EA044106B1 - ANTI-huTNFR1 THERAPY FOR NON-ALCOHOLIC STEATHOEPATITIS - Google Patents

Description

Field of technology to which the invention relates

The invention relates to a new treatment for non-alcoholic steatohepatitis (NASH) and related diseases.

Prior Art

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of diseases that occurs in the absence of alcohol abuse and includes non-alcoholic steatohepatitis (NASH). NAFLD shows increasing incidence in Western countries and is a critical contributor to the development of hepatocellular carcinoma.

One of the fundamental steps on the path from benign hepatic steatosis to progressive steatohepatitis is the occurrence of hepatocyte cell death, classified as apoptosis. Necroptosis has emerged as an alternative programmed cell death pathway and has been found to be upregulated in the livers of NASH patients (Gautheron et al. Cellular and Molecular Gastroenterology and Hepatology 2015, 1:264–266).

Aparicio-Vergara et al. (Hepatology 2013, 57(2):566-576) describe the role of TNFR1 ectodomain exfoliation in preventing the development of hepatic steatosis or insulin resistance. Failure to shed TNFR1 did not lead to obesity, insulin resistance, or hepatic steatosis in mice. However, mice containing the mutation without desquamation showed rapid progression towards NASH. Activation of TNFR1 ectodomain shedding has been found to play a key role in attenuating progression toward NASH.

Cubero et al. (Cell Death and Differentiation 2013, 20:1580-1592) describe that TNFR1 in hepatocytes and immune cells play different roles in their mode of action in chronic liver disease.

Tomita et al. (Gut 2006, 55: 415-424) describe that enhancement of the TNFa/TNFR-mediated signaling pathway may be critically involved in the pathogenesis of liver fibrosis in the NASH animal model.

Yaron Ilan (AASLD Liver Learning. Ilan Y. Nov 8 2014; 60709) discloses an oral anti-TNF immunotherapy for the treatment of fatty liver disease. An anti-TNF fusion protein (PRX-106), which binds TNFa, was used in a high-fat diet mouse model.

Antibodies to TNFR1 have been found to have agonistic potential, inducing a ligand-mimicking response. This response suggests that signal transduction is initiated by receptor aggregation by binding of multivalent TNF trimers.

However, TNFR1-selective inhibition can be achieved using TNFR1-specific antibodies. For example, the mouse monoclonal antibody, H398, and the antibody described in US 5,736,138, with selectivity for human TNFR1, demonstrated potent inhibition of TNF-mediated signal transduction and cytotoxicity (Moosmayer et al. 1995, Ther. Immunol. 2:31-40).

The humanized version of H398 is described in W02008/113515A2.

WO2012035141 discloses an anti-huTNFR1 antibody that is deficient in mediating effector function.

Monovalent antibodies against huTNFR1 are described in WO2017174586 A1.

Zettlitz et al. (LandesBioscience 2010, November/Dezember: 639-647) describe the preparation of a humanized TNFR1-specific antagonistic monoclonal antibody.

Richter et al. (PLOS One 2013, 8 (8): 1-13) describe the use of a humanized antagonistic anti-TNFR1 antibody to selectively inhibit the TNFR1 signaling pathway while reducing the pro-inflammatory activity of TNF while leaving TNFR2 intact.

Berger et al. (Protein Engineering, Design & Selection 2013, 26(10):581-587) describe an anti-TNFR1 scFv-HAS fusion protein as a selective antagonist of TNF action. Feagins et al. (Eur J Gastroenterol Hepatol. 2015, 27 (10): 1154-1160) describe that patients treated with tumor necrosis factor inhibitors (TNFi) develop non-alcoholic fatty liver disease (NASH or steatosis).

Therapeutic options for treating NASH are limited and limited to lifestyle modifications as specific medications are not yet available. Thus, there is a need to provide effective treatment for NASH and related diseases.

The essence of the invention

The object of the invention is to provide improved treatment for NASH and related disease states.

The problem is solved by the object of the invention.

The invention provides a novel medical use of antibodies that specifically recognize human tumor necrosis factor receptor-1 (huTNFR1) for the treatment of patients suffering from NASH and/or, in particular, any disease associated with NASH, including hepatic steatosis, NAFLD disease activity ( NAS), apoptosis, fibrosis and high levels of alanine transaminase (ALT) and insulin. Therefore, the invention provides a new method of treating patients suffering from NASH and related diseases.

In particular, the invention relates to an antibody that specifically recognizes huTNFR1 for use in the treatment of non-alcoholic steatohepatitis (NASH) and related diseases.

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In a particular aspect, the antibody is an isolated antibody.

In a particular aspect, the antibody is a monoclonal and/or recombinant antibody.

In a particular aspect, the antibody specifically recognizes an epitope in the membrane-distal CRD1 and/or subdomain A1 of CRD2 of huTNFR1, preferably specifically recognizing the epitope represented by amino acids 1 to 115 or 1 to 70 in the N-terminal region of huTNFR1. Specifically, the sequence of huTNFR1 is identified as SEQ ID NO: 32.

In a specific embodiment, the antibody is a monospecific, divalent full-length antibody or antigen-binding antibody fragment.

In another specific embodiment, the antibody is a monovalent huTNFR1 binder containing only one antigen binding site that has the specificity to bind huTNFR1. In particular, the antibody monovalently recognizes huTNFR1.

In a specific embodiment, the antibody is selected from the group of monovalent antibodies consisting of Fab molecules, scFv molecules, single variable domains, disulfide-stabilized Fvs (dsFvs), half-IgG1 antibodies and Fv domains, or functionally active derivatives of any of the above, preferably where the antibody construct is linked with a hydrophilic polymer such as PEG and/or fused to a polypeptide such as human (or murine) serum albumin, transferrin, albumin binding domains or peptides, Ig binding domains or peptides, PEG mimetic polypeptide extensions, Fc fragment antibodies, an Fc fragment of an antibody carrying mutations to provide preferential heterodimerization (over homodimerization), or a functional variant of any of the above polypeptides.

In particular, an antibody is any Fab, scFv, dsFv, or Fv domain that is fused to an Fc fragment of an antibody, wherein the Fc consists of a heterodimer of the CH2 and CH3 domains, wherein the CH2 and/or CH3 domains carry one or more point mutations that allow advantageous heterodimerization versus homodimerization. In particular, one or both CH3 domains in the Fc are modified to change the amino acid structure, for example, to produce an Fc containing a heterodimer of CH3/CH3 domains.

In particular, the antibody construct includes Fv domains fused to an Fc region or fragment of the antibody, with or without additional antibody domains, while maintaining the monovalent binding structure of the antibody. A specific example relates to a Fab fragment or an Fv fragment fused to an Fc or modified Fc.

A preferred antibody comprises a heavy and a light chain, wherein the heavy chain consists of a VH domain, a CH2 and CH3 domain, optionally further including one or more linkers, and the light chain consists of a VL domain, a CH2 and CH3 domain, optionally further including one or more linkers.

Specific embodiments include an Fc from human IgG1 in which the CH2-CH3 domains form a heterodimer as a result of one or more knobs-to-groove mutations, e.g., knob mutations modifying the surface of the CH3 beta sheets present on one monomer of the CH3 domain, which are T366W; and surface modifying pit mutations of CH3 beta sheets present on another CH3 domain monomer, which are selected from the group consisting of T366S, L368A, Y407V.

In particular, the antibody includes an Fc region that includes one or more mutations to reduce the effectiveness of effector function. In accordance with a particular aspect, the Fc region is glycoengineered to reduce modulation of effector function.

In a specific embodiment, the antibody construct comprises a human or artificial IgG1 Fc region that is a functional Fc variant of human IgG1 with at least any of 60%, 70%, 80%, 85%, or 90% sequence identity, which has been mutated to reduce effector function. Preferably the Fc region comprises a heavy chain with at least one mutation selected from the group consisting of E233P, L234V, L235A, 2G236, A327G, A330S and P331S, preferably including A327G/A330S/P331S (Kabat EU index numbering ). Preferably, at least two of these mutations, more preferably at least three, four, five or all six mutations are embedded in the Fc sequence. SEQ ID NO: 31 identifies the Fc sequence from human IgG1.

In particular, the antibody is PEGylated, HESylated or PSAylated.

Specifically, the antibody is PEGylated with PEG having a molecular weight ranging from 5,000 to 150,000 g/mol. Typical antibody constructs, such as Fabs, are PEGylated with PEG 40,000.

Specifically, the antibody is a half IgG1 antibody characterized by only one Fab portion, a hinge region, and one Fc portion, wherein the hinge region and/or Fc portion (specifically, the Fc of human IgG1) includes one or more mutations to to avoid heavy chain dimerization (Gu et al. (2015) PLoS One 10(1):e0116419), for example, those selected from the group consisting of mutation in the hinge region (SEQ ID NO: 33): C226S, C229S (EU numbering ) and mutations in

- 2 044106 parts Fc: P395A, F405R, Y407R, K409D (EU numbering).

Specifically, the antibody is an Fv-Fc fusion protein, wherein Fv consists of a VH/VL domain pair, and wherein VH is fused to the first strand of the CH2-CH3 domain via a first hinge/linker region, and VL is fused to the second strand of the CH2-CH3 domain. through the second hinge/linker area. Preferably, the first and second CH2-CH3 domain chains differ from each other by one or more point mutations, for example to provide preferential heterodimerization between the first and second CH2-CH3 domain chains, resulting in an Fv-Fc preparation that is characterized by an Fc heterodimer, e.g. through protrusion-to-recess mutations as above.

Specifically, the antibody comprises a disulfide-stabilized Fv (dsFv) that is characterized by one or more additional (artificial) interdomain disulfide bonds. Such disulfide bonds are formed by introducing one or more additional cysteine residues in any of the VH and VL domains at suitable positions that can be used as a bridge of disulfide bonds connecting the VH and VL domains, where the disulfide bonds are formed after reduction of the cysteines. In specific examples, a disulfide bond may be introduced into Fv at any of the following positions in VH and corresponding positions in VL: 44C in VH and 100C in VL, 108C in VH and 55C in VL, 106C in VH and 56C in VL, or 101C in VH and 46C to VL.

In particular, the antibody includes

a) a heavy chain variable domain (VH), including the complementarity determining regions (CDRs): VH-CDR1, VH-CDR2 and VH-CDR3; And

b) a light chain variable domain (VL), including CDRs: VL-CDR1, VL-CDR2 and VL-CDR3, where

i)

V H-CDR1 includes or consists of SEQ ID NO: 1;

V H-CDR2 includes or consists of SEQ ID NO: 2;

V H-CDR3 includes or consists of SEQ ID NO: 3;

V L-CDR1 includes or consists of SEQ ID NO: 4;

V L-CDR2 includes or consists of SEQ ID NO: 5;

VL-CDR3 includes or consists of SEQ ID NO: 6; or ii)

V H-CDR1 includes or consists of SEQ ID NO: 23;

V H-CDR2 includes or consists of SEQ ID NO: 24;

V H-CDR3 includes or consists of SEQ ID NO: 25;

V L-CDR1 includes or consists of SEQ ID NO: 26;

V L-CDR2 includes or consists of SEQ ID NO: 27;

V L-CDR3 includes or consists of SEQ ID NO: 28;

g where the numbering corresponds to the EU index according to Kabat;

or a functionally active variant of any of i) or ii) above, which includes 0, 1 or 2 (or up to 1, i.e. 0 or 1) point mutations in each of the CDR sequences and which specifically recognizes huTNFR1.

In particular, the antibody includes VH and VL, where VH-CDR1 includes or consists of SEQ ID NO: 1;

V H-CDR2 includes or consists of SEQ ID NO: 2;

V H-CDR3 includes or consists of SEQ ID NO: 3;

V L-CDR1 includes or consists of SEQ ID NO: 4;

V L-CDR2 includes or consists of SEQ ID NO: 5; And

V L-CDR3 includes or consists of SEQ ID NO: 6;

g where the numbering corresponds to the EU no Kabat index;

or a functionally active variant thereof comprising up to 1 (i.e., 0 or 1) point mutation in any one or more or each of the CDR sequences, and which specifically recognizes huTNFR1.

In particular, VH and VL sequences are characterized by VH- and VL-CDR sequences, where

i)

VH-CDR1 includes or consists of SEQ ID NO VH-CDR2 includes or consists of SEQ ID NO

VH-CDR3 includes or consists of SEQ ID NO

VL-CDR1 includes or consists of SEQ ID NO VL-CDR2 includes or consists of SEQ ID NO VL-CDR3 includes or consists of SEQ ID NO or ii)

VH-CDR1 includes or consists of SEQ ID NO: 1;

1;

10, where X at position 5 represents S;

3;

4;

5; And

11, where X at position 3 represents G;

VH-CDR2 includes or consists of SEQ ID NO: 10, where X at position 5 is S;

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VH-CDR3 includes or consists of SEQ ID NO: 3;

VL-CDR1 includes or consists of SEQ ID NO: 4;

VL-CDR2 includes or consists of SEQ ID NO: 5; And

VL-CDR3 includes or consists of SEQ ID NO: 11, where X at position 3 is S.

Specifically, the antibody comprises a VH sequence comprising or consisting of SEQ ID NO: 7 or 9; and a VL sequence comprising or consisting of SEQ ID NO: 8 or 10, or a functionally active variant thereof, comprising up to 1 point mutation in any one or more or each of the CDR sequences and at least 60% sequence identity in any one or several, or in each of the framework (FR) sequences FR1-4 from VH and VL.

Specific VH/VL combinations comprising an antigen binding site capable of specifically recognizing and binding to huTNFR1 are any of:

a) a VH sequence comprising or consisting of SEQ ID NO: 7; and a VL sequence comprising or consisting of SEQ ID NO: 8; or

b) a VL sequence comprising or consisting of SEQ ID NO: 9; and a VL sequence comprising or consisting of SEQ ID NO: 10.

Specifically, an antibody is a full-length or antigen-binding fragment of an antibody comprising or consisting of a Fab that includes:

a) a heavy chain (HC) sequence comprising or consisting of SEQ ID NO: 11; And

b) a light chain (LC) sequence comprising or consisting of SEQ ID NO: 12;

or a functionally active variant thereof comprising up to 1 point mutation in any one or more or each of the CDR sequences of the VH and VL domains contained in the HC and LC, respectively, and at least 60% sequence identity in any one or more, or in each of the FR sequences FR1-4 of the VH and VL domains.

Specifically, the antibody includes:

a) a HC sequence comprising or consisting of SEQ ID NO: 18; And

b) an LC sequence comprising or consisting of SEQ ID NO: 13;

or a functionally active variant thereof comprising up to 1 point mutation in any one or more or each of the CDR sequences of the VH and VL domains contained in the HC and LC, respectively, and at least 60% sequence identity in any one or more, or in each of the FR sequences FR1-4 of the VH and VL domains.

Specific functionally active antibody variants comprising the HC identified in SEQ ID NO: 18 and the LC identified in SEQ ID NO: 13 include

NS, consisting of:

a) VH comprising or consisting of SEQ ID NO: 19 or at least the CDR sequences contained in said VH sequence;

b) a linker sequence consisting of 4-10 amino acids, for example 4, 5, 6, 7, 8, 9 or 10 amino acids, preferably consisting of a series of glycines, serines or threonines, in any combination, such as, for example, a linker, consisting of SEQ ID NO: 15;

c) a CH2 domain comprising or consisting of SEQ ID NO: 16; And

d) a CH3 domain comprising or consisting of SEQ ID NO: 20; And

LC, consisting of:

a) VL comprising or consisting of SEQ ID NO: 14 or at least the CDR sequences contained in said VH sequence;

b) a linker sequence consisting of 4-10 amino acids, for example 4, 5, 6, 7, 8, 9 or 10 amino acids, preferably consisting of a series of glycines, serines or threonines, in any combination, such as, for example, a linker, consisting of SEQ ID NO: 15;

c) a CH2 domain comprising or consisting of SEQ ID NO: 16; And

d) a CH3 domain comprising or consisting of SEQ ID NO: 17.

In particular, such an antigen-binding antibody is encoded by one or more nucleic acid molecules comprising

a) NS encoded by the sequence SEQ ID NO: 22; And

b) LC encoded by the sequence SEQ ID NO: 21;

or a functionally active variant thereof comprising up to 1 point mutation in any one or more or each of the CDR sequences of the VH and VL domains contained in the HC and LC, respectively, and at least 60% sequence identity in any one or more, or in each of the FR sequences FR1-4 of the VH and VL domains.

According to a specific embodiment, the antibody includes an antigen binding site characterized by the following combination of six CDR sequences that include or consist of:

SEQ ID NO: 23: VH-CDR1;

SEQ ID NO: 24: VH-CDR2;

SEQ ID NO: 25: VH-CDR3;

SEQ ID NO: 26: VL-CDR1;

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SEQ ID NO: 27: VL-CDR2; And

SEQ ID NO: 28: VL-CDR3;

or a functionally active variant thereof comprising up to 1 point mutation in any one or more or each of the CDR sequences and specifically recognizing huTNFR1.

In particular, the antibody includes an antigen binding site included in the VH and VL domains, where

a) VH includes or consists of SEQ ID NO: 29; And

b) VL includes or consists of SEQ ID NO: 30;

or a functionally active variant thereof comprising 0, 1 or 2 (or up to 1) point mutations in any one or more or each of the CDR sequences of the VH and VL domains and at least 60% sequence identity in any one or more or in each of the FR sequences FR1 -4 VH and VL domains.

Specifically, the antibody comprises a VH and VL domain, wherein at least one of the VH and VL domains is a functional affinity maturation variant of the parent domain comprising at least one point mutation in any of the complementarity determining region (CDR) sequences, Where

a) the parent VH domain is characterized by the CDR sequences: SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25; And

b) the parent VL domain is characterized by the CDR sequences: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

In particular, said at least one point mutation is present in any of SEQ ID NO: 24 and/or SEQ ID NO: 28.

In particular, any of the typical antibodies (which are antibodies characterized by the sequences presented herein) can be used according to the invention. Likewise, any alternative antibodies that include the same antigen binding site and/or have the same target binding specificity can be used. Specific alternative antibodies are those that are functional variants of the generic antibodies, wherein any of the generic antibodies can be used as a parent to generate a variant that has the function of specifically recognizing the huTNFR1 target.

Specifically, an antibody is an antibody produced by affinity maturation of a parent antibody that is characterized by the sequences set forth herein, particularly wherein 1, 2, 3, 4, 5, or 6 CDR sequences are functionally active CDR variants comprising up to 1 point mutation compared to the corresponding CDR in the parent antibody.

In specific embodiments, a functionally active variant antibody comprises only 0, 1, 2, or 3 point mutations in each of the CDR sequences, preferably only 0, 1, or 2 point mutations in each of the CDR sequences, wherein the point mutation is any of a substitution, an insertion or deletions of one amino acid.

Any of the functionally active antibody variants (parent antibody) described herein are specifically characterized by huTNFR1 binding specificity. A functionally active variant may include one or more mutated FR sequences that include one or more, e.g., multiple point mutations, e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 point mutations to produce a variant sequence with at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity sequences compared to the corresponding FR sequence in the parent antibody.

Specifically, the antibody includes an antigen binding moiety that binds huTNFR1 with a KD of less than ^10'8 M or 5x10'9 M and a koff of less than 10 ^-3 s ^-1 . Binding affinity and binding characteristics (association and dissociation) are specifically determined in a standard monovalent binding assay, essentially eliminating the avidity effects of divalent binding. The standard test is based on piezoelectric microbalance (QCM) measurements at physiological temperature (about 37°C or at 37°C +/- 1°C). This affinity measurement is particularly performed in the Fab format. Thus, if the antibody is anything other than a Fab, the antigen binding site is specifically introduced into the corresponding Fab molecule for affinity measurement by QCM at 37°C. This ensures comparable affinity measurements of monovalent binders, regardless of avidity effects that may interfere with affinity measurements. A particularly preferred QCM is performed at moderate receptor densities. Specifically, the binding affinity of the antibody construct to huTNFR1 is determined for the Fab format using QCM at 37°C and a moderate receptor density in the range of 50-100 Hz, for example at approximately 50 Hz or at 50 Hz +/- 10 Hz or at 50 Hz +/- 5 Hz.

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In particular, KD is less than 4x10 ^-9 M or less than 3x10 ^-9 M or less than 2x10 ^-9 M or less than 10 ^-9

M or even less than 10 ^-10 M.

In particular, koff is less than 10 ^-3 , or less than 5x10 ^-4 s ^-1 , or less than 10 ^-4 s ^-1 , or less than 10 ^-5 s ^-1 .

In particular, the antigen binding fragment recognizes huTNFR1 with a kon of at least 105 M ^-1 s ^-1 .

In a specific aspect, the disease states are any of hepatic steatosis, inflamed liver, liver fibrosis (or apoptosis), and hepatocellular carcinoma. In particular, a patient with NASH who is at risk of developing or already suffering from any disease is treated. Several indicators of NASH or related diseases include NAFLD disease activity (NAS) and high serum ALT and insulin levels, which can be effectively reduced by the treatment described herein.

In particular, the patient also suffers from type II diabetes mellitus, type I diabetes mellitus, prediabetes, insulin resistance or obesity, whereby obesity is defined as a patient with a body mass index

In particular, the antibody is administered to the patient in an effective amount. In particular, this amount is effective in antagonizing TNFa/huTNFR1 signaling. It is particularly preferred that the antibody is an antagonist antibody, thereby avoiding significant TNFa/TNFR-mediated signaling and transduction measured in a cell-based assay. Any of the antibodies described herein and characterized by the antibody sequences set forth herein are specifically understood to be antagonistic antibodies.

In a specific aspect, the antibody directly inhibits TNF-huTNFR1 receptor interaction as determined by a cell-based assay, preferably by an assay for inhibiting TNFR1-mediated cell death in Kym-1 cells or by an assay for inhibiting the release of IL-6 or IL-8 from HeLa cells or HT1080 cells, respectively. In particular, in the assay for inhibition of TNFR1-mediated cell death in Kym-1 cells, the _IC50 value is less than 5.0x10 ^-9 M. In particular, in the assay for inhibition of IL-6 release from HeLa cells, the IC50 value is less than 4.0x10 ^-8 M or in the IL-8 release inhibition assay from HT1080 cells, the IC50 value is less than 2.0 x10 ^-8 M.

In accordance with a specific embodiment, an antibody is used that binds to huTNFR1 in a monovalent manner and reduces the risk of TNFmimetic agonist activity. Particularly preferred are antibodies with high binding affinity to TNFR1 and a low dissociation rate, which provides excellent inhibition of TNFR1-dependent TNF responses.

In particular, the antibody described herein is provided in a pharmaceutical composition containing the antibody and a pharmaceutically acceptable carrier and/or excipient. Due to the antagonistic properties of the antibody, the pharmaceutical formulation can contain high concentrations of the antibody while avoiding the side effects resulting from the agonist activity.

In particular, the pharmaceutical composition is formulated for parenteral administration, preferably by intravenous or subcutaneous administration.

In particular, the antibody described herein has low immunogenicity and can be reused without the formation of inhibitors such as anti-drug antibodies (ADA).

Surprisingly, the antibodies described herein, particularly monovalent antibodies, can be used to treat patients who develop ADA, for example, who have developed anti-immunoglobulin antibodies or immunotherapy antibodies. In the prior art, the presence of such ADAs would specifically preclude further immunotherapy with antibodies directed against TNFR1, since ADA has the ability to cross-link antibodies upon binding TNFR1 on the cell surface, thereby potentially acting agonistically on TNFR1 signaling. However, the antibodies described herein do not act agonistically (or are essentially non-agonistic) on TNFR1 signaling even in the presence of ADA.

In particular, the pharmaceutical composition described herein can be administered to patients who have developed ADAs, for example ADAs against anti-huTNFR1 antibodies or any IgG structures.

In particular, an effective amount of the antibody is administered to a patient suffering from NASH to reduce any one or more of

a) steatosis, triglyceride content, inflammation and/or apoptosis in liver tissue;

b) serum aminotransferase level;

c) insulin resistance and, possibly, positive dynamics of glucose tolerance; and/or

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d) NAFLD activity score.

In particular, the antibody is administered to a patient suffering from NASH at a dose of 0.05 to 20 mg/kg, preferably 0.2 to 6 mg/kg. The amount effective in humans can be derived from the therapeutically effective dose described in the mouse model (20 mg/kg). HED (human equivalent dose) is ~1-2 mg/kg.

Preferred doses of antibodies are, for example, from 0.5 to 1000 mg, preferably 1-400 mg. When administered subcutaneously, the preferred dose is 0.5 to 400 mg.

In a particular aspect, the antibody is administered to a patient in a therapeutically effective amount by systemic administration, preferably by intravenous infusion or bolus injection.

In accordance with a specific embodiment, the antibody is administered to the patient multiple times, for example, weekly, i.v. or p.c. by injection at a dose of, for example, 0.5-5 mg/kg, in particular about 2 mg/kg. The frequency and dose of the drug administered can be adapted to the disease state and response to therapy.

Specifically, the antibody is administered to a patient suffering from NASH in combination with dietary treatment. Antibody treatment may, in particular, be combined with anti-inflammatory drugs such as NSAP/NSAID, or with therapy using a farnesoid X receptor (FXR) agonist, glucagon-like peptide-1 receptor (GLP1R) agonist or peroxisome proliferator-activated receptor (PPAR) agonist. .

Unless otherwise stated, provisions in this document are numbered according to Kabat's EU suffix. An explanation of Kabat's numbering scheme can be found in Kabat, E. A., et al, Sequences of proteins of immunological interest (NIH publication no. 91-3242, 5th edition (1991)).

Blueprints

Fig. 1: B6-huTNFR1-k/i mice were fed a high-fat diet (HFD) for 32 weeks, including treatment with anti-TNFR1 or control antibody (Ab) for the last 8 weeks. Liver tissues from HFD mice treated with anti-TNFR1 Ab showed significant reductions in steatosis (A), triglyceride content (B), and NAFLD activity score (C) in the liver tissues compared with liver tissues from mice treated with control antibody. * P <0.05; **p <0.01.

Fig. 2: B6-huTNFR1-k/i mice were fed a high-fat diet (HFD) for 32 weeks, including treatment with anti-TNFR1 or control antibody (Ab) for the last 8 weeks. Liver tissues from HFD mice treated with anti-TNFR1 Ab showed improvement in liver fibrosis, assessed by Sirius red staining (A), which was significant compared with liver tissues from mice treated with control antibody (B). *p<0.05.

Fig. 3: B6-huTNFR1-k/i mice were fed a high-fat diet (FIFD) for 20 weeks, including anti-TNFR1 or control antibody (Ab) treatment for the last 4 weeks. Compared with control antibody, treatment with anti-TNFR1 antibody resulted in a significant reduction in caspase-3 activation in liver tissues. *p<0.05.

Fig. 4: B6-huTNFR1-k/i mice were fed a high-fat diet (FIFD) for 32 weeks, including treatment with anti-TNFR1 or control antibody (Ab) for the last 8 weeks. Compared with control antibody, treatment with anti-TNFR1 Ab resulted in significant improvements in ALT and serum insulin levels. * p <0.05

Fig. 5: Sequences

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

1: VH-CDR1;

2: VH-CDR2;

3: VH-CDR3;

4: VL-CDR1;

5: VL-CDR2;

6: VL-CDR3;

7: VH from IgG13.7/Fab13.7;

8: VL IgG13.7/Fab13.7;

9: VH ATROSAB/IZI06.1;

10: VL ATROSAB/IZI06.1;

11: (Fab13.7 heavy chain [bold = VH]);

12: (Fab13.7 light chain [bold=VL]);

13: VL1C (VL13.7-CH2-CH31; VL- and SS-containing chain):

14: VL13.7;

15: Linker;

16: CH2;

SEQ ID NO: 17: CH31: CH31 is an interleaved Ig constant domain that includes primarily residues derived from CH3, but also residues from CH1;

SEQ ID NO: 18: VHkC (VH13.7-CH2-CH3-kappa; chain containing VH and CLk):

SEQ ID NO: 19: VH13.7;

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SEQ ID NO:20:CH3k;

SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO

21: VL1C (VL13.7-CH2-CH31; chain containing VL and CH1):

22: VHkC (VH13.7-CH2-CH3-kappa; chain containing VH and CLk):

23: VH-CDR1 from ATROSAB;

24: VH-CDR2 from ATROSAB;

25: VH-CDR3 from ATROSAB;

26: VL-CDR1 from ATROSAB;

27: VL-CDR2 from ATROSAB;

28: VL-CDR3 from ATROSAB;

29:ATROSAB VH;

30:ATROSAB VL;

31: Human IgG1 Fc;

32: huTNFR1 sequence:

33: hinge area.

Fig. 6: Biochemical characterization of atrosimab (HC: SEQ ID NO: 18, LC: SEQ ID NO: 13). (a) Representative image of the molecular composition of atrosimab (white: Fc-derived Ig constant domains; bright gray: VH and CH1-derived sequences; dark gray: VL and CLk-derived sequences). Atrosimab was characterized by SEC (b) TSKgel SuperSW mAb HR, flow rate 0.5 ml/min, mobile phase Na2HPO4/NaH2PO ₄ ) and SDS-PAGE (c) NuPAGETM 4-12% Bis-TRIS Midi Gel) in reducing ( R) and non-reducing conditions (HP). M: Marker, (d) Thermal stability of atrosimab was analyzed by dynamic light scattering and visual interpretation of the data obtained. The stability of atrosimab after incubation in human plasma was analyzed by determining the residual binding activity to human TNFR1 in ELISA (e). Bars represent _EC50 values of three separate experiments (mean ± standard deviation). One sample incubated in PBS at 4°C and one sample frozen at -20°C immediately after dilution in human plasma served as controls.

Fig. 7: Antigen binding and interaction with Fc receptors and complement protein Clq. Equilibrium binding of atrosimab to human TNFR1-Fc was analyzed by ELISA ((a) n = 3, mean ± SD). Fab 13.7 (includes identical VH and VL) and ATROSAB (lower affinity bivalent version) served as controls. (b) Real-time binding kinetics were recorded by QCM at five concentrations from 128 nM to 4 nM (in 1:2 dilution steps) using a 1:1 binding algorithm for data analysis. (c) Interactions of immobilized atrosimab, as well as the two control proteins ATROSAB (inactive Fc) and rituximab (wild-type Fc portion) with human FcyRI, IIb and III, and the complement protein Clq were analyzed by ELISA (n = 2, average value ± standard deviation).

Fig. 8: Antagonistic biological activity of atrosimab and lack of agonism. Atrosimab demonstrated a complete lack of agonist activity in three different in vitro assays: (a) IL-6 release from HeLa cells, (b) IL-8 release from HT1080 cells, and in a cell death induction assay using Kym 1 cells (c). Parental Fab 13.7, which showed fully agonistic properties, and divalent IgG ATROSAB, which showed minor agonistic effects in (a) and (b), served as control proteins. The same set of proteins was assayed for their ability to inhibit cell surface TNFR1 activation in HeLa, HT1080, and Kym-1 cells, as measured by IL-6 release (d), IL-8 release (e), and cell death induction (f). , respectively. TNFR1 was activated using 0.1 nM TNF (d and f) or 0.01 nM TNF (f). All graphs represent the mean of three separate experiments, and error bars indicate SD.

Fig. 9: Lack of agonism of atrosimab in the presence of anti-human IgG antibodies. Activation of TNFR1 on the surface of HT1080 cells by atrosimab in the presence of a constant concentration (ca. 15.8 nM) of drug-specific antibodies was analyzed in an IL-8 release assay using three different mouse anti-human IgG sera (a, b and c). Mouse anti-human IgG sera, unstimulated cells, and TNF (33 nM) served as controls. The mean ± SD of three separate experiments is shown.

Fig. 10: Pharmacokinetic analysis of atrosimab. Circulating concentrations of atrosimab were determined in mouse serum after a bolus injection of 400 μg of protein in C57BL/6J knockout mice, which express the extracellular domain of human TNFR1 coupled to the mouse transmembrane and intracellular domain instead of the full mouse protein. Intact protein was determined by binding to human TNFR1-Fc in an ELISA. The graph shows the mean ± SD of five mice.

Detailed Description of the Invention

The use of the singular and the like used in the context of the description of the invention (especially in the context of the claims below) covers both the singular form and

- 8 044106 and plural, until otherwise stated herein or clearly inconsistent with the context.

The terms containing, having, including and containing are to be construed as open terms (i.e., meaning including without limitation) unless otherwise indicated. For purposes of the present invention, the term consisting of is considered the preferred embodiment of the term including. If a group is defined hereinafter to include at least a certain number of embodiments, this is also intended to include the group, which preferably consists only of those embodiments.

The term antibody in this context refers to polypeptides or proteins that consist of or include antibody domains, which are understood as constant and/or variable domains of immunoglobulin heavy and/or light chains with or without a linker sequence. Polypeptides are defined as antibody domains if they include a beta-barrel structure consisting of at least two beta strands of the antibody domain structure connected by a loop sequence.

Antibody domains may be of native structure or modified by mutagenesis or derivatization, for example, to modify antigen-binding properties or any other properties such as stability or functional properties such as binding to Fc receptors FcRn and/or Fcgamma receptor.

An antibody used herein includes at least one antigen binding site that specifically recognizes huTNFR1 or an epitope of huTNFR1. Thus, antibody binding to the huTNFR1 receptor can occur monovalently through only one huTNFR1-specific binding site per antibody, or bivalently through two huTNFR1-specific binding sites. In particular, the antigen binding site consists of one or two antibody domains. Any of the antibody variable domains, alone or in combination, such as a single VH domain or a combination of VH and VL domains, can be used to create an antigen binding site. In particular, the antigen binding site is formed by a combination of CDR sequences. Such a combination of CDR sequences is also understood as a CDR binding site, for example, an antigen binding pocket formed by three CDR sequences of one variable domain, such as a combination of CDRH1, CDRH2 and CDRH3, or a combination of CDRL1, CDRL2 and CDRL3, or six more CDR sequences of two variable domains, such as a combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3. Alternatively, an antigen binding site that is derived from a natural ligand to the receptor or from an artificial construct can be used. The CDR sequences mentioned herein are designated as follows:

VH domain CDR:

VH-CDR1 = CDRH1,

VH-CDR2 = CDRH2,

VH-CDR3 = CDRH3, VL domain CDR:

VL-CDR1 = CDRL1,

VL-CDR2 = CDRL2,

VL-CDR3 = CDRL3.

In particular, the CDR binding site of one antibody variable domain can be used as an antigen binding site, such as a heavy and light chain variable domain binding site (such as dAb, Fd, VL, Vkappa, Vlambda, VH, VHH), or a pair binding site antibody variable domains, such as the VH/VL pair.

Thus, an antibody containing a CDR binding site may contain a single antibody variable domain or a pair of variable binding domains, and optionally further contain other variable domains with the same or different antigen binding specificities, such as a bispecific or polyspecific antibody, where only one antigen binding site targets huTNFR1. and at least one other antigen binding site targets a target other than huTNFR1. Optionally, the antibody construct further includes antibody constant domains or combinations of antibody variable and/or constant domains with or without a linker sequence or hinge region.

Specific antibody formats may be used as described herein, for example, an antibody comprising or consisting of a single antibody variable domain, such as VH, VL, or VHH, or combinations of antibody variable and/or constant domains with or without a binding sequence or hinge regions including antibody variable domain pairs such as a VL/VH pair, an antibody containing or consisting of a VL/VH domain pair and antibody constant domains such as heavy chain antibodies, Fab, F(ab'), (Fab) ₂ , scFv, Fd, Fv or a full-length antibody, for example an IgG type (for example, an IgG1, IgG2, IgG3 or IgG4 subtype), an IgA1, IgA2, IgD, IgE or IgM antibody. The term full-length antibody can be used to refer to any antibody molecule containing at least a majority of the Fc domain and other domains typically found in a naturally occurring antibody monomer. This phrase is used herein to emphasize that a particular antibody molecule is not an antibody fragment.

Examples of monovalent monospecific binders are Fab, scFv, Fv, domain antibodies, IgG half-antibodies or monovalent IgG such as single-arm IgG consisting of a complete light chain, one complete heavy chain and an additional Fc chain lacking Fd (Fd = VHCH1 ), which can be prepared according to the protrusion-to-recess technique (or other asymmetric Fc parts) to avoid homodimerization of the Fc domains.

Typical di- or multivalent binders are full-length antibodies of any of the immunoglobulin types or an antigen-binding antibody fragment of any of the full-length antibodies that includes at least two antigen-binding sites, for example, any one or more Fab, F(ab'), (Fab)2, scFv or Fv.

The term Fv is understood herein to mean a region of variable domains that includes a CDR binding site, for example, VH, VL or VH/VL. The term Fv thus particularly refers to VH, VL or VH/VL, which is a VH domain linked to a VL domain by interaction between the beta sheet structure of both variable domains, with or without a linker.

As used herein, the term antibody specifically includes antibodies in isolated form in an antibody preparation that are substantially free of other antibodies directed against other target antigens or containing a different structural arrangement of antibody domains. However, the isolated antibody may be contained in a combination preparation containing a combination of the isolated antibody, for example, with at least one other antibody, such as monoclonal antibodies or antibody fragments having other specificities.

The term antibody shall apply to antibodies of animal origin, including human antibodies, such as antibodies from mammals such as human or mouse, or antibodies from birds such as chicken, and the term shall in particular include recombinant antibodies based on a sequence of animal origin, for example, human sequences like human antibodies. Human antibodies typically contain variable and constant regions derived from human germline immunoglobulin sequences. Preferably, the human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDR. Human antibodies include antibodies isolated from libraries of human immunoglobulins or animals transgenic for one or more human immunoglobulins.

However, the term antibody is further applied to chimeric antibodies with sequences originating from different species, for example, sequences of murine and human origin, or to humanized antibodies, which contain amino acid sequences of human origin and non-human amino acid sequences, for example, from rodents

The term antibody specifically refers to antibodies of any class or subclass. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be classified into the main antibody classes IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

The term further applies to monoclonal or polyclonal antibodies, in particular to recombinant antibody, which includes all antibodies and antibody structures that are produced, expressed, created or isolated by recombinant methods, such as antibodies derived from animals, for example mammals, including humans, which includes genes or sequences of various origins, such as murine, chimeric, humanized, or hybridoma-derived antibodies. Additional examples include antibodies isolated from a host cell transformed to express an antibody, or antibodies isolated from a recombinant combinatorial library of antibodies or antibody domains, or antibodies produced, expressed, generated or isolated by any other means that involve splicing of sequences antibody gene to other DNA sequences.

Antibody domains may have a native structure or be modified by mutagenesis or derivatization, for example, to modify antigen-binding properties or any other properties such as stability or functional properties such as binding to Fc receptors FcRn and/or Fcgamma receptor (FCGR).

Specific examples relate to non-naturally occurring antibodies that are artificial constructs engineered to specifically recognize a target huTNFR1 by at least one antigen binding site that includes one or more artificial CDR sequences, or engineered to produce non-naturally occurring antibody constructs. which have a structure different from any naturally occurring immunoglobulin structure.

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Specific examples of the antibodies described below do not occur in nature, for example, as provided in a combination formulation with another antibody or active agent, the combination of which does not occur in nature. Specific additional examples relate to an artificial derivative or variant of a naturally occurring antibody, or an optimized or affinity matured variant of a naturally occurring antibody, or an antibody with a framework region that is designed to improve the stability of the antibody. Through such optimization or design, the antibody includes one or more synthetic structures or sequences or characteristics that are not found in the context of the antibody in nature.

It is understood that the term antibody as used herein also refers to antibody derivatives, in particular, functionally active derivatives, also referred to herein as functional antibody variants.

Functionally active derivatives, in particular, are obtained by fusion or covalent chemical modification that does not change the primary amino acid sequence of the antibody itself. Derivatives may, for example, have the desired properties, including, for example, prolonging the half-life from the bloodstream, increasing stability, decreasing clearance, or changing immunogenicity. By derivative of a specific antibody is meant any combination of one or more antibody domains or antibodies and/or fusion protein, where any antibody domain can be fused at any position to one or more other proteins, such as other antibodies, for example, a binding structure including CDR loops, receptor polypeptide, as well as ligands, scaffold proteins, enzymes, toxins, etc. An antibody derivative can be produced by association or coupling with other substances by various chemical methods such as covalent coupling, electrostatic interaction, disulfide coupling, etc. Other substances associated with the antibody may be lipids, carbohydrates, nucleic acids, organic and inorganic molecules, or any combination thereof (eg, PEGs, prodrugs, or drugs). In a specific embodiment, the antibody is a derivative containing a drug, for example, to produce an antibody-drug conjugate.

The term derivative also includes fragments, variants, analogs or homologues of antibodies, for example, with a specific glycosylation pattern, for example, produced by glycoengineering, that are functional and can serve as functional variants, for example, to bind to a specific target.

The term glycoengineered in relation to antibody sequences shall refer to glycosylation variants having modified immunogenic properties, ADCC and/or CDC as a result of glycoengineering. All antibodies contain carbohydrate structures at conserved positions in the heavy chain constant regions, with each isotype having a different set of N-linked carbohydrate structures that have different effects on protein assembly, secretion, or functional activity. IgG1 antibodies are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are embedded between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is required for the antibody to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC). Removal of the Nglycan in N297, for example by mutating N297 to, for example, A or T299, typically results in aglycosylated antibodies with reduced ADCC.

Significant differences in antibody glycosylation occur between cell lines, and even minor differences are observed for a given cell line grown under different culture conditions. Expression in bacterial cells is usually achieved by an aglycosylated antibody.

Antibodies may lack an active Fc fragment and thus either consist of antibody domains that do not have an FCGR binding site, particularly including any antibody lacking a chain of CH2 and CH3 domains, or contain antibody domains that lack Fc effector function, such as modifications to reduce the effector functions of Fc, in particular to cancel or reduce the activity of ADCC and/or CDC. Such modifications may be caused by mutagenesis, for example, mutations in the FCGR binding site or derivatives or agents that affect the ADCC and/or CDC activity of the antibody, to achieve a decrease in Fc effector function or absence of Fc effector function, which is generally understood to refer to Fc effector function is less than 10% of the unmodified antibody (wild type), preferably less than 5%, as measured by ADCC and/or CDC activity.

Exemplary antibodies may contain an Fc fragment or at least a portion of an Fc fragment, for example, to maintain a binding site for FcRn, thereby producing an antibody with a significant half-life in vivo.

However, the Fc can be modified to produce a reduction in possible ADCC and/or CDC activity, for example by switching IgG1 to the IgG2 subtype or modifications to reduce binding to the Fc receptor, for example E233P and/or L234V and/or L235A, and/or D265G, and/or

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A327Q, and/or ADZOA, and/or G236, deletion and/or A327G, and/or A330S in human IgG1 Fc, where numbering corresponds to Kabat [EU index].

Additional examples relate to modification to reduce immunogenicity, for example by knocking out a glycosylation site such as N297Q, which provides impaired binding to the lectin receptor.

It is understood that the term antibody also refers to antibody variants, including antibodies with functionally active CDR variants of the parent CDR sequence and functionally active antibody variants of the parent antibody. For example, functional variants of those antibodies characterized by the binding CDR sequences and/or heavy and light chain sequences provided herein can be constructed and used as described later herein.

In particular, an antibody variant of a parent antibody may be produced by engineering at least one of the antibody sequences of the parent antibody, such as any of the antibody examples provided herein, for example, wherein the antibody variant includes at least 3 heavy chain variable region CDRs and, optionally, additionally at least 3 light chain variable region CDRs with at least one point mutation in at least one of the CDRs in either the FR regions or the heavy chain (HC) or light chain (LC) constant region , and still retains functional activity, as measured by specific binding to the huTNFR1 target.

In particular, an antibody variant is a mutant antibody or antibody fragment, e.g., produced by mutagenesis techniques, particularly to remove, exchange, introduce insertions into a specific amino acid sequence or region of the antibody, or to chemically derivatize an amino acid sequence, e.g., into constant domains to form stability, effector function or half-life of the antibody or in variable domains to improve antigen-binding properties, for example, using affinity maturation techniques available in the art. Any of the known mutagenesis methods can be used, including point mutations at the desired positions, for example, obtained by randomization methods. In some cases, positions are chosen randomly, for example with any of the possible amino acids or by selecting preferred amino acids to randomize antibody sequences. The term mutagenesis refers to any method recognized in the art for changing a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include PCR-based error-injection mutagenesis, saturation mutagenesis, or other site-directed mutagenesis.

A point mutation is specifically understood as the creation of a polynucleotide that results in the expression of an amino acid sequence that differs from the amino acid sequence that has not been subjected to the design, substitution or exchange, deletion or insertion of one or more single (non-consecutive) or pairs of amino acids with respect to different amino acids.

Specifically, a functionally active variant antibody is prepared by modifying a parent antibody or a sequence of a parent antibody by any one or more of insertion, deletion, or substitution of one or more amino acids, or chemical derivatization of one or more amino acid residues in an amino acid sequence or nucleotides in a nucleotide sequence, or at one or both distal ends of the sequence, for example, the N-terminal and/or C-terminal 1, 2, 3 or 4 amino acids of the CDR sequence and/or the central 1, 2, 3 or 4 amino acids (i.e. in the middle of the CDR sequence) , and the modification does not affect, in particular, does not impair the activity of this sequence. In the case where the binding site is specific for a selected target antigen, the functionally active antibody variant will still have a predetermined binding specificity or substantially the same biological activity, although this can be changed, for example, to change fine specificity for a particular epitope, affinity , avidity, Kon or Koff speed, etc. For example, an affinity mature antibody is specifically understood to be a functionally active variant of the antibody. Therefore, the modified CDR sequence in an affinity mature antibody is understood to be a functionally active variant of the CDR.

Affinity maturation is the process by which antibodies are produced with increased affinity for a target antigen. Any one or more methods for preparing and/or using affinity maturation libraries available in the art can be used to produce affinity matured antibodies in accordance with various embodiments of the invention disclosed herein. Examples of such methods and applications of affinity maturation include non-specific mutagenesis, passage of bacterial mutator strains, site-directed mutagenesis, hot spot targeting of mutagenesis, thrifty mutagenesis, antibody shuffling, light chain shuffling, heavy chain shuffling, CDR1 and/or CDR1 mutagenesis, and Methods for preparing and using affinity maturation libraries suitable for implementing the methods and uses in accordance with various embodiments of the invention disclosed herein include, for example, those disclosed in: Wark & Hudson, 2006, Advanced Drug Delivery Reviews 58: 657-670.

By structural changes to the antibody, including amino acid mutagenesis or resulting somatic mutation in immunoglobulin gene segments, variants of the antigen binding site are created and selected for greater affinity. Affinity-mature antibodies can exhibit several times greater affinity than the parent antibody. Single parent antibodies can undergo affinity maturation. Alternatively, pools of antibodies with similar binding affinities to the target antigen can be considered as parent structures that are varied to produce affinity-mature individual antibodies or affinity-mature pools of such antibodies.

A preferred embodiment of the affinity mature antibody described herein exhibits at least a 2-fold increase in binding affinity, preferably at least 5-fold, preferably at least 10-fold, preferably at least , 50x or preferably at least 100x magnification. Affinity maturation can be used in selection campaigns using appropriate libraries of parent molecules, with antibodies having intermediate binding affinity, to produce the antibody described herein. Alternatively, the affinity can be further increased by affinity maturation of the antibody described herein to obtain high KD values of less than 10 ^-10 M or even less than 10 ^-11 M.

In some embodiments, binding affinity is determined using an affinity ELISA assay. In some embodiments, binding affinity is determined using BIAcore, ForteBio, or MSD assays. In some embodiments, binding affinity is determined kinetically. In some embodiments, binding affinity is determined by an equilibrium/dissolution method. In some embodiments, binding affinity is determined using standard piezoelectric microbalancing (QCM) measurements, particularly under specified conditions that resemble physiological conditions (about 37° C., density about 50 Hz).

The specific function of the antibodies described herein is as an inhibitor (also called antagonist) of the TNF-huTNFR1 interaction. The term inhibitor, as used herein, is a substance that has the ability

a) modulate (eg, reduce or eliminate) TNFR1 signaling in vitro and/or in vivo, and/or

b) inhibit TNFR1-mediated cell death in vitro and/or in vivo, and/or

c) inhibit TNF-mediated cellular stimulation for the release of inflammatory cytokines in vitro and/or in vivo,

d) by inhibiting TNFR1 signaling through another mechanism.

In particular, the antibody, as used herein, prevents the binding of one or more TNF molecules to one or more TNFR1 molecules on the cell surface. For therapeutic use, without wishing to be bound by theory, it is believed that inhibitors of the TNF-huTNFR1 interaction may have the ability to inhibit huTNFR1 signaling in the presence of TNF or huTNFR1-mediated cell death in the presence of TNF or inhibit cellular stimulation to release inflammatory cytokines in the presence of TNF.

As used herein, signaling pathway activation and signaling is a biochemical process involving the transmission of extracellular stimuli through cell surface receptors through a specific and sequential series of molecules to genes in the nucleus, resulting in specific cellular responses to stimuli.

Inhibition of huTNFR1 interaction may result in down-modulation of the effects of TNFR1 signaling or signal transduction measured ex vivo in a cell-based assay or in vivo, depending on the dose. The functional activity of the antibody described herein is specifically characterized by an inhibitory function that inhibits the TNFhuTNFR1 interaction or the LTa-huTNFR1 interaction in vivo, as determined in an ex vivo cell-based assay. Additional analysis can be used to rule out significant adverse effects associated with TNFR1 receptor cross-linking, which may act agonistically on the TNF-TNFR1 interaction. A suitable assay is to determine the activity of the antibody or variant on HeLa or HT1080 cells by the absence of stimulatory activity for the production of the inflammatory cytokines IL-6 or IL-8, respectively. An example test is described in the examples section below.

Inhibition typically results in a reduction in the effects of huTNFR1 interaction or activity by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or to the maximum level. The methods for producing and characterizing antibodies described herein are well known in the art. In a preferred embodiment, antibody variants are generated and screened for predefined properties using one or more cellular assays using cells expressing huTNFRl or in vivo assays. For such assays, the antibody is typically added exogenously so that cells can be bound, for example, in the presence and absence of TNF to determine antagonistic and agonistic activity. These tests are usually based on immunoglobulin function; that is, the ability of an antibody to bind to huTNFR1 and mediate some biochemical event, for example blocking TNF binding to specified cells, for example, in a competitive binding assay, inhibiting TNF/receptor binding, reducing the expression of cytokines in the presence or absence of TNF, in particular inflammatory interleukins such such as IL-6 or IL-8, apoptosis, etc.

The antibody described herein preferably has only antagonistic activity against TNF (in particular, no detectable agonistic activity), thereby reducing the inflammatory response caused by increased levels of TNF in the circulation, which can lead to unwanted inflammatory reactions, apoptosis and necrosis or organ insufficiency. The preferred antibody has antagonistic activity corresponding to an IC ₅₀ of less than 100 nM, preferably less than 20 nM, more preferably less than 10 nM, most preferably in the single digit nanomolar range or less, as measured in a cell-based assay using TNF or LT-alpha with half-maximal saturation concentration, preferably in the range of 0.01-0.1 nM TNF and LT-alpha, respectively.

The potential agonist activity of a TNF mimetic can be measured in the same cell-based assay, but without the use of TNF. The antibody described herein preferably does not have significant agonist activity if incubation of HeLa or HT1080 cells in the absence of TNF results in no or only minor induction of the cytokine, e.g., elevated levels of IL-6 or IL-8 less than 0.5 ng/ml at concentration at least 5 nM or about 10 nM antibody. Preferably there is no or only slight or negative cytokine production, which can be measured as less than 10 pg/10 ⁵ cells. In a preferred example, cytokine expression and release is less than 2.5 pg/100,000 cells over 18 hours. Preferably, the agonist activity is thus below the baseline level or less than 2% of the response of a comparable TNF concentration, preferably less than 1% of the equivalent or maximum TNF response.

Percentage (%) amino acid sequence identity with respect to the antibody sequences described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular polypeptide sequence after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage sequence identity without considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

As used herein, the term antigen is interchangeable with the terms target or target antigen and refers to the entire target molecule or fragment of such a molecule recognized by the antibody binding site. In particular, antigen substructures, eg polypeptide or carbohydrate structure, commonly referred to as epitopes, eg viral epitopes or T-cell epitope, which are immunologically relevant, can be recognized by such a binding site. The huTNFR1 antigen is an antigen comprising receptor structures that is capable of specifically binding trimeric TNF or LTa as a mono- or multimeric cytokine receptor on the surface of most human cells.

As used herein, the term huTNFR1 refers to the TNF receptor, CD120a TNFR1 (p55/60, tumor necrosis factor receptor superfamily TNFRSF1A, member 1A [Homo sapiens (human)], Gene ID: 7132), expressed ubiquitously in most human cells. The specific exemplary sequence of huTNFR1 is provided as SEQ ID NO: 32.

As used herein, the term epitope specifically refers to a molecular structure that may entirely constitute a specific binding partner or be part of a specific binding partner at the binding site of an antibody. The epitope may consist of a carbohydrate, a peptide structure, a fatty acid, an organic, biochemical or inorganic substance, or derivatives thereof, and any combination thereof. If the epitope is included in a peptide structure, such as a peptide, polypeptide or protein, it will typically comprise at least 3 amino acids, preferably 5 to 40 amino acids, and more preferably 10 to 10 amino acids. Epitopes can be linear or conformational. A linear epitope consists of a single segment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes may be contiguous or overlapping.

Conformational epitopes consist of amino acids or carbohydrates joined together by folding the polypeptide to form a tertiary structure, and the amino acids are not necessarily adjacent to each other in a linear sequence.

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In particular, and with respect to polypeptide antigens, a conformational or discontinuous epitope is characterized by the presence of two or more distinct amino acid residues separated in primary sequence but assembled into a coherent structure on the surface of the molecule when the polypeptide is folded into the native protein/antigen.

As used herein, the term epitope will specifically refer to an epitope contained in huTNFR1, which is an epitope included in the membrane-distal CRD1 and the A1 subdomain of CDR2 in huTNFR1.

The term isolated or isolated as used herein in relation to a nucleic acid, antibody or other compound shall refer to such compound that has been sufficiently separated from the environment with which it would be naturally associated to exist in substantially pure form. Isolated does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not affect the fundamental activity and which may be present, for example, due to incomplete purification.

With respect to polypeptides or proteins, such as the isolated antibodies described herein, the term isolated will specifically refer to compounds that contain little or no material with which they are naturally associated, such as other compounds with which they occur in their natural environment or the environment in which they are obtained (for example, cell culture), when such production uses recombinant DNA technology practiced in vitro or in vivo. The isolated compounds may be formulated with diluents or adjuvants and may still be isolated for practical purposes - for example, the polypeptides or polynucleotides may be admixed with pharmaceutically acceptable carriers or excipients for diagnostic or therapeutic use.

As used herein, the term recombinant means genetically engineered or derived from genetic engineering. A recombinant host particularly includes an expression expression vector or a cloning vector, or the recombinant host has been genetically engineered to contain a recombinant nucleic acid sequence, particularly using a nucleotide sequence foreign to the host. The recombinant protein is produced by expressing the corresponding recombinant nucleic acid in the host.

An antibody further described herein may be a recombinant antibody. For this purpose, the term recombinant antibody as used herein includes antibodies that are produced, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas derived from them, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome, (c) antibodies isolated from a recombinant, combinatorial library of human antibodies or a library of antigen-binding antibody sequences, and (d) antibodies, obtained, expressed, created or isolated by any other means that involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies include antibodies engineered to incorporate rearrangements and mutations that occur, for example, during antibody maturation. In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques known to those skilled in the art may be used. Such methods are fully described in the literature. See, for example, Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982).

The antibody described herein is preferably provided as a recombinant protein produced by a recombinant expression system using a host cell, for example, by expression in the periplasmic space of E. coli or by expression as a secreted protein in a eukaryotic expression system, such as yeast or mammals, for example, CHO, HEK or in human host cell production lines.

Chinese hamster ovary (CHO) cells are most commonly used to produce antibodies. In addition to providing suitable glycosylation patterns, these cells enable the consistent generation of genetically stable, high-yielding clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media and enable the development of safe and reproducible biological processes.

Host cells are most preferred when they are generated, adapted and fully cultured under serum-free conditions and, optionally, in media free of any animal protein/peptide.

Specific binding, recognition or targeting as used herein means that a binder, such as an antibody or an antigen binding site of an antibody, exhibits detectable affinity for a target antigen or corresponding epitope in a heterogeneous population of molecules. Thus, under certain conditions (for example, in an immunoassay), the binder binds specifically to the target antigen and does not bind in significant amounts to other molecules present in the sample. Specific binding means that the binding is selective in terms of target identification, with high, medium or low binding affinity or avidity, depending on the choice. Selective binding is typically achieved if the binding constant or binding dynamics is at least 10-fold different (defined as a difference of at least 1 log), preferably different by at least 100-fold (defined as a difference at least 2 log) and more preferably different by at least 1000 times (defined as a difference of at least 3 log) compared to the other target. Differential binding can be determined by immunoassay, preferably immunoblotting, ELISA or other immunological methods. The specificity of an antibody molecule for a particular target can be determined using competition assays, for example, as described in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988). Selective binding can be developed or improved by recombinant antibody optimization techniques known in the art. For example, certain regions of the immunoglobulin chain variable regions described herein may be subject to one or more optimization strategies, including light chain shuffling, site-directed mutagenesis, CDR fusion, and site-directed mutagenesis of selected CDR regions and/or framework regions.

The use of the term having the same specificity, having the same binding site, or binding the same epitope indicates that equivalent monoclonal antibodies exhibit the same or substantially the same, i.e., similar immunoreactive (binding) characteristics and compete for binding to a preselected sequence of the target to be bound. The relative specificity of an antibody molecule for a particular target can be relatively determined by competition assays, for example, as described in Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

Competition as used herein means relative inhibition greater than about 30% as determined by a competitive ELISA assay or a ForteBio assay. It may be appropriate to establish a higher relative inhibition threshold as a criterion for what is an appropriate level of competition in a particular context, for example, when a competition assay is used to select or screen new antibodies developed with a putative antigen-binding function. Thus, for example, it is possible to establish criteria for competitive binding that exhibit a relative inhibition of at least 40%, or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even at least 100% before the antibody is considered sufficiently competitive.

As used herein, the term patient refers to humans and other mammals. In particular, the medical application described herein or the corresponding treatment method applies to a subject in need of prevention or treatment of NASH, or a disease associated with NASH. The subject may be a patient at risk of developing or suffering from NASH, including early or late stage disease. The term patient specifically includes subjects who are receiving prophylactic and/or therapeutic treatment. Thus, the term treatment includes both preventive and therapeutic treatment.

In particular, the term therapy refers to therapeutic measures that are intended to be administered to treat a disease or reduce the symptoms of a disease.

In particular, the term prophylaxis refers to preventive measures that are intended to reduce the risk of disease occurrence or disease recurrence.

The term therapeutically effective amount is used interchangeably herein with any of the terms effective amount or sufficient amount of a compound, for example, an antibody described herein is an amount or activity sufficient to, when administered to a subject, produce beneficial or desired results, including clinical results and, as such, its effective amount or synonym depends on the context in which it is used.

By effective amount is meant an amount of a compound that is sufficient to treat, prevent, or inhibit such diseases or disorders. In the context of a disease, therapeutically effective amounts of an antibody described herein are specifically used to treat, modulate, attenuate, reverse, or affect a disease or condition that benefits from inhibition of the TNF-TNFR1 interaction.

The amount of compound that will correspond to such an effective amount will vary depending on various factors, such as the drug or compound, the pharmaceutical composition, the route of administration, the type of disease or disorder, the characteristics of the subject or host being treated, and the like. ., but can nevertheless be determined by one of ordinary skill in the art.

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As further described herein, a method of treating a patient includes the step of administering a therapeutically effective amount of a huTNFR1 antibody as defined above to a patient in need thereof. A therapeutically effective amount is usually in the range of 0.5-500 mg, preferably 1-400 mg, even more preferably up to 300 mg, up to 200 mg, up to 100 mg or up to 10 mg, although higher doses may be indicated, e.g. patients with obesity or acute disease states.

A preferred pharmaceutical composition described herein comprises a therapeutically effective amount of a huTNFR1 antibody and optionally one or more additional components selected from the group consisting of a pharmaceutically acceptable carrier, pharmaceutically acceptable salts, an auxiliary agent, a stabilizer, a diluent and a solvent, or any combination thereof .

In one embodiment, the antibody described herein is the only therapeutically active agent administered to the patient. Alternatively, an antibody described herein is administered in combination with one or more other therapeutic agents, including, but not limited to, TNF antagonists, anti-inflammatory agents, cytokines, growth factors, or other therapeutic agents. An anti-TNFR1 antagonist antibody may be administered simultaneously or sequentially with one or more other treatment regimens, preferably an anti-TNF therapy such as an anti-TNF antibody. The antibody described herein is preferably administered to a patient as first-line treatment or as second-line therapy when anti-TNF therapy is ineffective, either acute or long-term treatment. A particularly preferred medical use is the treatment of a chronic disease.

In addition, a regimen for treating or preventing a subject with a therapeutically effective amount of an antibody described herein may consist of a single administration or, alternatively, include a series of applications. For example, the antibody can be administered at least once a year, at least once every six months, or at least once a month. However, in another embodiment, the antibody may be administered to a subject from about once a week to about daily for a given treatment. The length of the treatment period depends on many factors, such as the severity of the disease, acute or chronic disease, the age of the patient, and the concentration and activity of the antibody form. It should also be understood that the effective dosage used for treatment or prophylaxis may be increased or decreased during a particular treatment or prophylactic regimen. Changes in dosages may be effective and become apparent using standard diagnostic tests known in the art. In some cases, long-term administration may be required.

Non-alcoholic steatohepatitis (NASH) is a non-alcohol-related liver disease characterized by fatty degeneration of hepatocytes accompanied by intralobular inflammation and fibrosis. NASH is a common, often silent liver disease. This disease resembles alcoholic liver disease, but occurs in people who drink little or no alcohol. Three main features characterize NASH and distinguish it from other liver diseases of metabolic origin: abnormal accumulation of fat or deposition in the liver (hepatic steatosis), liver inflammation, and liver damage or damage to liver tissue (fibrosis).

NASH is a potentially serious disease that carries a significant risk of progression to end-stage liver disease, cirrhosis, and hepatocellular carcinoma. Some patients who develop cirrhosis are at risk for liver failure and may eventually require a liver transplant. NAFLD can be distinguished from NASH by the NAFLD Activity Score (NAS), liver biopsy histopathology score for steatosis (0 to 3), lobular inflammation (0 to 2), and hepatocellular ballooning (0 to 2). NAS <3 corresponds to NAFLD, 3-4 corresponds to borderline NASH, and >5 corresponds to NASH. The biopsy is also scored for fibrosis (0 to 4).

As used herein, a patient suffering from NASH is a patient with NASH, or has been diagnosed with NASH, or the patient is genetically predisposed to developing NASH, or the patient may be predisposed to developing NASH because he or she suffers from metabolic syndrome. obesity, diabetes or prediabetes. In other embodiments, a patient suffering from NASH is a patient who has been tested and found to have clinical signs consistent with NASH (abnormal fat accumulation in the liver, liver inflammation, and liver fibrosis), even though he or she may not yet exhibit any physical symptoms of NASH. In some cases, a patient suffering from NASH will develop symptoms of NASH even though a diagnosis has not yet been made.

Treatment of NASH may slow or stop the progression of NASH to cirrhosis. Some treatment regimens aim to delay the onset of a physical symptom or set of physical symptoms or clinical manifestations or signs associated with NASH. In some embodiments, the treatment results in improvement in at least one measurable physical symptom

- 17 044106 NASH symptoms, such as, for example, weight loss, weakness or fatigue. In other embodiments, treatment results in improvement in at least one clinical parameter or sign of NASH, such as, for example, abnormal liver fat accumulation, liver fibrosis as measured by biopsy, liver inflammation, abnormal liver enzyme levels (eg, ALT) inflammatory cytokine levels or NAS score. In other embodiments, the treatment results in the reduction, inhibition, or slowing of progression of NASH, either physically, for example, by stabilizing a measurable symptom or set of symptoms (such as fatigue, weight loss, or weakness), or clinically/physiologically, for example, by stabilizing a measurable parameter such such as abnormal fat accumulation in the liver, abnormal liver enzyme levels, abnormal levels of liver inflammatory markers, abnormal liver biopsy results, NAS score, or both. In another embodiment, the treatment may also result in preventing the cause and/or consequences or clinical manifestation of NASH or one of the symptoms resulting from NASH before the disease or disorder has fully manifested itself. In some embodiments, the treatment results in an increase in survival or survival time in a patient with NASH. In some embodiments, the treatment results in a reduction in the likelihood of a patient with NASH needing a liver transplant. In other embodiments, the treatment eliminates the need for the NASH patient to undergo a liver transplant. In other embodiments, the treatment results in a reduction in the likelihood of a patient with NASH developing cirrhosis. In other embodiments, the treatment results in the prevention of progression of liver cirrhosis as determined by histology.

Specific embodiments described herein relate to monoclonal antibodies (mAbs). Monoclonal antibodies are produced by cloning antibody genes into a monoclonal host cell or appropriate cell lines. Monoclonal antibodies can be produced by any method that produces antibody molecules from cell lines in culture, such as culturing recombinant eukaryotic (mammalian or insect) or prokaryotic (bacterial) host cells. Examples of suitable methods for producing monoclonal antibodies include the hybridoma methods of Kohler & Milstein (1975, Nature 256: 495-497) and the human B cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and Brodeur et al, 1987, Monoclonal Antibody Production Techniques and Applications, (Marcel Dekker, Inc., New York), pp. 51-63).

The antibodies described herein can be identified or obtained using the hybridoma method. In such a method, a mouse or other suitable host animal, such as a hamster, is immunized to recover lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with the myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell.

The culture medium in which hybridoma cells grow is analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

Monoclonal antibodies can then be purified from hybridoma supernatants for further testing for their specific binding to a target antigen and for antibody engineering, for example, for various diagnostic or therapeutic purposes.

huTNFR1-specific antibodies, in some cases, are generated by screening against the huTNFR1 antigen. To increase the likelihood of isolating differentially binding clones, multiple selection pressures should be applied through processive screening against different antigens or epitopes.

Screening methods for identifying antibodies with the desired selective binding properties can be implemented using display technologies using a library presenting antibody sequences or antigen-binding sequences (for example, using phages, bacterial, yeast or mammalian cells; or in vitro display systems that translate information of nucleic acids into corresponding (poly)peptides). Reactivity can be assessed based on ELISA, Western blotting or surface staining by flow cytometry, for example using standard assays.

The isolated antigen(s) can, for example, be used to select antibodies from a library of antibodies, for example, a library of antibodies presented by phages, phagemids or yeast.

Specific embodiments described herein relate to pharmaceutical compositions, such compositions may include an antibody described herein and a pharmaceutically acceptable carrier and/or excipient, particularly for the preparation of an artificial, non-naturally occurring composition. These pharmaceutical compositions can be administered in accordance with the present invention as a bolus injection or infusion or by continuous infusion. Pharmaceutical carriers suitable for facilitating such routes of administration are well known in the art.

- 18 044106 of this area.

Pharmaceutically acceptable carriers generally include any suitable solvents, dispersion media, coatings, antibacterial and fungicidal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible with the antibody or related composition or combination described herein. Additional examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and combinations of any of these.

In one such aspect, the antibody can be combined with one or more carriers suitable for the desired route of administration, the antibodies can be, for example, mixed with any of lactose, sucrose, starch, cellulose alkanoic acid esters, stearic acid, talc, stearate magnesium, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia gum, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinyl alcohol and optionally, additionally tableted or encapsulated for routine administration. Alternatively, the antibody can be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethylcellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, gum tragacanth and/or various buffers. The carrier may include a controlled release material or a time delayed release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, or other materials well known in the art.

Additional pharmaceutically acceptable carriers are known in the art and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES. Liquid formulations may be solutions, emulsions or suspensions and may include excipients such as suspending agents, solubilizers, surfactants, preservatives and chelating agents.

Pharmaceutical compositions are contemplated that include an antibody described herein and one or more therapeutically active agents. The stable antibody formulations described herein are prepared for storage by mixing said antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions. Formulations used for in vivo administration are sterile. This is easily achieved by filtration through sterile filter membranes or other methods. The antibody and other therapeutically active agents disclosed herein may also be formulated as immunoliposomes and/or microencapsulated.

Administration of a pharmaceutical composition containing an antibody described herein may be administered by a variety of routes, including orally, subcutaneously, intravenously, intranasally, intraotically, transdermally, transmucosally, topically, such as gels, ointments, lotions, creams, etc., intraperitoneal, intramuscular, intrapulmonary, for example, using inhalation technologies or pulmonary delivery systems, vaginal, parenteral, rectal or intraocular.

Examples of formulations used for parenteral administration include those suitable for subcutaneous, intramuscular or intravenous injection, such as, for example, a sterile solution, emulsion or suspension.

In one embodiment, an antibody described herein is the only therapeutically active agent administered to a subject, for example, as a disease modifying or preventing monotherapy.

In another embodiment, an antibody described herein is combined with other active agents, for example, in a mixture or set of parts to treat a subject in need of therapy or prevention, such as a disease-modifying or preventive combination therapy.

Combination with one or more other therapeutic or prophylactic agents may include standard treatments, such as antibiotics, steroidal and non-steroidal inflammatory inhibitors, and/or other antibody-based therapies. The combination may particularly include agents that are used to treat a primary disease where inflammatory processes may lead to secondary inflammatory, degenerative or malignant diseases. The underlying disease is, for example, NASH and the combination will, for example, include an NSAID or other new drugs such as FXR agonists, GLP1R agonists or PPAR agonists.

In combination therapy, the antibody may be administered as a mixture or concurrently with one or more other treatment regimens, for example, either before, concurrently, or after concomitant therapy.

The biological properties of an antibody or corresponding pharmaceutical described herein can be characterized ex vivo in experiments on cells, tissues and the whole body. As is known in the art, drugs are often tested in vivo in animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs and monkeys, to measure the effectiveness of the drug in treating a disease or disease model, or to measure pharmacokinetics.

- 19 044106 tics, pharmacodynamics, toxicity and other properties of the drug. Animals can be called disease models. Therapeutics are often tested in mice, including, but not limited to, nude mice, SCID mice, xenograft mice, and transgenic mice (including knockouts and knockouts). Such an experiment could provide meaningful data to determine the potential of an antibody to be used as a therapeutic or prophylactic agent with appropriate half-life, effector function, inhibitor activity, and/or immune response in passive immunotherapy. Any organism can be used for testing, preferably mammals. For example, because of their genetic similarity to human primates, monkeys may be suitable therapeutic models and thus may be used to test the efficacy, toxicity, pharmacokinetics, pharmacodynamics, half-life, or other properties of a subject agent or composition. Tests on humans are ultimately necessary for approval as medicines and so, of course, such experiments are contemplated. Thus, the antibody and corresponding pharmaceutical compositions described herein can be tested in humans to determine their therapeutic or prophylactic efficacy, toxicity, immunogenicity, pharmacokinetics and/or other clinical properties.

Therefore, the invention provides a new method of treating NASH, and in particular those diseases that are associated with NASH. Surprisingly, the anti-TNFR1 antibody demonstrated significant improvements in hepatic steatosis and histological disease activity in NASH, as demonstrated in a mouse model. Inhibition of TNFR1 resulted in a significant reduction in the percentage of hepatic steatosis as well as triglyceride levels. In addition, the NAFLD activity score, which accounts for steatosis, ballooning, and lobular inflammation, was significantly reduced by anti-TNFR1 antibody treatment. It was unexpected that selective inhibition of TNFR1 showed positive dynamics of liver fibrosis in a mouse model of NAFLD and was even able to treat liver fibrosis by reducing fibrosis in liver tissue. It was further shown that treatment with anti-TNFR1 antibody was able to reduce apoptosis in liver tissue. In addition, significant reductions in ALT and serum insulin levels were achieved. Thus, treatment with anti-TNFR1 antibody resulted in significant improvements in inflammation and insulin resistance in NAFLD.

It was particularly surprising that the anti-TNFR1 antibody demonstrated significant improvements in liver steatosis and histological disease activity in NASH in light of the prior art, since the development of NASH or steatosis has been reported as a side effect of TNFi therapy. Feagins et al. reported that patients with Crohn's disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis who were treated with infliximab, adalimumab or etanercept developed abnormal ALT levels during TNFi treatment and showed NAFLD (NASH or steatosis) on liver biopsy (Eur J Gastroenterol Hepat 2015, 27(10): 11541160). Thus, it was completely unexpected that selective inhibition of TNFR1 was effective against NASH.

Examples of antibodies described herein include, for example, full-length antibodies prepared in accordance with WO2012035141 and/or their parent mouse antibody H398 described in WO2008113515A2, functionally active variants with affinity mature variants and/or antibody fragments of any of the above or those antibodies which are monovalent anti-huTNFR1 binders and contain an antigen binding site of any of the above, such as the monovalent antibodies described in WO2017174586 A1.

The present invention is further illustrated by the following examples, without limitation.

Examples

Example 1: Positive dynamics of liver steatosis and histological activity of the disease in mice with experimental NAFLD.

Methods:

B6-huTNFR1-k/i mice were fed a high fat diet (HFD) consisting of 60% kcal fat + fructose/sucrose in drinking water (Kohli R et al, Hepatology 2010) for 24 weeks, supplemented with either anti- TNFR1 (atrosab) or control antibody (cetuximab, anti-EGFR antibody, Erbitux®, ImClone Systems, Bristol-Myers Squibb und Merck KGaA) (20 mg/kg body weight, 2x/w) for a subsequent 8 weeks.

The atrosab antibody used herein is a full length antibody prepared in accordance with WO2012035141 and contains an antigen binding site included in a combination of a VH and VL domain containing six CDR sequences which are:

SEQ ID NO: 23: VH-CDR1;

SEQ ID NO: 24: VH-CDR2;

SEQ ID NO: 25: VH-CDR3;

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SEQ ID NO: 26: VL-CDR1;

SEQ ID NO: 27: VL-CDR2;

SEQ ID NO: 28: VL-CDR3.

At the end of treatment, liver tissue from differentially treated mice was compared for hepatic steatosis, NAFLD activity score, apoptosis, and fibrosis.

Percentage of steatosis as well as NAFLD activity score (NAS) according to Kleiner et al. (Hepatology 2005) assessed by a pathologist. Triglyceride content was determined in homogenized liver tissues using an enzyme test (Roche Diagnostics). Apoptosis was examined by immunohistochemistry using an antibody for activated caspase-3 (Cell Signaling). Fibrosis was determined by Sirius Red staining according to the manufacturer's protocol (Sigma Aldrich) and quantified as described (Schindelin J et al., Nat Methods 2012).

Sera were analyzed for ALT levels using a UV kinetic assay (Beckman Coulter), and insulin levels were measured using a mouse ultrasensitive insulin ELISA kit (Crystal Chem.) according to the manufacturer's instructions.

Results:

Treatment with atrosab led to significant positive changes in hepatic steatosis and histological disease activity in mice with experimental NAFLD.

Liver tissues from HFD mice treated with anti-TNFR1 antibody (atrosab) or control antibody (cetuximab) were analyzed histologically for percentage of hepatic steatosis, triglyceride content, and disease activity as assessed by the NAFLD activity score (NAS). Compared with mice treated with control antibody, TNFR1 inhibition resulted in a significant reduction in the percentage of hepatic steatosis (P < 0.05; Fig. 1A) as well as triglyceride content (P < 0.01; Fig. 1B). In addition, the NAFLD activity score (NAS), which accounts for steatosis, ballooning, and lobular inflammation, was significantly (p < 0.05) decreased in Atrosab-treated compared with control mice (Figure 1C).

Example 2. Selective inhibition of TNFR1 is associated with positive dynamics of liver fibrosis in a mouse model with NAFLD.

Having demonstrated that selective inhibition of TNFR1 demonstrates positive dynamics of hepatic steatosis and NAS, the effect of atrosab on the reduction of fibrosis was analyzed. Positive dynamics of liver fibrosis were demonstrated when assessed by Sirius red staining in mice treated with anti-TNFR1 antibody compared with mice treated with control antibody (Fig. 2A). Quantification of fibrotic area revealed a significant (p < 0.05) reduction in fibrosis in liver tissues in mice treated with anti-TNFR1 antibody compared with control mice (Fig. 2B).

Example 3 Reduced apoptosis in liver tissues of NAFLD mice treated with anti-TNFR1 antibody.

In initial experiments, mice were fed an HFD for 20 weeks, including 4 weeks of treatment with anti-TNFR1 antibody or control antibody. It was demonstrated that treatment with anti-TNFR1 antibody was able to reduce apoptosis, as assessed by immunohistochemical analysis of active caspase-3 as early as a 4-week treatment period. Compared with control antibody, a significant (p < 0.05) decrease in active caspase-3 could be observed in the liver tissues of mice treated with anti-TNFR1 antibody (Figure 3).

Example 4: Positive dynamics of ALT and insulin levels in the serum of NAFLD mice treated with anti-TNFR1 antibody.

Along with the observation of positive histological dynamics in mice treated with anti-TNFR1 antibody (Figs. 1 and 2), a significant (p < 0.05) decrease in the levels of ALT (Fig. 4A) and insulin (Fig. 4B) in the serum of treated mice was demonstrated. anti-TNFR1 antibody compared to those treated with control antibody. Thus, selective anti-TNFR1 inhibition led to significant positive dynamics of inflammation and insulin resistance.

Example 5: Description of atrosimab - preparation and characterization.

Materials.

Atrosab antibody was prepared as described in Example 1. Human recombinant TNFR1Fc fusion protein was prepared as described in WO2012035141. Atrosimab (HC: SEQ ID NO: 18; LC: SEQ ID NO: 13) was prepared and purified following lentiviral transduction in CHO cells using Catalent (Catalent Pharma Solutions, Somerset, Ewing, NJ, USA). Anti-His-HRP (HIS-6 His-Probe-HRP, sc-8036) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-human IgG A (goat, polyclonal, 2010-01) was purchased from SouthernBiotech, and anti-human IgG B (goat, polyclonal, MBS571163), as well as anti-human IgG B (goat, polyclonal, MBS571678) were purchased from MyBioSource ( San Diego, California, USA). In addition, anti-human IgG antibody (Fab-specific, A 0293) and anti-human IgG antibody (Fc-specific, A 0170) were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Obtaining proteins.

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Fab 13.7 reference protein was prepared as described in WO2017174586 A1 in transiently transfected HEK 293E cells using polyethylenimine (linear, 25 kDa, Sigma-Aldrich, Taufkirchen, Germany) and purified by protein affinity chromatography strictly according to the manufacturer's recommendations (CaptureSelect ™ IgG-CH1 Affinity Matrix, 194320005, Thermo Fisher Scientific, Dreieich, Germany).

Protein characterization.

The purity and proper assembly of atrosimab was analyzed by SDS-PAGE using 4 μg of purified protein in the presence and absence of beta-mercaptoethanol as a reducing agent. Proteins in the gel were stained using Coomassie brilliant blue, and the stain was removed from the gels with water. Intact protein was analyzed by size exclusion chromatography using a Waters 2695 HPLC and a Phenomenex Yarra SEC-2000 column (300 x 7.8 mm). Standard proteins: thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), FLAG peptide (1 kDa).

Thermal stability.

The melting/aggregation temperature (Tm) of atrosimab was determined by dynamic light scattering (ZetaSizer Nano ZS, Malvern, Herrenberg, Germany) using 100 μg of purified protein in PBS. The applied temperature was increased in 1°C increments from 35 to 80°C with an equilibration time of 2 minutes before each measurement. Tm was determined by visual interpretation of the increasing signal (kilopulses per second).

Stability in plasma.

Purified atrosimab samples were diluted in human plasma to a concentration of 100 nM and incubated at 37°C for 1, 3 and 7 days. Subsequent analysis of residual binding capacity to human TNFR1 was performed by ELISA after serial dilution in 2% skim milk in PBS (2% MPBS) in steps of 1 to 3.16 (square root of 10). Control samples were incubated at 4°C in PBS for 7 days or directly frozen after dilution in human plasma.

Enzyme-linked immunosorbent assay (ELISA).

Microtiter plates were coated with 100 μl of TNFR1-Fc fusion protein (1 μg/ml in PBS) and incubated at 4°C overnight. Residual binding sites were blocked with 2% MPBS (skim milk in PBS, 200 μl per well) at room temperature for 2 hours and then washed twice with PBS. 100 μl of samples diluted in 2% MPBS were incubated at room temperature for 1 hour before the final incubation step with 100 μl of HRP-conjugated antibodies in 2% MPBS. Bound protein was determined using 100 μl of TMB substrate solution (1 mg/ml 3,3',5,5' tetramethylbenzidine [TMB], 0.006% _H2O2 in 100 mM Na-acetate buffer, pH 6 at room _temperature ), The HRP reaction was stopped by adding 50 μl of 1 M H ₂ SO ₄ and absorbance at 450 nm was measured using an Infinite microtiter plate reader (TECAN, Männedorf, Switzerland). Between each incubation step and before detection, the plates were washed twice with PBST and twice with PBS.

Affinity measurements using piezoelectric microweighing.

Real-time binding dynamics of protein-protein interactions were determined using piezoelectric microbalance measurements (Cell-200 C-Fast, Attana, Stockholm, Sweden). One of the binding partners (TNFR1-Fc) was chemically immobilized on the LNB Carboxyl Sensor chip (3623-3103, Attana, Stockholm, Sweden) according to the manufacturer's protocol at a moderate density of ~94 AGz. Binding experiments were performed with samples (analyte) diluted in PBST (PBS, 0.1% Tween 20) ranging from 128 nM to 4 nM (1:2 dilution steps) at pH 7.4 with a flow rate of 25 μl/ min at 37°C. The chip was regenerated with 25 μl of 20 mM glycine, pH 2.0. Every third measurement, the injection of running buffer was measured and subtracted from the binding curve before data analysis. Data were collected using software provided by Attana and analyzed using Attache Office Evaluation software (Attana, Stockholm, Sweden) and TraceDrawe (ridgview instruments, Vange, Sweden).

Interleukin Release Assay 2x104 HeLa or HT1080 cells per well were seeded in a 96-well microtiter plate and grown in 100 µl RPMI 1640 + 5% FCS overnight. The next day, supernatants were replaced to remove constitutively produced cytokines. Cells were incubated with serial dilutions of samples in RPMI 1640 + 5% FCS at 37°C, 5% CO2. For competition experiments, both analyte protein samples were prepared separately (either titrated or diluted to the same concentration) and then added to the plate. Unstimulated cells served as controls. After 16-20 hours, the plates were centrifuged at 500 g for 5 minutes and cell supernatants were analyzed directly by ELISA, which was performed according to the manufacturer's protocol. Supernatants were diluted in RPMI 1640 (no FCS) and antibodies were diluted in reagent diluent (0.1% BSA, 0.05% Tween 20, 20 mM TRIS, 150 mM NaCl, pH 7.5). Coated microtiter plates were blocked using 1% BSA

- 22 044106 (bovine serum albumin) in PBS, and washed, and detection and measurement were carried out as described above for ELISA. Sandwich ELISA kits for the determination of IL-6 and IL-8 in cell culture supernatant were purchased from ImmunoTools (Friesoythe, Germany).

Cytotoxicity/cell viability assay.

Cells (1 x ¹⁰⁴ per well) were seeded into 96-well microtiter plates and incubated overnight at 37°C and 5% CO2. Proteins were diluted in RPMI 1640 + 10% FCS and added to cells. For cytotoxicity assays, incubation was performed at 37°C, 5% CO2 for 24 hours, then the supernatant was discarded and 50 μl of crystal violet solution was added to the wells. The plates were then washed in ddH ₂ O for 20 times and dried. The remaining violet dye, obtained from living and adherent cells that were fixed with the methanol contained in the staining solution, was dissolved by adding 100 μL of methanol while shaking at room temperature for 10 minutes. The plates were measured using an Infinite microplate reader (Tecan, Maennedorf, Switzerland).

Pharmacokinetics.

C57BL/6J transgenic mice carrying the human TNFR-1 extracellular domain gene at a specific mouse gene locus (C57BL/6J-huTNFRSF1Aecdtm1UEG/izi, Dong et al., 2016) received an intravenous injection of 400 μg of atrosimab. Blood samples were collected at 3 min, 30 min, 1 h, 2 h, and 6 h, and after 3 and 7 days and immediately incubated on ice. The serum was separated by centrifugation (13,000 g, 4°C, 10 minutes) and stored at -20°C. The remaining serum protein was determined by binding ELISA as described above. The ELISA signal was interpolated from a freshly prepared standard binding curve of the protein being analyzed. Determined concentrations were plotted against time, and pharmacokinetic constants were obtained by analysis using the PKsolver add-in for Microsoft Excel.

Example 5.1: Biochemical characterization of Atrosimab.

A monovalent tumor necrosis factor receptor-1 (TNFR1)-specific antagonist, designated atrosimab, was prepared by fusing the variable domains of Fab 13.7 to the N-terminus of the heterodimerizing Fc module Fclk (one/kappa). This process resulted in an enlarged Fab-like monovalent molecule with the ability to interact with the neonatal Fc receptor to promote increased serum circulation (Figure 6a). The resulting drug candidate atrosimab was produced in CHO cells after several rounds of lentiviral transduction, purified by protein A affinity chromatography and sequential size exclusion chromatography (SEC) using Catalent Pharma Solutions (Catalent Pharma Solutions, Somerset, Ewing, NJ, USA).

Atrosimab showed a single peak in SEC at a retention time of 17.9 min with an interpolated molecular mass of 81 kDa and a Stokes radius rS of 3.5 nm (Figure 6b). In SDS-PAGE, two bands at 38 kDa and 43 kDa were detected under reducing conditions, as well as one band at 70 kDa under non-reducing conditions (Figure 6c). These data correspond well to the calculated molecular mass of 72 kDa (consisting of 35 kDa and 36 kDa chains) and indicate correct expression of both polypeptide chains and correct assembly of a functional heterodimeric protein. Additionally, atrosimab exhibited an aggregation point (Tm) of 64°C as determined by dynamic light scattering (DLS, Figure 6d), which is comparable to that of intact IgG molecules also analyzed by DLS (Martin et al., 2014 , Brader et al., 2015)). Finally, the binding activity of atrosimab to human TNFR1-Fc remained unchanged after incubation in human plasma for up to 7 days, indicating good stability in plasma (Fig. 6e).

Example 5.2: Atrosimab binding to TNFR1, Fcy receptors and complement protein Clq.

The interaction of atrosimab with its target receptor TNFR1 was analyzed by ELISA under equilibrium conditions, resulting in an EC ₅₀ value of 0.4 nM (Fig. 7a), representing a twofold reduction in binding activity compared to parental Fab 13.7 and a fourfold reduction compared with divalent IgG ATROSAB. Additionally, atrosimab bound in a real-time binding study using piezoelectric microbalancing to human TNFR1 immobilized at a moderate receptor density of ~94 AGz, with an apparent KD value of 2.7 nM and kon of 3.7x105 M ^-1 s ^-1 and koff 9 .8 x 10' ⁴ s ^-1 (Fig. 7b), which was analyzed using a 1:1 coupling algorithm. It should be noted that atrosimab includes an Fc region that is modified to reduce antibody-mediated effector functions (Armor et al, 1999). Accordingly, atrosimab showed almost complete absence of binding to human receptors Fcy Ia, IIb and IIIa, as well as to the complement protein Clq, as demonstrated by ELISA (Fig. 7c). Binding was reduced to a similar extent (FcyRI and FcyRIII) or even more pronounced (FcyRIIb and Clq) compared to the previously described anti-TNFR1 human IgG ATROSAB (Zettlitz et al., 2010), which carries identical Fc modifications to Fcy -receptor and Clq-binding.

Example 5.3: Atrosimab inhibits TNFR1 activation in vitro and does not have any agonist activity.

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Atrosimab demonstrated a complete absence of any agonistic bioactivity within the assayed concentration range between 50 pM and 500 nM in interleukin (IL) release experiments using HeLa cells for IL-6 and HT 1080 cells for IL-8, as well as cell death induction assays using Kym-1 cells (Fig. 3a-c). An identical loss of agonist activity was found in the parental Fab 13.7. In contrast, divalent IgG ATROSAB induced extreme release of IL-6 and IL-8 at concentrations ranging from 1 nM to 100 nM (Figure 8a and b), confirming previously published data (Richter et al., 2013). In contrast, the marginal agonistic activity of ATROSAB could not be detected in the Kym-1 cell death induction assay (Fig. 8c).

In addition, atrosimab inhibited TNFR1 activation induced by 0.1 nM TNF in the IL-6 release assay from HeLa and in the IL-8 release assay from HT1080 with EC ₅₀ values of 54.5 nM and 24.2 nM, respectively (Fig. 8d and e). Moreover, cell death induced by 0.1 nM TNF in Kym-1 cells was inhibited by atrosimab with an IC ₅₀ value of 16.2 nM (Fig. 8f). Compared to parental Fab 13.7, these data represent a 1.5- to 1.9-fold reduction in biological activity (Table 1). However, compared with the bioactivity of the divalent IgG ATROSAB, atrosimab demonstrated 3.0-fold, 3.5-fold, and 4.0-fold greater inhibition of TNF-mediated TNFR1 activation as determined in the IL6 release assay, IL-8 release assay, and induction assay. cell death, respectively (Table 1).

Table 1 Bioactivity of atrosimab1

Atrosimab Fab 13.7 ATROSAB IC50, IL-6 [nM] 54.5 37.1 164.7 IC50, IL-8 [nM] 24.2 12.7 84.1 IC50, cell death induction [nM] 16.2 9.5 64.4

To address the potential risk of secondary cross-linking of atrosimab mediated, for example, by anti-drug antibodies (ADA), the agonist potential of atrosimab was analyzed in the presence of three different mouse anti-human IgG sera in IL-8 release assays using HT1080 cells (Richter et al. 2013 ). ELISA demonstrated binding of mouse anti-human IgG sera to atrosimab, Fab 13.7, and ATROSAB (data not shown). Notably, atrosimab did not induce any IL-8 release within the concentration range analyzed (50 pM to 500 nM), which was also observed for parental Fab 13.7 (Fig. 9a-c). In contrast, divalent IgG ATROSAB induced a clearly increased release of IL-8 (Fig. 9a-c) compared to the marginal release observed in Fig. 9a-c. 8, indicating a clearly reduced propensity of Atrosimab to mediate any TNFR1 activation, even in the presence of drug-specific antibodies, compared to ATROSAB.

Example 5.4: Pharmacokinetics of atrosimab.

Finally, the pharmacokinetic properties of atrosimab were recorded following a bolus injection of 400 μg protein using C57BL/6J-huTNFRSF1Aecdtm1UEG/izi mice (Dong et al., 2016), which carry the human TNFR1 extracellular domain transgene (Figure 10, Table 2). Atrosimab was cleared from the circulation in mice with an initial half-life of 2.2 ± 1.2 hours and a terminal half-life of 41.8 ± 18.1 hours, resulting in an area under the curve of 5856.0 ± 1369.9 μg/mL x h.

table 2

Pharmacokinetic analysis of atrosimab2

11/2α (h) 2.2 = 1.2 11/2β (h) 41.8 Z 18.1 CO (µg/ml) 324.7 Z 53.5 AUC 0-t (µg/ml x h) θ 5856, Z 1369.9 Vss (µg/(µg/ml)) 3.4 Z 1.3 CL ((µg)/(µg/ml)/h) 0.29 Z 0.20

ti/ ₂ a, initial half-life; t _1/2 e, terminal half-life; _C0 , interpolated initial concentration; AUC 0-t, area under the curve until the laser detects the time point; Vss, volume of distribution (at steady state); CL, ground clearance.

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Claims (9)

Bibliography Armor KL, Clark MR, Hadley AG, Williamson LM. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Euro J Immunol. 1999 Aug;29(8):2613-24. Brader ML, Estey T, Bai S, Alston RW, Lucas KK, Lantz S, Landsman P, Maloney KM. Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies. Mol Pharm. 2015 Apr 6;12(4): 1005-17. doi: 10.1021/mp400666b. Dong Y, Fischer R, Naude PJ, Maier O, Nyakas C, Duffey M, Van der Zee EA, Dekens D, Douwenga W, Herrmann A, Guenzi E, Kontermann RE, Pfizenmaier K, Eisel UL. Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration. Proc Natl Acad Sci U S A2016; 113:12304-9; PMID:27791020; https://doi.org:10.1073/pnas,1605195113 Martin N, Ma D, Herbet A, Boquet D, Winnik FM, Tribet C. Prevention of thermally induced aggregation of IgG antibodies by noncovalent interaction with poly(acrylate) derivatives. Biomacromolecules. 2014 Aug ll;15(8):2952-62. doi: 10.1021/bm5005756. Zettlitz KA, Lorenz V, Landauer K, Miinkel S, Herrmann A, Scheurich P, Pfizenmaier K, Kontermann R. ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor onespecific antibody. MAbs. 2010 Nov-Dec;2(6):639-47. Epub 2010 Nov 1. CLAIM 1. Use of an antibody specifically recognizing human tumor necrosis factor receptor-1 (huTNFR1) in the treatment of non-alcoholic steatohepatitis (NASH) and liver fibrosis. 2. Use according to claim 1, wherein the antibody specifically recognizes an epitope in the membrane-distal CRD1 and/or the A1 subdomain of CRD2 in huTNFR1, preferably specifically recognizing the epitope represented by amino acids 1-115 in the N-terminal region of huTNFR1. 3. Use according to claim 1 or 2, wherein the antibody is a monoclonal antibody. 4. Use according to any one of claims 1-3, where the antibody is a monospecific, divalent full-length antibody. 5. The use of claim 4, wherein the antibody comprises an IgG1 Fc domain that is insufficient to provide effector function, preferably comprising at least one mutation selected from the group consisting of E233P, L234V, L235A, AG236, A327G, A330S and P331S preferably includes A327G/A330S/P331S, the numbering corresponding to the Kabat EU index. 6. Use according to any one of claims 1 to 3, wherein the antibody monovalently recognizes huTNFR1. 7. Use according to any one of claims 1 to 6, wherein the antibody comprises a) a heavy chain variable domain (VH), including the complementarity determining regions (CDRs): VH-CDR1, VH-CDR2 and VH-CDR3; And b) a light chain variable domain (VL), including CDRs: VL-CDR1, VL-CDR2 and VL-CDR3, where i) VH-CDR1 includes or consists of SEQ ID NO: 1; VH-CDR2 includes or consists of SEQ ID NO: 2, VH-CDR3 includes or consists of SEQ ID NO: 3, VL-CDR1 includes or consists of SEQ ID NO: 4, VL-CDR2 includes or consists of SEQ ID NO: 5, VL-CDR3 includes or consists of SEQ ID NO: 6; or ii) VH-CDR1 includes or consists of SEQ ID NO: 23, VH-CDR2 includes or consists of SEQ ID NO: 24, VH-CDR3 includes or consists of SEQ ID NO: 25, VL-CDR1 includes or consists of SEQ ID NO: 26, VL-CDR2 includes or consists of SEQ ID NO: 27, VL-CDR3 includes or consists of SEQ ID NO: 28, where the numbering corresponds to the Kabat EU index; or a functionally active variant of any of i) or ii) as above, which includes 0, 1 or 2 point mutations in each of the CDR sequences and which specifically recognizes huTNFR1. 8. Use according to any one of claims 1 to 7, wherein the antibody comprises a VH sequence comprising or consisting of SEQ ID NO: 7 or 9; and a VL sequence comprising or consisting of SEQ ID NO: 8 or 10, or a functionally active variant thereof, comprising up to 1 point mutation in each of the CDR sequences, and with at least 60% sequence identity in the framework (FR) sequences FR1- 4 from VH and VL. 9. Use according to any one of claims 1 to 8, wherein the antibody is used in an effective amount to counteract TNFa/huTNFR1 signaling. -