, 2006.

[0205] AAV particles are small (25 nm) viruses from the Parvoviridae family that consist of a non-enveloped, icosahedral capsid (protein shell) containing a linear, single-stranded DNA genome of approximately 4.8 kb. The AAV genome encodes several protein products: four nonstructural Rep proteins, three capsid proteins (VP1-VP3), and an assembly-activating protein (AAP). The AAV genes are flanked by two AAV-specific palindromic inverted terminal repeats (ITRs).

[0206] Thus, if the expression vector is an AAV vector, it may mean that the first and second expression cassettes are flanked by ITRs (e.g., ITR-first expression cassette-second expression cassette-ITR) so that when expressed in a cell together with the components required for AAV particle assembly, the expression cassettes are converted into a single-stranded genome packaged within an AAV particle.

[0207] AAV vectors may be modified, for example, to improve their function. Examples of modified AAV for clinical gene therapy are described in Kotterman and Schaffer, 2014.

[0208] AAV vectors have a packaging capacity of less than 5 kb, which can limit the size of genetic material (e.g., expression cassettes) that can be introduced into the viral genome. As shown herein, by using relatively small components such as Tn3 protein and scFv as binding members, expression cassettes encoding trimolecular complexes (e.g., as part of a dimer-inducing protein such as a split transcription factor) can be fitted into a single AAV vector. As further shown herein, the small size of the expression cassette encoding the trimolecular complex allows a transgene (e.g., as part of a third expression cassette) to be introduced into the same AAV vector as components of the split transcription factor, thereby allowing the split transcription factor to be delivered "in cis" with the transgene.

[0209] The present disclosure also includes methods for producing viral particles in vitro. In one embodiment, the method for producing viral particles includes transfecting host cells, such as mammalian cells, with a viral vector as described herein, expressing viral proteins necessary for forming particles within the cells, and culturing the transfected cells in a medium so that the cells produce viral particles. The viral particles may be released into the medium, or the method may further include lysing the particles and isolating the particles from the cell lysate. One example of a suitable mammalian cell is human embryonic kidney (HEK) 293 cells.

[0210] Typically, multiple plasmid expression vectors are used to produce the various protein components that make up the viral particle. Cell lines that constitutively express the viral packaging components are also available, allowing the use of fewer plasmids.

[0211] For example, the construction of AAV particles requires the Rep and Cap proteins, as well as additional genes from adenovirus that mediate AAV replication. The production of AAV particles is described, for example, in Robert et al. (2017).

[0212] An exemplary method for generating AAV particles is described in Robert et al. 2017. Briefly, this method involves transfecting mammalian cells, such as HEK293 cells, with three plasmids: one vector encodes the AAV rep and cap genes (pRepCap) using their endogenous promoters, one vector (pHelper) encodes three additional adenoviral helper genes (E4, E2A, and VA RNA) that are not present in HEK293 cells, and one vector (viral vector) (pAAV-GOI) contains one or more expression cassettes flanked by two ITRs. See Figure 2 in Robert et al.

[0213] After the viral particles are released, the medium containing the viral particles may be collected, and optionally the viral particles may be separated from the cell lysate. Optionally, the viral particles may be concentrated.

[0214] After production and optional concentration, the viral particles may be stored frozen, for example at -80°C, in preparation for use by administration to cells and / or for use in therapy.

[0215] The present disclosure also provides viral particles, e.g., AAV particles, produced by the methods described herein. As used herein, a viral particle comprises a viral genome packaged within a viral envelope that is capable of infecting a cell, e.g., a mammalian cell.

[0216] Disclosed herein are one or more viral particles comprising a viral genome, the viral genome comprising: i) a target protein capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; The target protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In one embodiment, the target protein is fused to a first constituent polypeptide and the binding member is fused to a second constituent polypeptide.

[0217] Also disclosed herein are one or more viral particles, the viral particles comprising: i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; The target protein is derived from a non-human protein, the small molecule is an inhibitor of the non-human target protein, and the first and second expression cassettes form part of the viral genome in one or more viral particles. In one embodiment, the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor. In one embodiment, the target protein is fused to a first constituent polypeptide and the binding member is fused to a second constituent polypeptide.

[0218] In some embodiments, the first and second expression cassettes form part of the viral genome of the same viral particle, while in other embodiments, the first expression cassette is located in a first viral genome of a first viral particle and the second expression cassette is located in a second viral genome of a second viral particle.

[0219] The expression cassette, target protein, binding member, small molecule, and first and second constituent polypeptides may be as defined above. Depending on the viral particle used, the viral genome may be a single-stranded or double-stranded nucleic acid, RNA or DNA. For example, when the viral particle is an AAV particle, the viral genome is a single-stranded DNA viral genome. The viral genome may encode a split protein as defined above.

[0220] Gene therapy The agents (i.e., one or more expression vectors, expression products, or viral particles, as well as small molecules) may be administered to a patient as part of a method for treating or preventing a disease. After the binding member binds to the T-SM complex, the recipient individual may experience a reduction in symptoms of the disorder or disease being treated. This may have a beneficial effect on the individual's condition.

[0221] The term "treatment," as used herein in reference to the treatment of a condition, generally relates to human care and therapy to achieve some desired therapeutic effect, such as, for example, inhibiting the progression of the condition, including slowing the rate of progression, halting the rate of progression, regressing the condition, ameliorating the condition, and curing the condition. Treatment as a preventative measure (i.e., prevention, prophylaxis) is also included.

[0222] "Prevention," in the context of this specification, should not be understood to be limited to complete success, i.e., complete protection or complete prevention. Rather, prevention, as used herein, refers to measures taken with the objective of preserving health by helping to delay, alleviating, or avoiding a particular condition before a symptomatic state is detected.

[0223] Therapeutic methods can include expressing in cells one or more dimer-inducing proteins, as further defined herein. The dimer-inducing protein, for example, includes a first constituent polypeptide and a second constituent polypeptide that dimerize to form a therapeutic polypeptide. In this manner, the addition of a small molecule can result in a therapeutic protein with increased activity, which can be used, for example, in methods of treating diseases where the therapeutic protein is deficient.

[0224] Disclosed is a method for modulating expression of a desired expression product in a cell, the method comprising: i) expressing a dimer-inducing protein as described herein in a cell, wherein the first and second constituent polypeptides dimerize to form a transcription factor, and the DNA binding domain binds to a target sequence in the cell such that the transcription factor can regulate (i.e., increase or decrease) expression of a desired expression product in the cell; and ii) administering a small molecule to the cell to regulate expression of the desired expression product.

[0225] Further disclosed herein is a dimer-inducing protein for use in a method of modulating expression of a desired expression product in a cell of a human or animal subject, the method comprising expressing a dimer-inducing protein as described herein in the cell, wherein a first constituent polypeptide and a second constituent polypeptide dimerize to form a transcription factor, and administering a small molecule to the cell to modulate (e.g., increase or decrease) expression of the desired expression product. Also disclosed herein is a small molecule for use in a method of modulating expression of a desired expression product in a cell of a human or animal subject, the method comprising expressing a dimer-inducing protein as described herein in the cell, wherein a first constituent polypeptide and a second constituent polypeptide dimerize to form a transcription factor, and administering a small molecule to the cell to modulate (e.g., increase or decrease) expression of the desired expression product.

[0226] The method can include administering one or more expression vectors or viral particles as described herein to express the dimer-inducing protein in the cell. In other embodiments, the method can include administering to the cell one or more expression vectors, e.g., an expression product produced from an mRNA encoding the dimer-inducing protein. Specific administration is subject to the judgment of the physician, who will further select the dosage using general knowledge common to physicians and administration regimens known to skilled practitioners.

[0227] The desired expression product may be RNA or may be peptidic (peptide, polypeptide, or protein). Preferably, the desired expression product is peptidic. The desired expression product may be a therapeutic protein, i.e., a protein that exerts a therapeutic effect in a subject.

[0228] The desired expression product may be part of an endogenous gene present in the genome of the target cell. For example, if the method is performed in a human cell, the desired expression product may be part of a human gene. Alternatively, the desired expression product may be part of a transgene, e.g., a therapeutic transgene, delivered to the target cell. Modulation of gene expression may be used in methods for treating or preventing disease. After expressing the split transcription factor and administering the small molecule, the recipient individual may show a reduction in symptoms of the disorder or disease being treated. This may have a beneficial effect on the individual's condition.

[0229] If the target sequence is part of a transgene delivered to the cell, the method may further include administering to the cell a third expression cassette encoding a desired expression product and including the target sequence. The transgene may include a promoter operably linked to the coding sequence of the desired expression product, and the desired expression product may be a therapeutic protein, such as a therapeutic antibody. An example of a therapeutic antibody is MEDI8852, which has a heavy chain amino acid sequence set forth as SEQ ID NO: 205 and a light chain amino acid sequence set forth as SEQ ID NO: 206. The third expression cassette may be part of the same expression vector or viral particle as one or both of the first and second expression cassettes. In other words, the transgene can be delivered to the cell in "cis" with the split transcription factor, such as within the same viral (e.g., AAV) particle. Alternatively, the third expression cassette may be part of a different expression vector or viral particle than one or both of the first and second expression cassettes. In other words, the transgene can be delivered to the cell in "trans" with the split transcription factor, such as in a separate viral (e.g., AAV) particle. As demonstrated herein, the split transcription factors of the present disclosure are suitable for delivery both "cis" and "trans" with the transgene.

[0230] The target sequence may be located in a promoter operably linked to the coding sequence of the desired expression product, or may be located proximal to the promoter, where "proximal" means that the target sequence is within 500 bp, 250 bp, 100 bp, 50 bp, or 25 bp of the sequence corresponding to the promoter.

[0231] Administration to cells may be by any suitable means. For example, the expression cassette may be delivered by viral means, such as as part of a viral particle as described herein, or by non-viral means. Non-viral means for delivery include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or polylipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and drug-enhanced DNA uptake. In one embodiment, the expression cassette is delivered as mRNA. In one embodiment, the expression cassette is delivered as a DNA plasmid.

[0232] In any of the in vivo methods disclosed herein, the small molecule may be administered orally to a human subject in an acceptable dosage form, such as a capsule, tablet, aqueous suspension, or solution. The amount used will depend on the host being treated and the particular method of administration. The small molecule may be administered as a single dose, multiple doses, or over an established period of time.

[0233] Where the method involves administering viral particles to cells, the unit dose may be calculated based on the dose of viral particles administered. A viral dose includes the number of specific viral particles, i.e., plaque-forming units (pfu) or viral genome copies (vgc). For embodiments involving AAV, a specific unit dose is 10 per kg of body weight. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 Contains viral genome copies (VGC). Particle dose may be somewhat higher (10-100 fold) due to the presence of particles defective for infection.

[0234] Without being bound by theory, infection and transduction of cells by viral particles (e.g., AAV particles) is believed to occur through a series of sequential events, including interaction of the viral capsid with receptors on the surface of the target cell, internalization by endocytosis, intracellular trafficking through endocytic / proteasomal compartments, endosomal escape, nuclear import, uncoating of the virion, and viral DNA double-strand conversion, resulting in the transcription and expression of proteins encoded by the viral genome within the viral particle.

[0235] While it is possible for one or more expression vectors, expression products, viral particles, and small molecules to be used (e.g., administered) alone, it is often preferable to present the individual components as a composition or formulation, e.g., together with a pharmaceutically acceptable carrier or diluent. For example, one or more viral particles may be administered as a pharmaceutical composition comprising one or more viral particles and a pharmaceutically acceptable carrier or diluent. As another example, a small molecule may be administered as a pharmaceutical composition comprising the small molecule and a pharmaceutically acceptable carrier or diluent.

[0236] The term "pharmaceutically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0237] The agents (i.e., one or more expression vectors, DNA plasmids, or viral particles, plus small molecules) may be administered simultaneously or sequentially, may be administered on different individual dosing schedules, and may be administered by different routes. For example, when administered sequentially, the agents can be administered closely spaced apart (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 hours or more, or even longer if necessary), the precise dosing regimen being commensurate with the properties of the agents being administered. In one embodiment, the small molecule is administered after the one or more expression vectors, DNA plasmids, or viral particles.

[0238] cell therapy Also provided are methods of cell therapy that involve administering to a patient cells that have been genetically modified to express an expression product, such as a dimer-inducing protein.

[0239] Cells, such as stem cells, may be used in cell therapy methods. One potential advantage of using stem cells is that they can be differentiated into other cell types in vitro and introduced into a mammal (such as the donor of the cells) by transplantation into the bone marrow. Suitable stem cells include embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, cardiac muscle stem cells, and mesenchymal stem cells.

[0240] For example, cell therapy may include administering one or more expression vectors described herein to cells (e.g., stem cells) in an ex vivo manner so that the cells express a dimer-inducing protein, and then administering the cells to a patient. After administering the cells expressing the dimer-inducing protein, a small molecule may be administered to the individual to induce dimerization of the first and second constituent polypeptides, reconstituting their function upon dimerization. For example, the first and second constituent polypeptides may dimerize to form a transcription factor, or the first and second constituent polypeptides may dimerize to form a CAR.

[0241] Disclosed are methods of treatment comprising administering to a patient cells expressing a dimer-inducing protein as defined herein, the method comprising: i) administering the cells to an individual; ii) administering the small molecule to the individual.

[0242] The dimer-inducing protein can be, for example, a split transcription factor, a split CAR, a split apoptotic protein, or a split therapeutic protein. The method of treatment can be a method of treating cancer.

[0243] Cell therapy can involve isolating cells from a patient, transfecting the cells ex vivo with one or more expression vectors, and administering the cells to the patient. A variety of cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994) and references cited therein for a description of methods for isolating and culturing cells from a patient).

[0244] For example, cell therapy may involve isolating cells from a patient, administering to the cells using an ex vivo method one or more expression vectors described herein such that the cells express a dimer-inducing protein, and re-administering the cells to the patient. After administering the cells expressing the dimer-inducing protein, a small molecule may be administered to the individual to induce dimerization of the first and second constituent polypeptides as described herein.

[0245] In one embodiment, the cell is an immune cell (such as a T cell), and the dimer-inducing protein expressed by the cell is a split CAR. Therapeutic methods for CAR T cell therapy are known in the art and are described, for example, in Miliotou and Papadopoulou, 2018.

[0246] Disclosed are methods of treatment comprising administering to such patients cells expressing a dimer-inducing protein as defined herein, wherein a first constituent polypeptide and a second constituent polypeptide dimerize to form a CAR, the method comprising: i) administering the cells to an individual; ii) administering the small molecule to the individual.

[0247] The method of treatment may be a method of treating cancer.

[0248] nucleic acid The present disclosure also provides nucleic acid molecules encoding binding members or dimer-inducing proteins as defined herein. The nucleic acid molecules may be isolated nucleic acid molecules. The nucleic acids encoding binding members and dimer-inducing proteins may have the required characteristics and sequence identity as described herein for expression vectors. Those skilled in the art will have no difficulty in preparing such nucleic acid molecules using methods known in the art.

[0249] In some embodiments, the nucleic acid molecule encodes the VH domain and / or VL domain of PRSIM_57, PRSIM_01, PRSIM_04, PRSIM_67, PRSIM_72, or PRSIM_75, the amino acid sequences of which VH or VL domains are defined herein.

[0250] In some embodiments, the nucleic acid molecule encodes a binding member of PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, PRSIM_47, PRSIM_57, PRSIM_01, PRSIM_04, PRSIM_67, PRSIM_72, or PRSIM_75, the amino acid sequences of which are defined herein.

[0251] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an exemplary nucleic acid sequence set forth in PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, PRSIM_47, PRSIM_57, PRSIM_01, PRSIM_04, PRSIM_67, PRSIM_72, or PRSIM_75. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, PRSIM_47, PRSIM_57, PRSIM_01, PRSIM_04, PRSIM_67, PRSIM_72, or PRSIM_75, the nucleic acid sequences of which exemplary binding members are set forth in the table below.

[0252] [Table 6]

[0253] In some embodiments, the nucleic acid molecule encodes a first and / or second constituent polypeptide, the amino acid sequences of which are defined herein, that is fused to a target protein or binding member as described above.

[0254] In some embodiments, the nucleic acid molecule encodes one or more of the DBD-T fusion proteins, TRD-BM fusion proteins, DBD-BM fusion proteins, and TRD-T fusion proteins described above, the amino acid sequences of which fusion proteins are defined herein.

[0255] In some embodiments, a nucleic acid molecule encoding a TRD-T fusion protein has a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 108. In some embodiments, a nucleic acid molecule encoding a TRD-T fusion protein has the nucleic acid sequence of SEQ ID NO: 108.

[0256] In some embodiments, a nucleic acid molecule encoding a DBD-T fusion protein has a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 109. In some embodiments, a nucleic acid molecule encoding a DBD-T fusion protein has the nucleic acid sequence of SEQ ID NO: 109.

[0257] In some embodiments, a nucleic acid molecule encoding a DBD-BM fusion protein has a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 110-120. In some embodiments, a nucleic acid molecule encoding a DBD-BM fusion protein has the nucleic acid sequence of any one of SEQ ID NOs: 110-120.

[0258] In some embodiments, a nucleic acid molecule encoding a TRD-BM fusion protein has a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 121-131. In some embodiments, a nucleic acid molecule encoding a TRD-BM fusion protein has the nucleic acid sequence of any one of SEQ ID NOs: 121-131.

[0259] In some embodiments, the nucleic acid molecule encodes a split CAR as defined herein. In some embodiments, the nucleic acid molecule encoding the split CAR has a nucleic acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 133 and the nucleic acid sequence encoding the antigen-specific recognition domain. In some embodiments, the nucleic acid molecule encoding the split CAR comprises a nucleic acid sequence encoding the antigen-specific recognition domain (e.g., scFv) located between positions 66 and 67, where the nucleotide numbering corresponds to SEQ ID NO: 133.

[0260] The isolated nucleic acid molecules may be used to express the binding members or dimer-inducing proteins disclosed herein. The nucleic acids will typically be provided in the form of one or more expression vectors, for example having the features of an expression vector described herein.

[0261] kit The present disclosure also provides kits comprising one or more expression vectors, one or more viral particles, cells, or one or more nucleic acids, all as defined herein, and a small molecule, also as defined herein. In some embodiments, the small molecule is simeprevir. When the one or more expression vectors or nucleic acids encode a polypeptide containing a DNA-binding domain derived from a CRISPR / Cas system, the kit may further comprise a guide RNA specific to the target sequence, or a nucleic acid encoding a guide RNA specific to the target sequence.

[0262] Sequence Identity and Variation Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG Package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty equal to 12 and a gap extension penalty equal to 4. While it may be preferable to use GAP, other algorithms may also be used, such as, for example, BLAST (using the method of Altschul et al. (1990)), FASTA (using the method of Pearson and Lipman (1988)), or the Smith-Waterman algorithm (Smith and Waterman (1981)), or the TBLASTN program of Altschul et al. (1990), supra, generally with default parameters. In particular, the psi-Blast algorithm may be used.

[0263] When the present disclosure refers to a particular amino acid sequence having at least 90% sequence identity to a reference amino acid sequence, it includes amino acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence identity to the reference amino acid sequence.

[0264] The term "sequence mutation," as used herein, is intended to encompass substitutions, deletions, and / or insertions of amino acid residues. Thus, a protein containing one or more amino acid sequence mutations compared to a reference sequence contains one or more substitutions, one or more deletions, and / or one or more insertions of amino acid residues compared to the reference sequence. The term "amino acid mutation" herein is also used interchangeably with "sequence mutation," unless the context clearly dictates otherwise.

[0265] In some embodiments where one or more amino acids are replaced with another amino acid, the substitutions may be conservative substitutions, for example, according to the following table: In some embodiments, amino acids in the same block in the middle column are replaced, i.e., a non-polar amino acid is replaced with another non-polar amino acid. In some embodiments, amino acids in the same row in the right-most column are replaced, i.e., a G is replaced with an A or a P.

[0266] [Table 7]

[0267] In some embodiments, substitutions may be functionally conservative substitutions, i.e., in some embodiments, the substitution may not affect (or not substantially affect) one or more functional properties (e.g., binding affinity) of the protein containing the substitution, compared to the equivalent unsubstituted protein.

[0268] Binding members may also comprise variants of the BC, DE or FG loops, Tn3, CDRs, VH domain, VL domain, and / or scFv sequence as disclosed herein. Suitable variants can be obtained by methods of sequence variation or mutation and screening. In preferred embodiments, binding members comprising one or more variant sequences retain one or more functional properties of the parent binding member, such as binding specificity and / or binding affinity for the T-SM complex. For example, binding members comprising one or more variant sequences preferably bind to the T-SM complex with the same affinity as, or higher affinity than, the (parent) binding member. A parent binding member is a binding member that does not contain the amino acid substitutions, deletions, and / or insertions incorporated into the variant binding member.

[0269] For example, a binding member may comprise a BC, DE or FG loop, Tn3, CDR, VH domain, VL domain, or scFv sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a BC, DE or FG loop, Tn3, CDR, VH domain, VL domain, or scFv sequence disclosed herein.

[0270] The binding member may comprise a BC, DE or FG loop, Tn3, CDR, VH domain, VL domain or scFv sequence that has one or more amino acid sequence mutations (additions, deletions, substitutions and / or insertions of amino acid residues) compared to the BC, DE or FG loop, Tn3, CDR, VH domain, VL domain or scFv sequence disclosed herein, preferably no more than 20 mutations, no more than 15 mutations, no more than 10 mutations, no more than 5 mutations, no more than 4 mutations, no more than 3 mutations, no more than 2 mutations or no more than 1 mutation. ***

[0271] The features disclosed in the above description, or the following claims, or the accompanying drawings are expressed in terms of their specific forms, or means for performing a disclosed function, or methods or processes for obtaining a disclosed result, and can be used to realize the disclosure in various of its forms, using such features individually or in any combination as appropriate.

[0272] While the present disclosure has been described in conjunction with the exemplary embodiments set forth above, many equivalent modifications and variations will become apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present disclosure set forth above are considered to be illustrative and not limiting. Various modifications may be made to the described embodiments without departing from the spirit and scope of the present disclosure.

[0273] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0274] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0275] Throughout this specification, including the claims which follow, unless the context otherwise requires, the words "comprise" and "include", and variations such as "comprises", "comprising", and "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0276] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value constitutes another embodiment by the use of "about." The term "about" in connection with numerical values ​​is optional and may mean, for example, ±10%. The present disclosure relates, for example, to the following: [1] i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; One or more expression vectors, wherein the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein. [2]

[0023] The one or more expression vectors according to [1], wherein the target protein is derived from a viral protease and the small molecule inhibitor is a viral protease inhibitor. [3] [2] The one or more expression vectors according to [2] above, wherein the viral protease is HCV NS3 / 4A protease or HIV protease. [4] The one or more expression vectors according to [3] above, wherein the viral protease is HCV NS3 / 4A protease. [5] 1. The one or more expression vectors of claim 1, wherein the small molecule is selected from the group consisting of simeprevir, boceprevir, telaprevir, asunaprevir, vaniprevir, voxilaprevir, glecaprevir, paritaprevir, and naraprevir, and optionally, the small molecule is selected from the group consisting of simeprevir, boceprevir, and telaprevir. [6] [5] One or more expression vectors according to [5], wherein the small molecule is simeprevir. [7] The one or more expression vectors according to any one of [1] to [6] above, wherein the target protein has an amino acid sequence that has at least 90% identity with SEQ ID NO:1. [8] The one or more expression vectors according to any one of [2] to [7] above, wherein the target protein has an attenuated activity compared to the protein from which it is derived, and optionally, the target protein has an attenuated viral activity compared to the viral protein from which it is derived. [9] [8] The one or more expression vectors described in [8], wherein the target protein comprises one or more amino acid mutations compared to the protein from which it is derived, and the one or more amino acid mutations attenuate the activity of the target protein, and optionally the target protein has attenuated viral activity compared to the viral protein from which it is derived.

[10] 10. The one or more expression vectors according to [9], wherein the target protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and the target protein contains amino acid mutations at one or more amino acids selected from positions 72, 96, 112, 114, 154, 160, and 164, wherein the numbering of the amino acids corresponds to SEQ ID NO: 1.

[11]

[10] The one or more expression vectors according to

[10] , wherein the target protein comprises an amino acid mutation at position 154, and optionally the amino acid mutation at position 154 is a mutation to alanine.

[12] The one or more expression vectors according to any one of [1] to

[11] above, wherein the target protein has the amino acid sequence set forth in SEQ ID NO:2.

[13]

[0023] The one or more expression vectors according to any one of [1] to

[11] , wherein the target protein has an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprises affinity-reducing amino acid mutations at one or more amino acids selected from positions 151 and 183, the numbering of the amino acids corresponding to SEQ ID NO: 1, and optionally, the amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine, and the amino acid mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine.

[14] the binding member binds to either the target protein alone and / or the small molecule alone, i) with at least 10-fold higher affinity, ii) with at least 50-fold higher affinity; iii) with at least 100-fold higher affinity, or iv) with at least 1000-fold higher affinity; One or more expression vectors according to any one of [1] to

[13] above, which bind to the T-SM complex.

[15] wherein the binding member is i) 500 nM, ii) 1 μM; iii) 10 μM; iv) 100 μM, or v) K greater than 1 mM D binds to the target protein or the small molecule with an affinity having a value Optionally, the affinity is measured using surface plasmon resonance.

[16] the binding member does not show significant binding to the target protein alone and / or the small molecule alone; or The one or more expression vectors according to any one of [1] to

[15] , wherein the binding member shows no or no detectable binding to the target protein alone and / or the small molecule alone.

[17] The one or more expression vectors described in any one of [1] to

[16] above, wherein the binding member specifically binds to the T-SM complex via an epitope that is present only in the T-SM complex and not present in the target protein alone or the small molecule alone.

[18] The one or more expression vectors according to any one of [1] to

[16] , wherein the formation of the T-SM complex induces a conformational change in the target protein, thereby resulting in the formation of the epitope to which the binding member specifically binds.

[19] wherein the binding member is i) 50 nM, ii) 25 nM; iii) 20 nM; iv) 15 nM, or v) K lower than 10 nM D binds to the T-SM complex with an affinity having a value Optionally, the affinity is measured using surface plasmon resonance.

[20] The one or more expression vectors according to any one of [1] to

[19] above, wherein the binding member is a Tn3 protein or an antibody molecule. [twenty one] 20. One or more expression vectors according to claim 20, wherein the binding member is a Tn3 protein. [twenty two] The Tn3 protein i) PRSIM_23, as set forth in SEQ ID NOs: 136, 137, and 138, respectively; ii) PRSIM_32, as set forth in SEQ ID NOs: 139, 140, and 141, respectively; iii) PRSIM_33, as set forth in SEQ ID NOs: 142, 143, and 144, respectively; iv) PRSIM_36, as set forth in SEQ ID NOs: 145, 146, and 147, respectively; or v) comprising the BC, DE and FG loops of PRSIM_47 as set forth in SEQ ID NOs: 148, 149 and 150, respectively; Optionally, the one or more expression vectors of

[21] , wherein the Tn3 protein comprises three, two, or one sequence mutation in the BC, DE, and / or EF loops. [twenty three] The Tn3 i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) One or more expression vectors according to

[22] above, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of PRSIM_47 set forth in SEQ ID NO: 9. [twenty four] The Tn3 i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) One or more expression vectors according to

[23] above, comprising the amino acid sequence of PRSIM_47 set forth in SEQ ID NO: 9. [twenty five]

[23] One or more expression vectors according to

[23] , wherein the Tn3 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of PRSIM_23 set forth in SEQ ID NO:5.

[26]

[24] One or more expression vectors according to

[24] , wherein the Tn3 comprises the amino acid sequence of PRSIM_23 set forth in SEQ ID NO: 5.

[27]

[20] One or more expression vectors according to

[20] , wherein the binding member is a single chain variable fragment (scFv).

[28] the scFv i) PRSIM_57, as set forth in SEQ ID NOs: 151, 152, 153, 154, 155 and 156, respectively; ii) PRSIM_01, as set forth in SEQ ID NOs: 151, 152, 198, 154, 155 and 156, respectively; iii) PRSIM_04, as set forth in SEQ ID NOs: 151, 152, 163, 154, 155 and 164, respectively; iv) PRSIM_67, as set forth in SEQ ID NOs: 165, 166, 167, 168, 169 and 170, respectively; v) PRSIM_72, as set forth in SEQ ID NOs: 171, 172, 173, 174, 175 and 176, respectively; or vi) heavy chain complementarity determining regions (HCDRs) 1 to 3 and light chain complementarity determining regions (LCDRs) of PRSIM_75 set forth in SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; The CDR sequences are defined according to the Kabat numbering scheme, Optionally, the one or more expression vectors of

[27] , wherein the scFv comprises three, two, or one sequence mutation in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[29] the scFv i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) One or more expression vectors according to

[28] above, comprising an amino acid sequence having at least 90% identity with the amino acid sequence of PRSIM_75 set forth in SEQ ID NO: 15.

[30] the scFv i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) One or more expression vectors according to

[29] above, comprising the amino acid sequence of PRSIM_75 set forth in SEQ ID NO: 15.

[31]

[29] One or more expression vectors according to

[29] , wherein the scFv comprises an amino acid sequence having at least 90% identity to the amino acid sequence of PRSIM_57 set forth in SEQ ID NO: 12.

[32]

[30] One or more expression vectors according to

[30] , wherein the scFv comprises the amino acid sequence of PRSIM_57 set forth in SEQ ID NO: 12.

[33] the target protein is fused to a first constituent polypeptide;

[0033] One or more expression vectors according to any one of [1] to

[32] above, wherein the binding member is fused to a second constituent polypeptide.

[34] The one or more expression vectors according to

[33] , wherein the one or more expression vectors encode a dimer-inducing protein.

[35] (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to the target protein to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain and is fused to the binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to the target protein to form a TRD-T fusion protein; the second component polypeptide comprises a DNA binding domain and is fused to the binding member to form a DBD-BM fusion protein;

[34] One or more expression vectors according to

[34] , wherein the first constituent polypeptide and the second constituent polypeptide form a transcription factor upon dimerization.

[36]

[35] One or more expression vectors according to

[35] , wherein the transcriptional regulatory domain is a transcriptional activation domain, or the transcriptional regulatory domain is a transcriptional repression domain.

[37] 36. The one or more expression vectors of claim 35 or 36, further comprising a third expression cassette encoding a desired expression product, wherein the DNA binding domain binds to a target sequence in the third expression cassette such that the transcription factor can regulate expression of the desired expression product, and optionally the target sequence is located in a promoter operably linked to a coding sequence of the desired expression product.

[38] 37. The one or more expression vectors of claim 37, wherein the desired expression product is a therapeutic protein, optionally wherein the therapeutic protein is a therapeutic antibody.

[39] the DBD-T fusion protein comprises the DNA-binding domain fused to two or more target proteins; or The one or more expression vectors according to any one of

[35] to

[38] , wherein the DBD-BM fusion protein comprises the DNA-binding domain fused to two or more binding members.

[40] (1) the first constituent polypeptide comprises a costimulatory domain and is fused to the target protein; the second component polypeptide comprises an intracellular signalling domain and is fused to the binding member; or (2) the first constituent polypeptide comprises an intracellular signaling domain and is fused to the target protein; 34. One or more expression vectors according to claim 34, wherein the second constituent polypeptide comprises a first costimulatory domain and is fused to the binding member.

[41] The first constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain; the second constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain; dimerization of the first and second constituent polypeptides to form a chimeric antigen receptor (CAR); Optionally, the target protein is fused to the C-terminus of the first costimulatory domain and / or the binding member is fused to the C-terminus of the second costimulatory domain.

[42] the first constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain; the second constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain; dimerization of the first and second constituent polypeptides to form a chimeric antigen receptor (CAR); Optionally, the binding member is fused to the C-terminus of the first costimulatory domain and / or the target protein is fused to the C-terminus of the second costimulatory domain.

[43] the first constituent polypeptide fused to the target protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70; the second component polypeptide fused to the binding member comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 200; Optionally, the antigen-specific recognition domain is located from the N-terminus to an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70.

[44] the first constituent polypeptide comprises a first caspase component; the second constituent polypeptide comprises a second caspase component; 34. The one or more expression vectors of claim 34, wherein the first and second constituent polypeptides form a caspase upon dimerization, and optionally the first and second caspase components comprise a caspase-9 activation domain.

[45] (1) the first and second expression cassettes are located on the same expression vector, and optionally the third expression cassette is located on the same expression vector or on a different expression vector; or (2) One or more expression vectors according to any one of [1] to

[44] , wherein the first expression cassette is located in a first expression vector, the second expression cassette is located in a second expression vector, and optionally, the third expression cassette is located in the first expression vector, the second expression vector, or a third expression vector.

[46] The one or more expression vectors according to any one of [1] to

[45] above, wherein each of the one or more expression vectors is a DNA plasmid.

[47] The one or more expression vectors according to any one of [1] to

[45] above, wherein each of the one or more expression vectors is a viral vector.

[48] 48. The one or more expression vectors according to claim 47, wherein the viral vector is selected from the list consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, and a picornavirus vector.

[49]

[48] ​​The one or more expression vectors according to

[48] , wherein the viral vector is an AAV vector.

[50] 1. A method for producing viral particles in vitro, comprising: transfecting a host cell with the viral vector according to any one of

[47] to

[49] above, and expressing a viral protein required for forming a viral particle in the host cell; and culturing the transfected cells in a medium such that the cells produce viral particles; Optionally, the method further comprises separating said viral particles from said medium and optionally concentrating said viral particles.

[51] a binding member that specifically binds to a complex between i) a target protein derived from a non-human protein and ii) a small molecule that is an inhibitor of said non-human protein, wherein said binding member binds to said complex with higher affinity than it binds to said target protein alone and / or said small molecule alone; Optionally, the non-human protein is a viral protease, optionally an HCV NS3 / 4A protease, further optionally, the viral protease has an amino acid sequence having at least 90% identity to SEQ ID NO:2; Further optionally, the binding member wherein said small molecule is simeprevir.

[52] the binding member is a Tn3 protein, and optionally the Tn3 protein comprises: i) PRSIM_23, as set forth in SEQ ID NOs: 136, 137, and 138, respectively; ii) PRSIM_32, as set forth in SEQ ID NOs: 139, 140, and 141, respectively; iii) PRSIM_33, as set forth in SEQ ID NOs: 142, 143, and 144, respectively; iv) PRSIM_36, as set forth in SEQ ID NOs: 145, 146, and 147, respectively; or v) comprising the BC, DE and FG loops of PRSIM_47 as set forth in SEQ ID NOs: 148, 149 and 150, respectively; Optionally, the Tn3 protein comprises three, two or one sequence mutation in the BC, DE and / or EF loops.

[53] The Tn3 i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) A binding member according to

[52] above, comprising an amino acid sequence which has at least 90% identity with the amino acid sequence of PRSIM_47 set forth in SEQ ID NO: 9.

[54] The Tn3 i) PRSIM_23 as set forth in SEQ ID NO: 5; ii) PRSIM_32 as set forth in SEQ ID NO: 6; iii) PRSIM_33 as set forth in SEQ ID NO: 7; iv) PRSIM_36 as set forth in SEQ ID NO: 8, or v) A binding member according to

[53] above, which comprises the amino acid sequence of PRSIM_47 as set forth in SEQ ID NO: 9.

[55] wherein the binding member is an scFv, and optionally the scFv comprises: i) PRSIM_57, as set forth in SEQ ID NOs: 151, 152, 153, 154, 155 and 156, respectively; ii) PRSIM_01, as set forth in SEQ ID NOs: 151, 152, 198, 154, 155 and 156, respectively; iii) PRSIM_04, as set forth in SEQ ID NOs: 151, 152, 163, 154, 155 and 164, respectively; iv) PRSIM_67, as set forth in SEQ ID NOs: 165, 166, 167, 168, 169 and 170, respectively; v) PRSIM_72, as set forth in SEQ ID NOs: 171, 172, 173, 174, 175 and 176, respectively; or vi) heavy chain complementarity determining regions (HCDRs) 1 to 3 and / or light chain complementarity determining regions (LCDRs) 1 to 3 of PRSIM_75 set forth in SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; The CDR sequences are defined according to the Kabat numbering scheme, Optionally, the binding member according to

[51] , wherein the scFv comprises 3, 2 or 1 sequence mutation in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3.

[56] i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) A binding member according to

[55] above, comprising an amino acid sequence which has at least 90% identity with the amino acid sequence of PRSIM_75 set forth in SEQ ID NO: 15.

[57] i) PRSIM_57 as set forth in SEQ ID NO: 12; ii) PRSIM_01 as set forth in SEQ ID NO: 10; iii) PRSIM_04 as set forth in SEQ ID NO: 11; iv) PRSIM_67 as set forth in SEQ ID NO: 13; v) PRSIM_72 as set forth in SEQ ID NO: 14, or vi) A binding member according to

[56] above, which comprises the amino acid sequence of PRSIM_75 as set forth in SEQ ID NO: 15.

[58] The binding member described in any one of

[51] to

[57] , wherein the binding member specifically binds to the T-SM by forming an interaction with at least one of the following residues of the target protein: Tyr71, Gly75, Thr76, Val93, Asp94, wherein the amino acid numbering corresponds to SEQ ID NO: 1, and optionally the binding member further forms an interaction with the quinolone moiety of simeprevir.

[59] A dimer-inducing protein, a first constituent polypeptide fused to a target protein; a second component polypeptide fused to a binding member; the target protein is capable of binding to a small molecule to form a complex (T-SM complex) between the target protein and the small molecule, and the binding member specifically binds to the T-SM complex such that it binds to the T-SM complex with higher affinity than it binds to both the target protein alone and / or the small molecule alone; 1. A dimer-inducing protein, wherein the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein, optionally wherein the non-human protein is a viral protease and the small molecule is a viral protease inhibitor.

[60] The dimer-inducing protein of

[59] , wherein the viral protease is HCV NS3 / 4A protease, and optionally, the viral protease has an amino acid sequence having at least 90% identity to SEQ ID NO:2.

[61] the small molecule is simeprevir; Optionally, the dimer-inducing protein according to

[59] or

[60] , wherein the target protein has an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 1 and comprises an amino acid mutation at one or more amino acids selected from positions 151 and 183 compared to SEQ ID NO: 1, wherein the numbering of the amino acids corresponds to SEQ ID NO: 1.

[62] The dimer-inducing protein according to any one of

[59] to

[61] above, wherein the binding member is as defined in any one of

[51] to

[58] above.

[63] (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to the target protein to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain and is fused to the binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein; the second component polypeptide comprises a DNA binding domain and is fused to the binding member to form a DBD-BM fusion protein; The dimer-inducing protein according to any one of

[59] to

[62] above, wherein the first constituent polypeptide and the second constituent polypeptide dimerize to form a transcription factor.

[64] the DBD-T fusion protein comprises the DNA-binding domain fused to two or more target proteins; or The dimer-inducing protein according to

[63] , wherein the DBD-BM fusion protein comprises the DNA-binding domain fused to two or more binding members.

[65] the DRD-T fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45; the TRD-BM fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 67; the DBD-BM fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46 to 56, and / or The dimer-inducing protein according to

[63] or

[64] , wherein the TRD-T fusion protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 44.

[66] (1) the first constituent polypeptide comprises a costimulatory domain and is fused to the target protein; the second component polypeptide comprises an intracellular signalling domain and is fused to the binding member; or (2) the first constituent polypeptide comprises an intracellular signaling domain and is fused to the target protein; The dimer-inducing protein according to any one of

[59] to

[62] , wherein the second constituent polypeptide comprises a first costimulatory domain and is fused to the binding member.

[67] The first constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain; the second constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain; dimerization of the first and second constituent polypeptides to form a chimeric antigen receptor (CAR); Optionally, the target protein is fused to the C-terminus of the first costimulatory domain and / or the binding member is fused to the C-terminus of the second costimulatory domain.

[66] (1) The dimer-inducing protein of claim 66.

[68] the first constituent polypeptide further comprises a transmembrane domain and a second costimulatory domain; the second constituent polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain; dimerization of the first and second constituent polypeptides to form a chimeric antigen receptor (CAR); Optionally, the binding member is fused to the C-terminus of the first costimulatory domain and / or the target protein is fused to the C-terminus of the second costimulatory domain.

[66] (2) A dimer-inducing protein according to the above.

[69] the first constituent polypeptide fused to the target protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 70; the second component polypeptide fused to the binding member comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 200; Optionally, the antigen-specific recognition domain is located from the N-terminus to an amino acid sequence having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 70. The dimer-inducing protein of

[67] .

[70] the first constituent polypeptide comprises a first caspase component; the second constituent polypeptide comprises a second caspase component; dimerization of the first and second constituent polypeptides to form a caspase; Optionally, the first and second caspase components comprise a caspase 9 activation domain.

[71] A cell expressing the dimer-inducing protein according to any one of

[59] to

[70] above.

[72] The cell according to

[71] above, wherein the cell is a stem cell or an immune cell.

[73] A method of genetically modifying a cell to produce the cell according to

[71] or

[72] , comprising administering to the cell one or more expression vectors according to any one of

[33] to

[44] , and optionally, the method being carried out in vitro or ex vivo.

[74] i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and / or the small molecule alone; the target protein is derived from a non-human protein and the small molecule is an inhibitor of the non-human protein, optionally the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor; the first and second expression cassettes form part of the viral genome in the one or more viral particles; Optionally, the one or more viral particles are AAV particles.

[75] 74. One or more viral particles according to claim 74, wherein the first and second expression cassettes form part of the same viral genome, or the first expression cassette forms part of a first viral genome of a first viral particle and the second expression cassette forms part of a second viral genome of a second viral particle.

[76] the viral protease is HCV NS3 / 4A protease, optionally wherein the viral protease has an amino acid sequence having at least 90% identity to SEQ ID NO: 1, and further optionally wherein the target protein has the amino acid sequence set forth in SEQ ID NO: 2; and / or

[74] or

[75] , wherein the small molecule is simeprevir.

[77]

[74] to

[76] . The one or more viral particles according to any one of

[74] to

[76] , wherein the binding member is as defined in any one of

[51] to

[56] .

[78] the target protein is fused to a first constituent polypeptide; the binding member is fused to a second constituent polypeptide; The one or more viral particles according to any one of

[74] to

[77] above, wherein the one or more expression vectors encode a dimer-inducing protein.

[79] (1) the first constituent polypeptide comprises a DNA-binding domain and is fused to the target protein to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain and is fused to the binding member to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to the target protein to form a TRD-T fusion protein; the second component polypeptide comprises a DNA binding domain and is fused to the binding member to form a DBD-BM fusion protein; dimerization of the first and second constituent polypeptides to form a transcription factor; optionally, further comprising a third expression cassette encoding a desired expression product, wherein said DNA binding domain binds to a target sequence within said third expression cassette such that said transcription factor can regulate expression of said desired expression product; 78. The one or more viral particles of claim 78, further optionally wherein the third expression cassette forms part of the same viral genome as the first and / or second expression cassette, or wherein the third expression cassette forms part of a third viral genome of a third viral particle.

[80] (1) the first constituent polypeptide comprises a first costimulatory domain, an antigen-specific recognition domain, and a transmembrane domain, and is fused to the target protein; the second component polypeptide comprises an intracellular signalling domain, a transmembrane domain and a second co-stimulatory domain and is fused to the binding member; or (2) the first constituent polypeptide comprises an intracellular signaling domain, a transmembrane domain, and a second costimulatory domain and is fused to the target protein; the second component polypeptide comprises a first costimulatory domain, an antigen-specific recognition domain, and a transmembrane domain, and is fused to the binding member; 78. The one or more viral particles according to claim 78, wherein the first and second constituent polypeptides dimerize to form a chimeric antigen receptor (CAR).

[81] One or more nucleic acids encoding the binding member or dimer-inducing protein according to any one of

[51] to

[70] above.

[82] One or more nucleic acids encoding the first and / or second constituent polypeptides fused to the target protein and / or the binding domain of the dimer-inducing protein according to any one of

[59] to

[70] .

[83] One or more expression vectors according to any one of [1] to

[49] above, for use in a method for treating a human or animal body.

[84] One or more virus particles according to any one of

[74] to

[80] above, for use in a method for treating the human or animal body.

[85] 1. A method for modulating expression of a desired expression product in a cell, comprising: i) expressing the dimer-inducing protein according to any one of

[63] to

[65] in the cell, wherein the DNA-binding domain binds to a target sequence in the cell so that the transcription factor can regulate the expression of the desired expression product in the cell; ii) administering said small molecule to said cell to modulate expression of said desired expression product; Optionally, the method comprises administering to the cell a third expression cassette encoding the desired expression product and comprising the target sequence, and further optionally, the target sequence is located in a promoter operably linked to the coding sequence of the desired expression product.

[86] The method according to

[85] , wherein the cells are part of a human patient and the method is carried out in vivo.

[87] The dimer-inducing protein according to any one of

[63] to

[65] above, which is used in a method for regulating expression of a desired expression product in a cell of a human or animal subject, wherein the first constituent polypeptide and the second constituent polypeptide form a transcription factor upon dimerization, wherein the method comprises: i) expressing the dimer-inducing protein according to any one of

[63] to

[65] in the cell, wherein the DNA-binding domain binds to a target sequence in the cell so that the transcription factor can regulate the expression of the desired expression product in the cell; ii) administering a small molecule to said cell to modulate expression of said desired expression product; Optionally, the method comprises administering to the cell a third expression cassette encoding the desired expression product and comprising the target sequence, and further optionally, the target sequence is located in a promoter operably linked to a coding sequence for the desired expression product of the dimeric-inducing protein.

[88] 1. A small molecule for use in a method of modulating expression of a desired expression product in a cell of a human or animal subject, said method comprising: i) expressing the dimer-inducing protein according to any one of

[63] to

[65] in the cell, wherein the DNA-binding domain binds to a target sequence in the cell so that the transcription factor can regulate the expression of the desired expression product in the cell; ii) administering said small molecule to said cell to modulate expression of said desired expression product; Optionally, the method comprises administering to the cell a third expression cassette encoding the desired expression product and comprising the target sequence, and further optionally, the target sequence is located in a promoter operably linked to the coding sequence of the desired expression product.

[89] The method according to

[85] or

[86] , the dimer-inducing protein used according to

[87] , or the small molecule used according to

[88] , wherein the dimer-inducing protein is expressed in the cell by administering to the cell one or more expression vectors according to

[35] to

[39] , or one or more viral particles according to

[79] .

[90] A therapeutic method comprising administering the cells according to

[71] or

[72] to an individual in need thereof, i) administering said cells to said individual; ii) administering said small molecule to said individual.

[91] The cell according to

[71] or

[72] above, used in a method for treating a human or animal body, wherein the method comprises: i) administering the cells to an individual; ii) administering said small molecule to said individual.

[92] 1. A small molecule for use in a method of treatment of the human or animal body, said method comprising: i) administering the cells according to

[71] or

[72] to an individual; ii) administering said small molecule to said individual.

[93] Use of the cells according to

[71] or

[72] for the manufacture of a medicament for treating a human or animal body, wherein the treatment comprises: i) administering the cells to an individual; ii) administering said small molecule to said individual.

[94] 1. Use of a small molecule for the manufacture of a medicament for treating the human or animal body, said treatment comprising: i) administering the cells according to

[71] or

[72] to an individual; ii) administering said small molecule to said individual.

[95] The method of

[90] , the cell for use according to

[91] , the small molecule for use according to

[92] , the use of the cell according to

[93] , or the use of the small molecule according to

[94] , wherein the cell is an immune cell, optionally the immune cell is a T cell.

[96] The method, cell for use, small molecule for use, use of the cell, or use of the small molecule according to

[95] , wherein the first constituent polypeptide and the second constituent polypeptide form a CAR upon dimerization.

[97] A kit comprising one or more expression vectors according to any one of [1] to

[49] above and the small molecule.

[98] A kit comprising the cells according to

[71] or

[72] and the small molecule.

[99] A kit comprising one or more virus particles according to any one of

[74] to

[80] above and the small molecule.

[0100] A kit comprising one or more nucleic acids according to

[81] or

[82] and the small molecule.

[0101] The kit according to any one of

[97] to

[0100] , wherein the small molecule is simeprevir.

[0102] i) a target protein capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone, A system wherein the target protein is a non-human protein and the small molecule is an inhibitor of the non-human protein, optionally wherein the non-human protein is derived from a viral protease and the small molecule is a viral protease inhibitor.

[0103] 1. A method of inducing degradation of a trimolecular complex, comprising administering to a cell containing said trimolecular complex a competing small molecule, the trimolecular complex is formed between a binding member that specifically binds to a complex formed by a target protein and a small molecule (a T-SM complex), and the binding member binds to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; The method wherein the competitor small molecule binds to the target protein in the T-SM complex and is capable of displacing the small molecule from the T-SM complex.

[0104] The method described in

[0103] above, wherein the cells are part of a human patient and the method is performed in vivo.

[0105] 1. A competitor small molecule for use in a method of inducing degradation of a trimolecular complex in a human body, said method comprising administering said competitor small molecule to a cell containing said trimolecular complex; the trimolecular complex is formed between a binding member that specifically binds to a complex of a target protein and a small molecule (a T-SM complex), the binding member binding to the T-SM complex with higher affinity than it binds to both the target protein alone and the small molecule alone; The competitor small molecule is capable of binding to the target protein in the T-SM complex and displacing the small molecule from the T-SM complex.

[0106] The method of any one of

[0103] and

[0104] , or the competitive small molecule used in accordance with

[0105] , wherein the target protein has an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1, and the small molecule is simeprevir.

[0107] The method or competitive small molecule used according to

[0106] above, wherein the target protein comprises affinity-reducing amino acid mutations at one or more amino acids selected from positions 151 and 183, the numbering of the amino acids corresponding to SEQ ID NO: 1, and optionally, the amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine, and the amino acid mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine.

[0108] The method or competitive small molecule used according to any one of

[0103] to

[0107] above, wherein the competitive small molecule is selected from the list consisting of asunaprevir, paritaprevir, vaniprevir, grazoprevir, danoprevir, and glecaprevir.

[0109] The method or competitor small molecule used in accordance with any one of

[0103] to

[0108] above, wherein the target protein and the binding member form part of a dimer-inducing protein, and optionally the dimer-inducing protein is as defined in any one of

[59] to

[70] above.

[0110] A target protein derived from HCV NS3 / 4A protease, wherein the target protein has an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 1 and contains amino acid mutations at one or more amino acids selected from positions 151 and 183 compared to SEQ ID NO: 1, wherein the amino acid numbering corresponds to SEQ ID NO: 1, and simeprevir is capable of binding to the target protein.

[0111] The target protein described in

[0110] above, wherein the amino acid mutation at position 151 is a mutation to aspartic acid, asparagine, or histidine, and the amino acid mutation at position 183 is a mutation to glutamic acid, glutamine, or alanine.

[0112] The target protein of

[0110] or

[0111] , wherein the target protein further comprises an amino acid mutation at position 154 compared to SEQ ID NO: 1, and optionally, the amino acid mutation at position 154 is a mutation to alanine.

[0113] The target protein according to any one of

[0110] to

[0112] , wherein the target protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 211, 213, and 215. [Example]

[0277] Example 1 - Materials and Methods Solvent-accessible surface area calculation The solvent-accessible surface area (SASA) of simeprevir was calculated using Visual Molecular Dynamics (VMD) software (University of Illinois at Urbana-Champaign) incorporating the measure_sasa command from the three-dimensional structure of the HCV NS3 / 4A PR:simeprevir complex available from the Protein Data Bank (PDB; http: / / www.rcsb.org / ) under PDB code 3KEE. The -restrict option and a radius of 1.4 Å were used to calculate the surface of simeprevir not bound to HCV NS3 / 4A PR, i.e., the solvent-accessible surface area.

[0278] Generation of biotinylated HCV NS3 / 4A protease The sequence used to design the HCV NS3 / 4A PR construct was derived from Uniprot entry A8DG50 (the genomic polyprotein of hepatitis C virus subtype 1a) with additional modifications from U.S. Patent No. 6,800,456. The protease domain corresponds to residues 1030–1206 of the polyprotein. A single chain consisting of an 11-residue peptide derived from the viral NS4A protein fused to the N-terminus of the NS3 protease (SEQ ID NO: 1) was used to generate a fully folded and activated polypeptide. This sequence (SEQ ID NO: 3) with an N-terminal hexahistidine (6His) and AviTag (enabling affinity purification and biotinylation, respectively) was purchased as a linear DNA string (GeneArt). In parallel, a DNA string encoding a sequence equivalent to the active site mutation S139A (SEQ ID NO: 4) was ordered. The DNA string was cloned into the pET-28a vector (for bacterial expression) using Gibson assembly. A second set of DNA strings was ordered, encoding human codon-optimized versions of His- and Avitag-tagged WT and S139 proteases, and these were cloned into mammalian expression vectors containing a CMV promoter. The sequence of the final construct was confirmed by Sanger sequencing of the entire coding sequence.

[0279] For bacterial expression, pET-28a plasmids were transformed into BL21(DE3) Escherichia coli (E. coli) cells and selected on kanamycin (50 μg / ml)-containing plates. For each expression, a single colony was used to inoculate 5 ml of 2xTY + 50 μg / ml kanamycin culture, which was grown overnight at 37°C. This culture was used to inoculate 500 ml of TB autoinduction medium (Formedium, supplemented with 10 ml / L glycerol and 100 μg / ml kanamycin) at a 1:500 dilution. The culture was grown at 37°C until the OD600 reached 1.3-1.5, then transferred to 20°C to induce expression for 20 hours. Cells were harvested by centrifugation, and the pellets were stored at -80°C.

[0280] For mammalian expression, plasmid DNA was prepared using the Qiagen Plasmid Plus Gigaprep kit. 6 Gigaprep DNA was transfected into Expi293F cells (ThermoFisher) by PEI-mediated delivery at a density of 1000 cells / mL and cultured in FreeStyle293 medium (ThermoFisher). Cells were cultured at 37°C, 5% CO2, 140 rpm, and 70% humidity for 6 days. Cells were harvested at 4,000g, and pellets were stored at -80°C.

[0281] To purify proteins, bacterial pellets from each 500 ml culture were thawed and resuspended in 50 ml of lysis buffer (2x DPBS, 200 mM NaCl, pH 7.4). Cells were lysed using a probe sonicator, and the lysate was clarified by centrifugation at 50,000 g for 40 min at 4°C. Mammalian cell pellets were lysed by resuspending them in detergent-containing lysis buffer (2x DPBS, 200 mM NaCl, 1 mM TCEP, EDTA-free protease inhibitor cOmplete, 25 U / ml Turbonuclease, and 1% Triton X-100, pH 7.4) and rotating at 10 rpm for 2 h at 4°C. Mammalian lysed samples were centrifuged at 50,000 g for 30 min at 4°C. All samples were filtered through a 0.22 μm bottle-top filter prior to column chromatography. The filtered supernatant was loaded onto a 5 ml HisTrap HP column (GE Healthcare) at a flow rate of 5 ml / min. The column was washed with 100 ml of wash buffer (2x DPBS, 200 mM additional NaCl, 20 mM imidazole, pH 7.4) and eluted with an imidazole gradient over 5 column volumes from 20 to 400 mM imidazole. Fractions were analyzed by SDS-PAGE, concentrated to pool the appropriate protein, and buffer exchanged into lysis buffer (2x DPBS, 200 mM NaCl, pH 7.4) using a HiPrep 26 / 10 desalting column (GE Healthcare). The desalted protein fractions were pooled, concentrated using a centrifugal concentrator, and purified on a HiLoad Superdex 75 26 / 600 pg column (GE Healthcare) equilibrated in 2x DPBS, 2 mM DTT, and 10 μM ZnCl2. Fractions were analyzed by SDS-PAGE, and fractions with purity greater than 95% were pooled, their concentrations measured by ultraviolet absorbance, and flash-frozen in liquid nitrogen before storage at −70° C. Final sample purity was confirmed by RP-HPLC on an XBridge BEH300, C4 (Waters).

[0282] The purified protein was biotinylated on the Avitag using MBP-tagged BirA enzyme, which was incubated with the sample for 2.5 hours at 22°C in the presence of ATP and biotin. Biotinylated proteins were purified by size-exclusion chromatography on a HiLoad Superdex 75 16 / 600 pg column (GE Healthcare) in 2x DPBS, 2 mM DTT, and 1 μM ZnCl. Fractions were analyzed by SDS-PAGE, and protease-containing fractions were pooled. The extent of biotinylation was confirmed by intact mass spectrometry on a Xevo G2-CS MS (Waters). The biotinylated protein was divided into aliquots, flash-frozen in liquid nitrogen, and stored at -70°C.

[0283] To generate a His- and Avitag-tagged NS3 / 4A S139A protease with additional mutations to reduce affinity for simeprevir, pET-28a, derived from the protease-encoding plasmid, was used as a template for site-directed mutagenesis using the Quikchange Lightning Site-Directed Mutagenesis Kit. The mutant forms of the protease construct were confirmed by Sanger sequencing of the entire coding sequence prior to expression. To overexpress BirA biotin-protein ligase with IPTG induction, the mutant proteins were transformed into a BL21(DE3) Escherichia coli (E. coli) derivative strain with the plasmid, allowing biotinylation during bacterial expression. An overnight culture was used to inoculate 50 ml of 2xTY + 50 μg / ml kanamycin at a 1:20 dilution. The culture was grown at 37°C to an OD600 of 0.6, then supplemented with 50 μM biotin and induced with 1 mM IPTG. The induced cultures were shifted to 25°C and expression was allowed to proceed for 20 hours. Cells were harvested by centrifugation, and the pellets were stored at -20°C. For purification, each pellet was resuspended in 20 ml of lysis buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, EDTA-free protease inhibitor cOmplete) and lysed by passage through a cell disruptor (Constant Systems) at 40,000 kpsi. Protein was purified using an automated two-step IMAC procedure, followed by buffer exchange on a desalting column. The sample was loaded onto the IMAC resin once, washed with lysis buffer supplemented with 20 mM imidazole, and eluted with a buffer containing 400 mM imidazole. The eluate was automatically captured and loaded onto a desalting column equilibrated with 50 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.5. The final protein sample was divided into aliquots, flash frozen in liquid nitrogen and stored at -70°C.

[0284] HCV NS3 / 4A PR protease activity assay To assess enzymatic activity, we measured the cleavage of a fluorogenic HCV protease FRET substrate bearing the EDANS-DABCYL donor-quencher pair using purified HCV NS3 / 4A PR and the S139A mutant (RET S1, AnaSpec). When in close proximity (10-100 Å), as in the case of the intact peptide, EDANS is excited at 340 nm, and the energy emitted from EDANS is quenched by DABCYL (at 490 nm). Cleavage of the peptide by HCV NS3 / 4A PR separates DABCYL from EDANS, allowing detection of fluorescence at 490 nm.

[0285] Serial dilutions of HCV NS3 / 4A PR and the active site mutant S139A in assay buffer (HEPES (pH 7.8), 5 mM DTT, 100 mM NaCl, 10% glycerol, 0.01% CHAPS) were incubated with fluorogenic substrate at room temperature. Fluorescence was measured after 3 hours using a PerkinElmer Envision plate reader (excitation 340 nm, emission 490 nm).

[0286] Isothermal calorimetry Isothermal calorimetry (ITC) was performed using an Auto-ITC 200 (Malvern) with a 0.4 μl pre-injection followed by 19 injections of 2 μl each at 120-second intervals. The solution rotation was set at 750 rpm, and the temperature was set at 37°C. Simeprevir (125 μM) was titrated into HCV NS3 / 4A PR (8 μM for WT and 8.2 μM for S139A mutant) or protein buffer (control). The protein buffer was concentrated with 2.5% DMSO to equalize the amount present in the simeprevir solution. Single replicates were run for WT and duplicates for the S139A mutant. Data were analyzed with ITC-PEAQ software (Malvern) using a single-site binding model and point-by-point reference subtraction.

[0287] Phage display selection scFv and Tn3 sequences were isolated from phage display selection using three phage display libraries: (i) Library 1, a Tn3 library based on the third FnIII module in human tenascin-C and developed as an alternative scaffold to FnIII ((Leahy et al. 1992), (Oganesyan et al. 2013), (Gilbreth et al. 2014)), (ii) Library 2, a restricted framework scFv library, and (iii) Library 3, a naive scFv library.

[0288] All phage selections were performed according to previously established protocols (Vaughan et al. 1996, Swers et al. 2013). Phage display selections were performed using biotinylated HCV NS3 / 4A PR (S139A) captured on streptavidin-coated magnetic beads (Promega). A total of four rounds of phage display selection were performed for each phage library using decreasing concentrations of biotinylated HCV NS3 / 4A PR and simeprevir (Figures 4A and 4B).

[0289] To ensure protease saturation, biotinylated HCV NS3 / 4A PR(S139A) antigen was preincubated with a 50-fold molar excess of simeprevir before selection began. Before each selection, the phage pool was incubated with streptavidin beads alone to remove any binding to streptavidin beads from the library. In rounds 1 and 2 of phage display selection, the biotinylated HCV NS3 / 4A PR(S139A) deselection step was not performed in the absence of simeprevir. However, in rounds 3 and 4, parallel selections were performed: one arm did not undergo the biotinylated HCV NS3 / 4A PR(S139A) deselection step, and the other arm preincubated phage particles with 250 nM biotinylated HCV NS3 / 4A PR(S139A) at room temperature for 15 minutes, followed by protease removal using streptavidin-coated beads. The resulting phage was then added to biotinylated HCV NS3 / 4A PR(S139A) coated on streptavidin beads in the presence of simeprevir for the selection protocol.

[0290] In each round, phage display selection was performed using the following concentrations of biotinylated HCV NS3 / 4A PR(S139A): Round 1: 250 nM biotinylated HCV NS3 / 4A PR(S139A) + 12.5 μM simeprevir Round 2: 100 nM biotinylated HCV NS3 / 4A PR(S139A) + 5 μM simeprevir Round 3: 25 nM biotinylated HCV NS3 / 4A PR(S139A) + 1.25 μM simeprevir Round 4: 25 nM biotinylated HCV NS3 / 4A PR(S139A) + 1.25 μM simeprevir.

[0291] The complex-bound phage were incubated with biotinylated HCV NS3 / 4A PR(S139A) in the presence of simeprevir, washed three times with D-PBS (Sigma), and then eluted with trypsin. The eluted phage were used to infect mid-logarithmic phage cultures of E. coli TG1 cells and plated on agar plates containing 100 μg / ml ampicillin and 2% (w / v) glucose.

[0292] Individual phage clones from rounds 3 and 4 were selected for DNA sequencing and screened for antigen binding by phage ELISA. The DNA sequence information is shown in Table 1.

[0293] Phage rescue Specific binding to HCV NS3 / 4A PR(S139A) was assessed by phage ELISA using single phagemid scFv or Tn3 clones induced for expression as described (Osbourn et al. 1996). Briefly, individual TG1 colonies encoding phage clones from round 3 and round 4 selection outputs, as well as negative control clones, were grown to logarithmic phase in 96-well plates at 37°C with shaking at 280 rpm in medium containing 100 μg / ml ampicillin and 2% (w / v) glucose. Helper phage was then added to each well, and the plates were incubated at 37°C for 1 hour with shaking at 150 rpm. The plates were then centrifuged at 4500 rpm for 10 minutes at room temperature, and the medium was removed and replaced with medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin. The plates were then incubated overnight at 25°C with shaking at 280 rpm. The next day, phage preps were blocked by adding an equal volume of 2x PBS containing 6% (w / v) nonfat dry milk (Marvel) to each well of the plate.

[0294] Phage ELISA Biotinylated HCV NS3 / 4A PR(S139A) was used to coat a 96-well streptavidin-coated plate at 5 μg / ml (1.875 μM) in the presence or absence of a threefold excess of simeprevir (5.6 μM). The coated plate was washed with PBS and blocked for 1 hour with PBS containing 3% (w / v) nonfat dry milk (Marvel). After this blocking step, the plate was washed three times with PBS, followed by the addition of blocked phage preps (generated as described in the phage rescue section). The phage preps were incubated with antigen for 1 hour at room temperature and then washed three times with PBS / Tween 20 (0.1% v / v). Phage specifically bound to the antigen-coated plate was detected using an anti-M13 phage-HRP-tagged antibody (GE Healthcare), followed by detection using 3,3',5,5'-tetramethylbenzidine (TMB; Sigma). The detection reaction was stopped using 0.5 M H2SO4, and the plate was read at 450 nm using a fluorescence plate reader. The fluorescence readout measured for each clone binding to biotinylated HCV NS3 / 4A PR(S139A) in the presence of simeprevir was compared to that of clones binding in the absence of simeprevir by dividing the signal observed in the presence of simeprevir by the signal observed in the absence of simeprevir. These data were plotted graphically (Figure 4B). In these data, the panel of scFv and Tn3 clones is designated PRSIM_xx, where xx denotes the clone number selected for further investigation. The selected clones had unique DNA sequences and showed no binding to HCV NS3 / 4A PR(S139A) in the absence of simeprevir, as measured by phage ELISA (except for the controls PRSIM 51, PRSIM 54, PRSIM 55, and PRSIM 85, which showed binding to HCV NS3 / 4A PR(S139A) in both the presence and absence of simeprevir).

[0295] Expression of scFv and Tn3 PRSIM binding molecules The scFv and Tn3 PRSIM binding molecules were purified from E. coli using a previous method (Vaughan et al., 1996) using nickel chelate chromatography followed by size-exclusion chromatography. To increase the expression levels of the most promising Tn3 PRSIM binding molecules, the encoding DNA sequences were subcloned into the pET16b vector using oligonucleotides Tn3_pETFwd2 (5'-CGATCATATGGACTACAAGGACGACGATGACAAGGGCAGCCGTCTGGATGCACCGAGCCAG-3' (SEQ ID NO: 183)) and Tn3_pETRev2 (5'-ATCGGGATCCCTACAGACCGGTTTTAAAGGTAATTTTTGCCGG-3' (SEQ ID NO: 184)) and expressed in the cytoplasm of BL21(DE3) E. coli (New England Biolabs). After dissolution in BugBuster plus Benzonase (EMD Millipore), Tn3-based PRSIM-binding molecules were purified to homogenization using nickel chelate chromatography followed by size-exclusion chromatography to yield monomeric proteins in PBS (pH 6.5).

[0296] Binding screening by homogeneous time-resolved fluorescence (HTRF) HCV NS3 / 4A PR(S139A)-selective scFv and Tn3 PRSIM binding molecules were identified in a homogeneous time-resolved fluorescence (HTRF®) assay performed in parallel with measurements of binding in the presence and absence of simeprevir. Serial dilutions of HCV NS3 / 4A PR(S139A) and purified PRSIM binding molecules were prepared in assay buffer (PBS containing 0.4 M potassium fluoride and 0.1% BSA). Streptavidin cryptate (Cisbio) was premixed in assay buffer with either anti-FLAG XL665 (detecting Tn3 molecules) or anti-c-myc XL665 (detecting scFv molecules). For each assay, 2.5 μl of sample was added dropwise to 2.5 μl of HCV NS3 / 4A PR(S139A) and 2.5 μl of premixed detection reagent. Additionally, either 2.5 μl of simeprevir or 2.5 μl of DMSO blank was added to each well. The background was defined by using wells without any sample. The assay plate was incubated overnight at 4°C, after which time-resolved fluorescence was read at emission wavelengths of 620 nm and 665 nm using a PerkinElmer Envision plate reader. Data were analyzed by calculating the % delta F value for each sample. Delta F was calculated according to Equation 1: Formula 1: % Delta F = ((665 nm / 620 nm ratio value of sample) - (665 nm / 620 nm ratio value of background) / (665 nm / 620 nm ratio value of background)) x 100

[0297] Selective binding molecules are defined as scFv and Tn3 PRSIM binding molecules that bind to HCV NS3 / 4A PR(S139A) in complex with simeprevir, but not to HCV NS3 / 4A PR(S139A).

[0298] Binding kinetics analysis The affinities of the scFv and Tn3 PRSIM binding molecules were measured using a Biacore 8K (GE Healthcare) at 25° C. The scFv and Tn3 PRSIM binding molecules were covalently immobilized onto a CM5 chip surface at a concentration of 1 μg / ml in 10 mM sodium acetate (pH 4.5) using standard amine coupling techniques.

[0299] HCV NS3 / 4A PR (S139A) or BSA control was diluted 1:4 (1.25–20 nM) ± 10 nM simeprevir in 10 mM Hepes (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, and 0.01% DMSO to ensure constant concentrations of simeprevir and DMSO. Samples were flowed over the chip at 50 μl / min using single-cycle kinetics with binding time of 120 s and dissociation time of 600 s. The chip surface was regenerated with two 20-s pulses of 10 mM glycine-HCl (pH 3.0). The final sensorgrams were analyzed using Biacore 8K Evaluation Software, and the affinity constant KD was calculated using a 1:1 binding model. The same method, with minor deviations, was used to measure the affinity of HCV NS3 / 4A PR mutants in PRSIM_23. The variants were diluted 1:4 (2.5–40 nM) ± simeprevir in 10 mM Hepes (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, and 0.08% DMSO to ensure constant concentrations of simeprevir and DMSO. Samples were flowed over the chip at 50 μl / min using single-cycle kinetics with binding at 180 s and dissociation at 600 s.

[0300] The effect of simeprevir concentration on the formation of HCV NS3 / 4A PR(S139A) / PRSIM binding complexes was also measured using a Biacore 8K. As before, PRSIM_57 and PRSIM_23 were covalently immobilized on a CM5 chip surface. Simeprevir was diluted 1:2 (0.0152–300 nM) in 10 mM Hepes (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, and 0.3% DMSO at a constant HCV NS3 / 4A PR(S139A) concentration of 40 nM. Samples were flowed over the chip at 50 μl / min using multicycle kinetics with 240 s binding and 600 s dissociation. Regeneration conditions were as described above. A titration curve for induction of HCV NS3 / 4A PR(S139A) / PRSIM dimerization by simeprevir was generated. The response of each simeprevir concentration at 225 seconds (15 seconds before binding ceased) was normalized as a percentage of the response of 300 nM simeprevir at 225 seconds and plotted against simeprevir concentration. Each data point represents the mean ± sem of three independent experiments. Nonlinear regression curve fitting was used to calculate the recorded EC 50 The same method was used for mutant HCV NS3 / 4A proteases, except that the concentration of 40 nM HCV NS3 / 4A PR(S139A) was kept constant and simeprevir was diluted 1:2 (0.0457–900 nM or 0.412–8,100 nM) in 10 mM Hepes (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, and 0.82% DMSO. Responses for each simeprevir concentration were normalized to the highest simeprevir concentration.

[0301] The affinity of simeprevir was measured at 25°C using an Octet RED384 (ForteBio). Biotinylated HCV NS3 / 4A PR (S139A), HCV NS3 / 4A K136D PR, HCV NS3 / 4A K136N PR, and HCV NS3 / 4A D168E PR were loaded onto a High Precision Streptavidin (SAX) biosensor at a concentration of 2 μg / ml in 10 mM Hepes (pH 7.4), 150 mM NaCl, 0.05% Surfactant P20, and 0.3% DMSO. Simeprevir was diluted 1:1 in the same buffer (46.88–3,000 nM), and binding was measured by immersing the loaded biosensor in the simeprevir sample for 180 seconds. For dissociation, the biosensor was immersed in buffer for 600 seconds. The traces were analyzed using ForteBio Data Analysis software and globally fitted using a 1:1 binding model.

[0302] Split NanoLuc reconstitution assay The ability of the PRSIM binding molecule to promote dimerization of two fused proteins was assessed using the NanoBiT system (Promega), which reconstitutes split Nanoluciferase (NanoLuc) and measures the luminescence generated upon application of the Nano-Glo NanoLuc substrate for cell imaging (Figure 8). In the NanoBiT system, one interacting partner is fused via a flexible linker to an 18 kDa fragment of NanoLuc termed LgBiT ("large bit") (SEQ ID NO: 16), and the other interacting partner is fused via an equivalent linker to a 1.3 kDa peptide, SmBiT ("small bit") (SEQ ID NO: 17). LgBiT and SmBiT have low affinity for each other (190 μM) in the absence of their interacting partners and do not reconstitute to form an active luciferase enzyme. When fused to a CID interacting protein and provided with an inducer, they reconstitute, allowing luminescence to be measured. The NanoBiT system provides two sets of regulatory proteins fused to LgBiT and SmBiT: the constitutively interacting PRKAR2A:PRKACA set, and the FRB:FKBP12 pair, whose dimerization can be induced by rapamycin.

[0303] To confirm the optimal orientation of the HCV NS3 / 4A PR(S139A) and PRSIM components, constructs were created in which HCV NS3 / 4A PR(S139A) was fused to either the N- or C-terminus of SmBiT (SEQ ID NOs: 18 and 19, respectively), and a similar set of constructs for each PRSIM binding module was fused to either the N- or C-terminus of LgBiT (SEQ ID NOs: 20-30 and 31-41, respectively). The NanoBiT kit (Promega) provides a set of vectors capable of generating these constructs. DNA strings encoding HCV NS3 / 4A PR(S139A) and PRSIM molecules were purchased from GeneArt, amplified by PCR using primers with extensions containing restriction sites compatible with the NanoBiT vector, and cloned by Gibson assembly. All constructs were verified by Sanger sequencing of the entire coding sequence.

[0304] All NanoBiT screens were performed on adherent HEK293 cells cultured in 96-well plates. Cells were enzymatically dissociated from tissue culture flasks and counted to a concentration of 2 × 10 4 Cells were plated at 1000 ng / well in white, opaque-bottom 96-well plates (Costar 3917). Plates were incubated overnight at 37°C in 5% CO2 to allow cells to adhere. On day 2, plasmids were co-transfected at a final concentration of 100 ng / well using Lipofectamine LTX (ThermoFisher) (50 ng / plasmid, one encoding an SmBiT fusion and the other encoding an LgBiT fusion). On day 3, wells were treated with 100 nM of the appropriate small molecule inducer (rapamycin (FRB:FKBP12) or simeprevir (HCV NS3 / 4A PR:PRSIM)) or solvent control, and luminescence was quantified on an Envision plate reader immediately after the addition of Nano-Glo Live Cell Substrate (Promega).

[0305] Transcriptional regulation assay We used the iDimerize regulated transcription system (Takara Bio Inc.) to test the ability of PRSIM-based CID to regulate gene expression. This system is based on the reconstitution of a split transcription factor, in which the DNA-binding domain (DBD) and activation domain (AD) are separated to prevent transcription. The DBD and AD are separately fused to the two protein components of the CID, such that only in the presence of a small-molecule inducer does the AD come into proximity with the DBD, allowing the transcription machinery to be recruited to promoters containing DBD recognition sites. The iDimerize regulated transcription system (Takara Bio Inc.) provides two vectors: pHet-Act1-2 and pZFHD1-luciferase. The pHet-Act1-2 vector encodes two fusion proteins, representing a positive control: one consisting of a fusion of FRB (T82L mutant; DmrC) with the activation domain (AD) from human p65 (SEQ ID NO: 42), and the other consisting of the DNA-binding domain (ZFHD1) (SEQ ID NO: 43) fused to three tandem copies of the FKBP12 (DmrA) DNA-binding domain. These sequences are placed in front of a CMV promoter and separated by an internal ribosome entry site (IRES). The ZFHD1 vector encodes luciferase in front of an inducible promoter consisting of 12 copies of the recognition sequence for the ZFHD1 DBD upstream of a minimal IL-2 promoter. Upon binding of the DBD to its recognition sequence and recruitment of the transcription machinery by the AD, transcription of the luciferase reporter gene is initiated. The DNA sequence encoding HCV NS3 / 4A PR(S139A) was purchased as a DNA string from GeneArt and cloned into the pHet-Act1-2 vector as either the N-terminal fusion partner to the activation domain (SEQ ID NO: 44) (replacing FRB) or the C-terminal fusion partner to the DNA-binding domain (SEQ ID NO: 45) (replacing FKBP12), with flexible linkers (TGGGGSGGGGS (SEQ ID NO: 185) and SA, respectively) between the fusion partners.Subsequently, sequences encoding one copy of the panel of 12 PRSIM molecules (Table 2) were purchased as DNA strings from GeneArt and cloned using Gibson assembly into the pHetAct1-2 construct containing the HCV NS3 / 4A PR(S139A) described above as a fusion partner to either the DBD (SEQ ID NOS: 46-56) or AD (SEQ ID NOS: 57-67), respectively. Equivalent constructs were generated, replacing the three copies of FKBP12 in pHet-Act1-2 with a single copy of FKBP12. The sequences of the constructs encoding both the activation domain and DNA-binding domain fusion proteins were confirmed by Sanger sequencing of the entire coding region.

[0306] The DNA sequence (SEQ ID NO: 68) encoding NanoLuc-PEST (Promega) was purchased as a DNA string from GeneArt and cloned downstream of the ZFHD1 inducible promoter of the pZFHD1-2 vector (Takara Bio Inc.) using Gibson assembly cloning. The nucleotide sequence of the final construct was confirmed by sequencing.

[0307] The DNA sequence encoding MEDI8852 (SEQ ID NO:237 and SEQ ID NO:238, separated by an internal ribosome entry site (IRES) sequence) was purchased as a DNA string from GeneArt and cloned downstream of the ZFHD1-inducible promoter in the pZFHD1-2 vector (Takara Bio Inc.) using Gibson assembly cloning. The nucleotide sequence of the final construct was confirmed by sequencing.

[0308] The sequences encoding the three HCV NS3 / 4A PR(S139A) mutants (Table 6) were purchased as DNA strings from GeneArt and cloned using Gibson assembly into the pHetAct1-2 HCV NS3 / 4A PR(S139A)-PRSIM_23 (three tandem copies) construct described above as fusion partners for AD (SEQ ID NOs: 211-216).

[0309] All transcriptional regulation assays were performed on adherent HEK293 cells cultured in 384-well plates. Cells were enzymatically dissociated from tissue culture flasks and counted to obtain 7.5 × 10 3 Cells were seeded at 1000 cells / well in a 384-well plate. The plate was incubated overnight at 37°C in 5% CO2 to allow cells to adhere. On day 2, cells were co-transfected with the pHet-Act1-2 plasmid (containing the FRB:FKBP12 control fusion protein (Clontech) or the HCV NS3 / 4A PR(S139A):PRSIM fusion protein) and the pZFHD1 plasmid (encoding either luciferase (Clontech) or NanoLuc-PEST (as described above)) using Lipofectamine LTX (ThermoFisher). On day 3, wells were treated with different concentrations of either the A / C heterodimerizer (for the FRB:FKBP12 control), simeprevir, or a solvent control. 24 hours later, luminescence was quantified on an Envision plate reader immediately after the addition of SteadyGlo luciferase substrate (Promega) or Nano-Glo Vivazine luciferase substrate (Promega). Alternatively, reverse transfection was performed on day 1, dimerizer was added on day 2, and luminescence was quantified 24 hours later on day 3.

[0310] Luminescence readouts were converted to fold change by dividing the signal in the presence of simeprevir by the signal in the absence of simeprevir.

[0311] To quantify antibody expression (MEDI8852) using a transcriptional regulation assay, cells were cotransfected with the pHet-Act1-2 plasmid (HCV NS3 / 4A PR(S139A):PRSIM_23) and the pZFHD1 plasmid (encoding MEDI8852), and 24 hours later, wells were treated with different concentrations of simeprevir. Antibody concentrations in the supernatants 48 hours after simeprevir addition were measured using an MSD kit (Singleplex Human / NHP IgG Isotyping Kit (Mesoscale)).

[0312] Split chimeric antigen receptor activation assay Chimeric antigen receptors (CARs), synthetic, genetically engineered versions of T cell receptors, can induce immune cell activation through target-specific recognition domains, such as single-chain variable antibody fragments (scFvs), in response to user-defined targets. These multidomain synthetic proteins are typically constructed by fusing the target recognition domain to a transmembrane domain, a T cell receptor costimulatory domain, and a C-terminal CD3 zeta cytoplasmic activation domain. Split CARs can be generated by expressing the target recognition domain / transmembrane domain / costimulatory domain and the CD3 zeta activation domain as two separate proteins. By adding appropriate heterodimer switch components to each protein, the CAR can be activated in the presence of the target protein through chemically induced heterodimerization.

[0313] We generated constructs encoding two split CARs using either the FRB:FKBP12 or HCV NS3 / 4A PR(S139A):PRSIM_23 heterodimerization components. Tricistronic constructs were generated for both split CARs. The three encoded fusion proteins were: 1) from N- to C-terminus, a signal peptide sequence, an scFv fragment recognizing the target antigen, a hinge domain from human IgG4, a transmembrane domain from CD28, the intracellular domain of the costimulatory protein 4-1BB activation domain, and either FKBP12 or HCV NS3 / 4A PR(S139A); 2) from N- to C-terminus, a signal peptide sequence, a hinge domain from human IgG4, a transmembrane domain from CD28, the intracellular domain of the costimulatory protein 4-1BB activation domain, and either FKBP12 or HCV NS3 / 4A PR(S139A), followed by the CD3 zeta domain; and 3) green fluorescent protein (GFP), used as a marker for transfected cells (Figure 15A). Fusion proteins 1 and 2 were linked via a P2A self-cleaving peptide, and proteins 2 and 3 were further linked via a T2A self-cleaving peptide. Tricistronic DNA sequences encoding the FRB:FKBP12-based split CAR and the HCV NS3 / 4A PR(S129A):PRSIM_23-based split CAR were purchased from GeneArt (Life Technologies) and cloned into a pCDH expression lentiviral vector (Systems Bioscience), and the sequences were confirmed by Sanger sequencing. The tricistronic DNA sequence of the FRB:FKBP12 split CAR (not including the scFv fragment recognizing the target antigen) is shown as SEQ ID NO: 132, and the tricistronic DNA sequence of the HCV NS3 / 4A PR(S139A):PRSIM_23 split CAR (also not including the scFv fragment recognizing the target antigen) is shown as SEQ ID NO: 133. A DNA sequence encoding an scFv fragment that recognizes the target antigen was inserted between nucleotides 66 and 67 of SEQ ID NOs: 132 and 133, respectively.

[0314] Lentiviral particles encoding each split CAR were generated using the pPACKH1 HIV Lentiviral Vector Packaging Kit (Systems Bioscience) according to the manufacturer's protocol. Jurkat cells were transduced with lentiviral particles for 24 hours in the presence of 8 μg / ml polybrene, after which the cells were transferred to fresh growth medium (RPMI-1640 + 10% fetal bovine serum) and grown for 5 days. To achieve comparable expression levels of both the FKBP12:FRB and HCV NS3 / 4A PR(S139A):PRSIM_23 CARs, pools of Jurkat cells transduced with split CARs were FACS-sorted based on GFP fluorescence prior to functional testing. The activity of split CAR-expressing Jurkat cells can be measured by interleukin-2 (IL-2) production after CAR stimulation (Smith-Garvin, Koretzky, and Jordan 2009). To promote CAR activation, we used a coculture assay in which CAR-expressing Jurkat cells were mixed at a 1:1 ratio with either HepG2 (antigen-positive) or A375 (antigen-negative) cells. Different concentrations of simeprevir or a solvent control (DMSO) were added to the cell mixture and incubated for 24 hours. After incubation, the cells were pelleted by centrifugation, and the supernatants were tested for IL-2 expression using a commercially available IL-2 ELISA (R&D Systems) according to the manufacturer's protocol.

[0315] AAV transduction experiments AAV expression vectors were generated by subcloning specific promoter and transgene elements into an intermediate vector derived from pAAV-CMV (Takara Bio Inc.), in which the CMV promoter downstream of the 5′ ITR had been removed and a WPRE element and SV40 polyA sequence had been inserted upstream of the 3′ ITR.

[0316] To generate AAVs encoding an inducible luciferase transgene, the ZHFD1-luciferase cassette was amplified by PCR from pZFHD1-luciferase provided by the iDimerize regulated transcription system (Takara Bio Inc.) and subcloned into an intermediate AAV vector. To generate AAVs encoding constitutively expressed huIL-2, a gene encoding human IL-2 (SEQ ID NO: 210) was subcloned downstream of the CAG promoter of the intermediate AAV vector (Figure 18A). To generate AAVs encoding the PRSIM_23 CID associated with the split transcription factor, a cassette encoding two fusion proteins (the DNA-binding domain of ZFHD1 fused to three copies of PRSIM_23 and the HCV NS3 / 4A PR(S139A) fused to AD) separated by the P2A self-cleaving peptide (SEQ ID NO: 208) was subcloned downstream of the hybrid EF1α-HTLV-1 promoter of the intermediate AAV vector. To generate an AAV encoding the PRSIM_23 CID split transcription factor plus an inducible IL-2 transgene, human IL-2 was subcloned into the ZFHD1-luciferase vector in place of the luciferase transgene, and the ZFHD1-huIL-2 cassette was amplified by PCR and inserted immediately downstream of the 5' ITR into the AAV vector encoding the PRSIM_23 CID split transcription factor construct (Figure 18C). All constructs were verified by Sanger sequencing.

[0317] Using standard helper-free techniques, 40 T-175cm plates containing HEK293 T-17 cells at 80% confluency were cultured. 2Recombinant AAV (rAAV) was generated by triple transfection of flasks. Briefly, using 90 μg of 40 kD linear polyethyleneimine (PEI), each flask was transfected with 15 μg of a helper plasmid (a plasmid containing adenovirus E2A and E4 genes), 7.5 μg of a plasmid carrying the AAV ITRs and encoding a transgene, and 7.5 μg of an AAV capsid plasmid (containing the AAV8 capsid and the corresponding Rep gene). Five days after transfection, the medium was collected from all flasks, treated with 2000 units of Benzonase nuclease, and incubated at 37°C for 1 hour. The medium was then filtered through a 0.22 μm filter and concentrated to a volume of 80 ml by tangential flow filtration (TFF). This volume was further concentrated and buffer exchanged with PBS using an Amicon 15ml 100kDa filter before being loaded onto a stepwise iodixanol gradient (15% / 25% / 40% / 60%) and spun at 69,000 rpm for 1.5 hours at 18°C ​​in an ultracentrifuge with a Ti70 rotor. Fractions were collected from the ultraclear tubes by piercing the tube with a 19-gauge syringe at the 60% layer below the clear band representing the virus. The purity of each fraction was assessed by SDS-PAGE and subsequent Sypro Ruby analysis. Pure fractions were combined, buffer exchanged with PBS in an Amicon 15ml 100kDa filter, concentrated to a final volume of 150µl, and stored in aliquots at -80°C to avoid repeated freeze / thaw cycles. The virus titer was measured using digital droplet PCR and a TaqMan probe specific for the ITR. Typical titers are 1-3 x 10 13 genome copies (GC) / ml.

[0318] All rAAV transduction assays were performed on adherent HEK293 cells cultured in 96-well plates. Cells were enzymatically dissociated from tissue culture flasks and counted to obtain 2.5 × 10 4Cells were plated in a 96-well plate at 1000 x 1000 cells / well. The plate was incubated overnight at 37°C in 5% CO2 to allow cells to adhere. On day 2, 2.5-5 x 10 cells were plated in a 96-well plate at 1000 x 1000 cells / well. 9 GC / ml (1–2 × 10 5 Cells were transduced with the relevant rAAV at a multiplicity of infection (MOI) of 1000 (corresponding to a multiplicity of infection (MOI) of 1000). After 48–72 h of incubation, cells were treated with different concentrations of simeprevir or a solvent control and incubated for an additional 24 h. For luminescence assays, SteadyGlo luciferase substrate (Promega) was added, and luminescence was quantified using an Envision plate reader. Luminescence readouts were converted to fold changes by dividing the signal in the presence of simeprevir by the signal in the absence of simeprevir. For IL-2 assays, supernatants were collected, and IL-2 was quantified using the V-PLEX Human IL-2 Kit (Meso Scale Discovery) according to the manufacturer's protocol.

[0319] Endogenous gene regulation assay To demonstrate that PRSIM-based CID can regulate endogenous genes, we used the activated CRISPR (CRISPRa) approach. CRISPRa relies on the use of an inactive Cas9 enzyme (dCas9), which lacks endonuclease activity, to bind to a target site within the promoter region of an endogenous gene via a single guide RNA. Upon recruitment of a transcriptional activator, transcription of the endogenous gene is initiated.

[0320] In this approach, dCas9 and the VPR activation domain (AD) are separated to prevent transcription. dCas9 and the AD are separately fused to the two protein components of the CID, allowing the AD to approach dCas9 only in the presence of a small molecule inducer, allowing it to recruit the transcription machinery to the promoter region of endogenous genes via a single guide RNA (sgRNA). In this example, we generated an activation plasmid consisting of two functional units: an AD (SEQ ID NO: 226) fused to HCV NS3 / 4A PR (S139A) and dCas9 (SEQ ID NO: 228) fused to three tandem copies of PRSIM-23. This sequence is placed in front of the CMV promoter and separated by an internal ribosome entry site (IRES). The gRNA plasmid was generated by Golden Gate assembly using BsaI. This gRNA plasmid encodes a human U6 promoter, an interleukin-2 (IL-2) target sequence (GTTACATTAGCCCACACTT, SEQ ID NO: 229), and a scaffold RNA sequence that enables Cas9 binding (Figure 19A).

[0321] Transcriptional regulation assays were performed on adherent HEK293 cells cultured in 96-well plates. Cells enzymatically dissociated from tissue culture flasks were counted and 2.5 × 10 4 Cells were plated at 1000 x gRNA / well. Plates were incubated overnight at 37°C in 5% CO2 to allow cells to adhere. On day 2, cells were co-transfected with the activation plasmid and gRNA plasmid at a 2:1 ratio of gRNA:activation plasmid DNA using Lipofectamine 3000 (ThermoFisher). On day 3, wells were incubated with 300 nM simeprevir or solvent control. After 72 hours of treatment (day 6), cell supernatants were harvested and IL-2 was quantified using the V-PLEX Human IL-2 Kit (Meso Scale Discovery) according to the manufacturer's protocol.

[0322] Molecular simulation to identify mutations predicted to reduce the affinity of simeprevir for the NS3 / 4A protease of hepatitis C virus (HCV) First, we used the Protein Preparation Wizard (Sastry et al., 2013) to prepare a co-crystal structure of HCV in complex with simeprevir, adding hydrogen atoms to fill missing side chains and provide appropriate ionization states for both the amino acids and simeprevir at physiological pH. We then used the FEP+ (module) in the Schrodinger 2019-2 (Moraca et al., 2019) release with the OPLS3e force field to predict the relative binding free energies of residues H57, K136, S139, and R155 of the HCV NS3 / 4A PR. Mutations predicted to reduce the affinity of HCV protease for simeprevir are listed in Table 4.

[0323] Generation of stable expression lines expressing GFP-PEST under the control of split transcription factors Monoclonal cell lines were generated using a CRISPR-mediated knock-in system (ORIGENE) to integrate a transgene into the AAVS1 locus according to the manufacturer's instructions (Figure 26B). First, HEK293 cells expressing GFP-PEST (SEQ ID NOs: 232, 233) under the control of an inducible promoter (minimal IL-2 promoter) were obtained by transient transfection with a pre-linearized pHet-ZFHD1-GFP-PEST plasmid. Transfected cells were selected by adding 800 μg / ml of geneticin to the growth medium (DMEM + 10% fetal bovine serum + 1% non-essential amino acids). Polyclonal cells were then transfected with the pHet-Act1-2-HCV NS3 / 4A PR(S139A)-PRSIM23 (three tandem copies) plasmid and FACS-sorted to isolate single-cell clones based on GFP fluorescence intensity in response to simeprevir treatment. The final monoclonal cell line was used as a basis for further generation of HEK293 cells expressing GFP-PEST under the control of the split transcription factor PRSIM_23 HCV NS3 / 4 PR WT and mutants.

[0324] The AAVS1 safe harbor CRISPR-mediated knock-in system uses two plasmids: the pCAS-Guide-AAVS1 vector, a CRISPR all-in-one vector, and a donor vector (pAAVS1-DNR-Puromycin) containing AAVS1 homology arms (SEQ ID NOs: 234 and 235). The AAVS1 targeting sequence (SEQ ID NO: 236) was previously cloned into the pCAS-Guide plasmid. The donor vector was modified by Gibson assembly by adding SbfI and HpaI restriction enzyme sites to allow for further subcloning of the heterodimerization components of HCV NS3 / 4A PR(S139A) and mutant: PRSIM_23. The pHet-Act1-2-HCV NS3 / 4A PR(S139A)-PRISM23 (three tandem copies) plasmid was then digested with SbfI and HpaI restriction enzymes (New England Biolabs) to obtain HCV NS3 / 4A PR(S139)-PRISM23 DNA, which was then subcloned into a donor vector by Gibson assembly. HCV NS3 / 4A PR mutants, including HCV NS3 / 4 PR(K136D) (SEQ ID NO: 211), HCV NS3 / 4 PR(D168E) (SEQ ID NO: 213), and HCV NS3 / 4 PR(K136N) (SEQ ID NO: 215), were subcloned from pHet-Act1-2-HCV NS3 / 4 PR(K136D / D168E or K136N)-PRISM23 into the pAAVS1-HCV NS3 / 4A PR(S139A)-PRISM23-puromycin plasmid by Gibson assembly using the SbfI and AfeI restriction enzyme sites. The nucleotide sequences were confirmed by Sanger sequencing.

[0325] Stable cells expressing GFP-PEST exclusively under the control of an inducible promoter were co-transfected with pAAVS1-HCV NS3 / 4A PR(S139A;K136D;D168E;K136N)-PRISM23-puromycin donor vector and pCAS-Guide-AAVS1 to allow targeted integration into the AAVS1 locus. Transfected cells were selected 48 hours posttransfection by adding 1 μg / ml puromycin to the growth medium (DMEM + 10% fetal bovine serum + 1% non-essential amino acids + 800 μg / ml Geneticin). After 14 days of selection, polyclonal cell lines were induced with 500 nM simeprevir and single-cell clones were isolated by FACS sorting based on GFP fluorescence intensity. The final monoclonal cell line (Figure 26C) was characterized by FACS based on the GFP signal in response to 500 nM simeprevir treatment.

[0326] Flow cytometry to measure the kinetics of GFP-PEST expression from the simeprevir-induced switch A monoclonal cell line expressing GFP-PEST under the control of a split transcription factor system was enzymatically removed from tissue culture flasks and plated onto 96-well collagen-coated plates. The following day, cells were treated with 100 nM simeprevir. 24 hours after treatment, cells were washed twice in growth medium without simeprevir and then maintained in medium without simeprevir. The GFP fluorescence of cells at various time points after simeprevir removal was measured by flow cytometry using a Fortessa Flow cytometer (BD Biosciences). For analysis, the GFP fluorescence (relative fluorescence units = RFU) of untreated cells was subtracted from all experimental values. Furthermore, RFU values ​​were normalized to the "0-hour" time point, obtained when simeprevir was removed.

[0327] Structural determination of the HCV NS3 / 4A PR(S139A):PRSIM57 complex A single-chain HCV protease construct (an 11-residue peptide derived from the viral NS4A protein fused to the N-terminus of the NS3 protease carrying the S139A mutation) was redesigned with an N-terminal hexahistidine (6His) followed by a tobacco etch virus (TEV) protease cleavage site (to allow affinity purification and tag removal, respectively) (SEQ ID NO: 218). A second construct was designed to express the PRSIM_57 scFv with an N-terminal pelB leader to direct secretion into the periplasm, and a C-terminal TEV site and 6His tag (SEQ ID NO: 221). Both sequences were purchased as linear DNA strings (GeneArt) and cloned into the pET-28a vector (for bacterial expression) using Gibson assembly. The sequence of the final construct was confirmed by Sanger sequencing of the entire coding sequence.

[0328] For expression, the pET-28a plasmid was transformed into BL21(DE3) Escherichia coli (E. coli) cells and selected on kanamycin (50 μg / ml)-containing plates. For each expression, a single colony was used to inoculate 5 ml of 2xTY + 50 μg / ml kanamycin culture, which was grown overnight at 37°C. This culture was used to inoculate 500 ml of TB autoinduction medium (Formedium, supplemented with 10 ml / L glycerol and 100 μg / ml kanamycin) at a 1:500 dilution. The culture was grown at 37°C until the OD600 reached 1.3-1.5 and then shifted to 25°C (HCV NS3 / 4A PR(S139A)) or 30°C (PRSIM_57) for 20 hours to induce expression. Cells were harvested by centrifugation, and the pellets were stored at -80°C.

[0329] To purify HCV NS3 / 4A PR(S139A) protein, bacterial pellets from each 500 ml culture were thawed and resuspended in 50 ml of lysis buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, pH 8.0). Cells were lysed by passage through a cell disruptor at 30,000 kpsi, and the lysate was clarified by centrifugation at 50,000 g for 30 minutes at 4°C. The clarified supernatant was loaded onto a 5 ml HisTrap HP column (GE Healthcare) at a flow rate of 5 ml / min. The column was washed sequentially with wash buffer (50 mM HEPES, 500 mM NaCl, 1 mM TCEP, 20 mM imidazole, pH 8.0, and 50 mM HEPES, 500 mM NaCl, 1 mM TCEP, 40 mM imidazole, pH 8.0) and eluted with an imidazole gradient from 40 to 400 mM imidazole over five column volumes. Fractions were analyzed by SDS-PAGE, concentrated to pool the correct protein, and buffer exchanged with 50 mM HEPES, 200 mM NaCl, 0.3 mM TCEP, 10 μM ZnCl, pH 7.5 (storage buffer) using a HiPrep 26 / 10 desalting column (GE Healthcare). The desalted protein fractions were treated with His-tagged TEV protease at 1:100 w / w overnight at 4°C. The sample was passed through a HisTrap HP column to remove TEV protease, and the resulting flow-through material was polished by loading onto a Superdex 75 26 / 600 column equilibrated with storage buffer.

[0330] The PRSIM-57 His-tagged scFv sample was released from the periplasm by osmotic shock of the cell pellet: cells were first resuspended in 300 ml of 50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0, then pelleted, resuspended in water, and osmotic shock to release the periplasmic contents. This sample was purified by loading onto a HisTrap Excel column and washing and eluting with the same buffer used for the HCV NS3 / 4A PR(S139A) construct. The eluted protein was buffer exchanged by loading onto a HiPrep 26 / 10 desalting column in 50 mM HEPES, 200 mM NaCl, pH 7.5, and treated overnight at 4°C with TEV protease at a 1:50 w / w ratio. The TEV digest was further purified by IMAC and size exclusion steps as well as protease and stored in 50 mM HEPES, 200 mM NaCl, pH 7.5.

[0331] To form the ternary complex of HCV NS3 / 4A PR(S139A), PRSIM_57, and simeprevir, a 50 μM HCV NS3 / 4A PR(S139A) was mixed with a 1.1-fold excess of PRSIM_57, and simeprevir was added to a final solution containing 3% DMSO for a final concentration of 100 μM. The sample was incubated at room temperature for 60 minutes to equilibrate and then loaded onto a Superdex 75 16 / 600 column at 0.75 ml / min in 20 mM HEPES, 200 mM NaCl, pH 7.5. Fractions containing the complex were pooled, concentrated to 12 mg / ml, divided into aliquots, flash-frozen in liquid nitrogen, and stored at -70°C. Aliquots of the complex were thawed and run on an HP-SEC column to confirm the integrity and monodispersity of the complex prior to crystallization.

[0332] The ternary complex was crystallized using the sitting-drop vapor diffusion method. Multiple proprietary crystallization screens were set at 277K and 293K. Hits from these screens were optimized using sitting-drop and hanging-drop vapor diffusion experiments, as needed. Final crystals were obtained at 293K from a reservoir solution consisting of 20-25% (w / v) PEG 8000, 100-300 mM magnesium chloride, and HEPES buffer (pH 7.0-8.0). Crystals were exposed to a reservoir cryoprotectant solution supplemented with 20% (v / v) ethylene glycol and subsequently directly frozen in liquid nitrogen.

[0333] Data collection was performed at the Diamond Light Source, beamline i04, at cryogenic temperatures. The CCP4 and autoBUSTER software packages were used to solve and refine the structure, and the program Coot was used to manually build the model. The structure was solved by molecular replacement using a model of HCV NS3 / 4A (S139A) from the Protein Data Bank.

[0334] In silico prediction of the stability of HCV NS3 / 4A PR(S193) mutants Changes in HCV protein stability in the mutations were calculated using the Schroedinger Residue Scanning tool (Schrodinger Release 2020-2; SiteMap, Schrodinger, LLC, New York, NY, 2020).

[0335] The Prime MM / GBSA energy function with an implicit solvent term was used for the calculations (Li et al., 2011). A 6 Å cutoff was used for refining the protein around the mutation. A negative value for the stability change is linked to an increased stability of the mutant.

[0336] PRISM-based kill switch cloning The sequence encoding the PRSIM23, HCV NS3 / 4A PR, and ΔCARD caspase-9 kill switch fusion protein, with a short GGGSG between the three fragments (SEQ ID NO: 223), was purchased from Geneart (Life Technologies) as a gene cloned into the vector pcDNA3.1. This fusion protein was subcloned into the EcoRI / NotI-digested lentiviral vector pCDH-EF1α-MCS-(PGK-GFP-T2A-Puro) (Systems Bioscience) using Gibson assembly cloning. To generate the caspase-9 S196A mutation, a DNA fragment in which the equivalent Ser371 in the kill switch construct was changed to Ala was synthesized by Geneart and cloned into the ClaI / NotI-cleaved kill switch vector (SEQ ID NO: 230). The gene sequence was confirmed by DNA sequencing.

[0337] Generation of PRISM-based kill-switch cell lines Lentiviral particles encoding the kill switch fusion protein (SEQ ID NO: 223) or the kill switch S196A mutant fusion protein (SEQ ID NO: 230) were generated using the pPACKH1 HIV lentiviral packaging kit (Systems Bioscience) according to the manufacturer's instructions. HEK293 cells were transduced in the presence of 8 μg / ml polybrene for 24 hours, after which the cells were transferred to fresh growth medium (DMEM + 10% fetal bovine serum + 1% non-essential amino acids). After 24 hours, transduced cells were selected by adding 2 μg / ml puromycin for 5 days. Prior to functional testing, pools of transduced cells were FACS-sorted based on GFP fluorescence, and high-expressing cell line pools and single-cell clones were isolated.

[0338] HCT116 and HT29 transduced cells were generated following the same protocol, except that McCoy's 5A medium plus 10% fetal bovine serum was used as the growth medium and supplemented with 2 μg / ml puromycin to select for transduced cells.

[0339] Additionally, the hESC line Sa121 (TakaraBio Europe) was transduced with lentiviral particles encoding the PRSIM-based kill switch fusion protein described above (SEQ ID NO: 223). Cells (passage 19) were cultured in a DEF-CS culture system at 3.5 × 10 5 cells / cm 2 Cells were plated at 100°C for 30 hours and transduced 30 hours later. Puromycin selection was initiated 24 hours after transduction and maintained under antibiotic selection until a stable cell pool was obtained.

[0340] Generation of stable iPS cell lines expressing a PRSIM-based kill switch A stable induced pluripotent stem cell (iPSC) line (a single clone (B-3 / 1F1) derived from fibroblasts of a healthy human donor from Astrazeneca's Research Specimen Collection Program) stably expressing a simeprevir-inducible kill switch was generated by CRISPR / Cas9 technology using AAV-encoding DNA as a template for targeted integration into the β2-microglobulin (B2M) locus.

[0341] A donor construct (Figure 33A) encoding a PRSIM-based kill switch (SEQ ID NO: 223) was synthesized and purchased from GenScript, Inc. and subcloned into the backbone of an AAV shuttle plasmid. The donor construct was packaged into adeno-associated virus (AAV) particles. Briefly, the donor plasmid was co-transfected with two helper plasmids, pAd5Helper and pR2C6, encoding the adenoviral components essential for AAV replication and AAV2 replication (rep) / AAV6 capsid (cap) proteins, respectively. After 72 hours, cells were harvested and disrupted by freeze-thawing. The cell lysate was digested with Benzonase (100 U / ml) at 37°C for 1 hour and centrifuged. The vector-containing supernatant was collected and applied to an iodixanol gradient, followed by ultracentrifugation. After ultracentrifugation, the vector-containing solution was collected and washed three times with 20 mL of PBS in a centrifugal concentration tube. Finally, the solution was concentrated to 1 mL. The genome copies of the vector contained in the solution were measured by qPCR.

[0342] iPSC cells (approximately 1.2 × 10) were seeded at 50–70% confluency onto vitronectin-coated 6-well plates. 6 Cells) were used for transfection / transduction. Cells were maintained in 2 mL of fresh StemFlex medium containing 1x RevitaCell (Life Technologies). 200 μL of Opti-MEM (Life Technologies) medium containing 220 nM CRISPR-ribonucleoprotein and 12 μL of RNAiMAX (Life Technologies) was applied to each well. Meanwhile, AAV vectors were applied at a multiplicity of infection (MOI) of 50,000. After 24 hours of incubation, the medium containing the RNP / AAV was replaced with fresh StemFlex medium.

[0343] Forty-eight hours after transfection, the medium was replaced with fresh StemFlex medium containing 5 μg / mL blasticidin S HCl (Life Technologies). The medium was replaced daily with fresh StemFlex medium containing blasticidin for an additional 3–4 days. Cells were then re-maintained in regular StemFlex medium.

[0344] To identify cells that were B2M-negative and therefore encoded a PRSIM-based kill switch, FACS was performed. Cells were detached from the plate using TrypLE Express (Life Technologies) and 1 × 10 cells were placed in FACS buffer (HBBS with 1% PBS and 1 × RevitaCell) containing 5% APC-conjugated anti-human B2M antibody (BioLegend, Inc.). 7 After a 10-minute incubation, the cells were washed twice with 10 volumes of FACS buffer and resuspended at a density of 2 × 10 cells / mL. 7 The cells were resuspended in FACS buffer at a density of 1000 cells / mL. B2M-negative cells were collected by FACS (FACSAria; BD Biosciences) and cultured for further experiments.

[0345] Subsequently, single-cell clones were isolated using single-cell printing. Cells were detached from the plate using TrypLE Express (Life Technologies) and plated in SCP buffer (HBBS with 1x RevitaCell) at 1.6 × 10 6 The cells were resuspended at a density of 1000 cells / ml. The cell suspension was loaded into the cartridge of a Cytena CloneSelect Single-Cell Printer (Cytena). Cells were seeded at 1 cell per well in Matrigel or vitronectin-coated 96-well plates containing 200 μL of fresh mTeSR (STEMCELL Technologies) or 1× RevitaCell (Life Technologies) in StemFlex medium. The day after SCP, the medium was replaced with fresh StemFlex medium.

[0346] Five single-cell clones were collected and expanded from the 96-well plate to a vitronectin-coated 24-well plate, and then further expanded and maintained on a vitronectin-coated 6-well plate. Approximately 5 × 10 cells were collected for each single-cell clone. 5 Cells were harvested. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen). The target region of the human B2M gene was amplified using SuperFi DNA polymerase (Life Technologies) with the following primers: PCR product was loaded onto a 1.2% agarose gel for electrophoresis. The gel was visualized to identify the gene knock-in status of single-cell clones by amplicon size (Figure 33B). Clones 1B7, 1D12, 1G8, and 2D8 were found to be biallelic at the B2M locus and were used to analyze the function of the kill switch activity.

[0347] [Table 8]

[0348] Kill-switch cell viability and caspase-3 functional assays HEK293, HCT116, or HT29 cells stably expressing a PRSIM-based kill switch fusion protein (SEQ ID NO: 223), or HEK293 cells stably expressing a PRSIM kill switch S196A mutant fusion protein (SEQ ID NO: 230) were plated on collagen-coated 96-well plates and treated with 100 nM simeprevir 24 hours later. Phase-contrast images were acquired at various time points using a 10x or 20x objective on an Incucyte Zoom (EssenBioscience).

[0349] Functional caspase-9 activates caspase-3, whose proteolytic activity can be measured by cleavage of the nonfluorescent substrate DEVD-AMC into the cleavage products DEVD and fluorescent AMC. The fluorescence signal of AMC at 430 nm is proportional to caspase-3 activity. For the caspase-3 assay, cells were plated in duplicate in six-well tissue-culture-treated plates. After 24 h, one duplicate well was treated with 10 nM simeprevir for 3 h. Total protein input was normalized to 50 μg by BCA assay (Life Technologies), and cell lysates were analyzed in triplicate using the BD Biosciences caspase-3 assay according to the manufacturer's instructions. Fluorescence was measured using an Envision plate reader (PerkinElmer) at 380 nm (Ex) and 430 nm (Em). For quantitation, the RFU (raw fluorescence value) of wells containing assay substrate alone was subtracted from all RFU derived from assay samples. Results were normalized to untransduced, simeprevir-treated cells. Analysis was performed in Prism (GraphPad) using one-way ANOVA followed by multiple comparisons.

[0350] PRSIM-based kill switch activity in ESC cells To verify the induction of the kill switch in Sa121 ES cells, 3.5 × 10 cells were cultured in 100% ES cells. 5 / cm 2 Two days later, kill-switch activity was induced by treatment with simeprevir at concentrations ranging from 10 nM to 1 μM. Cells were imaged using an Incucyte S3 (Essen Bioscience) at intervals ranging from 10 to 20 min, and kill-switch efficiency was quantified by image analysis of confluency.

[0351] Real-time cell analysis (RTCA) assay to detect simeprevir-induced kill switch activity in iPS cells Cells from each of the single-cell clones described above were plated at a density of 40,000 cells per well in a vitronectin-coated 96-well electronic microtiter plate (E-Plate® 96, ACEA Biosciences Inc.). The plate was connected to the xCelligence module and incubated in 5% CO2 to allow for the monitoring of cell proliferation index without disturbing normal cell growth. 2 The cells were incubated at 37°C in a humidified incubator. The cell proliferation index was measured and recorded every 15 minutes for 24 hours. Different concentrations of simeprevir were then added, and the cell proliferation index was measured every 5 minutes for 8 hours, followed by every 15 minutes for an additional 40 hours. All experiments were performed in triplicate wells for each clone and condition. The average cell index was quantified using xCELLigence RTCA Software Pro (ACEA Biosciences Inc.).

[0352] Example 2 - Identification of Simeprevir and HCV NS3 / 4A PR as the basis for a Chemically Induced Dimerization (CID) module To generate novel chemical dimerization inducers, we employed an approach in which the small molecule inducer is a clinically approved small molecule and one of the protein components is the target (target protein) of the small molecule. The second protein component (binding member) is derived from a library of binding molecules (Tn3 or scFv) and exhibits superior selectivity for the small molecule-bound target protein compared to the unbound target protein (Figure 1). Given that this small molecule is already deemed safe for use in humans at appropriate doses, we concluded that focusing on approved small molecules would significantly smooth the path to regulatory approval. Rather than using small molecules targeting human proteins, we instead focused on small molecules that bind to non-human proteins, such as antiviral compounds. We concluded that the advantage of this approach is that the target protein is not present in patients (non-infectious diseases), and therefore there is no competition for small molecule binding that could affect its pharmacokinetics, preventing the small molecule from eliciting any potentially harmful on-target pharmacological effects. To determine preferred small molecule / target protein pairs, the following criteria were considered: The criteria for an ideal small molecule are: · Approved Suitable for long-term administration (daily for >6 months) - Cell-permeable - Oral administration Not used as a first-line antiviral treatment The criteria for an ideal target protein are: -Being a monomer ·Small size (≦30kDa) The target protein (or a domain thereof) can be overexpressed in a non-toxic manner, or the target protein can be inactivated but retains SM binding. Can be expressed in the cytoplasm (i.e., not membrane-integrated or DNA-bound) Criteria for small molecule:target protein complexes include: There is reason to believe that the bound target protein will have a different epitope than the unbound target protein.

[0353] After extensive analysis, one of the preferred small molecule / target protein pairs identified was simeprevir and its target, the NS3 / 4A protease from hepatitis C virus (HCV NS3 / 4A PR). Simeprevir (Olysio®) is an orally administered small molecule that is cell membrane permeable and has a pharmacokinetic (PK) profile that supports once-daily dosing. Simeprevir has been used long-term (up to 39 months) in combination with ribavirin and pegylated interferon to treat HCV infection and is listed on the WHO Essential Medicines List, demonstrating its well-tolerated and widely administered nature. HCV NS3 / 4A PR is a monomer with a relatively small size (21 kDa), can be expressed in the cytoplasm, and does not exhibit DNA association. Furthermore, three-dimensional X-ray crystallography of the complex (PDB code: 3KEE) revealed that simeprevir binds within a shallow substrate-binding groove of HCV NS3 / 4A PR with an exposed surface area of ​​364 Å (Figure 2). We conclude that this relatively large exposed surface area is sufficiently different from that of unbound HCV NS3 / 4A PR to allow the identification of binding molecules specific to the complex.

[0354] Example 3 - Mutant HCV NS3 / 4A PR(S139A) retains binding to simeprevir despite significantly reduced activity HCV NS3 / 4A PR is an enzyme that cleaves at four junctions of the HCV polyprotein precursor and is known to cleave a limited number of endogenous human targets (Li, Sun, et al. 2005; Li, Foy, et al. 2005). We concluded that to limit its activity in human cells, it was necessary to identify a mutant HCV NS3 / 4A PR that was enzymatically inactive but retained simeprevir binding. An active site mutant (S139A) of HCV NS3 / 4A PR has previously been shown to exhibit significantly reduced activity compared to its wild-type counterpart (Sabariegos et al. 2009). To confirm this and investigate whether the mutant HCV NS3 / 4A PR retained simeprevir binding, we expressed the recombinant protein in Escherichia coli (E. coli) and purified it to homogeneity. Both the wild-type (SEQ ID NO: 3) and S139A mutant (SEQ ID NO: 4) HCV NS3 / 4A PR carrying an N-terminal hexahistidine and AviTag were separately expressed in 1-liter cultures of BL21(DE3) induced by autoinduction. The cultures were harvested, and the proteins were purified by a combination of immobilized metal affinity chromatography and size-exclusion chromatography. SDS-PAGE analysis of the final pooled sample demonstrated a purity level of >99% (Figure 3A). Aliquots of the purified proteins were site-specifically biotinylated at the AviTag using BirA enzyme and repurified by size-exclusion chromatography. Mass spectrometry analysis confirmed 100% biotinylation incorporation in both the wild-type and S139A HCV NS3 / 4A PR.

[0355] When these recombinant HCV NS3 / 4A PR WT and S139A proteins were tested for enzymatic activity in a fluorogenic peptide cleavage assay, we confirmed that the activity of the S139A mutant of HCV NS3 / 4A PR was significantly reduced. Enzymatic activity was undetectable at most concentrations tested, with minimal activity observed only at high concentrations ranging from nM to μM (Figure 3B).

[0356] Isothermal calorimetry was performed to assess the binding affinity of simeprevir to the WT and S139A HCV NS3 / 4A PR proteins. Very similar results were obtained for both proteins, with identical stoichiometry (approximately 0.6 simeprevir / NS3 binding site) and ΔH values ​​(approximately 22 kcal / mol) (Figure 3C). Although the calculated dissociation constant was very low (approximately 1 pM), the associated error was very large (10 nM), suggesting that the affinity is too high to be accurately measured using this technique without the use of a competing ligand. Nevertheless, the identical stoichiometry and ΔH values ​​suggest that there is very likely no significant difference in binding affinity between the WT and S139A HCV NS3 / 4A PR proteins.

[0357] Based on these data, we decided to proceed with the selection of HCV NS3 / 4A PR:simeprevir complex specific binding (PRSIM) molecules based on the S139A mutant protein.

[0358] Example 4 - Selection of HCV NS3 / 4A PR(S139A):Simeprevir Conjugate-Specific Binding (PRSIM) Molecules Biotinylated HCV NS3 / 4A PR(S139A) in the presence of simeprevir was subjected to four rounds of phage display selection. From the selection output of rounds 3 and 4, biotinylated HCV NS3 / 4A PR(S139A) was subjected to phage ELISA in both the presence and absence of simeprevir, and binding was measured by measuring the fluorescent signal (Figures 4A and 4B). The phage ELISA binding data was compared with the DNA sequence data of the same clones, and a panel of 34 scFv clones and 28 Tn3 clones with unique sequences that showed selective binding to biotinylated HCV NS3 / 4A PR(S139A) in the presence of simeprevir was selected and expressed for further biochemical studies (Tables 1A and 1B). Additionally, one scFv clone (PRSIM_51) and three Tn3 clones (PRSIM_54, PRSIM_55, and PRSIM_85) that showed binding to biotinylated HCV N3 / 4A protease (S139A) both in the presence and absence of simeprevir were also selected for further biochemical studies.

[0359] [Table 9]

[0360] [Table 10]

[0361] *All data were recorded in the presence of simeprevir except for data in parentheses which were measured in the absence of simeprevir.

[0362] Example 5 - A panel of PRSIM molecules is specific for the HCV NS3 / 4A PR(S139A):simeprevir complex The PRSIM binding proteins identified as complex-specific from the phage display selection were expressed and purified on a larger scale to provide sufficient material for further analysis. All HCV NS3 / 4A PR(S139A):simeprevir complex-specific PRSIM molecules were subjected to a homogeneous time-resolved fluorescence (HTRF) binding screen (Fig. 5), confirming that eight Tn3-based molecules and a panel of 14 scFv-based molecules were complex-specific, with no detectable binding to the HCV NS3 / 4A PR(S139A) protein alone (Table 1 (bold), Fig. 6).

[0363] To further characterize the PRSIM binding molecules, five scFv molecules (PRSIM_4, PRSIM_57, PRSIM_67, PRSIM_72, and PRSIM_75) and five Tn3 molecules (PRSIM_23, PRSIM_32, PRSIM_33, PRSIM_36, and PRSIM_47) were selected and the kinetics of HCV NS3 / 4A PR(S139A) protease binding in the presence or absence of simeprevir was measured using Biacore 8K (Table 2). All PRSIM binding molecules tested showed selectivity for simeprevir-bound HCV NS3 / 4A PR(S139A), with only three showing slight nonspecific binding to HCV NS3 / 4A PR(S139A) alone. PRSIM_57 (Figure 7A) and PRSIM_23 (Figure 7B) were selected for further characterization. HCV NS3 / 4A PR(S139A) had an affinity of 15.0 nM for PRSIM_57 (scFv) and 6.3 nM for PRSIM_23 (Tn3). The effect of simeprevir concentration on the formation of HCV NS3 / 4A PR(S139A) / PRSIM57 / 23 complexes was also evaluated (Figure 7C). Simeprevir showed almost identical EC2 activity to PRSIM_57 and PRSIM_23 in complexes with HCV NS3 / 4A PR(S139A). 50 and 4.57 nM and 4.03 nM, respectively. Table 2: Binding and kinetic constants for HCV NS3 / 4A PR(S139A) binding to PRSIM binding molecules in the presence or absence of simeprevir were measured. BSA in the presence of simeprevir was used as a control.

[0364] [Table 11]

[0365] Example 6 - PRSIM-based CID can regulate reconstitution of split proteins By isolating a PRSIM-binding molecule that specifically binds to the simeprevir:HCV NS3 / 4A PR(S139A) complex, we concluded that this system can be used to regulate split protein reconstitution. By providing temporal and spatial control of protein dimerization within the cell, CID can be applied within a range of post-translational contexts to control desired protein interactions or activities. Numerous examples exist of split proteins that gain activity upon reconstitution, one of which is split nanoluciferase, as provided by the NanoBiT system (Promega) (Figure 8). We adapted this system for PRSIM-based CID by fusing HCV NS3 / 4A PR(S139A) to SmBiT and PRSIM-binding members to LgBiT. A screen was performed to test the five Tn3 and six scFv PRSIM binding modules resulting from the phage selection process using both N- and C-terminal fusions to LgBiT and the equivalent N- and C-terminal fusions of HCV NS3 / 4A PR(S139A) fused to SmBiT. Cells were transfected with the appropriate plasmids, incubated for 24 hours, and then treated with 100 nM simeprevir or solvent control (or 100 nM rapamycin for the FRB:FKBP12 control provided in the kit). Luminescence was measured, and the fold change in signal in the presence of simeprevir relative to the signal obtained in the absence of simeprevir was calculated (Figure 9). Observing an overall trend, significant fold changes in luminescence were generally observed only when LgBiT was fused to the C-terminus of the PRSIM binding module. Significant signals above the associated background were observed for the following PRSIM binding modules: PRSIM_23 (31-fold), PRSIM_33 (9-fold), PRSIM_01 (16-fold), PRSIM_06 (11-fold), PRSIM_57 (14-fold), and PRSIM_75 (51-fold). This result indicates that a number of isolated PRSIM binding modules in the presence of simeprevir can specifically induce dimerization of split NanoLuc from the NanoBiT system.

[0366] Example 7 - PRSIM-based CID can regulate gene expression by reconstituting split transcription factors By fusing HCV NS3 / 4A PR(S139A) and PRSIM molecules to separate components of the split NanoLuc enzyme, we demonstrated that the PRSIM-based CID can reconstitute the activity of the split protein, leading us to conclude that the same CID can regulate transgene expression by fusing it to the two domains of a split transcription factor. To demonstrate this, we used the iDimerize regulated transcription system (Takara Bio Inc.), which provides two separate vectors: one (pHet-Act1-2) encodes FRB fused to the activation domain (AD) p65 and the DNA-binding domain (DBD) ZFHD1 fused to three copies of FKBP12, separated by an IRES sequence and located in front of the constitutive CMV promoter; the other (pZFHD1_Luciferase) encodes luciferase under the control of an inducible promoter containing 12 copies of the ZFHD1 recognition sequence upstream of a minimal IL-2 promoter. Transfection of both plasmids into cells resulted in expression of the FRB-AD and DBD-FKBP12 proteins, and although the DBD recognizes its target site on the inducible promoter, transcription initiation fails due to the absence of an AD proximal to the promoter. Only when the rapalog inducer "A / C heterodimerizer" is added does AD recruit the DBD bound to the promoter upstream of the luciferase gene, initiating expression.

[0367] The coding sequences for FRB and FKBP12 were replaced with sequences encoding one copy of HCV NS3 / 4A PR(S139A) and one of the 11 PRSIM molecules described below, fused either to the N-terminus of the activation domain or the C-terminus of the DNA-binding domain (Figure 10). After transfecting cells with pHet-Act1-2 (PRSIM) and pZFHD1_Luciferase constructs, the ability of the PRSIM-based CID to regulate luciferase gene expression in the presence of increasing concentrations of simeprevir was assessed. The different PRSIM-based CID constructs demonstrated dose-dependent gene expression regulation ranging from 1.4- to 146-fold (Figures 11A and 11B, Table 3), with six Tn3-based and five scFv-based PRSIM molecules demonstrating a greater than 10-fold increase in gene expression. The maximum fold change achieved with a Tn3-based clone based on PRSIM_23 fused to the activation domain was 106-fold. Interestingly, while the majority of PRSIM clones showed a preference for fusion to either the AD or DBD, PRSIM_23 was unique in that it was able to confer potent gene expression modulation to both (106-fold when fused to the AD and 88-fold when fused to the DBD). PRSIM_23 also exhibited the lowest EC50 (2 nM), indicating that a lower concentration of simeprevir was required to activate transcription. The clone that showed the highest fold change upon addition of simeprevir was PRSIM_57, an scFv-based clone fused to the DBD, which achieved 146-fold induction and a low EC50 value (3 nM).

[0368] [Table 12]

[0369] Direct comparison of the ability of HCV NS3 / 4A PR(S139A)-AD and DBD-PRSIM_23 or DBD-PRSIM_57-based constructs to modulate luciferase expression in the presence of simeprevir with the FRB:FKBP12:rapalog positive control revealed that PRSIM-based CID (100-fold increase) outperformed FRB:FKBP12-based CID (30-fold increase) (Figure 12A). Analysis of luminescence values ​​obtained in the absence of inducer (i.e., simeprevir or rapalog) revealed higher levels with FRB:FKBP12:rapalog-based CID, suggesting enhanced leakage levels with PRSIM-based CID (Figure 12B).

[0370] Example 8 - Increasing tandem copies of PRSIM fused to the DBD improves gene regulation To assess the effect of the copy number of the target protein fused to the DNA-binding domain, we generated pHet-Act1-2-based constructs encoding FRB-AD or HCV NS3 / 4A PR(S139A)-AD and DBD-FKBP12 or DBD-PRSIM_23 (the protein fused to the DBD was included as either a single copy or three tandem copies separated by short peptide linkers) (Figure 13). The ability of the PRSIM_23-based CID to regulate NanoLuc-PEST protein expression in the presence of simeprevir was compared with the FRB:FKBP12:rapalog positive control, and we found that the PRSIM_23-based CID outperformed the FRB:FKBP12-based CID when using either one copy or three copies of the DBD fusion partner (55-fold vs. 13-fold at one copy, and 100-fold vs. 55-fold at three copies) (Figure 14A).

[0371] Furthermore, when the effects of one, two, or three tandem copies of PRSIM_23 fused to the DBD were assessed in the same split transcription factor assay, measuring induction of firefly luciferase expression, a graded response was observed; one copy of PRSIM_23 resulted in a maximum fold change of 364.5, while two tandem PRSIM_23 molecules resulted in a maximum fold change of 2436, and a further increase of 4862-fold with three tandem PRSIM_23 molecules (Figure 14B).

[0372] This data suggests that the regulation of gene expression by inducible promoters can be improved by recruiting more copies of the activation domain, and that this is a general phenomenon and is not dependent on the CID used.

[0373] Example 9 - PRSIM-based CID can modulate the activity of split chimeric antigen receptors (CARs) Modulating CAR activity through chemically induced heterodimerization has previously been shown to be an effective method for modulating CAR function (Wu et al., 2015; Hill et al., 2018). We hypothesized that applying heterodimerizing PRSIM components to CARs might promote CAR modulation in a similar manner. We used the previously described FKBP12:FRB system (Wu et al., 2015) as a comparison for modulating CAR function. To test this, we engineered Jurkat T cells to express a CAR regulated by PRSIM and FKBP12:FRB using a lentiviral expression system (Figure 15A). Activation of the CAR by antigen binding resulted in the secretion of IL-2 in a dose-dependent manner in the presence of either the rapamycin analog AP2196 (a dimerizer of FKBP12:FRB) or simeprevir (a dimerizer of PRSIM) (Figure 15B). IL-2 expression can be rapidly quantified using an IL-2-specific ELISA (R&D Systems). The design of these systems requires that they promote T cell activation only upon antigen binding in the presence of an appropriate dimerizer. In both the PRSIM and FKBP12:FRB-regulated CAR systems, the addition of simeprevir or AP2196, respectively, resulted in dose-dependent activation of CAR-expressing Jurkat cells in the presence of antigen-positive HepG2 cells, as measured by IL-2 production (Figure 16). Importantly, neither CAR was activated in the presence of antigen-negative A375 cells (Figure 16). While both the FKBP12:FRB and PRSIM systems demonstrated dose-dependent activation, the PRSIM system demonstrated tighter control of CAR activity, as evidenced by lower background IL-2 levels and a greater dynamic range of CAR activity (Figure 16). Both systems exhibited similar maximal IL-2 expression levels.These data demonstrate that the PRSIM heterodimerization system can be used to mediate simeprevir-mediated modulation of intracellular signaling pathways initiated by CAR.

[0374] Example 10 - PRSIM-based CID can regulate gene expression of an antibody (MEDI8852) In addition to demonstrating gene regulation of two recombinant intracellular proteins (luciferase (Example 7) and NanoLuc-PEST (Example 8)) using PRSIM-based CID, we also investigated the regulation of gene expression of a secreted antibody (MEDI8852; SEQ ID NOs: 205 and 206). pHet-Act1-2-based constructs encoding HCV NS3 / 4A PR(S139A)-AD and DBD-PRSIM_23 (three tandem copies) and a construct encoding pZFHD1_MEDI8852 were generated. When cells were transfected with these two constructs, MEDI8852 expression was shown to be simeprevir dose-dependent, as measured using the Singleplex Human / NHP IgG Isotyping Kit (Mesoscale) (Figure 17).

[0375] Example 11 - PRSIM-based CID can regulate gene expression of proteins by adeno-associated viruses Recombinant adeno-associated virus (rAAV) vectors represent a well-studied platform that can deliver DNA encoding a PRSIM_23 / HCV NS3 / 4A PR(S139A)-based CID into cells and be used for controlled gene therapy. One such application is the delivery of the PRSIM_23 / HCV NS3 / 4A PR(S139A)-based split transcription factor components described in Example 7 into cells, either together with or separately from AAV particles, to regulate exogenous transgenes. In the context of the system described herein, the packaging capacity of AAV limits the size of transgenes that can be delivered in the same AAV vector to approximately 550 bp or in separate AAV particles to approximately 3.6 kb.

[0376] To demonstrate trans-delivery of the CID-encoding DNA and an inducible transgene, we generated two different AAV vectors: one encoding a PRSIM_23 / HCV NS3 / 4A PR(S139A)-based split transcription factor component, expression of which is driven by a constitutive EF1 / HTLV hybrid promoter, and the other encoding the firefly luciferase gene under the control of the inducible ZFHD1 promoter (Figure 18A). We generated AAV particles from these vectors and subsequently transduced HEK293 cells with two separate AAV8 preparations. Only when both AAV8 particle preps were added did we observe simeprevir dose-dependent regulation of luciferase gene expression by the PRSIM_23 / HCV NS3 / 4A PR(S139A)-based CID, resulting in a 228-fold induction of luciferase activity (Figure 18B).

[0377] To demonstrate the ability to deliver CID and an inducible transgene in cis, we generated an AAV8 vector encoding both the PRSIM_23 / HCV NS3 / 4A PR(S139A)-based transcription factor component and an inducible IL-2 transgene (Figure 18C). After transducing HEK293 cells with AAV8 particles generated using this AAV vector, we observed simeprevir dose-dependent regulation of PRSIM_23 / HCV NS3 / 4A PR(S139A)-based IL-2 gene expression, with a maximum level of approximately 3500 pg / ml of IL-2 (Figure 18D). At the highest concentration of simeprevir tested, the IL-2 expression level induced by PRSIM_23 / HCV NS3 / 4A PR(S139A)-based CID (3506 + / - 817 pg / ml) was similar to that obtained from a control AAV8 vector encoding an IL-2 transgene under the control of a constitutive CAG promoter (2606 + / - 189 pg / ml) (Figure 18E).

[0378] Thus, using either a single- or dual-AAV-based system, the ability of PRSIM-based CID to control gene expression by AAV transduction was demonstrated.

[0379] Example 12 - PRSIM-based CID can regulate transcription of endogenous genes Having demonstrated that PRSIM-based CIDs can regulate transgene expression by fusing them to two domains of a split transcription factor, we concluded that PRSIM-based CIDs can also regulate endogenous gene expression. Regulating endogenous gene activity using a chemically induced heterodimerization system has previously been shown to be an effective method of gene regulation (Foight et al. 2019). Therefore, we hypothesized that applying heterodimerizing PRSIM components to an activated CRISPR (CRISPRa) system could promote endogenous gene regulation in a similar manner.

[0380] To demonstrate this, we fused an inactive Streptococcus pyogenes Cas9 enzyme (dCas9) and an activation domain (AD) consisting of a fusion of three transcriptional activators (VP64, p65, and Rta, or VPR) to two protein components of the CID (three copies each of PRSIM_23 and HCV NS3 / 4A PR(S139A)), allowing the AD to approach dCas9 only in the presence of a small molecule inducer. Cotransfection of the PRSIM-based CID with a guide RNA (gRNA) targeting the interleukin-2 (IL-2) promoter enabled dCas9 to bind to its target site in the IL-2 promoter. Administration of the PRSIM dimerizer (simeprevir) subsequently recruited the AD and associated transcriptional machinery to the promoter region of the endogenous IL-2 gene and initiated transcription (Figure 19A). Thus, activation of the system can be ...

Claims

1. i) a first expression cassette encoding a target protein, wherein the target protein is capable of binding to a small molecule such that a complex (T-SM complex) is formed between the target protein and the small molecule; ii) a second expression cassette encoding a binding member that specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with a higher affinity than it binds to the target protein alone and to the small molecule alone; where: the target protein is derived from HCV NS3 / 4A protease and comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; the small molecule is simeprevir, and the binding member is a Tn3 protein comprising an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 5 to 9; One or more expression vectors.

2. 10. The one or more expression vectors of claim 1, wherein the target protein has attenuated viral activity compared to HCV NS3 / 4A protease having the amino acid sequence of SEQ ID NO:

1.

3. the target protein is fused to a first constituent polypeptide; 3. One or more expression vectors according to claim 1 or 2, wherein the binding member is fused to a second constituent polypeptide.

4. (1) the first constituent polypeptide comprises a DNA-binding domain (DBD) and is fused to the target protein (T) to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain (TRD) and is fused to the binding member (BM) to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to the target protein to form a TRD-T fusion protein; the second component polypeptide comprises a DNA binding domain and is fused to the binding member to form a DBD-BM fusion protein; dimerization of the first and second constituent polypeptides to form a transcription factor; 4. The one or more expression vectors of claim 3, wherein the transcriptional regulatory domain is a transcriptional activation domain or a transcriptional repression domain.

5. 5. The one or more expression vectors of claim 4, further comprising a third expression cassette encoding a desired expression product, wherein the DNA binding domain binds to a target sequence in the third expression cassette such that the transcription factor can regulate expression of the desired expression product, and the target sequence is located in a promoter operably linked to the coding sequence of the desired expression product.

6. 6. The one or more expression vectors of claim 5, wherein the desired expression product is a therapeutic protein, or wherein the desired expression product is a therapeutic protein and the therapeutic protein is a therapeutic antibody.

7. (1) the first constituent polypeptide comprises a first costimulatory domain and is fused to the target protein; the second component polypeptide comprises an intracellular signalling domain and is fused to the binding member; or (2) the first constituent polypeptide comprises an intracellular signaling domain and is fused to the target protein; 4. The one or more expression vectors of claim 3, wherein the second constituent polypeptide comprises a first costimulatory domain and is fused to the binding member.

8. the first constituent polypeptide comprises a first caspase component; the second constituent polypeptide comprises a second caspase component; 4. The one or more expression vectors of claim 3, wherein the first and second constituent polypeptides form a caspase upon dimerization.

9. 9. The one or more expression vectors of claim 8, wherein the first and second caspase components comprise a caspase 9 activation domain, or wherein the first and second caspase components comprise a caspase 9 activation domain and the first and second caspase components are identical.

10. 10. The one or more expression vectors according to any one of claims 1 to 9, wherein each of the one or more expression vectors is a DNA plasmid or a viral vector.

11. 1. A method for producing viral particles in vitro, comprising: transfecting a host cell with the viral vector of claim 10 to express viral proteins required for forming viral particles in the host cell; and culturing said transfected host cells in a medium such that said transfected host cells produce viral particles.

12. 12. The method of claim 11, further comprising separating the viral particles from the medium, or separating the viral particles from the medium and concentrating the viral particles.

13. A dimer-inducing protein, a first constituent polypeptide fused to a target protein (T); a second component polypeptide fused to a binding member; the target protein is capable of binding to a small molecule (SM) to form a complex (T-SM complex) between the target protein and the SM, and the binding member specifically binds to the T-SM complex such that it binds to the T-SM complex with a higher affinity than it binds to the target protein alone and to the small molecule alone; where: the target protein is derived from HCV NS3 / 4A protease and comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; the small molecule is simeprevir, and the binding member is a Tn3 protein comprising an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 5 to 9; Dimer-inducing proteins.

14. (1) the first constituent polypeptide comprises a DNA-binding domain (DBD) and is fused to the target protein (T) to form a DBD-T fusion protein; the second component polypeptide comprises a transcriptional regulatory domain (TRD) and is fused to the binding member (BM) to form a TRD-BM fusion protein; or (2) the first constituent polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein; the second component polypeptide comprises a DNA binding domain and is fused to the binding member to form a DBD-BM fusion protein; 14. The dimer-inducing protein of claim 13, wherein the first and second constituent polypeptides dimerize to form a transcription factor.

15. (1) the first constituent polypeptide comprises a first costimulatory domain and is fused to the target protein; the second component polypeptide comprises an intracellular signalling domain and is fused to the binding member; or (2) the first constituent polypeptide comprises an intracellular signaling domain and is fused to the target protein; 14. The dimer-inducing protein of claim 13, wherein the second constituent polypeptide comprises a first costimulatory domain and is fused to the binding member.

16. the first constituent polypeptide comprises a first caspase component; the second constituent polypeptide comprises a second caspase component; 14. The dimer-inducing protein of claim 13, wherein the first and second constituent polypeptides dimerize to form a caspase.

17. The dimer-inducing protein of claim 16, wherein the first and second caspase components comprise a caspase 9 activation domain, or the first and second caspase components comprise a caspase 9 activation domain and the first and second caspase components are identical.

18. A stem cell or immune cell expressing a dimer-inducing protein described in any one of claims 13 to 17.

19. A method of genetically modifying a cell to produce the cell of claim 18, comprising administering to the cell one or more expression vectors of any one of claims 3 to 9, the method being carried out in vitro or ex vivo.

20. One or more viral particles comprising one or more expression vectors according to any one of claims 3 to 9, One or more viral particles, wherein said first and said second expression cassettes form part of the viral genome within said one or more viral particles.

21. 21. One or more viral particles according to claim 20, wherein the viral particles are AAV particles.

22. A pharmaceutical composition comprising one or more expression vectors according to any one of claims 1 to 12 or one or more viral particles according to claim 20 or 21 for use in a method of treatment of the human or animal body.

23. 20. A pharmaceutical composition comprising the cells of claim 18 for use in a method of treatment of the human or animal body, said method comprising: i) administering the cells to an individual; ii) administering said small molecule to said individual.

24. an expression vector encoding an inducible caspase-9 (iCasp9) protein, the expression vector comprising an expression cassette encoding a target protein, a binding member, and a caspase-9 activation domain, wherein both the target protein and the binding member are fused to the caspase-9 activation domain; the target protein is capable of binding to the small molecule to form a complex (T-SM complex) between the target protein and the small molecule; specifically binds to the T-SM complex such that the binding member binds to the T-SM complex with higher affinity than it binds to the target protein alone and to the small molecule alone; where: the target protein is derived from HCV NS3 / 4A protease and comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; the small molecule is simeprevir, and the binding member is a Tn3 protein comprising an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NOs: 5 to 9; Expression vector.

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