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WO2024261323A1 - Commutateurs moléculaires - Google Patents

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Publication number
WO2024261323A1
WO2024261323A1 PCT/EP2024/067561 EP2024067561W WO2024261323A1 WO 2024261323 A1 WO2024261323 A1 WO 2024261323A1 EP 2024067561 W EP2024067561 W EP 2024067561W WO 2024261323 A1 WO2024261323 A1 WO 2024261323A1
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protein
cell
seq
expression
dimerization
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Natalie Jo Tigue
Lisa Marie Kitching VINALL
Anna Gudny SIGURDARDOTTIR
Christina Schindler
Stacey CHIN
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AstraZeneca AB
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AstraZeneca AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • compositions and methods that permit the controlled interaction of polypeptides to which a target protein and binding protein are fused.
  • the compositions and methods make use of a target protein that binds to the hepatitis C virus protease inhibitor grazoprevir to form a complex, and a Tn3 binding protein that specifically binds the complex.
  • the target protein is or is derived from a viral protease, such as the HCV NS3/4A protease.
  • dimerization-inducible proteins such as split transcription factors and split chimeric antigen receptors, that contain the target protein and Tn3 binding protein.
  • the methods and compositions described herein find application, for example, in cell and gene therapy methods that involve the controlled expression and/or activation of proteins.
  • PPIs Protein-protein interactions
  • gene transcription, protein folding, protein localisation, protein degradation and signal transduction all rely on the interaction or proximity of one protein to another, or indeed several others.
  • researchers can readily monitor the functional consequences of a PPI, enabling the dissection of complex biological mechanisms.
  • the ability to control biological functions are being utilised in cell and gene therapy to control therapeutic activity, enabling safer and more personalised therapies.
  • Cl Ds chemical inducers of dimerization
  • FKBP12 an immunosuppressive drug derived from Streptomyces hygroscopicus
  • FRB a domain from mTOR (mammalian target of rapamycin)
  • rapamycin along with other naturally-occurring Cl Ds, such as the plant hormones S-(+)-abscisic acid (ABA) and gibberellin (GA3-AM), is its co-operative binding mechanism whereby protein 2 can only bind to the protein 1 :CID complex (Banaszynski, Liu & Wandless, 2005).
  • WO 2021/009692 describes a system using a modified HCV NS3/4A protease as the target protein and the small molecule inhibitor of NS3/4A simeprevir as the CID. Summary
  • simeprevir has a short half-life in mice, which poses a challenge to pre-clinical development of simeprevir as a CID in pharmaceuticals.
  • grazoprevir another HCV NS3/4A protease inhibitor
  • has a superior pharmacokinetic profile in mice and have developed a new molecular switch for PPI control utilising grazoprevir as a CID.
  • Use of grazoprevir is also advantageous from a regulatory perspective, as unlike simeprevir, it has been investigated in a clinical trial as a monotherapy.
  • the inventors have also found that in some cases, grazoprevir can be substituted for glecaprevir without changing the other components of the molecular switch.
  • Tn3 protein that binds specifically to an HCV NS3/4A protease (“T”) complexed with a small molecule (“SM”) [T-SM complex], wherein the small molecule is grazoprevir, glecaprevir or an analogue or derivative thereof.
  • T HCV NS3/4A protease
  • SM small molecule
  • the HCV NS3/4A protease may have the sequence set forth in SEQ ID NO: 26, or may be a derivative thereof as described below.
  • the HCV NS3/4A protease is a small, monomeric protein that can be expressed cytoplasmically and has a limited number of endogenous human targets, therefore making it an ideal target protein.
  • the HCV NS3/4A protease is also, interchangeably, referred to herein as the “target protein” (“T”).
  • Potential off-target activity caused by overexpression of the HCV NS3/4A protease can be mitigated by using a modified target protein that has attenuated protease activity compared to the native NS3/4A protease from which it is derived.
  • the target protein has attenuated protease activity compared to the native HCV NS3/4A protease.
  • the target protein may contain one or more amino acid mutations compared to the HCV NS3/4A protease, for example at one or more amino acids selected from those at the positions corresponding to positions 72, 96, 112, 114, 154, 160 and 164 of SEQ ID NO: 26.
  • the target protein may have an amino acid mutation at the position corresponding to position 154 of SEQ ID NO: 26, such as a mutation to alanine.
  • positions 72, 96, 112, 114, 154, 160 and 164 of SEQ ID NO: 26 correspond to positions 57, 81 , 97, 99, 139, 145 and 149, respectively, of the full length NS3 protein set forth in SEQ ID NO: 34.
  • the target protein may comprise an affinity reducing amino acid substitution at one or both of the amino acid positions corresponding to positions 151 and 183 of SEQ ID NO: 26.
  • the affinity reducing amino acid mutation at the position corresponding to position 151 of SEQ ID NO: 26 is a mutation to aspartic acid, asparagine or histidine (e.g. aspartic acid or asparagine) and the affinity reducing mutation at the position corresponding to position 183 of SEQ ID NO: 26 is to glutamic acid, glutamine or alanine (e.g. glutamic acid).
  • the target protein may comprise the affinity reducing amino acid mutation in addition to other mutations described herein, such as the amino acid mutation at one or more amino acids selected from those at the positions corresponding to positions 72, 96, 112, 114, 154, 160 and 164 of SEQ ID NO: 26.
  • Binding of the Tn3 protein (also and interchangeably referred to herein as “the binding protein”) to the T-SM complex forms a tripartite complex made up of the binding protein, target protein and grazoprevir and the formation of this tripartite complex can be controlled by the presence of grazoprevir.
  • the controlled formation of the tripartite complex is useful as, for example, it permits the controlled interaction of polypeptides to which the target protein and binding member are fused.
  • split GFP split luciferases
  • split kinases e.g. NanoBiT
  • a further example describes a split transcription factor, whereby the distinct DNA binding domain (DBD) and transcription regulatory domain (TRD) (such as an activation domain (AD)) are separated such that the individual transcription factor domains alone cannot initiate transcription. Only when the two domains are brought into close proximity are they able to reconstitute the transcriptional activation of relevant genes (i.e. they form a functional “transcription factor”).
  • DBD DNA binding domain
  • TRD transcription regulatory domain
  • AD activation domain
  • Dimerization-deficient proteins are proteins that require dimerization for activity, but their endogenous dimerization capacity has been disabled e.g. via mutation or removal of the dimerization domain(s).
  • One such example is the iCasp9 molecule, a caspase-9 protein that has had its dimerization (CARD) domain removed.
  • Split complexes denote either single proteins or 2 or more different proteins that are not optimally functional or function differently, until they are brought into close proximity, or “clustered”.
  • CAR split chimeric antigen receptor
  • specific intracellular domains of the CAR that are responsible for the activation of cell signalling are physically separated such that full cellular activation is prevented. Once the domains are brought into close proximity, cell signalling is activated (i.e. they form a fully functional CAR).
  • a dimerization-inducible protein comprising: a) a first fusion protein, comprising a first component polypeptide fused to the binding protein of the first aspect; and b) a second fusion protein, comprising a second component polypeptide fused to an HCV NS3/4A protease.
  • the dimerization-inducible protein is a split transcription factor.
  • the first component polypeptide comprises a DNA binding domain and is fused to the target protein to form a DBD-T (DBD-target protein) fusion protein
  • the second component polypeptide comprises a transcriptional regulatory domain and is fused to the binding protein to form a TRD-BM (transcriptional regulatory domain-binding molecule) fusion protein
  • the first component 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 protein to form a DBD-BM fusion protein; wherein the first and second components form a transcription factor upon dimerization.
  • the dimerization-inducible protein is a split CAR.
  • the first component polypeptide comprises a first co-stimulatory domain and is fused to the target protein; and the second component polypeptide comprises an intracellular signalling domain and is fused to the binding protein; or (2) the first component polypeptide comprises an intracellular signalling domain and is fused to the target protein, and the second component polypeptide comprises a first co-stimulatory domain and is fused to the binding protein.
  • the component polypeptide comprising the first co-stimulatory domain further comprises an antigen-specific recognition domain and a transmembrane domain
  • the component polypeptide comprising the intracellular signalling domain further comprises a transmembrane domain and a second co-stimulatory domain.
  • the first and second component polypeptides form a chimeric antigen receptor (CAR) upon dimerization.
  • CAR chimeric antigen receptor
  • the dimerization-inducible protein is a dimerization-deficient caspase.
  • the first component polypeptide comprises a first caspase component and the second component polypeptide comprises a second caspase component, and the first and second component polypeptides form a caspase upon dimerization.
  • the dimerization-deficient caspase is iCasp9.
  • nucleic acid encoding a polypeptide comprising the binding protein of the first aspect, the BM-T fusion protein of the second aspect or the dimerization-inducible protein of the third aspect.
  • an expression vector comprising an expression cassette encoding: a) a first polypeptide comprising the binding protein of the first aspect; and b) a second polypeptide comprising the target protein.
  • an expression vector comprising: a) a first expression cassette encoding a first polypeptide comprising the binding protein of the first aspect; and b) a second expression cassette encoding a second polypeptide comprising the target protein.
  • a vector set comprising a first expression vector and a second expression vector, wherein the first expression vector comprises a first expression cassette and the second expression vector comprises a second expression cassette, and: a) the first expression cassette encodes a first polypeptide comprising the binding protein of the first aspect; and b) the second expression cassette encodes a second polypeptide comprising the target protein.
  • one or more expression vectors comprising: a) a first expression cassette encoding a first polypeptide comprising the binding protein of the first aspect; and b) a second expression cassette encoding a second polypeptide comprising the target protein.
  • the expression vector is a viral vector, such as an AAV vector.
  • a viral particle comprising a viral genome comprising an expression cassette as defined in the sixth or seventh aspect.
  • the approach described herein where the target protein and binding protein are fused to component polypeptides of a split transcription factor could have use in gene therapy methods that involve regulating the expression of a desired expression product (e.g. a desired polypeptide) in a cell.
  • a desired expression product e.g. a desired polypeptide
  • a method of regulating the expression of a desired expression product (or a target gene) in a cell comprising: i) expressing the dimerization-inducible protein defined herein in the cell, wherein the first and second components form a transcription factor upon dimerization, and wherein the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating expression of the desired expression product in the cell; and ii) administering grazoprevir or glecaprevir, or an analogue or derivative thereof, to the cell in order to regulate expression of the desired expression product.
  • the DNA binding domain target sequence is located in a promoter that is operably linked to a coding sequence for the desired expression product.
  • the method may involve delivery of the expression cassette or cassettes encoding the dimerization-inducible protein to control expression of a desired expression product that is also delivered exogenously to the cell.
  • the method comprises administering an additional expression cassette to a cell, wherein the additional expression cassette encodes the desired expression product, and wherein the additional expression cassette comprises the target sequence of the DNA binding domain.
  • the method may involve delivery of the expression cassette or cassettes encoding the dimerization-inducible protein to control 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).
  • the target sequence is located in the genome of the cell.
  • This method thus provides a method of treating a subject in need thereof by gene therapy, the method comprising administering to the subject the expression vector, vector set or one or more expression vectors of the sixth to tenth aspects, or the viral particle, viral particle set or one or more viral particles of the eleventh to thirteenth aspects, and optionally an additional expression cassette encoding the target sequence for the DNA binding domain, and grazoprevir or glecaprevir (or an analogue or derivative thereof).
  • the additional expression cassette may be encoded on an expression vector according to the sixth to tenth aspects, or on a separate expression vector, or in the genome of a viral particle according to the eleventh to thirteenth aspects, on in the genome of a separate viral particle.
  • grazoprevir or glecaprevir for use in a method of regulating the expression of a target gene in a cell in a subject, wherein the cell expresses a dimerization-inducible protein as described herein, wherein the first and second components form a transcription factor upon dimerization, and the DBD binds to a target sequence in the cell such that the transcription factor is capable of regulating the expression of the target gene, and the method comprises administering grazoprevir or glecaprevir (or an analogue or derivative thereof) to the subject.
  • the method may further comprise administering the expression vector, vector set or one or more expression vectors of the sixth to tenth aspects, and optionally an additional expression cassette encoding the target sequence for the DNA binding domain, to the subject.
  • the method may further comprise administering the viral particle, viral particle set or one or more viral particles of the eleventh to thirteenth aspects, and optionally an additional expression cassette encoding the target sequence for the DNA binding domain, to the subject.
  • the approach described herein could have use in methods of cellular therapy.
  • Such methods typically involve taking cells from an individual (autologous cells), modifying the cells ex vivo to express a particular protein, e.g. a dimerization-inducible protein, and administering the cells back into the individual.
  • the cells may be taken from a different individual to the subject to be treated (allogeneic cells). Allogeneic cells may be modified and administered to the subject to be treated in the same manner as autologous cells.
  • the present disclosure provides a method of treatment of a subject by cellular therapy, the method comprising: i) administering a cell comprising the expression cassette or cassettes encoding the dimerization-inducible protein as defined herein to a subject in need thereof; and ii) administering grazoprevir or glecaprevir, or an analogue or derivative thereof, to the subject.
  • the dimerization-inducible protein is a split CAR.
  • the expression vector, vector set or one or more expression vectors of the sixth to tenth aspects, or the viral particle, viral particle set or one or more viral particles of the eleventh to thirteenth aspects for use in a method of treatment of a subject, i.e. a method of treatment of a human or animal body.
  • This aspect may alternatively be seen as providing the expression vector, vector set or one or more expression vectors of the sixth to tenth aspects, or the viral particle, viral particle set or one or more viral particles of the eleventh to thirteenth aspects, for use in therapy.
  • provided herein is the expression vector, vector set or one or more expression vectors of the sixth to tenth aspects, or the viral particle, viral particle set or one or more viral particles of the eleventh to thirteenth aspects for use in gene therapy.
  • the cell of the fourteenth aspect for use in a method of treatment of a subject, i.e. a method of treatment of a human or animal body.
  • This aspect may alternatively be seen as providing the cell of the fourteenth aspect for use in therapy.
  • provided herein is the cell of the fourteenth aspect for use in cellular therapy.
  • the approach described herein could also have use in methods of inducing cell death in a target cell.
  • Such methods typically involve expression of a dimerization-inducible protein in the target cell, which dimerization-inducible protein induces cell death upon dimerization.
  • the dimerization-inducible protein for this purpose may be a caspase, the functionality of which is initiated only upon dimerization of the dimerization-inducible protein, e.g. the dimerization-inducible protein may be a dimerization deficient apoptotic protein, such as iCasp9.
  • a method of inducing death of a target cell wherein the target cell expresses a dimerization-inducible protein of the third aspect which, upon dimerization, forms a functional caspase, the method comprising contacting the cell with grazoprevir or glecaprevir, or an analogue or derivative thereof.
  • Such methods find particular utility in the context of cellular therapy, where overactivity of administered immune cells can result in cytokine release syndrome (CRS) and/or and immune-effector-cell-associated neurotoxicity syndrome (ICANS).
  • CRS cytokine release syndrome
  • ICANS immune-effector-cell-associated neurotoxicity syndrome
  • a dimerization-inducible protein which, upon dimerization, yields a functional caspase, can provide such a kill switch.
  • a method of inducing death of a target cell in a subject wherein the target cell expresses a dimerization-inducible protein of the third aspect which, upon dimerization, forms a functional caspase, the method comprising administering grazoprevir or glecaprevir, or an analogue or derivative thereof, to the subject.
  • grazoprevir or glecaprevir for use in a method of inducing death of a target cell in a subject, wherein the target cell expresses a dimerization-inducible protein of the third aspect which, upon dimerization, forms a functional caspase, and the method comprises administering grazoprevir or glecaprevor (or an analogue or derivative thereof) to the subject.
  • kits comprising grazoprevir or glecaprevir, or an analogue or derivative thereof, and: a) the expression vector, vector set or one or more vectors of any of the sixth to tenth aspects; b) the viral particle, viral particle set or one or more viral particles of any of the eleventh to thirteenth aspect; c) the cell of the fourth or fourteenth aspect; or d) the nucleic acid of the fifth aspect.
  • an additional small molecule (termed herein as a “competing small molecule”) to induce disassembly of a tripartite complex formed between the binding protein, target protein and grazoprevir/glecaprevir.
  • This may be useful, for example, where it is desirable to rapidly inactivate grazoprevir-induced dimerization, such as in order to turn off transgene expression or therapeutic activity associated with activity of a dimerization-inducible protein.
  • a method of inducing disassembly of a tripartite complex comprising administering a competing small molecule to a cell comprising the tripartite complex, wherein the tripartite complex is formed between a binding protein of the first aspect and a complex formed of a target protein and a small molecule (T-SM complex), and wherein the competing small molecule is capable of binding the target protein in the T- SM complex and displacing grazoprevir or glecaprevir from the T-SM complex.
  • T-SM complex small molecule
  • Methods of determining whether the competing small molecule is capable of binding to the target protein in the T-SM complex and displacing grazoprevir include assays where a pre-formed tripartite complex is generated and the ability of the binding protein to bind the T-SM complex is measured (e.g. by a homogeneous time-resolved florescence (HTFR) binding assay) as increasing concentrations of the competing small molecule are added.
  • HTFR time-resolved florescence
  • a competing small molecule may be capable of displacing grazoprevir from the T-SM complex if it is capable of inhibiting binding of the binding protein to the T-SM complex by at least 50 %, by at least 75 %, by at least 80 %, by at least 85 %, by at least 90 % or by at least 95 % when measured using the HTFR binding assay.
  • the competing small molecule may be another HCV NS3/4A protease inhibitor, such as simeprevir, asunaprevir, paritaprevir, vaniprevir or danoprevir.
  • Figure 1 shows a comparison of the pharmacokinetics in Balb/c mice of simeprevir dosed orally at 200 mg/kg and grazoprevir dosed orally at 20 mg/kg. Both the Cmax and exposure of unbound compound as determined by AUC are greater for grazoprevir compared to a 10-fold higher dose of simeprevir.
  • FIG. 2 shows the HTRF data obtained with a panel of Tn3 molecules that demonstrate HCV NS3/4A PR (S139A):grazoprevir-selective binding (and the HCV NS3/4A PR:simeprevir complex-specific Tn3 PRSIM23 as a control).
  • (Top) Tn3s bind to HCV NS3/4A PR (S139A) in the presence of grazoprevir but (Bottom) no binding is observed in the absence of grazoprevir.
  • Figure 3 shows the dose-dependent induction of complex formation between HCV NS3/4A PR (S139A) and selected Tn3 molecules with a panel of small molecule HCV protease inhibitors.
  • Figure 4A depicts a schematic of the split transcription factor assay.
  • HCV NS3/4A PR S139A
  • selected Tn3 molecules are fused to the DNA-binding domain (DBD) and activation domain (AD) of a split transcription factor.
  • DBD DNA-binding domain
  • AD activation domain
  • the transcription factor is reconstituted and activates expression of a luciferase reporter gene.
  • Figure 4B shows the dose-dependent activation of luciferase gene expression obtained from the split transcription factor assay.
  • Figure 5B shows the data obtained from transfection plasmids encoding one, two or three copies of PRGRZ103 Tn3 fused to the DBD, HCV NS3/4A PR (S139A) fused to the AD, and the inducible IL-2 transgene, indicating that an increase in copy number has an additive effect on the IL-2 expression induced by grazoprevir.
  • Figure 6A depicts a schematic of the split CAR and its activation.
  • Figure 6B shows the dose-dependent increase in IL-2 release, as a marker of T cell activation, from cells expressing a grazoprevir-based switch-containing split CAR in the presence of grazoprevir in antigen-positive HepG2 but not antigen-negative A375 cells.
  • Figure 7A shows the design of the grazoprevir-induced kill switch. Addition of grazoprevir results in formation of the PRGRZ103 T n3-HCV NS3/4A PR heterodimer, resulting in dimerization of caspase-9 (Casp9) activation domains and subsequent induction of apoptosis.
  • Figure 7B shows phase contrast images of HCT116 cells stably transduced with the kill switch showing rapid cell death upon treatment with grazoprevir
  • Figure 7C shows the normalized viability of HCT116 cells stably transduced with the kill switch 48 hours after grazoprevir dosing.
  • Figure 7D shows caspase-3 activity in kill switch-transduced HCT116 +/- 10 nM grazoprevir relative to treated untransduced HCT 116 cells.
  • FIG. 8 shows that the grazoprevir-based switch can be used to regulate a caspase-9- based kill switch in vivo.
  • Complete tumour regression is induced by a single 50 mg/kg dose of grazoprevir in a kill-switch-transduced HCT116 xenograft mouse model.
  • the dashed line represents the day of grazoprevir dosing. Each line represents an individual mouse.
  • Figure 9 depicts a schematic of the homology-directed repair template encoding CD47 and the grazoprevir-inducible kill switch, separated by a “self-cleaving” P2A peptide to enable CRISPR/Cas9 knock-in at the B2M locus.
  • LHA left homology arm
  • RHA right homology arm
  • hGH pA human growth hormone polyA sequence.
  • Figure 11 depicts killing of clone#76 ES cells by both cell density (quantified as % cytolysis) (A) and light microscopy (B) following treatment with grazoprevir.
  • Figure 12 depicts a time course of cell killing in ES-derived differentiated endothelial cells, following grazoprevir treatment.
  • binding protein refers to a Tn3 protein (or polypeptide) that specifically binds to the target protein-small molecule complex (T-SM complex).
  • T-SM complex target protein-small molecule complex
  • specific may refer to the situation in which the binding member will not show any significant binding to molecules other than the T-SM complex.
  • non-target molecules include the target protein alone and the small molecule alone, e.g. the target protein or grazoprevir when not part of the T-SM complex.
  • the binding member may be considered to not show any significant binding to a nontarget molecule if the extent of binding to a non-target molecule is less than about 10 % of the binding of the binding member to the T-SM complex as measured, e.g., by isothermal calorimetry, ELISA, surface plasmon resonance (SPR), Bio-Layer Interferometry (BLI), homogeneous time-resolved fluorescence (HTRF), MicroScale Thermophoresis (MST), or by a radioimmunoassay (RIA).
  • the extent of binding to a non-target molecule may be less than about 5 % or less than about 1 % of the binding of the binding member to the T -SM complex.
  • the binding member exhibits no significant binding to the target protein alone and/or grazoprevir alone. That is to say, the binding member may bind the target protein alone to less than 10 %, e.g. less than 5 % or less than 1 %, of the extent to which it binds the T-SM complex; and/or the binding member may bind grazoprevir alone to less than 10 %, e.g. less than 5 % or less than 1 %, of the extent to which it binds the T-SM complex.
  • the binding member described herein binds to the T-SM complex with an affinity that is at least 2-fold greater than the affinity towards another, non-target molecule, e.g. the target protein alone or grazoprevir alone. In some embodiments, the binding member binds to the T-SM complex with an affinity that is at least 3-, 5-, 10- or 20- fold greater than its affinity towards another, non- target molecule.
  • the binding specificity may be reflected in terms of binding affinity, where the binding member described herein binds to the T-SM complex with an affinity that is greater, e.g. at least 10-fold greater, than the affinity towards another, non-target molecule.
  • the binding protein may bind the T-SM complex with a higher affinity than it binds to either the target protein (i.e. an HCV NS3/4A protease) alone or grazoprevir alone. Binding affinity may be measured by surface plasmon resonance, e.g. Biacore.
  • the binding member binds to its target molecule with an affinity that is at least 10-, 50-, 100-, 1000- or 10000-fold greater than its affinity towards another, non-target molecule.
  • the binding member may bind to the T-SM complex with an affinity that is at least 10-, 50-, 100-, 1000- or 10000-fold greater than its affinity towards either the target protein alone and/or grazoprevir alone.
  • Binding affinity is typically measured by KD (the equilibrium dissociation constant between the 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 the T-SM complex with a KD of 1 nM would be considered to be binding the T-SM complex with an affinity that is greater than a binding member that binds with a KD of 100 nM. Similarly, a binding member that binds to the T-SM complex with a KD of 1 nM and to a non-target molecule with a KD of 100 nM would be considered to bind the T-SM complex with a greater affinity than the non-target molecule.
  • KD the equilibrium dissociation constant between the binding member and its target
  • the binding member may bind to the T-SM complex with an affinity having a KD equal to or lower than 50 nM, 25 nM, 20 nM, 15 nM or 10 nM.
  • the binding member may bind to the target protein alone or grazoprevir alone with an affinity having a KD equal to or higher than 500 nM, 1 pM, 10 pM, 100 pM, or 1 mM. Binding affinity may be measured by SPR, e.g. by Biacore.
  • the binding member may show minimal or no binding to the target protein alone and/or to grazoprevir alone when measured by SPR.
  • the binding member may thus exhibit no detectable binding to the target protein alone and/or to grazoprevir alone, by which is meant no binding of the binding protein to the target protein and/or grazoprevir can be detected by SPR. In some embodiments, the binding member may exhibit no binding at all to the target protein and/or grazoprevir.
  • the binding member specifically binds the T-SM complex at a site that is only present on the T -SM complex and not on the target protein alone or grazoprevir alone.
  • the binding member may bind to a site of the T -SM complex comprising at least a portion of the small molecule and a portion of the target protein.
  • the formation of a T-SM complex may induce a conformational change in the target protein that results in the formation of a new binding site that is specifically bound by the binding member.
  • Methods of determining the binding site in the T-SM complex of a binding member include X- ray crystallography, peptide scanning, site-directed mutagenesis mapping and mass spectrometry.
  • the binding member may specifically bind the T-SM by forming interactions with at least one of the following residues of the target protein: those corresponding to Tyr71 , Gly75, Thr76, Val93 and Asp94 of SEQ ID NO: 26.
  • the binding member may form interactions with 1 , 2, 3, 4 or most preferably all 5 of these residues.
  • the binding member may additionally specifically bind the T-SM complex by forming interactions with one or more functional groups of grazoprevir. At least some of these interactions may by hydrophobic interactions and/or water-mediated interactions. Interactions can be determined using e.g. X-ray crystallography.
  • the binding member is a Tn3 protein. That is to say, the binding member is a Tn3 protein that binds specifically to an HCV NS3/4A protease complexed with the small molecule, i.e. grazoprevir or glecaprevir, or an analogue or derivative thereof.
  • Tn3 proteins are termed herein HCV NS3/4A PR:grazoprevir complex-specific binding (PRGRZ) molecules.
  • PRGRZ PR:grazoprevir complex-specific binding
  • a number of suitable PRGRZ Tn3 proteins are demonstrated in the Examples.
  • the Tn3 protein binds specifically to the HCV NS3/4A protease complexed with grazoprevir.
  • the Tn3 proteins exemplified herein are capable of binding the HCV NS3/4A protease complexed with either grazoprevir or glecaprevir.
  • the Tn3 protein thus may bind specifically to an HCV NS3/4A protease complexed with grazoprevir or glecaprevir, in which case the Tn3 may bind more strongly to an NS3/4A-grazoprevir complex or an NS3/4A-glecaprevir complex.
  • Tn3 proteins are based on the structure of a type III fibronectin module (Fnlll) and are derived from the third Fnlll domain of human tenascin C. The generation and use of Tn3 proteins is described for example in WO 2009/058379, WO 2011/130324, WO 2011/130328 and Gilbreth et al. 2014.
  • Tn3 proteins and the native Fnlll domain from tenascin C are characterized by the same tridimensional structure, namely a beta-sandwich structure with three beta strands (A, B, and E) on one side and four beta strands (C, D, F, and G) on the other side, connected by six loop regions. These loop regions are designated according to the beta-strands connected to the N- and C-terminus of each loop.
  • the AB loop is located between beta strands A and B
  • the BC loop is located between strands B and C
  • the CD loop is located between beta strands C and D
  • the DE loop is located between beta strands D and E
  • the EF loop is located between beta strands E and F
  • the FG loop is located between beta strands F and G.
  • Fnlll domains possess solvent-exposed loops tolerant of randomization, which facilitates the generation of diverse pools of protein scaffolds capable of binding specific targets with high affinity.
  • a wild-type Tn3 protein may comprise the sequence of SEQ ID NO: 36.
  • the BC, DE and FG loops are located at positions 23 to 31 , 51 to 56 and 75 to 80, wherein the amino acid numbering corresponds to SEQ ID NO: 36.
  • the Tn3 protein may contain one, preferably two, more preferably three, even more preferably four of the stabilising mutations selected from the list consisting of I32F, D49K, E86I and T89K, wherein the amino acid numbering corresponds to SEQ ID NO: 36.
  • the amino acid sequence of a wildtype Tn3 protein comprising all four stabilising mutations is set forth in SEQ ID NO: 37.
  • the Tn3 protein may additionally contain one or more of the stabilising mutations described in Gilbreth et al. 2014 (see, in particular, Table 1 of Gilbreth et al. 2014).
  • Tn3 proteins can be subjected to directed evolution designed to randomize one or more of the loops which are analogous to the complementarity-determining regions (CDRs) of an antibody variable region.
  • CDRs complementarity-determining regions
  • the Tn3 protein that specifically binds to the T-SM complex described herein may comprise the BC, DE and FG loops of PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115.
  • the Tn3 protein may comprise the sequence of SEQ ID NO: 36 or SEQ ID NO: 37, where the BC, DE and FG loops located at positions 23 to 31 , 51 to 56, and 75 to 80, respectively, are substituted for the BC, DE and FG loops of PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115.
  • amino acid sequences of the BC, DE and FG loops of the PRGRZ Tn3 clones described herein could be compared to the amino acid sequences of the wild-type Tn3 protein, e.g. those amino acid sequences set forth in SEQ ID NO: 36 or 37.
  • Tn3 sequence, amino acid positions and sequences of the BC, DE and FG loops of PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 and PRGRZ115 are as set forth in Table 1 below.
  • the Tn3 protein comprises the BC, DE and FG loops of: a) PRGRZ093, set forth in SEQ ID NOs: 8, 9 and 10, respectively; b) PRGRZ094, set forth in SEQ ID NOs: 11 , 12 and 13, respectively; c) PRGRZ103, set forth in SEQ ID NOs: 14, 15 and 16, respectively; d) PRGRZ112, set forth in SEQ ID NOs: 17, 18 and 19, respectively; e) PRGRZ1 14, set forth in SEQ ID NOs: 20, 21 and 22, respectively; or f) PRGRZ115, set forth in SEQ ID NOs: 23, 24 and 25, respectively.
  • the Tn3 protein comprises a number of sequence alterations, e.g. one, two, three, four, or five sequence alterations, in any one or more of the BC, DE and EF loops defined above. In some embodiments, the Tn3 protein comprises a number of sequence alterations, e.g. one, two, three, four, or five sequence alterations, outside the BC, DE and EF loops defined above. Such sequence alterations may be amino acid insertions, deletions or substitutions, e.g. conservative substitutions. In particular embodiments, the Tn3 protein comprises the BC, DE and FG loops of PRGRZ103, set forth in SEQ ID NOs: 14, 15 and 16, respectively.
  • the Tn3 protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity with the amino acid sequence of: a) PRGRZ093, set forth in SEQ ID NO: 1 ; b) PRGRZ094, set forth in SEQ ID NO: 2; c) PRGRZ103, set forth in SEQ ID NO: 3; d) PRGRZ112, set forth in SEQ ID NO: 4; e) PRGRZ114, set forth in SEQ ID NO: 5; or f) PRGRZ115, set forth in SEQ ID NO: 6.
  • the Tn3 protein has or comprises an amino acid sequence of: a) PRGRZ093, set forth in SEQ ID NO: 1 ; b) PRGRZ094, set forth in SEQ ID NO: 2; c) PRGRZ103, set forth in SEQ ID NO: 3; d) PRGRZ112, set forth in SEQ ID NO: 4; e) PRGRZ114, set forth in SEQ ID NO: 5; or f) PRGRZ115, set forth in SEQ ID NO: 6.
  • the Tn3 protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity with the amino acid sequence of PRGRZ103, set forth in SEQ ID NO: 3.
  • the Tn3 protein has or comprises the amino acid sequence set forth in SEQ ID NO: 3.
  • the target protein described herein is the hepatitis C virus (HCV) NS3/4A protease (referred to herein as simply the “NS3/4A protease”).
  • HCV hepatitis C virus
  • the NS3/4A protease used herein may be a native (i.e. wild type) NS3/4A protease, or may be derived from a native NS3/4A protease.
  • the NS3/4A protease used is capable of binding grazoprevir and/or glecaprevir.
  • NS3/4A protease has a similar, but not necessarily identical, amino acid sequence to the native NS3/4A protease from which it is derived.
  • An NS3/4A protease that is derived from a native NS3/4A protease may have (i.e. consist of) or comprise 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.
  • An NS3/4A protease that is derived from a native NS3/4A protease may contain less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2 sequence alterations compared to the protein from which it is derived.
  • a target protein having the amino acid sequence set forth in SEQ ID NO: 27 is derived from the native NS3/4A protease having the sequence set forth in SEQ ID NO: 26.
  • the NS3/4A protease may have fewer amino acids (i.e. it may be a shorter protein) than the protein from which it is derived.
  • Viral proteases are enzymes encoded by the genetic material of viral pathogens. The normal function of these enzymes is to catalyse the cleavage of specific peptide bonds in viral polyprotein precursors or in cellular proteins.
  • viral proteases include those encoded by hepatitis C virus (HCV), human immunodeficiency virus (HIV), herpesvirus, retrovirus and human rhinovirus (HRV) families. Certain viral proteases, along with examples of small molecule inhibitors of these proteases, are described for example in Patick & Potts, 1998.
  • the HCV NS3/4A protease (HCV NS3/4A PR) is monomeric, relatively small in size (21 kDa), can be expressed cytoplasmically, and is not found associated with DNA, making it an ideal candidate for use herein.
  • the HCV NS3/4A protease may have the amino acid sequence of amino acid positions 1030-1206 of the amino acid sequence set forth in UniProt accession number A8DG50, the HCV polyprotein (version 2 of the sequence; sequence update 29 April 2008).
  • the terms “an HCV NS3/4A protease” and “the HCV NS3/4A protease” refer to a protein which is, or which is derived from a wild type HCV NS3/4A protease.
  • the HCV NS3/4A protease has or comprises the amino acid sequence set forth in SEQ ID NO: 26.
  • a target protein that is derived from the HCV NS3/4A protease may have or 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 set forth in SEQ ID NO: 26.
  • Table 2 HCV protease-inhibitors and their structures
  • the structures of the target proteins in complex with the respective small molecule are provided as PDB accession numbers, which correspond to the crystal structures available from the Protein Data Bank (PDB).
  • PDB accession numbers correspond to the crystal structures available from the Protein Data Bank (PDB).
  • the small molecule structures and chemical names are also provided as PDB accession numbers.
  • WO 2021/009692 describes a system in which simeprevir is used as a CID for controlling PPIs.
  • grazoprevir or glecaprevir is used, or a pharmacologically acceptable analogue or derivative of grazoprevir or glecaprevir.
  • small molecule refers to grazoprevir and glecaprevir and pharmacologically acceptable analogues or derivatives of grazoprevir or glecaprevir.
  • the small molecule is grazoprevir.
  • Grazoprevir is a small molecule that is administered orally, is cell-permeable, and has a pharmacokinetics (PK) profile that supports once-daily dosing. It is approved in combination with elbasvir (combination trade name Zepatier) for treatment of chronic hepatitis C, and has the following structure:
  • Glecaprevir is a small molecule that is administered orally, is cell-permeable, and has a pharmacokinetics (PK) profile that supports once-daily dosing. It is approved in combination with pibrentasvir (combination trade name Mavyret) for treatment of hepatitis C infection, and has the following structure:
  • Pharmacologically acceptable analogues and derivatives of grazoprevir and glecaprevir include compounds that differ from the “parent” small molecule (i.e. grazoprevir or glecaprevir having the structures set out above) but contain a similar antiviral activity as the parent small molecule and include tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other steroisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives (including prodrug forms) thereof where applicable, in context.
  • the target protein is a modified NS3/4A protease with attenuated protease activity compared to the native NS3/4A protease from which it is derived (e.g. the HCV NS3/4A protease of SEQ ID NO: 26).
  • Attenuated protease activity in this context means that the target protein has a lower enzymatic activity compared to the native NS3/4A protease.
  • Enzymatic activity can be tested, for example, using a fluorogenic peptide cleavage assay as described in WO 2021/009692 or in Sabariegos et al., 2009.
  • the fluorogenic peptide cleavage assay involves incubating the target protein with a fluorogenic protease FRET substrate containing a donor-quencher pair such that cleavage of the peptide separates the donor from the quencher, emitting energy that can be detected at a certain wavelength, e.g. 490 nm.
  • a modified NS3/4A protease is considered to have attenuated protease activity compared to the native NS3/4A protease if the target protein has an activity that is less than 10 % of the activity of the native NS3/4A protease as measured in an enzymatic activity assay, such as a fluorogenic peptide cleavage assay.
  • the target protein does not display any detectable viral activity when measured in an enzymatic activity assay, such as a fluorogenic peptide cleavage assay, when the target protein is at a concentration less than 1 nM, less than 10 nM, less than 100 nM, or less than 1 pM.
  • the target protein may comprise one or more amino acid mutations (e.g. substitutions, insertions and/or deletions) compared to the native NS3/4A protease from which it is derived (e.g. compared to SEQ ID NO: 26).
  • the target protein comprising the one or more amino acid mutations should retain its ability to form a tripartite complex with the small molecule and binding member, which can be determined e.g. using a homogeneous time-resolved fluorescence (HTRF) assay as described in WO 2021/009692.
  • HTRF time-resolved fluorescence
  • the target protein comprises one or more amino acid mutations compared to the native NS3/4A protease, 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 in the active site of the protease.
  • the HCV NS3/4A protease contains a catalytic triad involving the amino acid residues H57, D81 and S139 of the HCV NS3/4A protease, as described in 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: 26.
  • the target protein may contain an amino acid mutation at one or more amino acids selected from the position corresponding to positions 72, 96 and 154 of the HCV NS3/4A protease of SEQ ID NO: 26.
  • An amino acid position in the target protein that corresponds to a particular position in SEQ ID NO: 26 is the position in the target protein that aligns to the relevant position of SEQ ID NO: 26, if the sequence of the target protein is aligned with SEQ ID NO: 26.
  • the position in the target protein corresponding to position 154 of SEQ ID NO: 26 is the position in the target protein that aligns to position 154 of SEQ ID NO: 26.
  • the position corresponding to position 154 of SEQ ID NO: 26 may also be at position 154 in the target protein, but may be at a different position if the target protein contains an insertion or deletion mutation relative to SEQ ID NO: 26.
  • the target protein has attenuated enzymatic activity and contains an amino acid mutation (e.g. substitution) at one or more amino acids selected from the positions corresponding to positions 72, 96, 112, 114, 154, 160 and/or 164 of the HCV NS3/4A protease of SEQ ID NO: 26.
  • the target protein has attenuated enzymatic activity and comprises an amino acid mutation at the position corresponding to position 154 of the HCV NS3/4A protease of SEQ ID NO: 26, such as a mutation to alanine.
  • a mutation at this position is meant that the target protein contains a mutation at this position compared to the HCV NS3/4A protease of SEQ ID NO: 26.
  • the target protein has or comprises an amino acid sequence having at least 90 % identity to SEQ ID NO: 26, wherein the amino acid at the position corresponding to position 154 of SEQ ID NO: 26 is not serine.
  • the target protein has or comprises an amino acid sequence having at least 90 % identity to SEQ ID NO: 26, wherein the amino acid at the position corresponding to position 154 of SEQ ID NO: 26 is alanine.
  • the target protein has or comprises the amino acid sequence of SEQ ID NO: 27 (which corresponds to SEQ ID NO: 26 comprising the S154A mutation).
  • the full-length sequence of the NS3 protein is provided in SEQ ID NO: 34.
  • the amino acid mutation described here at position 154 of SEQ ID NO: 26 corresponds to the position 139 of SEQ ID NO: 34.
  • the target molecule is an HCV NS3/4A derivative comprising a mutation at the position corresponding to position 154 of SEQ ID NO: 26 as described above, and further comprises a mutation at one or more of the positions corresponding to positions 72, 96, 112, 114, 154, 160 and 164 of SEQ ID NO: 26.
  • the target protein and small molecule interact to form a complex between the target protein and the small molecule (SM) referred to herein as a T-SM complex.
  • the small molecule e.g. grazoprevir
  • the small molecule e.g.
  • grazoprevir binds to the target protein with a Kd between 25 nM and 200 nM, between 25 nM and 100 nM, or between 25 and 75 nM, when measured for example using surface plasmon resonance or bio-layer interferometry.
  • Reducing the binding affinity of the first small molecule (e.g. grazoprevir) to the HCV NS3/4A protease by introducing amino acid modification(s) in the target protein allows for the use of different small molecule inhibitors of the HCV NS3/4A protease to disrupt the tripartite complex formed between HCV NS3/4A protease (S139A), the small molecule (e.g.
  • the target protein e.g. HCV NS3/4A protease
  • the target protein comprises one or more affinity reducing amino acid mutations (e.g. substitutions) compared to the viral protease from which it is derived (e.g. SEQ ID NO: 26), such that the small molecule (e.g. grazoprevir) binds the target protein with a lower affinity than it binds the parent target protein from which it is derived.
  • the ‘parent target protein’ in this context lacks the one or more affinity reducing amino acid mutations but is otherwise identical to the target protein.
  • the parent target protein may be the native viral protease from which the target protein is derived (e.g.
  • the parent target protein may itself be derived from a viral protease (e.g. the parent target protein may be a modified viral protease, e.g. a modified HCV NS3/4A protease, such as the modified protease set forth in SEQ ID NO: 27).
  • the one or more affinity reducing amino acid mutations may result in the small molecule (e.g. grazoprevir) binding the target protein with at least a 1.5-fold lower affinity than it binds the parent target protein.
  • the one or more affinity reducing amino acid mutations may result in the small molecule (e.g. grazoprevir) binding the target protein with an affinity that is between 1.5-fold and 10-fold lower than the small molecule binds the parent target protein, or between 1.5-fold and 5-fold lower than binds the parent target protein.
  • the one or more affinity reducing amino acid mutations may result in the small molecule (e.g.
  • grazoprevir binding the target protein with a Kd between 25 nM and 200 nM, between 25 and 100 nM, or between 25 and 75 nM, optionally where affinity is measured using bio-layer interferometry, such as using an Octet RED384.
  • affinity is measured using bio-layer interferometry, such as using an Octet RED384.
  • amino acid substitutions at positions 151 and 183 of the HCV NS3/4A protease of SEQ ID NO: 26 were found to reduce the affinity of simeprevir to the HCV NS3/4A protease and allow a second small molecule to disrupt the tripartite complex formed between the HCV NS3/4A protease, simeprevir and a binding protein specific for the HCV NS3/4A-simeprevir complex.
  • the target protein has an affinity reducing amino acid mutation (e.g. substitution) at one or both amino acids selected from those at the positions corresponding to positions 151 and 183 of SEQ ID NO: 26.
  • the affinity reducing amino acid mutation at the position corresponding to position 151 of SEQ ID NO: 26 is a mutation to aspartic acid, asparagine or histidine, and/or the affinity reducing mutation at the position corresponding to position 183 of SEQ ID NO: 26 is to glutamic acid, glutamine or alanine.
  • the affinity reducing amino acid mutation at the position corresponding to position 151 of SEQ ID NO: 26 is a mutation to aspartic acid or asparagine and/or the affinity reducing mutation at the position corresponding to position 183 of SEQ ID NO: 26 is to glutamic acid.
  • the target protein may comprise the affinity reducing amino acid mutation in addition to another amino acid mutation described herein (e.g. in addition to one or more of the activity-attenuating amino acid mutations described above, such as a mutation at the position corresponding to position 154 of SEQ ID NO: 26, which may be a mutation to alanine).
  • the target protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 26, and comprises an amino acid at the position corresponding to position 151 of SEQ ID NO: 26 which is not lysine, and/or an amino acid at the position corresponding to position 183 of SEQ ID NO: 26 which is not aspartic acid.
  • the target protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 26, and comprises aspartic acid, asparagine or histidine (e.g. aspartic acid or asparagine) at the position corresponding to position 151 of SEQ ID NO: 26, and/or glutamic acid, glutamine or alanine (e.g. glutamic acid) at the position corresponding to position 183 of SEQ ID NO: 26.
  • aspartic acid, asparagine or histidine e.g. aspartic acid or asparagine
  • glutamic acid, glutamine or alanine e.g. glutamic acid
  • the target protein has or comprises the amino acid sequence of SEQ ID NO: 28 (which corresponds to SEQ ID NO: 26 comprising a K151 D mutation). In other embodiments, the target protein has or comprises the amino acid sequence of SEQ ID NO: 30 (which corresponds to SEQ ID NO: 26 comprising a D183E mutation). In other embodiments, the target protein has or comprises the amino acid sequence of SEQ ID NO: 32 (which corresponds to SEQ ID NO: 26 comprising K151 D and D183E mutations).
  • the target protein is derived from a viral protease having or comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the target protein differs from the viral protease in that it comprises alanine at the position corresponding to position 154 of SEQ ID NO: 26 and aspartic acid, asparagine or histidine (e.g. aspartic acid or asparagine) at the position corresponding to position 151 of SEQ ID NO: 26, 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 alterations (e.g. functionally conservative substitutions) relative to SEQ ID NO: 26.
  • the target protein has or comprises the amino acid sequence of SEQ ID NO: 29 (which corresponds to SEQ ID NO: 26 comprising K151 D and S154A mutations).
  • the target protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 26 and comprises alanine at the position corresponding to position 154 of SEQ ID NO: 26 and glutamic acid, glutamine or alanine (e.g. glutamic acid) at the position corresponding to position 183 of SEQ ID NO: 26.
  • the target protein has or comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 26, and comprises alanine at the position corresponding to position 154 of SEQ ID NO: 26, aspartic acid, asparagine or histidine (e.g. aspartic acid or asparagine) at position the position corresponding to position 151 of SEQ ID NO: 26, and glutamic acid, glutamine or alanine (e.g. glutamic acid) at the position corresponding to position 183 of SEQ ID NO: 26.
  • amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 26, and comprises alanine at the position corresponding to position 154 of SEQ
  • the target protein has or comprises the amino acid sequence of SEQ ID NO: 33 (which corresponds to SEQ ID NO: 26 comprising K151 D, S154A and D183E mutations).
  • polypeptide comprising a binding protein as described above fused to a target protein as described above.
  • the polypeptide may comprise a binding protein as described above fused to an HCV NS3/4A protease as described above.
  • the polypeptide is thus a fusion protein comprising the binding protein (Tn3 protein) and target protein (HCV NS3/4A protease) as described above.
  • a fusion protein is referred to herein as a binding molecule-target protein fusion (BM-T fusion)
  • BM-T fusion binding molecule-target protein fusion
  • the polypeptide of this aspect is a polypeptide comprising a BM-T fusion protein, as set out below. This aspect may alternatively be seen as providing a BM-T fusion protein.
  • the binding protein within the BM-T fusion may be any binding protein as described above.
  • the binding protein is a binding protein (i.e. Tn3 protein) that binds specifically to an HCV NS3/4A protease complexed with grazoprevir.
  • the target protein within the BM-T fusion may be any target protein as described above.
  • the binding protein and target protein may be fused directly to each other, i.e. without any intervening sequence, such that the C-terminal amino acid of the binding protein is fused to the N-terminal amino acid of the target protein, or vice versa.
  • the binding protein and target protein may be joined indirectly.
  • the binding protein and target protein may be fused via a linker, e.g. a linker as described below.
  • the target protein and binding protein may be arranged in either order, i.e. the target protein may be located N-terminal to the binding protein, or the binding protein may be located N-terminal to the target protein.
  • the BM-T fusion protein comprises one or more additional domains, i.e. one or more domains in addition to the binding protein (which, in the context of the BM-T fusion protein, may be referred to as the binding protein domain) and the target protein (which, in the context of the BM-T fusion protein, may be referred to as the target protein domain).
  • the BM-T fusion protein may comprise any number of additional domains, e.g. 1 , 2, 3, 4, 5, 6, 7 or 8 or more additional domains.
  • An additional domain may be located at the N-terminus of the BM-T fusion protein, at the C-terminus of the BM-T fusion protein, and/or between the target protein domain and the binding protein domain.
  • the fusion protein may be arranged, from N-terminus to C-terminus, as follows: N - binding protein domain - target protein domain - additional domain - C; N - binding protein domain - additional domain - target protein domain - C; N - target protein domain - binding protein domain - additional domain - C; N - target protein domain - additional domain - binding protein domain - C; N - additional domain - binding protein domain - target protein domain - C; or N - additional domain - target protein domain - binding protein domain - C.
  • the one or more additional domains may be fused to the target protein domain and/or the binding protein domain directly, as described above, or indirectly via a linker, such as one of the linkers described below.
  • the one or more additional domain is or comprises a caspase component or caspase domain.
  • the BM-T fusion protein may comprise one additional domain, which additional domain is or comprises a caspase domain.
  • the caspase domain is a domain of a dimerization-deficient caspase, as described below. That is to say, the caspase domain may be a caspase domain which is activated upon dimerization, such that when dimerized it is capable of inducing apoptosis. Thus the caspase domain may be a caspase activation domain.
  • the domain of a dimerizationdeficient caspase may be a domain of a dimerization-deficient caspase-9, in particular the activation domain of a caspase-9.
  • An exemplary caspase-9 activation domain is provided as amino acids residues 152-414 of the human caspase-9 amino acid sequence provided as NCBI accession number AAO21133.1 (version 1 ; last updated 1 December 2009), set out herein as SEQ ID NO: 35.
  • the caspase-9 activation domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % identity to SEQ ID NO: 35.
  • the BM-T fusion protein comprises a caspase-9 activation domain which is a variant of SEQ ID NO: 35 (i.e. a caspase activation domain with a sequence having at least 90 % identity to SEQ ID NO: 35)
  • the variant is a functional variant of SEQ ID NO: 35.
  • a functional variant of SEQ I D NO: 35 is a variant which retains the capability to induce apoptosis upon dimerization.
  • a functional variant of SEQ ID NO: 35 may retain at least 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % of the apoptosis-inducing activity of the native sequence of SEQ ID NO: 35.
  • the BM-T fusion protein comprises an N-terminal binding protein domain and a C-terminal caspase domain (such as one of those described above), and a target protein domain located in between.
  • An exemplary BM-T fusion protein comprising the PRGRZ103 Tn3 protein, the S154A NS3/4A target protein and the caspase-9 activation domain is set out in SEQ ID NO: 76.
  • the target protein is fused to a first component polypeptide and the binding member is fused to a second component polypeptide.
  • the first and second component polypeptides form part of a dimerization-inducible protein.
  • dimerization-inducible protein refers to a protein or complex comprising a first and second component polypeptide, wherein the first and second component polypeptides form a functional protein upon dimerization.
  • dimerizationinducible proteins includes “split proteins”, “dimerization-deficient proteins” and “split complexes”.
  • component polypeptide is intended to encompass both single-chain and multi-chain polypeptides.
  • the dimerization-inducible protein comprises (1) a first fusion protein, comprising a first component polypeptide fused to the binding protein described above; and (2) a second fusion protein, comprising a second component polypeptide fused to the target protein (e.g. HCV NS3/4A protease) as described above.
  • a first fusion protein comprising a first component polypeptide fused to the binding protein described above
  • a second fusion protein comprising a second component polypeptide fused to the target protein (e.g. HCV NS3/4A protease) as described above.
  • the first and second component polypeptides in the dimerization-inducible protein typically do not have activity or have less activity when separated, but upon dimerization are brought into close proximity and as such become active or have increased activity. Dimerization is induced by binding of the small molecule (e.g. grazoprevir) to the target protein, whereupon the binding protein binds the target protein-small molecule complex, bringing the first and second component polypeptides into proximity, resulting in dimerization.
  • the small molecule e.g. grazoprevir
  • the first and second fusion proteins contain complementary binding and target proteins, that is to say the binding protein of the first fusion protein specifically binds the target protein of the second fusion protein when the target protein of the second fusion protein is complexed with the small molecule, e.g. grazoprevir, or vice versa.
  • dimerization-inducible proteins include a split chimeric antigen receptor (split CAR; 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).
  • split CAR split CAR
  • 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
  • split reporter systems e.g. as described in Dixon et al., 2016).
  • the dimerization-inducible protein will have increased activity when the binding member is bound to the T-SM complex. Increased activity can be compared to the activity observed when the binding member is not bound to the T-SM complex (e.g. because one or more of the target protein, small molecule or binding member is not present).
  • the increased activity observed when the binding member is bound to the T-SM complex is at least a 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 as compared to activity observed when the binding member is not bound to the T-SM complex.
  • CAR activity can be determined by measuring the immune cell activation and/or proliferation in response to the antigen recognised by the CAR.
  • CAR activity can be measured by e.g. interleukin-2 (IL-2) production, e.g. by ELISA, after stimulation of the CAR by an antigen.
  • IL-2 interleukin-2
  • first and second component polypeptide form a kinase upon dimerization
  • activity of the kinase can be measured by incorporation of phosphate, e.g. radioactive 32 P, into a peptide substrate as described in Camacho-Soto et al., 2014.
  • phosphate e.g. radioactive 32 P
  • transcriptional activity can be determined by measuring expression of a downstream desired expression cassette modulated by the split transcription factor.
  • activity can be measured by using suitable assays for determining functional activity of the protein.
  • caspase activity can be measured using a caspase activity assay or by measuring apoptotic cell death.
  • reporter activity can be determined by measuring expression of the reporter, e.g. a luciferase.
  • the first component polypeptide may be fused to the C-terminus or the N-terminus of the target protein or binding member.
  • the second component polypeptide may be fused to the C-terminus or the N-terminus of the target protein or binding member.
  • the component polypeptides may be fused to the target protein or binding member via a peptide linker.
  • Suitable peptide linkers include those represented by [G]n, [S]n, [A]n, [GS]n, [GGS]n, [GGGS]n (SEQ ID NO: 38), [GGGGS]n (SEQ ID NO: 39), [GGSG]n (SEQ ID NO: 40), [GSGG]n (SEQ ID NO: 41), [SGGG]n (SEQ ID NO: 60), [SSGG]n (SEQ ID NO: 42), [SSSG]n (SEQ ID NO: 43), [GG]n, [GGGJn, [SA]n, [TGGGGSGGGGS]n (SEQ ID NO: 44), [SAGS]n (SEQ ID NO: 74) and combinations thereof, wherein n is an integer between 1 and 30.
  • n may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or any number up to 30.
  • the peptide linker may be contain any one of SEQ ID NOs: 38 to 44, or the other amino acids and short sequences set out above, as a repeating unit.
  • the component polypeptide may be fused to the target protein or binding member directly, e.g. in the format: first component polypeptide - peptide linker - target protein.
  • the component polypeptide may be fused to the target protein or binding member indirectly with one or more additional polypeptides separating the first component polypeptide from the target protein or binding member, e.g. first component polypeptide - additional polypeptide - peptide linker - target protein.
  • the first component polypeptide is fused to more than one target protein or binding member.
  • the second component polypeptide is fused to more than one target protein or binding member or a combination of both.
  • the first or second component polypeptide may be fused to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 binding members.
  • the first or second component polypeptide is fused to between 2 and 10, or between 2 and 5 binding members.
  • the first or second component polypeptide is fused to 3 binding members.
  • the first or second component polypeptide may be fused to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 target proteins.
  • the first or second component polypeptide is fused to between 2 and 10, or between 2 and 5 target proteins. In particular embodiments, the first or second component polypeptide is fused to 3 target proteins. Where multiple binding members or target proteins are present, they may be fused to each other by peptide linkers, e.g. those peptide linkers described above.
  • the first fusion protein of the dimerization-inducible protein is or comprises a BM-T fusion protein, as described above.
  • the second fusion protein of the dimerization-inducible protein is or comprises a BM-T fusion protein, as described above.
  • both the first fusion protein and the second fusion protein of the dimerization-inducible protein are or comprise a BM-T fusion protein, as described above. That is to say, the first and/or second fusion proteins may comprise a binding protein (or binding protein domain), a target protein (or target protein domain) and additional domain (which constitutes the first or second component polypeptide).
  • first and second fusion protein of the dimerization-inducible protein are identical, each constituting a BM-T fusion protein as described above, comprising the same component polypeptide as its additional domain.
  • Identical first and second fusion proteins may be particularly suitable for use as split caspases, as described further below.
  • the dimerization-inducible protein may be a split transcription factor.
  • the first component polypeptide comprises a DNA binding domain (DBD); and the second component polypeptide comprises a transcriptional regulatory domain (TRD), and wherein the first component polypeptide and second component polypeptide form a transcription factor upon dimerization.
  • DBD DNA binding domain
  • TRD transcriptional regulatory domain
  • form a transcription factor is meant that the first and second component polypeptides are brought into close enough proximity that they are able to reconstitute the transcriptional regulatory activity of desired expression products.
  • the dimerization-inducible protein will have increased transcriptional regulatory activity when the binding member is bound to the T-SM complex, compared to the transcriptional regulatory activity observed when the binding member is not bound to the T-SM complex.
  • the transcriptional regulatory domain may be a transcriptional activation domain that is capable of upregulating transcription of a gene that the split transcription factor binds to.
  • 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 The 5:3-28 (1998)), the replication and transcription activator (RTA; Lukac et al., J Virol. 73, 9348-61 (1999)), the HSV VP16 activation domain (see, e.g., Hagmann etal., J. Virol.
  • exemplary activation domains include, Oct 1 , Oct-2A, Sp1 , AP-2, and CTF1 (Seipel et al., EMBO J.
  • Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1 , C1 , AP1 , ARF-5,-6,-7, and -8, CPRF1 , CPRF4, MYC-RP/GP, and TRAB1 and a modified Cas9 transactivator protein (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.
  • the transcriptional activation domain may comprise any combination of the above exemplary activation domains. In some embodiments multiple transcriptional activation domains may be used, e.g.
  • the transcriptional activation domain is VPR, a tripartite activate made up of the VP64, p65 and Rta domains.
  • VPR a tripartite activate made up of the VP64, p65 and Rta domains.
  • An example of a TRD-T fusion protein comprising VPR is set forth in SEQ ID NO: 45 (NS4A/3 PR S139A-VPR). Generation and use of VPR as a transcriptional activator is described for example in Chavez etal. 2015.
  • the transcriptional activation domain is HSF-1 , optionally in combination with p65.
  • the human p65 activation domain may be used, which has the amino acid sequence set forth in SEQ ID NO: 72.
  • the transcriptional regulatory domain may be a transcriptional repression domain that is capable of downregulating transcription of a gene that the split transcription factor binds to.
  • Transcriptional repression domains include, but are not limited to, KRAB A/B, KOX, TGF-beta-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.
  • Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A (Chem et al. (1996) Plant Cell 8:305-321 ; and Wu et al. (2000) Plant J. 22:19-27).
  • the DNA binding domain may be any protein that binds to a target sequence in a sequence specific manner.
  • the DNA binding domain may be or may contain a transcription factor that binds to a target sequence in a sequence specific manner, or a DNA- binding fragment thereof. It is expected that any transcription factor, or DNA-binding fragment thereof, that is 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 may be or comprise a naturally occurring DNA-binding domain such as a binding domain from a human transcription factor.
  • the DNA-binding protein may be any of the human transcription factors described in Vaquerizas et al. (2009) (e.g.
  • the DNA- binding protein may be a member of the C2H2 zinc-finger family, the homeodomain family or the helix-loop-helix family or a DNA-binding fragment thereof.
  • the DNA binding domain may be zinc finger homeodomain transcription factor 1 (ZFHD1).
  • ZFHD1 contains zinc fingers 1 and 2 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. The amino acid sequence of ZFHD1 is set forth in SEQ ID NO: 73.
  • the DNA binding domain may be or comprise a DNA-binding domain such as a zinc finger DNA binding domain, a TALE DNA binding domain, a DNA binding domain from a meganuclease (e.g. based on Iscel) or a DNA binding domain from a CRISPR/Cas system.
  • These binding domains can be engineered to bind a target sequence of choice, e.g. a target sequence in a target gene that is naturally present (endogenous) in a cell or a target sequence that has been provided in trans (e.g. as part of a third expression cassette).
  • the engineering of zinc finger DNA binding domains to bind particular target sequences is described for example in US 6453242.
  • the DNA-binding domain is a TALE DNA binding domain.
  • the engineering of TALE DNA binding domain domains to bind particular target sequences is described for example in WO 2010/079430.
  • the DNA binding domain is an engineered DNA binding domain from a meganuclease.
  • the engineering of meganucleases to bind particular target sequence is described for example in WO 2007/047859.
  • a meganuclease may be engineered such that it no longer cleaves DNA.
  • the DNA binding domain is an engineered DNA binding domain from a CRISPR/Cas system.
  • the engineering of DNA binding domains from CRISPR/Cas systems to bind particular sequences is described for example in WO 2013/176772.
  • CRISPR/Cas systems generally involve an RNA-guided endonuclease (e.g. Cas9) that is directed to a specific DNA sequence through complementarity between the associated guide RNA (gRNA) and its target sequence.
  • the engineered DNA binding domain from a CRISPR/Cas system typically comprises a complex of a RNA-guided endonuclease (e.g. Cas9 or a variant thereof) and a guide RNA.
  • Variants of Cas9 have been generated that lack the endonucleolytic activity but retain the capacity to interact with DNA. See for example Chavez et al. 2015 which describes the use of nuclease-null (dCas9) variants in a method of transcriptional regulation.
  • the DNA-binding domain may include a nuclease null Cas9 variant which, upon addition of a particular gRNA specific for a target sequence, binds to the target sequence.
  • a nuclease null Cas9 variant which, upon addition of a particular gRNA specific for a target sequence, binds to the target sequence.
  • the use of a dCas9 variant as part of a split transcription factor is described in Hill et al. 2018 and WO 2018/213848.
  • the binding member may be fused to the transcriptional regulatory domain or to the DNA binding domain.
  • the first component polypeptide comprises a DNA binding domain and is fused to a target protein to form a DBD-T fusion protein; and the second component polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM fusion protein; or
  • the first component 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 a binding member to form a DBD-BM fusion protein, wherein the DNA binding domain, target protein, transcriptional regulatory domain and binding member are as defined above.
  • the first component polypeptide comprises a DNA binding domain and is fused to a target protein to form a DBD-T fusion protein
  • the target protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 26
  • the second component polypeptide comprises a transcriptional regulatory domain and is fused to a binding member to form a TRD-BM fusion protein
  • the first component polypeptide comprises a transcriptional regulatory domain and is fused to a target protein to form a TRD-T fusion protein, wherein the target protein has an amino acid sequence having at least 90 % identity to SEQ ID NO: 1
  • the second component polypeptide comprises a DNA binding domain and is fused to a binding member to form a DBD-BM fusion protein, wherein in either (1) or (2): a) the binding member comprises the BC, DE and FG loops, or Tn3 sequence, of PRGRZ093; b) the binding member comprises the BC, DE and FG loops, or Tn3 sequence, of PRGRZ094; c) the binding member comprises the BC, DE and FG loops, or Tn3 sequence, of PRGRZ103; d) the binding member comprises the BC, DE and FG loops, or Tn3 sequence, of PRGRZ112; e) the binding member comprises the BC, DE and FG loops, or Tn3 sequence, of PRGRZ114; or f)
  • 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: 46.
  • 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: 47-52.
  • 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: 53.
  • 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: 54-59.
  • Binding members may show a preference for fusion to either the DNA binding domain or the transcriptional regulatory domain, whereby transcriptional regulatory activity may be increased or decreased depending on if the particular binding member is fused to the DNA binding domain or transcriptional regulatory domain.
  • 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, for example via one or more of the peptide linkers set out above.
  • the linkers have the amino acid sequence TGGGGSGGGGS (SEQ ID NO: 44) or SA.
  • the methods and binding proteins described herein can also be applied to an activating CRISPR (CRISPRa) system.
  • CRISPRa activating CRISPR
  • This can be used, for example, to facilitate endogenous gene regulation.
  • the DBD-BM fusion protein can be guided to a target sequence through the use of particular guide RNAs that are specific for said target sequence.
  • split transcription factors comprising a DNA binding domain fused to multiple copies of the target protein or binding member exhibited increased expression relative to a split transcription factor comprising a DNA binding domain fused to a single copy of the target protein or binding member.
  • the DBD-T fusion protein comprises the DNA binding domain fused to multiple copies of the target protein (e.g. two, three, four, five or more target proteins); or the DBD-BM fusion protein comprises the DNA binding domain fused to multiple copies of the binding protein (e.g. two, three, four, five or more binding proteins).
  • the multiple binding members or multiple target proteins may be separated by a linker, for example by one or more peptide linkers as set out above.
  • the DBD-T fusion protein comprises a DNA binding domain fused to three target proteins
  • the DBD-BM fusion protein comprises a DNA binding domain fused to three binding members.
  • the first and/or second component polypeptide may additionally comprise nuclear localization signals (such as, for example, that from the SV40 medium T-antigen).
  • a split transcription factor may also be provided with a third expression cassette, wherein the third expression cassette encodes a desired expression product, wherein the DNA binding domain of the split transcription factor binds to a target sequence in the third expression cassette such that the transcription factor is capable of regulating expression of the desired expression product.
  • capable of regulating expression is meant that the DNA binding domain is able to bind the target sequence and upon forming a transcription factor with the transcriptional regulatory domain (i.e. upon dimerization of the dimerization-inducible protein), has transcriptional regulatory activity that regulates (increases or decreases) expression of the desired expression product.
  • the desired expression product can be RNA or peptidic (peptide, polypeptide or protein).
  • 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 the subject.
  • the target sequence may be located in or in close proximity to a promoter that is operably linked to a coding sequence for the desired expression product.
  • close proximity is meant that the target sequence is within 500 bp, within 250 bp, within 100 bp, within 50 bp, or within 25 bp of the sequence corresponding to the promoter.
  • the dimerization-inducible protein may be a split chimeric antigen receptor (split CAR).
  • CARs combine both antibody-like recognition with T cell-activating function. They are typically composed of an antigen-specific recognition domain, e.g. derived from an antibody, a transmembrane domain to anchor the CAR to an immune cell (e.g. T cell), a co-stimulatory domain and one or more intracellular signalling domains that induce persistence, trafficking and effector functions in transduced T cells.
  • an antigen-specific recognition domain e.g. derived from an antibody
  • T cell e.g. T cell
  • co-stimulatory domain e.g. T cell
  • intracellular signalling domains e.g. T cell
  • split CARs have been designed that require an exogenous, user-provided signal to activate the CAR, for example as described in Wu et al. 2015.
  • antigen binding and intracellular signalling components only assemble in the presence of a heterodimerizing small molecule, allowing the user to precisely control the timing, location and dosage of immune cell (e.g. T cell) activity.
  • immune cell e.g. T cell
  • split CARs are expected to mitigate toxicity, for example by inducing less off-target effects.
  • the dimerization-inducible protein comprises: a first component polypeptide comprising a co-stimulatory domain which is fused to the target protein as defined herein; and a second component polypeptide comprising an intracellular signalling domain which is fused to the binding member as defined herein.
  • the first component polypeptide set out above further comprises an antigen-specific recognition domain and a transmembrane domain and the second component polypeptide further comprises a transmembrane domain and a second co- stimulatory domain, such that the first and second component polypeptides form a chimeric antigen receptor (CAR) upon dimerization.
  • CAR chimeric antigen receptor
  • form a CAR is meant that the first and second component polypeptides are brought into close enough proximity that they are able to reconstitute a fully functional CAR.
  • the dimerization-inducible protein comprises: a first component polypeptide comprising an intracellular signalling domain and which is fused to the target protein as defined herein; and a second component polypeptide comprising a first co-stimulatory domain and which is fused to the binding member as defined herein.
  • the first component polypeptide set out above further comprises a transmembrane domain and a second co-stimulatory domain and the second component polypeptide further comprises an antigen-specific recognition domain and a transmembrane domain, wherein the first and second component polypeptides form a chimeric antigen receptor (CAR) upon dimerization.
  • CAR chimeric antigen receptor
  • the split CAR will have increased activity when the binding member is bound to the T- SM complex compared to the activity observed when the binding member is not bound to the T-SM complex.
  • the first component polypeptide comprises, from N-terminal to C-terminal: i) an antigen-specific recognition domain; ii) a transmembrane domain; and ii) a first co-stimulatory domain; and the second component polypeptide comprises, from N-terminal to C-terminal: i) a transmembrane domain; ii) a second co-stimulatory domain; and iii) an intracellular signalling domain, wherein the first component polypeptide and second component polypeptide form a CAR upon dimerization.
  • the first component polypeptide comprises from N-terminal to C-terminal: i) an antigen-specific recognition domain; ii) a transmembrane domain; and iii) a first co-stimulatory domain; and the second component polypeptide comprises from N-terminal to C-terminal: i) a transmembrane domain; ii) a second co-stimulatory domain; and iii) an intracellular signalling domain, wherein the binding member is fused to the C-terminus of the first co-stimulatory domain and the target protein is fused to the C-terminus of the second co-stimulatory domain.
  • the target protein and/or binding member may be fused directed to the respective co- stimulatory domains. More preferably, the target protein and binding member are separated from their respective co-stimulatory domains by peptide linkers.
  • the peptide linkers may be as further defined herein.
  • the target protein and binding member are separated from their respective co-stimulatory domains by a linker comprising the amino acid sequence set forth in SEQ ID NO: 61.
  • peptide linkers may separate the various domains in the first and second component polypeptides.
  • the transmembrane domain may be separated from the second co-stimulatory domain by a peptide linker, e.g.
  • a peptide linker comprising the amino acid sequence GS, and/or the second co-stimulatory domain may be separated from the intracellular signalling domain by a peptide linker, e.g. a peptide linker comprising the amino acid sequence set forth in SEQ ID NO: 61 .
  • Non-limiting examples of suitable co-stimulatory domains include, but are not limited to, activation domains from 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.
  • the first and second co-stimulatory domain is a 4-1 BB activation domain.
  • Non-limiting examples of suitable intracellular signalling domains include, but are not limited to, cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • Particular intracellular signalling domains are those that include signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM-containing signaling domains include those derived from FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signalling domain is derived from CD3 zeta (CD3Q.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions may be derived from (i.e. comprise at least the transmembrane region(s) of) 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 or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signalling domain of the CAR.
  • a glycineserine doublet provides a particularly suitable linker.
  • the transmembrane domain is derived from CD28.
  • the first and second component polypeptides may additionally include a hinge domain, such as an lgG4 or CD8a hinge domain, N-terminal to the transmembrane domains in the first and/or second polypeptides.
  • a hinge domain such as an lgG4 or CD8a hinge domain
  • Examples of hinge domains are described in, for example, Qin et al. 2017. In particular embodiments, the hinge domain is a human lgG4 hinge domain.
  • an antigen-specific recognition domain suitable for use in a dimerization-inducible protein of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art.
  • the antigen-binding domain is a single chain Fv (scFv).
  • Other antibody-based recognition domains e.g. cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use.
  • T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing v vP) are also suitable for use.
  • the antigen-specific recognition domain is a single chain Fv (scFv).
  • An scFv typically comprises 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: 61 .
  • an antigen-specific recognition domain suitable for use in a dimerization-inducible protein of the present disclosure can have a variety of antigen-binding specificities.
  • the antigen-binding domain is specific for an epitope present in an antigen that is expressed by (synthesized by) a cancer cell, i.e. a cancer cell associated antigen.
  • the cancer cell associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma cell, a glioblastoma cell, a medulloblastoma cell, a colorectal cancer cell, etc.
  • a cancer cell associated antigen may also be expressed by a non-cancerous cell.
  • the target protein used in the split-CAR is derived from an HCV NS3/4A protease.
  • the binding member is as described above, and in particular is or is based on PRGRZ103 (e.g. comprises the BC, DE and FG loops or Tn3 sequence of PRGRZ103, optionally with the sequence identity and/or alterations described herein).
  • the first component polypeptide comprises a first signal peptide located N-terminal to the antigen-specific recognition domain.
  • the first signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.
  • the second component polypeptide comprises a second signal peptide located N-terminal to the transmembrane domain.
  • the second signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 62.
  • an engineered immune cell comprising the split CAR disclosed herein.
  • the immune cell is a T-cell.
  • the immune cell is an NK cell.
  • a method of genetically modifying an immune cell to express a split CAR disclosed herein The method may be carried out ex vivo. The method may comprise administering the one or more expression vectors described herein to the immune cell such that the split CAR is expressed on the surface of the immune cell.
  • the dimerization-inducible protein may be a split reporter system.
  • the split reporter system may be an enzyme or fluorescent protein that provides an observable phenotype when the first and second component polypeptides dimerise.
  • the observable phenotype may be a colorimetic signal, a luminescent signal or a fluorescent signal.
  • split reporter systems are provided in Dixon et al. 2017.
  • the first component polypeptide comprises a first reporter component and the second component polypeptide comprises a second reporter component, wherein the first component polypeptide and second component polypeptide form a reporter system upon dimerization, optionally wherein the reporter system provides an increased colorimetric, luminescent, or fluorescent signal when the binding member is bound to the T- SM complex.
  • the dimerization-inducible protein may be a split apoptotic protein.
  • a split apoptotic protein is any protein that is capable of inducing apoptosis when the first and second component polypeptides dimerise.
  • An example of a split apoptotic protein is a split caspase or dimerization-deficient caspase (e.g. dimerization-deficient caspase-9 or dimerization-deficient caspase-3), that is capable of inducing apoptosis upon dimerization and as such can be used to kill specific cells that contain the split apoptotic protein (e.g. diseased cells, or therapeutic cells that have been administered for cell therapy purposes).
  • 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.
  • the first component polypeptide comprises a first caspase component; and the second component polypeptide comprises a second caspase component, wherein the first component polypeptide and second component polypeptide form a caspase upon dimerization.
  • the split caspase is capable of inducing cell death when the binding member is bound to the T-SM complex.
  • the first and second caspase components are identical, for example both caspase components comprise caspase-9 activation domains.
  • An exemplary caspase-9 activation domain is provided as amino acids residues 152-414 of the human caspase-9 amino acid sequence provided as NCBI accession number AAO21133.1 (version 1 ; last updated 1 December 2009), set out herein as SEQ ID NO: 35.
  • the first and second fusion proteins are both BM-T fusion proteins, each comprising a target protein domain, a binding protein domain and a caspase domain.
  • the first and second fusion proteins are identical.
  • the first and second caspase components may be encoded from the same expression cassette.
  • a split apoptotic protein may be encoded from one or more expression cassettes encoding the target protein, the binding member and the caspase-9 activation domain, where both the target protein and the binding member are fused to a caspase-9 activation domain.
  • a plurality of proteins comprising the target protein, binding member and caspase-9 activation domain are produced and dimerization of the caspase-9 activation domains (i.e. at least a first and a second caspase-9 activation domain) can be regulated through the addition of the small molecule, e.g. grazoprevir.
  • first and second fusion proteins are identical, it will be apparent to the skilled person that only a single expression cassette is required, comprising a gene encoding the fusion protein of the dimerization-inducible protein.
  • dimerization proteins contemplated for use herein include split therapeutic proteins, split TEV proteases and split Cas9.
  • a split therapeutic protein is any protein that is capable of exerting a therapeutic effect when the first and second component polypeptides of the split therapeutic protein dimerize.
  • nucleic acid molecule encoding a binding member or BM-T fusion protein as described herein, and a nucleic acid molecule or nucleic acid molecules encoding a dimerization-inducible protein as defined herein.
  • the nucleic acid molecule provided herein may be seen as comprising a nucleotide sequence encoding a binding member or BM-T fusion, and the nucleic acid molecule or nucleic acid molecules provided herein may be seen as comprising nucleotide sequences encoding a dimerization-inducible protein.
  • the nucleic acid molecule or molecules may be an isolated nucleic acid molecule or molecules. The skilled person would have no difficulty in preparing such nucleic acid molecules using methods well- known in the art.
  • the nucleic acid molecule encodes the T n3 protein PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115.
  • the amino acid sequences for those Tn3 proteins are defined herein.
  • the nucleic acid molecule or molecules comprise a nucleotide 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 with one or more of the exemplary nucleic acid sequences set forth for PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115 in the table below.
  • the nucleic acid molecule or molecules comprise a nucleotide sequence which is degenerate with one or more of the exemplary nucleic acid sequences set forth for PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115 in the table below.
  • the nucleic acid molecule or molecules comprise a nucleic acid sequence of PRGRZ093, PRGRZ094, PRGRZ103, PRGRZ112, PRGRZ114 or PRGRZ115 as set out in the table below.
  • the nucleotide sequences forthose exemplary binding members are set forth in Table 4 below:
  • the nucleic acid molecule or molecules encodes the first component polypeptide and/or second component polypeptide fused to the target protein or binding member as described above.
  • the amino acid sequences for those component polypeptides are defined herein.
  • the nucleic acid molecule or molecules encodes one or more of the DBD-T fusion protein, TRD-BM fusion protein, DBD-BM fusion protein, and TRD-T fusion protein as described above.
  • the amino acid sequences for those fusion proteins are defined herein.
  • nucleic acid molecule or molecules encode a split CAR as defined herein.
  • the nucleic acid molecule or molecules encode a split apoptotic protein as described herein, in particular a split caspase.
  • the nucleic acid molecule encodes a BM-T fusion protein comprising a caspase domain, as described above, wherein the caspase domain is dimerizable to form an active caspase.
  • the BM-T fusion protein constitutes a dimerization-inducible protein comprising identical first and second fusion proteins, as described above, and in such embodiments the nucleic acid molecule thus encodes a dimerization-inducible protein as described herein, in particular a split caspase.
  • nucleotide sequence encoding the binding protein, BM-T fusion protein or dimerization-inducible protein described herein may be provided in the context of an expression cassette, as described below. That is to say, the nucleic acid may comprise an expression cassette encoding the binding protein, BM-T fusion protein or dimerizationinducible protein described herein. An isolated nucleic acid molecule may be used to express a binding member, BM-T fusion protein or dimerization-inducible protein provided herein.
  • an “expression vector” as used herein is a DNA molecule used for expression of foreign genetic material in a cell. Any suitable vectors known in the art may be used. Suitable vectors include DNA plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). In certain embodiments, the expression vector is a viral vector as described in more detail below. In other embodiments, the expression vector is a DNA plasmid.
  • an “expression cassette” as used herein is a polynucleotide sequence that is capable of effecting transcription of an expression product, which may be a protein.
  • a “coding sequence” is intended to mean a portion of a gene’s polynucleotide sequence that encodes the expression product. Where the expression product is a protein, this sequence may be referred to as a “protein coding sequence”.
  • the protein coding sequence typically begins at the 5’ end with a start codon and ends at the 3’ end with a stop codon.
  • An expression cassette may be part of an expression vector, or part of a viral genome in a viral particle, as described in more detail below.
  • the expression cassette comprises a promoter operably linked to a protein coding sequence.
  • operably linked includes the situation where a selected coding sequence and promoter are covalently linked in such a way as to place the expression of the protein coding sequence under the influence or control of the promoter.
  • a promoter is operably linked to the protein coding sequence if the promoter is capable of effecting transcription of the protein coding sequence.
  • the resulting transcript may then be translated into a desired protein.
  • any suitable promoter known in the art may be used in the expression cassette providing it functions in the cell type being used.
  • the promoter may be a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • each coding sequence may be independently operably linked to its own promoter.
  • the coding sequence for one or more of the expression cassettes may be operably linked to the same promoter.
  • the promoter used in an expression cassette as provided herein may be a constitutive promoter or an inducible promoter.
  • An expression cassette may comprise additional elements useful for control of transcription, e.g. one or more enhancer sequences and one or more transcription termination (terminator) sequences.
  • first and second expression cassettes may be part of the same or different expression vectors.
  • first and second expression cassettes may be located on the same expression vector.
  • a first expression cassette is located on a first expression vector and a second expression cassette is located on a second expression vector.
  • the individual coding sequences may be separated by an internal ribosome entry site (IRES) or 2A element.
  • IRES or 2A elements allows multiple expression products to be expressed from a single mRNA transcript from a single promoter.
  • first and second coding sequences are separated by an IRES or 2A element, both the first and second coding sequences can be operably linked to the same promoter.
  • coding sequences for both parts of a dimerization-inducible protein as described herein may be encoded in a polycistronic (or bicistronic) expression cassette (i.e. an expression cassette which drives transcription of a single mRNA transcript encoding both components of the dimerization-inducible protein).
  • the expression vector provided herein may comprise a nucleic acid molecule as described above, or may comprise an expression cassette comprising a nucleic acid molecule as described above.
  • an expression vector comprising an expression cassette encoding a polypeptide comprising the binding protein or BM-T fusion protein as described herein.
  • the binding protein or BM-T fusion protein further comprises a first or second component polypeptide, as described above, and thus can constitute a first or second fusion protein of a dimerization-inducible protein as described herein.
  • the BM-T fusion protein additionally comprises a caspase domain as described above, wherein the caspase domain is dimerizable to form an active caspase.
  • the BM-T fusion protein constitutes part of a dimerizationinducible protein comprising identical first and second fusion proteins, as described above, and in such embodiments a single, monocistronic expression cassette thus encodes a dimerization-inducible protein as described herein, in particular a split caspase.
  • an expression vector comprising an expression cassette encoding the binding protein, BM-T fusion protein or dimerizationinducible protein flanked by targeting sequences, wherein the targeting sequences direct integration of the expression cassette into a target locus in a host cell genome.
  • the expression cassette may be flanked by sequences which, upon delivery of the vector into a cell, cause the expression cassette to be integrated into the genome of the cell at a desired site.
  • the targeting sequences may be homology arms, which are homologous to the sequences flanking the target locus, such that the expression cassette is inserted into the locus by homologous recombination. Such insertion may be referred to as “knock-in” of the expression cassette, and may be achieved, for example, using the CRISPR/Cas-9 system.
  • an expression vector encoding a split apoptotic protein e.g. a split caspase
  • the vector may comprise an expression cassette encoding a split caspase (as described above) flanked by targeting sequences.
  • the split caspase may be iCasp9.
  • the targeting locus is located in or at a target gene, which it is desired to knock out. In this way, insertion of the expression vector into the target gene can knock out the target gene, either by disrupting or replacing all or part of the target gene sequence.
  • the target gene is a gene associated with immune recognition of a cell, for instance a component of the MHC class I.
  • the target gene may be P-2-microglobulin (B2M).
  • B2M P-2-microglobulin
  • the vector comprises an expression cassette encoding a split caspase (e.g. iCasp9) targeted for integration at the B2M locus.
  • the vector may comprise an expression cassette encoding a split caspase (e.g. iCasp9) flanked by homology arms for the B2M gene locus.
  • homology arms for the B2M gene locus is meant sequences which are homologous to the B2M gene locus sequences, such that they target the expression cassette for genomic integration at the B2M gene locus and knock out the B2M gene.
  • an expression vector comprising a first expression cassette encoding a binding protein as defined herein and a second expression cassette encoding a target protein as defined herein.
  • the binding protein of the first expression cassette and/or the target protein of the second expression cassette may be encoded in the context of a BM-T fusion protein, as described herein, and/or a first or second fusion protein of a dimerization-inducible protein, as described herein.
  • the expression vector can thus encode a dimerization-inducible protein as described herein, comprising nonidentical first and second fusion proteins, each fusion protein being encoded in a separate expression cassette.
  • the first and second expression cassettes may utilise the same promoter (i.e. comprise separate copies of the same promoter) or may utilise different promoters.
  • an expression vector comprising a polycistronic (or bicistronic) expression cassette (as described above) encoding: (1) a first polypeptide comprising a binding protein as described herein, and (2) a second polypeptide comprising a target protein as described herein.
  • the binding protein of the first polypeptide and/or the target protein of the second polypeptide may be encoded in the context of a BM-T fusion protein, as described herein, and/or a first or second fusion protein of a dimerization-inducible protein, as described herein.
  • the expression vector can encode a dimerization - inducible protein as described herein, comprising non-identical first and second fusion proteins, within a single expression cassette.
  • an expression vector encoding a binding protein, BM-T fusion protein or dimerization-inducible protein, as described herein, and a second, unrelated protein.
  • the binding protein, BM-T fusion protein or dimerization inducible protein and the second protein may be encoded in separate expression cassettes, or in a polycistronic expression cassette encoding both proteins, as set out above.
  • the second protein is a protein associated with immune evasion, i.e. the expression of which by a cell enables the cell to avoid destruction by a host immune system.
  • the second protein is CD47, which, when expressed on a cell in vivo, prevents killing of the cell by NK cells.
  • the expression vector encodes a split caspase (e.g. iCasp9) and a protein associated with immune evasion.
  • the expression vector encodes a split caspase (e.g. iCasp9) and CD47.
  • the expression vector comprises a polycistronic expression cassette which encodes a split caspase (e.g. iCasp9) and CD47. In a polycistronic expression cassette, the split caspase (e.g. iCasp9) and immune evasion (e.g. CD47) genes may be separated by a 2A element.
  • the vector comprises a polycistronic expression cassette encoding a split caspase and an immune evasion protein, flanked by targeting sequences as described above.
  • the targeting sequences may be directed to a target gene associated with immune recognition, such as B2M, as described above.
  • the split caspase and immune evasion protein may be iCasp9 and CD47, respectively.
  • the vector comprises a polycistronic expression cassette encoding iCasp9 and CD47, wherein the expression cassette is flanked by targeting sequences for the B2M gene. An example of such a vector is shown in Figure 9.
  • the expression vectors described above may be provided in the context of a vector set, or vector pair.
  • the vector set or pair comprises a first expression vector comprising a first expression cassette and a second expression vector comprising a second expression cassette.
  • the first expression cassette encodes a polypeptide comprising a binding protein as described herein and the second expression cassette encodes a polypeptide comprising a target protein as described herein.
  • the binding protein and/or target protein may be encoded in the context of a BM-T fusion protein as described herein, and/or a first or second fusion protein of a dimerization-inducible protein as described herein.
  • the first expression cassette encodes a first fusion protein of a dimerization-inducible protein as described herein and the second expression cassette encodes a second fusion protein of a dimerization-inducible protein as described herein.
  • the vector set, or pair together encode a dimerization-inducible protein as described herein, and thus transfection of a cell with both the first and second expression vector of the vector set or pair can cause expression of the dimerization-inducible protein by the cell.
  • each vector may include a different selection marker.
  • one or more expression vectors comprising (1) a first expression cassette encoding a polypeptide comprising a binding protein as described herein; and (2) a second expression cassette encoding a polypeptide comprising a target protein as described herein.
  • the binding protein and/or target protein may be encoded in the context of a BM-T fusion protein as described herein, and/or a first or second fusion protein of a dimerization-inducible protein as described herein.
  • the first and second expression cassettes are comprised within a single expression vector, this may be an expression vector as described above, and where the first and second expression cassettes are comprised within more than one (e.g.
  • expression vectors these may be provided as an expression vector set or pair as described above.
  • the expression vectors are of the same type, i.e. both/all expression vectors are plasmids or viral vectors.
  • the expression vector is a viral vector.
  • Suitable viral vectors for use include adeno-associated virus (AAV) vectors, adenovirus vectors, herpes simplex virus vectors, retrovirus vectors, lentivirus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors and picornavirus vectors.
  • AAV adeno-associated virus
  • a viral vector means a DNA expression vector which comprises an expression cassette, or first and second expression cassettes as described above, such that the vector, or at least the expression cassettes, are converted into part of a viral genome, or incorporated into a viral genome, that is packaged in a viral particle when introduced into a cell alongside the necessary components for the assembly of the viral particle.
  • the viral vector comprises an additional expression cassette encoding a desired expression product which is not a binding protein, target protein or BM-T fusion protein, or a component of a dimerization-inducible protein, as described herein.
  • the expression vector is an adeno-associated virus (AAV) vector.
  • AAVs are one of the most actively investigated gene therapy vehicles and are characterized by excellent safety profile and high efficiency of transduction in a broad range of target tissues.
  • the use of AAVs as a vector for gene therapy is described in , for example, Naso et al. 2017 and Colella et al. 2018.
  • Various AAV serotypes including AAV1 , AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6.2FF, AAV8, AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can be used. Further examples of serotypes and their isolation are described in Srivastava, 2006.
  • the AAV particle is a small (25 nm) virus from the Parvoviridae family, and it is composed of a non-enveloped icosahedral capsid (protein shell) that contains a linear singlestranded DNA genome of around 4.8 kb.
  • the AAV genome encodes several protein products, namely, four non-structural Rep proteins, three capsid proteins (VP1-3), and the assemblyactivating protein (AAP).
  • the AAV genes are flanked by two AAV-specific palindromic inverted terminal repeats (ITRs).
  • the expression vector is an AAV vector
  • this may mean that the expression cassette, or first and second expression cassettes, are flanked by ITRs (e.g. ITR
  • the expression cassettes are converted into a single-stranded genome that is packaged in an AAV particle when introduced into a cell alongside the necessary components for the assembly of the AAV particle.
  • the AAV vector may be engineered, for example in order to improve its function. Examples of AAVs that have been engineered for clinical gene therapy are described in Kotterman and Schaffer, 2014.
  • AAV vectors have a packaging capacity of less than 5 kb, which can limit the size of the genetic material (e.g. expression cassettes) that can be introduced into the viral genome.
  • the small size of the expression cassette(s) encoding the tripartite complex allows for a transgene (e.g. as part of an additional expression cassette) to be introduced into the same AAV vector as the components of a split transcription factor, allowing the split transcription factor to be delivered “in cis” with the transgene.
  • a viral particle comprising a viral genome comprising an expression cassette as described above. That is to say, provided herein is a viral particle which contains a viral genome comprising an expression cassette encoding a binding protein, a binding protein and a target protein, a BM-T fusion protein or a dimerization-inducible protein as described above.
  • a viral particle comprising a viral genome comprising a first expression cassette and a second expression cassette, each as described above, the first expression cassette encoding a polypeptide comprising a binding protein as described herein and the second expression cassette encoding a polypeptide comprising a target protein as described herein.
  • the binding protein and/or target protein may be encoded in the context of a BM-T fusion protein as described herein, and/or a first or second fusion protein of a dimerization-inducible protein as described herein.
  • the first expression cassette encodes a first fusion protein of a dimerization-inducible protein as described herein and the second expression cassette encodes a second fusion protein of a dimerization-inducible protein as described herein.
  • the expression cassettes together encode a dimerization-inducible protein as described herein, and thus such a dimerization-inducible protein is encoded by the genome of the viral particle.
  • the viral particle pair or set comprises a first viral genome comprising a first expression cassette and a second viral particle comprising a second viral genome comprising a second expression cassette.
  • the first expression cassette encodes a polypeptide comprising a binding protein as described herein and the second expression cassette encodes a polypeptide comprising a target protein as described herein.
  • the binding protein and/or target protein may be encoded in the context of a BM-T fusion protein as described herein, and/or a first and/or second fusion protein of a dimerization-inducible protein as described herein.
  • the first expression cassette encodes a first fusion protein of a dimerization-inducible protein as described herein and the second expression cassette encodes a second fusion protein of a dimerization-inducible protein as described herein.
  • the viral particle set, or pair together encode a dimerization-inducible protein as described herein, and thus infection of a cell with both the first and second viral particle of the set or pair can cause expression of the dimerization-inducible protein by the cell.
  • one or more viral particles comprising one or more viral genomes encoding, between the one or more viral particles, a binding protein, a binding protein and a target protein, a BM-T fusion protein or a dimerization-inducible protein as described above.
  • one or more viral particles comprising: i) a first expression cassette encoding a target protein as described herein; and ii) a second expression cassette encoding a binding member as described herein, and wherein the first and second expression cassettes form part of a viral genome in the one or more viral particles.
  • the binding protein and/or target protein may be encoded in the context of a BM-T fusion protein as described herein, and/or a first or second fusion protein of a dimerization-inducible protein as described herein.
  • the first and second expression cassettes form part of the same viral genome of a viral particle.
  • 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.
  • the viral particle corresponds to the viral vector used to provide the viral genome encoding the binding protein, BM-T fusion protein, etc. That is to say that the viral particle is of a type for which the genome can be provided by the viral vector encoding the binding protein or suchlike.
  • the viral particle produced is an AAV.
  • the viral particle may, for instance, be an adeno-associated virus (AAV) particle, adenovirus particle, herpes simplex virus particle, retrovirus particle, lentivirus particle, alphavirus particle, flavivirus particle, rhabdovirus particle, measles virus particle, Newcastle disease virus particle, poxvirus particle or picornavirus particle.
  • the viral particle is an AAV particle.
  • AAV particles, including serotypes etc., are described above.
  • the viral genome may be a single-stranded or double-stranded nucleic acid and may be RNA or DNA.
  • the viral genome is a single stranded DNA viral genome.
  • the viral particle (optionally provided in the context of a pair or set of viral particles, or as one or more viral particles) is suitable for use in delivering its genome encoding the proteins as described above to a target cell for expression by the target cell.
  • the viral particle may be suitable for use in gene therapy, in which case it is suitable for administration to a subject (particularly a human subject) and in vivo delivery of its genome to target cells. In any event, the viral particle is generally replication deficient.
  • a method of making viral particles involves transfecting host cells such as mammalian cells with a viral vector as described herein and expressing viral proteins necessary for particle formation in the cells and culturing the transfected cells in a culture medium, such that the cells produce viral particles.
  • the viral particles may be released into the culture medium, or the method may additionally involve lysing and isolating particles from the cell lysates.
  • a suitable mammalian cell is a human embryonic kidney (HEK) 293 cell.
  • multiple plasmid expression vectors are utilised to generate the various protein components that generate the viral particles. It is also possible to make use of cell lines that constitutively express components for viral packaging, enabling the use of few plasmids. For example, construction of an AAV particle requires the Rep and Cap proteins and additional genes from adenovirus to mediate AAV replication. Making AAV particles is described for example in Robert et al. 2017. Briefly, this method involves transfection of a mammalian cell line, such as HEK293 cells, with three plasmids.
  • One vector encodes the rep and cap genes of AAV (pRepCap) using their endogenous promoters; one vector (pHelper) encodes three additional adenoviral helper genes (E4, E2A and VA RNAs) not present in HEK293 cells; and one vector (the viral vector) (pAAV-GOI) contains the one or more expression cassettes flanked by two ITRs (see Figure 2 of Robert et al.).
  • the culture medium comprising 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.
  • the viral particles may be stored, for example by freezing at -80°C ready for use by administering to a cell and/or use in therapy.
  • the disclosure also provides viral particles, such as AAV particles, for example those produced by the methods described herein.
  • a viral particle comprises a viral genome packaged within the viral envelope that is capable of infecting a cell, e.g. a mammalian cell.
  • cells comprising a nucleic acid, expression vector, expression vector set/pair, or one or more expression vectors as described herein.
  • such a cell does not express a protein encoded on the expression vector, it may be e.g. a cloning host.
  • such a cell expresses a binding protein, BM-T fusion protein and/or dimerization-inducible protein as described herein.
  • the cell comprises either the expression vector, expression vector set/pair, or one or more expression vectors as described herein, or the nucleic acid described herein comprising an expression cassette encoding a binding protein, BM-T fusion protein and/or dimerization-inducible protein as described herein.
  • the nucleic acid may in particular be a viral genome or part of a viral genome of a viral particle as described above, in which case the cell may have been infected with a viral particle or particles as described above.
  • the cell comprising the nucleic acid, expression vector, expression vector set/pair, or one or more expression vectors may comprise the nucleic acid or vector(s) extrachromasomally, or alternatively the nucleic acid or vector(s) may be integrated into the genome of the cell.
  • the cell provided herein is generally a mammalian cell, in particular a human cell.
  • a cell may be an in vitro cell (e.g. derived from a cell line) or an ex vivo cell isolated from a subject, e.g. a patient who is to receive cellular therapy or a donor of cells for cell therapy.
  • the cell is an immune cell (e.g. a T cell or NK cell) or a stem cell.
  • Suitable stem cells include embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, cardiac stem cells and mesenchymal stem cells.
  • the cell is an immune cell (e.g. T cell or NK cell) expressing a split CAR as described herein.
  • an immune cell e.g. T cell or NK cell
  • a split CAR as described herein.
  • the cell expresses a split transfection factor or reporter system as described herein.
  • the cell expresses a split apoptotic protein as described herein, in particular a split or dimerization-deficient caspase.
  • the cell may be an immune cell (e.g. T cell or NK cell).
  • the immune cell may further express an antigen receptor, e.g. a TCR or a CAR or a related receptor.
  • Such an immune cell may be for use in cellular therapy of a subject, as described further below.
  • the immune cell may be autologous or allogeneic to the subject to be treated.
  • the cell provided herein does not express a functional MHC class I.
  • such a cell may not express the B2M protein.
  • Cells which do not express B2M may be generated using a vector as described above, whereby the expression cassette encoding the binding protein, BM-T fusion protein or dimerization inducible protein is integrated into the cell’s genome at the B2M locus.
  • Such modification may in particular be made in a cell intended for use in cellular therapy, for example an immune cell.
  • Such a cell may express a split apoptotic protein, e.g. a split caspase such as iCasp9.
  • the cell provided herein additionally expresses a protein associated with immune evasion, e.g. CD47.
  • a protein associated with immune evasion e.g. CD47.
  • Expression of such a protein may be of particular value in a cell intended for use in cellular therapy, for example an immune cell.
  • a cell may express a split apoptotic protein, e.g. a split caspase such as iCasp9.
  • a split caspase such as iCasp9
  • Co-expression of these proteins may be achieved using an expression vector encoding both proteins, as described above.
  • the cell provided herein does not express a functional MHC class I, and does express a protein associated with immune evasion.
  • a cell may not express B2M, but express CD47.
  • Such cells may be generated using an expression vector encoding both proteins which also targets knock-out of B2M, as described above and shown in Figure 9. Modification of cells not to express a functional MHC class I and to express a protein associated with immune evasion may in particular be made in a cell intended for use in cellular therapy, e.g. an immune cell.
  • Such a cell may express a split apoptotic protein, e.g. a split caspase such as iCasp9.
  • Cells comprising an expression vector as provided herein may be generated by any suitable method, e.g. transformation, transfection or transduction. Such methods are well known in the art and are also further described below.
  • the genetic agents provided herein i.e. the nucleic acids, expression vectors or viral particles
  • a dimerization-inducible protein as described above may be administered to a subject in combination with the small molecule, e.g. grazoprevir as part of a method of treatment or a method of prophylaxis of a disease or disorder.
  • the small molecule e.g. grazoprevir
  • the expression vector, expression vector set and one or more expression vectors described above for use in therapy.
  • the viral particle, viral particle set and one or more viral particles described above for use in therapy.
  • the term “therapy” as used herein encompasses both treatment and prophylaxis of a disease or disorder.
  • Therapeutic use of a genetic agent as provided herein constitutes gene therapy.
  • Administration of the genetic agent(s) to the subject results in expression of the dimerization-inducible protein by cells of the subject.
  • Administration of the small molecule, e.g. grazoprevir, to the subject results in dimerization of the dimerization-inducible protein, which may have a beneficial effect on the disease condition in the individual, e.g. by causing the recipient individual to experience a reduction in symptoms of the disease or disorder being treated.
  • treatment pertains generally to treatment and therapy of a human, or alternative of a non-human animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e. , prophylaxis, prevention is also included.
  • prophylaxis in the context of the present specification should not be understood to circumscribe complete success i.e. complete protection or complete prevention. Rather prophylaxis in the present context refers to a measure which is administered in advance of detection of a symptomatic condition with the aim of preserving health by helping to delay, mitigate or avoid that particular condition.
  • the individual treated according to the therapeutic methods described herein may be referred to as a subject or (normally when referring to a human subject) a patient.
  • treatment using the genetic agents provided herein may involve expressing one or more dimerization-inducible proteins as defined herein in a cell.
  • the dimerization-inducible protein may, for example, comprise a first component polypeptide and a second component polypeptide that form a therapeutic polypeptide upon dimerization.
  • addition of the small molecule can result in the therapeutic protein having increased activity and can be used, for example, in a method of treatment of a disease where the therapeutic protein is deficient.
  • a method of regulating the expression of a target gene (which may alternatively be referred to as a target or desired expression product) in a cell, comprising (i) expressing a dimerization-inducible protein as described herein in the cell, wherein the first and second component polypeptides form a transcription factor upon dimerization, and wherein the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene in the cell, and (ii) contacting the cell with the small molecule, e.g. grazoprevir in order to regulate expression of the target gene.
  • the small molecule e.g. grazoprevir
  • This method may be performed in vitro, ex vivo or in vivo.
  • the cell When performed in vitro or ex vivo the cell can be modified to express the dimerization-inducible protein by transfection or transduction with the nucleic acid(s), expression vector(s) or viral particle(s) provided herein, and contacted with the small molecule, e.g. grazoprevir, simply by applying a solution of the small molecule to the cells.
  • the small molecule e.g. grazoprevir
  • the method may also be performed in the context of gene therapy, in which case the method is performed in vivo.
  • the method is performed in vivo, i.e. on a subject, the subject is administered the nucleic acid(s), expression vector(s) or viral particle(s) provided herein in order to cause expression of the dimerization-inducible protein in cells of the subject.
  • the subject is also administered the small molecule, e.g. grazoprevir, to cause dimerization of the dimerization-inducible protein, thereby regulating expression of the target gene.
  • the in vivo method may thus be seen as a method of regulating the expression of a target gene in a subject (or in a cell in a subject), comprising (i) expressing a dimerizationinducible protein as described herein in a cell in the subject, wherein the first and second component polypeptides form a transcription factor upon dimerization, and wherein the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene in the cell, and (ii) administering the small molecule, e.g. grazoprevir, to the subject.
  • a dimerizationinducible protein as described herein in a cell in the subject
  • the first and second component polypeptides form a transcription factor upon dimerization
  • the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene in the cell
  • administering the small molecule
  • the methods of gene therapy provided herein may be seen in a number of ways.
  • the small molecule e.g. grazoprevir for use in a method of regulating the expression of a target gene in a cell in a subject, wherein the cell expresses a dimerization-inducible protein as described herein in a cell in the subject, wherein the first and second component polypeptides form a transcription factor upon dimerization, and wherein the DNA binding domain binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene in the cell, and the method comprises administering the small molecule to the subject.
  • a dimerization-inducible protein for use in a method of regulating the expression of a target gene in a subject (or in a cell in a subject), the method comprising expressing the dimerization-inducible protein described herein in the cell, wherein the first and second component polypeptides form a transcription factor upon dimerization, and administering the small molecule, e.g. grazoprevir, to the cell in order to regulate (i.e. increase or decrease) expression of the target gene.
  • the small molecule e.g. grazoprevir
  • the method may comprise contacting a cell with one or more expression vectors or viral particles as described herein in order to express the dimerizationinducible protein in the cell.
  • the method may comprise contacting the cell with an expression product produced from the one or more expression vectors, e.g. mRNA encoding the dimerization-inducible protein.
  • contacting of the cell is achieved by administering the one or expression vectors (or expression products thereof, e.g. mRNA) or viral particles to a subject.
  • a method of regulating expression of a target gene in a subject comprising administering to the subject one or more nucleic acids, expression vectors or viral particles, as described above, which encode a dimerizationinducible protein as described above which forms a transcription factor upon dimerization, and wherein the DNA binding domain of the transcription factor binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene, such that the dimerization-inducible protein is expressed by cells within the subject; and administering the small molecule, e.g. grazoprevir, to the subject.
  • the small molecule e.g. grazoprevir
  • the small molecule e.g. grazoprevir
  • the small molecule in combination with (i) one or more nucleic acids as described above, (ii) one or more expression vectors as described above, or (iii) one or more viral particles as described above, for use in regulating expression of a target gene in a subject (or in a cell in a subject), wherein the one or more nucleic acids, expression vectors or viral particles encode a dimerization-inducible protein as described above which forms a transcription factor upon dimerization, and wherein the DNA binding domain of the transcription factor binds to a target sequence in the cell such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene.
  • the desired expression product (i.e. the product encoded by the target gene) can be RNA or a peptidic compound (peptide, polypeptide or protein).
  • 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 the subject.
  • the target gene may be an endogenous gene present in the genome of the target cell or subject.
  • the target gene may be a human gene.
  • the target gene may be a transgene (i.e. an exogenous gene) delivered to the target cell, e.g. a therapeutic transgene.
  • Regulating expression of the gene may be performed in a method of treatment or a method of prophylaxis of a disease.
  • the recipient individual may exhibit reduction in symptoms of the disease or disorder being treated. This may have a beneficial effect on the disease condition in the individual.
  • the method may further comprise contacting the cell with an additional (e.g. third) expression cassette, wherein the additional expression cassette encodes the desired expression product and wherein the additional expression cassette comprises the target sequence bound by the split transcription factor.
  • the transgene may comprise a promoter that is operably linked to a coding sequence for the desired expression product (i.e. that is operably linked to the target gene), which may be a therapeutic protein, e.g. a therapeutic antibody.
  • a therapeutic antibody is MEDI8852, having the heavy chain amino acid sequence set forth as SEQ ID NO: 64 and the light chain amino acid sequence set forth as SEQ ID NO: 65.
  • the additional expression cassette may be part of the same nucleic acid(s), expression vector(s) or viral particle(s) as the expression cassette or cassettes encoding the dimerizationinducible protein.
  • the transgene may be delivered “in cis” with the split transcription factor to the cell, such as within the same viral (e.g. AAV) particle.
  • the additional expression cassette may be part of a different nucleic acid, expression vector or viral particle to the expression cassette or cassettes encoding the split transcription factor.
  • the transgene may be delivered “in trans” with the split transcription factor to the cell, such as within separate viral (e.g. AAV) particles.
  • the split transcription factors provided herein are suitable for both “in cis” and “in trans” delivery with the transgene.
  • the methods further comprise contacting the cell with a nucleic acid, expression vector or viral particle comprising the target gene (in addition to the nucleic acid(s), expression vector(s) or viral particle(s)).
  • the methods further comprise administering to the subject a nucleic acid, expression vector or viral particle which comprises the target gene.
  • the small molecule e.g. grazoprevir
  • the small molecule e.g. grazoprevir
  • nucleic acid, expression vector or viral particle for use in regulating expression of a target gene in a subject (or in a cell in a subject), wherein the one or more nucleic acids, expression vectors or viral particles of (a) encode a dimerization-inducible protein as described above which forms a transcription factor upon dimerization, and wherein the additional nucleic acid, expression vector or viral particle of (b) comprises the target gene and a target sequence, wherein the DNA binding domain of the transcription factor binds the target sequence such that the transcription factor is capable of regulating (i.e. increasing or decreasing) expression of the target gene.
  • the target sequence may be located in or in close proximity to a promoter that is operably linked to the target gene.
  • close proximity is meant that the target sequence is within 500 bp, within 250 bp, within 100 bp, within 50 bp, or within 25 bp of the sequence corresponding to the promoter.
  • Delivery of genetic material to the cell may occur by any suitable means.
  • delivery may be by viral means, e.g. as part of a viral particle described herein, or by non-viral means.
  • Non-viral means of delivery include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, naked RNA, artificial virions, and agent-enhanced uptake of DNA.
  • the expression cassettes are delivered as mRNA.
  • the expression cassettes are delivered as DNA plasmids.
  • the nucleic acid(s), expression vector(s) or viral particles are administered to the subject.
  • the small molecule may be orally administered to a human subject, for example in an acceptable dosage form such as a capsule, tablet, aqueous suspension or solution.
  • an acceptable dosage form such as a capsule, tablet, aqueous suspension or solution.
  • the amount used will depend on the subject treated and the particular mode of administration.
  • the small molecule may be administered as a single dose, multiple doses or over an established period of time.
  • the unit dose may be calculated in terms of the dose of viral particles being administered.
  • Viral doses include a particular number of virus particles or plaque forming units (pfu) or viral genome copies (vgc).
  • particular unit doses include 10 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 viral genome copies (vgc) per kg of body weight.
  • Particle doses may be somewhat higher (10 to 100-fold) due to the presence of infection-defective particles.
  • viral particles e.g. AAV particles
  • infection and transduction of cells by viral particles is believed to occur by a series of sequential events as follows: interaction of the viral capsid with receptors on the surface of the target cell, internalization by endocytosis, intracellular trafficking through the endocytic/proteasomal compartment, endosomal escape, nuclear import, virion uncoating, and viral DNA double-strand conversion that leads to the transcription and expression of proteins encoded by the viral genome in the viral particle.
  • the one or more nucleic acid(s) expression vector(s), viral particles and the small molecule may be used (e.g., administered) alone, it is often preferable to present the individual components within a composition or formulation, e.g. with a pharmaceutically acceptable carrier or diluent.
  • the one or more viral particles may be administered as a pharmaceutical composition comprising the one or more viral particles and a pharmaceutically acceptable carrier or diluent.
  • grazoprevir may be administered as a pharmaceutical composition comprising both grazoprevir and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the agents for gene therapy may be administered to the subject simultaneously or sequentially and may be administered in individually varying dose schedules and via different routes.
  • the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the agent(s) being administered.
  • the small molecule is administered to the subject after administration of the one or more nucleic acids, expression vectors or viral particles.
  • Cellular therapy involves administering cells that have been genetically modified to express an expression product, such as a dimerizationinducible protein, to a patient.
  • an expression product such as a dimerizationinducible protein
  • stem cells such as stem cells may be used in methods of cellular therapy.
  • One potential advantage associated with using stem cells is that they can be differentiated into other cell types in vitro, and can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Suitable stem cells include embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, cardiac stem cells and mesenchymal stem cells.
  • the cellular therapy may involve administering the one or more nucleic acids, expression vectors or viral particles described herein to a cell (e.g. a stem cell) in an ex vivo method such that a dimerization-inducible protein is expressed by the cell and administering the cell to a patient.
  • a cell e.g. a stem cell
  • the small molecule e.g. grazoprevir
  • the first and second component polypeptides may form a transcription factor upon dimerization, or the first and second component polypeptides may form a CAR upon dimerization.
  • a cell which expresses a dimerization-inducible protein as described above for use in therapy.
  • a cell may be a stem cell, as described above.
  • the cell is an immune cell.
  • a method of treatment comprising administering a cell expressing a dimerization-inducible protein as defined herein to a patient, the method comprising: i) administering the cell to an individual; and ii) administering the small molecule, e.g. grazoprevir, to the individual.
  • the dimerization-inducible protein may be for example a split transcription factor, a split CAR, a split apoptotic protein or a split therapeutic protein.
  • the method of treatment may be a method of treating cancer.
  • Cellular therapy may involve isolating cells from a patient (or donor), transfecting the cells with one or more expression vectors ex vivo and administering the cells to the patient.
  • Various 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 the references cited therein for a discussion of how to isolate and culture cells from patients).
  • the cellular therapy may involve isolating a cell from a patient, administering the one or more expression vectors described herein to the cell in an ex vivo method such that a dimerization-inducible protein is expressed by the cell, and administering the cell back to the patient.
  • the small molecule may be administered to the individual in order to induce dimerization of the first and second component polypeptides as described herein.
  • Such methods may be applied in the context of cellular therapy by modifying a cell in vitro or ex vivo to express a dimerization-inducible protein which, upon dimerization, forms a transcription, using a method as described above, administering the cell to the subject and administering the small molecule (e.g. grazoprevir) to the subject.
  • a dimerization-inducible protein which, upon dimerization, forms a transcription
  • the cell is an immune cell (such as a T-cell or an NK cell) and the dimerization-inducible protein expressed by the cell is a split CAR.
  • an immune cell such as a T-cell or an NK cell
  • the dimerization-inducible protein expressed by the cell is a split CAR.
  • a method of treatment comprising administering a cell expressing the dimerization-inducible protein defined herein to a patient, wherein the first and second component polypeptide form a CAR upon dimerization, the method comprising: i) administering the cell to an individual; and ii) administering the small molecule (e.g. grazoprevir) to the individual.
  • the small molecule e.g. grazoprevir
  • the method of treatment may be a method of treating cancer.
  • a cell expressing a dimerization-inducible protein for use in a method of treatment of a subject wherein the dimerization-inducible protein is a split CAR, and the method comprises administering to the subject the cell and the small molecule, e.g. grazoprevir.
  • the small molecule e.g. grazoprevir
  • the treatment comprises administering to the subject grazoprevir and a cell expressing a dimerization-inducible protein, wherein the dimerizationinducible protein is a split CAR.
  • the small molecule e.g. grazoprevir
  • a cell expressing a dimerization-inducible protein for use in a method of treatment in a subject, wherein the dimerization-inducible protein is a split CAR.
  • the split CAR is generally expressed by an immune cell, as described above. Such methods may be for treating cancer.
  • the agents for cellular therapy using a split CAR or split transcription factor may be administered in the context of one or more pharmaceutical compositions, as described above.
  • the agents for cellular therapy using a split CAR or split transcription factor may also be administered to the subject simultaneously or sequentially and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the agent(s) being administered.
  • the small molecule is administered to the subject after administration of the cell expressing a split CAR or split transcription factor. Suitable dosing regimens may be determined by a physician.
  • Cells expressing a split apoptotic protein such as a split caspase, as described above, may also be used in cellular therapy.
  • a method of inducing death of a target cell in a subject wherein the target cell expresses a dimerization-inducible protein, the dimerizationinducible protein being a split apoptotic protein (e.g. split caspase) as described above, the method comprising contacting the cell with the small molecule, e.g. grazoprevir.
  • a method may be performed in vitro or ex vivo, e.g. for research purposes, or in vivo in the context of a method of treatment.
  • a method of inducing death of a target cell in a subject wherein the target cell expresses a dimerization-inducible protein, the dimerization-inducible protein being a split apoptotic protein (e.g. split caspase) as described above, the method comprising administering the small molecule, e.g. grazoprevir, to the subject.
  • a dimerization-inducible protein e.g. split caspase
  • the small molecule for use in a method of inducing death of a target cell in a subject, wherein the target cell expresses a dimerizationinducible protein, the dimerization-inducible protein being a split apoptotic protein (e.g. split caspase) as described above, the method comprising administering the small molecule to the subject.
  • the target cell may be an immune cell, e.g. a T cell or NK cell.
  • the target cell may express an antigen receptor, such as a CAR or a TCR.
  • the target cell may be modified to express both an antigen receptor and a split apoptotic protein.
  • the subject may be undergoing cellular immunotherapy, i.e. therapy with an immune cell expressing an antigen receptor.
  • the methods of inducing death of a target cell in a subject may thus be for the treatment of CRS or ICANS in a subject.
  • the subject may have been administered an immune cell expressing an antigen receptor and a split apoptotic protein, as described above.
  • a suitable small molecule e.g. grazoprevir
  • the small molecule may be administered in a pharmaceutical formulation.
  • an immune cell e.g. a T cell or NK cell
  • an antigen receptor e.g. a CAR or TCR
  • a split apoptotic protein as described herein e.g. a split caspase
  • Such therapy may in particular be cancer therapy.
  • a method of treatment comprising administering an immune cell (e.g. a T cell or NK cell) expressing an antigen receptor (e.g. a CAR or TCR) and a split apoptotic protein as described herein (e.g. a split caspase) to a subject in need thereof.
  • the subject may have cancer.
  • the antigen receptor may recognise a cancer antigen expressed by the cells of the cancer of the subject.
  • kits that comprise: (1) the expression vector, vector set or one or more vectors; viral particles, set of viral particles or one or more viral particles, cells; or one or more nucleic acids, as defined herein; and (2) the small molecule, e.g. grazoprevir.
  • the small molecule included in the kit is the molecule which forms a complex with the encoded target protein which is recognised by the encoded binding protein.
  • the kit may additionally include a guide RNA specific for the target sequence, or a nucleic acid encoding the guide RNA specific for the target sequence.
  • GAP Garnier GCG package, Accelerys Inc, San Diego USA
  • GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4.
  • Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990)), FASTA (which uses 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 employing default parameters.
  • the psi-Blast algorithm may be used.
  • this includes the amino acid sequence having 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % and 100 % sequence identity to the reference amino acid sequence (including as rounded to the nearest integer percentage).
  • sequence alterations are intended to encompass the substitution, deletion and/or insertion of an amino acid residue.
  • a protein containing one or more amino acid sequence alterations compared to a reference sequence contains one or more substitutions, one or more deletions and/or one or more insertions of an amino acid residue as compared to the reference sequence.
  • amino acid mutation is herein used interchangeably with “sequence alteration”, unless the context clearly identifies otherwise.
  • substitutions may be conservative substitutions, for example according to the following Table.
  • amino acids in the same block in the middle column are substituted, i.e. a non-polar amino acid is substituted for another non-polar amino acid for example.
  • amino acids in the same line in the rightmost column are substituted, i.e. G is substituted for A or P for example.
  • substitution(s) may be functionally conservative. That is, in some embodiments the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. binding affinity) of the protein comprising the substitution as compared to the equivalent unsubstituted protein.
  • the binding member may comprise a variant of a BC, DE or FG loop or T n3 sequence as disclosed herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening.
  • a binding member comprising one or more variant sequences relative to those specified herein is a functional variant, i.e. a variant that retains one or more of the functional characteristics of the parent binding member (defined herein), such as binding specificity and/or binding affinity for the T- SM complex.
  • a binding member comprising one or more variant sequences preferably binds to T-SM complex with the same affinity as, a higher affinity than, or a not substantially worse affinity than, the (parent) binding member.
  • variant binding member retains at least 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % of the affinity of the parent binding member.
  • the parent binding member is a binding member which does not comprise the amino acid substitution(s), deletion(s), and/or insertion(s) which has (have) been incorporated into the variant binding member.
  • a binding member may comprise BC, DE and FG loop sequences, or a Tn3 sequence, which 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 BC, DE and FG loop sequences, or a Tn3 sequence, as disclosed herein.
  • a binding member may comprise BC, DE or FG loop sequences, or a Tn3 sequence, which has one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with the BC, DE and FG loop sequences, and the Tn3 sequences, disclosed herein.
  • Example 1 Identification of grazoprevir and HCV NS3/4A PR as the basis for a chemical inducer of dimerization (CID) module
  • dimerization modules comprised of the catalytically inactive S139A mutant of the NS3/4A protease from hepatitis C virus (HCV NS3/4A PR (S139A), SEQ ID NO: 27), the antiviral compound simeprevir and proteins (termed PRSIM molecules) which bind selectively to the HCV NS3/4A PR (S139A):simeprevir complex (WO 2021/009692).
  • Grazoprevir is clinically approved for treatment of HCV in a fixed dose combination with the HCV NS5A inhibitor elbasvir. It is cell permeable, orally dosed and exhibits pharmacokinetics in humans which render it suitable for chronic daily dosing, all desired properties for a small molecule inducer. Furthermore, we have investigated the pharmacokinetics of grazoprevir in Balb/c mice, and have shown that grazoprevir exhibits superior pharmacokinetics in mice after oral dosing compared to simeprevir (Fig. 1), with a 10-fold lower dose of grazoprevir giving a 4.6- fold higher peak serum concentration (Cmax) and a 2-fold higher exposure of unbound compound based on AUC, thus facilitating in vivo studies in mice.
  • Fig. 1 simeprevir
  • Example 3 A panel of PRGRZ molecules are selective for the HCV NS3/4A PR ( S 139 A ) :grazoprevir complex
  • the PRGRZ binding proteins identified from phage display selections as complex-specific were expressed and purified at larger scale to provide sufficient material for further analysis.
  • a homogeneous time-resolved fluorescence (HTRF) binding screen was performed on all the HCV NS3/4A PR (S139A):grazoprevir complex-specific PRGRZ molecules and the PRGRZ molecules were confirmed as complex-specific with no detectable binding to the HCV NS3/4A PR (S139A) protein alone (Fig. 2).
  • HCV NS3/4A PR S139A:simeprevir complex-specific Tn3 protein PRSIM23 (SEQ ID NO: 7, WO 2021/009692) was used, which showed no binding to the HCV NS3/4A PR (S139A):grazoprevir complex.
  • HCV NS3/4A PR (S139A) protease binding in the presence or absence of grazoprevir were determined using Biacore 8K (Table 5). All the PRGRZ binding molecules tested showed selectivity for grazoprevir-bound HCV NS3/4A PR (S139A) with no binding observed to HCV NS3/4A PR (S139A) in the absence of grazoprevir.
  • Table 5 Binding and kinetic constants measured for the binding of HCV NS3/4A PR (S139A) to PRGRZ binding molecules in the presence of 10 nM grazoprevir.
  • N.D. indicates the values could not be determined due to absence of detectable binding
  • HCV NS3/4A PR (S139A):PRGRZ complex formed when grazoprevir was substituted with the alternative HCV PR inhibitor small molecules. All PRGRZ molecules exhibit a high level of selectivity for complex formation with HCV NS3/4A PR (S139A):grazoprevir compared to complexes of HCV NS3/4A PR (S139A) and simeprevir, paritaprevir, danoprevir, asunaprevir, or vaniprevir, with no or minimal complex formed.
  • HCV NS3/4A PR inhibitor small molecules with the exception of glecaprevir, such as in the case of a HCV-infected individual, would not be able to form an active HCV NS3/4A PR (S139A):PRGRZ103 complex.
  • PRGRZ-based CIDs can regulate gene expression via reconstitution of a split transcription factor
  • the PRGRZ-based CID constructs demonstrated dose-dependent gene expression regulation ranging from 160- to 2260-fold maximum induction with EC50s from ranging from 3.0 to 16.1 nM (Fig. 4B and Table 7).
  • the PRGRZ103-based CID demonstrated a 2-fold greater fold induction of luciferase activity compared to the PRSIM23-based switch induced by simeprevir, albeit with a 2-fold weaker EC50.
  • Table 7 EC50 and fold-change values for PRGRZ-based CIDs in a split transcription factor assay.
  • Example 5 Transgene expression can be tuned by changing the copy number of PRGRZ molecules fused to the DBD
  • a PRGRZ-based CID can regulate activity of a split chimeric antigen receptor (CAR)
  • Example 7 A PRGRZ-based CID can regulate the activity of an apoptotic protein to control cell death in tumour cell lines
  • the ability to “remotely control” therapeutic cells once they have been administered provides a safety net in the advent of uncontrolled proliferation or adverse event.
  • One way to control such cells is to endow them with a so-called “kill switch” such that they can be removed at will once they have performed their function or pose a safety risk.
  • a PRGRZ-based, grazoprevir-responsive Caspase-9-based kill switch was generated.
  • the homo-dimerisation CARD domain of Caspase-9 was replaced with both the PRGRZ103 and HCV NS3/4A PR (S139A) domains, separated by short linkers of SEQ ID NO: 75.
  • An active Caspase-9 homodimer can thus only be reconstituted by addition of grazoprevir (Fig. 7A).
  • Caspase-3 activity detected by cleavage of fluorogenic substrate Ac-DEVD-AMC, is significantly (p ⁇ 0.0001) up-regulated in 10 nM grazoprevir-treated kill switch-transduced HCT116 cells (Fig. 7D).
  • Example 8 A PRGRZ-based CID can regulate the activity of an apoptotic protein to control cell death in both undifferentiated and differentiated ES cells
  • Resulting cells were expected to express both CD47 (to prevent NK cell killing) and the kill switch, with concomitant lack of expression of B2M (to prevent recognition by T cells).
  • Individual clones were selected by fluorescence-activated cell sorting (FACS) for the presence of CD47 and absence of B2M. Furthermore, integration of the cassette at the B2M locus was detected by PCR amplification ( Figure 10). Based on this data clone #76 was chosen for further analysis.
  • both clone #76 and the wild-type ESC cell lines were treated with a single dose of 10 nM or 100 nM grazoprevir, or vehicle-only control (DMSO). Quantification of the cell number after 48 hours was achieved using the xCELLigence instrument which was then converted into percent cytolysis using untreated samples as the reference ( Figure 11 A).
  • clone #76 demonstrated significant cytolysis (>95 % cells) under both grazoprevir treatment conditions, but not with vehicle-only control treatment, demonstrating the efficacy of the grazoprevir-inducible kill switch. This killing could also be observed by light microscopy when overgrown ES cell cultures of wild-type and clone #76 were treated with 10 nM Grazoprevir every 48 h for 3 rounds over a period of 7 days. In contrast to the wild-type cells, no clone #76 cells were detected after the 3rd round of grazoprevir treatment, confirming that sustained activation of the highly potent kill switch can kill 100 % of cells without the cells becoming resistant to grazoprevir ( Figure 11 B).
  • PRGRZ-based kill switch can efficiently and rapidly eliminate many cell types, including both undifferentiated and differentiated ES cells in vitro and in vivo, thereby providing a means for the rapid removal of therapeutic cells in patients.
  • the VSGB 2.0 model a next generation energy model for high resolution protein structure modeling. Proteins. 2011 ;79(10):2794-2812. Li, Kui, Eileen Foy, Josephine C. Ferreon, Mitsuyasu Nakamura, Allan C. M. Ferreon, Masanori Ikeda, Stuart C. Ray, Michael Gale, and Stanley M. Lemon. 2005.
  • HIV protease inhibitors a review of molecular selectivity and toxicity.
  • Upper case sequences are protein sequences.
  • Lower case sequences are nucleic acid sequences.
  • KHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 66 - PRGRZ093 agactggacgccctagccagatcgaagtgaaggacgtgaccgacaccaccgctctgatcacctggacagacccctactacg tgatctacagcttcgagctgacctacggcatcaaggacgtgcccggcgatagaaccaccatcaagctggatagcgacgtgctgt actactccatcggcaacctgaagcctgacaccgagtacgaggtgtccctgatcagcaacaccgacggcctgagagtgcggagagcctaatcctgccaagatcaccttcaagaccggcctgggaccggcctggccaagatcaccttcaagaccggc

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Abstract

L'invention concerne des compositions et des procédés qui permettent l'interaction contrôlée de polypeptides auxquels une protéine cible et une protéine de liaison sont fusionnées. Les compositions et les procédés utilisent une protéine cible qui se lie au grazoprévir, un inhibiteur de la protéase du virus de l'hépatite C, pour former un complexe, et une protéine de liaison Tn3 qui se lie spécifiquement au complexe. La protéine cible est ou est dérivée d'une protéase virale, telle que la protéase NS3/4A du VHC.
PCT/EP2024/067561 2023-06-23 2024-06-21 Commutateurs moléculaires Pending WO2024261323A1 (fr)

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