WO2020021090A1 - Agents cytotoxiques conditionnels - Google Patents

Agents cytotoxiques conditionnels Download PDF

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Publication number
WO2020021090A1
WO2020021090A1 PCT/EP2019/070229 EP2019070229W WO2020021090A1 WO 2020021090 A1 WO2020021090 A1 WO 2020021090A1 EP 2019070229 W EP2019070229 W EP 2019070229W WO 2020021090 A1 WO2020021090 A1 WO 2020021090A1
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Prior art keywords
virus
polypeptide
protease
sequence
smash
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PCT/EP2019/070229
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English (en)
Inventor
Karl-Klaus Conzelmann
Alexander Ghanem
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Conzelmann Karl Klaus
Alexander Ghanem
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Application filed by Conzelmann Karl Klaus, Alexander Ghanem filed Critical Conzelmann Karl Klaus
Priority to EP19742617.4A priority Critical patent/EP3830136A1/fr
Publication of WO2020021090A1 publication Critical patent/WO2020021090A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to a first polypeptide consisting of (i) an amino acid sequence of a Flaviviridae NS3 protease, a mutant thereof, or a fragment of said sequence or of said mutant, wherein said mutant exhibits at least 80% sequence identity to said sequence, and wherein said mutant and said fragment have substantially the same capability of binding an inhibitor of said protease as said amino acid sequence; (ii) the amino acid sequence of SEQ ID NO:1 , or a variant thereof, wherein said variant exhibits at least 80% sequence identity to said sequence of SEQ ID NO:1 , and (iii) an optional amino acid sequence of 1 to 20, preferably 10 to 12 residues between (i) and (ii), wherein the amino acid sequence of (i) is located N- terminally of the amino acid sequence of (ii), and wherein said first polypeptide triggers cell death upon administration of said inhibitor to a cell comprising said polypeptide.
  • Negative strand RNA viruses comprise numerous medically important viruses for which no specific antiviral treatment is available. Especially there is no way to pharmacologically control or eliminate most viruses of the Rhabdoviridae, Bornaviridae, and Paramyxoviridae families. In addition to the natural pathogens, genetically modified viruses of these families are in extensive use as vectors or tracers in basic research, or as biomedical tools in the form of live vaccines, oncolytic viruses, tracers, or other formulations.
  • Rabies virus of the Lyssavirus genus, is the medically most important member and a prototype of the Rhabdoviridae family. Natural variants or genetically engineered recombinant rabies viruses are in use as live rabies vaccines for wildlife, are under investigation as multivalent vaccines against rabies and other pathogens, and serve as vectors and tracers in basic research, including its gold-standard role in neuronal tracing and analysis of neuronal connections.
  • Lyssavirus infection is not leading to rapid overt cell damage, but rather causes persistent infections.
  • This non-cytolytic feature of rabies and other lyssaviruses limits approaches in which viral cytopathogenicity is highly desirable.
  • These include oncolytic virotherapy which aims at the elimination of malignant or undesired cells by direct or indirect lysis, and/or stimulating an anti-cancer immune response.
  • cytopathic viruses can induce a more efficient and comprehensive immune response, which is desirable in vaccine formulations as well as in oncolytic virotherapy.
  • Cytopathic viruses are needed for high content- and high throughput screens for essential host interaction targets (i.e. genes that support virus growth, also known as proviral host genes), antiviral host gene products (i.e. genes which defend cells against infection), and immune stimulatory pro-inflammatory and antiviral compounds.
  • essential host interaction targets i.e. genes that support virus growth, also known as proviral host genes
  • antiviral host gene products i.e. genes which defend cells against infection
  • immune stimulatory pro-inflammatory and antiviral compounds i.e. genes that support virus growth, also known as proviral host genes
  • Reliable and fast elimination of virus-infected cells and biological selection of non-permissive cells would greatly facilitate and speed up identification of druggable host cell and virus targets which are especially important for the rational development of rabies antiviral treatment.
  • the gained knowledge of anti- infectious, anti-cancer and immune-modulating therapeutic targets of the host cell and virus would enable the development of corresponding pharmaceuticals.
  • conditionally cytopathic viruses in which cell death can deliberately be induced and at any time, e.g. by addition of a drug, are highly desired in various fields, including vaccines, immune modulation, oncolytic virotherapy, as well as genetic and pharmaceutical screening.
  • SMASh small molecule-assisted shutdown
  • Rabies virus live vaccines, virotherapy Rabies is among the longest known and feared infectious diseases for humans and animals .
  • the causative agent is the highly neurotropic rabies virus (RABV), the prototype ofthe Lyssavirus genus in the Rhabdoviridae family (Walker et al. 2018).
  • RABV has a broad animal reservoir, including terrestrial animals and bats, with dogs being mainly responsible for the estimated 59,000 human deaths each year, most of which are children (Fooks et al. 2017).
  • Other members of the Lyssavirus genus also known as“rabies-related viruses” are mostly associated with bat reservoirs, but can also cause rabies-like encephalitis and death in terrestrial animals and humans (Luis et al. 2013).
  • Attenuated live vaccines are preferred over inactivated vaccines and are often needed, because they can evoke a long lasting protective immune response usually based on both humoral and cellular reactions, and they are more cost-effective.
  • Live attenuated viruses without producing disease manifestations have proven effective vaccines against DNA and positive strand RNA viruses causing smallpox, yellow fever and poliomyelitis.
  • NNSV non-segmented negative strand RNA viruses
  • VSV vesicular stomatitis virus
  • rVSV-ZEBOV Ebola virus glycoprotein
  • Live rabies vaccines are currently licensed only for wildlife animal immunization, as they still have undesired residual pathogenicity.
  • Oncolytic virotherapy and immune modulatory virotherapy exploits the cytotoxic and/or immune-stimulatory effect of viruses on cells and is emerging as a viable treatment option for cancer.
  • the rational engineering of viruses containing specificity elements that confer safety and cancer-specific infection or replication, including measles virus and VSV is ongoing (Miest and Cattaneo 2014; Buijs et al. 2015).
  • Rhabdoviruses are also under investigation as oncolytic tools, in particular the vesicular stomatitis virus (VSV) of the Vesiculovirus genus, which has already entered clinical trials.
  • VSV vesicular stomatitis virus
  • VSV-M(M51 R) or -M(DM51 ) are more immunogenic but not cytopathic per se (Ahmed, Cramer, and Lyles 2004).
  • RABV is an example of a non-cytopathic member of the Rhabdoviridae family, which so far has largely precluded its use as oncolytic tool.
  • the non-cytopathic nature of natural RABV is important for completion of the natural infection, which typically involves infection of peripheral neurons, and relies on the integrity of the neuronal network to reach the central nervous system.
  • a variety of viral traits (known as stealth strategy), including mechanisms preventing premature neuronal damage and dampening innate immune responses, provide for the necessary time to reach the CNS and replicate in the CNS (Ghanem and Conzelmann 2016; Schnell et al. 2010; Lafon 2011 ).
  • RABV stealth behavior
  • available technical possibilities including e.g. specific cell targeting of RABV or of single round G-deficient AG RABV (Ghanem and Conzelmann 2016), and making RABV sensitive to type I interferon signaling, which is often defective in cancer cells, would make RABV a perfect tool for targeting and infection of cancer cells, if only a means to kill the infected cancer cells would be available
  • virotherapy is selective targeting of RABV to cells infected with another pathogen, and their elimination, as illustrated in vitro for HIV-1 infected and Env-expressing cells (Mebatsion et al. 1997).
  • a therapeutic approach was impractical at that time, because of the non-cytopathic nature of RABV.
  • the present invention provides a first polypeptide consisting of (i) an amino acid sequence of a Flaviviridae NS3 protease, a mutant thereof, or a fragment of said sequence or of said mutant, wherein said mutant exhibits at least 80% sequence identity to said sequence, and wherein said mutant and said fragment have substantially the same capability of binding an inhibitor of said protease as said amino acid sequence; (ii) the amino acid sequence of SEQ ID NO:1 , or a variant thereof, wherein said variant exhibits at least 80% sequence identity to said sequence of SEQ ID NO: 1 , wherein the amino acid sequence of (i) is located N-terminally of the amino acid sequence of (ii) and wherein said first polypeptide triggers cell death upon administration of said inhibitor to a cell comprising said polypeptide.
  • a first polypeptide consisting of (i) an amino acid sequence of a Flaviviridae NS3 protease, a mutant thereof, or a fragment of said sequence or of said mutant, wherein said mutant exhibits at least 80% sequence identity to said sequence, and wherein said mutant and said fragment have substantially the same capability of binding an inhibitor of said protease as said amino acid sequence; (ii) the amino acid sequence of SEQ ID NO;1 , or a variant thereof, wherein said variant exhibits at least 80% sequence identity to said sequence of SEQ ID NO:1 , and (iii) an optional amino acid sequence of 1 to 20, preferably 10 to 12 residues between (i) and (ii), wherein the amino acid sequence of (i) is located N-terminally of the amino acid sequence of (ii), and wherein s aid first polypeptide triggers cell death upon administration of said inhibitor to a cell comprising said polypeptide.
  • polypeptide has its art-established meaning. As such, it refers to a polycondensate of amino acids, a-amino acids being the preferred building blocks. Particularly preferred are the twenty naturally occurring proteinogenic amino acids. Having said that, modified a-amino acids as well as a-amino acids which do not belong to the mentioned twenty proteinogenic amino acids may be present.
  • Such unusual a-amino acids include D-amino acids, in particular the D-amino acid counterparts of the mentioned twenty naturally occurring proteinogenic amino acids, and furthermore homo-amino acids (side chains are extended by the addition of one or more methylene groups), a-methyl amino acids (the hydrogen bound to the C-a carbon has been replaced with a methyl group), citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, ornithine, naphthylalanine and methionine sulfoxide.
  • b- or g-amino acids may be present.
  • protease has its art-established meaning. It relates to any enzyme capable of cleaving a polypeptide.
  • said protease originates from a member of the Flaviviridae family.
  • Flaviviridae are a family of viruses characterized by a genome which is single-stranded RNA. As such, they belong to the RNA viruses.
  • Important general belonging to the family of Flaviviridae are Flavivirus, Pegivirus, Pestivirus and Hepacivirus.
  • a prominent member of the Hepacivirus genus is hepatitis C virus (HCV).
  • the term“Flaviviridae protease” refers to a protease which occurs naturally in the virus belonging to the Flaviviridae family, namely the NS3 protease.
  • the term“NS3 protease” also embraces the NS3/4A protease, wherein the NS3 protease (as such an active protease) is associated with the virus-encoded co-factor NS4A which increases protease activity.
  • the mentioned optional co-factor (designated NS4A for example in HCV) may be different and bear different names in different viruses. For example, in Dengue virus and Zika virus it is called NS2B. Presence of said co-factor in its entirety is not required.
  • An N-terminal part thereof is comprised in preferred constructs of the invention such as SEQ ID NO: 3.
  • mutants and fragments of said protease are also embraced by the invention. Mutants may be substitution mutants, insertion mutants and deletion mutants. As such, the term“mutant”, to the extent it embraces N-terminal and C-terminal deletion mutants also includes fragments. Yet, fragments are recognized as a separate category in this disclosure and are discussed further below.
  • said first polypeptide does not comprise a protein of interest (POI).
  • POI protein of interest
  • mutants and fragments exhibit protease activity
  • the relevant functional property retained by mutants and fragments of said Flaviviridae protease is the capability of binding an inhibitor of said protease.
  • the requirement to maintain the capability to bind said ligand also implies that catalytic activity is maintained (in the state where the inhibitor is not bound). Otherwise, and also in case of allosteric inhibitors, there is no such requirement.
  • said Flaviviridae protease is catalytically inactive.
  • polypeptides of the invention may comprise a POI as disclosed above.
  • the present invention provides also a second polypeptide, wherein said second polypeptide comprises or consists of said first polypeptide as defined above and a protein of interest (POI), wherein the protase comprised in said first polypeptide is catalytically inactive, and wherein said POI is connected to the remainder of said second polypeptide via a second peptide linker which preferably comprises a site which is cleavable by said protease.
  • POI protein of interest
  • Said protein of interest (POI) in said second polypeptide may be located N-terminally or C- terminally with respect to the said first polypeptide.
  • Preferred in that said POI is located N- terminally with respect to the said first polypeptide.
  • the binding site of the inhibitor is preferably located on a solvent-exposed surface of said protease and has a rather shallow pocket.
  • the binding site is primarily formed by the side chains of the conserved residues Phe-154, Ala-157, and Leu-135 which define the key properties of the pharmacophore comprised in inhibitors.
  • Residues 41-43, 57, 81 , 132-139, 155-159, and 168 are likely to stabilize ligand binding.
  • the catalytic triad is His-57, Asp-81 , and Ser-139.
  • Residue numbers refer to the HCV NS3 sequence given in SEQ ID NO: 2.
  • the available protease inhibitors have generally been derived from the decapeptide (P6-P4') substrate and its cleaved products.
  • the scissile peptide bond has been substituted with a-ketoamide for boceprevir and telaprevir, sulfonate for danoprevir, or carboxylate for BI201335; see, e.g. Meeprasert et al. (2014).
  • This publication also contains information about the three-dimensional structure of the protease which permits rational design of inhibitors.
  • sequence identity between said mutant and the cognate wildtype protease are at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and including full identity (100%).
  • sequence identity levels apply preferably also to said fragment, i.e. deletion of flanking sequences not exceeding 20% of the total sequence. Having said that, and in those cases where the part of the sequence which upon folding gives rise to the inhibitor binding pocket defines a folding unit which is shorter than 80% of the wildtype protease sequence, also shorter fragments are envisaged.
  • the capability of binding an inhibitor can be assessed by a skilled person.
  • identification of ligands binding and/or inhibiting NS3 protease can be done by fluorometric assays, in which a fluorescent protein is released by the protease activity from a fusion protein linked to a solid substrate (Berdichevsky et al. 2003).
  • binding of substrates or inhibitors to NS3 can be determined using labeled substrates/inhibitors without or with crosslinking (Ray and Das 2011 ).
  • the amino acid sequence of SEQ ID NO: 1 is the sequence of SEQ ID NO: 1 as disclosed in WO 2017/004022, which document is herewith incorporated by reference in its entirety. To explain further, the mentioned sequence of SEQ ID NO: 1 as disclosed in WO 2017/004022 includes and defines a so-called“degron”. This is an amino acid sequence which is capable of promoting degradation of a polypeptide.
  • variants thereof exhibiting at least 80% sequence identity, preferably at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity as well as full identity (100%) are envisaged.
  • the sequence of SEQ ID NO: 1 is located downstream , i.e. C-terminal of the protease sequence,
  • the position of the protease with the first polypeptide is N-terminal.
  • Either term (“N-terminal” and“upstream (location of the protease) or“C-terminal’’ and“downstream”) include the option of a direct fusion of the amino acid sequence of (i) to the amino acid sequence of (ii), but are not limited thereto.
  • linkers (iii) with a maximum length of twenty amino acids may occur between the protease sequence and SEQ ID NO: 1.
  • shorter linker length such as 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s) are preferred.
  • Preferred amino acids comprised in the linker sequence include glycine.
  • a particularly preferred sequence (iii) linking (i) and (ii) is the undecapeptide of SEQ ID NO: 4 (NSSPPAVTLTH).
  • WO 2017/004022 discloses on the one hand the mentioned degron sequence and on the other hand a so-called degron fusion protein, said degron fusion protein comprising four components (a polypeptide of interest; said degron; an active protease; and a linker which is cleavable by said protease), the present inventors surprisingly discovered the first polypeptide in accordance with the first aspect of the present invention has an unexpected and surprising functional property. Importantly, said first polypeptide does not exhibit all four components of the“degron fusion protein” of WO 2017/004022, namely, only two, i.e., lacking the linker and the POI.
  • the second polypeptide does comprise a POI and a linker connecting it to the remainder of said second polypeptide, wherein said linker may be cleavable by an active Flaviviridae NS3 protease, but is not necessarily cleavable.
  • the protease as comprised in said second polypeptide is preferably catalytically inactive, though, but is not necessarily catalytically inactive.
  • the surprising property is that upon binding of an inhibitor to said protease, the first polypeptide becomes cytotoxic or cytopathic (which terms are used equivalently in this disclosure). As such, it is capable of triggering cell death, preferably, but not necessarily via the apoptotic route, once an inhibitor has been added to a cell comprising said polypeptide.
  • binding of the ligand to said protease involves a structural change of the first polypeptide which structural change allows interaction with death receptors, death-inducing signaling complexes (DISC), apoptosomes, or apoptotic initiator caspases such as Caspase 8 or 10, which leads to activation of caspases.
  • DISC death-inducing signaling complexes
  • apoptosomes apoptosomes
  • apoptotic initiator caspases such as Caspase 8 or 10, which leads to activation of caspases.
  • cytotoxicity and“cytopathic effect” are used equivalently herein to characterize the properties of the first polypeptide upon ligand binding.
  • said protease is catalytically inactive;
  • said first polypeptide does not comprise a site which is cleavable by said protease; and/or
  • said first polypeptide further consists of one or both of (iv) one or two terminal amino acid sequences which do not define a folding unit and/or each of which is 30 or less amino acids in length; and (v) one or more peptide tags such as tags suitable for identification, monitoring and/or purification of said polypeptide, each of said tags preferably being 20 or less amino acids in length.
  • a preferred terminal amino acid (iv) is the sequence of SEQ ID NO: 5 (GCWIVGRIVLSGKPAIIPDREVLY), said sequence being located downstream of SEQ ID NO: 1 or a homolog thereof as defined herein above. Also preferred is that the sequence of SEQ ID NO: 5 is the only terminal amino acid in accordance with (iv).
  • Part (a) of this preferred embodiment further illustrates the distinction from WO 2017/004022. While in accordance with this prior art document such a cleavable site is indispensable for switching off the conditional degradability conferred by the degron to a polypeptide of interest, this has no relevance for the present invention to the extent it relates to said first polypeptide. I.e., the feature of degradation is not important for the toxicity/action of the first polypeptide .
  • Part (b) of this preferred embodiment refers to additional structural elements present in the first polypeptide.
  • Typical elements are those in accordance with items (iv) and (v).
  • Lengths shorter than 30 or 20 amino acids, respectively, are deliberately envisaged such as 29, 28, 27, 26, 25, 24, 23, 22, 21 ,19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s).
  • the mentioned peptide tags include tags for analytical and preparative approaches, which include a His-tag, a Strep-tag, a TAP -tag, an S-tag, an SBP-tag, an Arg-tag, a calmodulin binding peptide tag, a cellulose-binding domain tag, a DsbA tag, a c-myc tag, a glutathione S- transferase tag, a FLAG tag, a HA-tag, a HAT -tag, a maltose-binding protein tag, a NusA tag, or a thioredoxin tag.
  • First polypeptides according to the present invention may contain in addition, or alternatively, a detectable label which may be a fluorescent, bioluminescent, chemiluminescent, colorimetric, or isotopic label.
  • a detectable label which may be a fluorescent, bioluminescent, chemiluminescent, colorimetric, or isotopic label.
  • the detectable label is a fluorescent protein or bioluminescent protein.
  • said FlavMridae protease is a Hepacivirus protease, preferably a Hepatitis C virus (HCV) protease, more preferably a HCV NS3 protease, most preferably a protease comprising or consisting the sequence of SEQ ID NO:2, a Flavivirus protease, a Pestivirus NS3 protease or a Pegivirus protease, preferably a Zika virus protease.
  • HCV Hepatitis C virus
  • a preferred example of such protease is the nonstructural protein 3 (NS3) protease, or the NS3/4A protease, of hepatitis C virus (HCV), which belongs to the Flaviviridae family.
  • the HCV NS3 proteases are well characterized and can be inhibited by small, cell permeable and orally applicable molecules like Asunaprevir (ASV; BMS-650032), Danoprevir (ITMN- 191/R7227), Simeprevir (Olysio®), Paritaprevir, Vaniprevir, Grazoprevir (MK-5172), Voxilaprevir (GS-9857), Boceprevir, Telaprevir, Ciluprevir, Faldaprevir, Sovaprevir, ABT-450, or BILN-2061.
  • Asunaprevir ASV; BMS-650032
  • Danoprevir ITMN- 191/R7227
  • Simeprevir Olysio®
  • HCV-derived protease fusion constructs as described by (Chung et al. 2015) and in WO 2017/004022 A2 (these are also referred to as SMASh tags), in combination with HCV protease inhibitors such as described above represent a preferred protease/inhibitor combinations for generating and modulating conditionally cytopathic viruses (CCPV), including CCP rhabdoviruses, while emphasizing, as noted above, that in none of these prior art documents there is any recognition of a cytopathic or cytotoxic effect.
  • CCPV conditionally cytopathic viruses
  • Constructs including the NS3/4A protease, the degron, a cleavage site and an optional further, artificial or synthetic, linker can be derived from any isolate or recombinant HCV, of any genotype (GT 1-7 so far defined).
  • Such constructs, i.e. tags which comprise protease, degron and cleavage site are considered in the prior art, however, only in the context of a fusion construct which in addition comprises a protein of interest, and which requires a proteolytically active form of the NS3 protease.
  • First polypeptides need not be physically derived from HCV or any other virus, but may be produced synthetically or recombinantly.
  • the HCV nonstructural protein 3 (NS3) protease consists of an N-terminal serine protease domain and a C-terminal helicase domain.
  • the protease domain of NS3 may form a heterodimer with the HCV nonstructural protein 4A (NS4A), which is a co-factor that further activates proteolytic activity.
  • An NS3 protease according to the present invention may comprise the entire NS3 protein or a proteolytically active fragment thereof and may further comprise the NS4A protein. It may comprise additional sequences, including tags for serological detection, including His-tag or Flag-tag, or sequences encoding fluorescent or luminescent proteins for detection like eGFP of Firefly luciferase.
  • the Hepacivirus genus comprises other animal and human viruses , including for example Hepacivirus G (Norway rat hepacivirus 1 ; New York city rat hepacivirus 1 ), which is particularly useful as rodent model for HCV, furthermore Hepacivirus A (equine hepacivirus), -B (GBV-B), -D (Guereza hepacivirus), -E (rodent hepacivirus-339), -F (rodent hepacivirus-NLR07), -H (Norway rat hepacivirus 2), -I (rodent hepacivirus-SAR-3/RSA/2008), -J (rodent hepacivirus- RMU10-3382/GER/2010), -K, -L, and -M, (bat hepaciviruses-PDB), and -
  • First polypeptides comprising a protease of any of the members of the Hepacivirus genus are also considered and are furthermore suitable for generation of CCPVs.
  • first polypeptides comprising a protease from other members of the Flaviviridae family are considered.
  • Such other members include, but are not limited to the Pestivirus genus which includes the so far approved species of bovine viral diarrhea virus 1 and 2 (BVDV 1/2), border disease virus (BDV), or classical swine fever virus (CSFV), and the suggested species of border disease virus 2 (BDV-2) and bovine viral diarrhoea virus 3 (BVDV-3; HoBi group).
  • cp cytopathic
  • noncp non-cytopathic
  • First polypeptides comprising a protease of the Flavivirus genus, including but not limited to species like Zika virus (ZIKV), Dengue virus, Japanese encephalitis virus, Saint Louis encephalitis virus, Yaounde virus, Yellow fever, tick-borne encephalitis virus (TBEV), or Langat virus, are also considered.
  • ZIKV Zika virus
  • Dengue virus Japanese encephalitis virus
  • Saint Louis encephalitis virus Saint Louis encephalitis virus
  • Yaounde virus Yellow fever
  • tick-borne encephalitis virus (TBEV) tick-borne encephalitis virus
  • first polypeptides comprising a protease from the genus Pegivirus of the Flaviviridae family are provided.
  • Said genus includes Pegivirus A (GBV-A), -B (GBV-D), -C (GBV-C, previously known as human hepatitis G virus (HGV)), -D (Theiler’s disease- associated virus), -E (Equine pegivirus), -F and -G and I (Bat pegiviruses), -H (Human pegivirus 2), -J (Rodent pegivirus), and -K (Porcine pegivirus).
  • said polypeptide comprises or consists of the sequence of SEQ ID NO: 3.
  • SEQ ID NO:3 is also given below:
  • NS3 protease (SEQ ID NO: 2): boldface
  • NS3 helicase sequence i.e. the part indicated by“ [ -dele tion- ]” comprises 416 amino acids (i.e. most of said helicase):
  • sequence of SEQ ID NO: 3 is that of the NS3 protease, followed by the sequence of NS3 helicase (wherein a large part thereof is deleted), followed by the N-terminal part of the NS4A protein.
  • said protease is a HCV genotype 1 (GT1 ) protease. Also, preferred is that said protease is HCV GT2, GT3, GT4, GT5, GT6 or GT7 protease.
  • GT1 HCV genotype 1
  • said protease is HCV GT2, GT3, GT4, GT5, GT6 or GT7 protease.
  • Hepacivirus G (Norway rat Hepacivirus 1 ; also known as New York City rat Hepacivirus 1 ).
  • Pegivirus proteases are proteases from Dengue virus and yellow fever virus. Particularly preferred is that (a) said protease is a HCV protease and said inhibitor is selected from Asunaprevir, Danoprevir, Simeprevir, Paritaprevir, Vaniprevir, Grazoprevir, Volixaprevir (GS-9857) Boceprevir, Telaprevir, Ciluprevir, Faldaprevir, Sovaprevir, ABT-450 and BILN- 2061 ; or (b) said protease is a Zika virus protease and said inhibitor is bromocriptine or novobiocin.
  • said protease is a HCV protease and said inhibitor is selected from Asunaprevir, Danoprevir, Simeprevir, Paritaprevir, Vaniprevir, Grazoprevir, Volixaprevir (GS-9857) Boceprevir, Telaprevir, Ciluprevir, Faldaprevir, Sova
  • Novobiocin is also known in the art as albamycin or cathomycin.
  • said polypeptide further consists of an amino acid sequence of a protein of interest (POI), wherein preferably said POI is fused N-terminally to the amino acid sequence of (i).
  • POI protein of interest
  • said POI may be fused C-terminally to the amino acid sequence of (ii).
  • a POI may be part of said first polypeptide. Having said that, and in line with what has been stated further above, absence of a site which is cleavable by said protease may be preferred in certain embodiments. Importantly, to the extent a protein of interest is comprised in said first polypeptide, it is a feature of this invention that said first polypeptide does not have to comprise and/or does not comprise any site which is cleavable by said protease.
  • the protein of interest fused to a first polypeptide according to the present invention may be any natural or synthetic protein, protein domain, or peptide, including membrane proteins, cytoplasmic proteins, enzymes, transcription factors, ligands, interaction domains.
  • said POI is selected from (a) marker proteins such as YFP; (b) immunomodulatory proteins including GM-CSF, interferons, interleukins, inhibitors of immune checkpoints including antibodies, and bi- or multispecific antibodies recruiting immune cells such as T cells and macrophages; and (c) viral proteins, viral proteins being particularly preferred.
  • marker proteins such as YFP
  • immunomodulatory proteins including GM-CSF, interferons, interleukins, inhibitors of immune checkpoints including antibodies, and bi- or multispecific antibodies recruiting immune cells such as T cells and macrophages
  • viral proteins viral proteins being particularly preferred.
  • said amino acid sequence of (i) is an active protease.
  • protease activity is not a requirement. What matters is the capability of the first polypeptide of binding a protease inhibitor, preferably such that binding causes a toxic configuration of the first polypeptide. Yet, and in accordance with the above-disclosed preferred embodiment, said protease is furthermore enzymatically active and exhibits protease activity.
  • the present invention provides a nucleic acid which is (a) a first nucleic acid comprising or consisting of a first nucleotide sequence encoding the first polypeptide as defined above; or (b) a second nude id acid comprising or consisting of a second nucleotide sequence encoding the second polypeptide as defined above.
  • Said nucleic acid which may be said first nucleic acid or said second nucleid acid is an alternative means of conferring conditional cytopathic effects.
  • said nucleic acid may be transcribed and translated (if said nucleic acid is DNA) or translated (if said nucleic acid is RNA) and give rise to a polypeptide which polypeptide is a first polypeptide in accordance with the present invention or a second polypeptide in accordance with the present invention which in turn may be administered.
  • said nucleic acid may be introduced into a target cell wherein said target cell produces the polypeptide encoded by the nucleic acid. In either case, conditional cytotoxicity is conferred by the polypeptide and may be triggered by administration of an inhibitor of said protease, mutant thereof or fragment thereof.
  • Administration of said polypeptide or said nucleic acid may be done by using any of the art-established methods of administering polypeptides and nucleic acids to cells, respectively.
  • a conceivable method would be microinjection, transfection, transduction, electroporation, or linking to cell membrane permeable peptides, proteins or agents.
  • a preferred means of administration is via a virus or virus vector in accordance with the present invention. Said virus is described in more detail below.
  • said nucleic acid comprises a promoter operably linked to the sequence encoding the polypeptide of the first aspect of the invention.
  • the present invention provides a first virus comprising the first nucleic acid in accordance with the second aspect and a second virus comprising the second nucleic acid in accordance with the second aspect.
  • Viruses in accordance with the present invention are also referred to as conditionally cytopathic viruses (CCPV).
  • CCPV conditionally cytopathic viruses
  • the term “conditional” refers to the drug dependency: only upon administration of an inhibitor of said protease, the cytopathic effect occurs, or an enhanced cytopathic effect occurs compared to the parental virus not carrying said first nucleic acid.
  • the enhancement is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold or at least 10-fold.
  • Administration of said inhibitor kills cells infected by said virus. As such, any unintended spreading of said virus may be stopped.
  • CCPV conditionally cytopathic viruses
  • cytopathic viruses can be grown to high titers in the absence of the drug, and applied in prophylactic and therapeutic applications, including, but not limited to, vaccination, immune modulation, or oncolytic virotherapy, and at will instructed to become cytotoxic.
  • CCPV represent perfect tools in high content- or high throughput genetic screens for experimental identification of pro- and antiviral genes and compounds, as they provide the opportunity of efficient biological selection of non- or poorly permissive cells.
  • the taxonomic category to which said virus belongs is not particularly limited. Preferred taxa are disclosed further below.
  • the present invention provides the use of (a) a first agent selected from (aa) the first polypeptide in accordance with the first aspect; (ab) the nucleic acid in accordance with the second aspect; and (ac) the virus in accordance with the third aspect; (b) a second agent selected from (ba) the second polypeptide of as defined above; (bb) the second nucleic acid as defined above; (be) the second virus as defined above; (c) a third agent selected from (ca) a third polypeptide, wherein said third polypeptide comprises or consists of said first polypeptide as defined in accordance with the first aspect and a POI, preferably as defined herein above, wherein said POI is connected to the remainder of said third polypeptide via a third peptide linker which comprises a site which is cleavable by said protease; (cb) a third nucleic acid comprising or consisting of a third nucleotide sequence encoding said third polypeptide
  • third agent which third agent may be selected from a third polypeptide, a third nucleic acid and a third virus.
  • third constructs extend to the fusion constructs of WO 2017/004022.
  • the prior art fails to recognize to a conditional cytopathic effect conferred by such constructs.
  • the POI, and a proteolytically active NS3 protease which are optional in first agents, are compulsory.
  • embodiments of the third polypeptide may comprise a protease which is preferably inactive and/or a linker connecting the POI to the remainder of the third polypeptide which linker is not cleavable by an active Fiaviviridae in this third protease.
  • composition is distinct from the agents in that it provides for a POI, wherein said POI is present as a separate constituent of said composition in accordance with (c).
  • cell death occurs via the apoptotic route.
  • Other envisaged routes are via pyroptosis and necroptosis.
  • pyroptosis and necroptosis are also envisaged routes.
  • killing by alternative routes may be observed with constructs of the present invention.
  • the present invention provides an in vitro or ex vivo method of inducing death of a cell, said method comprising administering (a) a first agent, a second agent, a third agent or a composition as defined in accordance with the fourth aspect; and (b) said inhibitor to said cell.
  • administering implies conditions which allow internalization of said first agent, said second agent or said composition by said cell.
  • Preferred conditions are infection of cell cultures or organoids with a CCPV and addition of the inhibitor after establishment of the infection (e.g. 24 hrs or later post infection with a rabies virus or 12 hrs or later post infection with; a VSV), or infection of organisms with the CCPV by oral or parenteral route, or targeted infection or injection of a virus into a tumor, and application of an inhibitor by the oral, parenteral or local injection route.
  • Said cell may be any cell such as normal cells of any tissue type, including immune cells, or neuronal and glial cells as well as malignant cells such as cancer cells or pathogen-infected or pathogen-affected cells, or stem cells.
  • a virus it is understood that said cell is preferably amenable to infection by said virus.
  • Said cell may also be in a context of a tissue, provided that the above-disclosed restriction (in vitro or in vivo) applies.
  • Cells and tissues may be, for example, of human, murine or canine origin, and of any animal wildlife or livestock species origin.
  • step (b) between administering in accordance with step (a) and adding said inhibitor in accordance with step (b), sufficient time is allowed to a lapse that conditionally cytotoxic constructs are formed and accumulate.
  • Preferred time spans between infection and application of the inhibitor without implying a limitation may extend from about 6 hours to about 6 days or more, such as about 1 , about 2, about 3, about 4 or about 5 days.
  • said nucleic acid comprises a promoter and optionally an enhancer which are operatively linked to said first or second nucleotide sequence, respectively; and/or (b) said nucleic acid is (ba) a plasmid; (bb) a viral genome; (be) a cellular genome; (bd) an episomal nucleic acid; or (be) a mobile nucleic acid element including transposons or retrotransposons.
  • viruses are preferred vehicles for conferring conditional cytotoxicity, but also plasmids.
  • an expression vector for example, a bacterial plasmid vector or a viral expression vector.
  • Exemplary viral vectors include rabies virus, vesicular stomatitis virus, adenovirus, retrovirus (e.g., y-retrovirus and lentivirus), poxvirus, adeno-associated virus, baculovirus, or herpes simplex virus vectors.
  • said virus belongs to the Mononegavirales, preferably to the Rhabdoviridae, and more preferably is a rabies virus (RABV); and/or (b) said virus is a pseudotyped virus.
  • RABV rabies virus
  • said virus belonging to the Rhabdoviridae is a VSV and/or (b) said virus is a pseudotyped virus.
  • Rabies virus is an example of the Rhabdoviridae family, and the prototype of the Lyssavirus genus.
  • Other major rhabdovirus genera are Vesiculovirus, Ephemerovirus, Novirhabdovirus, Cytorhabdovirus, and Nucleorhabdovirus which are long established, and a number of smaller genera, which were newly classified (Afonso et al. 2016).
  • ICTV Virus taxonomy classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. (2012) Ed: King, A.M.Q., Adams, M.J., Carstens, E.B. and Lefkowitz, E.J. San Diego, Updates available: http://www.ictvonline.orq/virusTaxonomv.asp.
  • Rhabdoviruses share a highly similar genetic and morphological organization. Morphologically, rhabdoviruses are characterized by a typical rod- or bullet-shaped morphology of the enveloped virions.
  • the genomes of the prototype viruses RABV and VSV comprise a minimal set of only 5 essential genes (3 -N-P-M-G-L-5’) encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), the transmembrane spike glycoprotein (G), and the “large” protein (L), which is the catalytic subunit of the viral RNA polymerase.
  • RNAs are tightly enclosed by the N protein in a highly stable helical ribonucleoprotein (RNP) which makes the RNA inaccessible to small molecules like RNAses and miRNAs or siRNAs.
  • RNPs represent the exclusive templates for transcription and replication by the viral RNA polymerase complexes (L+P, and L+P+N, respectively) and only for these processes is the N-RNA complex transiently opened to allow access to the RNA.
  • Rhabdovirus gene expression involves the sequential and polar transcription of subgenomic 5’- cap modified and polyadenylated mRNAs from the genome RNP.
  • RNAs are produced from the 3’- ends of the genome and antigenome RNPs, the leader and trailer RNAs, respectively, which have 5’-triphosphate ends like the genome and antigenome RNAs (Lamb 2007).
  • the genomes of other rhabdoviruses may comprise additional genes, often located between G and L, or between P and M genes.
  • the incorporation of extra genes into the genomes of recombinant RABV or VSV, having conserved gene order or a modified gene order, is similarly being utilized for expression of heterologous viral or cellular factors.
  • Genetic engineering of RABV and other rhabdoviruses relies on the expression of cDNA- encoded antigenome RNA, and N, P, and L proteins, to allow formation of an infectious RNP as described by (Schnell, Mebatsion, and Conzel ann 1994).
  • Rabies virus (suggested binomial species name is Rabies Lyssavirus) is the prototype species of the Lyssavirus genus which represents the largest genus of the family by number of individual species.
  • Lyssavirus genus also known as“rabies-related viruses” are mostly associated with bat reservoirs, but can also cause rabies-like encephalitis and death in terrestrial animals and humans (Luis et al. 2013).
  • Rabies Lyssavirus thirteen lyssavirus species have been identified: Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus 1 (EBLV-1 ) and 2 (EBLV-2), Australian bat lyssavirus (ABLV), West Caucasian bat virus (WCBV), Irkut virus (IRKV), Aravan virus (ARAV), Khujand virus (KHUV), Shimoni bat virus (SHIBV), Ikoma lyssavirus (IKOV) and Bokeloh bat lyssavirus (BBLV).
  • LBV Lagos bat virus
  • MOKV Mokola virus
  • DMVV Duvenhage virus
  • EBLV-1 European bat lyssa
  • lyssavirus species from Europe and elsewhere are continuously being identified (Gunawardena et al. 2016). As most of these viruses were isolated from bats, lyssaviruses may have originated in Chiroptera (Fooks et al. 2017) .
  • the Vesiculovirus genus includes animal viruses like the prototypic vesicular stomatitis virus (VSV), which can induce severe disease in cattle and pigs causing enormous economic loss.
  • VSV prototypic vesicular stomatitis virus
  • Chandipura virus A closely related vesiculovirus, Chandipura virus, has emerged in India as serious human pathogen associated with severe acute encephalitis (for review, see (Basak et al. 2007).
  • Natural and attenuated VSVs, including noncytotoxic variants like VSVAM51 are potent oncolytic oncolytic viruses (Miest and Cattaneo 2014).
  • other vesiculoviruses have been identified which display strong oncolytic activities in humans and animals, including Maraba virus (Hummel et al. 2017).
  • the present invention further enhances the anti-cancer effectiveness of these viruses.
  • BEFV bovine ephemeral fever virus
  • NGS new generation sequencing
  • Bas-Congo virus which was recently found to cause hemorrhagic disease in humans
  • Ekpoma virus which is related to the Tibro avirus genus, and which was identified in healthy individuals
  • the Novirhabdovirus genus includes typical rhabdoviruses from fish, such as viral hemorrhagic septicemia virus (VHSV) which infects numerous marine and freshwater fish species .
  • VHSV viral hemorrhagic septicemia virus
  • rhabdoviruses that infect plants are separated into the genera Cytorhabdovirus and Nucleorhabdovirus, based on their sites of replication and morphogenesis (Jackson et al. 2005). They include important plant pathogens such as lettuce necrotic yellows virus (LNYV), or sonchus yellow net virus (SYNV). Plant rhabdoviruses are also transmitted by insect vectors. This has led to the suggestion that the entire family evolved from a common ancestral insect virus .
  • the order Mononegavirales comprises the families Bornaviridae, Paramyxoviridae (e.g. measles virus), Pneumoviridae, Filoviridae, Mymonaviridae, Nyamiviridae, and Sunviridae.
  • the segmented negative strand RNA viruses comprise the families Orthomyxoviridae (e.g. Influenza A virus), Arenaviridae, and Bunyaviridae.
  • Bornaviridae including human and animal Borna disease viruses (BDV)
  • BDV Borna disease viruses
  • viruses are DNA-, RNA-, and Retroviruses including Adenoviruses, Lentiviruses, Parvoviruses, Herpesviruses, and Poxviruses.
  • Agents and compositions in accordance with the invention can be delivered by plasmid or other DNA or RNA transfection in a way resulting in stable genome-integrated DNA or non- integrative expression, or transient expression in cells.
  • the nucleic acids may contain promoter, transcription enhancer elements, transcription termination signal, polyadenylation sequences, or exogenous nucleic acid, or any combination thereof to facilitate expression.
  • the envelope proteins of RABV and other rhabdoviruses can be easily exchanged with other viral envelope or cellular transmembrane proteins, either in trans (so-called pseudotype viruses) or in cis, by exchanging the virus genes (surrogate viruses).
  • pseudotype viruses pseudotype viruses
  • different envelope proteins can be co-incorporated into the viral envelope.
  • Examples include viral spike proteins like HIV-1 Env, ASLV EnvA, HCV E2/3, VSV G, EBOV Gp, Measles F and H, Hendra virus G, and natural and genetically engineered G proteins from different rabies virus strains and lyssavirus isolates, as well as cellular transmembrane proteins like CD4 and CXCR54 or CCR5, or fluorescent reporter proteins (Finke and Conzelmann 2005; Ghanem and Conzelmann 2016).
  • the use of differently pseudotyped or different surrogate rabies or rhabdovirus CCPV therefore allows extending the screens to investigate requirements for entry of heterologous viruses as well, or to study cellular vesicle transport and fusion mechanisms relevant for the virus life cycle.
  • the tropism of life vaccines, immunomodulatory viruses, and oncolytic viruses can be determined or adjusted by preparing pseudotype or surrogate CCPV. This allows specific targeting of relevant cell types, such as antigen presenting cells (A PC), immune cells, or malignant or cancerous cells (Miest and Cattaneo 2014).
  • a PC antigen presenting cells
  • immune cells immune cells
  • malignant or cancerous cells Malignant or cancerous cells
  • said first or second virus is a Lyssavirus or a Vesiculovirus such as VSV, Maraba virus, or Isfahan virus.
  • said first or second virus may further be any RNA-, DNA-, Retro- or Hepadnavirus, including segmented and unsegmented negative strand RNA viruses, Picornaviruses, Reoviruses like Rotavirus, Parvoviruses like Adeno-associated viruses (AAV), Poxviruses like vaccinia virus, Adenoviruses, Herpesviruses, Retrovirus- and Lentiviruses.
  • AAV Adeno-associated viruses
  • Poxviruses like vaccinia virus, Adenoviruses, Herpesviruses, Retrovirus- and Lentiviruses.
  • said POI is a protein of said virus, preferably a protein which is essential for the life cycle of said virus such as a nucleoprotein (N) or phosphoprotein (P) protein, more preferably an N protein.
  • N nucleoprotein
  • P phosphoprotein
  • said second polypeptide is expressed as fusion with the rabies virus nucleoprotein (N), which in the rabies context provides the most specific killing, or with the phosphoprotein (P), which provides the most efficient killing.
  • Specific killing means the CCP virus is not cytotoxic in untreated cells, but toxic in drug-treated cells; most efficient killing means highly toxic in presence of drug.
  • said amino acid sequence of (i), said amino acid sequence of (if), said POI, and said second peptide linker are arranged in said second polypeptide in the following order from N-terminus to C-terminus: (a) POI - second linker - amino acid sequence of (i) - amino acid sequence of (ii); or (b) amino acid sequence of (i) - amino acid sequence of (ii) - second linker - POI.
  • the present invention provides a kit comprising (a) a first or second agent as defined in accordance with the fouth aspect; (b) an inhibitor of said protease.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a first agent, a second agent, a third agent or a composition as defined in accordance with the first aspect and optionally an inhibitor of said protease.
  • an inhibitor is comprised in said pharmaceutical composition, it is understood that preference is given to a pharmaceutical composition comprising two distinct units, a first unit comprising said first agent, said second agent or said composition, and a second unit comprising said inhibitor. In typical uses, said inhibitor will be administrated after administration of the first unit.
  • said pharmaceutical composition may comprise further therapeutic agents, such as cytokines, antibodies, immune checkpoint antagonists, chimeric antigen receptor (CAR) T cells, miRNAs, and cytostatic medicals including for example 5-fluorouracil, cisplatin, methotrexate, cyclophosphamide, Paclitaxel, etoposide and/or bleomycin.
  • cytokines such as cytokines, antibodies, immune checkpoint antagonists, chimeric antigen receptor (CAR) T cells, miRNAs, and cytostatic medicals including for example 5-fluorouracil, cisplatin, methotrexate, cyclophosphamide, Paclitaxel, etoposide and/or bleomycin.
  • CAR chimeric antigen receptor
  • the pharmaceutical composition comprises in terms of active agents only those which are expressly recited.
  • the pharmaceutical composition in accordance with the present invention may comprise carriers, excipients and/or diluents.
  • suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
  • compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration.
  • the compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Pharmaceutically active matter in accordance with the invention may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • a dose is preferably a daily dose. If the regimen is a continuous infusion, it should be in the range of 1 ng to 10 g units per kilogram of body weight per minute.
  • the first unit is said composition as defined in accordance with the fourth aspect, the first unit may be split as well, noting that said composition comprises two separate constituents.
  • the present invention provides a live vaccine comprising the virus in accordance with the third aspect or the third virus as defined in accordance with the fourth aspect.
  • the term“vaccine” includes prophylactic as well as therapeutic vaccines. Preferred individuals to be vaccinated include humans, dogs, rodents such as mice as well as wildlife and livestock animals.
  • said vaccine comprises a third virus as defined in accordance with the fourth aspect
  • said POI is an essential protein of said virus.
  • the live vaccine is preferably parenterally or orally. In case of rabies virus vaccines, oral administration is particularly preferred.
  • This preferred embodiment provides for double-safety switch.
  • the cell is killed via the surprisingly discovered cytotoxic effect in accordance with the present invention, and furthermore the essential protein of said virus is targeted for degradation since the degron sequence is not cleaved off in the presence of an inhibitor.
  • the third virus of said live vaccine comprises or encodes a fusion construct comprising four elements which are the protease in accordance with the invention, the degron sequence, a POI which is an essential protein of the virus, and a site which is cleavable by said protease (in the absence of an inhibitor).
  • Preferred architectures of such four component constructs are disclosed herein above.
  • Such double-safety measures limit potential biohazard and will increase the acceptance of live replicating viruses in a variety of applications, said applications including, but not limited to live vaccines.
  • Preferred essential proteins are proteins required for replication, assembly or spread of said virus.
  • an essential protein examples include, in case of a virus which belongs to the Mononegavirales, the N- and the P-protein.
  • the present invention provides a first agent, a second agent, a third agent or a composition as defined in accordance with the fourth aspect for use in a method of killing diseased cells in an individual affected by a disease.
  • said disease is hyperproliferation, cancer or infection by a pathogen, said pathogen preferably being a virus such as HIV, in particular HIV-1.
  • targeting of cancer cells can be done by using pseudotypes directed against Tumor surface markers (EGFR) or indirectly, by using IFN-sensitive viruses in cancers in which IFN signalling is abolished.
  • Targeting virus (HIV-1 ) infected cells can be performed as described in (Mebatsion et al. 1997) i.e.: the CCPV is pseudotyped with the receptor for the envelope/fusion protein of another virus, resulting in“reversed fusion”.
  • said method of killing is oncolytic virotherapy
  • said POI is selected from immunomodulatory proteins including GM-CSF, interferons, interleukins, inhibitors of immune checkpoints, including antibodies, and bi- or multispecific antibodies recruiting immune cells such as T cells and macrophages.
  • Said POIs can be fused to the first or second polypeptide via a non-cleavable or cleavable linker, or directly.
  • the POIs can also be expressed independent from the cytotoxic tag. In this case, cell killing via the tag and inhibitor is independent of the expression of the immunomodulatory POI.
  • Said immunomodulatory proteins may be the only POIs employed in accordance with this preferred embodiment.
  • a second POI may be present.
  • This second POI may be, for example, an essential protein of the virus used.
  • the two POIs may be comprised in one single fusion construct.
  • the POI which is an immunomodulatory protein may be provided separately, i.e. in accordance with item (cc) of the fourth aspect of the present invention.
  • the present invention provides use of a first or second virus in accordance with the third aspect or a third virus as defined in accordance with item (cc) of the fourth aspect in a screen for one or more of antiviral cellular genes, antiviral agents, proviral cellular genes and proviral agents.
  • non-infectable cells are surviving and can be selected for analysis of the host genes required for infection and viral gene expression (Marceau et al. 2016; Zhang et al. 2016).
  • cells transduced with whole genome libraries of individual genes, or a selected sub-library, such as an interferon-induced set of genes, or guide RNAs targeting all promoters and activating transcription of host genes can be challenged with a CCP virus.
  • Cells expressing antiviral gene products will be protected from infection or virus growth, and can be selected for the analysis of genes involved, as described e.g. in (Schoggins et al. 2014).
  • pools with DNA bar-coded substances, compounds, drugs, natural products, extracts or small molecules can be analyzed for pro- and antiviral compounds in high-content screens.
  • This technology involves the conjugation of chemical compounds or substances to DNA fragments that serve as identification bar codes and enables the mass creation and interrogation of compound libraries (Mullard 2016).
  • the present invention provides an in vitro or ex vivo method of screening for a cell which is not amenable to killing by the method in accordance with the fifth aspect, said method comprising: (a) applying the method in accordance with the fifth aspect to candidate cells; and (b) identifying surviving cells.
  • Possible reasons for a cell not being amenable to killing may be that the cell is not amenable to infection by a first or second virus in accordance with the present invention, or said virus is not capable of replicating in said cell.
  • the present invention provides an in vitro or ex vivo cell expressing a first, second, or third agent as defined in accordance with the fourth aspect.
  • the cell may be a human, mammalian, vertebrate cell of any tissue, and developmental stage, including pluripotent stem cells.
  • the cell may be comprised in multicellular cellular tissue or in organoids.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C,
  • FIG. 1-1 Small molecule-assisted shutoff (SMASh) system: (a) working principle of drug- dependent protein degradation (b) Composition of the C-terminal SMASh-tag, (c) composition of the N-terminal SMASh-tag.
  • SMASh Small molecule-assisted shutoff
  • Figures taken from Chung et al. (2015) (a, b: Fig. 1 , c: Suppl. Fig. 2.).
  • Figure b) was taken from Chung et al. (2015).
  • Figure 1-2 (A) Organization of recombinant RABV expressing SMASh-tagged (red) fusion proteins.
  • VSV vesicular stomatitis virus
  • Figure 2 (A) Growth curves of SAD N-SMASh and SAD P-SMASh in BSR cell culture, and (B) ASV-mediated degradation of SMASh-tagged proteins.
  • AS V and DNV are equally effective in killing CCCP-ibfected cells.
  • Monolayers of HEK293T cells were infected for 24 hours with SAD P- SMASh-eGFP and incubated with increasing concentrations of ASV or DNV, and 24h later stained with crystal violet to reveal intact cell monolayers. ASV or DNV doses above 0.1 mM are sufficient to cause complete monolayer destruction.
  • E/F Cell viability of P-SMASh (E) and N-SMASh (F)-encoding RABV.
  • HEK 293T cells were infected with the respective RABVs and one day after infection were treated with 3uM DNV. Viability of cells was analyzed at the indicated time points after DNV treatment with the RealTime-GloTM MT Cell Viability Assay.
  • FIG. 5A Killing of SAD-P-SMASh infected cells by NS3 inhibitor depends on accumulation of tagged proteins.
  • HEK293T cells were infected at MOI of 3 and ASV (3 pM) added after indicated time points. Cells were harvest and lysed at the same time (30 hpi). Substantial amounts of cleaved PARP and Casp9 are observed from 8 h.p.i. on, but reduction of procaspase 9 is observed already at earlier time points. Note that ASV at early time points prevents virus replication and accumulation of P-SMASh (not shown) and thereby of N protein at early time points.
  • FIG. 5B Protease activity of the SMASh tag is not required for drug-dependent cell killing.
  • Rabies viruses expressing YFP-SMASh fusion proteins in which the protease catalytic residue Ser139 was mutated to Ala is cytotoxic after addition of an NS3 protease inhibitor (Danoprevir).
  • HEK293T cells were infected with SAD viruses containing functional NS3 protease in their SMASh tag (SAD YFP- SMASh) or nonfunctional NS3 protease mutant (YFP-SMASh S139A) at a MOI of 3, 24 hours post infection 3 pM DNV or DMSO was added and after further 24 h lysates were prepared for Western blot.
  • Inactive protease is indicated by uncleaved AFP-SMASh protein in the absence of DNV.
  • Figure 5C Viruses encoding proteoiytically active (YFP-SMASh) and inactive (YFP- SMASh(S139A)) SMASh tags cause comparable drug-dependent cell death.
  • HEK 293T cells were infected with the respective viruses and one day after infection were treated with 3uM DNV. Viability of cells was analyzed at the indicated time points post DNV treatment with the RealTime-GloTM MT Cell Viability Assay.
  • Figure 5D A SMASh tag lacking the upstream POI is conditionally toxic.
  • SMASh-only tag (SMASH-tag lacking POI) was used to infect HEK293T cells for a day and then treated with 3uM DNV. WB showing cleaved PARP after addition of DNV.
  • FIG. 6 ASV-mediated cell death is intrinsic and involves activation of multiple caspases.
  • A Only virus-infected cells are killed, not bystander. BSR cells were infected with a non-spreading (single round) virus SADAG-eGFP P-SMASh at a MOI of 1 , and 3 mM ASV added 24 h.p.L The image was taken at 24 h post ASV. Note that only infected (green) cells are rounded and dying, while the morphology of bystander cells remains intact.
  • B Luciferase assays reveal activation of multiple caspases in DNV- treated SAD P-SMASh-infected HEK293T cells.
  • C Cleavage of Caspases 10, 9, and 3 after DNV treatment reveals active intrinsic and extrinsic apoptosis pathways.
  • FIG. 7 ASV-mediated cell death is partially preventable by pan-caspase inhibitor Z-
  • VAD-FMK SAD P-SMASh infected cells were treated at 22.5 h.p.i. with the indicated compounds. 24 hpi, ASV (3mM) or DMSO was added. 50 pM Z-VAD- FMK was added 1.5 h before ASV treatment 5 mM staurosporin was used as positive control Cells were lysed 24 h post ASV treatment. Note that staurosporine is not a great inducer of apoptosis in HEK293T cells.
  • Figure 9 (A)NS3 inhibitor-mediated killing of mouse melanoma cell line MC38 infected with SAD P-SMASh-eGFP.
  • Figure 11a Conditional cell killing by YFP-SMASh protein expressed from transfected pCAGGS YFP-SMASh expression plasmid.
  • HEK293T in 24 well plates were transfected with 500 ng pCAGGS YFP-SMASH, 3 mM DNV in DMSO (+) or DMSO alone (-) were added 24 h post transfection, and lysates for WB were prepared 28 h later.
  • Figure 11b Virus-independent conditional cell killing by SMASh proteins expressed from transfected plasmids.
  • HEK293T in 24 well plates were transfected with 500 ng of pCAGGS N-SMASh or pCAGGS YFP-SMASh plasmids or infected with SAD viruses expressing the corresponding fusion protens.
  • Three 3 mM DNV in DMSO (+) or DMSO alone (-) were added 24 h post transfection/infection, and lysates for WB were prepared 6 h later. With equal expression of proteins, DNA plasmids and RNA viruses induce equal killing
  • Figure 12 (A) Killing of SAD P-SMASh-infected Cas9-expressing HEK293T cells by addition of ASV. Live cells are visualized by Trypan Blue staining. (B) Scheme of high-content gene knock out screen and positive selection of non-infectable or non-killable cells.
  • Figure 13 (A) Flavivirus genes and gene products. (B) Sequence of the wild-type HCV
  • Plasmids pCS6-YFP-SMASh and pCS6-SMASh-YFP encoding C- and N-terminal SMASh-tags were obtained from addgene ⁇ #68853 and #68852, respectively).
  • Fig. 1 (assembled from Fig. 1 and Suppl.
  • Fig. 2 of Chung et al., 2015) shows the principle of drug-dependent degradation of SMASh-tagged proteins (a) and the amino acid sequences employed in C-terminai (b) and N- terminal (c) fusion proteins.
  • RABV SAD L16 cDNA encoding a P protein fused at the C-terminus with the SMASh-tag (SAD P-SMASh).
  • viruses were constructed encoding an N-smash protein (SAD N-SMASh), and a SMASh-L protein (SAD SMASh-L)).
  • the N, P, and L proteins were chosen because they have essential roles in the virus life cycle and/or in host interaction, and their drug-induced degradation should have severe effects on transcription, replication and interferon escape (P), replication (N), and RNA overall synthesis (L).
  • a RABV was constructed carrying an extra gene encoding a YFP-SMASh fusion protein (SAD YFP-SMASh) as obtained from addgene (Fig. 1-2).
  • SAD YFP-SMASh YFP-SMASh fusion protein
  • full-length versions and single round delta G (AG) versions of SMASH-tagged SAD viruses encoding an extra untagged eGFP (between G and L genes or instead of the G gene, respectively) were constructed.
  • a CVS-N2c AG virus carrying a SMASh-tagged P (N2c- AG P-SMASh-eGFP) was constructed. All smash-tagged RABV viruses were rescued as described before (Ghanem, Kern, and Conzelmann 2012).
  • VSV Mq SMASh-P eGFP essential virus protein
  • VSV Mq YFP-SMASh non-essential reporter gene
  • Figure 1-2 (B) the previously described full length plasmid pVSV * Mq was used as a backbone .
  • Mq denotes a modified M gene encoding a mutant M protein which is characterized by the amino acid changes M33A, M51 R, V221 F and S226R, that are known to abolish the host shutoff activity of the protein (Hoffmann et al. , 2010)
  • the star ( * ) denotes a GFP gene located between G and L genes
  • the YFP-SMASh gene described above was introduced in place of the GFP gene of VSV * Mq giving rise to VSV Mq YFP-SMASh, and a SMASh-P fusion construct was used to replace the authentic P ORF, giving rise to VSV * Mq SMASh-P..
  • Recombinant VS viruses were rescued by transfecting plasmids comprising the respective VSV full-length genomes and VSV N-, P-, and L-encoding helper plasmids (addgene plasmids #64087, #64088, and #64085) into HEK293T cells.
  • Virus-nfected cultures were identified by fluorescence microscopy.
  • RABV SAD P-smash, SAD N-smash (Fig. 2), and SAD YFP-SMASh (not shown) were found to replicate similar to the parental virus SAD L16 in BSR cells in the absence of an NS3 protease inhibitor while SAD SMASh-L was slightly attenuated (not shown).
  • SMASh- tagged VSV replicated like parental VSV Mq (not shown)
  • RABV and VSV encoding SMASh-tagged viral and non-viral proteins can be grown to high titers in the absence of a drug.
  • SAD N-SMASh or -P-SMASh viruses The difference between SAD N-SMASh or -P-SMASh viruses and SAD YFP-SMASh is that in the absence of ASV growth of the former is inhibited, and therefore the SMASh-tagged proteins cannot accumulate, while in case of SAD YFP-SMASh or VSV-YFP-SMASh the steadily growing virus is increasingly producing a SMASh-tagged protein.
  • the viruses were grown initially in the absence of an NS3 protease inhibitor, to allow accumulation for one day, and then treated with ASV or DNV. This led to a rapid destruction of hamster BSR T7/5 cell cultures infected with both N-SMASh and P-SMASh-expressing viruses (Fig. 4a), and cleavage of PARP in the dying cells (Fig. 4b).
  • NS3 inhibitor treatment of SAD L16 infections did not result in cell death.
  • proteolytic activity of the NS3 protease is in fact not required for cell killing in accordance with the present invention.
  • protease active and- inactive SMASh tags display equal drug-dependent toxicity, as verified in RealTime-GloTM MT Cell viability assays.
  • Cells infected with virus encoding the protease-inactive SMASh tag (YFP-SMASh(S139A)) killed cells as effectively and with the same kinetics compared to the proteolytically active tag. (Fig. 5C).
  • the POI which is the target of SMASh-tag-dependent regulation as described by Chung et al., is not necessary for drug-dependent cell toxicity of the SMASh-tag.
  • SMASh-tag mediated cell death involves intrinsic cell death rather than release of a soluble cytotoxic substance like TNF.
  • Addition of ASV to cell cultures infected at a MOI of ⁇ 1 reveals death of green fluorescent cells only (Fig. 6A, red arrows), while non-infected, non-fluorescent bystander cells maintain a healthy morphology (Fig. 6A, white arrows).
  • apoptosis seems to be the prominent mode of cell death.
  • activation of multiple caspases, including Casp8, Casp9, and the executioner Casp3 was induced by addition of the NS3 inhibitor, but not by the DMSO solvent, in cells infected previously with SAD P-SMASh (Fig. 6B).
  • pan-caspase inhibitor Z-VAD-FMK could at least partially prevent cleavage of PARP, CasptO, and Casp3 (Fig. 7) indicating that apoptosis is contributing to cell death induced by the SMASh-tag/NS3 inhibitor drug combination.
  • mouse cancer in vivo model cell lines including B16 melanoma (not shown) and MC38 colon adenocarcinoma cells (Fig. 9) were killed by SAD P-SMASh- (Fig. 9) and other SMASh-expressing viruses (not shown) by addition of the NS3 inhibitor ASV or DNV.
  • SAD eGFP already has some intrinsic oncolytic activity in MC38 cells, as indicated by partial PARP cleavage in SAD eGFP infected cells or SAD P-SMASh eGFP in the absence of an NS3 inhibitor.
  • DNV strongly enhances the cytopathic effect of SAD P-SMASh eGFP, as illustrated by more efficient PARP cleavage.
  • N2A cells expressisng the G protein (N2AN2cG-IRES-Crimson cells) the G-defective virus could spread and express genes, as indicated by GFP fluorescence (Fig. 10). Addition of ASV lead to killing of the cells, while DMSO treated cells survived.
  • Virus-independent killing of cells by SMASh-tag expression plasmids and NS 3 inhibitors As an alternative to virus-mediated expression of gene products, cells can be transfected with expression plasmids encoding the gene product under the control of a promoter which is active in the transfected cell.
  • NS3 inhibitor-mediated toxicity of SMASh-tag constructs is independent of the individual virus used, including rabies virus or VSV, pCAGGs N-SMASh, and pCAGGS-YFP-SMASh, in which the N- and YPF-SMASh genes are transcribed from a Chicken actin promoter, was transfected into HEK293T cells.
  • Viruses with a cytopathic phenotype, and in particular with a conditional cytopathic phenotype as described here, are highly desirable in genetic knock-out experiments involving biological selection of non-permissive (non-infectable) cells.
  • the recently introduced CRISPR/Cas9 technology for genome editing in mammalian cells allows knock-out of genes with minimal off- target effects and reliable identification of loss-of-function phenotypes.
  • a single-guide RNA directs a bacterial Cas9 nuclease to cause double- stranded cleavage of matching target DNA sequences (Jinek et al. 2012).
  • sgRNA libraries which comprises sgRNAs targeting >20.000 genes in the human and mouse genomes (Sanjana, Shalem, and Zhang 2014).
  • the sgRNA library as provided by Addgene, was amplified in E.coli Endura and packaged in VSV-G pseudotyped Lentiviruses as suggested by the provider.
  • a Cas9-expressing HEK 293T cell line was generated by transduction of cells with a Cas9-encoding Lentivirus (LV-Cas9-blast). Individual cell clones were selected according to their ability to c leave a GFP cDNA upon transfection of a corresponding sgRNA.
  • the Cas9- transduced cell line 293T-Cas9 was fully susceptible to ASV killing; in fact not a single cell survived ASV/CCPV treatment (Fig. 12 A).
  • a genome wide KO screen was performed with the LV-GeCKOv2 library generated as described above (Fig. 12 B).
  • 4.3 x 10 8 293T-Cas9 cells in 10 T175 flasks were transduced with 2.1 x 10 7 colony forming units (cfu) LV-guide human A puro (MOI ⁇ O.11 ) and grown in the presence of puromycin to select for LV-transduced cells.
  • the selected cells (6x10 8 ) were then plated into forty 15 cm-dishes and infected at an MOI of 2 with SAD P-SMASh, and 24 h.p.i treated with ASV to induce killing of virus-infected cells. Five days post ASV treatment, individual surviving colonies were observed (as indicated in Fig. 12B right part).
  • Surviving colonies either should either be non-permissive for CCPV infection, as an essential gene for entry or replication of RABV is knocked out, or a gene essential for the killing pathway by which ASV induces cell death.
  • This pilot screen verified the immediate and superb suitability of CCPV for high content genetic screens. It is obvious that this system is useful to identify potential host genes essentially required for virus entry or propagation, such as essential receptors or factors for replication. The genes to be identified provide immediate targets for development of antiviral therapies.
  • glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity. J Gen Virol. 2010;91 :2782-2793
  • RV Rabies virus glycoprotein expression levels are not critical for pathogenicity of RV, J Virol, 85: 697-704.
  • ⁇ CRISPR screen defines a signal peptide processing pathway required by flaviviruses', Nature, 535: 164-8.

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Abstract

La présente invention concerne un polypeptide capable de déclencher la mort cellulaire lors de l'administration d'un inhibiteur à petites molécules à une cellule comprenant ledit polypeptide. Le polypeptide peut être administré par un virus ou des systèmes d'administration de protéines ou d'acides nucléiques non viraux et l'inhibiteur par voie orale ou parentérale. L'invention est particulièrement utile pour une virothérapie oncolytique ainsi que d'autres utilisations de la mort cellulaire dépendante d'un médicament.
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WO2021233989A1 (fr) 2020-05-20 2021-11-25 Hennrich Alexandru Adrian Vecteur de vaccin viral pour l'immunisation contre un bêta-coronavirus
CN113975278A (zh) * 2021-10-29 2022-01-28 中国科学院昆明动物研究所 溴隐亭在制备治疗非洲猪瘟的产品中的应用

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KR20210051468A (ko) * 2019-10-30 2021-05-10 주식회사 바이오쓰리에스 바이러스성 출혈성 패혈증 바이러스를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물
KR102301833B1 (ko) 2019-10-30 2021-09-14 주식회사 바이오쓰리에스 바이러스성 출혈성 패혈증 바이러스를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물
WO2021233989A1 (fr) 2020-05-20 2021-11-25 Hennrich Alexandru Adrian Vecteur de vaccin viral pour l'immunisation contre un bêta-coronavirus
CN113975278A (zh) * 2021-10-29 2022-01-28 中国科学院昆明动物研究所 溴隐亭在制备治疗非洲猪瘟的产品中的应用

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