CN111201241A

CN111201241A - Treatment of inflammatory diseases with inhibitors of C5a activity

Info

Publication number: CN111201241A
Application number: CN201880041303.4A
Authority: CN
Inventors: 郭仁峰; 尼尔斯·R·里德曼
Original assignee: InflaRx GmbH
Current assignee: InflaRx GmbH
Priority date: 2017-06-23
Filing date: 2018-06-13
Publication date: 2020-05-26
Also published as: SG11201912882QA; TW201904611A; KR20200020727A; IL271074A; TWI786132B; JP2020524696A; EP3642230A1; CA3066689A1; WO2018234118A1; CA3066689C; AU2018286754A1

Abstract

The present invention relates to inhibitors of C5a activity and their use in treating a neutrophilic inflammatory disease in the skin of a subject.

Description

Treatment of inflammatory diseases with inhibitors of C5a activity

Technical Field

The present invention relates to inhibitors of C5a activity and their use in treating a neutrophilic inflammatory disorder in the skin of a subject.

Background

Target in inflammation C5a

C5a is a cleavage product of its "parent molecule" C5 spanning 74 amino acids, representing one end of the complement activation cascade. It can be produced by activating at least three well-described pathways (the alternative, classical and MBL pathways). All pathways fuse at the level of C3, forming a C5 convertase or an alternative C5 convertase, cleaving C5 into C5a and C5 b. The latter binds to C6, C7, C8 and multiple C9 molecules, eventually leading to the formation of pores in, for example, bacterial membranes (end-tapping membrane complex ═ MAC). C5a is produced when the complement system is activated in the context of inflammation and other immune and inflammatory conditions/diseases.

Among complement activation products, C5a is one of the most potent inflammatory peptides, with a broad spectrum of functions (Guo and Ward, 2005). C5a functions through the high affinity C5a receptor (C5aR and C5L2) (Ward, 2009). C5aR belongs to the rhodopsin family of G protein-coupled receptors with seven transmembrane segments; C5L2 has a similar structure, but does not appear to be G protein coupled. It is currently believed that C5a exerts biological functions primarily through C5a-C5aR interactions, as the biological response of C5a-C5L2 interactions is rarely found. However, recent reports indicate that signaling is also through C5L2 activation (ritttirsch et al, 2008).

C5aR is widely expressed in myeloid lineage cells, including neutrophils, eosinophils, basophils and monocytes, as well as non-myeloid lineage cells in many organs, especially lung and liver, suggesting the importance of C5a/C5aR signaling. The extensive up-regulation of C5aR expression occurs during the onset of sepsis and blockade of C5a/C5aR interaction by anti-C5 a or anti-C5 aR antibodies or C5aR antagonists is highly protective in rodent models of sepsis (Czermak et al, 1999; Huber-Lang et al, 2001; Riedemann et al, 2002).

C5a has multiple biological functions (Guo and Ward, 2005). C5a is a strong chemoattractant for neutrophils and also has chemotactic activity for monocytes and macrophages. C5a causes an oxidative burst (depletion) in neutrophilsO₂) C5a has been shown to be involved in regulating cytokine expression from various cell types and to enhance expression of adhesion molecules expressed on neutrophils high doses of C5a may lead to a "desensitization" of neutrophil non-specific chemotaxis leading to a wide range of dysfunctions in the experimental setting of sepsis A number of inflammatory diseases may be attributed to the effects of C5a including sepsis, acute lung injury, inflammatory bowel disease, rheumatoid arthritis etc. in the experimental setting of sepsis exposure of neutrophils to C5a may lead to neutrophil dysfunction and signaling pathways leading to NADPH oxidase assembly deficiency, MAPK signaling cascade arrest, oxidative burst, phagocytosis and a dramatic decrease in chemotaxis (Guo et al, 2006; Huber-Lang et al, 2002), thymocyte apoptosis and neutrophil delayed apoptosis are two important pathogenic events in the development of NADPH oxidase, which are dependent on the presence of C5a, during experimental C5a, regulation of C5 on the experimental conditions, activation of macrophage cellular activity in C5-Lang et al, 2002, TNF-IL-5 activation, and macrophage migration of inflammatory cytokines appear to be responsible for the development of acute inflammatory cytokine receptor cascade, macrophage activation, macrophage migration, macrophage activation, and inflammatory response promotion of TNF-5 activation, macrophage activation, cell activation, macrophage activation, cell activation, macrophage activation, cell activation, macrophage activation, cell activation.

In the immune regulatory network of adaptive immunity, C5a affects the crosstalk between Dendritic Cells (DCs) and δ ∈ T cells, which may lead to the production of large amounts of inflammatory mediators, such as IL-17(Xu et al, 2010). The important role of C5a in the generation of a pathogenic Th17 response in Systemic Lupus Erythematosus (SLE) has been identified and defined (Pawaria et al, 2014). In addition, C5a is reported to be a key regulator of Treg cells with a strong inhibitory effect on Treg proliferation and induction (Strainic et al, 2013). Given that Treg and TH17 are indispensable participants in the context of autoimmune diseases, inhibition of C5a signaling is expected to significantly reduce the hyperactive immune state in autoimmune diseases.

IFX-1

IFX-1 is a chimeric monoclonal IgG4 antibody that specifically binds to the soluble human complement lysate C5a. IFX-1 consists of 1328 amino acids and has a molecular weight of about 148472 daltons. The CDR and FR sequences of IFX-1 are disclosed in Table 3 below.

IFX-1 is expressed as a recombinant protein in mammalian CHO cell lines and is ultimately formulated in phosphate buffered saline for intravenous administration. The binding of this antibody to human C5a facilitates the efficient blocking of the biological effects induced by C5a by hindering the binding and reaction of C5a to its corresponding cell-bound receptor.

Various non-clinical studies performed to assess pharmacological and toxicological aspects of IFX-1 can be divided into in vitro/ex vivo tests and in vivo studies, including GLP toxicology studies in cynomolgus macaques (using IFX-1). No non-clinical tests and studies were performed to find that IFX-1 had any toxicological or safety issues. Phase I testing of humans showed no clinically relevant time or dose related changes in safety laboratory parameters, vital signs and ECG parameters.

In vitro analysis of IFX-1 showed that it has strong binding ability to soluble human C5a and high blocking activity against C5 a-induced biological effects such as lysozyme release from human neutrophils or CD11b upregulation in human whole blood neutrophils. An IFX-1 antibody has the ability to neutralize the effects of 2 molecules of C5a with nearly 100% efficiency in an in vitro experimental setting. Clinical trials of IFX-1 are underway to test its clinical efficacy in several inflammatory diseases, including septic organ dysfunction and complex cardiac surgery.

Neutrophils

Neutrophils are terminally differentiated cells with a short circulatory life, and are the most abundant leukocytes in the human body. As a first line of defense against invading microorganisms, neutrophils are characterized by phagocytic function, releasing lytic enzymes from their granules and producing reactive oxygen species upon stimulation. In addition to microbial classes, other stimuli such as immune complexes can also induce respiratory bursts of neutrophils, leading to enhanced inflammation and inflammatory cell recruitment (Kaplan, 2013).

After infiltration of neutrophils into inflamed tissues, they are involved in many other cell types, such as macrophages, Dendritic Cells (DCs), natural killer cells, lymphocytes and mesenchymal stem cells, and regulate innate and adaptive immune responses. For example, neutrophils can regulate maturation of DCs and proliferation and polarization of T cells, and they can also directly trigger antigen-specific T helper type 1 and T helper type 17 cells (Abi Abdallah et al, 2011). Various stimuli induce neutrophil degranulation, including C5a, formyl-methionyl-leucyl-phenylalanine (FMLP), lipopolysaccharide, platelet activating factor and Tumor Necrosis Factor (TNF) (Kaplan, 2013). The contents and oxidative substances released by degranulation and cytokines and chemokines produced by neutrophil activation are the main inflammatory mediators responsible for tissue damage, and it is believed that this mechanism can be attributed to various types of inflammatory tissue damage.

Hidradenitis Suppurativa (HS)

HS is a chronic, devastating skin disease affecting areas rich in the apocrine gland, which is considered to be one of the neutrophil-associated inflammatory diseases of the skin. Nodules appear at the affected area and as the pus is released, the nodules gradually swell and rupture. This process occurs repeatedly, resulting in sinus formation and scarring (Jemec, 2004). This course of disease presents a frustrating situation for both the patient and the physician. The prevalence of time points is reported to be 1% to 4% (Jemec et al, 1996).

The exact pathophysiology of HS is not well defined, smoking, eating habits and genetic susceptibility are all related to HS (Kurzen et al, 2008; slow et al, 2003). the percentage of NK cells increases and the percentage of CD4 lymphocytes decreases compared to healthy controls, which may suggest that the disorder is autoimmune prone IL-1 β and IL-17 have been found to be upregulated in HS lesions, which is associated with activation of the inflamed bodies (Lima et al, 2016). Hidradenitis Suppurativa (HS) is manifested by a large number of neutrophil infiltration into inflamed skin, especially in the late stages of the disease (Lima et al, 2016; Marzano, 2016). activated neutrophils may be an important type of effector cell, causing tissue damage through direct deleterious or indirect regulation of other effector cells (e.g. activated T cells and TH17 in this disease setting).

In a prospective placebo-controlled study, positive results of administration of TNF antagonists further confirm this hypothesis, which led to approval of adalimumab, an antibody to tumor necrosis factor α, for moderate to severe HS patients.

Considering the paradox that both anti-infection (antibiotics) and pro-infection (anti-TNF, steroid corticosteroids, immunosuppressive drugs) therapies may help, HS may appear as an auto-inflammatory disease based on congenital immunodeficiency of the hair follicle (Revuz, 2009), supported by the fact that proinflammatory cytokines such as Interleukin (IL) -1 β and tumor necrosis factor- α tumor necrosis factor in the skin around the lesion and lesion are significantly increased (Wollina et al, 2013).

Dermatosis of neutral tropism

Neutrophilic Dermatosis (ND) is a group of conditions characterized by skin lesions, the histological examination of which shows a strong inflammatory infiltration consisting mainly of neutrophils, with no signs of infection. ND mainly includes Sweet Syndrome (SS), Pyoderma Gangrenosum (PG), subepithelial impetigo dermatosis (SPD), other well-defined entities, and their atypical or transitional forms (Prat et al, 2014). Recently Hidradenitis Suppurativa (HS) has been classified into the ND family based on the large neutrophil infiltration observed in inflamed skin (Lima et al, 2016; Marzano, 2016).

Pyoderma Gangrenosum (PG) and Hidradenitis Suppurativa (HS) are the prototypes of neutrophilic dermatosis, which are considered to be autoinflammatory diseases, both characterized by the accumulation of neutrophils in the skin (Braun-Falco et al, 2012; Marzano et al, 2014). Autoinflammatory syndrome represents an emerging inflammatory condition distinct from autoimmune, allergic and infectious diseases. From a pathophysiological point of view, all autoinflammatory syndromes, such as PAPA (suppurative arthritis, PG and acne), PASH (PG, acne and suppurative hidradenitis) or PASH (suppurative arthritis, acne, PG and suppurative hidradenitis) have a common mechanism, including overactivation of the innate immune system and inflammation of the skin with "sterile" neutrophil enrichment (Cugno et al, 2017).

BADAS (bowel-related skin disease-arthritis syndrome) is characterized by fever, flu-like symptoms, arthritis and inflammatory skin infections. The latter are characterized by lesions causing different neutrophilic dermatosis, such as papules and plaques (Sweet syndrome), pustules and ulcers (pyoderma gangrenosum) or nodules, abscesses or fistulas (hidradenitis suppurativa). In addition, acne and neutrophilic granulocytic panniculitis may also be combined. Patients often experience symmetric non-erosive polyarthritis, which is mainly related to the facet joints (Cugno et al, 2018).

Synovitis, acne, impetigo, hyperostosis, and osteomyelitis (SAPHO) syndromes were initially described in 1987. SAPHO syndrome is a rare disease and can be misdiagnosed. Although the pathogenesis remains elusive, it is increasingly recognized that SAPHO shares similarities with other autoinflammatory diseases (Cugno et al, 2018).

Neutrophil and autoimmune diseases

Autoimmune diseases are defined as the lack of differentiation of self and non-self molecules, resulting in inappropriate recognition of self molecules and tissues as foreign structures, with concomitant immune attack on the host organs. The pathogenesis of autoimmune diseases can generally be divided into two phases, the immune phase and the effector phase. The immune phase is characterized by the presence of autoreactive T lymphocytes. Those T cells then trigger a secondary phase of response leading to tissue damage by activating various other cell types (B cells, cytotoxic T cells, NK cells, neutrophils, macrophages, osteoclasts, fibroblasts, etc.). Activation of these effector cells by autoreactive T cells can be considered to be the effector phase, which can be mediated by various levels, including autoantibody production, cytokine networks, or direct cell-cell contact (Nemeth and Mocsai, 2012).

The role of neutrophils in the pathophysiological development of autoimmune diseases is limited but is gaining increasing attention. Neutrophils may be involved in multiple steps of the autoimmune disease process, including antigen presentation, modulation of other immune cell type activities, and direct tissue damage. Neutrophils expose/release autoantigens upon activation or death due to apoptosis, or during Neutrophil Extracellular Trap (NET) formation. They may also promote tissue deposition of autoantibodies, or, as effector cell types, they may themselves induce tissue damage. Accumulating studies have shown that neutrophils play a positive role in the development of autoimmune diseases, such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), bullous pemphigoid, acquired epidermolysis bullosa, ANCA-associated vasculitis, familial mediterranean fever, coldpirine-associated periodic syndrome (CAPS), and gout, among others (Nemeth and Mocsai, 2012; Nemeth et al, 2016). Skin inflammation is one of the most common syndromes of these autoimmune diseases, since skin is readily the target of the immune response. However, rheumatoid neutrophilic dermatosis is a rare skin manifestation in patients with severe rheumatoid arthritis. It mainly affects patients with severe seropositive rheumatoid arthritis, mainly women (ratio 2: 1), but is also observed in seronegative rheumatoid arthritis (Cugno et al, 2018).

Problem of the invention

As mentioned above, there is a need in the art for effective therapies for the treatment of neutrophilic dermatoses such as Hidradenitis Suppurativa (HS) and neutrophilic autoimmune diseases of the skin.

The inventors have now surprisingly found that molecules inhibiting C5a signalling, such as anti-C5 a antibodies, are very suitable for the treatment of hidradenitis suppurativa. The present inventors also studied the physiological mechanisms leading to neutrophil activation and found that C5a is a key driver of neutrophil activation.

Thus, the inventors contemplate that inhibition of C5a activity would be a suitable therapeutic approach for the treatment of various neutrophilic disorders, particularly neutrophilic inflammatory diseases of the skin.

Disclosure of Invention

In a first aspect, the present invention relates to a compound for use in the treatment of a neutrophilic inflammatory disease of the skin of a subject, wherein the compound is an inhibitor of C5a activity, and wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever, cold imidacloprid-related disorders; gout and senitler syndrome.

In a second aspect, the present invention relates to a method for treating a neutrophilic inflammatory disease in the skin of a subject, the method comprising the steps of:

administering to a subject in need thereof a therapeutically effective amount of a compound, wherein the compound is an inhibitor of C5a activity, and wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); acquired epidermolysis bullosa, persistent erythema upliftum (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

In a third aspect, the present invention relates to the use of a compound for the manufacture of a pharmaceutical composition for the treatment of neutrophilic inflammatory diseases of the skin, wherein said compound is an inhibitor of C5a activity, and wherein said neutrophilic inflammatory diseases of the skin are selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

This summary does not necessarily describe all features of the invention. Other embodiments will become apparent upon reading the following detailed description.

Drawings

FIG. 1: blocking activity of IFX-1 on recombinant human C5a (rhC5a) induced upregulation of CD11b in blood neutrophils. IFX-1-004 and IFX-1-012 represent two different production batches. Human whole blood was incubated with buffer, antibody alone, rhC5a alone, or a combination of different concentrations of antibody and rhC 5a. After incubation, cells were incubated with anti-mouse CD11 b: FITC staining and analysis of CD11b MFI by flow cytometry. Results are expressed as mean ± SD. The percent blocking activity of IFX-1 on C5 a-induced CD11b expression is indicated (arrow). Statistical differences were calculated by one-way anova, with p values of p < 0.05 being statistically significant.

FIG. 2: IFX-1 blocking activity on endogenous C5a (eC5a) driven CD11b upregulation in neutrophils. Zymosan activated human plasma (ZAP) was used as the source of eC 5a. Whole blood was incubated with buffer, IFX-1 alone, ZAP alone, or a combination of IFX-1 and ZAP. After incubation, cells were incubated with anti-mouse CD11 b: FITC staining and analysis by flow cytometry. Results are expressed as mean ± SD. The percent blocking activity of IFX-1 on eC5 a-induced CD11b expression is indicated (arrows). Statistical differences were calculated by one-way anova, with p values of p < 0.05 being statistically significant.

FIG. 3: zymosan activates blood neutrophils and IFX-1 blocking activity. Whole blood was incubated with HBSS, rhC5a and zymosan a alone or a combination of different concentrations of IFX-1 and rhC5a or zymosan a. After incubation, cells were incubated with anti-mouse CD11 b: FITC staining and analysis of CD11b MFI by flow cytometry. Results are expressed as mean ± SD. The percent blocking activity of IFX-1 on C5 a-induced CD11b expression is indicated (arrow). Statistical differences were calculated by one-way anova, with p values of p < 0.05 being statistically significant.

FIG. 4: IFX-1 inhibits zymosan-induced IL-8 production in human whole blood. IL-8 concentrations were obtained by ELISA after incubation of human whole blood with different concentrations of zymosan A (as shown on the x-axis) in the presence of IFX-1 (open circles) or in the absence of IFX-1 (closed circles). Results are expressed as mean ± SD.

FIG. 5: concentrations of C3a (A), C5a (B) and C5B-9(C) in the plasma of 14 healthy controls and 54 Hidradenitis Suppurativa (HS) patients. Circles indicate outliers and asterisks indicate extreme values. P values represent significant differences between patients and controls.

FIG. 6: the effect of HS plasma on blood neutrophil activation and the potential role of C5a. HS plasma samples were incubated with human whole blood in the presence and absence of IFX-1 and CD11b expression in blood neutrophils was determined by flow cytometry. The levels of C5a in the control and HS samples are marked in the embedded tables.

FIG. 7: HiSCR response in HS patients after IFX-1 treatment. HiSCR responders were defined as having an inflammatory lesion count (abscess + inflammatory nodules) that was > 50% reduced and no increase in abscesses or draining fistulas compared to baseline.

Figure 8 blocking of C5 a-induced up-regulation of CD11b by different anti-C5 aR antibodies. Whole blood as a source of neutrophils was incubated with (added to) the plasma sample in the absence or presence of the respective anti-C5 aR antibody. The blocking activity of each inhibitor is expressed as a percentage of the corresponding sample.

FIG. 9 blocking C5 a-induced upregulation of CD11b by the C5aR antagonist PMX-53. Whole blood as a source of neutrophils was incubated with (added to) a plasma sample in the absence or presence of low (a) or high (B) concentrations of the C5aR antagonist PMX-53. Blocking activity of PMX-53 is expressed as a percentage of the corresponding sample.

FIG. 10 blocking of C5 a-induced upregulation of CD11b by C5a by antibody IFX-1. Whole blood as a source of neutrophils was incubated with (added to) the plasma sample in the absence or presence of IFX-1. The blocking activity of IFX-1 is expressed as a percentage of the corresponding sample.

Figure 11 blocking of C5 a-induced CD11b upregulation by the C5aR inhibitor Avacopan. Whole blood as a source of neutrophils was incubated with (added to) the plasma sample in the absence or presence of Avacopan. The blockade activity of Avacopan is expressed as a percentage of the corresponding sample.

Detailed Description

Definition of

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Preferably, the terms used herein are as defined in "A multilingual collaboration of biotechnology employees: (IUPAC Recommendations), ", Leuenberger, H.G.W, Nagel, B. and

H. edit (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland ".

Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise" and "comprise" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification, reference is made to documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence submissions, etc.). Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some documents cited herein are characterized as "incorporated by reference". In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited in this specification, the text of this specification controls.

The sequence is as follows: all sequences referred to herein are disclosed in the accompanying sequence listing, the entire contents and disclosure of which are a part of this specification.

In the context of the present invention, C5a particularly refers to human C5a. Human C5a is a 74 amino acid peptide having the following amino acid sequence:

the amino acid sequence of HUMAN C5 can be found under accession number UniProtKB P01031(CO5_ HUMAN).

As used herein, the term "inhibitor of C5a activity" refers to any compound that reduces C5a activity in any way. The activity may be reduced by directly or indirectly reducing the concentration of C5a, or by reducing the activity of C5a, or by preventing C5a from acting on one or more of its receptors (e.g., on C5aR or C5L2), or by reducing the concentration or activity of one or more of its receptors of C5a.

In the context of the present invention, the expression "C5 a receptor" refers to any potential C5a binding ligand on the cell surface, in particular to any receptor protein to which C5a can bind and trigger a response on the receptor (e.g. activation or inhibition of the receptor). The term "C5 a receptor" especially includes the two receptors C5aR and C5L 2. Alternative names for C5aR are C5aR1 and CD 88. An alternative name for C5L2 is C5aR 2.

Some embodiments of the invention relate to C5a inhibitors that interfere with the C5a receptor (e.g., by binding to the C5a receptor or by blocking expression of the C5a receptor). In these cases, the term "C5 a receptor" may refer to (i) C5aR or (ii) C5L2 or (iii) both C5aR and C5L 2. This means that some C5a inhibitors interfere with only one C5a receptor (i.e., C5aR or C5L2), while other C5a inhibitors interfere with both C5a receptors (i.e., C5aR and C5L 2).

In the context of the present invention, the expression "protein ligand" refers to any molecule consisting of amino acids linked by peptide bonds and capable of specifically binding to another molecule, irrespective of the overall size of the molecule. Thus, the expression "protein ligand" includes oligopeptides (> 100 amino acids) and polypeptides (> 100 amino acids). The expression "protein ligand" also includes cyclic peptides, irrespective of their size. The expression "protein ligand" includes in particular antibodies, antigen-binding fragments of antibodies, antibody-like proteins and peptide mimetics.

As used herein, a dissociation constant K if a first compound (e.g., a protein ligand or aptamer) is separated from a second compound (e.g., a target protein)_dIs 1mM or less than 1mM, preferably 100. mu.M or less than 100. mu.M, preferably 50. mu.M or less than 50. mu.M, preferably 30. mu.M or less than 30. mu.M, preferably 20. mu.M or less than 20. mu.M, preferably 10. mu.M or less than 10. mu.M, preferably 5. mu.M or less than 5. mu.M, more preferably 1. mu.M or less than 1. mu.M, more preferably 900nM or less than 900nM, more preferably 800nM or less than 800nM, more preferably 700nM or less than 700nM, more preferably 600nM or less than 600nM, more preferably 500nM or less than 500nM, more preferably 400nM or less than 400nM, more preferably 300nM or less than 300nM, more preferably 200nM or less than 200nM, even more preferably 100nM or less than 100nM, even more preferably 90nM or less than 90nM, even more preferably 80nM or less than 80nM, even more preferably 90nM or lessA first compound is considered to be "binding" to a second compound, selected from 70nM or less than 70nM, even more preferably 60nM or less than 60nM, even more preferably 50nM or less than 50nM, even more preferably 40nM or less than 40nM, even more preferably 30nM or less than 30nM, even more preferably 20nM or less than 20nM, even more preferably 10nM or less than 10 nM.

The term "binding" according to the present invention preferably relates to specific binding. By "specifically binds" is meant that a compound (e.g., a protein ligand or aptamer) binds more strongly to its particular target, such as an epitope, than to another target. If the compound has a dissociation constant (K) lower than that for the second target_d) Binding to the first target is stronger than binding to the second target. Preferred compounds have dissociation constants (K) for their targets to which they specifically bind_d) Dissociation constant (K) of target to which it is not specifically bound_d) More than 10 times lower, preferably more than 20 times lower, more preferably more than 50 times lower, even more preferably more than 100 times lower, 200 times lower, 500 times lower or 1000 times lower.

As used herein, the term "K_d"(usually measured in" mol/L ", sometimes abbreviated as" M ") is intended to denote the dissociation equilibrium constant for a particular interaction between a compound (e.g., a protein ligand) and a target molecule.

Method for determining the binding affinity of a compound, i.e. for determining the dissociation constant K_dThe methods of (a) are known to those of ordinary skill in the art and may be selected from, for example, the following methods known in the art: surface Plasmon Resonance (SPR) -based techniques, biolayer interferometry (BLI), enzyme-linked immunosorbent assays (ELISA), flow cytometry, Isothermal Titration Calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and Enhanced Chemiluminescence (ECL). In general, the dissociation constant K_dDetermined at 20 deg.C, 25 deg.C, 30 deg.C or 37 deg.C. K as defined herein, if not otherwise specified_dValues were determined by ELISA at 20 ℃.

An "epitope", also called an antigenic determinant, is a part of a macromolecule that can be recognized by the immune system, in particular by antibodies, B cells or T cells. As used herein, an "epitope" is a portion of a macromolecule that is capable of binding a compound (e.g., an antibody or antigen-binding fragment thereof) as described herein. In this context, the term "binding" preferably relates to specific binding. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the conformational epitope is lost and binding to the non-conformational epitope is not lost in the presence of denaturing solvents.

A "paratope" is the portion of an antibody that binds to an epitope. In the context of the present invention, a "paratope" is the portion of a compound (e.g., a protein ligand) described herein that binds an epitope.

The term "antibody" generally refers to a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" also includes all recombinant forms of antibodies, particularly of antibodies described herein, such as antibodies expressed in prokaryotes, unglycosylated antibodies, antibodies expressed in eukaryotes (e.g., CHO cells), glycosylated antibodies, and any antigen-binding antibody fragments and derivatives, as described below. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH or V)_H) And a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL or V)_L) And a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

As used herein, surgeryThe term "antigen-binding fragment" (or simply "binding portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341: 544-546) consisting of the VH domain; (vi) an isolated Complementarity Determining Region (CDR); and (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by a synthetic linker using recombinant methods, making them a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Another example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are also disclosed in US2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies. Other examples of "antigen-binding fragments" are so-called miniantibodies derived from a single CDR. For example, Heap et al, 2005, describes a minibody of 17 amino acid residues derived from the heavy chain CDR3 of an antibody directed against the gp120 envelope glycoprotein of HIV-1 (Heap C.J. et al (2005) Analysis of a 17-aminoacid residue, virus-neutral microbiological.J.Gen.Virol.86: 1791-1800). Other examples include small antibody mimetics comprising two or more CDR regions fused to each other, preferably through a homologous framework region. This involves passing through a homologous V_HV connected with FR2_HCDR1 and V_LSmall antibody mimetics of CDR3 have been described by Qiu et al (Qiu X. -Q. et al (2007) Small antibody mimicry comprising two complementary-determining regions and a frame region for tumor targeting. Nature Biotechnology 25 (8): 921-929).

Thus, as used herein, the term "antibody or antigen-binding fragment thereof" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that comprise an antigen binding site that immunospecifically binds an antigen. Also included are immunoglobulin-like proteins that are screened for specific binding to a target molecule or target epitope by techniques including, for example, phage display. The immunoglobulin molecules of the invention may be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG1, IgG2, preferably IgG2a and IgG2b, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

Antibodies and antigen binding fragments thereof useful in the present invention may be from any animal source, including birds and mammals. Preferably, the antibody or fragment is from a human, chimpanzee, rodent (e.g., mouse, rat, guinea pig or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat or dog. Particularly preferably, the antibody is of human or murine origin. Antibodies of the invention also include chimeric molecules in which an antibody constant region from one species, preferably human, is combined with an antigen binding site from another species, e.g., mouse. Furthermore, the antibodies of the invention include humanized molecules in which the antigen binding site of an antibody derived from a non-human species (e.g., from a mouse) is combined with constant and framework regions of human origin.

As exemplified herein, the antibodies of the invention can be obtained directly from a hybridoma expressing the antibody, or can be cloned and recombinantly expressed in a host cell (e.g., a CHO cell or a lymphocyte). Other examples of host cells are microorganisms, such as E.coli, and fungi, such as yeast. Alternatively, they may be produced recombinantly in transgenic non-human animals or plants.

The term "chimeric antibody" refers to an antibody in which one portion of each amino acid sequence of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining fragments of the chains are homologous to the corresponding sequence in another species or class. Typically, the variable regions of the light and heavy chains mimic the variable regions of an antibody derived from one mammal, while the constant portions are homologous to sequences of an antibody derived from another mammal. A clear advantage of such chimeric forms is that the variable regions can be conveniently obtained from sources known today using readily available B cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. In the case where the variable region has the advantage of being easy to prepare, and the specificity is not affected by the source, the constant region from a human is less likely to elicit an immune response in a human subject when the antibody is injected than the constant region from a non-human source. However, the definition is not limited to this particular example.

The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise the entire variable domain fused to a constant domain, or only the Complementarity Determining Regions (CDRs) grafted onto the appropriate framework regions in the variable domain. The antigen binding site may be wild-type or may be modified by one or more amino acid substitutions, for example to more closely resemble a human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (e.g., a humanized mouse antibody that comprises all six CDRs from a mouse antibody). Other forms have one or more CDRs that are altered relative to the original antibody.

Such as Almagro and Fransson, 2008, Frontiers in Bioscience, 13: 1619-1633, various methods for humanizing antibodies are known to those skilled in the art, the contents of which are incorporated herein by reference in their entirety. A brief overview of review articles written by Almagro and Fransson is outlined in US2012/0231008a1, which is a national phase application of international patent application WO2011/063980a 1. The contents of US2012/0231008a1 and WO2011/063980a1 are herein incorporated by reference in their entirety.

As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). The human antibodies of the invention include antibodies isolated from a human immunoglobulin library or from animals transgenic for one or more than one human immunoglobulin and which do not express endogenous immunoglobulins, as described in U.S. patent No. 5939598 to Kucherlapati and Jakobovits.

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. In one embodiment, the monoclonal antibody is produced by a hybridoma that includes a B cell obtained from a non-human animal, such as a mouse, fused to an immortalized cell.

As used herein, the term "recombinant antibody" includes all antibodies produced, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from hybridomas prepared from animals transgenic for immunoglobulin genes or transchromosomes (e.g., mice), (b) antibodies isolated from host cells transformed to express the antibodies, such as from transfectomas, (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies produced, expressed, produced or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express the antibody, such as CHO cells, NS/0 cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, "heterologous antibody" is defined with respect to the transgenic organism producing such antibody. The term means that the amino acid sequence or coding nucleic acid sequence of the antibody corresponding to a sequence present in an organism does not constitute a sequence of a transgenic organism and is generally derived from a species other than the transgenic organism.

As used herein, "hybrid antibody" refers to an antibody having light and heavy chains of different organism origin. For example, an antibody having a human heavy chain linked to a murine light chain is a hybrid antibody.

Thus, "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, recombinant antibodies, heterologous antibodies, hybrid antibodies, chimeric antibodies, humanized (particularly CDR-grafted) antibodies, deimmunized antibodies, or human antibodies, Fab fragments, Fab 'fragments, F (ab')₂Fragments, fragments produced by Fab expression libraries, Fd, Fv, disulfide linked Fv (dsfv), single chain antibodies (e.g., scFv), diabodies, or tetrabodies (Holliger p. et al (1993) proc. natl.acad.sci.u.s.a.90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including anti-Id antibodies such as the antibodies described herein), and epitope-binding fragments of any of the above.

The antibodies described herein are preferably isolated. As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds C5a is substantially free of antibodies that specifically bind to antigens other than C5 a). However, an isolated antibody that specifically binds to an epitope, isoform or variant of human C5a may have cross-reactivity with other related antigens, e.g., from other species (e.g., homologs of C5a species, e.g., rat C5a +). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies relates to antibodies having different specificities and combined in a well-defined composition.

As used herein, the term "naturally occurring" as applied to an object means that the object may exist in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from nature and not intentionally modified by man is naturally occurring.

As used herein, the term "nucleic acid aptamer" refers to a nucleic acid molecule that has been engineered to bind to a target molecule by repeated cycles of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) (for reviews, see Brody E.N. and Gold L. (2000), Aptamers as therapeutic and diagnostic agents.J. Biotechnol.74 (1): 5-13). The aptamer may be a DNA or RNA molecule. The aptamer may comprise modifications, for example, modified nucleotides, such as 2' -fluoro substituted pyrimidines, and/or may comprise one or more nucleotides having an L-ribose unit (or L-deoxyribose) instead of a standard D-ribose unit (or D-deoxyribose unit).

As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g., by mutagenesis of loops) to specifically bind a target molecule, typically such antibody-like proteins comprise at least one variable peptide loop that is attached at both ends to a protein scaffold, this dual structural constraint greatly increases the level of binding affinity of the antibody-like protein, making it comparable to an antibody, the variable peptide loop is typically 10 to 20 amino acids in length the scaffold protein can be any protein with good solubility, preferably the scaffold protein is a small globular protein, antibody-like proteins include, but are not limited to, affibody, avidin, α body, anti-carrier protein, affinity multimer, DARP protein (designed ankyrin repeat protein), fynomer, Kunitz peptide and monomers (see: Binz h.k. et al (2005) Engineering binding proteins from a library of antibody binding proteins, sometimes called a large antibody library, and can be isolated from a library of antibody-like proteins, such as a large antibody-like protein, 1258.

As used herein, a "peptidomimetic" is a small protein-like chain designed to mimic a peptide. Peptidomimetics typically result from the modification of an existing peptide to alter the properties of the molecule. For example, they may result from modifications that alter the stability or biological activity of the molecule. This may have an effect on the development of drug-like compounds from existing peptides. These modifications involve changes to the peptide that do not occur naturally (e.g., backbone changes and incorporation of unnatural amino acids).

In the context of the present invention, the term "small molecule" refers to a molecule having a molecular weight of 2kDa or less than 2kDa, preferably having a molecular weight of 1kDa or less than 1 kDa. The term "small molecule" particularly refers to a molecule that is neither an oligopeptide nor an oligonucleotide.

In the context of the present invention, the general expression "wherein a competes with B for binding to C" (e.g. in the expression "wherein the antibody or antigen-binding fragment thereof competes with one of the antibodies indicated in (a) for binding to C5 a") is used to define the binding characteristics of the compounds listed at position a. The compound A is combined with C, the compound B is also combined with C, but the compound A and the compound B cannot be combined with C at the same time; i.e., a and B bind to the same epitope on C (or at least bind to overlapping epitopes). This binding competition can be determined by competitive ELISA or Surface Plasmon Resonance (SPR) -based techniques or any other technique listed above in the context of determining binding affinity. Unless otherwise specifically indicated, the competitive binding properties of the compounds were determined by ELISA using equimolar concentrations of the two competitive compounds at 20 ℃.

As used herein, "neutrophilic inflammatory disease of the skin" refers to any disease associated with inflammation of the skin and with neutrophil infiltration (e.g., into the epidermis) of the skin of an individual afflicted with the disease. The term "neutrophilic inflammatory disease of the skin" refers in particular to Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

As used herein, the expression "HS-related disease" includes, but is not limited to Pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); and subepithelial impetigo dermatosis (SPD).

IFX-1 (alternative name: CaCP 29; Inflarx GmbH, Germany) is an antibody that specifically binds to C5a. The CDR sequences and FR sequences of IFX-1 are disclosed in WO2015/140304A1 (Table 3), the contents of which are incorporated herein by reference in their entirety.

INab708(Inflarx GmbH, Germany) is another antibody that specifically binds to C5a. The CDR sequences and FR sequences of INab708 are also disclosed in WO2015/140304A1 (Table 3), the contents of which are incorporated by reference in their entirety.

MEDI-7814(MedImmune) is a recombinant humanized anti-C5 a antibody. The crystal structure of human C5a bound to MEDI7814 was obtained as 4UU9 (DOI: 10.2210/pdb4UU9/pdb) in the RCSB protein database.

ALXN-1007(Alexion) is a humanized anti-C5 a antibody.

NOX-D21(Noxxon) is a pegylated mixed L-RNA/DNA aptamer (Spiegelmer)^TM) A 40kDa PEG-aminohexyl-GCG AUG (dU) GG UGG UGA AGG GUU GUU GGG (dU) GU CGA CGC A (dC) G C (SEQ ID NO: 34). NOX-D21 targets C5a (Hyzewicz J, Tanihata J, Kuraoka M, Nitahara-Kasahara Y, Beylier T, Rueg UT, Vater A and Takeda S.2017.Low-Intensity training and the C5a complementary Antagonst NOX-D21 resin the mdx photophenylethour modulationMotion of inflammation, am, j, pathol, 187 (5): 1147-; electronic publishing before printing: year 2017, month 3, day 18).

Ekulizumab (alternative name: Soliris)^TM5G 1-1; h5G1.1; alexion Pharmaceuticals) is a recombinant humanized monoclonal IgG2/4 κ antibody produced by murine myeloma cell culture and purified by standard bioprocessing techniques. Eculizumab specifically binds to human C5. Eculizumab comprises human constant regions from the human IgG2 sequence and human IgG4 sequence and murine complementarity determining regions grafted to the human framework light and heavy chain variable regions. Eculizumab consists of two 448 amino acid heavy chains and two 214 amino acid light chains, and has a molecular weight of about 148 kDa. The heavy and light chains of eculizumab are disclosed in, e.g., WO2016/061066a1 as SEQ ID NOs: 1 and SEQ ID NO: 34. nucleic acids encoding the heavy and light chains of eculizumab are disclosed, for example, in U.S. patent No. 6355245.

ALXN1210 (alternative name: BNJ 441; Alexion Pharmaceuticals) is an anti-C5 antibody. The heavy and light chains of ALXN1210 are disclosed in WO2016/209956a1 as SEQ ID NO: 14 and SEQ ID NO: 11.

ALXN5500(Alexion) is a humanized anti-C5 antibody. It is the next generation eculizumab candidate.

LFG316 (alternative name: Tesidolumab, NOV-4; Morphosys, Novartis) is an anti-C5 antibody.

Coversin^TM(alternative names: EV 576; PAS-coverin; rEV 576; tissue-targeted coverin^TM-Akari; akari Therapeutics, Evolutec) is a recombinant protein molecule (16.7kDa) derived from the salivary molecule of Ornithodoros moubata, which helps the parasite feed without eliciting a host immune response. The amino acid sequence of the EV576 protein (i.e. Coversin) and its encoding nucleotide sequence are shown in figure 2 of WO 2008/029167. Coversin^TMIn combination with C5.

RA101495(Ra Pharma) is a macrocyclic synthetic peptide inhibitor of C5 (Ricardo A, Arata M, DeMarcos, Dhamnoshar K, Hammer R, Fridkis-Hareli M, Rajagopal V, Seyb K, Tang G-Q, Tobe S and Treco D.2015.preclinical Evaluation of RA101495, aPotent Cyclic peptide inhibition of C5 for the Treatment of the therapy of partial nocturin hemoglobin.blood 126: 939).

(alternative names: anti-C5 aptamer; ARC-187; ARC-1905; Avacincaptad sodium pegol; OphthoTech, Inc., Archemix, Inc.) is a pegylated RNA aptamer that inhibits complement factor C5. The nucleotide sequence of ARC1905 (i.e., Zimura) is described, for example, in WO2005/079363A2 as SEQ ID NO: 67, the structure of which is shown in FIG. 22 of WO2005/079363A 2.

AMY-201(Amyndas pharmaceuticals) is an engineered form of factor H, which is directly linked to regulatory and surface recognition domains; therefore, it is a miniature FH molecule.

Mirococept (alternative names: APT070 and APT 070C; original manufacturers: Adprotecch; developers: Inflazyme pharmaceuticals) consists of the first three short consensus domains of human complement receptor 1, which are produced in recombinant bacteria and modified with membrane-targeting amphipathic peptides based on naturally occurring membrane-bound myristoyl electrostatic switching peptides (Souza DG, Esser D, Bradford R, Vieira AT, and Teixeira MM.2005.APT070(Mirococept), a membrane-localized peptide-containing composition inhibitor, inhibition of fluorescence reactivity with mutation, Br J Pharmaco 1145 (8): 1027 1034).

BikacioMab (Novelmed) is the F (ab) of anti-factor Bb antibody NM001₂And (3) fragment. Antibody NM001 was produced by hybridoma cell line 1D3 and deposited under ATCC accession number PTA-8543.

Lampalizumab (alternative name: anti-factor D Fab; FCFD 4514S; RG 7417; TNX-234; original developer: Tanox, developer: Genentech) is a humanized anti-factor D Fab fragment that inhibits factor D and the alternative complement pathway by binding to the exosite of factor D.

ALN-CC5(Alnylam) is an RNAi therapeutic agent that targets C5 in humans, primates and rodents. Exemplary iRNA compositions targeting the C5 gene are described in WO 2016/044419.

Avacopan (also known as CCX 168; Chemocentryx) is a small molecule (MW: 581.66g/mol) having the structure of formula I:

the IUPAC/chemical name of Avacopan is (2R, 3S) -2- [4- (cyclopentylamino) phenyl ] -1- (2-fluoro-6-methylbenzoyl) -N- [ 4-methyl-3- (trifluoromethyl) phenyl ] piperidine-3-carboxamide. Avacopan is a selective inhibitor of C5 aR. In the context of the present invention, the term "Avacopan" refers to compounds according to formula I and physiologically acceptable salts thereof.

Compounds similar to Avacopan that are also suitable for use in the practice of the present invention are disclosed in international patent applications WO 2010/075257a1 and WO 2011/163640a1, the contents of which are incorporated herein by reference in their entirety. Thus, in some embodiments, the inhibitor of C5a activity is a compound having formula II:

wherein

C¹Selected from aryl and heteroaryl, wherein heteroaryl has 1 to 3 heteroatoms selected from N, O and S as ring members; and wherein aryl and heteroaryl are optionally substituted with 1 to 3R¹Substituent group substitution;

C²selected from aryl and heteroaryl, wherein heteroaryl has 1 to 3 heteroatoms selected from N, O and S as ring members; and wherein aryl and heteroaryl are optionally substituted with 1 to 3R²Substituent group substitution;

C³is selected from C_1-8Alkyl or heteroalkyl, C_3-8Cycloalkyl radical, C_3-8cycloalkyl-C_1-4Alkyl, aryl-C_1-4Alkyl, heteroaryl-C_1-4Alkyl, heterocycloalkyl or heterocycloalkyl-C_1-4Alkyl, wherein heterocycloalkyl or moiety has 1 to 3 heteroatoms selected from N, O and S, and wherein heteroaryl has 1 to 3 heteroatoms selected fromA heteroatom from N, O and S as a ring member, and each C³Optionally substituted with 1 to 3R³Substituent group substitution;

each R¹Independently selected from halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)2R^c、-NR^a-C(O)NR^aR^b、-NR^aC(O)NR^aR^b、-NR^aR^b、-OR^aand-S (O)₂NR^aR^b(ii) a Wherein R is^aAnd R^bEach independently selected from hydrogen and C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and each R is^aAnd R^bOptionally substituted with one or two oxo; each R^cIndependently selected from C_1-8Alkyl or heteroalkyl, C_1-8Haloalkyl, C_3-6Cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R^a、R^bAnd R^cOptionally further substituted with 1 to 3 halogens, hydroxy, methyl, amino, alkylamino and dialkylamino groups; optionally, when two R are¹When the substituents are on adjacent atoms, they combine to form a fused five-or six-membered carbocyclic or heterocyclic ring;

each R²Independently selected from halogen, -CN, -NO₂、-R^f、-CO₂R^d、-CONR^dR^e、-C(O)R^d、-OC(O)NR^dR^e、-NR^eC(O)R^d、-NR^eC(O)₂R^f、-NR^dC(O)NR^dR^e、-NR^dC(O)NR^dR^e、-NR^dR^e、-OR^dand-S (O)₂NR^dR^e(ii) a Wherein R is^dAnd R^eEach is independentSelected from hydrogen and C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and each R is^dAnd R^eOptionally substituted with one or two oxo; each R is independently selected from C_1-8Alkyl or heteroalkyl, C_1-8Haloalkyl, C_3-6Cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and wherein R^d、R^eAnd R^fOptionally further substituted with 1 to 3 halogens, hydroxy, methyl, amino, alkylamino and dialkylamino groups; optionally, when two R are²When the groups are on adjacent atoms, they combine to form a five or six membered ring;

each R³Independently selected from halogen, -CN, -Rⁱ、-CO₂R^g、-CONR^gR^h、-C(O)R^g、-C(O)Rⁱ、-OC(O)NR^gR^h、-NR^hC(O)R^g、-NR^hCO₂Rⁱ、-NR^gC(O)NR^gR^h、-NR^gR^h、-OR^g、-OR^j、-S(O)₂NR^gR^h、-X⁴-R^j、-NH-X⁴-R^j、-O-X⁴-R^j、-X⁴-NR^gR^h、-X⁴-NHR^j、-X⁴-CONR^gR^h、-X⁴-NR^hC(O)R^g、-X⁴-CO₂R^g、-O-X⁴-CO₂R^g、-NH-X⁴-CO₂R^g、-X⁴-NR^hCO₂Rⁱ、-O-X⁴-NR^hCO₂Rⁱ、-NHR^jand-NHCH₂R^jWherein X is⁴Is C_1-4An alkylene group; r^gAnd R^hEach independently selected from hydrogen and C_1-8Alkyl or heteroalkyl, C_3-6Cycloalkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a four-, five-, or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and each R is^gAnd R^hOptionally substituted with one or two oxo; each RⁱIndependently selected from C_1-8Alkyl or heteroalkyl, C_1-8Haloalkyl, C_3-6Cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and each R^jIs selected from C_3-6Cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl and S, S-dioxo-tetrahydrothiopyranyl, and wherein R^g、R^h、RⁱAnd R^jOptionally further substituted with 1 to 3 halogens, methyl, CF3, hydroxy, C_1-4Alkoxy radical, C_1-4alkoxy-C_1-4Alkyl, -C (O) O-C_1-8Alkyl, amino, alkylamino and dialkylamino, and optionally, when two R are present³When the groups are on adjacent atoms, they combine to form a five or six membered ring; and

x is hydrogen or CH₃。

Compounds similar to Avacopan but with improved solubility characteristics are disclosed in WO 2017/176620 a2, the contents of which are incorporated herein by reference in their entirety. Thus, in some other embodiments, the inhibitor of C5a activity is a compound of formula III below:

wherein:

R¹selected from H, -O-CH₂-O-P(O)OR^aOR^b、-O-C(O)-C_1-6alkylene-L²-X¹、O-P(O)OR^aOR^band-O-C (O) -A¹-(C_1-3Alkylene radical)_n-C_4-7Heterocyclic group, wherein C_4-7Heterocyclyl is optionally substituted with 1 to 6R^cSubstituted by groups;

A¹is selected from C_6-10Aryl radical, C_3-10Cycloalkyl radical, C_5-10Heteroaryl and C_5-10Heterocyclyl, each of which is optionally substituted by 1 to 5R, which are the same or different^xSubstitution;

n is 0 or 1;

L²independently selected from the group consisting of a bond, -O-C (O) -C_1-6alkylene-and-NR^d-C(O)-C_1-6Alkylene-;

X¹independently selected from-NR^eR^f、-P(O)OR^aOR^b、-O-P(O)OR^aOR^band-CO₂H；

R²Selected from H, -L³-C_1-6alkylene-L⁴-X²、-L³-(C_1-6Alkylene radical)_m-A²-X²、-P(O)OR^aOC(O)-C_1-6Alkyl, -P (O) OR^aNR^gR^hand-P (O) OR^aOR^b；

L³Independently selected from-C (O) -O-and-C (O) -;

L⁴independently selected from the group consisting of a bond, -O-C (O) -C_2-6Alkenylene-, -O-C (O) -C_1-6alkylene-and-NR^d-C(O)-C_1-6Alkylene-in which-NR^d-C(O)-C_1-6alkylene-and-O-C (O) -C_1-6C in alkylene-radical_1-6Alkylene is optionally substituted by NR^eR^fSubstitution;

X²independently selected from-NR^kR¹、-P(O)OR^aOR^b、-O-P(O)OR^aOR^band-CO₂H；

m is 0 or 1;

A²is selected from C_6-10Aryl radical, C_3-10Cycloalkyl radical, C_5-10Heteroaryl and C_5-10Heterocyclyl, each of which is optionally substituted by 1 to 5R, which are the same or different^xSubstitution;

R³is H or-L⁵-p(O)OR^aOR^bWherein L is⁵Independently selected from the group consisting of a bond and-CH₂-O-；

Each R^xIndependently selected from halogen, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Heteroalkyl, CN, NR^yR^z、SR^yAnd OR^y；

Each R^cIndependently selected from halogen, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Heteroalkyl, CN, NR^yR^z、SR^yAnd OR^y；

R^a、R^b、R^d、R^e、R^f、R^g、R^k、R^l、R^yAnd R^zEach independently selected from H and C_1-6An alkyl group.

Each R^hIndependently selected from H and C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with 1 to 5 independently selected CO₂H、NRⁱR^j、C_6-10Aryl radical, C_3-10Cycloalkyl radical, C_5-10Heteroaryl and C_5-10Substituent of heterocyclic radical, wherein RⁱAnd R^jEach independently is H or C_1-6An alkyl group;

wherein R is¹、R²And R³Are H, and R¹、R²And R³Is not H.

PMX-53 is a potent antagonist of C5aR (CD 88). It is a cyclic peptide consisting of six amino acids and has the following sequence: Ac-Phe-ring (Orn-Pro-D-Cha-Trp-Arg), wherein a lactam bridge is formed between Orn-2 and Arg-6. Since PMX-53 comprises at least one D-amino acid (i.e., D-Cha), it is not included in the sequence listing accompanying this application. PMX-53 is commercially available under catalog number 5473 from bio-technique GmbH (Wiesbaden-Nordenstadt, Germany).

Compounds similar to PMX-53 also suitable for use in the practice of the present invention are disclosed in International patent applications WO 99/00406A 1, WO 03/033528A 1 and WO 2008/009062A 1, the entire contents of which are incorporated herein by reference. Thus, in some embodiments, the inhibitor of C5a activity is a cyclic peptide or peptidomimetic of formula IV.

Wherein A is H, alkyl, aryl, NH₂NH-alkyl, N (alkyl)₂NH-aryl, NH-acyl, NH-benzoyl, NHSO₃、NHSO₂Alkyl, NHSO₂-aryl, OH, O-alkyl or O-aryl;

b is alkyl, aryl, phenyl, benzyl, naphthyl or indolyl, or a side chain of a D-amino acid or an L-amino acid, but not of glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L-homophenylalanine, L-tyrosine or L-homophenyltyrosine;

c is the side chain of a D-amino acid, L-amino acid or homo-amino acid, but not of isoleucine, phenylalanine or cyclohexylalanine;

d is a side chain of a neutral D-amino acid, but not a glycine or D-alanine side chain, a bulky planar side chain, or a bulky charged side chain;

e is a bulky substituent other than the side chain of D-tryptophan, L-N-methyltryptophan, L-homophenylalanine, L-2-naphthyl L-tetrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L-fluorenylalanine, or L-histidine;

f is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine or bioisosteres thereof; and

X¹is- (CH)₂)_nNH-or (CH)₂)_nS-, wherein n is an integer of 1 to 4; - (CH)₂)₂O-；-(CH₂)₃O；-(CH₂)₃-；-(CH₂)₄-，-CH₂-COCHRNH-; or-CH₂-CHCOCHRNH-, wherein R is the side chain of any common or unusual amino acid.

The term "unusual amino acid" includes, but is not limited to, D-amino acids, homo-amino acids, N-alkyl amino acids, dehydro-amino acids, aromatic amino acids other than phenylalanine, tyrosine, and tryptophan, anthranilic acid, m-or p-aminobenzoic acid, ornithine, citrulline, canavanine, norleucine, delta-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine, and α -disubstituted amino acids.

Specific antagonists of C5aR (CD88) suitable for use in the practice of the invention include PMX95, PMX218, PMX200, PMX273, PMX205 and PMX201 as disclosed in WO 2008/009062 a 1.

Clone S5/1 is a monoclonal antibody recognizing the human C5a receptor (CD 88). Clone S5/1 was generated for a synthetic peptide containing the N-terminal domain of C5aR (Met1-Asn 31). This antibody has been shown to inhibit the binding of C5a to its receptor. It is commercially available under catalog number HM2094 from Hycult Biotech (Uden, the Netherlands).

Clone 7H110 is a monoclonal mouse antibody that recognizes the human C5a receptor (CD 88). It is commercially available under catalog number C2439-60N from Biomol GmbH (Hamburg, Germany).

As used herein, "patient" refers to any mammal or bird that may benefit from treatment with a compound described herein (i.e., an inhibitor of C5a activity described herein). Preferably, the "patient" is selected from a laboratory animal (e.g. mouse or rat), livestock (including, for example, guinea pig, rabbit, chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat or dog) or primate, including chimpanzees and humans. Particularly preferably, the "patient" is a human.

As used herein, "treatment" or "treating" of a disease or disorder refers to the completion of one or more than one of: (a) reducing the severity and/or duration of the disease; (b) limiting or preventing the development of symptoms characteristic of the condition being treated; (c) inhibiting the worsening of symptoms characteristic of the condition being treated; (d) limiting or preventing relapse of a disorder in a patient previously suffering from the disorder; and (e) limiting or preventing relapse of symptoms in a patient who has previously had symptoms of the disorder.

As used herein, "preventing" or "prevention" of a disease or disorder refers to preventing the occurrence of the disease or disorder in a subject.

An "effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary depending upon factors such as the nature of the therapeutic agent, the route of administration, the size and type of animal receiving the therapeutic agent, and the purpose of administration. The effective amount in each case can be determined empirically by the skilled worker according to established methods in the art.

"pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Embodiments of the invention

The invention will now be further described. In the following paragraphs, the different aspects of the invention are defined in more detail. Each aspect defined below may be combined with one or more other aspects, unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a first aspect, the present invention relates to a compound for use in the treatment of a neutrophilic inflammatory disease of the skin of a subject, wherein the compound is an inhibitor of C5a activity, and wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

administering to a subject in need thereof a therapeutically effective amount of a compound, wherein the compound is an inhibitor of C5a activity, and wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

In some embodiments of any aspect of the invention, the inhibitor of C5a activity:

-reducing the concentration of C5 (e.g., by inhibiting the formation and/or activity of C3 convertase, by inhibiting the formation and/or activity of C5 convertase, by inhibiting transcription of the C5 gene, by blocking translation of C5 mRNA, by increasing degradation of C5 mRNA, by increasing degradation of C5 protein, or by preventing secretion of C5 from the liver);

inhibition of cleavage of C5 to C5a and C5b (e.g., by inhibition of C5 convertase or by binding to the cleavage site on C5, thereby blocking cleavage);

-decreasing the concentration of C5a (e.g. by increasing degradation of C5a protein);

inhibit binding between C5a and C5a receptors (e.g. by binding to C5a or by binding to C5a receptors);

-reducing the concentration of C5a receptor (e.g., by inhibiting transcription of the C5a receptor gene; by blocking translation of the C5a receptor mRNA; by increasing degradation of the C5a receptor mRNA; by increasing degradation of the C5a receptor protein); and/or

-inhibiting the activity of the C5a receptor.

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is selected from a protein ligand (as defined above); an oligonucleotide; and small molecules (as defined above). An oligonucleotide that acts as an inhibitor of C5a activity may achieve its inhibitory effect, for example, by binding to a nucleic acid molecule (thereby inhibiting transcription and/or translation) or by binding to a protein (e.g., when the oligonucleotide is an aptamer).

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is a protein ligand that specifically binds to C5 protein, or C5a protein, or C5a receptor protein. In other embodiments, the protein ligand is selected from the group consisting of:

(i) antibodies (e.g., anti-C5 antibody, anti-C5 a antibody, anti-C5 aR antibody, or anti-C5L 2 antibody),

(ii) an antigen-binding fragment of an antibody,

(iii) an antibody-like protein which is capable of binding to a protein,

(iv) an inhibitory variant of C5a wherein,

(v) inhibitory variants of the C5a receptor (e.g. decoy receptors),

(vi) proteins that act on the complement pathway (e.g., Coversin); and

(i) (vii) peptides (e.g., RA101495(Ra Pharma, Cambridge, MA); PMX-53(bio-techne GmbH (Wiesbaden-Nordenstadt, Germany)).

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is a protein ligand or oligonucleotide, preferably a protein ligand, that specifically binds to a conformational epitope formed by the amino acid sequence NDETCEQRA of human C5a (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3). Binding is represented by the amino acid sequence according to SEQ ID NO: 2 and SEQ ID NO: 3 refers to the binding of a protein ligand or oligonucleotide to a protein according to SEQ ID NO: 2 and at least one amino acid within the amino acid sequence according to SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof. SEQ ID NO: 2 corresponds to amino acids 30-38 of human C5a. SEQ ID NO: 3 corresponds to amino acids 66-72 of human C5a.

In some embodiments of any aspect of the invention, a protein ligand or oligonucleotide, preferably a protein ligand, binds to at least one amino acid within the amino acid sequence according to DETCEQR (SEQ ID NO: 4). SEQ ID NO: 4 corresponds to amino acids 31-37 of human C5a.

In some embodiments of any aspect of the invention, the protein ligand or oligonucleotide, preferably the protein ligand, binds to at least one amino acid within the amino acid sequence according to HKDMq (SEQ ID NO: 5), more preferably to at least one amino acid within the amino acid sequence KDM. SEQ ID NO: 5 corresponds to amino acids 67-71 of human C5 a; the sequence KDM corresponds to amino acids 68-70 of human C5a.

In some embodiments of any aspect of the invention, a protein ligand or oligonucleotide, preferably a protein ligand, binds to at least one amino acid within the amino acid sequence DETCECQR (SEQ ID NO: 4) and binds to at least one amino acid within the amino acid sequence HKDMQ (SEQ ID NO: 5).

In some embodiments of any aspect of the invention, a protein ligand or oligonucleotide, preferably a protein ligand, binds to at least one amino acid within the amino acid sequence DETCECQR (SEQ ID NO: 4) and binds to at least one amino acid within the amino acid sequence KDM.

In some embodiments of any aspect of the invention, the two sequences forming the conformational epitope of C5a (e.g., the pair of sequences according to SEQ ID NOS: 2 and 3; SEQ ID NOS: 4 and 5; or SEQ ID NO: 4 and sequence KDM) are separated by 1 to 50 contiguous amino acids that are not involved in binding to a binding moiety of the invention. Hereinafter, amino acids not involved in binding to the binding moieties of the invention are referred to as "non-binding amino acids". The two sequences forming the conformational epitope are preferably separated by 6 to 45 consecutive non-binding amino acids, more preferably by 12 to 40 consecutive non-binding amino acids, more preferably by 18 to 35 consecutive non-binding amino acids, more preferably by 24 to 30 consecutive non-binding amino acids, more preferably by 25 to 29 consecutive non-binding amino acids, even more preferably by 26 to 28 consecutive non-binding amino acids, most preferably by 27 consecutive non-binding amino acids.

In some embodiments of any aspect of the invention, the protein ligand or oligonucleotide that specifically binds to a conformational epitope of C5a, preferably the protein ligand, has a K of the binding constant to human C5a_dValues of 10nM or less than 10nM, preferably 9nM or less than 9nM, more preferably 8nM or less than 8nM, more preferably 7nM or less than 7nM, more preferably 6nM or less than 6nM, more preferably 5nM or less than 5nM, more preferably 4nM or less than 4nM, more preferably 3nM or less than 3nM, more preferably 2nM or less than 2nM, and even more preferably 1nM or less than 1 nM. In some embodiments of any of the aspects of the invention, the dissociation constant, K, of the binding moiety to human C5a_dFrom 1pM (picomolar) to 5nM (nanomolar), more preferably from 2pM to 4nM, more preferably from 5pM to 3nM, more preferably from 10pM to 2nM, more preferably from 50pM to 1nM, more preferably from 100pM to 900pM, more preferably from 200pM to 800pM, more preferably from 300pM to 700pM, even more preferably from 400pM to 600 pM.

In some embodiments of any aspect of the invention, the protein ligand or oligonucleotide, preferably a protein ligand, that specifically binds C5a, exhibits at least 75% blocking activity, preferably at least 80% blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, more preferably at least 95% blocking activity, for a biological effect induced by a molecule of C5a, particularly human C5a. These specific blocking activities refer to binding moieties that, in those embodiments, comprise a single paratope that binds to C5a, preferably human C5a. In embodiments wherein the binding moiety comprises two or more C5a specific paratopes, the blocking activity of at least 75%, preferably at least 80%, more preferably at least 85%, etc., is achieved when one binding moiety molecule is contacted with a number of C5a molecules equal to the number of C5a specific paratopes present in the binding moiety. In other words, when the paratope of the binding moiety described herein and C5a are present at equimolar concentrations, the binding moiety exhibits at least 75% blocking activity, preferably at least 80% blocking activity, more preferably at least 85% blocking activity, more preferably at least 90% blocking activity, more preferably at least 95% blocking activity for a biological effect induced by C5a. The preferred biological effect to be blocked is C5 a-induced lysozyme release from human whole blood cells. Assays for determining this C5 a-induced lysozyme release and its blockade are described, for example, in WO2011/063980a1 and the corresponding US national phase application US2012/0231008a 1.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

(i) SEQ ID NO: 6, the heavy chain CDR3 sequence; or

(ii) SEQ ID NO: 7, the heavy chain CDR3 sequence;

wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition.

(iii) SEQ ID NO: 8, a light chain CDR3 sequence; or

(iv) SEQ ID NO: 9, a light chain CDR3 sequence;

wherein the light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition.

(i) SEQ ID NO: 6 and the heavy chain CDR3 sequence shown in SEQ ID NO: 8, a light chain CDR3 sequence; or

(ii) SEQ ID NO: 7 and the heavy chain CDR3 sequence shown in SEQ ID NO: 9, a light chain CDR3 sequence;

wherein the heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition; and is

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises at least one of the following sequences:

(v) according to SEQ ID NO: 10, a heavy chain CDR2 sequence;

(vi) according to SEQ ID NO: 11, a heavy chain CDR2 sequence;

(vii) according to SEQ ID NO: 12, a light chain CDR2 sequence;

(viii) according to SEQ ID NO: 13, a light chain CDR2 sequence;

(ix) according to SEQ ID NO: 14, a heavy chain CDR1 sequence;

(x) According to SEQ ID NO: 15, a heavy chain CDR1 sequence;

(xi) According to SEQ ID NO: 16, a light chain CDR1 sequence; or

(xii) According to SEQ ID NO: 17, the light chain CDR1 sequence;

wherein the heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition;

wherein the light chain CDR2 sequence optionally comprises 1, 2, or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2, or 3 amino acid deletion and/or a1, 2, or 3 amino acid addition;

wherein the heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition; and is

Wherein the light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition.

In a specific embodiment, the sequence as set forth in SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. and SEQ ID NO: 17, the total number of these optional changes enumerated above, i.e., the total number of exchanges, deletions, and additions, is 1 or 2.

In particular embodiments, the total number of exchanges, deletions, and additions of all CDRs present in the antibody or antigen-binding fragment thereof is from 1 to 5 (e.g., 1, 2, 3, 4, or 5).

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising one of the heavy chain CDR3, heavy chain CDR2, and heavy chain CDR1 sequence sets A through H listed in Table 1 below,

wherein each heavy chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition;

wherein each heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition; and is

Wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition:

table 1: heavy chain CDR sequence sets suitable for use in antibodies or fragments thereof of the invention

Labeling of heavy chain sets	CDR3 sequence	CDR2 sequence	CDR1 sequence
				A	SEQ ID NO：6	SEQ ID NO：10	SEQ ID NO：14
B	SEQ ID NO：6	SEQ ID NO：10	SEQ ID NO：15
				C	SEQ ID NO：6	SEQ ID NO：11	SEQ ID NO：14
D	SEQ ID NO：6	SEQ ID NO：11	SEQ ID NO：15
				E	SEQ ID NO：7	SEQ ID NO：10	SEQ ID NO：14
F	SEQ ID NO：7	SEQ ID NO：10	SEQ ID NO：15
				G	SEQ ID NO：7	SEQ ID NO：11	SEQ ID NO：14
H	SEQ ID NO：7	SEQ ID NO：11	SEQ ID NO：15

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising one of the light chain CDR3, light chain CDR2, and light chain CDR1 sequence sets I through IV set forth in Table 2 below,

wherein each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition;

wherein each light chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition; and is

Wherein each light chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably conservative amino acid exchanges, 1, 2 or 3 amino acid deletions and/or 1, 2 or 3 amino acid additions.

Table 2: light chain CDR sequence sets suitable for use in antibodies or fragments thereof of the invention

Since the CDR2 light chain sequence of antibody IFX-1 (SEQ ID NO: 12) is identical to the CDR2 light chain sequence of antibody INab708 (SEQ ID NO: 13), the sequences include the amino acid sequences set forth in SEQ ID NO: 13 to a set comprising SEQ ID NO: the set of 12 is redundant. Thus, the table lists only four sets of light chain CDR sequences.

Numbering of light chain sets	CDR3 sequence	CDR2 sequence	CDR1 sequence
				I	SEQ ID NO：8	SEQ ID NO：12	SEQ ID NO：16
II	SEQ ID NO：8	SEQ ID NO：12	SEQ ID NO：17
				III	SEQ ID NO：9	SEQ ID NO：12	SEQ ID NO：16
IV	SEQ ID NO：9	SEQ ID NO：12	SEQ ID NO：17

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof comprising one of the heavy chain CDR sets a-H listed in table 1 above and one of the light chain CDR sets I-IV listed in table 2 above, i.e. one of the combinations of the following sequence sets: A-I, A-II, A-HI, A-IV, B-I, B-II, B-III, B-IV, C-I, C-II, C-III, C-IV, D-I, D-II, D-IH, D-IV, E-I, E-II, E-III, E-IV, F-I, F-II, F-III, F-IV, G-I, G-II, G-HI, G-IV, H-I, H-II, H-III, or H-IV (with combinations A-I and H-IV being particularly preferred),

wherein each heavy chain CDR2 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition;

wherein each heavy chain CDR1 sequence optionally comprises 1, 2 or 3 amino acid exchanges, preferably a conservative amino acid exchange, a1, 2 or 3 amino acid deletion and/or a1, 2 or 3 amino acid addition;

wherein each light chain CDR3 sequence optionally comprises 1, 2 or 3 amino acid exchanges, in particular conservative amino acid exchanges, 1, 2 or 3 amino acid deletions and/or 1, 2 or 3 amino acid additions;

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof, comprising a VH domain comprising, consisting essentially of, or consisting of the sequence of seq id no: (i) the VH domain of IFX-1 or (ii) the VH domain of INab 708.

Table 3 below shows the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequences that define the VH domains of IFX-1 and INab 708.

In some embodiments of any aspect of the invention, the protein ligand is an antibody or antigen-binding fragment thereof, comprising a VL domain comprising, consisting essentially of, or consisting of the sequence of seq id no: (i) the VL domain of IFX-1 or (ii) the VL domain of INab 708.

Table 3 below shows the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequences that define the VL domain of IFX-1 and INab 708.

Table 3: CDR and FR sequences of antibodies IFX-1 and INab708 (Chothia Classification model)

In some embodiments of any aspect of the invention, the inhibitor of C5a activity is an oligonucleotide that specifically binds to C5, or to C5a, or to the C5a receptor. In other embodiments, the oligonucleotide is an aptamer. The aptamer may be selected from the group consisting of DNA aptamers, D-RNA aptamers, and L-RNA aptamers (e.g., Spiegelmers^TM)。

In some embodiments of any aspect of the invention, the inhibitor of C5a activity reduces the expression of C5 protein or C5a receptor protein. In a further embodiment, the inhibitor of C5a activity that reduces expression of C5 protein or C5a receptor protein is an oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, siRNA and miRNA.

In some embodiments of any aspect of the invention, the C5a receptor is C5aR and/or C5L 2. In a preferred embodiment of any aspect of the invention, the C5a receptor is C5aR (also known as CD88 or C5aR 1).

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is selected from:

(a) IFX-1, INab708, MEDI-7814, ALXN-1007, or NOX-D21, or an antigen-binding fragment thereof;

(b) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to C5a with one of the antibodies set forth in (a);

(c) eculizumab, ALXN1210, ALXN5500, or LFG316, or an antigen binding fragment thereof;

(d) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to C5 with one of the antibodies set forth in (C);

(e) coversin or RA 101495;

(f) an antibody or antigen-binding fragment thereof or a protein or macrocyclic peptide, wherein the antibody or antigen-binding fragment thereof or protein or macrocyclic peptide competes for binding to C5 with one of the proteins or peptides set forth in (e);

(g)Zimura；

(h) an antibody or antigen-binding fragment thereof or aptamer, wherein said antibody or antigen-binding fragment thereof or aptamer competes for binding to C5 with Zimura;

(i) AMY-201 or Mirococept;

(j) (ii) an antibody or antigen-binding fragment thereof or protein, wherein the antibody or antigen-binding fragment thereof or protein competes for binding to C3b with one of the proteins set forth in (i);

(k)Bikaciomab；

(l) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with Bikaciomab for binding to factor B;

(m)Lampalizumab；

(n) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with Lampalizumab for binding to factor D;

(o)ALN-CC5；

(p) Avacopan or a compound according to formula II or formula III or PMX-53 or a compound according to formula IV;

(q) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with Avacopan or PMX-53 for binding to C5 aR;

(r) clone S5/1 or clone 7H110, or an antigen-binding fragment thereof; and

(s) an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes for binding to C5aR with one of the antibodies set forth in (r).

In some embodiments of any aspect of the invention, the neutrophilic inflammatory disease of the skin is:

-auto-inflammatory diseases (more precisely: neutrophilic auto-inflammatory diseases of the skin); or

Autoimmune diseases with cutaneous inflammation (more precisely: autoimmune diseases with cutaneous neutrophilic inflammation).

In some embodiments of any aspect of the invention, the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); and SAPHO (synovitis, acne, impetigo, hyperostosis, and osteomyelitis) syndrome.

In some embodiments of any aspect of the invention, the cutaneous neutrophilic inflammatory disease is HS or is selected from Pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS) and subepithelial impetigo dermatosis (SPD).

In some embodiments of any aspect of the invention, the cutaneous neutrophilic inflammatory disease is an autoimmune disease with skin inflammation selected from the group consisting of rheumatoid neutrophilic dermatosis, familial mediterranean fever, a cold imidacloprid-related disorder, gout, and schnithler syndrome.

In some embodiments of the first and third aspects of the invention, the compound is administered at a dose of 800mg once weekly or at a dose of 800mg twice weekly. In other embodiments of the first or third aspect:

-the inhibitor of C5a activity is a compound that specifically binds to C5a (preferably selected from IFX-1, INAb708, MEDI-7814, ALXN-1007, NOX-D21 and antigen-binding fragments thereof; more preferably, the inhibitor of C5a activity is selected from IFX-1, INAb708, MEDI-7814, ALXN-1007 and antigen-binding fragments thereof; even more preferably, the inhibitor of C5a activity is selected from IFX-1 and antigen-binding fragments thereof; most preferably, the inhibitor of C5a activity is IFX-1); and is

-the neutrophilic inflammatory disease of the skin is Hidradenitis Suppurativa (HS); and is

-the compound is administered at a dose of 800mg once weekly or at a dose of 800mg twice weekly.

In other embodiments of the first or third aspect, the inhibitor of C5a activity is administered intravenously. In other embodiments of the first or third aspect, the inhibitor of C5a activity is administered twice weekly at a dose of 800mg for the first week of treatment and the inhibitor of C5a is administered once weekly at a dose of 800mg for the second and subsequent weeks of treatment. In other embodiments of the first or third aspect, the total duration of treatment is 5 weeks to 12 weeks (e.g., 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks).

In some embodiments of the second aspect of the invention, the compound is administered at a dose of 800mg once weekly or at a dose of 800mg twice weekly. In other embodiments of the second aspect:

In other embodiments of the second aspect, the inhibitor of C5a activity is administered intravenously. In other embodiments of the second aspect, the compound is administered twice weekly at a dose of 800mg for the first week of treatment and once weekly at a dose of 800mg for the second and subsequent weeks of treatment. In other embodiments of the second aspect, the total duration of treatment is 5 weeks to 12 weeks (e.g., 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks).

Pharmaceutical compositions and modes of administration

In the practice of any aspect of the invention, a compound (e.g., an inhibitor of C5a activity as described herein) or a pharmaceutical composition comprising the compound may be administered to a patient by any route established in the art that provides sufficient levels of the compound in the patient. Can be administered systemically or locally. Such administration may be parenteral, transmucosal, e.g., oral, nasal, rectal, intravaginal, sublingual, submucosal, transdermal, or by inhalation. Preferably, administration is by parenteral administration, such as by intravenous or intraperitoneal injection, and also includes, but is not limited to, intraarterial, intramuscular, intradermal, and subcutaneous administration. If a compound described herein (e.g., an inhibitor of C5a activity described herein) or a pharmaceutical composition comprising the compound is administered topically, it can be injected directly into the organ or tissue to be treated.

Pharmaceutical compositions suitable for oral administration may be in the form of capsules or tablets; powder or granules; solutions, syrups or suspensions (in aqueous or non-aqueous liquids); an edible foam or snack; or in the form of an emulsion. Tablets or hard gelatine capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Gelatin soft capsules may contain vegetable oils, waxes, fats, semi-solid or liquid polyols, and the like. Solutions and syrups may contain water, polyols and sugars.

Active agents intended for oral administration may be coated with or mixed with materials that delay disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be employed). Thus, a sustained release of the active agent can be achieved over several hours and, if desired, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at specific gastrointestinal locations due to specific pH or enzymatic conditions.

Pharmaceutical compositions suitable for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for an extended period of time. Pharmaceutical compositions suitable for topical administration may be provided in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical application to the skin, mouth, eyes or other external tissues, a topical ointment or cream is preferably used. When formulated in an ointment, the active ingredient may be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eye include eye drops. In these compositions, the active ingredient may be dissolved or suspended in a suitable carrier, for example, an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the mouth include lozenges, tablets and mouthwashes.

Pharmaceutical compositions suitable for nasal administration may comprise a solid carrier, for example a powder (preferably having a particle size of from 20 microns to 500 microns). The powder may be administered by inhalation of snuff, i.e. a rapid inhalation through the nose from a powder container held close to the nose. Alternatively, compositions for nasal administration may comprise a liquid carrier, such as a nasal spray or nasal drops. These compositions may comprise aqueous or oily solutions of the active ingredient. Compositions for administration by inhalation may be provided by specially adapted devices, including but not limited to pressurized nebulizers, or insufflators, which may be configured to provide a predetermined dose of the active ingredient. The pharmaceutical composition may also be administered to the lungs via nasal cavity.

Pharmaceutical compositions suitable for rectal administration may be presented as suppositories or as enemas. Pharmaceutical compositions suitable for vaginal administration may be provided in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or suspensions, which may contain antioxidants, buffers, bacteriostats and solutes that render the composition substantially isotonic with the blood of the intended recipient. Other components that may be present in such compositions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. Compositions suitable for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition ready for use only after addition of the sterile liquid carrier, for example sterile physiological saline for injection. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In a preferred embodiment, the compounds described herein (e.g., the inhibitors of C5a activity described herein) are formulated according to conventional procedures as pharmaceutical compositions suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet holding the amount of surfactant. When the composition is to be administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administration of the composition by injection, an ampoule of sterile saline may be provided so that the ingredients may be mixed prior to administration.

In another embodiment, for example, a compound (e.g., an inhibitor of C5a activity as described herein) or a pharmaceutical composition comprising the compound can be delivered in a controlled release system. For example, the compounds may be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Sefton (1987) CRC crit. Ref. biomed. Eng.14: 201; Buchwald et al (1980) Surgery 88: 507; Saudek et al (1989) N. Eng.J. Med.321: 574). In another embodiment, the compounds may be delivered in vesicles, particularly liposomes (see Langer (1990) Science 249: 1527-. In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release (1974) Langer and Wise (eds.), CRC Press: Boca Raton, Fla.; Controlled Drug Bioavailability, Drug product Design and Performance, (1984) Smolen and Ball (eds.), Wiley: N.Y., Ranger and Peppas (1953) J.Macromol.Sci.Rev.Macromol.Chem.23: 61; see also Levy et al (1985) Science 228: 190; During et al (1989) Ann.Neurol.25: 351; Howard et al (1989) J.Neurosurg.71: 105).

In yet another embodiment, the Controlled Release system can be placed in the vicinity of the therapeutic target, i.e., the target cell, tissue or organ, so that only a fraction of the systemic dose is required (see, e.g., Goodson (1984)115-138in medical applications of Controlled Release, Vol.2). Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-.

In a particular embodiment, it may be desirable to administer a compound described herein (e.g., an inhibitor of C5a activity described herein) or a pharmaceutical composition comprising the compound topically to an area in need of treatment. This may be achieved, for example and without limitation, by: local infusion during surgery, local administration, for example in combination with a postoperative wound dressing, injection, by means of a catheter, suppository, or implant, which is a porous, non-porous or gelatinous material, including membranes, for example, silicone membranes or fibers.

The selection of a preferred effective dose will be determined by the skilled artisan based on several factors known to those of ordinary skill in the art. Such factors include the particular form of the pharmaceutical composition, e.g., the polypeptide or carrier and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be determined during routine development normally used to obtain regulatory approval for a pharmaceutical compound. Other factors that take into account dosage include the disease or condition to be prevented and/or treated, or the benefit to be obtained, in normal individuals, the weight of the patient, the route of administration, whether the administration is short-term or long-term, concomitant medication, and other factors known to affect the efficacy of the administered drug. Thus, the precise dosage should be determined according to standard clinical techniques, at the discretion of the practitioner and at the discretion of each patient, for example according to the condition and immune status of the individual patient.

Examples

The following examples are provided to further illustrate the invention. However, the present invention is not limited thereto, and the following examples show the utility of the present invention based only on the above description.

1. Method of producing a composite material

1.1Preparation of zymosan A stock solution and zymosan A activated plasma (ZAP)

Zymosan A was dissolved in 50ml of sterile saline to 2mg/ml and boiled at 100 ℃ for 1 hour. After centrifugation, the supernatant was discarded and the pellet was resuspended in 50ml of sterile saline. After the second centrifugation step, the pellet was resuspended in 5ml sterile saline to obtain a 20mg/ml stock solution. The stock solution was aliquoted and stored at-20 ℃ until use. To activate the plasma, zymosan A stock solution was mixed with 100. mu.l plasma and incubated at 37 ℃ for 30 minutes. After incubation, the tubes were centrifuged, and the supernatant was aliquoted and stored at-20 ℃ until use.

1.2 determination of CD11b Using rhC5a or ZAP as stimulus

Human whole blood was stimulated with rhC5a or ZAP. To test the blocking activity of IFX-1 and irrelevant control IgG4 on rhC5a, the antibodies were diluted to final Ab/Ag molar ratios of 1: 1 and 0.5: 1. To test the blocking activity of IFX-1 against eC5a, IFX-1 was diluted to a final Ab/Ag molar ratio of about 4: 1/3: 1/2: 1/1: 1/0.5: 1. Blood with buffer only was used as a non-stimulated control to assess baseline CD11b expression. Blood with individual antibodies was used to determine the effect on CD11b expression of the antibodies under non-stimulated conditions. The complete mixture (Ab/Ag/blood) was incubated at 37 ℃ for 20min to assess C5 a-induced CD11b upregulation. After addition of anti-mouse CD11 b: after FITC, the samples were incubated on ice for 30min to minimize background staining. Granulocytes were gated and the Mean Fluorescence Intensity (MFI) of FITC-labeled granulocytes (expressing CD11 b) was detected by flow cytometry.

1.3 determination of CD11b in Whole blood Using rhC5a or zymosan A

Human blood was stimulated with rhC5a or zymosan A and the whole mixture (Ab/Ag/blood) was incubated at 37 ℃ for 20min to stimulate C5 a-induced CD11b upregulation. After incubation, 2 μ l of anti-mouse CD11 b: FITC or isotype: FITC control, and the samples were incubated on ice for 30min to minimize background staining. After lysis, the cells were analyzed using a flow cytometer. On FSC/SSC dot plots, granulocytes were gated and the Mean Fluorescence Intensity (MFI) of FITC-labeled granulocytes (expressing CD11 b) was examined for the entire sample set.

1.4 cytokine IL-8 ELISA

The human IL-8 ELISA was performed according to the recommendations in the manual "assay procedures" section (eBioscience Inc., san Diego, Calif.). Briefly, 100. mu.l of 1 × capture antibody was used to coat overnight at 4 ℃. Plates were sealed using 200 μ l of 1x assay dilution for 1 hour at room temperature. The standard stock solution was diluted to the desired concentration with 1x assay diluent and then subjected to 6 consecutive 1: 2 dilutions. The sample supernatant was diluted as needed in 1x assay dilution. According to the "assay procedure", 100. mu.l of standard and sample dilutions were added to the coated plates and incubated at room temperature for 1h, then with 100. mu.l of 1 × detection antibody (room temperature, 1h) and 100. mu.l of 1 × avidin-HRP (room temperature, 30 min). The development was carried out at room temperature in the dark for 10min with 100. mu.l of TMB substrate solution, and then stopped with 100. mu.l of stop solution. The absorbance was read using a microplate reader at 450nm over 30 min. The zero standard value (blank) was subtracted from all standards and samples. The cytokine concentration of the samples was calculated using the log (x)/log (y) standard curve of the included standard samples.

1.5 C5a ELISA

Purified anti-human C5a monoclonal antibody (InflaRx GmbH, jena, germany) was coated overnight on ELISA plates at a final concentration of 0.5 μ g/mL. After blocking with assay diluent (1 xPBS containing 0.05

% tween

20 and 2% heat-inactivated FBS), calibration samples (recombinant human C5a, Sigma, Taufkirchen, germany) and samples diluted in assay diluent were incubated for 90 min at room temperature. Mouse anti-human C5/C5a antibody clone 561(HycultBiotech, Uden, netherlands) diluted to 2 μ g/mL in assay dilution was used as the primary detection antibody to incubate at room temperature for 60 minutes, then with horseradish peroxidase-labeled secondary antibody (goat anti-mouse IgG2a polyclonal antibody, southern biotech, burminhan usa) diluted to 0.05 μ g/mL in assay dilution for 30 minutes at room temperature. The color development was carried out with tetramethylbenzidine substrate solution (TMB, Biozol, Eching, Germany) and was stopped with 3.7N sulfuric acid. By having a Tecan Magellan^TMTecan (C)

The reader (Tecan Group, Maennedorf, Switzerland) reads the absorbance at OD 450 nm. The internally developed C5a was validated by ELISA according to EMA guidelines for bioanalytical method validation.

Intra-and inter-assay precision tested at five different concentrations showed Coefficients of Variance (CV) of 0.65% to 4.96% and 1.50% to 4.88% for six and 18 replicates, respectively. The recovery analysis of the buffer to which recombinant human C5a was added showed that the lower limit recovery was 86.98. + -. 1.20% (mean. + -. SD) and the upper limit recovery was 91.50. + -. 3.29%. No cross-reactivity of C3, C3a, and C4 was detected, and cross-reactivity of C5b-6 was < 0.01%. The human IgG4 antibody did not interfere with the assay. The mean level of C5a in citrate plasma from 20 human volunteers was 17.08 ng/mL. + -. 6.96ng/mL, ranging from 7.52ng/mL to 30.17 ng/mL.

1.6 determination of complement activation products

The concentrations of complement activation products C3a, C5a, and tapping membrane complex C5b-9 were determined by ELISA. C3a ELISA (BDOptEIA)^TMHuman C3a ELISA kit, BD Bioscience, germany) was performed according to the manufacturer's instructions. Using BDOptEIA-based^TMInflarX-validated C5b-9 ELISA from the human C5b-9 ELISA kit (BD Bioscience) determined C5b-9 concentrations. C5a concentrations were measured using a C5a ELISA established and validated by InflaRx described above.

1.7 statistical analysis

All results are expressed as mean ± standard deviation. After baseline correction, statistical differences between groups were calculated by one-way analysis of variance including Tukey's multiple comparison test or by student's t-test for both groups. A p-value of 0.05 was used in the calculation to determine if there was a significant difference between the two groups. Use of

(California, USA) for graph creation and statistical analysis.

2. Preclinical relevant data

2.1C5 a-induced neutrophil activation and IFX-1 blockade

Since the upregulation of CD11b is a sensitive marker of neutrophil activation, the level of neutrophil CD11b was used to assess neutrophil activation. In this study, the blocking activity of IFX-1 against recombinant human C5a (rhC5a) was evaluated using a human whole blood model. Human whole blood was incubated with buffer, antibody alone, rhC5a alone, or a combination of different concentrations of antibody and rhC 5a. After incubation, cells were incubated with anti-mouse CD11 b: FITC staining and CD11bMFI was analyzed by flow cytometry to examine the activation level of blood neutrophils. As shown in fig. 1, recombinant human C5a strongly stimulated up-regulation of CD11b in human neutrophils. This effect was completely blocked in the presence of anti-human C5a antibody IFX-1. This inhibition was highly specific and the unrelated human IgG4 antibody did not show any blocking activity.

As a source of endogenous C5a (eC5a), Zymosan Activated Plasma (ZAP) was used to stimulate blood neutrophils. The amount of eC5a in ZAP was measured using a commercial C5a ELISA kit. The data presented here (fig. 2) indicate that the level of CD11b upregulation induced by eC5a in ZAPs is comparable to the level of CD11b upregulation induced by rhC 5a. The presence of IFX-1 significantly reduced the expression of human neutrophil CD11b even at an Ab: Ag molar ratio of 0.5: 1. According to the Ab: Ag ratio, IFX-1 had 100% to 82% overall blocking activity on ZAP-induced upregulation of CD11 b. Despite the high levels of eC3a and other complement activation products present in ZAP, IFX-1 can specifically block up to 100% of CD11b upregulation. It can therefore be concluded that eC5a is the only driver of neutrophil activation following ZAP stimulation, whereas IFX-1 can completely block it.

2.2Blockade of C5a attenuated the zymosan-induced inflammatory response in human whole blood

Zymosan a, as an active fungal cell wall component, can induce a strong inflammatory response in human whole blood characterized by activation of neutrophils and elevated levels of cytokines and chemokines. In this study, zymosan A human whole blood will be incorporated in the presence or absence of IFX-1 and CD11b expression by blood neutrophils is measured by flow cytometry. As shown in figure 3, CD11b in blood neutrophils was strongly upregulated when zymosan was present in human whole blood. Zymosan-stimulated CD11b upregulation could be inhibited by 79% to 93%, depending on the concentration of IFX-1 added. As a positive control, rhC5 a-stimulated CD11b upregulation was blocked by IFX-1100%. Thus, it was confirmed that CD11b upregulation of blood neutrophils was mainly caused by eC5a upon zymosan A stimulation. Furthermore, it can be concluded that, once eC5a is produced in whole blood by zymosan A stimulation, eC5a first binds to IFX-1, thereby blocking access of IFX-1 to its native receptor.

In the same experimental setup, the level of IL-8 was measured and used to assess the inflammatory response. In the absence of IFX-1, the IL-8 concentration after various doses of zymosan A stimulation ranged from 458pg/ml to 3218 pg/ml. As shown in FIG. 4, the presence of IFX-1 significantly reduced IL-8 production after stimulation with various concentrations of zymosan A, and reductions of up to 54% were observed. Thus, in the inflammatory whole blood environment, the zymosan-induced inflammatory response is largely dependent on the presence of C5a.

3. Clinically relevant data

3.1 data obtained from clinical samples

3.1.1Complement activation in HS patients

A total of 54 HS patients and 14 healthy volunteers participated in the study. Patients were followed by the department of infectious disease immunology at the department of outpatient service of the university of greek ATTIKON hospital. The study was approved by the hospital ethics committee. All patients provided written informed consent. The diagnosis of HS is based on the following criteria: a) early onset after puberty; b) the presence of subcutaneous nodules in the area of the skin rich in the apical plasma glands; and c) a compatible history of repeated drainage of pus from the affected area.

Circulating concentrations of complement factors C3a and C5a and tapping membrane complex sC5b-9 in the plasma of 54 patients and 14 healthy controls and in the pus of 7 patients were determined. As shown in fig. 5, circulating C5a in patient plasma was significantly greater than circulating C5a in control plasma (P < 0.01), and the differences in C3a and C5b-9 between patients and controls were of similar significance. Thus, it can be concluded that complement activation is present in HS. Given the important role of complement activation in innate and adaptive immunity, the inventors believe that targeting complement activation may be a novel therapeutic strategy for the treatment of HS.

However, it is not clear from the above results which of C3a, C5a or C5-9b or other complement activation products will be the most promising targets for this new therapeutic strategy, and whether targeting only one of these factors is sufficient, or whether two or more factors involved in complement activation must be targeted.

3.1.2Blocking HS plasma-induced neutrophil CD11b upregulation

To determine the effect of C5a on neutrophil activation in HS plasma samples, HS plasma samples with high levels of C5a were selected and evaluated by using the human whole blood model. As shown in fig. 6, HS plasma samples with high C5a levels (pat.088 and pat.092) significantly upregulated CD11b expression of blood neutrophils compared to control plasma samples with low C5a levels (Ctrl 008 and Ctrl 012). Recombinant human C5a was used as a positive control, while plasma from healthy volunteers was selected as a negative control. HS plasma-induced up-regulation of CD11b can be inhibited by IFX-1100%, indicating that C5a is the most important activator in HS plasma to trigger neutrophil activation. The inventors concluded from these novel results that blocking of C5a in HS patients was sufficient to achieve strong inhibition of neutrophil activation.

3.2 data obtained from clinical trials

3.2.1Design of experiments

A phase II patency marker test was performed on 11 patients with moderate to severe hidradenitis suppurativa in the department of the university of greek ATTIKON hospital.

The main objective of this trial was to investigate the safety and tolerability of taking IFX-1 within 8 weeks. The secondary objective of this trial was to assess the pharmacokinetics and pharmacodynamics of IFX-1 and generate preliminary data regarding the efficacy of IFX-1 in clinical endpoints (e.g., HiSCR, DLQI, VAS for disease states, VAS for pain, HS-PGA, modified Sartorius score) to derive further hypotheses. The enrolled patients received 800mg IFX-1 treatment for the first week and 800mg IFX-1 treatment once a week thereafter for 8 weeks; i.e., IFX-1 was administered as nine intravenous 800mg IFX-1 doses on

days

1, 4, 8, 15, 22, 29, 36, 43 and 50. All patients received an additional 12 weeks of follow-up.

Inclusion criteria in screening:

1. male or female patient aged 18 years or more

2. Written informed consent

3. Diagnosis of HS for at least 1 year

4. At least 2 different anatomical regions have HS lesions, one of which is hurley phase ii or III

5. Total AN (abscess and nodule) count of not less than 3

6. Patients who have failed primary or secondary biological therapy or who are otherwise refractory to treatment with other biologies.

Note that: primary failure is defined as no effect for at least 12 weeks of treatment with the biologic compound, secondary failure is defined as an initial response and subsequent relapse achieved after at least 12 weeks of treatment with the biologic compound.

7. Failure of previous antibacterial therapy

Exclusion criteria at screening:

1. the body weight is more than 150kg or less than 60kg

2. Fistula drainage count at baseline greater than 30

3. Planning for operative management in the next 24 weeks

4. An HS outbreak occurred within the last 14 days, resulting in intravenous antimicrobial therapy

5. Any other diseases and conditions that may interfere with the study product assessment, outcome assessment, or study satisfaction:

a) active infection

b) Severe congestive heart failure (i.e., NYHA class IV)

c) Depression (depression)

d) History of systemic lupus erythematosus or rheumatoid arthritis

e) Any immunodeficiency disease

f) Active hematologic or solid malignancy

g) The patient must not have any other active skin disease or condition (e.g., bacterial, fungal, or viral infection) that might interfere with HS assessment.

6. One of the following abnormal laboratory results:

a) white blood cell count < 2500/mm³

b) Neutrophil count < 1000/mm³

c) Serum creatinine > 3x Normal Upper Limit (UNL)

d) Total bilirubin > 2XUNL

e) Alanine aminotransferase (ALAT) > 2XUNL

f) Positive screening for hepatitis B, hepatitis C or HIV1/2

7. The last 3 months before any biological compound is taken

8. The intake of the steroid corticosteroid is determined as the daily intake of prednisone or more than an equivalent amount of 1mg/kg in the last three weeks;

9. taking immunosuppressive drugs (e.g. cyclosporine, tacrolimus) within the last 30 days

10. General exclusion criteria:

a) pregnant (women with fertility must undergo urine pregnancy test) or lactating women

b) Fertile women (defined as within two years after the last menstruation) are reluctant to take appropriate contraceptive measures (e.g., etonogestrel implant, injection, oral contraceptive, intrauterine device, partner vasectomy, abstinence) when participating in the test

c) Participation in any interventional clinical trial within the last three months

d) Known abuse of intravenous drugs

e) Staff at the study site, spouse/partner or relative of any researcher (e.g. researcher, subordinate researcher or research nurse) or relationship with a guarantor

3.2.2Results of clinical trials

HS patients were well tolerated by IFX-1. No serious adverse events associated with the drug were reported during the treatment period.

Efficacy parameters commonly used in the clinical response to hidradenitis suppurativa (HiSCR). HiSCR is defined by the status of three types of lesions (defining criteria): abscesses (with or without fluctuation, pressure pain or pain), inflammatory nodules (pressure pain, erythema, pyogenic granulomatosis), and draining fistulas (sinuses, communicating with the skin surface, draining pus). The definition of proposed treatment responders (HiSCR acquirers) is: (i) at least a 50% reduction in AN, (ii) no increase in the number of abscesses, and (iii) no increase in the number of draining fistulas from baseline. Hisscr has recently been demonstrated to be a responsive clinically meaningful endpoint for HS inflammatory responses (Kimball et al, 2014).

The HiSCR response over an 8-week treatment period was studied in this study, and until day 56, 8 of the 11 patients who had received treatment developed a response, representing a response rate of 72.7% with a 95% confidence interval of 43% to 91%. To compare these results to historical data, a literature search was conducted to observe a placebo-controlled clinical study using hisscr as an efficacy parameter. Table 4 below summarizes the five studies that have been recently completed:

table 4. complete clinical study using hisscr as efficacy parameter.

¹Siebole EMA assessment report:

http：//www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Assessment_Report_-_Variation/human/000481/WC500195564.pdf

²anakinra study (Tzanetakou et al, 2016).

³XBotech (http:// investors. XBiotech. com/phoenix. zhtml&p＝irol-newsArticle&ID＝2246777)

In the placebo group of these studies, a total of 179 patients were treated with a response rate of 19.0% with a 95% confidence interval of 14% to 25%. Since the confidence intervals (e.g., historical placebo patients and patients receiving IFX-1 treatment) do not overlap, it can be concluded that IFX-1 has a significant therapeutic effect.

Image recordings of the affected areas confirm these findings, i.e. a substantial reduction in skin inflammation, as evidenced by a visual reduction in inflammatory swelling and redness after treatment.

Thus, anti-C5 a represents a powerful anti-inflammatory agent in the context of HS disease. This clinical finding indicates that the blockade of C5a is very effective in reducing neutrophil activation, thereby effectively alleviating a cutaneous neutrophilic inflammatory disease.

4. Blockade of activated complement factor-induced CD11b upregulation in hidradenitis suppurativa patients by C5a-C5aR axis inhibition

4.1 purpose

The purpose of the following study was to demonstrate the blocking effect of anti-human C5a monoclonal antibody IFX-1, anti-human C5a receptor C5aR (CD88) antibody and C5aR antagonist and C5aR inhibitor on plasma-induced CD11b upregulation in patients with Hidradenitis Suppurativa (HS) on the surface of neutrophils.

4.2 principle of measurement

The accumulation of neutrophils at the site of inflammation depends on the expression of adhesion molecules, including CD11b (also known as integrin α M) (Larson and Springer, 1990; Carlos and Harlan, 1990). the upregulation of CD11b/CD18 in the intracellular pool and mobilization to the surface of neutrophils is essential for the rolling action and migration of human neutrophils (Smith et al, 1989). thus, increased expression of CD11b/CD18 reflects an inflammatory triggering event. human CD11b assays using flow cytometry were performed to detect FITC-coupled anti-CD 11b antibodies on the surface of neutrophils.the expression of activated complement products, in particular the increased endogenous C5a (eC5a) in HS patient plasma samples, could strongly upregulate CD11b expression by binding of C5a to its receptor C5aR (CD 36) on neutrophils.thus, it is expected that the blockade of the C5-a-C5 axis on the surface of neutrophils 8938 would be attenuated.

IFX-1 has been shown to control the inflammatory response leading to tissue and organ damage as the first anti-human C5a monoclonal antibody to enter clinical development. The antibody is currently in phase IIb studies to evaluate its effect on patients with moderate or severe hidradenitis suppurativa. It specifically and directly neutralizes terminal complement anaphylatoxin C5a and blocks the deleterious effects of C5a as a key inflammatory mediator in acute and chronic inflammatory diseases (Klos et al, 2009; Guo and Ward, 2005; Riedemann et al, 2017).

C5a functions by interacting with high affinity C5a receptors (C5aR and C5L2) (Guo and Ward, 2005). C5aR belongs to the rhodopsin family of G protein-coupled receptors, with seven transmembrane segments, whereas C5L2 is not G protein-coupled. It is generally believed that C5a-C5aR signaling is important in the pathogenesis of pro-inflammatory outcomes (Ward, 2009). Thus, targeting C5aR is another strategy for inhibiting complement-dependent inflammatory diseases. A series of small molecules derived from the C-terminus of C5a were developed as C5aR antagonists. Among these, the lead compound cyclohexapeptide PMX-53(AcF- [ OP (D-Cha) WR ]) (Finch et al, 1999) has been shown to reduce injury in a number of animal models of inflammation following intravenous, subcutaneous, intraperitoneal, and oral administration (Proctor et al 2006). Due to their structural similarity to C5a, such antagonists compete with C5a for the C5a receptor on neutrophils (March et al, 2004). Furthermore, anti-C5 aR antibodies can block the binding of C5a to C5aR, thereby reducing the accumulation and activation of myeloid suppressor cells and neutrophils (Markiewski et al, 2008). Two commercially available monoclonal anti-C5 aR antibodies, clone S5/1 and 7H110, were tested in this study. They were produced in mice by synthetic peptides directed against the N-terminal ectodomain comprising C5aR (Met1-Asn31) and synthetic peptides comprising recombinant human C5aR (Met1-Val350), respectively, two antibodies being described as neutralizing antibodies. Avacopan (CCX168) is an orally administered small molecule drug candidate that selectively inhibits complement C5a receptor (C5aR) and is being developed for inflammatory and autoimmune diseases (Bekker et al, 2008; Jayne et al, 2017).

The inhibitory effect of these blockers targeting the C5a-C5aR axis was monitored by flow cytometry against the block of neutrophil CD11b upregulation.

4.3 Experimental details

4.3.1Sample (I)

Plasma samples from two HS patients (pat.088 and pat.092) and two healthy controls (Ctrl 009 and Ctrl 010) were included in this study, according to definitions and diagnostic criteria approved by the foundation of hidradenitis suppurativa in 2009. Elevated levels of C3a, C5a, and C5b-9 indicate that the complement system is activated in the pathogenesis of HS (see table 5 below). The level of C5a in the 088 and 092 patients (93.77 ng/mL and 70.01ng/mL, respectively) was significantly higher than that in the healthy controls (21.02ng/mL and 11.77 ng/mL).

TABLE 5 complement factor concentrations measured in plasma samples of subjects

Patient numbering	Substrate	C5a[ng/ml]	C3a[ng/ml]	C5b-9[ng/ml]
					Pat.088	Blood plasma	93.77	13177.50	Maximum > value
Pat.092	Blood plasma	70.01	8801.00	276.12
					Ctrl 009	Blood plasma	21.02	1818.68	122.76
Ctrl 010	Blood plasma	11.77	2625.40	129.52

4.3.2Reagent

● AnalaR Water, VWR (Darmstadt, Germany), Cat No. 102923C, NORMAPUR for analysis, sterile filtration

● ACD, Sigma Aldrich (Taufkirchen, Germany), Cat No. C3821-50ML

● reagent for flow cytometry

○ FACS Flow sheath fluid, BD Bioscience (NJ, USA), Cat No. 342003

○ FACS shutdown fluid, BD Bioscience (NJ, USA), Cat No. 334224

○ FACS Wash solution, BD Bioscience (NJ, USA), Cat No. 340345

○ rat anti-mouse CD11b FITC, BD Bioscience (NJ, USA), Cat No. 553310, 0.5mg/mL

○ 10 FACS lysate, BD Bioscience (NJ, USA), Cat No. 349202 → working fluid 1 FACS lysate (diluted 1: 10 in AnalaR water)

○ staining buffer 1% Heat-inactivated FBS + 0.1% sodium azide in 1XPBS solution

● FBS, Thermo Fisher Scientific (Darmstadt, Germany), Cat No. 10099133 Heat-inactivation: 56 ℃ for 30min

● PBS powder, Sigma Aldrich (Taufkirchen, Germany), Cat No. P3813-10PAK

● sodium azide, VWR (Darmstadt, Germany), Cat No. 1.06688.0250

● recombinant human C5a (rhC5a), Hycult Biotech (Uden, Netherlands), Cat # HC2101 expressed in E.coli, dissolved in sterile AnalaR water

● 0.9.9% sterile sodium chloride (saline), B.Braun (Melsungen, Germany), catalog No. 3200950

● IFX-1, anti-human C5a antibody used as a control, InflaRx (Jena, Germany), 10mg/mL in PBS + 0.05% Tween 80

● PMX-53, bio-technique (Wiesbaden-Nordenstadt, Germany), Cat. No. 5473

● anti-C5 aR (CD88) antibody clone S5/1, Hycult Biotech (Uden, Netherlands), Cat # HM2094

● anti-C5 aR (CD88) antibody clone 7H110, biomol (Hamburg, Germany), Cat # C2439-60N

● Avacopan, MedKoo Biosciences Inc. (Morrisville, USA), Cat. No. 319575

● human blood from healthy donors containing 12% ACD (for immediate use)

● human plasma pool (citrate plasma) of Yana university Hospital.

4.3.3Device

● flow cytometer (FACS Canto II with DIVA software V6.1.2)

4.3.4Procedure for measuring the movement of a moving object

a) Human CD11b potency assay (flow cytometry assay)

Two patient plasma samples Pat.088 and Pat.092 (5. mu.L) were incubated with fresh human blood (60. mu.L as a source of neutrophils) in a total volume of 100. mu.L in the absence or presence of a C5a-C5aR axis blocker (anti-C5 a antibody IFX-1, anti-C5 aR antibody clone S5/1 and clone 7H110, C5aR antagonist PMX-53 and C5aR inhibitor Avacopan; 10. mu.L). Two control plasma samples (Ctrl 009 and

Ctrl

010, 5 μ L) prepared according to the procedure applied to the patient samples were used as control for non-specific activation. Blood plus saline alone (40 μ L) or a pool of normal human plasma (hupP 5 μ L + saline 35 μ L) served as unstimulated controls to determine baseline expression of CD11 b. Blood samples with a pool of normal human plasma and the addition of recombinant human C5a (rhC5a) simulated the stimulated condition. All samples were incubated at 37 ℃ for 20 minutes to activate CD11b upregulation. After cooling on ice, 2 μ L FITC-conjugated anti-mouse CD11b antibody was added to the sample. The labeled samples were placed on ice in the dark for an additional 30 minutes to minimize background signal. Erythrocytes were then lysed with 1xFACS lysate for 10min at room temperature. The remaining cells were washed twice with 2mL of staining buffer. After centrifugation at 2500rpm for 3min, the cells were resuspended in 0.5mL staining buffer and prepared for FACS analysis. Gates were set against FSC and SSC to analyze only neutrophils sized cells.

The percent Blocking Activity (BA) of the C5a-C5aR axis blocking test material was calculated using the following formula. Where MFI is the mean fluorescence intensity emitted by FITC bound to CD11b on neutrophils.

BA(％)＝(MFI_{Patient plasma}-MFI_{Patient plasma with test material added})÷(MFI_{Patient plasma}-MFI_huPP)×100

b) Statistical analysis

Graphs were generated using GraphPad Prism 7 (ca, usa).

4.4 results and discussion

Expression of integrin CD11b on neutrophils

Plasma samples from two healthy donors (Ctrl 009 and Ctrl 010) and two patients diagnosed with HS (pat.088 and pat.092) were used to assess CD11b expression on neutrophils. The Mean Fluorescence Intensity (MFI) of the control samples was 2156.9. + -. 114.3, which falls within the unstimulated baseline expression range for CD11b (MFI. ltoreq.3500). In comparison, HS patient samples (Pat.088, Pat.092) or 20nM recombinant human C5a (rhC5a) induced a 2.3-fold to 3.9-fold increase in CD11b expression (FIG. 8, FIG. 9, FIG. 10 and FIG. 11; Table 6, Table 7 and Table 8). These data indicate that significant upregulation of CD11b expression is mediated by inflammatory factors in HS patients. It is postulated that complement activation products, particularly C5a, play a major role in CD11b upregulation (table 5).

TABLE 6 blocking activity of anti-C5 aR antibody clones S5/1 and 7H110 and the C5aR antagonist PMX-53 on complement factor-induced CD11b upregulation.

TABLE 7 blocking activity of anti-C5 a antibodies IFX-1 and C5aR antagonist PMX-53 on complement factor-induced CD11b upregulation.

Anti-human C5aR monoclonal antibody, C5aR antagonist and C5aR inhibitor inhibit CD11b up-regulation

As shown in fig. 8, rhC5a (20nM) and two HS patient plasma samples with high levels of eC5a (pat.088 and pat.092) strongly up-regulated CD11b expression on blood neutrophils, while the presence of C5 aR-targeted blocking antibody at a final concentration of 50nM significantly attenuated CD11b expression driven by rhC5a or HS patient plasma samples. By using the anti-C5 aR antibody, clone 7H110 and clone S5/1, high blocking activity of 71% to 79% was obtained (fig. 8, table 9 and table 6). In contrast, the small hexapeptide C5aR antagonist PMX-53 was not as effective as the C5aR specific monoclonal antibody. The blocking activity produced by the same concentration of PMX-53(50nM) ranged from 50% to 57% (FIG. 9A, tables 9 and 6). To examine whether the up-regulation of CD11b could be abolished by inhibition of C5aR, the blocking activity of PMX-53(20 μ M) at high concentration was further evaluated under the same experimental conditions. As a result, the up-regulation of CD11B induced by HS patient plasma samples as well as rhC5a was completely abolished in the presence of high levels of PMX-53 (fig. 9B, tables 9 and 7). Similar data were also obtained in the presence of the C5aR inhibitor, Avacopan (figure 11, table 8 and table 9). In the presence of 100 μ M Avacopan, the blocking activity was 77% to 80%, whereas in the presence of 500 μ MAvacopan, the induction of CD11b upregulation by HS patient plasma was completely blocked (fig. 11, table 8 and table 9).

These results indicate that the C5a/C5aR axis is primarily responsible for CD11b upregulation of blood neutrophils, and that C5aR may serve as a potential target for blocking C5a activity.

Table 8 blocking activity of the C5aR inhibitor Avacopan on complement factor-induced CD11b upregulation.

Table 9. blocking activity (%) of all C5a-C5aR axis blocking test molecules summarized against CD11b upregulation induced by HS patient plasma or healthy human plasma supplemented with rhC 5a.

Anti-human C5a monoclonal antibody IFX-1 completely blocks HS-induced CD11b up-regulation

By using the same experimental setup as above, rhC5a and HS patient plasma samples (pat.088 and pat.092) strongly activated CD11b expression on blood neutrophils as expected, and addition of IFX-1 at concentrations as low as 20nM completely abolished the up-regulation of CD11b (fig. 10). These results indicate that IFX-1 is more effective in inhibiting the C5a/C5aR driven inflammatory response.

4.5 conclusion

Taken together, our results indicate that the method of targeting C5a, i.e., administration of the anti-C5 a monoclonal antibody IFX-1, effectively abolished C5 a-mediated up-regulation of CD11 b. In addition, anti-C5 aR antibodies, C5aR antagonists, and C5aR inhibitors also showed strong blocking activity under the same experimental conditions. Thus, targeting C5aR with blocking antibodies or antagonists represents another strategy to block the C5a/C5aR axis under inflammatory conditions (e.g., HS).

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Claims

1. A compound for use in treating a neutrophilic inflammatory disorder in the skin of a subject, wherein the compound is an inhibitor of C5a activity, and

wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); SAPHO (synovitis, acne, impetigo, hypertrophic bone and osteitis) syndrome; rheumatoid neutrophilic dermatosis; familial mediterranean fever; a cold imidacloprid-related disorder; gout and senitler syndrome.

2. The compound for use according to claim 1, wherein the inhibitor of C5a activity:

-lowering the concentration of C5;

-inhibiting the cleavage of C5 into C5a and C5 b;

-lowering the concentration of C5 a;

-inhibiting the binding between C5a and C5a receptors;

-lowering the concentration of C5a receptors; and/or

-inhibiting the activity of the C5a receptor.

3. The compound for use according to claim 1 or 2, wherein the inhibitor of C5a activity is a protein ligand that specifically binds to C5, or to C5a, or to the C5a receptor.

4. A compound for use according to claim 3, wherein the protein ligand is selected from the group consisting of:

(i) an antibody, which is capable of binding to a target,

(ii) an antigen-binding fragment of an antibody,

(iii) an antibody-like protein which is capable of binding to a protein,

(iv) an inhibitory variant of C5a wherein,

(v) an inhibitory variant of the C5a receptor,

(vi) proteins that act on the complement pathway; and

(vii) a peptide.

5. The compound for use according to claim 1 or 2, wherein the inhibitor of C5a activity is an oligonucleotide that specifically binds to C5, or to C5a, or to the C5a receptor.

6. The compound for use according to claim 5, wherein the oligonucleotide is selected from the group consisting of a DNA aptamer, a D-RNA aptamer and an L-RNA aptamer.

7. The compound for use according to claim 1 or 2, wherein the inhibitor of C5a activity reduces the expression of the C5 protein or the C5a receptor protein.

8. The compound for use according to claim 7, wherein said inhibitor of C5a activity is an oligonucleotide selected from the group consisting of antisense DNA, antisense RNA, siRNA and miRNA.

9. The compound for use according to any one of claims 2 to 8, wherein the C5a receptor is C5aR and/or C5L 2.

10. A compound for use according to any one of claims 1 to 9, wherein the inhibitor of C5a activity is selected from:

(e) coversin or RA 101495;

(g)Zimura；

(i) AMY-201 or Mirococept;

(k)Bikaciomab；

(m)Lampalizumab；

(o)ALN-CC5；

(p) Avacopan or a compound according to formula II or formula III; or PMX-53 or a compound according to formula IV;

(r) clone S5/1 or clone 7H110, or an antigen-binding fragment thereof; and

11. The compound for use according to any one of claims 1 to 10, wherein the neutrophilic inflammatory disease of the skin is selected from Hidradenitis Suppurativa (HS); pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS); subcorneal impetigo dermatosis (SPD); epidermolysis bullosa acquisita; persistent Elevated Erythema (EED); neutrophilic panniculitis; bowel-related skin disease-arthritis syndrome (BADAS); and SAPHO (synovitis, acne, impetigo, hyperostosis, and osteomyelitis) syndrome.

12. Compound for use according to any one of claims 1 to 10, wherein said cutaneous neutrophilic inflammatory disease is HS or is selected from Pyoderma Gangrenosum (PG); PAPA (suppurative arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); passh (suppurative arthritis, acne, PG and hidradenitis suppurativa); sweet Syndrome (SS) and subepithelial impetigo dermatosis (SPD).

13. The compound for use according to any one of claims 1 to 10, wherein the cutaneous neutrophilic inflammatory disease is an autoimmune disease with skin inflammation selected from the group consisting of rheumatoid neutrophilic dermatosis, familial mediterranean fever, a cold imidacloprid-related disorder, gout, and schnithler's syndrome.

14. The compound for use according to any one of claims 1 to 13, wherein the compound is administered at a dose of 800mg once weekly or at a dose of 800mg twice weekly.