WO2021255217A1 - Amatoxin and amatoxin conjugates for use in inhibition of rna virus replication - Google Patents

Abstract

The present application relates to the use of amanitins, amatoxins and amatoxin conjugates for inhibition of RNA virus replication and propagation, in particular for inhibition of Coronavirus replication and propagation, and thus for prevention and treatment of Coronavirus-associated and/or Coronavirus-caused disorders and diseases.

Description

Amatoxin and Amatoxin Conjugates for Use in Inhibition of RNA virus Replication

Field of the invention

The present application relates to the use of amanitins, amatoxins and amatoxin conjugates for inhibition of RNA virus replication and propagation, in particular of Coronavirus replication and propagation, and thus for prevention and treatment of Coronavirus-associated and/or Coronavirus-caused disorders and diseases.

Background

An RNA virus is a virus that has RNA (ribonucleic acid) as its genomic material. RNA viruses are viruses that belong to group III, group IV or group V of the Baltimore classification system of classifying viruses, not considering viruses with DNA intermediates in their life cycle as RNA viruses. RNA-viruses are encompassing viruses having a double- stranded (ds)RNA genome (group III), viruses having a positive-sense single-stranded (ss)RNA genome (group IV, the genome itself acting as messenger RNA), and viruses having a negative-sense single-stranded (ss)RNA genome (group V, the genome used as a template for mRNA synthesis).

Coronaviruses (CoVs) are enveloped positive-sense RNA viruses, which are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses represent important pathogens for humans and vertebrates, and can infect respiratory, gastrointestinal, hepatic, central nervous system and other organs. The outbreaks of the severe acute respiratory syndrome (SARS), caused by SARS-CoV, in 2002/2003, the Middle East respiratory syndrome (MERS), caused by MERS- CoV, in 2012, and the pandemic outbreak of Coronvirus Disease (COVID-19), caused by SARS-CoV-2, in 2019/2020 have demonstrated the significant pathogenic potential of these viral agents (Chen etal , 2020; Fehr and Perlman, 2015).

Coronaviruses, belonging to the Coronaviridae family, are divided into 4 genera, a-, b-, d- and g-coronaviruses, while the b-coronaviruses are further divided into A, B, C, and D lineages. The seven CoVs known so far that can infect humans (HCoVs) include HCoV-229E and HCoV-NL63 in the a-coronaviruses, HCoV-OC43 and HCoV-HKU 1 in the b-corona- viruses lineage A, SARS-CoV and SARS-CoV-2 in the b-coronaviruses lineage B (b-B coronaviruses), and MERS-CoV in the b-coronaviruses lineage C (Xia et al , 2020). Human Coronaviruses were identified for the first time in 1965 in patients suffering from respiratory disease (Modrow etal , 2003).

Coronavirus virions are spherical with diameters of approximately 125 nm according to cryo- electron tomography and cryo-electron microscopy studies. The most prominent feature of Coronaviruses is the club-shaped spike projections emanating from the surface of the virion. These spikes give the viral particles the appearance of a “solar corona“, from which the name of this virus family has been derived. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses.

Coronavirus particles contain four main structural proteins: the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, all of which are encoded within the 3' end of the viral genome. The S protein (-150 kDa) utilizes an N-terminal signal sequence to gain access to the endoplasmic reticulum (ER), and is heavily N-linked glycosylated. Homotrimers of the virus-encoded S protein make up the distinctive spike structure on the surface of the virus. The M protein has three transmembrane domains and supports shaping the virion by promoting membrane curvature, and binds to the nucleocapsid. The E protein plays a role in virus assembly and release, and is involved in viral pathogenesis. The N protein comprises two domains, both of which can interact with the viral RNA genome via different mechanisms. A fifth structural protein, the hemagglutinin-esterase (HE), is present in a subset of b-coronaviruses (Chen etal , 2020; Fehr and Perlman, 2015). Of note, the S protein is also present in the cell membrane of Coronavirus-infected cells (Modrow et al, 2003).

Coronaviruses contain a non-segmented, single-stranded, positive-sense RNA genome of ~30 kb. The genome contains a 5 '-cap structure and a 3'-poly-(A) tail, allowing it to act as an mRNA for direct translation of the replicase polyproteins. The replicase gene encoding the nonstructural proteins (nsps) occupies two-thirds of the genome, about 20 kb, as opposed to the structural and accessory proteins, which make up only about 10 kb of the viral genome. The 5' end of the genome contains a leader sequence and untranslated region (UTR) that comprises multiple stem loop structures required for RNA replication and transcription. Additionally, at the beginning of each structural or accessory gene are transcriptional regulatory sequences (TRSs) that are required for expression of each of these genes. A 3' UTR also contains RNA structures required for replication and synthesis of viral RNA. The organization of the coronavirus genome follows the structural order of 5'-leader-UTR- replicase-S (Spike)-E (Envelope)-M (Membrane)-N (Nucleocapsid)-3' UTR-poly (A) tail with accessory genes interspersed within the structural genes at the 3' end of the genome. The accessory proteins are almost exclusively non-essential for replication in tissue culture; however, some have been shown to have important roles in viral pathogenesis (Fehr and Perlman, 2015).

Regarding the whole genome, SARS-CoV-2 maintains -80% nucleotide identity to the original SARS epidemic human virus SARS-CoV; its closest whole genome relatives are two bat SARS-like CoVs (ZC45 and ZXC21) that share -89% sequence identity with SARS- CoV-2 (Gralinski and Menachery, 2020).

Coronavirus cellular infection begins with viral entry, in which the viral particle recognizes a host cell receptor and fuses its membrane with the host cell membrane (Belouzard et al, 2012). These two steps are mediated by the Coronavirus spike (S) protein. The Coronavirus spike contains three segments: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail. The ectodomain consists of a receptor-binding subunit SI and a membrane-fusion subunit S2. Electron microscopy studies revealed that the spike is a clove shaped trimer with three SI heads and a trimeric S2 stalk. During virus entry, SI binds to a receptor on the host cell surface for viral attachment, and S2 fuses the host and viral membranes, allowing viral genomes to enter host cells. Receptor binding and membrane fusion are the initial and critical steps in the Coronavirus infection cycle; they also have been suggested as primary targets for human inventions (Li, 2016; Tang et al , 2020).

Both SARS-CoV and SARS-CoV-2 utilize the C-terminal domain of the SI subunit to bind angiotensin-converting enzyme 2 (ACE2) as a receptor, which is, for example, abundantly detected on lung and small intestine cells (Tang et al, 2020).

Fusion of the viral and cellular membranes via the S2 subunit requires an acid dependent proteolytic cleavage of the S protein by a cathepsin, TMPRRS2 (transmembrane protease serine subtype 2) or another protease. The fusion generally occurs within acidified endosomes, but some Coronaviruses can fuse at the plasma membrane. As a result of membrane fusion, the viral genome is released into the cytoplasm.

The next step in the Coronavirus lifecycle is the translation of the replicase gene from the viral genomic RNA. Viral RNA synthesis follows the translation and assembly of the viral replicase complexes. Viral RNA synthesis by RNA-dependent RNA polymerase produces both genomic and sub-genomic RNAs. Sub-genomic RNAs serve as mRNAs for the structural and accessory genes which reside downstream of the replicase polyproteins.

Following replication and sub-genomic RNA synthesis, the viral structural proteins, S, E, and M are translated and inserted into the endoplasmic reticulum (ER). These proteins move along the secretory pathway into the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). There, viral genomes encapsidated by N protein bud into membranes of the ERGIC containing viral structural proteins, forming mature virions. Following assembly, virions are transported to the cell surface in vesicles and released by exocytosis (see Fig. 1) (Fehr and Perlman, 2015).

Prior to the SARS outbreak in 2002/2003 in China, Coronaviruses were only thought to cause mild, self-limiting respiratory infections in humans. Two of the human Coronaviruses then known are a-coronaviruses, HCoV-229E and HCoV-NL63, while the other two are b- coronaviruses, HCoV-OC43 and HCoV-HKU 1. These viruses are endemic in the human populations, causing 15-30% of respiratory tract infections. Coronaviruses now have been realized to pose serious health threats to humans and other animals. From 2002 to 2003, SARS-CoV causing severe acute respiratory syndrome infected more than 8,000 people, with a mortality rate of ~10%. This rate was much higher in elderly individuals over 60 years of age, with mortality rates approaching 50%. SARS-CoV has been shown to primarily infect epithelial cells within the lung.

In 2012, MERS-CoV causing Middle East respiratory syndrome has infected more than 1,700 people, with a fatality rate of ~36%. MERS-CoV has been suggested to originate from bats, but the spillover to humans has been traced to dromedary camels. MERS-CoV utilizes Di- peptidyl peptidase 4 (DPP4) as its cellular receptor in humans.

Since 2013, porcine epidemic diarrhea coronavirus (PEDV) has swept throughout the United States, causing an almost 100% fatality rate in piglets and wiping out more than 10% of America’s pig population in less than a year.

SARS-CoV-2, causing the current COVID-19 pandemia that started in late 2019 from China, has been responsible for more than 600,000 deaths in the USA alone so far. SARS-CoV-2 and SARS-CoV are closely related and originated in bats, who most likely serve as reservoir host for these two viruses.

In general, Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and animals, threatening human and veterinary health and causing tremendous economic loss (Fehr and Perlman, 2015; Li, 2016; Walls et al., 2020).

Various pharmaceutical agents are currently in develoment or under preclinical or clinical review for treatment of human Coronavirus infection, including, for example, Remdesivir, Chloroquine and Hydroxychloroquine, Lopinavir/Ritonavir, and Nitazoxanide (for review see McCreary and Pogue, 2020). However, neither any pharmaceutical therapeutic drug nor any specific preventive vaccine has been approved so far for treatment and prevention of Coronavirus-associated diseases, in particular of COVID-19 (Sahin etal , 2020).

As possible sites of intervention, various stages of the Coronaviral life cycle have been suggested, including initial attachment of the spike protein with the cellular receptor, fusion of viral and cellular membranes, or viral genomic replication and RNA translation (Tang et al, 2020).

Amanitins are cyclic peptides composed of 8 amino acids originally found in Amanita phalloides mushrooms which specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells, and thereby also the transcription and protein biosynthesis of the affected cells (see Fig. 2). Though not covalently bound, the complex between amanitin and RNA- polymerase II is very tight (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, thus making recovery of an affected cell unlikely. When the inhibition of transcription lasts sufficiently long, the cell will undergo programmed cell death (apoptosis).

Albeit no influence of amanitin on the replication of a number of DNA or RNA-containing viruses had been found before, amanitin was described to specifically inhibit influenza virus replication some time ago (Rott and Scholtissek, 1970). Further findings demonstrated that DNA transcription mediated by DNA-dependent RNA polymerase II is essential for early- phase influenza virus replication (Mahy et al, 1972) and that DNA-dependent RNA polymerase II participates in replication of these RNA viruses (Spooner and Barry, 1977). The influenza virus genome consists of several single-stranded RNA segments, each of which is associated with a protein complex, with the 3'- and 5 '-ends bound to the influenza RNA- dependent RNA polymerase (RdRp) and the remainder associated with the viral nucleo- protein. More recently it was shown that during transcription of influenzaviral mRNA, this ribonucleoprotein complex “steals“ short, 5 '-capped transcripts produced by the cellular DNA dependent RNA polymerase II and uses them to prime transcription of viral mRNA, a process termed “cap-snatching“ (De Vlugt et al, 2018).

In contrast to influenza virus, whose RNA transcription and replication occurs in the cell nucleus (Modrow et al, 2003) and requires DNA-dependent RNA polymerase II activity and a cap-snatching step (De Vlugt et al, 2018), the RNA transcription and replication of Corona- viruses occurs in the cellular cytoplasm, employs the viral RNA-dependent RNA polymerase, and is not known to require DNA-dependent RNA polymerase II activity and a cap-snatching mechanism (Tang et al, 2020; Modrow et al, 2003). Surprisingly, the inventors of the present application found that amanitins and amatoxins inhibit viral replication of Coronaviruses, in particular of SARS-CoV-2. The observation of this inhibitory effect of amanitins and amatoxins has been unexpected in view of the prior art.

Summary of the Invention

In view of the prior art, it was one object of the present invention to provide for the use of an amatoxin for inhibition of RNA virus replication and propagation, in particular of Coronavirus replication, and to provide for an amatoxin for use in the treatment of Coronavirus-associated diseases.

It was one further object of the present invention to provide for the use of a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for inhibition of RNA virus replication and propagation, in particular of Coronavirus replication, and to provide for such a conjugate for use in the treatment of Coronavirus-associated diseases.

These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.

The invention and general advantages of its features will be discussed in detail below.

Description of the Figures

Fig. 1. Model of Coronavirus life cycle. As the virus binds to its receptor (7), it can achieve entry via two routes: plasma membrane or endosome. For SARS-CoV, the presence of exogeneous and membrane bound proteases, such as trypsin and TMPRSS2, triggers the early fusion pathway (2a); otherwise, it will be endocytosed (2b, 3). For MERS-CoV, if furin cleaved the S protein at S1/S2 during biosynthesis, exogeneous and membrane bound proteases, such as trypsin and TMPRSS2, will trigger early entry (2a); otherwise, it will be cleaved at the S1/S2 site (2b) causing the virus to be endocytosed (3). For both CoV types, within the endosome, the low pH activates cathepsin L (4), further cleaving S2, triggering the fusion pathway and releasing the CoV genome. Upon viral entry, copies of the genome are made in the cytoplasm (5), where components of the spike protein are synthesized in the rough endoplasmic reticulum (ER) (6). The structural proteins are assembled in the ER-Golgi intermediate compartment (ERGIC), where the spike protein can be precleaved by furin, depending on cell type (7), followed by release of the virus from the cell (8, 9). For SARS- CoV-2, current studies are indicating that SARS-CoV-2 can utilize membrane-bound TMPRSS2 or endosomal cathepsin L for entry, and that the S protein is processed during biosynthesis (from Tang etal ., 2020).

Fig. 2. Structural formulae of various amatoxins. The numbers in bold type (1 to 8) designate the standard numbering of the eight amino acids forming the amatoxin. The standard designations of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters a to g, Greek letters a to d, and numbers from G to 7’, respectively).

Fig. 3. Inhibitory effect of Amanitin on viral replication of SARS-CoV-2 in VERO cell culture. Amanitin inhibits replication of SARS-CoV-2 with an IC50 of 6.2 mM. Circles, upper panel: SARS-CoV-2 inhibition [%]; circles, lower panel: SARS-CoV-2 copy number [copies/ml]; squares, toxicity [%].

Detailed Description of the Invention

Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure and avoid lengthy repetitions.

According to one aspect of the present invention, the present invention relates to the use of an amanitin or amatoxin for inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication. The term “inhibition of Coronavirus replication” or “inhibiting Coronavirus replication” as used according to the invention referes to a reduction of about 10%, 25%, 30%, 40%, 45%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, or of at least 10%, 12.5%, 25%, 50%, 75%, 80% in the replication rate compared to a control which was not treated or contacted with amanitin, or an amatoxin. Inhibition of coronavirus replication may e.g. be assessed by the number of viral particles released from a cell using a particle gel assay (see e.g. Yan et ah, Methods Mol Biol. 2017 ; 1540: 193-202) or the amount of viral RNA that can be detected within a cell, or body fluid, such as saliva, blood, plasma, urine, cerebrospinal fluid, or in vitro the amount of viral RNA detectable in the cell supernatant. Detection of coronavirus replication may e.g. be done by quantitative RT-PCR according to the method disclosed in Chu et al. Lancet Microbe. 2020 May;l(l):el4-e23.

According to one aspect, the present invention relates to the use of amatoxin for inhibition of an RNA-dependent RNA polymerase, preferably of a viral RNA-dependent RNA polymerase.

In the context of the present invention the term “RNA virus” refers to a virus that belongs to group III, group IV or group V of the Baltimore virus classification system and that is depending on the use of an RNA-dependent RNA polymerase for viral replication. An RNA-dependent RNA polymerase (RdRP), such as e.g. a viral RNA-dependent RNA polymerase, or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template, in contrast to a typical DNA-dependent RNA polymerase, which catalyzes the transcription of RNA from a DNA template. RdRP is an essential protein encoded in the genomes of all RNA viruses with no DNA replication stage, z.e., of the RNA viruses. It catalyses synthesis of the RNA strand complementary to a given RNA template.

Amatoxins are cyclic peptides composed of 8 amino acids that are found in Amanita phalloides mushrooms (see Fig. 2). Amatoxins specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells, and thereby also the transcription and protein biosynthesis of the affected cells. Inhibition of transcription in a cell causes stop of growth and proliferation. Though not covalently bound, the complex between amanitin and RNA- polymerase II is very tight (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, thus making recovery of an affected cell unlikely. When the inhibition of transcription lasts sufficiently long, the cell will undergo programmed cell death (apoptosis).

In the context of the present invention the term “amatoxin” includes all cyclic peptides composed of 8 amino acids as isolated from the genus Amanita and described in Wieland, T. et al. (Wieland el al , 1978), further all chemical derivatives thereof; further all semisynthetic analogs thereof; further all synthetic analogs thereof built from building blocks according to the master structure of the natural compounds (cyclic, 8 amino acids), further all synthetic or semisynthetic analogs containing non-hydroxylated amino acids instead of the hydroxylated amino acids, further all synthetic or semisynthetic analogs, in which the sulfoxide moiety is replaced by a sulfone, thioether, or by atoms different from sulfur, e.g ., a carbon atom as in a carbanalog of amanitin.

As used herein, a “derivative” of a compound refers to a species having a chemical structure that is similar to the compound, yet containing at least one chemical group not present in the compound and/or deficient of at least one chemical group that is present in the compound. The compound to which the derivative is compared is known as the “parent” compound. Typically, a “derivative” may be produced from the parent compound in one or more chemical reaction steps. As used herein, an “analogue” of a compound is structurally related but not identical to the compound and exhibits at least one activity of the compound. The compound to which the analogue is compared is known as the “parent” compound. The afore-mentioned activities include, without limitation: binding activity to another compound; inhibitory activity, e.g. enzyme inhibitory activity; toxic effects; activating activity, e.g. enzyme-activating activity. It is not required that the analogue exhibits such an activity to the same extent as the parent compound. A compound is regarded as an analogue within the context of the present application, if it exhibits the relevant activity to a degree of at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%_, more preferably at least 40%, and more preferably at least 50%) of the activity of the parent compound. Thus, an “analogue of an amatoxin”, as it is used herein, refers to a compound that is structurally related to any one of a-amanitin, b-amanitin, g- amanitin, e- amanitin, amanin, amaninamide, amanullin, and amanullinic acid and that exhibits at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, and more preferably at least 50%) of the inhibitory activity against mammalian RNA polymerase II as compared to at least one of a-amanitin, b-amanitin, g-amanitin, e-amanitin, amanin, amaninamide, amanullin, and amanullinic acid. An “analogue of an amatoxin” suitable for use in the present invention may even exhibit a greater inhibitory activity against mammalian RNA polymerase II than any one of a-amanitin, b-amanitin, g-amanitin, e-amanitin, amanin, amaninamide, amanullin, or amanullinic acid. The inhibitory activity might be measured by determining the concentration at which 50% inhibition occurs (IC50 value). The inhibitory activity against mammalian RNA polymerase II can be determined indirectly by measuring the inhibitory activity on cell proliferation.

A “semisynthetic analogue” refers to an analogue that has been obtained by chemical synthesis using compounds from natural sources (e.g. plant materials, bacterial cultures, fungal cultures or cell cultures) as starting material. Typically, a “semisynthetic analogue” of the present invention has been synthesized starting from a compound isolated from a mushroom of the Amanitaceae family. In contrast, a “synthetic analogue” refers to an analogue synthesized by so-called total synthesis from small (typically petrochemical) building blocks. Usually, this total synthesis is carried out without the aid of biological processes. According to some embodiments of the present invention, the amatoxin can be selected from the group consisting of a-amanitin, b-amanitin, amanin, amaninamide and analogues, derivatives and salts thereof.

Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase IF Preferred amatoxins are those with a functional group (e.g. a carboxylic group, an amino group, a hydroxy group, a thiol or a thiol-capturing group) that can be reacted with linker molecules or target-binding moieties as defined below.

In the context of the present invention, the term “amanitins” particularly refers to bicyclic structure that are based on an aspartic acid or asparagine residue in position 1, a proline residue, particularly a hydroxyproline residue in position 2, an isoleucine, hydroxyisoleucine or dihydroxyisoleucine in position 3, a tryptophan or hydroxytryptophan residue in position 4, glycine residues in positions 5 and 7, an isoleucine residue in position 6, and a cysteine residue in position 8, particularly a derivative of cysteine that is oxidized to a sulfoxide or sulfone derivative (for the numbering and representative examples of amanitins, see Figure 1), and furthermore includes all chemical derivatives thereof; further all semisynthetic analogues thereof; further all synthetic analogues thereof built from building blocks according to the master structure of the natural compounds (cyclic, 8 amino acids), further all synthetic or semisynthetic analogues containing non-hydroxylated amino acids instead of the hydroxylated amino acids, further all synthetic or semisynthetic analogues, in each case wherein any such derivative or analogue is functionally active by inhibiting mammalian RNA polymerase II.

According to one aspect, the present invention relates to the use of amatoxin for inhibition of human Coronavirus replication.

According to one aspect of the present invention, the present invention relates to the use of an amatoxin for the treatment of Coronavirus-associated diseases. For example, Coronavirus- associated diseases include without limitation pneumonia, cardiovascular disease, chronic obstructive pulmonary disease, pulmonary fibrosis, chronic liver disease, cytokine storm, Lymphopenia, thrombocytopenia. According to one aspect of the present invention, the present invention relates to an amatoxin for use in the treatment of Coronavirus-associated diseases, in particular for the treatment of human Coronavirus-associated diseases, e.g. such as those disclosed above.

According to one aspect of the present invention, the present invention relates to amatoxin for use in the inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication.

According to a further aspect, the present invention relates to the use of a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication.

According to one aspect of the present invention, the present invention relates to the use of a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for the treatment of Coronavirus-associated diseases.

According to one aspect of the present invention, the present invention relates to a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for use in the treatment of Coronavirus-associated diseases.

The term “target-binding moiety”, as used herein, refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. Preferred target binding moieties in the context of the present application are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. “Target-binding moieties” suitable for use in the present invention typically have a molecular mass of 10 000 Da (10 kDa) or more.

The conjugate for use in the treatment of Coronavirus-associated diseases preferably comprises a target-binding moiety which is selected from the group consisting of (i) an antibody, preferably a monoclonal antibody, (ii) an antigen-binding fragment thereof, preferably a variable domain (Fv), a Fab fragment or an F(ab)2 fragment,

(iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and

(iv) an antibody-like protein.

According to a preferred embodiment, the target-binding moiety of the invention is an antibody, preferably a monoclonal antibody.

Said antibody, or antigen-binding fragment thereof or antigen-binding derivative thereof, can be a murine, a chimeric, a humanized or a human antibody, or antigen-binding fragment or antigen-binding derivative thereof, respectively. The term “humanized” as used herein refers to antibody which resemble human antibodies and which have been designed to generate antibody molecules with minimal immunogenicity when applied to humans, while ideally still retaining specificity and affinity of the non-human parental antibody (for review see Almagro and Fransson 2008). Using these methods, e.g., the framework regions of a mouse mAb are replaced by corresponding human framework regions (so-called CDR grafting). W0200907861 discloses the generation of humanized forms of mouse antibodies by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA technology. US6548640 by Medical Research Council describes CDR grafting techniques, and US5859205 by Celltech describes the production of humanised antibodies.

As used herein, the term “chimeric antibody” relates to an antibody consisting of the antibody’s original antigen-binding variable domains with the constant domains being derived from a different species. Since antibodies, in particular monoclonal antibodies, originally most often have been derived from mouse, typically chimeric antibodies are containing human constant domains and mouse variable domains, in order to reduce immunogenicity in humans.

The antibodies, the antibody fragments or antibody derivatives thereof, disclosed herein can comprise humanized sequences, in particular of the preferred VH- and VL-based antigen binding region which maintain appropriate ligand affinity. The amino acid sequence modifications to obtain said humanized sequences may occur in the CDR regions and/or in the framework regions of the original antibody and/or in antibody constant region sequences. The spike (S) protein of Coronavirus has been shown to be present in the cell membrane of Coronavirus-infected cells (Modrow el al , 2003).

Therefore, for targeting Coronavirus-infected cells, or cells in which Coronavirus replicates, said antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof, respectively, may e.g. be specific for the Coronavirus spike (S) protein, the SI subunit or S2 subunit of the spike (S) protein, angiotensin-converting enzyme 2 (ACE2), or the TMPRRS2 (transmembrane protease serine subtype 2) protein. For example, antibodies which may be used according to the invention include antibodies specific for the spike (S) protein such as bamlanivimab (LY-CoV555, LY-3819253), etesevimab (LY-C0VOI6), casirivimab.

Corresponding anti-ACE2 antibodies which may e.g. be used according to the invention include those disclosed in W002/098906, corresponding anti-TMPRRS2 antibodies which may e.g. be used according to the invention include those disclosed in WO2019/147831.

According to one embodiment, said antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof as disclosed herein may e.g. bind to spike variants, which comprise one or more amino acid subsitutions, deletions, polymorphisms or insertions. For example, said antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof according to the invention may bind to spike (S) protein variants of SARS-CoV2 strains Alpha, Beta, Gamma, Epsilon, Delta, Kappa, Eta (nomenclature according to WHO labels), whereby the above strains comprise at least the following mutations in the spike protein S:

Alpha: 69-70del, N501Y, P681H,

Beta: K417N, E484K, N501Y,

Gamma: K417T, E484K, N501Y Epsilon: L452R,

Delta: L452R, T478K, P681R,

Kappa: L452R, E484Q, P681R,

Eta: E484K, F888L, whereby “del” denotes a deletion of the respective amino acids in the S protein. Amino acid numbering is made in reference to GenBank entry QHU36824.1 (SEQ ID NO: 1). Alternative nomenclature for the WHO labels for the spike protein S variants as disclosed above may e.g. also be referred to as B.l.1.7 (corresponding to Alpha), B.1.351 (corresponding to Beta), P.l (corresponding to Gamma), B.1.429, B.1.427 (both corresponding to Epsilon), B.1.617 (corresponding to Delta), B.1.617.1 (corresponding to Kappa) and B.1.525 (corresponding to Eta).

The spike protein S variants as disclosed above may e.g. comprise additional mutations, for example the Alpha variant may comprise in addition to the mutations disclosed above one or more, e.g. one, two, three, or more, or all of the mutations A570D, D614G, T716I, S982A, D1118H, the Beta variant variant may comprise in addition to the mutations disclosed above one or more, e.g. one, two, three, or more, or all of the mutations L18F, D80A, D215G, R246I, D614G, A701V, the Gamma variant may comprise in addition to the mutations disclosed above one or more, e.g. one, two, three, or more, or all of the mutations L18F, T20N, P26S, D138Y, R190S, D614G, H655Y, T1027I, V1176F, the Delta variant may comprise in addition to the mutations disclosed above one or more, e.g. one, two, three, or more, or all of the mutations T19R, Deletion 157-158, D614G, D950N. The spike protein S variant may e.g. also comprise a combination of deletions of the variants disclosed above, e.g. 69-70del, E484K, N501Y, P681H.

In one embodiment, the antibody, antigen-binding fragment thereof, or antigen-binding derivative thereof as disclosed above may e.g. bind to the spike protein S variants as disclosed above, which comprise the additional mutations and/or deletions as disclosed above.

As used herein, the term “antibody” shall refer to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes or fragments of immunoglobulin genes or cDNAs derived from the same. Said immunoglobulin genes include the light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma and mu constant region genes as well as any of the many different variable region genes.

The basic immunoglobulin (antibody) structural unit is usually a tetramer composed of two identical pairs of polypeptide chains, the light chains (L, having a molecular weight of about 25 kDa) and the heavy chains (H, having a molecular weight of about 50-70 kDa). Each heavy chain is comprised of a heavy chain variable region (abbreviated as VH or V_H) and a heavy chain constant region (abbreviated as CH or C_H). The heavy chain constant region is comprised of three domains, namely CHI, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL or V_L) and a light chain constant region (abbreviated as CL or C_L). The VH and VL regions can be further subdivided into regions of hypervariability, which are also called complementarity determining regions (CDR) interspersed with regions that are more conserved called framework regions (FR). Each VH and VL region is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen.

The CDRs are most important for binding of the antibody or the antigen binding portion thereof. The FRs can be replaced by other sequences, provided the three-dimensional structure which is required for binding of the antigen is retained. Structural changes of the construct most often lead to a loss of sufficient binding to the antigen.

The term “antigen binding portion“ of the (monoclonal) antibody refers to one or more fragments of an antibody which retain the ability to specifically bind antigen in its native form. Examples of antigen binding portions of the antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfid bridge at the hinge region, an Fd fragment consisting of the VH and CHI domain, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and a dAb fragment which consists of a VH domain and an isolated complementarity determining region (CDR).

The antibody, or antibody fragment or antibody derivative thereof, according to the present invention can be a monoclonal antibody. The antibody can be of the IgA, IgD, IgE, IgG or IgM isotype.

As used herein, the term “monoclonal antibody (mAh)” shall refer to an antibody composition having a homogenous antibody population, z.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof. Particularly preferred, such antibody is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.

As used herein, the term “fragment” shall refer to fragments of such antibody retaining target binding capacities, e.g., a CDR (complementarity determining region), a hypervariable region, a variable domain (Fv), an IgG heavy chain (consisting of VH, CHI, hinge, CH2 and CH3 regions), an IgG light chain (consisting of VL and CL regions), and/or a Fab and/or F(ab)2.

As used herein, the term “derivative” shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and/or F(ab)2, as well as bi-, tri- or higher specific antibody constructs. All these items are explained below.

Other antibody derivatives known to the skilled person are Diabodies, Camelid Antibodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerised constructs comprising CH3+VL+VH, other scaffold protein formats comprising CDRs, and antibody conjugates (e.g., antibody, or fragments or derivatives thereof, linked to a drug, a toxin, a cytokine, an aptamer, a nucleic acid such as a desoxyribonucleic acid (DNA) or ribonucleic acid (RNA), a therapeutic polypeptide, a radioisotope or a label). Said scaffold protein formats may comprise, for example, antibody-like proteins such as ankyrin and affilin proteins and others.

As used herein, the term “antibody-like protein” refers to a protein that has been engineered (e.g. by mutagenesis of Ig loops) to specifically bind to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein may be any protein having good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include without limitation affibodies, anticalins, and designed ankyrin repeat proteins (Binz et al., 2005). Antibody-like proteins can be derived from large libraries of mutants, e.g. by panning from large phage display libraries, and can be isolated in analogy to regular antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins.

As used herein, the term “Fab” relates to an IgG fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody.

As used herein, the term “F(ab)₂” relates to an IgG fragment consisting of two Fab fragments connected to one another by disulfide bonds.

As used herein, the term “scFv” relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually comprising serine (S) and/or glycine (G) residues. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.

Modified antibody formats are for example bi- or trispecific antibody constructs, antibody- based fusion proteins, immunoconjugates and the like.

IgG, scFv, Fab and/or F(ab)₂ are antibody formats which are well known to the skilled person. Related enabling techniques are available from respective textbooks.

A “linker” in the context of the present application refers to a molecule that increases the distance between two components, e.g. to alleviate steric interference between the target binding moiety and the amatoxin, which may otherwise decrease the ability of the amatoxin to interact with RNA polymerase II. The linker may serve another purpose as it may facilitate the release of the amatoxin specifically in the cell being targeted by the target binding moiety. It is preferred that the linker and preferably the bond between the linker and the amatoxin on one side and the bond between the linker and the target binding moiety or antibody on the other side is stable under the physiological conditions outside the cell, e.g. the blood, while it can be cleaved inside the cell, in particular inside the target cell, e.g. cancer cell. To provide this selective stability, the linker may comprise functionalities that are preferably pH-sensitive or protease sensitive. Alternatively, the bond linking the linker to the target binding moiety may provide the selective stability. Preferably a linker has a length of at least 1, preferably of 1-30 atoms length ( e.g ., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), wherein one side of the linker has been reacted with the amatoxin and, the other side with a target-binding moiety. In the context of the present invention, a linker preferably is a C 1.30-alkyl, C 1.30-heteroalkyl, C2-30- alkenyl, C2-3o-heteroalkenyl, C2-3o-alkynyl, C2-3o-heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or a heteroaralkyl group, optionally substituted. The linker may contain one or more structural elements such as amide, ester, ether, thioether, disulfide, hydrocarbon moieties and the like. The linker may also contain combinations of two or more of these structural elements. Each one of these structural elements may be present in the linker more than once, e.g. twice, three times, four times, five times, or six times. In some embodiments the linker may comprise a disulfide bond. It is understood that the linker has to be attached either in a single step or in two or more subsequent steps to the amatoxin and the target binding moiety. To that end the linker to be will carry two groups, preferably at a proximal and distal end, which can (i) form a covalent bond to a group, preferably an activated group on an amatoxin or the target binding-peptide or (ii) which is or can be activated to form a covalent bond with a group on an amatoxin. Accordingly, if the linker is present, it is preferred that chemical groups are at the distal and proximal end of the linker, which are the result of such a coupling reaction, e.g. an ester, an ether, a urethane, a peptide bond etc. The presence of a “linker” is optional, i.e. the toxin may be directly linked to a residue of the target-binding moiety in some embodiments of the target-binding moiety toxin conjugate.

In the context of the present invention, said linker can be a stable or a cleavable linker. In the context of the present invention, the term “stable linker” refers to a linker that is stable (i) in the presence of enzymes, particularly of lysosomal peptidases, such as Cathepsin B, and (ii) in an intracellular reducing environment.

In particular embodiments, the stable linker does not contain (i) an enzyme-cleavable substructure, particularly no dipeptide sequence cleavable by Cathepsin B), and/or (ii) a disulfide group. In particular such embodiments, the linker has a length of up to 12 atoms, particularly from 2 to 10, more particularly from 4 to 9, and most particularly from 6 to 8 atoms. In particular embodiments, the a stable linker of the invention comprises a moiety selected from the following group of moieties:

(amatoxin side) -(CEb^- (target-binding moiety side);

(amatoxin side) -(CEb^- (target-binding moiety side);

(amatoxin side) -(CEE - (target-binding moiety side);

(amatoxin side) -(CEb^- (target-binding moiety side);

(amatoxin side) -(CEE (target-binding moiety side);

(amatoxin side) -(CEE)_?- (target-binding moiety side);

(amatoxin side) -(CEE (target-binding moiety side);

(amatoxin side) -(CEb^- (target-binding moiety side);

(amatoxin side) -(CH₂)_IO- (target-binding moiety side);

(amatoxin side) -(CH₂)n- (target-binding moiety side);

(amatoxin side) -(CH₂)i₂- (target-binding moiety side);

(amatoxin side) -(CH₂)i₆- (target-binding moiety side);

(amatoxin side) -(CH2)2-0-(CH2)2-0-(CH2)2- (target-binding moiety side);

(amatoxin side) -(CH2)2-0-(CH2)2-0-(CH2)2-0-(CH2)2- (target-binding moiety side); and (amatoxin side) -(CH2)2-0-(CH2)2-0-(CH2)2-0-(CH2)2-0-(CH2)2- (target-binding moiety side), wherein the stable linker additionally may comprise a thiol reactive group for coupling to cysteine residues on the target-binding moiety. The thiol reactive group may e.g. be selected from bromo acetamide, iodo acetamide, methylsulfonylbenzothiazole, 4,6-dichloro-l,3,5- triazin-2-ylamino group methyl-sulfonyl phenyltetrazole or methyl sulfonyl phenyloxadiazole, pyridine-2-thiol, 5- nitropyridine-2-thiol, methanethiosulfonate, or a maleimide.

According to a preferred embodiment, the thiol -reactive group of the stable linker of the invention is maleimide.

The term “cleavable linker” as used in the present invention is understood as comprising at least one cleavage site. As used herein, the term “cleavage site” shall refer to a moiety that is susceptible to specific cleavage at a defined position under particular conditions. Said conditions are, e.g., specific enzymes or a reductive environment in specific body or cell compartments. An enzymatically cleavable moiety according to the invention may also be referred to as “cleavable by an enzyme”. Enzymatic cleavage of the linker results in the intracellular release of the toxin cargo conjugated to the targeting moiety or antibody as disclosed herein, or a metabolite thereof after internalization (see Dubowchik et al., Bioconjug Chem. 13 (2002) 855-69).

Said cleavable linker can be selected from the group consisting of an enzymatically cleavable linker, preferably a protease-cleavable linker, and a chemically cleavable linker, preferably a linker comprising a disulfide bridge.

According to preferred embodiments of the present invention, the cleavage site is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, said enzymatically cleavable moiety comprises a valine-alanine (Val-Ala), valine-citrulline (Val- Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, a phenylalanine-lysine- glycine-proline-leucin-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or a b-glucuronide or b-galactoside.

According to some embodiments, said cleavage site can be cleavable by at least one protease selected from the group consisting of cysteine protease, metalloprotease, serine protease, threonine protease, and aspartic protease.

Cysteine proteases, also known as thiol proteases, are proteases that share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

Metalloproteases are proteases whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt. The metal ion is coordinated to the protein via three ligands. The ligands co-ordinating the metal ion can vary with histidine, glutamate, aspartate, lysine, and arginine. The fourth coordination position is taken up by a labile water molecule.

Serine proteases are enzymes that cleave peptide bonds in proteins; serine serves as the nucleophilic amino acid at the enzyme's active site. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

Threonine proteases are a family of proteolytic enzymes harbouring a threonine (Thr) residue within the active site. The prototype members of this class of enzymes are the catalytic subunits of the proteasome, however, the acyltransferases convergently evolved the same active site geometry and mechanism.

Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.

In particular embodiments of the present invention, the cleavable site is cleavable by at least one agent selected from the group consisting of Cathepsin A or B, matrix metalloproteinases (MMPs), elastases, b -glucuronidase and b-galactosidase, preferably Cathepsin B.

In particularly preferred embodiments, the enzymatically cleavable linker according to the invention comprises a dipeptide selected from Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit, particularly wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptides and the amatoxin:

Phe-Ala-PAB Val-Ala-PAB

Phe-Cit-PAB Val-Cit-PAB

Accordingly, the conjugates of the invention as disclosed herein may comprise an enzymatically cleavable linker which comprises any one of the dipeptides-PAB moieties Phe- Lys-PAB, Val-LysPAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit-PAB, or Val-Cit-PAB as disclosed above. Peferably, the cleavable linker of the conjugates of the invention comprises the dipeptide-PAB moiety Val-Ala-PAB.

Val-Ala-PAB whereby the PAB moiety is linked to the amatoxin.

According to some embodiments, the linker of the invention as disclosed above comprises a thiol reactive group, selected from bromo acetamide, iodo acetamide, methylsulfonylbenzothiazole, 4,6-dichloro-l,3,5-triazin-2-ylamino group methyl-sulfonyl phenyltetrazole or methyl sulfonyl phenyl oxadi azole, pyridine-2 -thiol, 5- nitropyridine-2- thiol, methanethiosulfonate, or a maleimide.

According to a preferred embodiment the thiol reactive group is a maleimide (meleimidyl moiety).

According to a particularly preferred embodiment, the linker prior to the coupling to a target binding moiety of the invention comprises the structure:

According to a particularly preferred embodiment, the linker of the invention comprises the structure

wherein the maleimidyl moiety is covalently bound to a cysteine residue on the antibody according to the invention and the PAB moiety is bound to amanitin or an amatoxin.

According to preferred embodiments of the present invention, in said conjugate as described, said linker, if present, or said target binding moiety is connected to said amatoxin via (i) the g C-atom of amatoxin amino acid 1, or (ii) the d C-atom of amatoxin amino acid 3, or (iii) the 6’-C-atom of amatoxin amino acid 4.

According to preferred embodiments, the conjugate of the invention comprises a linker- amatoxin conjugate selected from

(VI)

According to a preferred embodiment of the present invention, said linker, if present, or said amatoxin is connected to said antibody via any of the natural Cys residues of said antibody, preferably via a disulfide linkage.

According to particularly preferred embodiments, the target-binding moiety of the invention is an antibody as disclosed herein, preferably a monoclonal antibody, which has been genetically engineered to comprise a heavy chain 118Cys, a heavy chain 239Cys, or heavy chain 265Cys according to the EU numbering system (Edelman et ah, Proc. Natl. Acad. Sci. EISA; 63 (l):78-85 (1969)), preferably a heavy chain 265Cys according to the EEi numbering system, and wherein said linker, if present, or said amatoxin is connected to said antibody via said heavy chain 118Cys, or said heavy chain 239Cys, respectively, preferably to heavy chain 265Cys residue.. Said cysteine-engineered antibodies may e.g. be obtained according to methods disclosed in Junutula et al. Nat Biotechnol. 2008 Aug;26(8):925-32, or e.g. as disclosed in WO2016/ 142049 Al the content of which is incorporated herein by reference. The use of said cysteine-engineered antibodies may be particularly advantageous to obtain conjugates according to the invention as disclosed herein which have a controlled drug-to- antibody ratio (DAR) of about 1 to about 4, depending of the number of cysteines introduced into the heavy chain, preferably the drug-to-antibody ratio is about 2 (DAR=2 (e.g. one linker-compound conjugate per heavy chain of the antibody) which e.g. can result in an improved therapeutic index of said conjugates compared to conjugates having a higher DAR, or e.g. of conjugate preparations which comprise a mixture of conjugate species having a DAR of about 1 to about 6, 8, or 10. The term “therapeutic index” as used herein is a quantitative measurement of the relative safety of a drug and may e.g. be defined as TI=TD50:ED50, whereby TD50 refers to the toxic dose of drug in 50% of subjects and ED50 refers to the efficacious dose in 50% of subjects. Accordingly, a higher therapeutic index is preferable to a lower one in that for example a subject in need thereof would have to take a higher dose of a drug, such as the the inventive conjugates to reach the toxic threshold than the dose taken to elicit the therapeutic effect.

According to one embodiment, the cysteine-engineered antibodies as disclosed above may additionally comprise the amino acid substitutions L234A, L235A (according to the EU numbering system) in its Fc region, as disclosed in WO 2020/086776 A1 the content of which is incorporated herein by reference. Cysteine-engineered antibodies of the invention as disclosed above comprising the mutation L234A, L235A in their Fc do not bind to FcgR, or Clq and corresponding antibodies harboring said mutations in their Fc region are devoid of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The use of corresponding antibdoies according to the invention which harbor the above mutations in their Fc region in addition to e.g. Cys265, may be advantageous in cases in which ADCC and CDC reduce the therapeutic index of the conjugates of the invention.

Furthermore, the conjugate according to the present invention can have a cytotoxic activity of an ICso better than 10x1 O^'9 M, 9x1 O^'9 M, 8x1 O^'9 M, 7x1 O^'9 M, 6x1 O^'9 M, 5x1 O^'9 M, 4x1 O^'9 M, 3x1 O^'9 M, 2x1 O^'9 M, preferably better than 10x1 O^'10 M, 9x1 O^'10 M, 8x1 O^'10 M, 7x1 O^'10 M, 6x1 O^'10 M, 5x1 O^'10 M, 4x1 O^'10 M, 3x1 O^'10 M, 2x1 O^'10 M, and more preferably better than lOxlO^'11 M, 9xl0 ^u M, dcΐq^'11 M, 7X10^'11 M, όcΐq^'11 M, 5X10^'11 M, 4X10^'11 M, 3X10^'11 M, 2xl0 ^u M, or lxlO^'11 M.

According to one embodiment, the present invention pertains to the use of a conjugate according to the invention as disclosed herein in the treatment of Coronavirus-associated diseases, or for inhibiting Coronavirus replication, wherein said conjugate comprises an antibody which is specific for the Coronavirus spike (S) protein and is selected from the group comprising bamlanivimab (LY-CoV555, LY-3819253), etesevimab (LY-C0VOI6), or casirivimab. Said antibodies may e.g. be engineered to comprise Fc mutations as disclosed above, e.g. Cys265, or Cys265 and L234A, L235A as disclosed above. According to one embodiment, the present invention pertains to the use of a conjugate according to the invention as disclosed herein in the treatment of Coronavirus-associated diseases, or for inhibiting Coronavirus replication, wherein said conjugate comprises an antibody which is specific for the ACE-2 protein and is selected from antibodies as disclosed in W002/098906. Said antibodies may e.g. be engineered to comprise Fc mutations as disclosed above, e.g. Cys265, or Cys265 and L234A, L235A as disclosed above.

According to one embodiment, the present invention pertains to the use of a conjugate according to the invention as disclosed herein in the treatment of Coronavirus-associated diseases, or for inhibiting Coronavirus replication, wherein said antibody, is specific for the TMPRRS2 protein and is selected from antibodies as disclosed in WO2019/147831, such as e.g. H1H7017N, H1H11729P. Said antibodies may e.g. be engineered to comprise Fc mutations as disclosed above, e.g. Cys265, or Cys265 and L234A, L235A as disclosed above.

According to a further aspect of the present invention, the present invention relates to a pharmaceutical formulation or composition comprising said amatoxin, or said conjugate, for use in the treatment of Coronavirus-associated diseases.

Said pharmaceutical formulation or composition may further comprise one or more excipients, preferably selected from the group consisiting of pharmaceutically acceptable buffers, surfactants, diluents, carriers, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and/or preservatives.

In aqueous form, said pharmaceutical formulation or composition may be ready for administration, while in lyophilised form said formulation can be transferred into liquid form prior to administration, e.g ., by addition of water for injection which may or may not comprise a preservative such as for example, but not limited to, benzyl alcohol, antioxidants like vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium, the amino acids cysteine and methionine, citric acid and sodium citrate, synthetic preservatives like the parabens methyl paraben and propyl paraben.

Said pharmaceutical formulation may further comprise one or more stabilizer, which may be, e.g ., an amino acid, a sugar polyol, a disaccharide and/or a polysaccharide. Said pharmaceutical formulation may further comprise one or more surfactant, one or more isotonizing agents, and/or one or more metal ion chelator, and/or one or more preservative.

The pharmaceutical formulation as described herein can be suitable for at least intravenous, intramuscular or subcutaneous administration, or inhalation. Alternatively, said conjugate according to the present invention may be provided in a depot formulation which allows the sustained release of the biologically active agent over a certain period of time.

In still another aspect of the present invention, a primary packaging, such as a prefilled syringe or pen, a vial, an infusion bag, or an inhaler or nebulizer, or a cartridge therefor, is provided, which comprises said formulation according to the previous aspect of the invention.

The prefilled syringe or pen may contain the formulation either in lyophilised form (which has then to be solubilised, e.g., with water for injection, prior to administration), or in aqueous form. Said syringe or pen is often a disposable article for single use only, and may have a volume between 0.1 and 20 ml. However, the syringe or pen may also be a multi-use or multi-dose syringe or pen.

Said vial may also contain the formulation in lyophilised form or in aqueous form and may serve as a single or multiple use device. As a multiple use device, said vial can have a bigger volume.

According to the present invention, said Coronavirus-associated diseases may be selected from the group consisting of Coronavirus-associated respiratory disease, gastrointestinal disease, hepatic disease, kidney failure, central nervous system disorder, MERS, SARS and COVID-19, in particular wherein the Coronavirus-associated disease is COVID-19.

According to the present invention, said Coronavirus can be any Coronavirus being a member of the Coronaviridae family. Preferably, said Coronavirus is a human Coronavirus. Further preferably, said Coronavirus is a human Coronavirus selected from the group consisting of the human Coronavirus types HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV- HKU1, MERS-CoV, SARS-CoV and SARS-CoV-2. Most preferably, said Coronavirus is SARS-CoV-2. According to one embodiment, the present invention pertains to a method of treating a patient in need thereof inflicted with a Coronavirus infection, wherein the method comprises administering a therapeutically effective amount of a pharmaceutical formulation as disclosed herein or administering a therapeutically effective amount of composition comprising an amatoxin, or a conjugate as disclosed herein to said patient. The term “therapeutically effective amount” refers to an amount which inhibits Coronavirus replication by at least 10%, 12.5%, 25%, 50%, 75%, 80%. The therapeutically effective amount required for inhibiting Coronavirus replication by at least 10%, 12.5%, 25%, 50%, 75%, 80% may vary depending on the age of the patient, body weight, disease severty, or viral load (viral titer) of said patient.

According to one embodiment, the method of treatment as disclosed above, may additionally comprise the administration of tocilizumab, sarilumab, silituximab in cases in which the patient inflicted with a Coronavirus infection is experiencing a cytokine storm to prevent or reduce the risk of e.g. renal failure, acute liver injury, cholestasis, or a stress-related or Takotsubo-like cardiomyopathy.

Sequences

Examples

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

Example 1: Inhibitory effect of Amanitin on viral replication of SARS-CoV-2

The inhibitory effect of amanitin on viral replication of SARS-CoV-2 was tested in VERO cell culture. For this assessment, 1.5xl0⁴ VERO cells (African green monkey kidney cell line; Ammerman et al. 2008) were plated in 100 mΐ DMEM in 96-well plates. After plating, the cells were infected with SARS-CoV-2 from a clinical isolate, and a-amanitin was added at various concentrations. The inhibitory effect of a-amanitin on SARS-CoV-2 replication was assessed by determining the virus load by PCR assay. Toxicity of the amanitin was assessed on non-infected cells by a commercial MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) cell viability assay.

Amanitin was shown to inhibit viral replication of SARS-CoV-2 on VERO cells with an IC50 of 6.2 mM (Fig. 3; circles, upper panel: SARS-CoV-2 inhibition [%]; circles, lower panel: SARS-CoV-2 copy number [copies/ml]; squares, toxicity [%]). A basic background toxicity of amanitin on said cells, as assessed by MTT viability assay on non-infected cells, was shown to increase from about 10 mM. However, surprisingly, even at amanitin concentrations of up to 100 mM, the cellular toxicity remained rather low. References:

Ammerman NC et al. (2008). Growth and maintenance of Vero cell lines. Curr. Protoc. Microbiol . (doi : 10.1002/9780471729259.mca04es 11 ).

Belouzard S et al. (2012). Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses Vol 4: 1011-1033.

Chen Y et al. (2020). Emerging Coronaviruses: genome structure, replication, and pathogenesis. J. Med. Virol. Vol 92: 418-423.

Chu et al. (2020) Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. Lancet Microbe. May;l(l):el4- e23

De Vlugt C et al. (2018). Insight into Influenza: A Virus Cap-Snatching. Viruses Vol 10: 641-647.

Dubowchik et al. (2002), Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem. 13: 855-69.

Edelman et al. (1969), The covalent structure of an entire gammaG immunoglobulin molecule Proc. Natl. Acad. Sci. USA; 63 (l):78-85

Fehr AR and Perlman S (2015). Coronaviruses: An Overview of Their Replication and Pathogenesis. Chapter 1. Coronaviruses 1282: 1-23 (doi: 10.1007/978-1-4939-2438-7-1).

Gralinski LE and Menachery VD (2020). Return of the Coronavirus: 2019-nCoV. Viruses Vol 12: 135 (doi:10.3390/vl2020135).

Li F (2016). Structure, function, and evolution of Coronavirus spike proteins. Annu. Rev. Virol. Vol 3: 237-61. Mahy BWJ et al. (1972). Inhibition of Influenza Virus replication by a-amanitin: mode of action. Proc. Nat. Acad. Sci. USA Vol. 69 (6): 1421-1424.

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Claims

What is claimed is:

1. Use of amatoxin for inhibition of a viral RNA-dependent RNA polymerase.

2. Use of amatoxin for inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication.

3. Use of amatoxin for inhibition of human Coronavirus replication.

4. Use of amatoxin in the treatment of Coronavirus-associated diseases, in particular for the treatment of human Coronavirus-associated diseases.

5. Amatoxin for use in the inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication.

6. Amatoxin for use in the treatment of Coronavirus-associated diseases, in particular for the treatment of human Coronavirus-associated diseases.

7. Use of a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for inhibition of RNA virus replication, in particular for inhibition of Coronavirus replication.

8. Use of a conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for the treatment of Coronavirus-associated diseases.

9. A conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for use in the inhibition of RNA virus replication, in particular for use in the inhibition of Coronavirus replication.

10. A conjugate comprising (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety with said at least one amatoxin, for use in the treatment of Coronavirus-associated diseases.

11. Use of a conjugate according to any one of claims 7 or 8, or the conjugate for use according to any one of claims 9 or 10, wherein said target binding moiety is selected from the group consisting of

(i) an antibody, preferably a monoclonal antibody,

(ii) an antigen-binding fragment thereof, preferably a variable domain (Fv), a Fab fragment or an F(ab)2 fragment,

(iii) an antigen-binding derivative thereof, preferably a single-chain Fv (scFv), and

(iv) an antibody-like protein.

12. Use of a conjugate according to claim 11, or the conjugate for use according to claim 11, wherein said antibody, or antigen-binding fragment thereof or antigen-binding derivative thereof, is a murine, a chimeric, a humanized or a human antibody, or antigen-binding fragment or antigen-binding derivative thereof, respectively.

13. Use of a conjugate according to any one of claims 11 or 12, or the conjugate for use according to any one of claims 11 or 12, wherein said antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof, respectively, is specific for the Coronavirus spike (S) protein, the SI subunit or S2 subunit of the spike (S) protein, angiotensin-converting enzyme 2 (ACE2), or the TMPRRS2 protein.

14. A pharmaceutical formulation comprising the amatoxin according to any one of claims 5 or 6, or , or the conjugate according to any one of claims 9 - 13 , for use in the treatment of Coronavirus-associated diseases, in particular for use in the treatment of human Coronavirus-associated diseases.

15. Use of an amatoxin according to claim 4, or use of a conjugate according to claim 10, or amatoxin for use according to claim 6, or conjugate for use according to any one of claims 10 - 13, or pharmaceutical formulation according to claim 14, wherein the Coronavirus-associated disease is selected from the group consisting of Coronavirus- associated respiratory disease, gastrointestinal disease, hepatic disease, kidney failure, central nervous system disorder, MERS, SARS and COVID-19, in particular wherein the Coronavirus-associated disease is COVID-19.

16. Use of an amatoxin according to any one of claims 1 to 4, or use of a conjugate according to any one of claims 7 or 8, or amatoxin for use according to claim 5, or claim 6, or conjugate for use according to any one of claims 9 - 10, or pharmaceutical formulation according to claim 14, wherein the Coronavirus is a human Coronavirus, preferably selected from the group consisting of the human Coronavirus types HCoV- 229E, HCoV-NL63, HCoV-OC43, HCoV-HKUl, MERS-CoV, SARS-CoV and SARS-CoV-2.

17. The pharmaceutical formulation for use according to claim 14, wherein said formulation further comprises one or more excipients, preferably selected from the group consisiting of pharmaceutically acceptable buffers, surfactants, diluents, carriers, fillers, binders, lubricants, glidants, disintegrants, adsorbents, and/or preservatives.

18. The conjugate for use according to any one of claims 10 - 13, wherein said linker is a stable or a cleavable linker, particularly wherein said cleavable linker is selected from the group consisting of an enzymatically cleavable linker, preferably a protease- cleavable linker, and a chemically cleavable linker, preferably a linker comprising a disulfide bridge.

19. The amatoxin for use according to claim 5 or claim 6, or the conjugate for use according to any one of claims 10 - 16 or 18, wherein said amatoxin can be selected from the group consisting of a-amanitin, b-amanitin, amanin, amaninamide and analogues, derivatives and salts thereof.