阅读说明：本技术 非酒精性脂肪性肝炎的抗huTNFR1疗法 (anti-huTNFR 1 therapy for non-alcoholic steatohepatitis ) 是由 H·班泰 K·普菲泽迈尔 A·赫尔曼 于 2018-11-27 设计创作，主要内容包括：一种特异性识别人肿瘤坏死因子1(hu TNFR1)的抗体,用于治疗非酒精性脂肪性肝炎(NASH)及与其相关疾病状况。(An antibody that specifically recognizes human tumor necrosis factor 1(hu TNFR1) for use in the treatment of nonalcoholic steatohepatitis (NASH) and disease states associated therewith.)

1. An antibody that specifically recognizes human tumor necrosis factor 1(huTNFR1) for use in the treatment of nonalcoholic steatohepatitis (NASH) and disease conditions associated therewith.

2. The antibody for use according to claim 1, which specifically recognizes an epitope within the distal CRD1 and/or subdomain a1 of CRD2 of membrane-huTNFR 1, preferably an epitope represented by amino acids 1 to 115 in the N-terminal region of huTNFR 1.

3. The antibody for use according to claim 1 or 2, which is a monoclonal antibody.

4. The antibody for use according to any one of claims 1 to 3, which is a monospecific, bivalent full-length antibody.

5. The antibody for use according to claim 4, said antibody comprising an IgG1 Fc domain, the IgG1 Fc domain lacking mediation of effector function, preferably comprising at least one mutation selected from the group consisting of E233P, L234V, L235A, Δ G236, A327G, A330S and P331S, preferably comprising A327G/A330S/P331S, wherein numbering is according to the Kabat EU index.

6. The antibody for use according to any one of claims 1 to 3, which antibody monovalently recognizes huTNFR 1.

7. The antibody for use according to any one of claims 1 to 6, which comprises:

a) a heavy chain variable domain (VH) comprising Complementarity Determining Regions (CDRs): VH-CDR1, VH-CDR2 and VH-CDR 3; and

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

wherein

i)

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

VH-CDR2 comprises or consists of SEQ ID NO: 2;

VH-CDR3 comprises or consists of SEQ ID NO 3;

VL-CDR1 comprises or consists of SEQ ID NO. 4;

VL-CDR2 comprises or consists of SEQ ID NO 5;

VL-CDR3 comprises or consists of SEQ ID NO 6;

or

ii)

VH-CDR1 comprises or consists of SEQ ID NO: 23;

VH-CDR2 comprises or consists of SEQ ID NO: 24;

VH-CDR3 comprises or consists of SEQ ID NO: 25;

VL-CDR1 comprises or consists of SEQ ID NO: 26;

VL-CDR2 comprises or consists of SEQ ID NO: 27;

VL-CDR3 comprises or consists of SEQ ID NO 28;

wherein numbering is according to the Kabat EU index;

or a functionally active variant of any of i) or ii) above, which comprises 0, 1 or 2 point mutations in each CDR sequence and which specifically recognizes huTNFR 1.

8. The antibody for use according to any one of claims 1 to 7, which antibody comprises a VH sequence and a VL sequence, or a functionally active variant thereof; the VH sequence comprises or consists of SEQ ID NO 7 or 9; the VL sequence comprises or consists of SEQ ID NO 8 or 10, the functionally active variant comprises at most 1 point mutation in each CDR sequence and has at least 60% sequence identity in the Framework (FR) sequences FR1-4 of VH and VL.

9. The antibody for use according to any one of claims 1 to 8, wherein the disease condition is any of hepatic steatosis, liver inflammation, liver fibrosis and hepatocellular carcinoma.

10. The antibody for use according to any one of claims 1 to 9, wherein the antibody is administered to a patient suffering from NASH in an amount effective to antagonize TNFa/huTNFR1 signaling.

11. The antibody for use according to any one of claims 1 to 10, wherein the antibody is administered to a patient suffering from NASH in an amount effective to reduce any one or more of:

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

b) serum transaminase levels;

c) insulin resistance and optionally improved glucose tolerance; and/or

d) NAFLD activity score.

12. The antibody for use according to any one of claims 1 to 11, wherein a dose ranging from 0.05mg/kg to 20mg/kg of the antibody is administered to a patient suffering from NASH.

13. The antibody for use according to any one of claims 1 to 12, wherein the antibody is administered to a patient suffering from NASH in combination with anti-inflammatory drug therapy, or therapy with a Farnesoid X Receptor (FXR) agonist, a glucagon-like peptide-1 receptor (GLP1R) agonist, or a peroxisome proliferator-activated receptor (PPAR) agonist.

14. The antibody for use according to any one of claims 1 to 13, wherein the antibody is administered to a patient who also suffers from type II diabetes, type I diabetes, pre-diabetes, insulin resistance or obesity, wherein obesity is defined as a patient having a body mass index of at least 30.

15. The antibody for use according to any one of claims 1 to 14, wherein a pharmaceutical formulation comprising an effective amount of the antibody is used.

Technical Field

The present invention relates to a novel treatment for nonalcoholic steatohepatitis (NASH) and disease conditions associated therewith.

Background

Nonalcoholic fatty liver disease (NAFLD) represents a range of diseases that occur without alcohol abuse and includes nonalcoholic steatohepatitis (NASH). NAFLD shows an increasing incidence in western countries and contributes to the development of hepatocellular carcinoma to a large extent.

One of the basic steps from benign hepatic steatosis to progressive steatohepatitis is the occurrence of hepatocyte death, which is classified as apoptosis. Necroptosis (Neoptosis) has become an alternative programmed cell death pathway and has been found to be activated in the liver of NASH patients (Gautheron et al. cellular and molecular biology and Hepatology 2015,1: 264-.

Apariciio-Vergara et al (Hepatology 2013,57(2):566-576) describe the role of TNFR1 ectodomain shedding in preventing the development of hepatic steatosis or insulin resistance. Failure of TNFR1 to shed does not cause obesity, insulin resistance or hepatic steatosis in mice. However, mice containing non-shedding mutations show rapid progression towards NASH. Activation of TNFR1 ectodomain shedding was found to play a key role in slowing the progression of NASH.

Cubero et al (Cell Death and Differentiation 2013,20: 1580) -1592) describe TNFR1 in hepatocytes and immune cells as having different effects in the mode of action of chronic liver disease.

Tomita et al (Gut 2006, 55:415-424) describe that the enhancement of TNFa/TNFR mediated signalling pathways may be strongly correlated with the pathogenesis of liver fibrosis in an animal model of NASH.

Yaron Ilan (AASLD Liver learning. Ilan Y. Nov 82014; 60709) discloses an anti-TNF based oral immunotherapy for the treatment of fatty Liver disease. An anti-TNF fusion protein that binds TNF α (PRX-106) was used in a mouse model on a high fat diet.

Antibodies to TNFR1 were found to have agonistic potential by inducing responses mimicking the ligand. This response suggests that signal transduction is initiated by receptor aggregation that binds multivalent TNF trimers.

However, selective inhibition of TNFR1 can be achieved with TNFR 1-specific antibodies. For example, monoclonal murine antibody H398, as well as the antibodies described in US5736138, are selective for human TNFR1, showing inhibitory potential for TNF-mediated signal transduction and cytotoxicity (Moosmayer et al 1995, the r. Immunol.2: 31-40).

WO2008/113515A2 describes humanized versions of H398.

WO2012035141 discloses anti-huTNFR 1 antibodies lacking mediated effector function.

Monovalent anti-huTNFR 1 antibodies are described in WO2017174586a 1.

Zettlitz et al (Landes bioscience 2010, November/Dezember:639-647) describe the generation of humanized TNFR 1-specific antagonistic monoclonal antibodies.

Richter et al (PLOS One 2013,8(8):1-13) describe the selective inhibition of TNFR1 signaling using humanized antagonistic anti-TNFR 1 antibodies to reduce the pro-inflammatory activity of TNF while leaving TNFR2 unaffected.

Berger et al (Protein Engineering, Design & Selection 2013,26(10):581-587) describe anti-TNFR 1 scFv-HAS fusion proteins as selective antagonists of TNF action. Feagins et al (Eur JGastroenterol hepatol.2015,27(10):1154-1160) describe the development of nonalcoholic fatty liver disease (NASH or steatosis) in patients treated with tumor necrosis factor inhibitor (TNFa).

The therapeutic possibilities for treating NASH are limited or restricted by lifestyle changes, since no specific drugs exist so far. Therefore, there is a need to provide effective treatment of NASH and its associated disease activity.

Disclosure of Invention

It is an object of the present invention to provide an improved treatment for NASH and corresponding disease conditions.

The object is solved by the subject matter of the invention.

The present invention provides a novel medical use of an antibody that specifically recognizes human tumor necrosis factor 1(huTNFR1) for treating a patient suffering from NASH and/or any disease condition associated with NASH in particular, including hepatic steatosis, NAFLD disease activity (NAS), apoptosis, fibrosis, high alanine Aminotransferase (ALT) and insulin levels. Thus, the present invention provides a new medical treatment for patients suffering from NASH and disease conditions associated therewith.

In particular, the invention provides antibodies that specifically recognize huTNFR1 for use in the treatment of nonalcoholic steatohepatitis (NASH) and disease conditions associated therewith.

According to a particular aspect, the antibody is an isolated antibody.

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

According to a particular aspect, the antibody specifically recognizes an epitope within the membrane (distal to CRD1 and/or subdomain a1 of CRD2 of huTNFR1), preferably an epitope that appears as amino acids 1 to 115, or 1 to 70, in the N-terminal region of huTNFR 1. Specifically, the sequence of huTNFR1 was identified as SEQ ID NO 32.

According to particular embodiments, the antibody is a monospecific, bivalent full-length antibody, or an antigen-binding antibody fragment.

According to another specific example, the antibody is a monovalent binder (binder) of huTNFR1, comprising only one antigen binding site with specificity for binding huTNFR 1. Specifically, the antibody monovalently recognizes huTNFR 1.

According to a particular embodiment, the antibody is selected from the group of "monovalent antibodies" consisting of Fab molecules, scFv molecules, single variable domains, disulfide stabilized Fv (dsfv), half IgG1 antibodies and Fv domains, or functionally active derivatives of any of the foregoing, preferably wherein the antibody construct is coupled to a hydrophilic polymer (such as PEG) and/or fused to a polypeptide, such as human (or mouse) serum albumin, transferrin, an albumin binding domain or peptide, an Ig-binding domain or peptide, a PEG mimetic polypeptide extension, an antibody Fc fragment carrying mutations to allow for preferred heterodimerization (as compared to homodimerization), or a functional variant of any of the foregoing polypeptides.

In particular, the antibody is any of a Fab, scFv, dsFv or Fv domain fused to an antibody Fc fragment, wherein Fc consists of a heterodimer of CH2 and CH3 domains, wherein the CH2 and/or CH3 domains carry one or more heterodimerization point mutations that allow preferential over homodimerization. In particular, one or both of the CH3 domains in the Fc are modified to alter the amino acid structure, e.g., to obtain an Fc that contains a heterodimer of CH3/CH3 domains.

In particular, the antibody constructs include Fv domains fused to an Fc region or fragment of an antibody, with or without other antibody domains, while retaining the monovalent binding structure of the antibody. Specific examples relate to Fab or Fv moieties fused to Fc or modified Fc.

Preferred antibodies comprise a heavy chain and a light chain, wherein the heavy chain consists of a VH domain, CH2 and CH3 domains, optionally further comprising one or more linkers (linkers); and the light chain consists of a VH domain, CH2 and CH3 domains, optionally further comprising one or more linkers.

Drawings

FIG. 1: B6-huTNFR 1-k/i-mice received High Fat Diet (HFD) for 32 weeks, including the last 8 weeks of treatment with anti-TNFR 1 or control antibody (Ab). Liver tissue of HFD mice treated with anti-TNFR 1-Ab showed significant reductions in steatosis (a), triglyceride content (B), and NAFLD activity score (C) in liver tissue compared to liver tissue from mice treated with control antibody. P < 0.05; p < 0.01;

FIG. 2: FIG. 1: B6-huTNFR 1-k/i-mice received High Fat Diet (HFD) for 32 weeks, including the last 8 weeks of treatment with anti-TNFR 1 or control antibody (Ab). Liver tissue (a) of HFD mice treated with anti-TNFR 1-Ab was assessed by Sirius Red staining (Sirius Red staining) compared to liver tissue (B) from mice treated with control antibody, showing improvement in liver fibrosis. P < 0.05;

FIG. 3: B6-huTNFR 1-k/i-mice received High Fat Diet (HFD) for 32 weeks, including the last 4 weeks of treatment with anti-TNFR 1 or control antibody (Ab). anti-TNFR 1 antibody treatment resulted in a significant reduction in activated caspase-3 (caspase-3) in liver tissue compared to control antibodies. P < 0.05;

FIG. 4: B6-huTNFR 1-k/i-mice received High Fat Diet (HFD) for 32 weeks, including the last 8 weeks of treatment with anti-TNFR 1 or control antibody (Ab). anti-TNFR 1-Ab treatment resulted in significant improvement in ALT and insulin serum levels compared to control antibodies. P < 0.05;

FIG. 5: sequence of

SEQ ID NO:1：VH-CDR1

SEQ ID NO:2：VH-CDR2

SEQ ID NO:3：VH-CDR3

SEQ ID NO:4：VL-CDR1

SEQ ID NO:5：VL-CDR2

SEQ ID NO:6：VL-CDR3

7, SEQ ID NO: VH of IgG13.7/Fab13.7

8, SEQ ID NO: VL of IgG13.7/Fab13.7

9 of SEQ ID NO: VH of ATROSAB/IZI 06.1.1

SEQ ID NO 10: VL of ATROSAB/IZI 06.1.1

11, SEQ ID NO: (Fab13.7 heavy chain [ VH in bold ])

SEQ ID NO 12: (Fab13.7 light chain [ VL in bold ])

13 in SEQ ID NO: VL1C (VL13.7-CH2-CH 31; chain containing VL and CH 1);

SEQ ID NO:14：VL13.7

15, SEQ ID NO: connecting body

SEQ ID NO:16：CH2

17 in SEQ ID NO: CH 31: CH31 is interspersed with Ig constant domains, and contains mainly residues from CH3, as well as residues from CH 1;

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

SEQ ID NO:19：VH13.7

SEQ ID NO:20：CH3k

21, SEQ ID NO: VL1C (VL13.7-CH2-CH 31; chain containing VL and CH 1):

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

23, SEQ ID NO: VH-CDR1 of ATROSAB

24, SEQ ID NO: VH-CDR2 of ATROSAB

25 in SEQ ID NO: VH-CDR3 of ATROSAB

26, SEQ ID NO: VL-CDR1 of ATROSAB

27 of SEQ ID NO: VL-CDR2 of ATROSAB

28, SEQ ID NO: VL-CDR3 of ATROSAB

SEQ ID NO:29：ATROSAB VH

SEQ ID NO:30：ATROSAB VL

31, SEQ ID NO: human IgG1 Fc

32 in SEQ ID NO: huTNFR1 sequence;

33, SEQ ID NO: a hinge region;

FIG. 6: biochemical characterization of Atrosimab (HC: SEQ ID NO:18, LC: SEQ ID NO: 13). (a) Representative cartoon of the molecular components of Atrosimab (white: constant Ig domain derived from Fc; light gray: VH and sequence derived from CH 1; dark gray: VL and sequence derived from CL κ). Atrosimab is characterized by SEC (b) TSKgel SuperSWmAb HR, flow rate 0.5ml/min, mobile phase Na2HPO4/NaH2PO4) and SDS-PAGE (c) NuPAGETM 4-12% Bis-TRIS Midi Gel under reducing (R) or non-reducing (NR) conditions. M: and (4) marking. (d) The thermal stability of the Atrosimab was analyzed by dynamic light scattering and visual interpretation of the resulting data points. Stability of the Atrosimab in human plasma after incubation was analyzed by detection of residual binding activity to human TNFR1 in ELISA (e). Bars represent EC50 values (mean ± SD) for three separate experiments. One sample was incubated in PBS at 4 ℃ and one sample was diluted in human plasma and frozen directly to-20 ℃ as a control;

FIG. 7: the antigen binds to and interacts with Fc receptors and C1q complement proteins. Equilibrium binding of Atrosimab to human TNFR1-Fc was analyzed by ELISA ((a) n-3, mean ± SD). Fab13.7 (containing the same VH and VL) and ATROSAB (lower affinity bivalent version) served as controls. (b) Recording real-time binding kinetics by QCM at five concentrations (between 128nM and 4nM (1:2 dilution step)) using the 1:1 binding algorithm for data analysis, (C) analyzing the interaction of immobilized Atrosimab and two control proteins, ATROSAB (silent Fc) and Rituximab (Rituximab) (wild-type Fc part), with human Fc γ RI, IIb and III and complement protein C1q by ELISA (n ═ 2, mean ± SD);

FIG. 8: lack of antagonistic biological activity and agonism of Atrosimab. Atrosimab showed a complete loss of agonistic activity in three different in vitro experiments: (a) IL-6 released by HeLa cells, (b) IL-8 released by HT1080 cells and in cell death induction experiments using Kym 1 cells (c). The parent Fab13.7, as a control protein, showed full agonistic properties as well as bivalent IgG ATROSAB, showing slight agonistic effects in (a) and (b). The same histones were assayed for their potential to inhibit activation of TNFR1 at the cell surface of 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 with 0.1nM TNF (d and e) or 0.01nM TNF (f). All graphs represent the mean of three separate experiments, error bars indicate SD;

FIG. 9: absence of Atrosimab agonism in the presence of anti-human IgG antibodies. Activation of TNFR1 on the surface of HT1080 cells was analyzed by Atrosimab at a constant concentration of drug-specific antibody (approximately 15.8nM) in an IL-8 release assay using three different mouse anti-human IgG sera (a, b, and c). Mouse anti-human IgG serum alone, unstimulated cells and TNF (33nM) served as controls. Shown are the mean ± SD of three separate experiments;

FIG. 10: pharmacokinetic analysis of Atrosimab. Circulating concentrations of Atrosimab in the mouse serum were determined after bolus injection of 400. mu.g of protein in C57BL/6J knock-in mice, which expressed the extracellular domain of human TNFR1 linked to the mouse transmembrane and intracellular domains rather than the intact murine protein. The intact protein that binds to human TNFR1-Fc was determined in an ELISA. The figure shows the mean ± SD of five mice.

Specific examples include human IgG1 Fc, in which the CH2-CH3 domain is mutated by one or more "knob-to-hole (knob-to-hole)" mutations to form heterodimers, e.g.,

the "knob" mutation modifies the surface of the CH3 β -sheet, which is present on one CH3 domain monomer, which is T366W; and

the "hole" mutation modifies the surface of the CH3 β -sheet, which is present on another CH3 domain monomer, selected from the group consisting of T366S, L368A, Y407V.

In particular, the antibodies include an Fc region that includes one or more mutations to down-regulate effector function. According to particular aspects, the Fc region is glycoengineered to down-regulate effector function.

According to particular embodiments, the antibody construct comprises a human or artificial IgG1 Fc region that is a functional variant of human IgG1 Fc having at least any one of 60%, 70%, 80%, 85%, or 90% sequence identity that is mutated to down-regulate effector function. Preferably, the Fc region comprises a heavy chain, with at least one mutation selected from the group consisting of E233P, L234V, L235A, Δ G236, a327G, a330S and P331S, preferably including a327G/a330S/P331S (Kabat EU index numbering). Preferably, at least two of the mutations, more preferably at least three, four, five or all of the six mutations, are engineered into the Fc sequence. SEQ ID NO 31 identifies the sequence of human IgG1 Fc.

Specifically, the antibody is PEGylated (PEGylated), HES (HESylated), or PSA (PSAYlated).

Specifically, antibodies were pegylated (pegylated) with PEG with molecular weights ranging between 5,000 and 150,000g/mol, and exemplary antibody constructs, such as Fab, were pegylated with PEG 40,000.

In particular, the antibody is a half antibody IgG1, characterized by only One Fab portion, hinge region and One Fc portion, wherein the hinge region and/or Fc portion (in particular human IgG1 Fc) comprises One or more mutations to avoid heavy chain dimerization (Gu etal (2015) PLoS One 10(1): e0116419), e.g., selected from the group consisting of:

-mutation in the hinge region (SEQ ID NO: 33): C226S, C229S (EU numbering), and

-mutation in the Fc part: P395A, F405R, Y407R and K409D (EU numbering).

In particular, the antibody is an Fv-Fc fusion protein, wherein the Fv consists of a VH/VL domain pair, and wherein VH is fused to a first CH2-CH3 domain chain through a first hinge/linker region, and VL is fused to a second CH2-CH3 domain chain through a second hinge/linker region. Preferably, the first and second CH2-CH3 domain chains differ from each other in one or more point mutations, e.g., allowing for preferential heterodimerization between the first and second CH2-CH3 domain chains, thereby obtaining an Fv-Fc preparation characterized as an Fc heterodimer, e.g., by a mutation of "protuberance-into-hole" as described above.

In particular, antibodies include disulfide stabilized fv (dsfv) characterized by one or more additional (artificial) interdomain disulfide bonds. Such a disulfide bond is obtained by introducing one or more additional cysteine residues into either the VH or VL domains at appropriate positions that can serve as bridge piers for disulfide bridges between the VH and VL domains, which disulfide bonds are available upon reduction of the cysteine. According to a specific example, disulfide bonds can be introduced into Fv at any of the following positions in VH and at the 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 in VL.

Specifically, the antibody comprises

a) A heavy chain variable domain (VH) comprising Complementarity Determining Regions (CDRs): VH-CDR1, VH-CDR2 and VH-CDR 3; and

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

wherein

i)

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

VH-CDR2 comprises or consists of SEQ ID NO 2

VH-CDR3 comprises or consists of SEQ ID NO 3

VL-CDR1 comprising or consisting of SEQ ID NO. 4

VL-CDR2 comprising or consisting of SEQ ID NO 5

VL-CDR3 comprises or consists of SEQ ID NO 6;

or

ii)

VH-CDR1 comprises or consists of SEQ ID NO: 23;

VH-CDR2 comprises or consists of SEQ ID NO:24

VH-CDR3 comprises or consists of SEQ ID NO:25

VL-CDR1 comprising or consisting of SEQ ID NO 26

VL-CDR2 comprising or consisting of SEQ ID NO 27

VL-CDR3 comprises or consists of SEQ ID NO 28;

wherein numbering is according to the Kabat EU index;

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

Specifically, the antibody includes VH and VL,

wherein

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

VH-CDR2 comprises or consists of SEQ ID NO: 2;

VH-CDR3 comprises or consists of SEQ ID NO 3;

VL-CDR1 comprises or consists of SEQ ID NO. 4;

VL-CDR2 comprises or consists of SEQ ID NO 5; and

VL-CDR3 comprises or consists of SEQ ID NO 6;

wherein numbering is according to the Kabat EU index;

or a functionally active variant thereof, comprises 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 huenfr 1.

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

i)

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

VH-CDR2 comprises or consists of SEQ ID NO 10, wherein X at position 5 is S;

VH-CDR3 comprises or consists of SEQ ID NO 3;

VL-CDR1 comprises or consists of SEQ ID NO. 4;

VL-CDR2 comprises or consists of SEQ ID NO 5; and

VL-CDR3 comprises or consists of SEQ ID NO. 11, wherein X at position 3 is G.

Or ii)

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

VH-CDR2 comprises or consists of SEQ ID NO 10, wherein X at position 5 is S;

VH-CDR3 comprises or consists of SEQ ID NO 3;

VL-CDR1 comprises or consists of SEQ ID NO. 4;

VL-CDR2 comprises or consists of SEQ ID NO 5; and

VL-CDR3 comprises or consists of SEQ ID NO. 11, wherein X at position 3 is S.

In particular, the antibody comprises a VH sequence comprising or consisting of SEQ ID NO 7 or 9 and a VL sequence or functionally active variants thereof; the VL sequence comprises or consists of SEQ ID NO 8 or 10, the functionally active variant comprises at most 1 point mutation in any one or more, or each, CDR sequence and has at least 60% sequence identity in any one or more, or each, of the Framework (FR) sequences FR1-4 of VH and VL.

A particular VH/VL combination comprising an antigen binding site capable of specifically recognizing and binding huTNFR1 is any one 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.

In particular, the antibody is a full-length or antigen-binding antibody fragment comprising or consisting of a Fab comprising:

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 at most 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 having at least 60% sequence identity in any one or more, or each, of the FR sequences FR1-4 of the VH and VL domains.

Specifically, antibodies include:

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 at most 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 having at least 60% sequence identity in any one or more, or each, of the FR sequences FR1-4 of the VH and VL domains.

Specific functionally active variants of the antibody include HC recognized by SEQ ID NO. 18 and LC recognized by SEQ ID NO. 13, including:

HC, consisting of:

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

b) a linker sequence consisting of 4-10 amino acids, e.g. 4, 5, 6, 7, 8, 9 or 10 amino acids, preferably a number of glycines, serines or threonines in any combination, such as, e.g., 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

an LC consisting of:

a) a VL comprising or consisting of SEQ ID NO. 14, or a CDR sequence comprised at least in said VH sequence;

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

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

d) comprises SEQ ID NO 17 or the CH3 domain consisting of SEQ ID NO 17.

In particular, such antigen-binding antibodies are encoded by one or more nucleic acid molecules comprising:

a) HC of coding sequence SEQ ID NO. 22; and

b) LC of coding sequence SEQ ID NO. 21;

or a functionally active variant thereof comprising at most 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 having at least 60% sequence identity in any one or more, each, of the FR sequences FR1-4 of the VH and VL domains.

According to a particular embodiment, the antibody comprises an antigen binding site characterized by a combination of the following six CDR sequences, the antibody comprising or consisting 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；

27 of SEQ ID NO: VL-CDR 2; and

SEQ ID NO:28：VL-CDR3；

or a functionally active variant thereof which comprises up to 1 point mutation in any one or more, or each, CDR sequence and which specifically recognizes huenfr 1.

In particular, the antibodies comprise antigen binding sites incorporated in the VH and VL domains, wherein

a) A VH comprising or consisting of SEQ ID NO: 29; and

b) VL comprising or consisting 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 having at least 60% sequence identity in any one or more, or each, of the FR sequences FR1-4 of the VH and VL domains.

In particular, the antibody comprises VH and VL domains, wherein at least one of the VH and VL domains is a functional variant of affinity maturation of the parent domain, comprising at least one point mutation in a Complementarity Determining Region (CDR) sequence, wherein

a) The parent VH domain is characterized by the CDR sequences: 23, 24 and 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, the at least one point mutation is in any of SEQ ID NO. 24 and/or SEQ ID NO. 28.

In particular, any of the exemplary antibodies (those antibodies characterized by the sequences provided herein) can be used according to the present invention. Likewise, any alternative antibody that includes the same antigen binding site and/or has the same target binding specificity may be used. Particular surrogate antibodies are those that are functional variants of the exemplary antibody, wherein any of the exemplary antibodies can be used as a "parent" to generate variants that have the function of specifically recognizing the huTNFR1 target.

In particular, the antibody is an affinity matured antibody of a parent antibody, characterized by the sequences provided herein, in particular, wherein 1, 2, 3, 4, 5 or 6 of the CDR sequences are functionally active CDR variants comprising at most 1 point mutation compared to the respective CDR in the parent antibody.

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

Any functionally active variant of the antibody described herein (parent antibody) is specifically characterized by huTNFR1 binding specificity. Functionally active variants may comprise one or more mutated FR sequences comprising one or more, e.g., several point mutations, e.g., at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 point mutations, to obtain variant sequences having at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity compared to the respective FR sequences in the parent antibody.

In particular, the antibodies include an antigen binding portion that binds to K of huTNFR1_DLess than 10^-8M or 5 × 10^-9M, and k_offLess than 10^-3s^-1. In standard assays for determining monovalent binding, the affinity of the binding as well as the binding properties (binding and separation) are specifically determined, substantially excluding the binding force effects of bivalent binding. The standard test is based on the measurement of the temperature at physiological temperature (about 37 ℃ C., or at 37 ℃ C. +/-ion-A) by means of a Quartz Crystal Microbalance (QCM)Measurement at 1 ℃ C.). Such affinity measurements are performed in particular in the Fab format. Thus, if the antibody is any other antibody than a Fab molecule, an antigen binding site is specifically introduced into the respective Fab molecule for affinity measurement by QCM at 37 ℃. This ensures comparability of the affinity measurements of the monovalent binders despite binding force effects that may interfere with the affinity measurements. In particular, preferred QCMs are performed at moderate receptor densities. Specifically, the affinity of the antibody construct binding to huTNFR1 was determined by QCM in Fab format at 37 ℃ for binding of the Fab format antibody construct to huTNFR1, while the intermediate receptor density was in the range of 50-100Hz (e.g., at about 50Hz, or at 50Hz +/-10Hz, or at 50Hz +/-5 Hz).

In particular, K_DLess than 4 × 10^-9M, or less than 3 × 10^-9M, or less than 2 × 10^-9M, or less than 10^-9M, or even less than 10^-10M。

Specifically, and k_offLess than 10^-3Or less than 5 × 10^-4s^-1Or less than 10^-4s^-1Or less than 10^-5s^-1。

In particular, the antigen binding portion recognition has a value of at least 10^-5M^-1s^-1K of (a)_on huTNFR 1.

According to a particular aspect, the disease condition is any of hepatic steatosis, inflamed liver, liver fibrosis (or apoptosis) and hepatocellular carcinoma. In particular, NASH patients at risk of developing or already having any disease condition are treated. Several indicators of NASH or related disease conditions, including NAFLD disease activity (NAS), as well as high ALT and insulin serum levels, can be effectively reduced by the treatments described herein.

In particular, the patient also suffers from type II diabetes, type I diabetes, pre-diabetes, insulin resistance or obesity, wherein obesity is defined as a patient body mass index ≥ 30.

In particular, the antibody is administered to the patient in an effective amount. In particular, the amount is effective to antagonize TNFa/huTNFR1 signaling. It is particularly preferred that the antibody is an antagonistic antibody, thereby avoiding a large number of TNFa/TNFR mediated signaling and signal transduction as measured in cell-based assays. Any antibody described herein and characterized by the antibody sequences provided herein is specifically understood to be an antagonist antibody.

According to particular aspects, the antibody directly inhibits TNF-huTNFR1 receptor interaction, as determined in a cell-based assay, preferably by an assay for TNFR 1-mediated inhibition of cell death in Kym-1 cells, or by an assay for inhibiting release of IL-6 or IL-8 by HeLa cells or HT1080 cells. Specifically, in the assay of TNFR1 mediated inhibition of cell death in Kym-1 cells, IC₅₀A value of less than 5.0 × 10^-9And M. In particular, IC in assays for inhibiting IL-6 release from HeLa cells₅₀A value of less than 4.0 × 10^-8M, or in assays inhibiting IL-8 release from HT1080 cells, IC₅₀A value of less than 2.0 × 10^-8M。

According to particular embodiments, the antibodies are used to bind to huTNFR1 through a monovalent interaction and reduce the risk of exhibiting TNF-mimetic agonistic activity. Particularly preferred are antibodies with high affinity for TNFR1 binding and a low off-rate (off) that provide excellent inhibition of TNFR 1-dependent TNF responses.

In particular, the antibodies described herein are provided in a pharmaceutical formulation comprising the antibody and a pharmaceutically acceptable carrier and/or excipient. Due to the antagonistic properties of the antibodies, the pharmaceutical preparation may comprise high antibody concentrations while avoiding side effects caused by agonistic activity.

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

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

It has surprisingly been found that the antibodies described herein, particularly monovalent antibodies, can be used to treat patients undergoing ADA, for example, antibodies or antibody immunotherapy against immunoglobulins have been developed. In the prior art, the presence of such ADA would specifically exclude further immunotherapy with antibodies against TNFR1, as ADA has the potential to cross-link antibodies when binding TNFR1 on the cell surface, thereby possibly agonizing TNFR1 signaling. However, the antibodies described herein do not (or do not substantially) agonize (agonise) TNFR1 signaling even in the presence of ADA.

In particular, the pharmaceutical formulations described herein may be administered to a patient who has already had ADA, for example, ADA directed to an anti-huTNFR 1 antibody or any IgG structure.

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

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

b) serum transaminase levels;

c) insulin resistance and optionally improved glucose tolerance; and/or

d) NAFLD activity fraction.

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

Preferred antibody doses are, for example, in the range 0.5 to 1000mg, preferably 1-400 mg. If administered subcutaneously, the preferred dosage range is 0.5 to 400 mg.

According to a particular aspect, the antibody is administered systemically, preferably by intravenous infusion or bolus injection (bolus injection), to the patient in a therapeutically effective amount.

According to a particular embodiment, the patient is administered with the antibody repeatedly on a regular basis, for example, once a week, intravenously or subcutaneously, at a dose of, for example, 0.5-5mg/kg, particularly about 2 mg/kg. The frequency and dosage of the drug administered may be adapted to the disease state and response to treatment.

In particular, the antibody is administered in combination with dietary therapy to patients suffering from NASH. Antibody therapy may be particularly combined with anti-inflammatory drugs such as NSAP/NSAIDs or therapies using Farnesoid X Receptor (FXR) agonists, glucagon-like peptide-1 receptor (GLP1R) agonists, or peroxisome proliferator-activated receptor (PPAR) agonists.

Unless otherwise indicated, positions herein are numbered according to the EU index of Kabat. An explanation of the Kabat numbering scheme can be found in Kabat, EA, et al, Sequences of proteins of immunological interest (NIH publication No. 91-3242, 5 th edition (1991)).