Combination of HB-EGF inhibitors and corticosteroids for the treatment of renal diseases associated with activation of the HB-EGF / EGFR pathway

Abstract

The present invention relates to a combination of a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor for use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway.

Description

[Technical Field]

[0001] The present invention relates to the treatment of renal diseases associated with activation of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) / epidermal growth factor receptor (EGFR) pathway, and more particularly to the use of HB-EGF inhibitors in combination with corticosteroids. [Background technology]

[0002] Increased expression of pro-HB-EGF and / or activation of the HB-EGF / EGFR pathway associated with HB-EGF release lead to harmful cell proliferation that causes numerous pathological conditions, particularly renal diseases such as crescentic glomerulonephritis (CGN) and other related disorders.

[0003] Crescent glomerulonephritis (CGN) is a rare kidney disease that destroys the glomeruli, the filtering units of the kidney. CGN is caused by various immune-inflammatory attacks, including immune complexes from anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV), anti-glomerular basement membrane (GBM) autoantibodies, systemic lupus erythematosus, and infection (Couser et al., Am. J. Kidney Dis., 1988, 11, 449-464; Jennette et al., Nephrol. Dial. Transplant., 2001, 16, 80-82). Treatment is based solely on immunosuppression, with the latest options being a combination of rituximab (anti-CD20) and corticosteroids (CTCs), along with avacopan, a complement C5a receptor inhibitor for AAV (Chevet et al., Rheumatology (Oxford), 2022, doi:10.1093 / rheumatology / keac623). However, CGN still leaves unaddressed medical needs. The mortality rate for patients with chronic kidney disease (CGN) is up to 20% within one year (Heijl et al., RMD Open, 2017, 3, e000435; Wallace et al., Semin. Arthritis Rheum., 2016, 45, 483-489), 90% of patients have chronic renal failure, and the risk of end-stage renal disease is up to 70% (Chen et al., Am. J. Kidney Dis., 2016, 67, 376-383).

[0004] CGN is characterized by the dedifferentiation and proliferation of glomerular structure-forming cells: podocytes and parietal epithelial cells (PECs) (Bollee et al., Nat. Med., 2011, 17, 1242-; Lazareth et al., Nat. Commun., 2019, 10; Shankland et al., Nature Reviews Nephrology, 2014, 10, 158-173). Podocytes surround glomerular capillaries and are involved in the integrity of the blood / urinary filtration barrier. PECs line Bowman's capsule around the glomerulus. During immune attacks that lead to CGN (Continuous Nephrogenesis), podocytes and PECs overexpress the growth factor HB-EGF (heparin-binding EGF-like growth factor) (Bollee et al., Nat. Med., 2011, 17, 1242-1250; Flamant et al., Nephrology Dialysis Transplantation, 2012, 27, 1297-1304). HB-EGF binds to the EGF receptor (EGFR). EGFR is phosphorylated, activating STAT3 signaling. As a result, podocytes and PECs undergo dedifferentiation and proliferate, leading to progressive glomerular occlusion. Mice in which the Hb-egf gene, selective Egfr gene, or Stat3 gene were knocked out in podocytes were shown to be resistant to the development of CGN despite immune attack (Bollee et al., Nat. Med., 2011, 17, 1242-1250; Dai et al., Kidney Int., 2013, 84, 950-961, Henique et al. Nat Commun., 2017, 8(1):1829).The diagnosis of CGN depends on clinical symptoms (asthenia, weakness, fever, nausea, vomiting, anorexia, arthralgia, abdominal pain, and hypertension), inflammatory glomerulonephritis with hematuria, hyperproteinuria, hypoalbuminemia, and elevated plasma creatinine and urea nitrogen levels, as well as progressive renal failure over several days to months (O'Brien, F. Rapidly Progressive Glomerulonephritis (RPGN) (Crescentic Glomerulonephritis). MSD Manual Professional Version. 2022). The disease develops into severe oliguria. Serology can identify causative antibodies (ANCA, anti-GBM, anti-DNA histone, anti-streptrysin O.). Histology of renal biopsy reveals classic crescent glomerular lesions. Podocyte and PEC proliferation leads to the formation of crescent-shaped lesions that gradually invade the glomeruli. Ultimately, crescent glomeruli can lead to scarring lesions such as focal segmental glomerulosclerosis (FSGS), in which collagen deposits are replaced by crescent cells. Interestingly, at early diagnosis, most glomeruli are still healthy and functional, but their PECs and podocytes are already positive for HB-EGF expression (Bollee et al., Nat. Med., 2011, 17, 1242-1250). The involvement of HB-EGF in pathogenic dedifferentiation, migration, and proliferation of PECs and podocytes has been demonstrated (Bollee et al., Nat. Med., 2011, 17, 1242-1250, Lazareth et al. Nat Commun. 2019;10(1):3303). This suggests that early treatment with HB-EGF inhibitors can rescue and preserve these healthy glomeruli and renal function (Flamant et al., Nephrology Dialysis Transplantation, 2012, 27, 1297-1304).

[0005] Activation of the HB-EGF / EGFR pathway has also been shown to be involved in other kidney diseases such as diabetic nephropathy (Miyazama et al., Kidney International, 2013, 84, 1176-1188), autosomal dominant polycystic kidney disease, renal tubulointerstitial fibrosis (Overstreet et al., FASEB, 2017, 10, 4407-4421), chronic kidney disease, and hypertensive nephropathy (Zeng et al., American Journal of Physiology - Renal Physiology, 2016, 311, F695-F707).

[0006] Therefore, HB-EGF emerges as a potential new target for the treatment of CGN (Bollee et al., Nat. Med., 2011, 17, 1242-1250; Flamant et al., Nephrology Dialysis Transplantation, 2012, 27, 1297-1304) and other renal diseases involving activation of the HB-EGF / EGFR pathway.

[0007] Currently, there are two types of HB-EGF inhibitors that can be used to block EGFR stimulation by HB-EGF: an antibody against HB-EGF, and an HB-EGF ligand derived from diphtheria toxin (DT), the natural ligand for HB-EGF (Gillet & Barbier, Diphtheria toxin. in The Comprehensive Sourcebook of Bacterial Protein Toxins 111-132 (Academic Press, 2015). doi:10.1016 / B978-0-12-800188-2.00004-5). HB-EGF blocking antibodies are disclosed in WO2009 / 040134;WO2011 / 21381;WO2009 / 72628;EP2221374;EP2281001;EP2093237;EP2078731;EP2039704; and WO2008 / 053270. CRM197 is a native mutant of DT proposed for the treatment of ovarian cancer (US7700546;US2006 / 0270600). Synthetic variants of the DT cell receptor binding domain (R or DTR) engineered to have a smaller size, better affinity for HB-EGF, and reduced antigenicity, immunogenicity, and toxicity compared to CRM197 are disclosed in WO2013 / 140335.

[0008] The current treatment of CGN relies primarily on CTCS and potent immunosuppression with anti-CD20 (rituximab) or cyclophosphamide or other anti-mitotic agents (O'Brien et al., MSD Manual Professional Version., 2022; Yates et al., Annals of the Rheumatic Diseases, 2016, 75, 1583-1594). Immunosuppression aims to reduce circulating autoantibodies and / or immune complexes that cause glomerular deposition in the glomeruli. Immunosuppression can also reduce periglomerular infiltration of immune cells involved in immune attack. However, while maintenance therapy with prednisone at 7.5–10 mg / day is recommended, initial treatment for patients at diagnosis includes intravenous administration of methylprednisolone 1 g once daily for 3–5 days, followed by oral administration of prednisone 1 mg / kg once daily, gradually decreasing over 5 months (O'Brien et al., MSD Manual Professional Version., 2022; Yates et al., Annals of the Rheumatic Diseases, 2016, 75, 1583-1594; Walsh et al. N Engl J Med. 2020;382(7):622-631; Furuta et al. Ann Rheum Dis. 2024;83(1):96-102.) (Figure 1). These high starting doses for CTC are recommended in the acute phase of CGN and relapse. These extremely aggressive dosages cause serious adverse effects (Stone et al., Semin. Arthritis Rheum., 2022, 55, 152010), such as infections, myopathy, neuropsychotoxicity, and cutaneous toxicity, leading to increased morbidity and reduced quality of life, and explaining the high mortality rate (10-20%) in patients in the first few years of treatment (Heijl et al., RMD Open, 2017, 3, e000435; Wallace et al., Semin. Arthritis Rheum., 2016, 45, 483-489). As a result, current treatments for CGN are unsatisfactory, and the disease remains an unmet medical need.

[0009] Therefore, improved treatment for related renal diseases associated with activation of the GCN and HB-EGF / EGFR pathways is still needed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Using a porcine model of crescentic glomerulonephritis (CGN), the inventors demonstrated that an HB-EGF inhibitor (DTR8) can reduce the high starting dose of corticosteroids used in the acute or recurrent phase, thereby reducing the progression of CGN glomerular lesions and the decline in renal function. These results suggest that the combination of an HB-EGF inhibitor and a corticosteroid provides an alternative treatment for GCN and other renal diseases associated with activation of the HB-EGF / EGFR pathway. The DTR8 inhibitor enhances the effects of the corticosteroid. Therefore, combination therapy favorably enables the use of low doses of corticosteroids to avoid the adverse effects associated with high doses of corticosteroids, while simultaneously providing an efficient treatment of the disease, as demonstrated by its ability to limit the progression of glomerular lesions and the decline in renal function. [Means for solving the problem]

[0011] Therefore, the present invention relates to a combination of a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor for use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway.

[0012] In some embodiments, the combination therapy includes a lower dose of corticosteroid compared to the starting dose of the corticosteroid used in the reference.

[0013] In some embodiments, combination therapy is for the treatment of the acute phase or relapse of the disease.

[0014] In some embodiments, the combination therapy is for the treatment of crescentic glomerulonephritis, preferably for the treatment of the acute or recurrent phase of crescentic glomerulonephritis.

[0015] In some embodiments, combination therapy reduces the severity of glomerular lesions and / or the decline in renal function.

[0016] In some embodiments, the combination therapy comprises an HB-EGF inhibitor which is a recombinant protein containing an isolated R domain of diphtheria toxin, comprising at least substitutions to the amino acid sequence of SEQ ID NO: L390T and Y380K; L390T and Y380E; Y380K and Q387E; or Y380E and Q387K. In some specific embodiments, the recombinant protein further comprises at least one substitution to SEQ ID NO: F389Y, A395T, N399K, T436H, V452T, V483Q, H492E, S494K, E497D, G510A, and T517E. In some more specific embodiments, the recombinant protein includes substitutions Y380K, Q387E, L390T, A395T, F389Y, G510A, N399K, V452T, T517E, V483Q, H492E, and S494K relative to SEQ ID NO: 1. In some specific embodiments, the recombinant protein includes an amino acid sequence having at least 70% identity with residues 380-535 of SEQ ID NO: 1. In some preferred embodiments, the recombinant protein includes the amino acid sequence of SEQ ID NO: 2.

[0017] In some embodiments, the combination therapy includes a corticosteroid selected from the group consisting of prednisolone, betamethasone, prednisone, triamcinolone, methylprednisolone, dexamethasone, and mixtures thereof.

[0018] In some specific embodiments, the combination therapy includes a low dose of corticosteroids, less than 1 mg / kg per day.

[0019] In some embodiments, the combination therapy includes a dose of 0.6 mg / kg of an HB-EGF inhibitor per day.

[0020] In some specific embodiments, the combination therapy includes daily administration of a low dose of a corticosteroid agent and / or an HB-EGF inhibitor over at least 3 weeks.

[0021] In some specific embodiments, the combination therapy includes daily administration of a low dose of a corticosteroid agent and / or an HB-EGF inhibitor for up to 6 months, preferably up to 5 months, preferably up to 4 months, and even more preferably up to 3 months.

[0022] In some embodiments, the combination therapy includes simultaneous or separate administration of an HB-EGF inhibitor and a corticosteroid agent.

[0023] In some embodiments, the combination therapy includes oral or injection, particularly intravenous or subcutaneous, administration of an HB-EGF inhibitor and / or a corticosteroid agent.

[0024] Another aspect of the present invention relates to a product containing a corticosteroid agent and an inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF) as a combined preparation for simultaneous use, separate use, or sequential use in the treatment of kidney diseases related to the activation of the HB-EGF / EGF receptor pathway according to the present disclosure.

[0025] The present invention also relates to a pharmaceutical composition for treating kidney diseases related to the activation of the HB-EGF / EGF receptor pathway, comprising an HB-EGF inhibitor and a low dose of a corticosteroid agent according to the present disclosure.

Mode for Carrying Out the Invention

[0026] The present invention relates to a combination of a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor for use in the treatment of renal diseases associated with activation of the HB-EGF / EGF pathway. The present invention encompasses products containing a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor as a combination formulation for concurrent, individual, or sequential use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway, as disclosed herein. The present invention also encompasses pharmaceutical compositions for the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway, comprising an HB-EGF inhibitor and a low dose of a corticosteroid, as disclosed herein. The combination therapy according to the present invention is advantageously effective using a low dose of a corticosteroid compared to the high starting dose used in the acute or recurrent phase of the disease. Thus, the combination therapy avoids the adverse effects associated with current corticosteroid therapies for the disease.

[0027] HB-EGF inhibitors Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a growth factor expressed on the surface of cells in organisms in the form of a precursor membrane protein, pro-HB-EGF, which is a natural diphtheria toxin receptor (Gillet & Barbier, Diphtheria toxin. in The Comprehensive Sourcebook of Bacterial Protein Toxins 111-132 (Academic Press, 2015). doi:10.1016 / B978-0-12-800188-2.00004-5). During inflammatory processes involving HB-EGF, the pro-HB-EGF membrane protein is cleaved by proteases, and HB-EGF is released from the cell surface in the form of a secreted protein. This then binds to the ErbB1 (HER1 or EGFR) and ErbB4 (HER4) subunits of the EGF receptor family (EGFR family or EGFR), which is expressed in substantially all tissues of organisms, in an autocrine or paracrine manner. In addition to HB-EGF, at least 10 EGFR ligands are present, namely EGF, TGF-α, amphiregulin, betacerulin, epigen, epiregulin, and neuregulin-1, -2, -3, and -4. Local conditions determine the stimulation of EGFR by one or another of these ligands.

[0028] HB-EGF inhibitors refer to drugs that can block EGFR stimulation by HB-EGF, i.e., inhibit the HB-EGF / EGFR pathway. Inhibition of the HB-EGF pathway can be determined by standard assays well known in the art, for example, by measuring the inhibition of HB-EGF-induced proliferative activity in HB-EGF-dependent cell lines, such as those disclosed in WO2013 / 140335. Various HB-EGF inhibitors well known in the art may be used in combination therapies according to the present invention.

[0029] "A," "an," and "the" refer to multiple objects unless the context otherwise clearly indicates otherwise. Thus, the terms "a" (or "an"), "one or more," or "at least one" are interchangeable herein, and unless otherwise specified, "or" means "and / or."

[0030] The first type of HB-EGF inhibitor is an antibody against HB-EGF. Such HB-EGF blocking antibodies are disclosed in WO2009 / 040134;WO2011 / 21381;WO2009 / 72628;EP2221374;EP2281001;EP2093237;EP2078731;EP2039704; and WO2008 / 053270. As used herein, the term “antibody” encompasses the antigen-binding fragment of an antibody.

[0031] Another type of HB-EGF inhibitor is a ligand for HB-EGF derived from diphtheria toxin (DT), a natural ligand for HB-EGF. Diphtheria toxin (DT) is an exotoxin consisting of 535 amino acids (SEQ ID NO: 1), composed of fragment A (N-terminus) and fragment B (C-terminus) (Gillet & Barbier, Diphtheria toxin. in The Comprehensive Sourcebook of Bacterial Protein Toxins 111-132 (Academic Press, 2015)). doi:10.1016 / B978-0-12-800188-2.00004-5. Fragment A contains a catalytic domain (C or DTA / DT-A; residues 1-193), and fragment B contains a transition domain (T; N-terminus; residues 202-378) and a cell receptor binding domain (R or DTR; C-terminus; residues 379-386-535). DT binds to the cell surface via the pro-HB-EGF binding of its R domain, and after activation of DT by proteolytic cleavage between fragments A and B, the T domain transitions fragment A into the cytoplasm. This enables the process. Once in the cytoplasm, the catalytic domain held by fragment A blocks protein synthesis by inactivating the EF2 elongation factor, thus causing cell death. Studies of native and synthetic mutants of DT have shown that the binding of DT to the pro-HB-EGF receptor is lost by substitution of residue S508 (S508F) or residues in the loop region K516-F530 of DTR (K516A, K516E, F530A, F530S, and S525F), but not by deletion of four C-terminal residues (residues E532-S535; Shen et al., J. Biol. Chem., 1994, 269, 29077-29084).

[0032] CRM197 is a native mutant of DT containing the G52E mutation, resulting in significantly reduced catalytic activity (US7700546; US2006 / 0270600; Giannini et al., NAR, 1984, 12, 4063-4069).

[0033] Isolated DTR domain mutants lacking a fusion partner sequence are disclosed in WO2013 / 140335. These isolated DTR domain mutants are directly and in large quantities expressed in the form of small-sized (approximately 17500 Da) soluble recombinant DTR proteins with pro-HB-EGF and affinity for HB-EGF. Surprisingly, these DTR mutants have been modified to produce improved recombinant DTR proteins with both reduced antigenicity and immunogenicity and significantly increased affinity compared to CRM197.

[0034] In some embodiments, the HB-EGF inhibitor for combination therapy according to the present invention is a recombinant protein comprising substitution of at least one, preferably at least two, of residues Y380, P382, Q387, P388 and / or L390 of the R domain with another amino acid selected from the group consisting of S, T, N, C, Y, Q, R, K, H, D, and E.

[0035] Amino acids are indicated using single-letter codes. Their positions are shown by alignment with wild-type diphtheria toxin in Sequence ID No. 1.

[0036] In this application, the terms "DTR" or "DTR domain" mean the R domain of diphtheria toxin corresponding to residues 380-385 to 531-535 of the amino acid sequence of wild-type diphtheria toxin (SEQ ID NO: 1). The expression "DTR protein" means a recombinant protein containing an isolated DTR domain, i.e., a recombinant protein lacking the T domain or C domain and T domain sequence of diphtheria toxin, and the sequence of a protein or protein domain that can improve the stability or purification of the R domain, at its N-terminus and C-terminus. A recombinant protein containing at least one, preferably at least two, substitutions as defined above is a wild-type DTR recombinant protein (DTR) that does not contain these substitutions. WT In contrast, these are called mutant DTR proteins or DTR proteins with mutations introduced.

[0037] The similarity of amino acid sequences to a reference sequence is assessed according to the percentage of amino acid residues that are identical or differ via conservative substitutions when the two sequences are aligned to obtain the greatest possible match between them. If only identical residues are considered and the percentage of identical residues is determined, the identity of the amino acid sequence to the reference sequence is referenced. For the purposes of this invention, the expression "conservative substitution in the amino acid sequence of a protein" means substituting one amino acid with another natural or synthetic amino acid that has similar chemical or physical properties (size, charge, or polarity) and does not adversely affect the biological activity of the protein. Thus, two amino acid sequences of proteins are similar if they differ from each other by amino acid substitutions at positions that do not adversely affect the biological activity of the protein, or by deletions and / or insertions of amino acids or a small number of amino acids (generally fewer than five). Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved by various methods known to those skilled in the art, for example, by using publicly available computer software such as the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pile-up program, or by using any of the sequence comparison algorithms such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-410), FASTA, or CLUSTALW. When using such software, default parameters are preferably used on a comparison window consisting of residues 380-535 of the amino acid sequence of SEQ ID NO: 1. The BLASTP program uses a word length (W) of 3 and an expected value (E) of 10 by default.

[0038] In some specific embodiments, the HB-EGF inhibitor for combination therapy according to the present invention comprises at least substitutions to the amino acid sequence of SEQ ID NO: 1: Y380K and L390T; Y380K and Q387E; Y380E and L390T or Y380E and Q387K; preferably, a recombinant protein comprising an amino acid sequence comprising the isolated R domain of diphtheria toxin comprising at least substitutions to the amino acid sequence of SEQ ID NO: 1: Y380K or Y380E and L390T. Preferably, the recombinant protein further comprises at least one substitution selected from the group consisting of F389Y, A395T, N399K, T436H, V452T, V483Q, H492E, S494K, E497D, G510A and T517E. Preferably, the recombinant protein comprises an amino acid sequence having at least 70% identity with residues 380-535 of SEQ ID NO: 1.

[0039] In some preferred embodiments, the HB-EGF inhibitor for combination therapy according to the present invention is a recombinant protein comprising substitutions Y380K, Q387E, L390T, A395T, F389Y, G510A, N399K, V452T, T517E, V483Q, H492E, and S494K relative to SEQ ID NO: 1. Preferably, the recombinant protein comprises the amino acid sequence of SEQ ID NO: 2. This protein is called DTR8. It has a molecular weight of 17327 Da.

[0040] These recombinant DTR proteins, ligands for HB-EGF and pro-HB-EGF, offer the following advantages:

[0041] -These are wild-type forms of DTR WTIt exhibits significantly increased solubility compared to wild-type DTR protein (DTR) produced in the same expression system. By substituting at least one, preferably at least two, of residues Y380, P382, Q387, P388 and / or L390 with different hydrophilic, polar, or charged amino acid residues, the solubility of DTR protein in aqueous solution can be considerably increased. Therefore, they can be extracted and purified from host cells without the use of surfactants, chaotropic agents, or denaturants. In comparison, wild-type DTR protein produced in the same expression system (DTR) WT ) is insoluble and cannot be solubilized using surfactants suitable for therapeutic use. DTR WT Proteins are solubilized in the presence of 0.5% sarcosyl or sodium dodecyl sulfate, but sarcosyl or sodium dodecyl sulfate are surfactants unsuitable for therapeutic use at these concentrations.

[0042] -Those are DTR WT It is far easier to produce them in recombinant form than in other forms. They are produced directly without the use of fusion partners to improve the stability or purification of the R domain. They can be produced in E. coli in a folded, soluble form and in large quantities (tens of mg / l cultures under unoptimized laboratory conditions) after final purification, following a standard fermentation procedure.

[0043] -They have an affinity for HB-EGF and pro-HB-EGF that is at least 10 times greater than the affinity for CRM197. Surprisingly, wild-type DTR protein (DTR WT) has an affinity for HB-EGF and pro-HB-EGF that is two-fold lower than CRM197. However, the mutant DTR protein has a considerably higher affinity for HB-EGF and pro-HB-EGF than CRM197. The protein called DTR8 (SEQ ID NO: 2) has an affinity 1400-fold higher than CRM197. This means that DTR8 can be used at a much lower dose than CRM197 for the same therapeutic effect. As a result, the risk of side effects generally associated with the presence of low-affinity binding to other proteins or ligands of the organism is thus considerably reduced compared to CRM197. These interactions are eliminated by using low doses on the order of pM concentrations, which should apply in the case of DTR8.

[0044] - They are less immunogenic compared to CRM197 and DTR WT Ten out of the 26 individual CD4-T cell epitopes identified in DTR WT are deleted from the DTR8 protein by site-directed mutagenesis, including 7 out of the 9 immunodominant epitopes.

[0045] - They have significantly reduced antigenicity compared to CRM197 and DTR WT Serum from individuals vaccinated against diphtheria toxin preferentially recognizes the catalytic domain of the toxin. This domain is present in the CRM197 molecule and not in the mutagenized DTR proteins according to the invention. Furthermore, the mutagenized DTR proteins according to the invention are not recognized as well by antibodies from vaccinated subjects that recognize DTR WT as DTR WT ​​​​​​​​- They are small in size; they have a sequence of 157 amino acids and a MW of approximately 17,500 Da, i.e., 1 / 3.4 the size of CRM197 and 1 / 8.8 the size of anti-HB-EGF antibodies currently used in clinical protocols to block the HB-EGF pathway. As a result of their small size, recombinant proteins are more effective in treating renal diseases associated with activation of the HB-EGF pathway, because recombinant proteins can diffuse more easily into tissues, pass through the glomerular filtration barrier, and thereby penetrate the renal glomeruli.

[0048] In some embodiments of the present invention, the combination includes a nucleic acid molecule that encodes an inhibitor.

[0049] Nucleic acids can be recombinant, synthetic, or semi-synthetic nucleic acids that can be expressed in target cells for in vivo protein production. These nucleic acids may be DNA, RNA, or a mixture of molecules, and may be single-stranded and / or double-stranded, which may be further modified and / or included in any suitable expression vector. The nucleic acids may contain coding sequences optimized for the cells in which the protein is expressed. As used herein, the terms “vector” and “expression vector” mean a vehicle capable of introducing a DNA or RNA sequence (e.g., a foreign gene) into host cells to transform the host and promote the expression of the introduced sequence (e.g., transcription and translation (DNA) or translation (RNA)). Expression vectors as defined herein are suitable for protein expression in target cells and are particularly suitable for gene therapy. Such vectors known in the art include viral vectors and non-viral vectors. Non-viral vectors include plasmids. Examples of viral vectors include adenoviruses, adeno-associated viruses, lentiviruses, and the like.

[0050] In some more specific embodiments, the combination includes an expression vector containing the nucleic acid molecule.

[0051] Corticosteroids In the present invention, the terms "corticosteroid" and "corticoid" are used interchangeably and refer to corticosteroid drugs. Non-limiting examples of corticosteroids that can be used in the combination therapy of the present invention include prednisolone, betamethasone, prednisone, triamcinolone, methylprednisolone, dexamethasone, derivatives thereof, and mixtures thereof.

[0052] In some embodiments, the corticosteroid for combination therapy according to the present invention is selected from the group consisting of prednisolone, betamethasone, prednisone, triamcinolone, methylprednisolone, dexamethasone, their derivatives, and mixtures thereof. In some specific embodiments, the corticosteroid included in the combination for use according to the present invention is prednisolone or prednisone.

[0053] Combination therapy As used herein, the terms “combination,” “therapeutic combination,” “combination administration,” or “combination therapy” in the context of the present invention refer to administering a corticosteroid and an HB-EGF inhibitor to a patient in order to obtain therapeutic benefits.

[0054] As used herein, “treatment” or “to treat” is defined as applying or administering a combination of a corticosteroid and an HB-EGF inhibitor to a patient with renal disease involving activation of the HB-EGF / EGFR pathway for the purpose of curing, resolving, alleviating, reducing, altering, correcting, improving, or influencing the disease or any symptoms thereof, or applying or administering such therapeutic combination to tissues or cell lines isolated from such patient. The terms “treatment” or “to treat” are also used in the context of administering a therapeutic agent prophylactically. In particular, the terms “to treat” or “to treat” refer to reducing or alleviating at least one adverse clinical symptom associated with renal disease involving activation of the HB-EGF / EGFR pathway. Adverse clinical symptoms include, in particular, nephritis syndrome and renal failure. Nephritis syndrome includes, in particular, hematuria, hyperproteinuria, hypoalbuminemia, hypercreatinine and urea nitrogen.

[0055] In some embodiments, the terms “treat” or “treatment” refer to delaying or reversing the progression of glomerular lesions and / or reducing the severity of glomerular lesions. The progression or severity of glomerular lesions may be determined by standard assays known in the art and disclosed in the examples of this application, such as the percentage of glomeruli occluded by fibrosis (grade 4 FSGS lesions). Combination treatment reduces the percentage of glomeruli occluded by fibrosis compared to an untreated control or a control treated with a corticosteroid alone or an HB-EGF inhibitor alone (Figure 2).

[0056] In some embodiments, the terms “treat” or “treatment” refer to delaying or reversing the decline in renal function. The progression or severity of glomerular lesions may be determined by standard assays, well known in the art and disclosed in the examples of this application, e.g., blood markers of renal function, particularly blood chemistry markers of urinary function: creatininemia, blood urea nitrogen, albuminemia, and combinations thereof. The percentage of individuals with normal blood markers of renal function is increased by combination treatment compared to vehicle controls or controls treated with corticosteroids alone or HB-EGF inhibitors alone (Figure 3). Similarly, combination treatment reduces the reduction in disease-induced albuminemia compared to DTR8 or corticosteroid treatment alone or vehicle controls (Figure 4).

[0057] As used herein, the terms “patient,” “individual,” or “subject” include human and other mammalian subjects receiving either prophylactic or therapeutic treatment. Preferably, the patient, individual, or subject according to the present invention is human.

[0058] The combination therapy according to the present invention may be used to treat various kidney diseases associated with (or involving) activation of the HB-EGF / EGFR pathway, particularly crescentic glomerulonephritis (CGN) and related diseases, such as focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, autosomal dominant polycystic kidney disease (ADPKD), renal interstitial fibrosis (IF), chronic kidney disease (CKD), and hypertensive glomerulosclerosis (HTN); more preferably, various kidney diseases selected from the group consisting of crescentic glomerulonephritis (CGN) and related diseases, such as focal segmental glomerulosclerosis (FSGS).

[0059] The terms "crescentic glomerulonephritis" (CGN) and "rapidly progressive glomerulonephritis" (RPGN) are used interchangeably in this application and refer to the same disease (MSD Manual). CGN is characterized by crescent-shaped glomeruli. When collagen deposits replace crescent-shaped glomeruli, the crescent shape is called "sclerosing," or the disease is called focal segmental glomerulosclerosis (FSGS). Therefore, FSGS is a progressive form of CGN (or CGN-related disease) as included in this invention.

[0060] Combination therapy may be used for maintenance of diseases such as crescentic glomerulonephritis (CGN), or for treatment of acute or relapsed conditions.

[0061] In some embodiments, the combination therapy is used for the treatment of the acute or recurrent phase of the disease, preferably for the treatment of the acute or recurrent phase of crescentic glomerulonephritis (CGN).

[0062] Preferably, the combined treatment according to the present invention reduces the severity of glomerular lesions and / or reduces the decline in renal function.

[0063] The HB-EGF inhibitor and the corticosteroid may be in the same pharmaceutical composition or in separate compositions. In various embodiments of the present invention, the pharmaceutical composition or combination of pharmaceutical compositions comprises a therapeutically effective dose of the HB-EGF inhibitor and the corticosteroid. In the context of the present invention, a therapeutically effective dose means a dose sufficient to reverse, alleviate or inhibit the progression of the disorder or condition to which such terms apply, or to reverse, alleviate or inhibit the progression of one or more symptoms of the disorder or condition to which such terms apply. The terms “effective dose” or “effective dosage” are defined as a dose sufficient to achieve, or at least partially achieve, the desired effect.

[0064] The effective dose is determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration, including sex, age, and weight, concomitant medications, and other factors recognized by those skilled in the medical field.

[0065] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0066] A "pharmaceutically acceptable carrier" means a vehicle that, when administered to a mammal, particularly a human, as necessary, does not cause adverse reactions, allergic reactions, or other undesirable reactions. A pharmaceutically acceptable carrier or excipient means any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0067] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline (such as sodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a mixture of such salts), or a dry composition, especially a lyophilized composition, to which sterile water or saline may be added as needed to form an injectable solution. Suitable pharmaceutical forms for injection include sterile aqueous solutions or suspensions. The solution or suspension may contain corticosteroids, proteins, nucleic acids, and vector-compatible additives. In all cases, the form must be sterile and fluid enough to be easily drawn with a syringe. It must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi. Examples of suitable solutions are buffers, such as phosphate-buffered saline (PBS) or Ringer's lactate solution.

[0068] Therapeutic combinations may include at least one other therapeutic agent for treating the disease, such as an anti-inflammatory agent, an immunosuppressant, or another therapeutic agent. Specific examples of such other therapeutic agents include cyclophosphamide, methotrexate, mycophenolate mofetil, anti-CD20 agents such as rituximab, and complement C5a receptor inhibitors such as avacopan.

[0069] The therapeutic combination of the present invention is generally administered in a dose and duration effective to induce a therapeutic effect in a patient, following known procedures. The administration of the combination according to the present invention can be carried out by any method that allows for the delivery of the compounds to the site of action. HB-EGF inhibitors and corticosteroids may be administered in single or multiple doses by any acceptable mode of administration of drugs having similar utility. The pharmaceutical composition may be administered by any convenient route, such as in a non-limiting manner by infusion or bolus injection, or by absorption via the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa). Administration may be systemic, topical, or systemic in combination with topical administration; systemic includes parenteral and oral administration, and topical includes topical and topical areas. Systemic administration is preferably parenteral, such as subcutaneous (SC) and intramuscular (IM), intravascular, such as intravenous (IV) or intra-arterial; intraperitoneal (IP); intradermal (ID), etc. In some preferred embodiments, administration is oral or parenteral, and parenteral is preferably intravascular, intramuscular, or subcutaneous.

[0070] The use of the term “combined” does not restrict the order of administration of therapies (e.g., corticosteroids and HB-EGF inhibitors) to the target population. Therapies may be administered before (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to patients with a disease associated with activation of the HB-EGF / EGFR pathway. These therapies are administered to the patient in an order and time interval such that they can act together. In certain embodiments, therapies are administered to the subject in an order and time interval such that they provide greater benefits than if they were administered in any other way. Any additional therapies may be administered in any order with other additional therapies.

[0071] In some embodiments, the HB-EGF inhibitor and the corticosteroid are administered simultaneously.

[0072] In other embodiments, the HB-EGF inhibitor and the corticosteroid are administered separately.

[0073] In some embodiments, corticosteroids and / or HB-EGF inhibitors are administered orally or by injection, particularly IV, IM, or SC. In some specific embodiments, corticosteroids are administered orally or by intravenous injection, and HB-EGF inhibitors are administered intravenously or by subcutaneous injection.

[0074] In some embodiments, the therapeutic combination includes an HB-EGF inhibitor at a dose of 0.6 mg / kg per day. The daily dose of the inhibitor may be a single dose or two separate doses, preferably a single dose. In some specific embodiments, the HB-EGF inhibitor is DTR8, and preferably the therapeutic combination includes DTR8 at a dose of 0.6 mg / kg per day, more preferably a single dose.

[0075] Combination therapy allows for the administration of reduced doses of corticosteroids, particularly at the onset (acute phase) or relapse phase of the disease, thereby avoiding undesirable side effects associated with high doses of corticosteroids in current therapies for the disease. In particular, combination therapy avoids the use of high starting doses of corticosteroids at the onset or relapse of the disease. The starting dose of corticosteroids administered at the onset of the disease typically corresponds to 1 g / day IV for 3 to 5 days, as shown in Figure 1 illustrating the reference treatment. The low dose of corticosteroids in the combination therapy of the present invention is at least 3 times, and particularly at least 5, 10, 15, 20, 25, 30, and 100 times, lower than the starting dose of corticosteroids administered at the onset of the disease in the reference treatment. Preferably, the low dose of corticosteroids in the combination therapy of the present invention is lower than the initial dose of corticosteroids administered orally after the starting dose (intravenous administration) in the reference treatment. The initial dose administered after the starting dose is typically 0.5–1 mg / kg (maximum 80 mg) orally for 3–4 weeks, as shown in Figure 1 illustrating the reference treatment.

[0076] In some specific embodiments, the low dose of corticosteroids is less than 80 mg per day, typically 30 mg / day administered orally after the initial dose (intravenous administration), and reduced to 7.5 mg / day by 3 months and 5 mg / day by 6 months.

[0077] In some specific embodiments, the low dose of corticosteroid is less than 1 mg / kg per day and is preferably administered for at least 3 weeks at the onset or relapse of the disease. In some specific embodiments, the low dose of corticosteroid is about 0.5 mg / kg per day and is preferably administered for at least 3 weeks at the onset or relapse of the disease.

[0078] In some specific embodiments, the low dose of corticosteroid is less than 1 mg / kg per day and is administered at the onset or relapse of the disease, preferably for a maximum of 6 months, preferably for a maximum of 5 months, preferably for a maximum of 4 months, and more preferably for a maximum of 3 months. In some specific embodiments, the low dose of corticosteroid is about 0.5 mg / kg per day and is administered at the onset or relapse of the disease, preferably for a maximum of 6 months, preferably for a maximum of 5 months, preferably for a maximum of 4 months, and more preferably for a maximum of 3 months.

[0079] In some specific embodiments, low doses of corticosteroids are administered intravenously or orally.

[0080] In some specific embodiments, the corticosteroid is prednisolone, prednisone, or a derivative thereof.

[0081] In some specific embodiments, a low dose of a corticosteroid and / or a dose of an HB-EGF inhibitor is administered daily for at least three weeks.

[0082] In some specific embodiments, a low dose of a corticosteroid and / or a dose of an HB-EGF inhibitor is administered daily for up to 6 months, preferably up to 5 months, preferably up to 4 months, and more preferably up to 3 months.

[0083] An example of combination therapy includes DTR8 and prednisolone administered at the onset or recurrence of the disease, preferably DTR8 administered via saccharin or intravenous administration at a dose of 0.6 mg / kg / day, and prednisolone or prednisone administered via saccharin or orally at a maximum dose of 1 mg / kg / day, preferably up to 0.5 mg / kg / day, and particularly 0.37 mg / kg / day, administered via saccharin or orally. The combination therapy is preferably administered for at least 3 weeks.

[0084] An example of combination therapy includes DTR8 and prednisolone administered at the onset or recurrence of the disease, preferably DTR8 administered via SC or IV at 0.6 mg / kg / day, and prednisolone or prednisone administered via SC or orally at a maximum of 1 mg / kg / day, preferably up to 0.5 mg / kg / day, and particularly 0.37 mg / kg / day, administered via SC or orally. The combination therapy is preferably administered for a maximum of 6 months, preferably up to 5 months, preferably up to 4 months, and even more preferably up to 3 months.

[0085] The present invention also provides a method for treating renal disease involving HB-EGF / EGFR activation, comprising administering a therapeutically effective dose of the therapeutic combination according to the present disclosure to a patient.

[0086] The present invention also provides the use of therapeutic combinations according to the present disclosure for the treatment of renal diseases involving activation of the HB-EGF / EGFR pathway according to the present disclosure.

[0087] The present invention provides the use of a combination drug comprising an HB-EGF inhibitor and a corticosteroid according to the present invention in the manufacture of a drug for treating renal disease associated with HB-EGF / EGFR activation according to the present invention.

[0088] The present invention provides a pharmaceutical composition for treating renal disease involving HB-EGF / EGFR activation according to the present invention, comprising a therapeutic combination of an HB-EGF inhibitor and a corticosteroid as active ingredients.

[0089] The present invention provides a pharmaceutical composition comprising a therapeutic combination according to the present invention, comprising an HB-EGF inhibitor and a corticosteroid, for treating renal disease involving HB-EGF / EGFR activation according to the present invention.

[0090] In the various therapeutic uses and methods used herein, the therapeutically combined HB-EGF inhibitor and corticosteroid may be in the same pharmaceutical composition or in separate pharmaceutical compositions.

[0091] Another aspect of the present invention relates to a product containing a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor as a combination formulation for concurrent, individual, or sequential use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway as disclosed herein.

[0092] The present invention also relates to a pharmaceutical composition for treating renal diseases associated with activation of the HB-EGF / EGF receptor pathway, comprising an HB-EGF inhibitor and a corticosteroid at a lower dose compared to the starting dose used in reference treatment with a corticosteroid according to the present disclosure.

[0093] The present invention also relates to a pharmaceutical composition comprising the HB-EGF inhibitor DTR8 (SEQ ID NO: 2) and a corticosteroid according to the present disclosure.

[0094] Various embodiments of this disclosure can be combined with one another, and this disclosure encompasses various combinations of embodiments of this disclosure.

[0095] Unless otherwise indicated, the implementation of this invention will employ prior art within the scope of the art, which is fully described in the literature.

[0096] The present invention is illustrated by the following embodiments, which are not limited to the present invention, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0097] [Figure 1] Comparison of corticosteroid dosages for the treatment of CGN using prior art (reference treatment) and the combination therapy of the present invention (O'Brien et al., MSD Manual Professional Version., 2022; Yates et al., Annals of the Rheumatic Diseases, 75, 1583-1594, 2016). [Figure 2]Figure 2 shows the percentage of glomeruli with grade 4 FSGS lesions on kidney sections from experimental animals, stained with Masson's trichrome and observed under a light microscope. Grade 4 FSGS lesions are defined as covering 75%–100% of the glomerulus. Data were analyzed using the Kruskal-Wallis test with corrections for multiple comparisons. *p<0.05. [Figure 3] Figure 3 shows the percentage of animals in each experimental group with normal levels of blood chemistry markers of urinary function: creatininemia, serum urea nitrogen, and albuminemia. Animals were counted when the markers were within the normal range for all tested samples (days 0, 1, 4, 8, 14, and 21). [Figure 4] Figure 4 shows the percentage change in albuminemia in animals in each experimental group. The percentage change was calculated from the value at the end of the experiment (day 21) compared to the value on day 0 before injection of NTS (nephrotoxic serum that induces disease). [Examples]

[0098] Materials and methods animal Female wild-type domestic pigs (Sus scrofa domesticus) were used in this study. All animal experiment procedures followed European Community guidelines (L358-86 / 609EEC) and were approved by the Department of Higher Education and Scientific Research (APAFIS#13259-2017121809512560v3). The animals were individually housed at the Center for Biomedical Research facilities of the National Veterinary School in Maison-Alfort, France (Approval E940462).

[0099] Nephrotoxic serum (NTS) Sheep nephrotoxic serum containing polyclonal anti-porcine glomerular basement membrane (GBM) antibodies was obtained by heterologous immunization. Whole kidneys were obtained from healthy pigs. Cortical tissue was dissected and digested in type I collagenase (17100-017, GIBCO). The tissue samples were then filtered through several sieves. The well-purified samples (over 80% glomerular) were mixed, centrifuged, and resuspended in ultrapure water to induce hypotonic cell swelling and lysis, and intracellular proteins were removed from the supernatant. The remaining proteins, essentially GBM-derived, were dissociated from the cell fragments by sonication on ice (Vibra-Cell, Sonics & Materials, Inc., Newtown, CT, USA). Six sheep were immunized with GBM extract via four weekly intradermal injections (1 mg of protein in a complete Freund's adjuvant), followed by a single intravenous infusion (1 mg of protein, without Freund's adjuvant) four weeks after the final intradermal injection.

[0100] Serum was collected from living sheep one week after euthanasia. Each individual serum was tested for GBM specificity in pig kidney tissue sections by immunofluorescence. Specific serums were pooled and centrifuged. Finally, the supernatant was thermally inactivated at 56°C in a water bath for complement inactivation and filtered twice consecutively through 0.45 and 0.20 μm filters. Aliquots of NTS were frozen (-80°C) until experiment time.

[0101] Induction of crescentic glomerulonephritis The porcine model for CGN was adapted from the accelerated anti-GBM CGN model described in rodents (Bollee et al., Nat. med., 2011, 17, 1242-1250). Two-to-three-month-old female pigs (average body weight 27 kg) were pre-immunized seven days before the start of the protocol by subcutaneous injection of 500 μg of purified sheep IgG (I5131, Sigma) diluted in 1 mL of sterile PBS and 1 mL of incomplete Freund's adjuvant (F5881, Sigma). Each individual was given intravenous injection of NTS for two consecutive days (0.1 mL / kg body weight on day 0 and 0.2 mL / kg on day 1) to induce the accelerated CGN model.

[0102] Pigs were administered intramuscularly at a dose of 9 mg of DTR8 twice daily, starting in the afternoon of day 1, or / or an oral dose of 10 mg of prednisolone (Sandoz) in an apple as a vehicle. The vehicle-treated group (placebo group) received both intramuscular injection of buffer and an empty apple. The DTR8-treated group received an empty apple as a vehicle control for CTC, and the CTC-treated group received intramuscular injection of buffer as a vehicle control for DTR8 injection. Animals were euthanized on day 21, and kidney samples were collected.

[0103] Biochemical measurements of blood and urine All clinical chemistry measurements were performed at the MEDICAL Biology Laboratory of the CEA Saclay Center using a Cobas 6000 / C501 analyzer. Blood samples were collected on days 0, 1, 4, 8, 14, and 21. Urine samples were collected on days 0, 8, 14, and 21. Creatinemia was measured using the creatininase / creatinase method. Serum urea was measured using the kinetic urease-glutamate dehydrogenase method. Albuminemia was measured using the bromocresol green method. Total urine protein concentration was quantified by turbidimetry using the EDTA / benzethonium method.

[0104] histology Pig kidney samples were fixed in 10% formalin, embedded in paraffin, and then 4 μm thick sections were treated for Masson's trichrome staining. Approximately 100–200 glomerular sections were counted per individual to determine the percentage of healthy glomeruli, crescents, and FSGS lesions. The severity of FSGS lesions was assessed by the surface of glomerular sections exhibiting pathological features. Grade 4 FSGS lesions were defined as those covering 75%–100% of the glomerulus. Data were analyzed using the Kruskal-Wallis test with correction for multiple comparisons using GraphPad Prism software (GraphPad Software, La Jolla, CA).

[0105] result Establishment of a CGN model in pigs CGN was induced in pigs by injecting them with sheep antiserum against purified porcine glomerular basement membrane (GBM), as previously described in mice (Bollee et al., Nat. Med., 2011, 17, 1242-1250). This antiserum, named nephrotoxic serum (NTS), mimics anti-GBM autoantibodies found in patients with Goodpasture syndrome, one of the four major causes of CGN. Control animals were given sheep-derived serum collected before immunization with GBM instead of NTS. Immunofluorescence (IF) detection of sheep IgG on kidney sections from NTS-treated pigs showed their deposition at the interface between glomerular capillaries and podocytes separated by GBM. After 4–8 days, the pigs gradually developed nephrotic syndrome with proteinuria, hypoalbuminemia, hematuria, subcutaneous edema, and ascites.

[0106] Twenty-one days after disease induction, the animals were sacrificed and their kidneys were subjected to histological analysis. Masson's trichrome staining revealed the presence of crescent-shaped cells and fibrous cells, Bowman's capsule destruction with varying degrees of FSGS, tubular damage, and interstitial fibrosis. These features are typical of human CGN. Interestingly, healthy glomeruli coexist with altered glomeruli, as observed in patient biopsies at disease onset. Healthy control pigs that received preimmunized serum showed no lesions at all. Immunohistochemistry using anti-HB-EGF antibody confirmed HB-EGF expression in crescent-shaped lesions, in contrast to normal glomeruli from healthy control animals. However, as expected, healthy glomeruli from CGN animals also expressed HB-EGF (Bollee et al., Nat. Med., 2011, 17, 1242-1250). 86% of animals administered the vehicle showed altered levels of urinary function blood markers (albuminemia, creatininemia, and blood urea nitrogen) (Figure 3). The animals developed proteinuria, and albuminemia decreased by at least 20% by day 21 compared to day 0 (Figure 4).

[0107] Overall, the data indicate that pigs treated with NTS develop a set of histopathological features characteristic of chronic neuropathy (CGN). In conclusion, this pig model is well-suited for evaluating innovative therapies, such as the combination of DTR8 and low-dose CTC, to induce rapid glomerular protection against the cellular and functional consequences of immune injury in CGN.

[0108] Comparative evaluation of DTR8, CTC, and combinations thereof as treatments for CGN. Table 1 summarizes the study design. Four groups of animals were given NTS and treated as shown. A dose of 9 mg of DTR8 (SEQ ID NO: 2) was administered twice daily for 3 weeks via the intramuscular (IM) route to approximately 30 kg pigs. The IM route was chosen because the SC route (twice daily administration) was not feasible in pigs. This dose was administered for 20 hours per day at 100 × K d To achieve the above plasma concentrations, the following were estimated according to pharmacokinetic experiments and simulations (2 pM). <K d <4 pM (WO2013 / 140335). DTR8 is a globular protein with a density of 17.3 kDa. Therefore, it is thought to be filtered by glomeruli with both altered and unaltered GBM (Choi et al., Nat. Biotechnol., 2009, 25, 1165-1170). At such concentrations, podocytes and PECs located within the filtration chamber and expressing HB-EGF would be sufficiently exposed to the protein. An oral dose of 10 mg / day of prednisolone over 3 weeks corresponds to approximately 1 / 3 of the oral dose given in patients at the onset of the disease (after an initial dose of 1 g / day (IV) over 3-5 days), or 3-4 times the maintenance dose given after 5 months of treatment (O'Brien et al., MSD Manual Professional Version., 2022). This is consistent with the recommended dose of corticosteroids for maintenance therapy. This dose was chosen to evaluate whether the therapeutic effect of DTR8 compensates for the reduction in corticosteroids at the time of onset.

[0109] [Table 1]

[0110] The combination of DTR8 and CTC reduces the severity of FSGS lesions compared to CTC or DTR8 alone. Most interestingly, combined treatment with DTR8 and CTC dramatically reduced the severity of FSGS lesions (p<0.05). Figure 2 shows the percentage of glomeruli with grade 4 FSGS lesions on kidney sections from experimental animals, stained with Masson's trichrome and observed under a light microscope. Grade 4 FSGS lesions are defined as those covering 75%–100% of the glomerulus. Data were analyzed using the Kruskal-Wallis test with corrections for multiple comparisons. On average, 10% of vehicle-treated animals had glomeruli showing terminal FSGS lesions (grade 4 FSGS lesions) where the entire glomerulus was occluded by fibrosis. This percentage was 8% in DTR8 animals, 4% in CTC animals, and 2% in combined treatment animals. These results indicate a significant reduction in the severity of FSGS lesions when DTR8 is combined with CTC, even at lower doses of CTC than those typically given at the onset of the disease. Therefore, adding DTR8 to the treatment of CGN allows for a reduction in the dose of CTC.

[0111] The combination of DTR8 and CTC increases the proportion of animals without urinary blood chemistry abnormalities. During the experiment, blood samples were collected from animals on days 0, 1, 4, 8, 14, and 21. Serum creatinine, blood urea nitrogen, and albumin levels were monitored. Most vehicle-treated animals experienced a progressive decrease in albuminemia, reaching levels below or well below normal. Similarly, abnormalities in serum creatinine and blood urea nitrogen were observed in these animals. Figure 3 shows the percentage of animals in each experimental group that maintained normal levels for these three markers of renal function. The combined treatment protected 43% of animals from urinary blood chemistry abnormalities. In contrast, only 30% of CTC-treated animals, 20% of DTR8-treated animals, and 14% of vehicle-treated animals lacked these functional abnormalities. In summary, combining DTR8 and CTC treatment provided better protection of renal function in animals subjected to immune attack against the glomeruli than each treatment alone.

[0112] The combination of DTR8 and CTC reduces albuminemia fluctuations more effectively than DTR8 or CTC alone. The glomerular filtration barrier retains nanoparticles or proteins larger than 6–8 nm or approximately 70 kDa (Du et al., Nat. Nanotechnol., 2017, 12, 1096-1102; Lawrence and al., Proc. Natl. Acad. Sci., 2017, 114, 2958-2963). Therefore, albumin is retained in circulation. A decrease in serum albumin levels is associated with changes in the filtration barrier. To assess the level of such functional changes in renal filtration cutoff, the percentage change in serum albumin between day 0 and day 21 was calculated for each animal (Figure 4). In vehicle-treated animals, the median albuminemia decreased by 35%. In contrast, in animals treated with a combination of DTR8 and CTC, the reduction was only 24%. DTR8 alone did not improve albuminemia (median reduction of 37%), and CTC alone resulted in slight improvement (median reduction of 30%), but less than when combined with DTR8. Overall, the results indicate that the combination of DTR8 and CTC treatment reduced changes in albuminemia in CGN animals more effectively than CTC or DTR8 alone, with the latter being ineffective.

[0113] (summary) Overall, the results indicate that DTR8 administration improves the histological signs and functional parameters of the kidney, reducing the need for high doses of cytotoxic tetracycline (CTC) during the initial treatment of chronic neuropathy (CGN). In particular, combined treatment with DTR8 and lower doses of CTC than those used at disease onset not only statistically significantly reduced the severity of FSGS lesions (p<0.05) but also resulted in a higher proportion of animals without abnormal urinary blood chemistry (43% in the DTR8+CTC group vs. 30% in the CTC group, 20% in the DTR8 group, and 14% in the vehicle group). These are proof-of-concept studies demonstrating the efficacy of DTR8 as an HB-EGF inhibitor for treating CGN at disease onset and its ability to enable reduced CTC dosage during early treatment, in a large animal model.

[0114] The sequence disclosed in this application Sequence ID 1: Wild-type diphtheria toxin GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKY DAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGT EEFIKRFGDGASRVVLSLFPAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYE YMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTTKKIESLKEHGPIKNKMSESPNKTVSE EKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKT TAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNF VESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKI TAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVG NGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0115] அக்க்கு நுர்க்கு2:DTR8 GKSPGHKTEPYTHDGYTVSWKTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISTNGRKIRMRCRAIDGDVTFCRKPSKSPVYVGNGQHANLHVAFERKSSE KIHSNEISSSDSIAVLGYQKEVDHTKVNSKLSLFFEIKS

Claims

1. A combination of a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor selected from anti-HB-EGF antibodies and diphtheria toxin-derived HB-EGF ligands, for use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway.

2. The combination for use according to claim 1, wherein the disease is selected from the group consisting of crescentic glomerulonephritis (CGN) and related diseases such as focal segmental glomerulosclerosis (FSGS), renal interstitial fibrosis (IF), chronic kidney disease (CKD), and hypertensive arteriolar nephrosclerosis (HTN).

3. The combination for use according to claim 1 or 2, wherein the treatment by the combination comprises the administration of a low dose of a corticosteroid agent consisting of less than 1 mg / kg per day, preferably less than 80 mg per day.

4. A combination of uses according to any one of claims 1 to 3 for use in treating the acute phase or recurrence of the aforementioned disease.

5. The combination for use according to any one of claims 1 to 4, wherein the disease is crescentic glomerulonephritis (CGN) or focal segmental glomerulosclerosis (FSGS).

6. The combination for use according to any one of claims 1 to 5, wherein the treatment reduces the severity of the glomerular lesion and / or reduces the decline in renal function.

7. The combination for use according to any one of claims 1 to 6, wherein the HB-EGF inhibitor is a recombinant protein comprising the isolated R domain of the diphtheria toxin, comprising at least substitutions to the amino acid sequence of SEQ ID NO: L390T and Y380K; L390T and Y380E; Y380K and Q387E; or Y380E and Q387K.

8. The recombinant protein comprises substitutions Y380K, Q387E, L390T, A395T, F389Y, G510A, N399K, V452T, T517E, V483Q, H492E, and S494K relative to SEQ ID NO: 1, as per the combination for use described in claim 7.

9. The recombinant protein comprises the amino acid sequence of SEQ ID NO: 2, the combination for use according to claim 8.

10. The corticosteroid agent is selected from the group consisting of prednisolone, betamethasone, prednisone, triamcinolone, methylprednisolone, dexamethasone, and mixtures thereof, as per any one of claims 1 to 9.

11. A combination for use according to any one of the preceding claims, comprising a dose of 0.6 mg / kg of an HB-EGF inhibitor per day.

12. The combination for use according to any one of claims 2 to 11, wherein the low dose of the corticosteroid and / or the dose of the HB-EGF inhibitor is administered daily for at least three weeks and / or up to six months.

13. The combination for use according to any one of the preceding claims, wherein the corticosteroid and / or HB-EGF inhibitor is administered orally or by injection, particularly intravenously or subcutaneously.

14. In particular, a product containing a corticosteroid and a heparin-binding epidermal growth factor-like growth factor (HB-EGF) inhibitor selected from anti-HB-EGF antibodies and diphtheria toxin-derived HB-EGF ligands, as a combination formulation for simultaneous, individual, or sequential use in the treatment of renal diseases associated with activation of the HB-EGF / EGF receptor pathway, selected from the group consisting of crescentic glomerulonephritis (CGN) and related diseases such as focal segmental glomerulosclerosis (FSGS), renal interstitial fibrosis (IF), chronic kidney disease (CKD), and hypertensive arteriolar nephrosis (HTN).

15. A pharmaceutical composition for treating renal disease associated with activation of the HB-EGF / EGF receptor pathway, comprising an HB-EGF inhibitor and a corticosteroid at a lower dose compared to the starting dose of a corticosteroid used in a reference treatment.

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