Simmons Ref.: P73697PC

HYBRID SOLUBLE sgp130-SPIKE-NANOBODY FUSION PROTEINS

BACKGROUND OF THE INVENTON

The present invention is related to the field of biomedical science and provides polypeptides comprising cytokine binding domains and at least one single domain nanobody, preferably at least one single domain antibody, capable of binding interleukin ( I L)-6: soluble interleukin (IL)-6 receptor (slL-6R) complexes. Moreover, provided herein are polypeptides that are further capable of binding to any additional target site of interest, preferably to a receptor binding domain of interest, more preferably to the receptor binding domain (RBD) of the SARS-CoV2 spike protein. The invention further relates to the use of said polypeptides for use in medical applications, preferably for preventing and/or treating inflammatory processes or viral infections.

Introduction

IL-6-Trans-Signalling and inflammatory processes

IL-6 is a cytokine involved in a plethora of processes including inflammation, acute phase response, infection, neurodegenerative processes, autoimmunity and trauma. The cytokine exerts its effects by different mechanisms depending on the target cells. IL-6 classic signaling, which mainly controls acute inflammatory responses, such as the acute phase reaction, is induced by binding of IL-6 to membrane-bound IL-6R on target cells. This event is followed by recruitment of the ubiquitously expressed signal-transducing co-receptor gp130 and initiation of downstream signaling cascades leading to activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), protein kinase B (PKB or AKT) and mitogen-activated protein kinase (MAPK) pathways. Increased or dysregulated IL-6 trans-signaling is observed in many chronic inflammatory diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and cancer. IL-6 trans- signaling occurs via complex formation of IL-6 with soluble forms of the IL-6R (slL-6R). These complexes can activate downstream signaling cascades in all cells expressing gp130. It was shown that some dendritic cells do not express gp130 but IL-6R. These cells are able to induce a third mechanism of IL-6 signal transduction, namely IL-6 transpresentation. For IL-6 trans-presentation, IL-6 binds to membrane-bound IL-6 receptor on a transmitter cell. The resulting complex engages gp130 on a different, so-called receiver cell and activates signal transduction through gp130 dimerization. Targeting IL-6 signaling is of interest for many diseases. However, global IL-6 blockade, e.g., via IL-6 antibodies, is associated with many side effects (including an increased risk of bacterial and viral infections) due to the multitude of functions of this cytokine. Hence, in order to efficiently target distinct IL-6 functions specific for a disease, it is of utmost importance to understand the mode of IL-6 signaling in the disease process and the ability to target only this specific signaling mode. IL-6 has three binding sites for its receptors. Site I recognizes IL-6R, while sites II and III establish the contact to gp130. IL-6 or IL-6R antibodies usually target the site I interface formed between IL-6 and the IL-6R and affect classic and trans-signaling. The fusion protein sgp130Fc targets sites II and III and does in principle not affect IL-6 classicsignaling, unless extremely high concentrations are used that allow quantitative complexing of IL-6 with slL-6R and, thus, can reduce free IL-6 levels.

Therefore, in contrast to neutralizing antibodies, the control of bacterial and viral infections is not expected to be affected by sgp130Fc, which is supported by mouse experiments. A recent phase II study with soluble gp130 (Olamkicept) (EudraCT, Number 2016-000205- 36) demonstrated the safety and efficacy of sgp130 in the treatment of Crohn’s disease and ulcerative colitis. However, sgp130Fc inhibits IL-11 trans-signaling with similar potency as IL-6 trans-signaling. The physiological role of IL-11 trans-signaling is currently not understood. There is clear evidence for the presence of IL-1 1/slL-1 1 R complexes in human serum and that these complexes are able to activate cells via IL-1 1 trans-signaling. In addition, leukemia inhibitory factor (LIF) and oncostatin M (OSM) signaling are also affected by high concentrations of sgp130Fc. Moreover, sgp130Fc is a very large molecule. It is a protein dimer of approximately 186 kDa plus 20 glycosylation chains, leading to an apparent molecular weight of about 240 kDa. Efforts have been made to reduce the size of the molecule using also naturally occuring sgp130 monomers (120 kDa) or the short sgp130- RAPS variant consisting of only the ligand-binding domains D1 -D3 of gp130 (50 kDa), but the inhibitory capacities and binding affinities of these proteins were 10-1 ,000 fold lower than those of sgp130Fc dimers.

It was further reported in the art that the single domain antibody VHH6 stabilizes IL-6:slL- 6R complexes, leading to enhanced IL-6 trans-signaling (Baran et. al, 2018). In contrast, in the context of the present invention, it has surprisingly be found that the fusion of said single domain nanobody (VHH6) to cytokine binding domains, in particular the fusion of VHH6 to cytokine binding domains of a gp130 protein, reveals a novel class of molecules suitable for inhibiting IL-6 trans-signalling and, thereby, suitable as novel agents for medicinal applications. One object of the present invention is, therefore, to provide novel polypeptides which are capable of selectively inhibiting IL-6-trans-signalling, with reduced effects on other IL-6 family cytokines including IL-1 1 , LIF and OSM and which are, thereby, useful and applicable in medical applications.

These objects have been solved by the presently claimed subject-matter. In particular, the present invention provides a novel class of polypeptides, comprising at least three cytokine binding domains and at least one single domain nanobody, preferably at least one single domain antibody. In particular, the present invention provides chimeric soluble gp130-based IL-6 trans-signaling inhibitors (cs-130Fc). These cs-130Fc variants can comprise or be composed of cytokine binding domains of sgp130 fused to at least one single domain nanobody, preferably to at least one single domain antibody, specifically binding to I L-6 :sl L- 6R complexes (VHH6).

IL-6-Trans-Signalling and COVID-19 related CRS

Since its emergence in 2019, SARS-CoV-2 spread globally and as of 5th of May 2021 a total 155 million infections were recorded (https://coronavirus.jhu.edu/), threatening to overwhelm healthcare systems in many countries. The COVID19 disease resulting from SARS-CoV-2 infection leads to a broad variety of outcomes ranging from very mild cases to life threatening respiratory failure, shock or multi-organ failure. Even though SARS-CoV- 2 infection in many cases leads to no or only mild symptoms, millions of hospitalizations and mortalities are associated with COVID19 worldwide https://coronavirus.jhu.edu/). Mortality rates vary considerably between studies, however, a clear age dependency was observed with regards to the development of severe COVID19. Severe COVID19 causes a hyper-inflammatory syndrome culminating in respiratory dysfunction and multi-organ damage. The SARS-CoV2 induced hyper-inflammatory syndrome is often compared to cytokine-induced conditions known from other diseases including sepsis, acute respiratory distress syndrome and chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Interleukin (IL)-6 and the soluble IL-6 receptor (slL-6R) were identified among the key players in the COVID-19 induced cytokine release syndrome. In 2017, the IL-6R antibody tocilizumab was approved for the treatment of CAR T-cell-induced cytokine storm. Tocilizumab and Sarilumab bind to the soluble and membrane-bound IL-6R (Nishimoto et aL, 2008, Boyce, et al. 2018), whereas Siltuximab binds to IL-6 (Rossi et al. 2010). However, all antibodies prevent binding of IL-6 to IL-6R and equally well inhibit classic and trans-signaling (Kang, Tanaka et al. 2019). In classic signaling, IL-6 initially binds to the membrane-bound IL-6R followed by full-receptor complex formation with the signaltransducing receptor chain gp130. In trans-signaling, complexes of IL-6 and slL-6R bind to membrane gp130 (Scheller, Garbers et al. 2014). In inflammation, membrane-bound IL-6R is proteolytically cleaved into the slL-6R by a disintegrin and metalloproteases proteases (ADAM), mainly ADAM10 and 17 (Mullberg et al. 1993, Matthews et al. 2003). Consequently, serum levels of IL-6 and slL-6R concomitantly rise during inflammatory conditions (Akira, Taga et al. 1993, Mitsuyama, Toyonaga et al. 1995, Montero-Julian 2001 , Jones 2005, Baran et aL, 2018). Of note, whereas IL-6 classic signaling is considered as beneficial, IL-6 trans-signaling has been shown to be the mostly detrimental driving force of ongoing inflammatory reactions (Scheller et al. 2014, Schumacher 2019) including autoimmune disease (reviewed in Hirano et al. 2010), sepsis (Calandra et al. 1991 , Hack et al. 1989), cytokine release syndrome (Tanaka, et al. 2016) and COVID19, (Chen et aL, 2021 , Patra et al 2020). Hence, antibody I L-6R-in hibitors are of great interest for the treatment of the COVID19 induced hyperinflammatory syndrome (Patra 2020, Chen, Biggs et al. 2021 ). In fact, the IL-6R antibodies tocilizumab and sarilumab demonstrated beneficial effects on the survival rates in severe COVID-19 cases in preclinical and clinical studies (Guaraldi, Meschiari et al. 2020, Jordan, Zakowski et al. 2020, Meleveedu, Miskovsky et al. 2020, Investigators, Gordon et al. 2021 ). However, IL-6 is required to control viral infection, hence global blockade of IL-6 signaling e.g. through tocilizumab is associated with an increased risk of especially airway infections. This might be detrimental for the treatment of COVID- 19 with IL-6 inhibitors due to a potential increase in viral replication due to IL-6 blockade. Whereas antibodies did not differentiate between classic and trans-signaling, soluble forms of gp130 are selective binders of IL-6: slL-6R complexes, thereby only interfering with IL-6 trans-signaling. Therefore, inhibition of IL-6 trans-signaling by sgp130 molecules might offer an attractive alternative inhibitory pathway for cytokine release syndrome after CAR T cell therapy and severe cases of SARS-CoV2 infections. In mice, sgp130 prevents death caused by cecal ligation puncture-induced septic shock syndrome and at least bacterial infections are better controllable after selective inhibition of IL-6 trans-signaling compared to inhibition of both IL-6 classic and trans-signaling by monoclonal IL-6 antibodies. However, the combination of IL-6 blockade with agents reducing uncontrolled viral infection may prove beneficial in the treatment of severe COVID-19 cases. The spike protein of SARS-Cov2 binds to human ACE2 on the cell surface to facilitate viral cell entry. Hence, preventive vaccination strategies as well as most efforts on the development of therapeutic antibodies focuses on the inhibition of the interaction of the receptor binding domain of the spike protein (S-RBD) with ACE2. There are more than 50 monoclonal antibodies against SARS-CoV-2 in various developmental stages many of those directed against the spike protein. Among them, the dimeric single domain antibody VHH72 was shown to efficiently block viral cell entry, whereas monomeric VHH72 was less effective. It is therefore another object of the present invention to provide a polypeptides, further comprising at least one protein domain which is capable of specifically binding to a target site of interest, such as, for example a (receptor) binding domain (RBD). This at least one protein domain may, for example, be the nanobody VHH72, capable of binding the receptor binding domain (RBD) of the SARS-CoV2 spike protein.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the present invention is described in detail. The features of the present invention are described in individual paragraphs. This, however, does not mean that a feature described in a paragraph stands isolated from a feature or features described in other paragraphs. Rather, a feature described in a paragraph can be combined with a feature or features described in other paragraphs.

In a first aspect, the present invention relates to a polypeptide, comprising at least three cytokine-binding domains and at least one single domain nanobody, wherein the at least one single domain nanobody specifically binds to an interleukin (IL)-6: soluble interleukin ( I L)-6 receptor (si L-6R) complex (VHH6). In the context of the present invention, the at least one single domain nanobody is preferably a single domain antibody.

The term “comprise/s/ing”, as used herein, is meant to include or encompass the disclosed features and further features which are not specifically mentioned. The term “comprise/es/ing” is also meant in the sense of “consist/s/ing of” the indicated features, thus not including further features except the indicated features. Thus, the subject-matter of the present invention may be characterized by additional features in addition to the features as indicated.

The term “nanobody” as it is used herein is interchangeable as a "single domain antibody", and defines a recombinant, antigen-specific antibody consisting of only one single monomeric variable antibody domain (normally, these correspond to the variable region (VHH) of a heavy-chain antibody). Nanobodies can be derived from naturally occurring heavy chain antibodies. Due to their small size they offer several advantages over conventional antibodies. The nanobody VHH6, to which reference is made herein, inter alia is known to the skilled person in art. The single domain antibody VHH6 is thereby recognizing the interface formed by IL-6 in complex with the IL-6R. In one embodiment, the nanobody VHH6 comprises the sequence of SEQ ID NO.: 3 (PMID: 28134246):

DVQFVESGGGSVHAGGSLRLNCATSGYIYSTYCMGWFRQAPGKEREGVAHIYTNSGR T YYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAARPSIRCASFSATEYKDWGQ GTQVTVSS (SEQ ID NO.: 3).

In another embodiment, the nanobody VHH6 is composed of SEQ ID NO: 3.

“Cytokine-binding domains” as they are described herein are protein domains, which are capable to bind specific cytokines, such as IL-6. Related thereto, in one embodiment, the cytokine-binding domains are derived from the signal-transducing transmembrane coreceptor gp130 protein. As stated above, gp130 is a ubiquitously expressed signaltransducing co-receptor and is involved in downstream signaling cascades leading to activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), protein kinase B (PKB or AKT) and mitogen-activated protein kinase (MAPK) pathways. sgp130 naturally comprises 6 (D1 -D6) extracellular domains followed by a transmembrane and an intracellular domain in its native (wild type) form (see also Fig 13A). Related thereto, in one embodiment, the cytokine-binding domains of the polypeptide of the present invention (D1 -D3) comprises a truncated version of the natural occurring gp130 protein. In one preferred embodiment, the cytokine-binding domains of the polypeptide of the present invention comprise protein domains/units, of SEQ ID NO: 1.

Accordingly, the polypeptide of the present invention may comprise at least three cytokinebinding domains from the signal-transducing transmembrane co-receptor gp130 protein. In a preferred embodiment, the cytokine-binding domains of the polypeptide of the present invention comprises or are composed of the cytokine-binding domains D1 -D3 according to SEQ ID NO: 1 . In another embodiment, the polypeptide of the invention may comprise at least four, at least five, at least six or more cytokine binding domains from the signaltransducing transmembrane co-receptor gp130.

In one embodiment, the cytokine-binding domain(s) comprise(s) the following sequence (SEQ ID NO: 1 ):

MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDY FHV

NANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYG ITIISGL PPEKPKNLSCIVNEGKKMRCEWDRGRETHLETNFTLKSEWATHKFADCKAKRDTPTSC TVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKL T WTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMK E DGKGYWSDWSEEASGITYEDRPSK (SEQ ID NO: 1 ).

In another embodiment, the cytokine-binding domains D1 -D3 comprise or are composed of an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 1 . In a preferred embodiment, the at least three binding domains comprise or are composed of the amino acid sequences of SEQ ID NO: 1 .

In other preferred embodiments, the cytokine-binding domains D1 -D3 comprise or are composed of any of the following amino acid sequences SEQ ID NO: 26 or SEQ ID NO. 27. gp130 ^{T102Y/Q113F/N114L} D1 -D3 (cytokine binding domains)

MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDY FHV NANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLYCNILTFGQLEFLVYGITI ISGLP PEKPKNLSCIVNEGKKMRCEWDRGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCT VDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLT W TNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKE DG KGYWSDWSEEASGITYEDRPSK (SEQ ID NO: 26). gp130 ^{T102Y/Q113F/N114L7R281 Q} D1 -D3 (cytokine binding domains)

MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDY FHV NANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLYCNILTFGQLEFLVYGITI ISGLP PEKPKNLSCIVNEGKKMRCEWDRGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCT VDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLT W TNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTQSSFTVQDLKPFTEYVFRIRCMKE DG KGYWSDWSEEASGITYEDRPSK (SEQ ID NO: 27).

In yet another preferred embodiment, the polypeptide of the present invention is characterized by its binding affinity to its binding site. The measurement of binding affinities in the context of polypeptide - binding target sites, for example, by surface plasmon resonance biosensors or other high-throughput affinity-based technologies, is routine practice in the art and well known to the skilled person (Myszka and Rich, 2000; Zhu and Cuozzo, 2009). Accordingly, in a preferred embodiment, the polypeptide of the invention has a binding affinity to its binding site that is characterized by a dissociation rate constant (k _O ff) in the range of below 10 ^-5 1/s, preferably in the range of between 1 x 10 ^-6 to 1 x 10 ^-8 1/s. In yet another preferred embodiment, the dissociation rate constant (k _O ff) may be in the range of 3.0 to 4.0 x 10 ⁸ 1 /s.

The k _O ff value can be determined by routine assays well known in the art or described herein, e.g., stopped flow, biolayer interference or surface plasmon resonance. The K _O ft values displayed by the polypeptides of the present invention are demonstrated and described in detail in the examples provided herein.

In one embodiment, the polypeptide of the present invention comprises the amino acid sequence according to SEQ ID NO: 7 (cs-130Fc). In a preferred embodiment, the polypeptide of the present invention is composed of the amino acid sequence of SEQ ID NO: 7 (cs-130Fc). In another preferred embodiment, the polypeptide of the present invention comprises or is composed of an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 7 (cs-130Fc).

In a further embodiment, the polypeptide of the present invention optionally further comprises at least one protein domain of interest capable of specifically binding to a target site of interest. Preferably, this target site of interest may be a (receptor) binding domain (RBD) of interest. More preferably the at least one protein domain of interest is a single domain nanobody, preferably a single domain antibody, directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein (VHH72). The single domain antibody VHH72 was also previously described in the art and shown to efficiently block viral cell entry during infection with SARS-CoV-2 (Wrapp et al., 2020) by interacting with the viral spike protein (S protein).

In one preferred embodiment, the at least one single domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein (VHH72) is defined by SEQ ID NO: 2.

QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYW GQGTQVTVSS (SEQ ID NO: 2)

In another embodiment, the nanobody VHH72 is composed of SEQ ID NO: 2. In another preferred embodiment, the at least one single domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein (VHH72) comprises or is composed of an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 2, preferably wherein the single domain nanobody comprises or is composed of the amino acid sequence of SEQ ID NO: 2.

In another embodiment, at least one single domain nanobody directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein (VHH72) comprises the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the polypeptide of the present invention comprises the amino acid sequence according to SEQ ID NO: 8 (c19-s130Fc). In a preferred embodiment, the polypeptide of the present invention is composed of the amino acid sequence of SEQ ID NO: 8 (c19-s130Fc). In another preferred embodiment, the polypeptide of the present invention comprises or is composed of an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 8 (c19-s130Fc).

Moreover, the polypeptide of the present invention can further comprise additional structures. Related thereto, the polypeptide of the present invention optionally further comprises an Fc constant region of an IgG antibody. In another embodiment, the polypeptide of the present invention optionally further comprises an Fc constant region of any other immunoglobulin classes or sub-classes. In one specific embodiment, the Fc constant region of the polypeptide of the invention comprises an amino acid sequence of SEQ ID NO: 5. In another preferred embodiment, the Fc constant region of the polypeptide of the invention is composed of an amino acid sequence of SEQ ID NO: 5. In other preferred embodiments, the Fc constant region of the polypeptide of the invention comprises or is composed of an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 5.

These additional moieties, such as e.g. Fc constant regions, can be utilized for example for purification procedures of the polypeptide of the present invention. The term "purification", in particular purification by means of protein tags, is clearly and unambiguously known to the skilled person. Methods include all possible techniques that are known to the skilled person in art, such as methods relying on characteristics as solubility, size, charge, and specific binding affinity. Non-limiting examples are for example salting out, ammonium sulfate or ethanol precipitation, dialysis, chromatography (such as protein A purification, gel-filtration chromatography, ion-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, lectin chromatography high-pressure, liquid chromatography (HPLC), electrophoresis, and/or centrifugation.

In one preferred embodiment, the polypeptide of the present invention comprises a TEV protease site for cleavage. The TEV site is a specific sequence that can be recognized and cleaved by so-called TEV proteases. This method is well known to the expert in the field. For example, the TEV site can be used to cleave the Fc part of the polypeptide of the present invention. In one embodiment, the polypeptide of the present invention comprises a TEV protease cleavage site comprising an amino acid sequence according to SEQ ID NO: 4. In one specific embodiment, the TEV protease cleavage site is composed of an amino acid sequence according to SEQ ID NO: 4, preferably comprising the amino acid sequence ENLYFQS. Equally preferred is that cleavage site comprises or is composed of functionally equivalent and/or structurally similar variants of SEQ ID NO: 4.

Moreover, the polypeptide of the present invention can additionally comprise any other moieties, for example protein tags, which are known the skilled person in art. The term “tag” as it is used herein means any naturally occurring or artificial polypeptide or another molecule structure, which allows purification and/or detection of any polynucleotide of the present invention and/or further improves the pharmacodynamics and/or pharmacokinetic properties of any polypeptide of the invention.

These protein tags can for example be used to facilitate purification of the polypeptide. Nonlimiting examples related thereto are for example GFP-tags or derivatives thereof, (poly)HIS-tags, Myc-Tags, Strep-Tags, polyarginine-Tags, Flag-Tags, TAP-Tags, glutathione S-transferase (GST)-Tags, HA-Tags, calmodulin-binding peptide (CBP)-Tags, maltose-binding protein (MBP)-Tags, V5-Tags, HSV-Tags, Protein C-Tags, Luciferase- Tags, or any other common polypeptide tags. Such tags can be removed prior to final preparation of a polypeptide.

Additionally, the polypeptide of the present invention can also comprise one or more linker(s). The term “linker” as it is used herein, is to be understood as any natural occurring and/or artificial peptide sequence, including, but not limited to for example amino acid residues from about 2 - 50 amino acid residues. Therefore, in one embodiment the at least one single domain nanobody is fused via a linker region to the cytokine binding domains, preferably via a flexible linker sequence, more preferably by linker containing or composed of the sequence (GGGGGS)2GGGGTG). In another embodiment, the polypeptide of the present invention optionally further comprises at least one protein domain of interest capable of specifically binding to a target site of interest, preferably wherein the at least one protein domain is capable of specifically binding to a (receptor) binding domain (RBD) of interest, more preferably wherein the at least one protein domain of interest is a single domain nanobody, preferably a single domain antibody, directed against the receptor binding domain (RBD) of the SARS-CoV2 spike protein (VHH72).

Accordingly, the present invention thereby provides a further novel class of polypeptides with dual or even multiple target binding sites and, as a consequence, with similar or different binding affinities, dependent on the individual binding target sites. That is, in a preferred embodiment, the invention provides a polypeptide which has the capacity of specifically binding, via one binding site, to an interleukin (IL)-6: soluble interleukin (IL)-6 receptor (slL-6R) complex, as well specifically binding to another target site of interest, via another binding site. In a preferred embodiment, the polypeptide specifically binds, via its second binding site, to a particular receptor binding domain (RBD), preferably to the receptor binding domain (RBD) of the SARS-CoV2 spike protein.

Hence, in a further preferred embodiment, the polypeptide of the invention has a binding affinity to its target binding sites that is characterized by an equilibrium dissociation constant (K _D ) in the range of 50-100 pM for complexes of IL-6 and the slL-6R and a K _D of < than 0.8 to 1 pM for the receptor binding domain (RBD) of the SARS-CoV2 spike protein.

In the context of the present invention, binding to IL-6 also includes binding to an interleukin (IL)-6: soluble interleukin (IL)-6 receptor (slL-6R) complex and vice versa.

In a second aspect, the present invention relates to an expression vector comprising one or more a nucleic acid sequence encoding for any of the polypeptides of the present invention provided herein. An “expression vector” or an “expression construct” is usually a plasmid or virus designed for gene expression in any possible cell. The expression vector is usually used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Related to the present invention, any common expression vector, which is known to the skilled person in the art can be used for this purpose. In a third aspect of the present invention, a host cell or a cell-free expression system comprising the expression vector is provided herein. “Expression systems” are used for the production of proteins and include e.g. usage of eukaryotic cells. Accordingly, the expression system for the production of any of the polypeptides of the invention can for example use eukaryotic cells, which include, but are not limited to those e.g. hamster cell lines (CHO and their derivatives), mouse cell lines (such as C127, NS0, SP2/0, YB2/0, XB2/09 and derivatives of all of them), or human cell lines (such as HEK and their derivatives, e.g. HT-1080, PER. C6, or Huh-7). Also included are cell lines from monkeys, such as e.g. Vero cells and their derivatives and insect cells, such as SF-9 cells and their derivatives. As it is used herein “derivative” and “derivatives” is to be understood as all descendant cell lines that have been derived from them or have emerged from them with modification or further development. Polypeptide expression using cellular systems can be performed by using diverse transfection systems. Non-limiting examples are for example lipid-based transfection or viral transduction techniques, which are very well known to a skilled person in the art.

“Cell-free expression” or “In-vitro translation” are characterized by in vitro protein expression, including the production of recombinant polypeptides in solution, using biomolecular translation machinery extracted from cells.

Furthermore, a fourth aspect of the present invention relates to the use of any of the polypeptides of the present invention described herein for inhibiting mammalian cell proliferation. As surprisingly found in the context of the present invention, the polypeptides described herein are capable of inhibiting mammalian cell proliferation (see e.g. Fig. 6). In one preferred embodiment, the mammalian cell is a T cell. In a more preferred embodiment, the mammalian cell is a Th17 cell. Nevertheless, it is highlighted that inhibiting of mammalian cell proliferation is not restricted to those cells. Any other cells, which is relying on IL-6 as cytokine in view of cellular proliferation, can be targeted by the polypeptide of the invention, in particular by using the polypeptides of the present invention for inhibiting cellular proliferation of mammalian cells.

In the context of the present invention, it has been found that the functional capacities of the polypeptides are particularly favorable if the polypeptide has the form of a dimer.

Accordingly, in one preferred embodiment, the polypeptide of the present invention is in the form of a dimer. In one embodiment, the polypeptide of the present invention is in the form of a monomer. In order to form dimers, the two polypeptides of the present invention are linked to each other through a simple covalent bond, a flexible peptide linker or via one or more disulfide bridges.

In one embodiment, the inhibiting of the proliferation comprises interference with interleukin ( I L)-6 trans-signaling, preferably wherein the polypeptide acts as an inhibitor on IL-6 transsignaling induced STAT3 and/or ERK phosphorylation. As it is stated above, any other cell, which is relying on IL-6 trans-signaling for cellular proliferation can be targeted by the polypeptides of the present invention. STAT3 is a transcription factor which can be phosphorylated by cytokine stimulation thereby driving the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. ERK stands for "extracellular-signal regulated kinases". These are serine/threonine kinases that belong to the group of mitogen-activated protein kinases (MAP kinases). ERK can be activated by various extracellular signals (such as cytokines) or pathogens and controls important cell functions such as proliferation and differentiation.

As suggested, targeting IL-6 trans-signalling plays an important and necessary role in the treatment of several diseases and disorders. Related thereto, any polypeptide of the present invention can be used as a medicine. “A medicine” as it is used herein can be understood as any substance and/or drug, curing or improving human’s or animal’s health condition. Accordingly, in another embodiment of the present invention, any polypeptide of the present invention can be used in a method of preventing and/or treating an inflammatory process, comprising administering said polypeptide in a therapeutically effective amount to a subject in need thereof.

A “therapeutically effective amount” means an amount that is effective in prevention and/or therapy, or an amount sufficient to provide a preventive and/or therapeutic effect. An amount that is effective in therapy is an amount which produces a biological activity and will depend, among other things, on the individual.

Furthermore, in another embodiment, the inflammatory process is an autoimmune disease, preferably wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease and T 1 D, more preferably wherein the inflammatory disease is inflammatory bowel disease.

Non-limiting examples of inflammatory processes are for example autoimmune diseases, such as chronic inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), type 1 diabetes, chronic inflammation of the thyroid gland (Hashimoto's thyroiditis, circular hair loss (alopecia areata), multiple sclerosis, rheumatoid arthritis, psoriasis, vitiligo, gluten intolerance (coeliac disease), ankylosing spondylitis (Bekhterev's disease), juvenile rheumatoid arthritis, polyneuropathy, rheumatic fever, myasthenia gravis, autoimmune hepatitis, lupus erythematosus, Sjogren's syndrome, Guillain-Barre syndrome. Autoimmune-related chronic inflammation can also occur in diseases of the heart, respiratory tract or kidney, as well as in cancer.

In the context of the present invention, it has surprisingly been found that the novel class of the polypeptides described herein are capable of inhibiting viral entry into mammalian cells, as shown in detail by the examples of the present application (see, for example, Figure 15, Figure 16, Figure 17 and Figure 18).

In another embodiment of the present invention the polypeptide of the invention can be used for inhibiting viral entry into mammalian cells, preferably for inhibiting viral entry of SARS-CoV-2. In another embodiment, the polypeptide(s) of the invention can be used for inhibiting viral entry into mammalian cells, wherein viral entry of any virus using S-protein- related cell entry is inhibited. In one embodiment, the viral entry of SARS-CoV virus is inhibited.

Related thereto, the polypeptide of the invention can be used in a method for preventing and/or treating viral infection, preferably SARS-CoV-2 viral infection and/or SARS-CoV-2 viral infection mediated diseases, comprising administering said polypeptide in a therapeutically effective amount to a subject in need thereof. In one specific embodiment, the polypeptide of the invention for use in a method for preventing and/or treating viral infection comprises the amino acid sequence of SEQ ID NO: 8. In another preferred embodiment, the polypeptide of the invention for use in a method for preventing and/or treating viral infection is defined by SEQ ID NO: 8. In another preferred embodiment, the polypeptide of the present invention comprises an amino acid sequence with at least 90 %, 95 %, 98 % or 99 % sequence identity to SEQ ID NO: 8.

In one preferred embodiment, SARS-CoV-2 viral infection mediated disease is COVID-19. Non-limiting examples of other viral infection mediated diseases are for example “Long Covid Syndrome”, or cytokine release storm (CRS) disease caused by viral infection. As indicated above, infections with SARS-CoV-2 accompanies an unprecedented spike in cytokines levels termed cytokines release syndrome (CRS) in critically ill patients, which can have dramatic consequences for the patient. Administration of the compositions may be effected by different ways of administration. Nonlimiting examples include, but are not limited to, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, pulmonal, inhalative administration. The dosage regimen will be determined by the attending physician and other clinical factors. As well known the skilled person in art, the dosages for any one patient can vary and depend on many factors, including for example size, age, sex, time and router of administration and stage of the disease.

The invention is further explained by the attached figures and examples, which are intended to illustrate, but not to limit the present invention.

FIGURES

Figure 1. Schematic illustration of cs-130Fc variant design. Protein domains are depicted as (not size-proportional) boxes (A) Domains D1 -D3 (yellow) of sgp130Fc, incorporating sites II and III required for cytokine binding, were fused via a long flexible linker to a single domain antibody (VHH6) recognizing the complex of IL-6 and slL-6R. A TEV protease recognition sequence connects VHH6 to the Fc fragment of a human IgG antibody. Due to the presence of the Fc fragment, all depicted proteins are disulfide linked dimers. Disulfide bonds connecting the Fc fragments are depicted as yellow lines. (B) Overview of different cs-130Fc variants. TEV protease recognition sequences ( | ) and positions of amino acid substitutions are indicated.

Figure 2. cs-130Fc variants are readily expressed and can be purified from CHO-K1 cells. (A) CHO-K1 cells were transfected with expression plasmids encoding the displayed proteins. Cells were harvested and lysed 48 h post transfection. Supernatants and lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an antibody against human IgG-Fc. Western blots shown are representative of three different experiments with similar outcomes. (B) SDS-PAGE of purified sgp130Fc and cs-130Fc variants (10 pig of total protein) stained with Coomassie brilliant blue. (C) Following Fc affinity purification, cs-130Fc was processed with TEV protease, and the resulting protein fragments were separated via size exclusion chromatography (SEC). Three molecular species corresponding to different peaks observed (labeled 1 -3) were isolated and analyzed by SDS-PAGE and Coomassie staining. BT, before TEV; PT, post TEV cleavage. (D) Coomassie staining of SDS-PAGE analysis of purified sgp130Fc and cs-130Fc variants. 5 pg total protein, non-reduced or reduced (Red. +), were loaded. (E) SEC-MALS analysis of affinity purified sgp130Fc and cs-130Fc variants. Between 10 and 50 pg were injected per sample on a Superpose 6 Increase column. Solid lines under the peaks correspond to molecular weight. (F) Conjugate analysis of cs-130Fc data from (E). Solid lines under the peak correspond to total mass (red), protein mass (black) and sugar mass (blue).

Figure 3. cs-130Fc variants are biologically active. (A) Surface plasmon resonance analysis of hyper-IL-6-Fc binding to sgp130Fc and cs-130Fc variants. Hyper-IL-6-Fc was immobilized on a CM-5 chip and increasing concentrations of inhibitors were injected. Sensorgrams in response units (RU) over time are depicted as blue lines, global fit data are displayed as black lines. Residuals are shown below the sensorgrams. (B) A normalized overlay of the dissociation phases of sgp130Fc and cs-130Fc for their interaction with hyper-IL-6 for the highest concentration 80 nM is shown. (C) Ba/F3-gp130 cells were stimulated with 50 ng/ml IL-6 and 100 ng/ml slL-6R in the presence of increasing sgp130Fc or cs-130Fc variant concentrations. At 72 h post stimulation, cellular proliferation was detected using a CellTiter-Blue assay. Normalization of relative proliferation was performed as described in methods. Assays are representative of three independent experiments. (D) Western blot analysis of Ba/F3-gp130 cells stimulated for 30 min with 50 ng/ml IL-6 and 100 ng/ml slL-6R in the presence of the indicated concentrations of sgp130Fc, cs-130 or cs- 130Fc variants. Unstimulated controls are indicated (“-“ lane). Prior to stimulation, IL-6, sIL- 6R and inhibitors were incubated separately for 30 min. Western blots were stained for phosphorylated (p)STAT3 and STAT3. Western blots are representative of three independent experiments. Normalized band intensity data are means ± SD from all experiments. *P< 0.05, **P< 0.01 , ***p< 0.001 0.0001 by analysis of variance (ANOVA) test with Dunnett’s correction.

Figure 4. cs-130Fc variants are poor inhibitors of IL-11 trans-signaling. (A) Surface plasmon resonance analysis of hyper-IL-11 -Fc binding to sgp130Fc and cs-130Fc variants. Hyper-IL-11 -Fc was immobilized on a CM-5 chip and increasing concentrations of inhibitors were injected. Sensograms are depicted as blue lines, global fit data are displayed as black lines. (B) Ba/F3-gp130 cells were stimulated with 10 ng/ml IL-1 1 and 100 ng/ml slL-11 R in the presence of increasing sgp130Fc or cs-130Fc concentrations. At 72 h post stimulation, cellular proliferation was detected using a CellTiter-Blue assay. Normalization of relative proliferation was performed as described in methods. Assays are representative of three independent experiments. (C) Western blot analysis of Ba/F3-gp130 cells stimulated for 30 min with 10 ng/ml hyper- IL-1 1 -Fc in the presence of the indicated concentrations of sgp130Fc or cs-130Fc variants. Unstimulated controls are indicated (“-“ lane). Prior to stimulation, IL-1 1 , slL-1 1 R and inhibitors were incubated for 30 min. Western blots were stained for phosphorylated (p)STAT3 and STAT3. Blots are representative of three independent experiments. Normalized band intensity data are means ± SD from all experiments. *P< 0.05, **P< 0.01 , ***p< 0.001 and 0.0001 by analysis of variance (ANOVA) test with Dunnett’s correction.

Figure 5: Affinity-enhancing mutations improve inhibitory capacity and selectivity of cs-130Fc variants. Ba/F3-gp130 cells were stimulated with 50 ng/ml IL-6 and 100 ng/ml slL-6R (A) or 10 ng/ml IL-11 and 100 ng/ml slL-11 R (B) in the presence of increasing inhibitor concentrations. At 72 h post stimulation, cellular proliferation was detected using a CellTiter-Blue assay. Normalization of relative proliferation was performed as described in methods. Assays are representative of three independent experiments.

Figure 6: cs-130Fc variants efficiently block Th17 cell expansion. Naive CD4 ⁺ T-cells were cultured in Iscove’s Modified Dulbecco’s Medium) supplemented with [TGFp (1 ng/mL), IL-23 (20 ng/mL), IL-6 (50 ng/mL), slL-6R (100 ng/mL), and anti-IL-2 (10 ng/mL)] in the presence of increasing inhibitor concentrations. To monitor Th17 cell expansion, IL-17 production was detected by flow cytometry. Histograms (A) are representative of three replicates. Dose-response-curves of the inhibitors (B) were plotted as percentage inhibition normalised to controls. Normalized data are means ± SD from all replicates. To calculate IC ₅ o values, a non-linear regression analysis was performed.

Figure 7: cs-130 variants inhibit IL-6 trans-signaling induced ERK phosphorylation. Western blot analysis of Ba/F3-gp130 cells stimulated for 30 min with 50 ng/ml IL-6 and 100 ng/ml slL-6R in the presence of the indicated concentrations of sgp130Fc, cs-130 or cs- 130Fc variants. Prior to stimulation, IL-6, slL-6R and inhibitors were incubated separately for 30 min. Western blots were stained for phosphorylated (p)ERK and ERK. Western blots are representative of N = 2 independent experiments. Both replicates are shown.

Figure 8: cs-130(Fc) variants do not affect IL-6 classic signaling. Ba/F3-gp130-IL-6R cells were stimulated with 10 ng/ml IL-6 in the presence of increasing inhibitor concentrations. As a control for the inhibition of IL-6 classic-signaling, the clinically approved IL-6R neutralizing antibody tocilizumab was used. At 72 h post stimulation, cellular proliferation was detected using a CellTiter-Blue assay. Normalization of relative proliferation was performed as described in methods. Assays are representative of three independent experiments. Figure 9: cs-130Fc variants have reduced effect on IL-11 trans-signaling. Western blot analysis of Ba/F3-gp130 cells stimulated for 30 min with 400 ng/ml IL-1 1 and 800 ng/ml sIL- 1 1 R in the presence of the indicated inhibitor concentrations. Prior to stimulation, IL-1 1 , sIL- 1 1 R and inhibitors were incubated separately for 30 min. Western blots were stained for phosphorylated (p)ERK and ERK. Western blots are representative of N = 3 independent experiments. Normalized band intensity data are means ± SD from all experiments. *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 by analysis of variance (ANOVA) test with Dunnett’s correction.

Figure 10: cs-130 ^{T102Y/Q113F/N114L} Fc is readily purified via affinity chromatography. SDSPAGE analysis of purified CS-130T102Y/Q113F/N1 14LFc. After affinity purification of cs-130 ^{T102Y/Q113F/N114L} Fc, the Fc fragment was removed by TEV protease-mediated proteolysis. The resulting fragments were separated by SEC and analyzed by SDS-PAGE and stained with Coomassie brilliant blue.

Figure 11 : Flow cytometric analysis of cs-130Fc variant mediated inhibition of Th17 cell expansion. Representative flow cytometry plots (n= 3) of naive CD4 ⁺ T-cells (CD4 ⁺ CD25 CD44 ^l0 CD62L ^hi ) from IL-6ra ^/_ mice cultured in vitro under polarising conditions for Th17 cells [TGF (1 ng/mL), IL-23 (20 ng/mL), IL-6 (50 ng/mL), SIL-6R (100 ng/mL), and anti-IL-2 (10 ng/mL)] in the presence of increasing inhibitor concentrations, as indicated, for 4 days. Th17 cell differentiation was determined by flow cytometry after stimulation with PMA, ionomycin and monensin for 4 hours.

Figure 12: cs-130 variants prevent IL-17 secretion from naive T cells stimulated under polarizing conditions for TH17 expansion. (A) ELISA data (n= 3) of naive CD4 ⁺ T-cells (CD4 ⁺ CD25 CD44 ^l0 CD62L ^hi ) from IL-6ra ^/_ mice cultured in vitro under polarising conditions for Th17 cells [TGF (1 ng/mL), IL-23 (20 ng/mL), IL-6 (50 ng/mL), SIL-6R (100 ng/mL), and anti-IL-2 (10 ng/mL)] in the presence of increasing inhibitor concentrations, as indicated, for 4 days. IL-17 secretion was determined by ELISA after stimulation with PMA, ionomycin and monensin for 4 hours. (B) Non-linear regression analysis of ELISA data from (A) was performed to determine IC50 values.

Figure 13: Expression and purification of recombinant proteins. (A) Schematic overview of recombinant proteins utilized in this study. (B) Western blotting analysis of supernatants and lysates of HEK-293T cells expressing the indicated proteins. (C) SDSPAGE analysis of purified proteins followed by Coomassie staining. (D) SDS-PAGE analysis of purified proteins in presence (+) of absence of p-mercaptoethanol. Figure 14: c19s-130Fc blocks IL-6 trans-signaling. (A) Surface plasmon resonance analysis of hyper-IL-6-TS binding to Cigs130Fc. Cigs130Fc was immobilized on a Protein A chip and increasing concentrations of hyper-IL-6-TS were injected. Sensorgrams in response units (RU) over time are depicted as coloured lines, global fit data are displayed as black lines. (B) Ba/F3-gp130 cells were stimulated with 100 ng/ml IL-6 and 200 ng/ml slL-6R in the presence of increasing CigS-130Fc, cs-130Fc or VHH72-Fc concentrations. At 72 h post stimulation, cellular proliferation was detected using a CellTiter-Blue assay. Normalization of relative proliferation was performed as described in methods. Assays are representative of three independent experiments. (C) Western blot analysis of Ba/F3-gp130 cells stimulated for 30 min with 10 nM IL-6 and 2 nM slL-6R in the presence of the indicated concentrations of CigS-130Fc. Prior to stimulation, IL-6, slL-6R and inhibitors were incubated separately for 30 min. Western blots were stained for phosphorylated (p)STAT3, STAT3, (p)ERK and ERK. Western blots are representative of three independent experiments. Controls for unstimulated cells (-), in absence of CigS-130Fc (+) and stimulation with hyper- IL-6-Fc (Hy) were included. (D) Western blot analysis of Vero cells stimulated for 30 min with 20 nM IL-6 and 4 nM slL-6R in the presence of the indicated concentrations of Cigsl 30Fc. Prior to stimulation, IL-6, slL-6R and inhibitors were incubated separately for 30 min. Western blots were stained for phosphorylated (p)STAT3, STAT3, pSTATI and STAT1. Western blots are representative of three independent experiments. Controls for unstimulated cells (-), in absence of CigS-130Fc (+), 80 nM cs-130Fc and stimulation with hyper-IL-6-Fc (HIL-6) were included.

Figure 15: Cigs130Fc binds to S-RBD and blocks its binding to ACE2 (A) Surface plasmon resonance analysis of c19s-130Fc binding to S-RBD. c19s-130Fc was captured on a Protein A chip and increasing concentrations of S-RBD were injected. Sensorgrams in response units (RU) over time are depicted as coloured lines, global fit data are displayed as black lines. (B) Surface plasmon resonance analysis of VHH72-Fc binding to S-RBD. VHH72-Fc was captured on a Protein A chip and increasing concentrations of S-RBD were injected. Sensorgrams in response units (RU) over time are depicted as coloured lines, global fit data are displayed as black lines. (C) Surface plasmon resonance analysis of ACE2 binding to S-RBD. ACE2 was immobilized on a CM-5 chip and increasing concentrations of S-RBD were injected. Sensorgrams in response units (RU) over time are depicted as coloured lines, global fit data are displayed as black lines. (D) Surface plasmon resonance analysis of ACE2 binding to S-RBD in presence of c19s-130Fc. ACE2 was immobilized on a CM-5 chip and 125 nM S-RBD were injected in the presence of increasing concentrations of increasing concentrations c19s-130Fc (coloured lines). Figure 16: Ci9S-130Fc limits SARS-CoV-2 mediated cytopathic effects on Vero cells. Vero cells were treated with indicated concentration of CigS-130-Fc, VHH72-Fc and Cs-130- Fc and infected afterwards with a MOI of 0.03 SARS-CoV-2. (A) Brightfield images were taken on day 3 post infection (one representative of n=4 is shown, scale bar = 1 mm) (B-D) CPE Score (circle) and TOX Score (square) of c19s-130-Fc (B) VHH72-Fc (C) and cs-130- Fc (D) were determined from brightfield images from a by ‘CPETOXnet’ and are shown in a concentration dependent manner (n=4). (E) CPE Score (left panels) and TOX Score (right panels) of the negative, positive and toxic controls calculated by ‘CPETOXnet’ on images 3 days post infection with MOI of 0.03 or with Staurosporine (5 pM) treated cells.

Figure 17: CigS-130-Fc inhibits SARS-CoV-2 infection of Vero cells. (A-H) Vero cells were treated with indicated concentration of CigS-130Fc, VHH72-Fc and cs-130Fc, infected afterwards with a MOI of 0.03 SARS-CoV-2 and were stained with anti-SARS-CoV-2 nucleocapsid antibody 2 days after infection. (A) Representative fluorescence images are shown (n = 4, scale bar = 1 mm). (B) IF Score was determined from fluorescence images from panel A using ‘IFnet’ and are shown in a concentration dependent manner (n=4). (C) IF signal of the negative and positive control images calculated by ‘IFnet’ (n=24). (D-H) An entry assay was performed by adding Monensin on different timepoints after infection to the cells. (D) The positive control is shown (n = 12). (E-H) In a concentration dependent manner, the results of stopping the infection after 5 (E), 15 (F), 45 (G) and 135 min (H) are shown (n = 4).

Figure 18: c19s130Fc prevents S-RBD binding to overexpressed ACE2 in Ba/F3 cells. (A) Flow cytometric analysis of cell surface expression of hACE2-gp130 (red population) in Ba/F3-gp130 cells detected by hACE2 antibody. The blue population indicates Ba/F3- gp130 cells incubated without hACE antibody (control). (B) Flow cytometric analysis of S- RBD binding to Ba/F3-ACE2 cells. Following incubation with S-RBD, S-RBD binding was detected (blue area) using an anti-spike S1 antibody (Sino Biological). Ba/F3-gp130- hACE2-gp130 cells without treatment served as controls (red area). (C to E) S-RBD binding in the presence of increasing concentrations of c19s130Fc, VHH72Fc, or cs130Fc. A total of 20,000 events were recorded, and the cell count was normalized. Histograms are representative of results from 3 independent experiments. EXAMPLES

Methods

Molecular cloning

A cDNA for human IL-11 was synthesized (Biocat GmbH, Heidelberg Germany) and subcloned into pcDNA3.1 via standard PCR methods. For cloning of cs-130Fc variants the cDNA encoding single domain antibody VHH6 was chemically synthesized by Biocat (Heidelberg, Germany). VHH6 was subsequently subcloned in pcDNA3.1-nHyper-IL-6-Fc (Lamertz et aL, 2018) to generate pcDNA3.1-IL-6-VHH6-Fc. Domains D1 -D3 of sgp130Fc were PCR-amplified using the following oligonucleotides:

D1 -D3 _fw:AAGCTTGCCACCATGCTGACC

D1 -D3_rv:CTCGAGCTTAGAGGGTCTGTCCTCGTAGG

D2_fwSOE:GATATGTGGAAAAGACATTTCTTCTGGACTGCCCCCCGAGAAGCCCAA G DeltaD1_rvSOE:CAGGTTTAGCAGCGGCGGC D1_rv:CTCGAGGCCGCTGATGATTGTGATGCC.

Amplicons were subcloned via Hindlll and Xhol sites to generate pcDNA3.1-cs-130Fc. An expression plasmid for CGFPS-130FC was generated by fusion of coding sequences of the D1 -D3 domains of sgp130 with cDNA encoding a nanobody against GFP (Engelowski et aL, 2018). The resulting PCDNA3.1 -CGFPS-130 was used to subclone CGFPS-130 via Hindlll and Notl sites into pcDNA3.1- cs-130Fc to generate CGFPS-130FC. The inactivating mutations Y190K/F191 E or the activity-boosting mutations T102Y/Q113F/N114L were introduced into pcDNA3.1 - cs-130Fc via site directed mutagenesis using the following oligonucleotides:gp130 ^Y190K/F191 Erv: ACCCACACTTCGATGTTCACCTCCTTCACGGTGCTGTAGTCCACGG, gp130 ^{Y190K/F191 E} fw:

CCGTGGACTACAGCACCGTGAAGGAGGTGAACATCGAAGTGTGGGT,

T102Y_fw: CAGCCTGAACATCCAGCTGTATTGCAACATCCTGACCTTCG,

T02Y_rv: CGAAGGTCAGGATGTTGCAATACAGCTGGATGTTCAGGCTG,

Q113F/N114L_fw:

GCTGATGATTGTGATGCCGTACACTAGGAATTCCAGCTGGCCGAAGGTCAGGATG, Q113F/N114L_rv: CATCCTGACCTTCGGCCAGCTGGAATTCCTAGTGTACGGCATCACAATCATCAGC.

For Cloning of c19s-130Fc variants. The cDNA encoding single domain antibody VHH6 (5) and SARS VHH72 (6) connected with a linker was chemically synthesized by Biocat (Heidelberg, Germany). The cDNA encoding residues 1 -615 of ACE2 (Q9BYF1 ) and residues 319-591 of SARS-CoV-2 RBD (P0DTC2) were chemically synthesized by Biocat. VHH72 with a TEV site (ENLYFQS) was subcloned via Agel and Notl in pcDNA3.1 vector (Invitrogen) containing N-terminal signal peptide and a Myc tag (EQKLISEEDL) and a C- terminal human Fc tag, thereby generating pcDNA3.1 -myc-VHH72-TEV-Fc for secreted expression. A linker followed by VHH6, (GGGGS)3, VHH72 and a TEV site was subcloned via Xhol and Notl into the pcDNA3.1 -cs-130Fc expression vector to create c19s-130Fc. The ACE2 sequence was amplified by PCR using the following forward (5'- AGTCCTTAAGCCACCATGTCAAGC TCTTCCTGGC-3') and reverse (5'- TGCGTATGCGG CCGCGTCTGCATATGGACTCCAG-3') primers. The fragment was cloned between the Aflll and Notl sites to generate pcDNA3.1 -ACE2-Fc. pcDNA3.1 -RBD- TwinStrep expression vector was cloned via Hind 111 and Notl out of the cDNA encoding the residues 319-591 of SARS-CoV-2 RBD to extend with TwinStrep tag (WSHPQFEK) connected with (GGS)3 linker.

Cells and reagents. The generation of Ba/F3-gp130 and Ba/F3-gp130-IL-6R cells has been described elsewhere (Chalaris et aL, 2007). For seeding and subcultivation of Vero cells, cells were first washed with PBS and then incubated in the presence of trypsin/EDTA solution (Genaxxon bioscience cat. #4261 .0110) until cells detached. Cell lines were grown in DMEM high glucose culture medium (GIBCO®, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (GIBCO®, Life Technologies), 60 mg/l penicillin and 100 mg/l streptomycin (Genaxxon bioscience GmbH, Ulm, Germany) at 37°C with 5% CO2. Proliferation of Ba/F3-gp130 cells was maintained in the presence of hyper-IL-6 (H- IL-6), a fusion protein of IL-6 and slL-6R, which mimics the IL-6 trans-signaling complex (Fischer et aL, 1997). Expression and purification of human hyper-IL-6 and human IL-6 was performed as described previously (Fischer et aL, 1997). The Expi-293F™ cells (ThermoFisher Scientific) were cultured in 30 mL Expi293™ expression medium without antibiotics until they reached a density of 3-5 x106 c/ml in a 37 °C incubator with 8% CO2 on an orbital shaker at 125 rpm. The Expi293-F™ cells were sub-cultured in shaker flask until they reached a density of 3-5 x10 ⁶ c/mL Antibodies directed against STAT3 phosphorylated at Tyr705 (clone D3A7) and STAT3 (Fischer et aL, 1997) were obtained from Cell Signaling Technology (Frankfurt, Germany). Rabbit anti-human IgG Fc (#31423) and peroxidaseconjugated secondary mAbs (#31432, #31462) were obtained from Pierce (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies directed against pERK (#4370) and ERK (#4695) were obtained from Cell Signaling. Recombinant sgp130Fc and IL-6 were produced and purified as described previously (Modares et aL, 2019). Rabbit anti-human IgG Fc HRP conjugate was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant soluble sgp130 ^{T102Y/Q113F/N114L} -Fc and slL-6R were obtained from CONARIS Research Institute AG (Kiel, Germany). An expression plasmid for IL-11(A11)Hise lacking the first 1 1 amino acids of IL-11 , was kindly provided by Prof. Dr. Christoph Garbers (Otto- von-Guericke-University, Magdeburg). IL-1 1 was expressed as a soluble protein in E. coli or Expi293 cells and purified via immobilized metal affinity chromatography. The slL-1 1 R was obtained from Bio-Techne (Wiesbaden, Germany).

Transfection, transduction and selection of cells. CHO-K1 cells were cultured in DMEM medium. For expression of recombinant proteins, 5x10 ⁵ CHO-K1 cells were transfected with Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and 5 pg plasmid DNA encoding cs-130Fc variants. At 5 hours after transfection, the medium was exchanged to DMEM medium without transfection reagent. For the generation of stable CHO-K1 cell lines, G418 was added to the medium 48 h after transfection. Single clones were selected via limiting dilution. Positive clones expressing Fc fusion proteins were identified by Western blotting using anti-human Fc antibodies.

Proliferation assays. Ba/F3-gp130 and Ba/F3-gp130-IL-6R cells were washed and 5,000 cells of each cell line were cultured for three days in a final volume of 100 pl in the presence of cytokines and inhibitors. The CellTiter-Blue® Reagent was used to determine cellular viability by recording the fluorescence (excitation 560 nm, emission 590 nm) using an Infinite M200 PRO plate reader (Tecan, Crailsheim, Germany) immediately after adding 20 pl of reagent per well (time point 0) and up to 120 min thereafter.

Stimulation of Ba/F3 cells assays and lysate preparation. 10 ⁶ Ba/F3-gp130 cells/ml and variants thereof were washed and starved in serum-free medium for 5 h. Vero cells were seeded at a density of 8x10 ⁵ cells per 60 mm dish 24 h prior stimulation and also washed five times with PBS before starving in serum-free DMEM for at least 5 h. Prior to stimulation, cytokines and inhibitors were pre-incubated at room temperature for 30 min. Subsequently, cells were stimulated with the indicated cytokines and inhibitor combinations for 30 min, harvested by centrifugation at 4°C for 5 min at 1500 x g, frozen and lysed. Protein concentration of cell lysates was determined by the BCA Protein Assay (Pierce, Thermo Scientific). Analysis of STAT3 activation was performed by Western blotting of 25-75 pg of total protein from total cell lysates and subsequent detection steps using the antipSTAT3 (Tyr705) (1 :1000) and anti-STAT3 (1 :2000) antibodies described above.

Western blotting. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were blocked and probed with the indicated primary antibodies. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies or fluorescence-labeled secondary antibodies (1 :10.000 dilution). The Immobilon™ Western Reagents (Millipore Corporation, Billerica, MA, USA) and the ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Gottingen, Germany) or the Odyssey Fc Imaging System (LI-CORE Biosciences, Bad Homburg, Germany) were used for signal detection. Control STAT3 blots were produced on the same membrane either following stripping or simultaneously to pSTAT3 imaging using the Odyssey Fc Imaging System.

Expression and purification of recombinant proteins. Inhibitory proteins were produced in stably transfected CHO-K1 cells (see above) using a roller bottle system. Culture supernatants from roller bottles were harvested and centrifuged at 1 ,000 x g and 4°C for 30 min, followed by centrifugation of the resulting supernatant at 10,000 x g at 4°C for 30 min. Mammalian expression plasmids encoding VHH72-Fc, c19s-130Fc, residues 319-591 of SARS-CoV-2 RBD, residues 1 -615 of ACE2 and were transfected into Expi-293F™ cells using ExpiFectamine™. Reaching 4.5-5.5x10 ⁶ c/ml, the cells were diluted to a final density of 3x10 ⁶ c/ml in 30 ml Expi293™ expression medium for transfection. 30 pg of the plasmid expression vectors were used for transfection. Henceforth, the culture was harvested by centrifugation at 450 x g at 4 °C for 5 min, followed by centrifugation of the resulting supernatant at 4000 x g at 4 °C for 20 min. The supernatant of the second centrifugation step was filtered (bottle top filter, 0.45-pm pore diameter; Nalgene; Rochester, NY) and purified by affinity chromatography. Before chromatography, the pH values of the filtered cell culture supernatants were adjusted to 7.4. Supernatant was loaded on a protein-A column (HiTrap protein A HP; GE Healthcare) at a flow rate of 2 ml/min. The column was then washed with 30 column volumes of PBS. Proteins were eluted at pH 3.2-3.5 using a 50 mM citric acid buffer. Fractions of 1 ml were collected. Fractions containing the protein peak were pooled, and the pH was adjusted to pH 7 with 1 M Tris. Proteins containing a C- terminal Twin-Strep-Tag (SARS-CoV-2 RBD) was purified using Strep-Tactin resin (IBA cat. #2-5025-001 ) according to the manufacturer’s instructions. Proteins were buffer exchanged to PBS using illustra NAP 25 (GE Healthcare Life Sciences, Munich, Germany) columns. Protein concentration was determined by measuring absorbance at 280 nm, and samples were flash-frozen in liquid nitrogen. Protein quality was assessed by SDS-PAGE and Coomassie staining. Following affinity purification, cs-130Fc variants were processed with TEV protease (ratio of proteimTEV protease of 1 :10) overnight at 4°C. After protease processing, the resulting protein fragments were separated by size exclusion chromatography (gel filtration) using a Superdex® 200 Increase 10/300 GL column (GE Healthcare Life Sciences, Munich, Germany). Surface plasmon resonance. For surface plasmon resonance experiments, a Biacore X100 instrument (GE Healthcare Life Sciences) was used. Fc-tagged VHH72-Fc or c19s- 130Fc were captured to a single flow cell of an ProtA sensorchip at a level of ~ 300 or 650 response units (RUs), respectively per cycle. Three samples containing only running buffer were injected over both ligand and reference flow cell, followed by S-RBD serially diluted from 500-3.9 nM, with a replicate of the 125 nM concentration. The analyte S-RBD was injected at a flow rate of 30 pl/min. ACE2 was immobilized in 10 mM acetate buffer (pH 4.5) by amine coupling on a CM5 chip (2500 RU). After immobilization, S-RBD was injected at a flow rate of 30 pl/min at increasing concentrations (2-250 nM). Association was monitored in periods of 60 sec, and the dissociation was measured for 600 sec. Immobilized ACE2 was regenerated with 2 M NaCI to remove bound S-RBD in multiple cycle measurement. ACE2 was immobilized on a CM-5 chip and 125 nM S-RBD were injected in the presence of increasing concentrations of increasing concentrations c19s-130Fc. Experiments were carried out at 25 °C in PBS pH 7.4, composed of 137 mM NaCI, 2.7 mM KCI, 12 mM HPO42- und H2PO4-, and 0.05% (v/v) surfactant P20 (GE Healthcare). Further analysis was performed in single cycle mode using CM5 sensor chips. For every single experiment a new sensor chip was used because of the high affinity protein protein interaction which makes the regeneration of chips inefficient. Experiments were carried out at 25°C in HBS- P + buffer (GE-Healthcare), containing 10 mM HEPES, pH 7.4, 0.15 M NaCI, 0.05% (v/v) surfactant P20 (GE Healthcare). Hyper-IL-6-Fc or hyper-IL-1 1 -Fc were immobilized by amine coupling on a CM5 chip (1200 RU). After immobilization, sgp130 and cs-130 variants were injected at a flow rate of 30 pl/min at increasing concentrations (5-80 nM). Association of cs-130Fc variants in each defined concentration was monitored in periods of 60 sec, and the global dissociation was measured at the end of the final injection in periods of 600 sec. Where applicable, final graphs were fitted using 1 :1 binding model. Due to the complexity of the data obtained as was also reported previously (Adams et aL, 2017) and its extremely low dissociation rate constant we have limited the kinetic characterization to the dissociation rate constant k _O ft where an obvious difference was observed. This is in accordance with the SPR analysis Adams et aL, performed on the interaction of an IL-6-SIL-6R fusion protein with the VHH6 sdAb that is part of cs-130Fc.

SEC-MALS (Size Exclusion Chromatography coupled to Multi-Angle Light Scattering). The determination of the absolute weight-averaged molar mass (M _w ) and its distribution was taken out using a Multi-Angle Light Scattering (MALS) system from Wyatt Technology. An 18-angle light scattering detector (model DAWN) and a refractive index detector (model Optilab T-rEX, Wyatt Technology) were coupled to an Agilent 1260 Infinity II HPLC system, consisting of a quaternary pump (model G711 1 B, incl. degasser), a variable wavelength detector (VWD, model G7114A) as well as an autosampler (model G7129A). Separation was achieved using a GE Superose 6 Increase column with a flow rate of 0.5 mL/min. PBS (pH 7.0) was used as the mobile phase. Between 10 and 50 pg were injected per sample. The control of the HPLC and the analysis of the light scattering as well as the concentration data were carried out using the software ASTRA 7 from Wyatt Technology. Measurements were performed by representatives of Wyatt Technology.

In vitro T-cell cultures. Murine CD4 ⁺ T-cells were enriched by negative magnetic selection (Miltenyi Biotec) before purification of naive (CD4 ⁺ CD25-CD44 ^l0 CD62L ^hi ) T-cells. T-cell purity was > 92%. Naive CD4 ⁺ T-cells were cultured in IMDM medium already containing 4 mM L-glutamine and 25 mM HEPES and supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 50 mM b-mercaptoethanol (all from Thermo Fisher Scientific). 1 .5X10 ⁵ CD4 ⁺ T-cells were cultured in 96-well U-shape bottom plates coated with anti-CD3 (1 mg/mL; 145- 2C11 ), added the previous day and incubated at 4°C overnight, and soluble anti-CD28 (5 mg/mL; 37.51 ). Recombinant mouse cytokines were included at the following concentrations to promote differentiation of naive CD4+ T-cells into a defined Th17 lineage: TGFb (1 ng/mL), IL-23 (20 ng/mL), IL-6 (50 ng/mL), slL-6R (100 ng/mL) and anti-IL-2 (10 ng/mL). Increasing inhibitor concentrations were added as indicated. 1 .5 x 10 ⁵ cells were plated and grown at 37°C with 5% CO2 for 4 days before phenotypic characterisation of 40000 cells by flow cytometry.

Methods related to SARS-CoV-2 S-Protein binding studies:

Viruses. SARS-CoV-2 was used as described previously (Sequence Accession Number: EPI ISL 425126). SARS-CoV-2 virus stock was obtained in Vero cells by infection at a multiplicity of infection (MOI) of 0.001 . After 72h, the supernatant was collected and stored at -80°C until usage.

SARS-CoV-2 Infection of Vero cells. Vero cells were cultured as previously described. Vero cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) with the addition of 10% foetal calf serum (FCS), minimal essential amino acids, and Penicillin/Streptomycin at 37 °C and 5% CO2. 3 x 104 cells were seeded per well in a 96 well plate one day before infection. On the next day, the medium was changed to the cell culture medium containing different dilutions of c19s-130-Fc, VHH-72-Fc, cs-130-Fc dissolved in PBS and Staurosporine (5pM) as a toxic control. Moreover a 6 or 12 serial 2-fold dilution was used. The cells were infected with SARS-CoV-2 20 min later with a MOI of 0.03. For the entry assay Monensin was added 5, 15, 45 and 135min after infection to the wells to stop the further entry of SARS-CoV-2 in the cells. An overlay composed DMEM with 1 % methylcellulose was added 2h post infection. The IC50 was measured using GraphPad Prism.

Immunfluorescence. Two days after infection, the supernatant was discarded, and 4% Formalin was added for 30 min. Hank’s buffer containing Triton-X was applied to the cells for 20 min followed by 10% FCS in PBS for 1 h to block unspecific binding sites. The cells were stained with a SARS-CoV-2 Nucleocapsid antibody (2019 nCoV) (Sino Biology Inc., Eschborn, Germany) for 1 h. Following washing, Fluorescein (FITC) conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Immuno Research, Cambridgeshire, UK) was added for 1 h. The cells were washed again and analyzed with the Nikon Eclipse TS100 fluorescence microscope. Pictures were taken with the software NIS-Elements F4.30.01 .

Quantification. The images were quantified using Deep Transfer Learning as previously described. ResNet18 was retrained to classify SARS-CoV-2 infected cell cultures. For recognizing of CPE and toxic effects we used the ‘CPETOXnet’ (Werner et al. 2021) with three different classification (CPE, TOX and No CPE). For quantification of SARS-CoV-2 immunofluorescence pictures we used ‘IFnet’ with two different classifications (IF Signal and No Signal).

Statistical Analyses. Data are expressed as mean ± SEM.

Example 1 : Design and production of miniaturized sgp130 variants. Albeit sgp130Fc is a very potent IL-6 trans-signaling inhibitor, it is a rather large dimer of approximately 240 kDa (186 kDa protein plus glycosylation), even compared to, e.g., therapeutic IgG antibodies (150 kDa). For antibodies and their fragments, a clear correlation between biodistribution and molecular weight has been demonstrated. Several smaller variants of sgp130 including monomeric variants lacking the Fc part or the natural short sgp130-RAPS isoform were characterized previously. These studies demonstrated that, for unknown reasons, the activity of sgp130-based trans-signaling inhibitors inversely correlates with their molecular weight. sgp130Fc inhibits IL-6 trans-signaling with an IC ₅ o of 77 pM, the IC ₅ o of monomeric sgp130 is above 700 pM (Jostock et aL, 2001 ) and the IC ₅ o for sgp130-RAPS is well over 70 nM. It was aimed to obtain novel fusion proteins with biological activities comparable to sgp130Fc but much lower molecular weights. To this end, miniaturized sgp130 variants were designed composed of combinations of the cytokine binding domains D1 and D2-D3, respectively. To improve their inhibitory potential, these sgp130 domains were fused with a previously described single domain antibody (sdAb, VHH, nanobody) recognizing the interface formed by IL-6 in complex with the IL-6R (VHH6). The sdAb recognizes only the complex of IL-6 and slL-6R, but not the individual components. The sdAb binds the complex of IL-6 and the slL-6R with nanomolar affinity and, based on the dissociation rate constant, stabilizes the complex. By combining the sgp130 ligand-binding domains and the sdAb, it was expected to obtain avidity effects restoring the biological activity lost through miniaturization of sgp130. Fusion proteins were designed as follows: The first three domains of sgp130 also included in the sgp130-RAPS variant (Sommer et aL, 2014) were connected to a flexible linker, followed by the sdAb VHH6 and a tobacco etch virus (TEV) protease recognition site. An Fc domain was used to generate dimeric inhibitors that, similar to the natural membrane-bound receptor gp130, are able to contact complexes of I L-6:sl L-6R via site II and site III, thereby completely enclosing them. The aim was to enabling the inhibitors to contact site II and III residues simultaneously through dimeric design and hence obtain more efficient inhibitors through avidity effects. The resulting fusion protein labeled cs-130Fc has a theoretical molecular weight of 157 kDa compared to 186 kDa for sgp130Fc (Fig. 1 A). After TEV cleavage, the resulting cs-130 was expected to have a molecular weight of about 52 kDa. A variety of fusion proteins were generated to investigate the effects of the domains D1 -D3 of sgp130 and the sdAb components in terms of inhibitory function (Fig. 1 B). To evaluate effects of the sdAb component, CGFPS- 130FC was generated, which contains a sdAb directed against instead of the I L-6/s I L-6R complex. In additional variants, mutations were introduced in the D1 and D2 domains of sgp130 to reduce or increase binding affinity to the IL-6:slL-6R complex termed cs-130 ^{Y190K/F191 E} Fc (Kurth et aL, 1999) and cs-130 ^{T102Y/Q113F/N114L} Fc, respectively (Fig. 1 B). cs-130Fc variants containing domains D1 -D3 of sgp130Fc were readily expressed and secreted by CHO-K1 cells (Fig. 2A). sgp130Fc and cs-130Fc variants were detected at slightly higher molecular weights (120 and 100 kDa, respectively) than the theoretical molecular weight (93 and 78 kDa, respectively), which is likely due to glycosylation. However, miniaturized variants containing only the D1 or the D2-D3 domains were poorly expressed and secreted. Consequently, only variants containing domains D1 -D3 of sgp130 were considered for further characterization. cs-130Fc variants were expressed by stably transfected CHO-K1 cells and affinity-purified from cell culture supernatants. Following affinity purification, proteins were pure, as demonstrated by SDS-PAGE analysis and subsequent Coomassie staining (Fig. 2B). Purified cs-130Fc was processed with TEV protease to remove the Fc segment. Proteolytic processing yielded three molecular species with molecular weights of approximately 100, 70 and 25 kDa consistent with the presence of unprocessed protein (78 kDa), cs-130 (52 kDa) and the Fc fragment (26 kDa), respectively (Fig. 2C, inset). The resulting fragments were separated by size exclusion chromatography (SEC). Further analysis using non-reducing SDS-PAGE (Fig. 2D) confirmed the absence of the Fc sequence from the 70 kDa cs-130 fragment. In the absence of reducing agent, sgp130Fc and cs-130Fc fusion proteins migrated corresponding to dimeric proteins with molecular weights above 170 kDa. In the presence of reducing agent, molecular weights of both proteins dropped to approximately 120 and 100 kDa, corresponding to monomeric sgp130Fc and cs-130Fc. Following Fc removal, cs-130 displayed a reduction of molecular weight to 70 kDa independent of the presence of reducing agent. This further indicated the successful removal of the Fc fragment. The quality and molecular mass of the purified proteins was subsequently assessed in solution via analytical size exclusion chromatography coupled to multi angle light scattering (SEC- MALS) (Fig. 2E). All samples displayed minor peaks corresponding to aggregated protein (<10%), while the main peaks contained approximately 90% of the sample. For all proteins, molecular masses close to the theoretical masses were determined (summarized in Table 1 ). Minor differences between estimated molecular weight and theoretical molecular weight of approximately 20% are likely due to glycosylation. For cs-130, an additional conjugate analysis using dn/dc values for proteins and sugars was performed to determine the composition of the protein. A total mass of 75 kDa was determined, composed of 58 kDa protein and 17 kDa sugar components (Fig. 2F). The protein mass determined was in accordance with the theoretical molecular weight of 52 kDa.

Example 2: cs-130Fc and cs-130 fusion proteins display improved IL-6 transsignaling inhibition in comparison to sgp130. The kinetics of cs-130Fc binding to the IL- 6/slL-6R fusion protein hyper-IL-6 binding were characterized using surface plasmon resonance. Hyper-IL-6 was immobilized on a CM5 chip, and inhibitory proteins were injected at increasing concentrations. Binding of the inhibitory proteins resulted in the sensorgrams displayed in Fig. 3A. Striking differences in the dissociation rate constants were observed (Fig. 3 A and B). For sgp130Fc a k _O ft of 7.2x10 ^-5 s ^-1 was determined, while cs-130Fc shown a dissociation rate constant of 3.6x10 ^-8 s ^-1 which is significantly lower than sgp130Fc. This indicates that incorporation of VHH6 strongly increased the stability of the hyper-IL-6/cs-130Fc complexes, which is in good agreement with the previously described stabilization effect of VHH6 on IL-6/slL-6R complexes. Next, the capacity of cs-130 variants to interfere with IL-6 trans-signaling was examined in a model system of Ba/F3 cells stably transduced with gp130 (Ba/F3-gp130). These cells respond to IL-6 trans-signaling with STAT3 activation and proliferation. To test dimeric and monomeric cs-130 variants, Ba/F3- gp130 cells were stimulated with 50 ng/ml IL-6 and 100 ng/ml slL-6R or with 10 ng/ml hyper- IL-6-Fc in the presence of increasing concentrations of the inhibitory proteins (Fig. 3C). sgp130Fc potently inhibited proliferation with an IC ₅ o of 0.87 nM (Table 2), while cs-130Fc displayed improved inhibitory activity with an IC ₅ o value of 0.27 nM. The negative control proteins CGFPS-130FC (comprising a GFP-specific nanobody instead of VHH6) and cs- 130 ^Y i90K/Fi9iEp _c (comprising two affinity-reducing point mutations in the CBM of sgp130Fc) had markedly reduced biological activities with IC50 values of 6.54 nM and 7.55 nM, respectively. Also monomeric cs-130 potently inhibited IL-6:slL-6R induced proliferation with an IC ₅ o of 1 .46 nM comparable to sgp130Fc. All IC ₅ o values are summarized in Table 2. For monomeric sgp130 and dimeric sgp130Fc, an approximately 10-fold difference in biological activity has been described previously (Jostock et al, 2001 ). The apparent differences between IC ₅ o values of dimeric cs-130Fc and its monomeric counterpart cs-130 are comparable to the differences of sgp130 dimers and monomers, and may be explained by avidity effects. Subsequently, it was investigated whether the above findings could be confirmed in orthogonal assays. STAT3 and ERK phosphorylation were analyzed as the important second messenger of IL-6 trans-signaling. STAT3 and ERK phosphorylation was analyzed in Ba/F3- gp130 cells. Cells were stimulated with 50 ng/ml IL-6 and 100 ng/ml sIL- 6R in the presence of increasing concentration of inhibitory proteins. Concentrationdependent inhibition of STAT3 phosphorylation was observed for all inhibitory proteins (Fig. 3D). sgp130Fc completely suppressed STAT3 phosphorylation at a concentration of 10 nM. At 1 nM sgp130Fc, STAT3 phosphorylation was strongly reduced, while only a minor reduction was observed at 0.1 nM. Similarly to the effects observed in proliferation assays, cs-130Fc displayed potent inhibitory potential on STAT3 phosphorylation comparable to sgp130Fc. Monomeric cs-130 inhibited STAT3 phosphorylation at concentrations of 10 and 1 nM, while at 0.1 nM in contrast to sgp130Fc and its dimeric counterpart no inhibition was observed anymore. The affinity-compromised variants C-GFPS-130FC and cs-130 ^{Y190K/F191 E} Fc displayed inhibitory activity, however, higher concentrations were required to obtain full inhibition (Fig. 3D). In addition, a trend indicating a possible reduction of ERK phosphorylation was observed (Fig. 7). In contrast to other IL-6 signaling inhibitors like monoclonal antibodies, sgp130Fc is specific for trans-signaling and only affects IL-6 classic signaling at very high doses when sIL- 6R is in excess over IL-6 (Garbers et aL, 201 1 ). To exclude an effect on classic signaling, we tested cs-130Fc variants on Ba/F3-gp130-IL-6R cells (additionally stably transduced with IL-6R), which were stimulated with IL-6. Proliferation assays with Ba/F3-gp130-IL-6R cells revealed that sgp130Fc and cs-130Fc variants did not inhibit IL-6 classic-signaling at the concentrations tested (and in the absence of slL-6R), while tocilizumab, a monoclonal antibody directed against site 1 of the IL-6R, did inhibit signaling as expected (Fig 8). Example 3: cs-130Fc variants do not inhibit IL-11 trans-signaling. Next, affinity constants for the interaction of the different inhibitory proteins with hyper-IL-11 -Fc, a fusion protein of IL-11 and slL-11 R analogous to hyper-IL-6 were determined, of which two fusion proteins are dimerized by an Fc part. Hyper-IL-11 -Fc was immobilized on a CM-5 chip, and binding of injected inhibitors was analyzed via surface plasmon resonance (Fig. 4A). Observed kinetic parameters are summarized in Table 3. It was found that sgp130Fc bound to hyper-IL-1 1 -Fc with approximately 28-fold higher affinity (K _d = 0.3 nM) than cs-130Fc (K _d = 8.6 nM). sgp130 and its derivatives efficiently block IL-6 trans-signaling but are not restricted to IL-6. sgp130Fc also inhibits IL-1 1 trans-signaling and, albeit to a much lesser extent, also affects LIF and OSM signaling. An IL-11 trans-signaling model was chosen to investigate whether the incorporation of the I L-6/sl L-6R single domain antibody VHH6 into the cs-130Fc variants resulted in improved IL-6 trans-signaling specificity over sgp130Fc. First, proliferation assays were performed using Ba/F3-gp130 cells stimulated with IL-11 and slL-1 1 R (Fig. 4B). It was found that sgp130Fc was 32 times more potent in blocking IL- 1 1 trans-signaling than cs-130Fc with IC ₅ o values of 0.2 and 7.1 nM, respectively (Table 2). Moreover, cs-130Fc and CGFPS-130FC displayed very similar IC ₅ o values of 7.1 and 16.7 nM for IL-11 trans-signaling, indicating that VHH6 had no effects on the formation or stabilization of the IL-11/slL-11 R complex. Under the conditions used, monomeric cs-130 had no effect on IL-11 trans-signaling, similarly to mutationally inactivated cs- 13O ^{YI9OK/FI91 E} FC. These findings were confirmed by analysis of the phosphorylation status of STAT3 and ERK following stimulation of Ba/F3-gp130 cells with 400 ng/ml IL-1 1 and 800 ng/ml slL-1 1 R (Fig. 4C, Fig. 9). Analogous to the effects observed in proliferation assays, sgp130Fc was most potent in blocking IL-11 trans-signaling induced STAT3 and ERK phosphorylation. STAT3 phosphorylation was completely inhibited by 10 and 1 nM sgp130Fc. cs-130Fc only induced a reduction of STAT3 phosphorylation at 10 nM concentrations similar to the CGFPS-130FC control, cs-130 and cs-130 ^{Y190K/F191 E} Fc had no significant effect on IL-1 1 trans-signaling-induced STAT3 phosphorylation.

Example 4: Affinity-enhancing mutations improve inhibitory capacity and selectivity of cs-130Fc variants. sgp130Fc is a very well-characterized IL-6 trans-signaling inhibitor. IL-6 interacts with the IL-6R via its binding site I and with gp130 via sites II and III. The identity of residues at the interfaces of these contact sites was found by crystallographic studies and extensive mutagenesis. Tenhumberg et al. described several mutations in sgp130 associated with improved affinity of sgp130 towards IL-6. The mutations T102Y, Q1 13F and N1 14L at site III of sgp130 were introduced into cs-130Fc to further improve its affinity towards IL-6. The resulting cs- ^{130T102Y/Q113F/N114L} Fc was expressed and purified by affinity chromatography (Fig. 10). Monomeric cs-130 ^{T102Y/Q113F/N114L} was then generated by TEV protease cleavage of the Fc fragment and isolated by SEC (Fig. 10). Both the dimeric cs-130 ^{T102Y/Q113F/N114L} Fc and the monomeric cs-130 ^{T102Y/Q113F/N114L} efficiently inhibited IL-6 trans-signaling-induced proliferation of Ba/F3-gp130 cells with IC ₅ o values of 0.36 and 0.48 nM, respectively (Fig. 5A and Table 2)). In comparison, for sgp130Fc an IC ₅ o of 0.87 nM was determined. In addition, no effects of cs-130 ^{T102Y/Q113F/N114L} Fc and its monomeric form cs- 13o ^{T1O2Y/Q113F/N114L} were observed on IL-1 1 trans-signaling or IL-6 classic-signaling- induced proliferation at any concentration (up to 100 nmol/L) (Fig. 5B, Fig. 10).

Example 5: cs-130Fc variants inhibit Th17 cell formation in a dose dependent manner. IL-6 trans-signaling mediated expansion of Th17 cells is a crucial factor in the pathology of a variety of autoimmune diseases including rheumatoid arthritis. Inflammation results in reduced surface of IL-6R levels on activated Th (CD4+CD44hiCD62Llo) cells and T cell activation is linked with a downregulation of IL-6R. Activated neutrophils, monocytes and T cells can release soluble IL-6R through IL-6R shedding leading to an increase in local soluble IL-6R levels. The increased slL-6R levels enable IL-6 trans-signaling mediated activation of cells lacking surface IL-6R. In line with this IL-6 trans-signaling promotes the local expansion of TH17 cells, which in turn can be inhibited by the IL-6 trans-signaling inhibitor sgp130Fc. Since it was observed that cs-130Fc variants efficiently blocked IL-6 trans-signaling without affecting IL-6 classic-signaling in the Ba/F3 test system, it was next investigated whether they can also inhibit IL-6:slL-6R induced Th17 expansion. To assess the effect of cs-130Fc variants on Th17 expansion, naive CD4 ⁺ T-cells from IL-6ra ^/_ mice were treated using a combination of IL-6 and slL-6R, transforming growth factor (TGF)P, anti-IL-2 and IL-23 under anti-CD3/CD28 stimulation. Th17 cell expansion was monitored by flow cytometry based on IL-17 production (Fig. 6 and Fig. 11 ). sgp130Fc, cs-130Fc and cs-130 displayed comparable inhibition of Th17 cell expansion. With IC ₅ o values of 9.8 and 10.5 nM, sgp130Fc and cs-130Fc were equally potent, while sgp130Fc was twice as active than cs-130 (20 pM). During control experiments using CGFPS-130FC and cs-130 ^{190K/F191 E} Fc no effect on Th17 expansion was observed. In addition to TH17 expansion we also monitored the effect of inhibitory proteins on the secretion of IL-17 (Fig. 12). To this end IL- 17 secreted by T cells treated with increasing inhibitor concentrations as described above for cell expansion assays was quantified by ELISA. Very similar IC ₅ o values for sgp130Fc, cs-130Fc and cs-130 were observed with regards to their inhibition of IL-17 secretion of 1 .99 nM, 1 .54 nM and 2.04 nM, respectively.

Example 6: Modular architecture of the bispecific inhibitor c19s-130Fc. Several variants of soluble gp130 were described to selectively inhibit IL-6 trans-signaling. Sgp130Fc consists of all six extracellular domains of gp130 fused to the Fc-part of an IgG antibody. Dimerization of sgp130 increased the affinity towards IL-6:slL-6R by a factor of

10 compared to monomeric sgp130. Interestingly, only the first three extracellular domains of gp130 are needed for cytokine binding, however, sgp130 variants consisting only of these three domains (sgp130RAPS, sgp130-ELP, sgp130E10) showed markedly reduced binding affinities for IL-6:slL-6R compared to sgp130. Recently, we generated the high affinity sgp130 variant cs130, which consists of the first three extracellular domains D1 -D3 of sgp130 fused to the nanobody VHH6 which showed sgp130Fc like binding affinities and inhibitory capacity. VHH6 specifically binds to IL-6:slL-6R complexes, without inhibitory capacity. In particular, fusion of VHH6 to sgp130D1 -D3 resulted in equally potent but much smaller IL-6 trans-signaling inhibitor compared to sgp130Fc. Like IL-6, IL-11 signals via soluble and membrane-bound IL-1 1 R and homodimeric gp130 and sgp130Fc inhibits IL-6 and IL-11 trans-signaling with comparable efficacy. Due to VHH6, cs130 did not inhibit IL-

1 1 trans-signaling. Due to the smaller size and modular architecture of cs130, we wondered, if this design enables further upgrading into bispecific binding of I L-6:sl L-6R complexes and S-RBD from Sars-CoV2. To minimize the size of the resulting bispecific inhibitor, we selected to fuse the SARS-CoV-2 S-RBD VHH (VHH72) (Wrapp et al. 2020) to cs-130 connected via a flexible linker sequence (GGGGS)2GGGGTG)) (Fig. 13A). The resulting c19s130Fc plus the control proteins VHH72 fused to an IgG Fc (VHH72-Fc) and cs-130Fc were produced in and purified from supernatants of HEK293T cells (Fig. 13B). Following affinity purification, proteins were pure, as demonstrated by SDS-PAGE analysis and subsequent Coomassie staining (Fig. 13C). The disulfide mediated dimerization of all proteins was assessed by non-reducing SDS-PAGE. In this analysis all inhibitory proteins demonstrated a shift to higher molecular weight in the absence of reducing agent (Fig. 13D) confirming disulfide mediated dimerization. Monomeric c19s130 were not produced, because it was shown previously that VHH72 was only inhibiting cellular virus entry in dimeric form.

Example 7: C19s-130Fc efficiently inhibits IL-6 trans-signaling. First of all, the affinity of c19s-130Fc, cs-130Fc and VHH72Fc for Hyper IL-6 were determined and compared in surface plasmon resonance (SPR) experiments. The trans-signaling designer cytokine Hyper IL-6 is a fusion protein composed of IL-6 and the slL-6R connected via a flexible peptide linker (Fischer et aL, 1997). CigS-130Fc displayed very high affinities of 55 pM, for Hyper IL-6 (Fig 14A). The kinetic analysis of the interaction revealed the formation of a very stable complex characterized by a very low k _O ft rate of 7.1 x 10 ^-5 1/s. Next, the inhibitory potential of the c19s130Fc towards IL-6 trans-signaling was analyzed. To this end, Ba/F3 cells stably transduced with gp130 (Ba/F3-gp130) were stimulated with 100 ng/ml IL-6 and 200 ng/ml slL-6R, which induced STAT3/ERK phosphorylation-dependent cellular proliferation. This cell-based assay served as a surrogate model for the induction of IL-6 trans-signaling. A concentration dependent inhibition of IL-6 trans-signaling through c19s- 130Fc and cs-130Fc was observed, while no effect was found for VHH72-Fc (Fig. 14B). IC50 values of 0.62 and 0.51 nM were determined for c19s-130Fc and cs-130Fc, respectively. In an orthogonal experimental setup, I L-6:sl L-6R stimulated Ba/F3-gp130 cells and Vero cells were utilized to examine the effect of c19s-130Fc on STAT3 phosphorylation. In line with the proliferation data, c19s-130Fc and cs130Fc inhibited STAT3 and ERK phosphorylation at concentrations above 5 nM in Ba/F3-gp130 cells (Fig 14C). In Vero cells STAT1 and STAT3 phosphorylation were blocked at concentrations between 0.5 and 10 nM (Fig. 14D). Taken together, c19s-130Fc and cs-130Fc but not VHH72-Fc are highly potent inhibitors of IL-6 trans-signaling as shown in biophysical and cell-based assays.

Example 8: C19s-130Fc binds to SARS-CoV-2 S-RBD and prevents viral entry. To determine the activity of the SARS-CoV-2-neutralizing entity in c19s130Fc, the binding affinity with the S-RBD was first determined to be 880 nM using SPR (Fig. 15A). Next, an affinity of 680 nM was found for the interaction of VHH72Fc with S-RBD (Fig. 15B), which deviates from the previously described affinity of 39 nM (44). The differences in binding affinities can primarily be attributed to a lower association rate constant (1.6 x 10 ⁵ 1/Ms [1 per molar times second]) and a higher dissociation rate constant (0.1 1/s). This can be explained by differences in the S-RBD protein compositions used. The previously described affinity of 39 nM was measured toward S-RBD subdomain 1 (S-RBD-SD1 ), in contrast to S-RBD. This may lead to an altered dissociation rate constant. For c19s130Fc, highly comparable S-RBD binding kinetics with an affinity of 880 nM were found. Hence, VHH72 fully retains its activity in c19s130Fc. Further, the affinity of purified S-RBD towards immobilized ACE2Fc was analyzed via SPR (Fig. 15C). An affinity of 52 nM was detected, which is in good agreement with the previously described affinity of 44 nM (Shang et al. 2020). In competition assays with immobilized ACE2-Fc, S-RBD, and increasing concentrations of c19s130Fc, an inhibitor dependent reduction of the binding of S-RBD to ACE2-Fc was found (Fig. 15D). A concentration of 78 nM c19s130Fc resulted in a reduction of ACE2:S-RBD by approximately 40%, as apparent by a reduction of the maximal binding response from 98.96 response units (RU) to 60.28 RU. This suggested that c19s130Fc binding to SARS-CoV-2 S-RBD prevents spike protein binding to ACE2 and, hence, neutralizes this key interaction required for viral cell entry. Next, the effect of c19s130Fc on SARS-CoV-2-mediated cytopathic effects (CPE) on Vero cells was analyzed. Vero cells can be efficiently infected with SARSCoV-2 (Rosa et al. 2021 ) and serve as a model system for viral infection. Following incubation with SARS-CoV-2, a reduction of virus-induced cytophathic effects was found in the presence of c19s130Fc and VHH72Fc but not cs130Fc (Fig. 16A-E). IC50 values of 8.1 ± 0.8 nM and 32.3 ± 18.6 nM were determined for c19s130Fc and VHH72Fc, respectively (Fig. 16B and C). As for SPR-based affinity assays, IC50 values for VHH72Fc reported previously by Wrapp et al. (2.5 nM) differed slightly in pseudovirus neutralization assays. In addition to CPE assays, the ability of c19s130Fc to prevent infection of Vero cells by SARS-CoV-2 was investigated. Virally infected Vero cells were visualized via immunofluorescence (IF) using anti-SARS-CoV-2 nucleocapsid antibodies (Werner et al. 2021 ). Vero cells treated with c19s130Fc and VHH72Fc showed reduced SARS-CoV-2 cell entry with comparable IC50 values of 15.1 ± 3.7 nM and 20.7 ±

1.6 nM, respectively (Fig. 17A to C). No effect was observed for cs130Fc. Next, timedependent effects of the inhibitors on viral entry during early phases of infection were investigated. Vero cells were incubated with SARS-CoV-2 for 5, 15, 45, or 135 min, and virus infection and uptake were then stopped by the addition of monensin. Total viral entry increased from 5 min to 135 min of incubation (Fig. 17D). Both c19s130Fc and VHH72Fc reduced SARS-CoV-2 uptake with very comparable IC50 values of 14.2 ± 5.4 nM, 1 1.8 ±

3.6 nM, 9.1 ± 1.4 nM, and 8.2 ± 1.4 nM for c19s130Fc and 16.2 ± 4.3 nM, 9.8 ± 1.2 nM, 1 1 .9 ± 0.8 nM, and 10.7 ± 3.5 nM for VHH72Fc (Fig. 17E to H). These IC50 values are very comparable to the IC50 values determined for viral uptake after 48 h. Hence, the inhibitory proteins seem to be stable and maintain activity for at least 48 h in a cell culture setting. To further confirm the effect of c19s130Fc on SARS-CoV-2 cell entry, ACE2(1-615) was stably expressed on Ba/F3 cells (Ba/F3-ACE2). ACE2 was detected on the cell surface by flow cytometry (Fig. 18A). Following incubation of Ba/F3-ACE2 cells with S-RBD, surface binding of S-RBD was detected by flow cytometry (Fig. 18B). In the presence of c19s130Fc and VHH72Fc but not cs130Fc, a concentration-dependent reduction of surface-attached S- RBD was observed (Fig. 18C to E), indicating that c19s130Fc and VHH72Fc prevent the binding of the SARS-CoV-2 spike protein to ACE2. The data showed that c19s130Fc efficiently neutralized SARS-CoV-2 binding to ACE2 and blocked viral cell entry and infection.

Tables and Legends

Table 1 : SEC-MALS analysis of purified proteins. The absolute weight-averaged molar mass (Mw) of affinity purified sgp130Fc and cs-130Fc variants was analyzed by SEC- MALS. Between 10 and 50 pg were injected per sample on a Superpose 6 Increase column in PBS (pH 7.0) at a flow rate of 0.5 ml/min and analyzed using a Multi-Angle Light Scattering (MALS) system (Wyatt Technology). Inhibitor MW (kDa) SEC-MALS MW

(theoretical) (kDa) sgp130Fc 240 240.4 (± 0.2%)

CS-130FC 157 184.7 (± 0.2%) cs-130 52 75.0 (± 1.1%)

CGFPS-130FC 153 179.9 (± 0.2%) cs-130 ^{Y190K/F191 E} Fc 157 191 .7 (± 0.6%)

Table 2: Inhibitory profile of sgp130 and chimeric soluble sgp130 fusion proteins

Ba/F3-gp130 cells were stimulated with the indicated concentrations of cytokines and their respective soluble a-receptors in the presence of decreasing inhibitor concentrations. IC ₅ o values were determined from three independent experiments.

IC50

Inhibitor Stimulation ng/ml nM cytokine (ng/ml) sgp130Fc IL-6/SIL-6R (50/100) 226.1 ± 176.3 0.87 ± 0.68 sgp130 ^{T102Y/Q113F/N114L} Fc SIL-6R/IL-6 (50/100) 70.1 ± 34.0 0.27 ± 0.18

CS-130FC SIL-6R/IL-6 (50/100) 86.4 ± 73.7 0.46 ± 0.39 cs-130 SIL-6R/IL-6 (50/100) 102.0 ± 102.1 1.46 ± 1.46

C _G FPS-130FC SIL-6R/IL-6 (50/100) 1243.0 ± 957.6 6.54 ± 5.04 cs-130 ^{Y190K/F191 E} Fc SIL-6R/IL-6 (50/100) 1963.3 ± 1153.9 7.55 ± 6.07 cs-130 ^{T102Y/Q113F/N114L} Fc SIL-6R/IL-6 (50/100) 68.2 ± 29.5 0.36 ± 0.16 cs-13O ^T1 O2Y/Q113F/N114L _s l L-6R/I L-6 (50/100) 33.5 ± 14.9 0.48 ± 0.08 sgp130Fc slL-11 R/IL-11 (10/100) 56.4 ± 5.8 0.22 ± 0.02 sgp130 ^{T102Y/Q113F/N114L} Fc slL-11 R/IL-11 (10/100) No inhibition No inhibition

CS-130FC slL-11 R/IL-11 (10/100) 1355.9 ± 697.1 7.14 ± 3.77 cs-130 slL-11 R/IL-11 (10/100) No inhibition No inhibition

CGFPS-130FC slL-11 R/IL-11 (10/100) 3181 .5 ± 2834.8 16.67 ± 14.92 cs-130 ^{Y190K/F191 E} Fc slL-11 R/IL-11 (10/100) No inhibition No inhibition cs-130 ^{T102Y/Q113F/N114L} Fc slL-11 R/IL-11 (10/100) No inhibition No inhibition cs-130 ^{T102Y/Q113F/N114L} slL-11 R/IL-11 (10/100) No inhibition No inhibition

Table 3: Kinetic parameters of inhibitor interaction with hyper-IL-11-Fc. Surface plasmon resonance analysis of hyper-IL-11 -Fc binding to purified sgp130Fc and cs-130Fc. Hyper-IL-11 -Fc was immobilized on a CM-5 chip and increasing concentrations of inhibitors (5-80 nM) were injected at a flow rate of 30 pl/min . Measurements were carried out on a a Biacore X100 instrument. kon T(kon) koff (1/s) T(koff) Kd (M)

(1/M.s) sgp130Fc 8.6E+5 1.1 E+2 2.7E-6 2.4 3.1 E-11

CS-130FC 4.6E+4 4.3E+2 3.9E-4 7.7E-6 8.6E-9

REFERENCES

Baran et. aL, The balance of interleukin (IL)-6, IL-6:soluble IL-6 receptor (slL-6R), and IL- 6:slL-6R:sgp130 complexes allows simultaneous classic and trans-signalling, J Biol Chem. 2018 May 4; 293(18): 6762-6775

L. Lamertz, et. aL, Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses, Sci Signal, 2018 Oct 2;1 1 (550):eaar7388.doi: 10.1 126/scisignaLaar7388.

E. Engelowski et. aL, Synthetic cytokine receptors transmit biological signals using artificial ligands. Nat Commun 9, 2034 (2018).

A. Chalaris et. aL, Apoptosis is a natural stimulus of IL6R shedding and contributes to the proinflammatory trans-signaling function of neutrophils. Blood 110, 1748-1755 (2007).

M. Fischer et. aL, A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat Biotechnol 15, 142-145 (1997).

R. Adams et. aL, Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein protein interaction and modulates its downstream signaling. Scientific reports 7, 37716 (2017).

T. Jostock et. aL, Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses. European journal of biochemistry / FEBS 268, 160-167 (2001 ).

J. Sommer et. aL, Alternative intronic polyadenylation generates the interleukin-6 trans- signaling inhibitor sgp130-E10. J Biol Chem 289, 22140-22150 (2014). C. Garbers et. aL, Inhibition of classic signaling is a novel function of soluble glycoprotein 130 (sgp130), which is controlled by the ratio of interleukin 6 and soluble interleukin 6 receptor. J Biol Chem 286, 42959-42970 (201 1 ).

S. Tenhumberg et. aL, Structure-guided Optimization of the lnterleukin-6 Trans-signaling Antagonist sgp130. Journal of Biological Chemistry 283, 27200-27207 (2008).

N. F. Modares et. aL, IL-6 trans-signaling controls liver regeneration after partial hepatectomy. Hepatology, (2019).

G. Guaraldi et. aL, Tocilizumab in patients with severe COVID-19: a retrospective cohort study. Lancet Rheumatol 2, e474-e484 (2020).

S. C. Jordan et. aL, Compassionate Use of Tocilizumab for Treatment of SARS-CoV-2 Pneumonia. Clin Infect Dis 71 , 3168-3173 (2020).

K. S. Meleveedu et. aL, Tocilizumab for severe COVID-19 related illness - A community academic medical center experience. Cytokine X 2, 100035 (2020).

L. Y. C. Chen et. alt., Soluble lnterleukin-6 Receptor in the COVID-19 Cytokine Storm Syndrome. Cell Rep Med, 100269 (2021 ).

R.-C. Investigators, A. C. Gordon et. aL, lnterleukin-6 Receptor Antagonists in Critically III Patients with Covid-19. April 22, 2021 , N Engl J Med 2021 ; 384:1491 -1502, DOI: 10.1056/NEJMoa2100433

M. Kopf et. aL, Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368, 339-342 (1994).

Z. Geng et. aL, Tocilizumab and the risk of respiratory adverse events in patients with rheumatoid arthritis: a systematic review and meta-analysis of randomised controlled trials. Clin Exp Rheumatol 37, 318-323 (2019).

A. Pawar et. aL, Risk of serious infections in tocilizumab versus other biologic drugs in patients with rheumatoid arthritis: a multidatabase cohort study. Ann Rheum Dis 78, 456- 464 (2019). T. Barkhausen et. aL, Selective blockade of interleukin-6 trans-signaling improves survival in a murine polymicrobial sepsis model. Crit Care Med 39, 1407-1413 (2011 ).

J. Shang et. aL, Structural basis of receptor recognition by SARS-CoV-2. Nature 581 , 221 - 224 (2020).

N. C. Kyriakidis et. aL, SARS-CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates. NPJ Vaccines 6, 28 (2021 ).

P. Deb et. aL, An update to monoclonal antibody as therapeutic option against COVID-19. Biosaf Health s, 87-91 (2021 ).

D. Wrapp et. aL, Structural Basis for Potent Neutralization of Betacoronaviruses by SingleDomain Camelid Antibodies. Cell 181 , 1004-1015 e1015 (2020).

Rafael B Rosa et. aL, In Vitro and In Vivo Models for Studying SARS-CoV-2, the Etiological Agent Responsible for COVID-19 Pandemic. Viruses, 2021 Feb 27;13(3):379.

Werner Julia et. aL, Deep Transfer Learning Approach for Automatic Recognition of Drug Toxicity and Inhibition of SARS-CoV-2. Viruses 2021 , 13(4), 610.

N. Schumacher, S. Rose-John, ADAM17 Activity and IL-6 Trans-Signaling in Inflammation and Cancer. Cancers (Basel) 1 1 , (2019).

J. Scheller et. aL, lnterleukin-6: from basic biology to selective blockade of pro-inflammatory activities.

Tanaka et. aL, Immunotherapeutic implications of IL-6 blockade for cytokine storm. Immunotherapy. 2016 Jul;8(8):959-70. doi: 10.2217/imt-2016-0020.

Kang et aL, 2019, Targeting lnterleukin-6 Signaling in Clinic. Immunity. 2019 Apr 16;50(4):1007-1023. doi: 10.1016/j.immuni.2O19.03.026.

Rossi et. aL, A phase l/ll study of siltuximab (CNTO 328), an anti-interleukin-6 monoclonal antibody, in metastatic renal cell cancer. Br J Cancer. 2010 Oct 12;103(8):1 154-62. doi: 10.1038/sj.bjc.6605872. Nishimoto et aL, Humanized antihuman IL-6 receptor antibody, tocilizumab. Handb Exp Pharmacol. 2008;(181 ):151 -60. doi: 10.1007/978-3-540-73259-4_7.

Boyce et aL, Sarilumab: Review of a Second IL-6 Receptor Antagonist Indicated for the Treatment of Rheumatoid Arthritis. Ann Pharmacother 2018 Aug;52(8):780-791 . doi: 10.1 177/1060028018761599.

Mullberg et aL, The soluble interleukin-6 receptor is generated by shedding. Eur J Immunol. 1993 Feb;23(2):473-80. doi: 10.1002/eji.1830230226.

Matthews et aL, Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE). J Biol Chem. 2003 Oct 3;278(40):38829-39. fmadoi: 10.1074/jbc.M210584200. Epub 2003 Jun 27.

Hirano et. aL, Interleukin 6 in autoimmune and inflammatory diseases: a personal memoir. J-Stage 2010 Volume 86 Issue 7 Pages 717-730. https://doi.org/10.2183/pjab.86.717.

Akira et. aL, lnterleukin-6 in biology and medicine. Adv Immunol. 1993;54:1 -78. doi: 10.1016/s0065-2776(08)60532-5.

Mitsuyama et. aL, Soluble interleukin-6 receptors in inflammatory bowel disease: relation to circulating interleukin-6. Gut. 1995 Jan;36(1 ):45-9. doi: 10.1136/gut.36.1 .45.

Montero-Julian et. aL, The soluble IL-6 receptors: serum levels and biological function. Cell Mol Biol (Noisy-le-grand). 2001 Jun;47(4):583-97.

Jones et. aL, IL-6 transsignaling: the in vivo consequences. J Interferon Cytokine Res. 2005 May;25(5):241 -53. fdoi: 10.1089/jir.2005.25.241 .

Calandra et. aL, High circulating levels of interleukin-6 in patients with septic shock: evolution during sepsis, prognostic value, and interplay with other cytokines. The Swiss- Dutch J5 Immunoglobulin Study Group. Am J Med. 1991 Jul;91 (1 ):23-9. doi: 10.1016/0002- 9343(91 )90069-a.

Hack et aL, Increased plasma levels of interleukin-6 in sepsis. Blood. 1989 Oct;74(5): 1704- 10. Myszka and Rich, Implementing surface plasmon resonance biosensors in drug discovery.

PSTT Vol. 3, No. 9, 2000: 310-317.

Zhu and Cuozzo, High-throughput Affinity- Based Technologies for Small-Molecule Drug Discovery. Journal of Biomolecular Screening, 2009: 1157-1164.