JP7502865B2 - Treatment of inflammatory diseases with inhibitors of C5A activity - Google Patents

Description

TECHNICAL FIELD The present invention relates to inhibitors of C5a activity and their use in treating skin, neutrophilic, and inflammatory diseases in a subject.

Background of the Invention Targeting C5a in Inflammation
C5a is a 74 amino acid cleavage product of its "mother molecule" C5 and represents one end point of the complement activation cascade. It can be generated through the activation of at least three well described pathways (alternative, classical and MBL pathways). All pathways integrate at the level of C3 to form C5 or alternative C5 convertase, leading to the cleavage of C5 into C5a and C5b. The latter combines with C6, C7, C8 and multiple C9 molecules, ultimately leading to the formation of pores, for example in bacterial membranes (terminal membrane attack complex = MAC). C5a is generated when the complement system is activated in the context of inflammation and other immunological and inflammatory disorders/diseases.

Among the products of complement activation, C5a is one of the most potent inflammatory peptides and has a wide range of functions (Guo and Ward, 2005). C5a exerts its effects through the high-affinity C5a receptors (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. Currently, it is believed that C5a exerts its biological functions mainly through the C5a-C5aR interaction, as few biological responses have been seen to the C5a-C5L2 interaction. However, recent reports have demonstrated signaling also through C5L2 activation (Rittirsch and others, 2008).

C5aR is widely expressed on myeloid cells, including neutrophils, eosinophils, basophils, and monocytes, and on nonmyeloid cells in many organs, especially the lung and liver, demonstrating the importance of C5a/C5aR signaling. Widespread upregulation of C5aR expression occurs during the development of sepsis, and blockade of the C5a/C5aR interaction with anti-C5a or anti-C5aR antibodies, or C5aR antagonists, is highly protective in rodent models of sepsis (Czermak and others, 1999; Huber-Lang and others, 2001; Riedemann and others, 2002).

C5a has various biological functions (Guo and Ward, 2005). C5a is a potent chemoattractant for neutrophils and also has chemotactic activity for monocytes and macrophages. C5a causes oxidative burst ( _O2 consumption) in neutrophils and enhances phagocytosis and release of granular enzymes. C5a has also been found to be a vasodilator. C5a has been shown to be involved in regulating cytokine expression from various cell types and to enhance the expression of adhesion molecule expression in neutrophils. High doses of C5a can cause nonspecific chemotactic "desensitization" of neutrophils, thereby causing widespread functional impairment. Many inflammatory diseases, including sepsis, acute lung injury, inflammatory bowel disease, rheumatoid arthritis, etc., are attributed to the effects of C5a. In experimental sepsis situations, exposure of neutrophils to C5a can cause neutrophil dysfunction and paralysis of signaling pathways, resulting in assembly defects of NADPH oxidase, paralysis of MAPK signaling cascade, and significant depression of oxidative burst, phagocytosis, and chemotaxis (Guo and others, 2006; Huber-Lang and others, 2002). Thymocyte apoptosis and delayed neutrophil apoptosis are two important pathogenic events for the development of sepsis, which depend on the presence of C5a. During experimental sepsis, C5a upregulates β2-integrin expression in neutrophils and promotes cell migration to organs, which is one of the main causes of multiple organ dysfunction (MOF). It is also found that C5a is due to the activation of coagulation pathways that occurs in experimental sepsis. C5a stimulates the synthesis and release of inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IL-8, and macrophage migration inhibitory factor (MIF), from human leukocytes. Because complement activation is an event that occurs during the development of acute inflammation, C5a may begin to act before the appearance of most inflammatory "cytokine storms." C5a appears to play a key role in modulating and amplifying the potency of cytokine networks and the formation of the systemic inflammatory response syndrome (SIRS).

In the immunological regulatory network leading to adaptive immunity, C5a influences the crosstalk between dendritic cells (DCs) and δε T cells, which can lead to the massive production of inflammatory mediators, such as IL-17 (Xu and others, 2010). A key role for C5a has been established and defined in the generation of pathogenic Th17 responses in systemic lupus erythematosus (SLE) (Pawaria and others, 2014). In addition, it has been reported that C5a is a key regulator on Treg cells, providing a strong suppressive effect on Treg proliferation and induction (Strainic and others, 2013). Considering the fact that Tregs and Th17 are important players in the autoimmune disease context, inhibition of C5a signaling is expected to significantly reduce the overactive immune state in autoimmune diseases.

IFX-1
IFX-1 is a chimeric monoclonal IgG4 antibody that specifically binds to the soluble human complement split product C5a. IFX-1 is composed of 1328 amino acids and has an approximate molecular weight of 148,472 daltons. The CDR and FR sequences of IFX-1 are set forth in Table 3 below.

IFX-1 is expressed in mammalian CHO cell lines as a recombinant protein and finally formulated in phosphate buffered saline solution for intravenous administration. Binding of this antibody to human C5a facilitates highly effective blockade of C5a-induced biological effects by abolishing C5a binding to and reaction with its corresponding cell-bound receptor.

Various non-clinical studies, which can be categorized into in vitro/ex vivo studies (using IFX-1) and in vivo studies, including GLP toxicology tests in cynomolgus monkeys, have been conducted to evaluate the pharmacological and toxicological aspects of IFX-1. The non-clinical tests and studies conducted did not indicate any toxicological or safety concerns for IFX-1. Human Phase I studies showed that safety laboratory parameters, vital signs and ECG parameters did not show any clinically relevant time- or dose-dependent changes.

In vitro analysis of IFX-1 demonstrates a strong binding capacity to soluble human C5a as well as high blocking activity of C5a-induced biological effects, such as lysozyme release from human neutrophils or CD11b upregulation in neutrophils in human whole blood. One IFX-1 antibody reaches the capacity to neutralize the effect of bimolecular C5a with nearly 100% efficiency in experimental in vitro situations. Clinical trials with IFX-1 are ongoing to test its clinical efficacy in several inflammatory diseases, including septic organ dysfunction, and in complex cardiac surgery.

Neutrophils Neutrophils, terminally differentiated cells with a short life span in the circulation, are the most abundant white blood cells in the human body. As the first line of defense against invading microorganisms, neutrophils are characterized by their ability to act as phagocytes, release lytic enzymes from granules, and generate reactive oxygen species upon stimulation. In addition to microbial products, other stimuli such as immune complexes can also induce a respiratory burst in neutrophils, resulting in enhanced inflammation and recruitment of inflammatory cells (Kaplan, 2013).

After infiltrating inflamed tissues, neutrophils commit to numerous other cell types, such as macrophages, dendritic cells (DCs), natural killer cells, lymphocytes, and mesenchymal stem cells, to control innate and adaptive immune responses. For example, neutrophils can regulate DC maturation and T cell proliferation and polarization, and they can also directly prime antigen-specific T helper type 1 and T helper type 17 cells (Abi Abdallah and others, 2011). A variety of stimuli, including C5a, formyl-methionyl-leucyl-phenylalanine (FMLP), lipopolysaccharide, platelet-activating factor, and tumor necrosis factor (TNF), induce neutrophil degranulation (Kaplan, 2013). Contents released from degranulation and oxidative species along with cytokines and chemokines resulting from neutrophil activation are the major inflammatory mediators that cause tissue damage, and this mechanism is believed to be responsible for many types of inflammatory tissue injury.

Hidradenitis suppurativa (HS)
HS is a chronic, devastating skin disorder affecting areas rich in apocrine glands and is considered a neutrophil-associated cutaneous inflammatory disease. Nodules appear in the affected areas, which progress to swelling and rupture with the release of pus. This process occurs repeatedly, leading to the formation of sinus tracts and scarring (Jemec, 2004). The course of the disease creates a frustrating situation for the patient as well as the physician. The point prevalence is reported to range between 1% and 4% (Jemec and others, 1996).

The exact pathophysiology of HS is not clearly defined. Smoking, dietary habits, and genetic predisposition have all been associated with HS (Kurzen and others, 2008; Slade and others, 2003). Compared to healthy controls, the percentage of NK cells is increased and the percentage of CD4- lymphocytes is decreased, possibly implying the presence of an autoimmune predilection for the disorder. IL-1β and IL-17 have been found to be upregulated in HS lesions, which are associated with inflammasome activation (Lima and others, 2016). Hidradenitis suppurativa (HS) is seen in inflamed skin, with numerous neutrophil infiltrates, especially in the later stages of the disease (Lima and others, 2016; Marzano, 2016). Activated neutrophils may be an important effector cell type in this disease setting, causing tissue damage through direct deleterious effects or indirect regulatory effects on other effector cells such as activated T cells and TH17.

Hypotheses regarding the relevance of some autoimmune or autoinflammatory mechanisms in the pathogenesis of HS have been formulated over the past few years. The hypotheses are further strengthened by the positive results from the administration of TNF antagonists in prospective placebo-controlled trials leading to the approval of adalimumab (an antibody against tumor necrosis factor alpha) in patients with moderate to severe HS. One major yet unsolved question is how neutrophils are recruited to affected skin lesions and to what extent activated neutrophils contribute to the development of the disease.

The wide range of possible pathogenic mechanisms suggested by various studies may imply that HS is associated with host mechanisms rather than exogenous factors. Considering the paradox that both anti-infective (antibiotics) and pro-infective (anti-TNF, corticosteroids, immunosuppressants) treatments may be helpful, HS may present as an autoinflammatory disease based on defects in hair follicle innate immunity (Revuz, 2009), supported by the fact that proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), are significantly increased in the lesions and perilesional skin (Wollina and others, 2013).

Neutrophilic dermatoses Neutrophilic dermatoses (ND) are a group of disorders characterized by skin lesions in which histological examination reveals an intense inflammatory infiltrate composed mainly of neutrophils without evidence of infection. ND primarily includes Sweet syndrome (SS), pyoderma gangrenosum (PG), subcorneal pustulosis (SPD), and other distinct entities, as well as atypical or transitional forms of these (Prat and others, 2014). Hidradenitis suppurativa (HS) was recently assigned to the family of ND based on the numerous neutrophil infiltrates observed in inflamed skin (Lima and others, 2016; Marzano, 2016).

Pyoderma gangrenosum (PG) and HS are prototypic neutrophilic dermatoses characterized by the accumulation of neutrophils in the skin and considered to be autoinflammatory diseases in nature (Braun-Falco and others, 2012; Marzano and others, 2014). Autoinflammatory syndromes represent a new group of inflammatory conditions distinct from autoimmune, allergic, and infectious diseases. From a pathophysiological point of view, all autoinflammatory syndromes, e.g., PAPA (septic arthritis, PG and acne), PASH (PG, acne and HS) or PAPASH (septic arthritis, acne, PG and HS) share a common mechanism consisting of overactivation of the innate immune system and “sterile” neutrophil-rich skin inflammation (Cugno and others, 2017).

BADAS (bowel-associated dermatosis-arthritis syndrome) is characterized by fever, flu-like symptoms, arthritis, and inflammatory skin involvement. The latter is characterized by lesions reminiscent of various neutrophilic dermatoses, such as papules and plaques (Sweet syndrome), pustules and ulcers (pyoderma gangrenosum) or nodules, abscesses, or fistulas (hidradenitis suppurativa). In addition, acne and neutrophilic panniculitis may be associated. Patients usually experience a symmetric nonerosive polyarthritis that mainly involves small joints (Cugno and others, 2018).

Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome was first described in 1987. SAPHO syndrome is a rare condition, likely due to misdiagnosis. Although its pathogenesis remains elusive, there is growing understanding that SAPHO shares similarities with other autoinflammatory diseases (Cugno and others, 2018).

Neutrophils and Autoimmune Diseases Autoimmune diseases are defined by an incomplete discrimination of self and non-self molecules, leading to inappropriate recognition of self molecules and tissues as foreign structures and a concomitant immune attack on host organs. The pathogenesis of autoimmune diseases can generally be divided into two phases: the immunization phase and the effector phase. The immunization phase is characterized by the appearance of autoreactive T lymphocytes. These T cells then trigger a secondary response that leads to the tissue damage phase by activating various other cell types (B cells, cytotoxic T cells, NK cells, neutrophils, macrophages, osteoclasts, fibroblasts, etc.). The activation of these effector cells by autoreactive T cells can be considered as an effector phase that can be mediated by multiple 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 limitedly defined but increasingly recognized. Neutrophils can participate in multiple steps of the autoimmune disease process, including antigen presentation, control of the activity of other immune cell types, and direct tissue damage. Neutrophils can expose/release autoantigens when activated, when dying by apoptosis, or during the formation of neutrophil extracellular traps (NETs). They can also contribute to tissue deposition of autoantibodies or, as an effector cell type, induce tissue damage themselves. Accumulating studies have demonstrated that neutrophils play an active role in the development of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), bullous pemphigoid, epidermolysis bullosa acquisita, ANCA-associated vasculitis, familial Mediterranean fever, cryopyrin-associated periodic disorder (CAPS), and gout (Nemeth and Mocsai, 2012; Nemeth and others, 2016). Cutaneous inflammation is one of the most frequent syndromes exhibited by these autoimmune diseases, as the skin is an easy target for immune responses. However, rheumatic neutrophilic dermatosis is a rare cutaneous manifestation in patients with severe rheumatoid arthritis. It mainly affects patients with severe seropositive rheumatoid arthritis, mainly women (ratio 2:1), but has also been observed in seronegative rheumatoid arthritis (Cugno and others, 2018).

Technical Problem Underlying the Present Invention As explained above, there was a need in the prior art for an effective therapy for the treatment of neutrophilic dermatoses, such as hidradenitis suppurativa (HS), and cutaneous neutrophilic autoimmune diseases.

The inventors have now surprisingly found that molecules that inhibit C5a signaling, such as anti-C5a antibodies, are highly suitable for the treatment of hidradenitis suppurativa. The inventors have further investigated the physiological mechanisms leading to neutrophil activation and found that C5a is an important driver of neutrophil activation.

The inventors therefore anticipate that inhibiting C5a activity may be a suitable therapeutic approach for the treatment of a variety of neutrophilic disorders, particularly skin, neutrophilic, and inflammatory diseases.

SUMMARY OF THE DISCLOSURE In a first aspect, the present invention provides a compound for use in treating a skin, neutrophilic, inflammatory disease in a subject, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa). ); Sweet's syndrome (SS); subcorneal pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatus diurnitiva (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's syndrome.

In a second aspect, the present invention provides a method for the treatment of a skin, neutrophilic, inflammatory disease in a subject, comprising administering to a subject in need thereof a therapeutic amount of a compound, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne) The present invention relates to a method for treating a skin disease selected from the group consisting of: ne, PG and hidradenitis suppurativa; Sweet's syndrome (SS); subkeratotic pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutentiata (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's 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 a skin, neutrophilic, inflammatory disease, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa ulcer); Sweet's syndrome (SS); subcorneal pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutentiata (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's syndrome.

This summary does not necessarily describe all features of the present invention. Other aspects will become apparent from the detailed description that follows.

Blocking activity of IFX-1 on recombinant human C5a (rhC5a)-induced CD11b upregulation 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 antibody concentrations and rhC5a. After incubation, cells were stained with anti-mouse CD11b:FITC and CD11b MFI was analyzed by flow cytometry. Results are shown as mean ± SD. Percentage of IFX-1 blocking activity of C5a-induced CD11b expression is marked (arrows). Statistical differences were calculated by one-way ANOVA and a p-value of p<0.05 was statistically significant.

Blocking activity of IFX-1 on endogenous C5a (eC5a)-driven CD11b upregulation in neutrophils. Zymosan-activated human plasma (ZAP) was used as a source of eC5a. Whole blood was incubated with buffer, IFX-1 alone, ZAP alone, or a combination of IFX-1 and ZAP. After incubation, cells were stained with anti-mouse CD11b:FITC and analyzed by flow cytometry. Results are shown as mean ± SD. Percentage of IFX-1 blocking activity of eC5a-induced CD11b expression is marked (arrows). Statistical differences were calculated by one-way ANOVA, and a p-value of p<0.05 was statistically significant.

Activation of blood neutrophils by zymosan and IFX-1 blocking activity. Whole blood was incubated with HBSS, rhC5a and zymosan A alone or with different IFX-1 concentrations and combinations of rhC5a or zymosan A. After incubation, cells were stained with anti-mouse CD11b:FITC and CD11b MFI was analyzed by flow cytometry. Results are shown as mean ± SD. Percentage of IFX-1 blocking activity of C5a-induced CD11b expression is marked (arrows). Statistical differences were calculated by one-way ANOVA and a p-value of p<0.05 was statistically significant.

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

Concentrations of C3a (A), C5a (B) and C5b-9 (C) in plasma of 14 healthy controls and 54 patients with hidradenitis suppurativa (HS). Circles indicate outliers and asterisks indicate extreme values. P values represent significant differences between patients and controls.

Effect of HS plasma on blood neutrophil activation and the possible role of C5a. HS plasma samples were incubated with human whole blood in the presence and absence of IFX-1, and CD11b expression on blood neutrophils was determined by flow cytometric analysis. C5a levels in control and HS samples are displayed in the embedded table.

HiSCR Responses Following IFX-1 Treatment in HS Patients. HiSCR responders are defined as a > 50% reduction in inflammatory lesion counts (abscesses + inflammatory nodules) and no increase in abscesses or draining fistulas compared to baseline.

Blockade of C5a-induced CD11b upregulation via various anti-C5aR antibodies. Whole blood, as a source of neutrophils, was incubated with plasma samples in the absence or presence of each anti-C5aR antibody (spiked-). The blocking activity of each inhibitor was expressed as a percentage relative to the corresponding sample.

Blockade of C5a-induced CD11b upregulation via the C5aR antagonist PMX-53. Whole blood, as a source of neutrophils, was incubated with plasma samples in the absence or presence of low (A) or excess (B) concentrations of the C5aR antagonist PMX-53 (spiked-). The blocking activity of PMX-53 was expressed as a percentage relative to the corresponding sample.

Blockade of C5a-induced CD11b upregulation via the C5a blocking antibody IFX-1. Whole blood, as a source of neutrophils, was incubated with plasma samples in the absence or presence of IFX-1 (spiked-). The blocking activity of IFX-1 was expressed as a percentage relative to the corresponding sample.

Blockade of C5a-induced CD11b upregulation via the C5aR inhibitor Avacopan. Whole blood, as a source of neutrophils, was incubated with plasma samples in the absence or presence of Avacopan (spiked-). Blocking activity of Avacopan was expressed as a percentage relative to the corresponding samples.

DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT Definitions Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as they can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

Preferably, the terms used herein are defined as set forth in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). Throughout this specification and the appended claims, unless the context requires otherwise, the terms “comprise” and variations such as “comprises” and “comprising” are understood to mean 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.

Several documents (e.g., patents, patent applications, scientific publications, manufacturers' manuals, instructions, GenBank Accession Number sequence deposits, etc.) are cited throughout the text of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such incorporated document and a definition or teaching cited herein, the text of this application controls.

SEQUENCES: All sequences referred to in this specification are set forth in the attached Sequence Listing, which, together with its entire contents and description, is hereby incorporated by reference in its entirety.

In the context of the present invention, C5a refers in particular to human C5a, which is a 74 amino acid peptide having the following amino acid sequence:
(SEQ ID NO: 1)

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 in some way the activity of C5a. This reduction in activity can be accomplished by directly or indirectly lowering the concentration of C5a, or by reducing the activity of C5a, or by preventing C5a from exerting its effect on one or more of its receptors (e.g., on C5aR or C5L2), or by reducing the concentration or activity of one or more receptors for C5a.

In the context of the present invention, the expression "C5a receptor" refers to any potential C5a-binding ligand on the cell surface, in particular any receptor protein to which C5a may bind and trigger a reaction, such as activation or inhibition of the receptor. The term "C5a receptor" includes in particular the two receptors C5aR and C5L2. Alternative names for C5aR are C5aR1 and CD88. Alternative names for C5L2 are C5aR2.

Certain aspects of the present invention refer to inhibitors of C5a that block the C5a receptor (e.g., by binding to the C5a receptor or by blocking expression of the C5a receptor). In these contexts, the term "C5a receptor" can refer to (i) C5aR or (ii) C5L2 or (iii) both C5aR and C5L2. This means that some inhibitors of C5a block only one of the C5a receptors (i.e., either C5aR or C5L2), while other inhibitors of C5a block both C5a receptors (i.e., both C5aR and C5L2).

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

As used herein, a first compound (e.g., a protein ligand or nucleic acid aptamer) has a dissociation constant K to a second compound of 1 mM or less, preferably 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, even more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, and even more preferably 10 nM or less. When it has _d , it is considered to be "bound" to a second compound (eg, a target protein).

The term "binding" according to the present invention preferably relates to specific binding. "Specific binding" means that a compound (e.g., a protein ligand or a nucleic acid aptamer) binds more strongly to a target, e.g., an epitope, to which it is specific, compared to binding to another target. When a compound binds to a first target with a lower dissociation constant (K _d ) than the dissociation constant for the second target, it binds to the first target more strongly than the second target. Preferably, the dissociation constant (K _d ) for the target to which the compound specifically binds is 10 times or more lower, preferably 20 times or more lower, more preferably 50 times or more lower, and even more preferably 100 times, 200 times, 500 times or more lower than the dissociation constant (K _d ) for the target to which the compound does not specifically bind.

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

Methods for determining the binding affinity of a compound, i.e. for determining the dissociation constant _Kd , are known to those skilled in the art and can be selected, for example, from the following methods known in the art: surface plasmon resonance (SPR)-based techniques, biolayer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Typically, the dissociation constant _Kd is determined at 20°C, 25°C, 30°C or 37°C. Unless otherwise specifically indicated, the _Kd values described herein are determined by ELISA at 20°C.

An "epitope", also known as an antigenic determinant, is a portion of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. As used herein, an "epitope" is a portion of a macromolecule that can bind to a compound described herein (e.g., an antibody or an antigen-binding fragment thereof). In this context, the term "binding" preferably relates to specific binding. Epitopes usually consist of chemically active surface groupings 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 can be distinguished in that the binding to the former but not the latter is 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 described herein (e.g., a protein ligand) that binds to an epitope.

The term "antibody" generally refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. The term "antibody" also includes all recombinant forms of antibodies, particularly those described herein, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, antibodies expressed in eukaryotes (e.g., CHO cells), glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described below. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH or _VH ) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL or _VL ) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. 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 (C1q).

As used herein, the term "antigen-binding fragment" of an antibody (or simply "binding portion") 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 within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (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, optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows the use of recombinant methods to produce 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: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. A further 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 the 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 further described in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. A further example of an "antigen-binding fragment" is the so-called microantibody, which is derived from a single CDR. For example, Heap et al., 2005, describes a 17 amino acid residue microantibody derived from the heavy chain CDR3 of an antibody against the gp120 envelope glycoprotein of HIV-1 (Heap CJ et al. (2005) Analysis of a 17-amino acid residue, virus-neutralizing microantibody. J. Gen. Virol. 86:1791-1800). Other examples include small antibody mimetics that contain two or more CDR regions fused together, preferably by cognate framework regions. Such a small antibody mimetics comprising a _VH CDR1 and a _VL CDR3 linked by a cognate _VH FR2 has been described by Qiu et al., 2007 (Qiu X.-Q. et al. (2007) Small antibody mimetics comprising two complementary-determining regions and a framework 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 contain an antigen-binding site that immunospecifically binds to an antigen. Also included are immunoglobulin-like proteins selected through techniques, including, for example, phage display, that specifically bind to a target molecule or target epitope. The immunoglobulin molecules of the present invention may be 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 molecule.

The antibodies and antigen-binding fragments thereof that can be used in the present invention may be from any animal origin, including birds and mammals. Preferably, the antibodies or fragments are from human, chimpanzee, rodent (e.g. mouse, rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog origin. It is particularly preferred that the antibodies are of human or murine origin. The antibodies of the present 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 present invention include humanized molecules in which an antibody antigen-binding site from a non-human species (e.g. mouse) is combined with constant and framework regions of human origin.

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

The term "chimeric antibody" refers to an antibody in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequence in antibodies originating from a particular species or belonging to a particular class, while the remaining segments of the chains are homologous to the corresponding sequence in another species or class. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies originating from one species of mammal, while the constant portions are homologous to the sequences of antibodies originating from another species. One distinct advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources, for example using readily available B-cells or hybridomas from non-human host organisms, in combination with constant regions originating from human cell preparations. The variable regions have the advantage of ease of preparation and specificity is not affected by the source, but constant regions that are human are less likely to provoke an immune response from a human subject when the antibody is injected than constant regions 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 substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused onto a constant domain or only the complementarity determining regions (CDRs) grafted onto 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, e.g., to more closely resemble a human immunoglobulin. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibodies). Other forms have one or more CDRs that have been altered relative to the original antibody.

Various methods for humanizing antibodies are known to those skilled in the art, as described by Almagro & Fransson, 2008, Frontiers in Bioscience, 13:1619-1633, the contents of which are incorporated herein by reference in their entirety. The review article by Almagro & Fransson is briefly summarized in US 2012/0231008 A1, which is a national phase matter of International Patent Application WO 2011/063980 A1. The contents of US 2012/0231008 A1 and WO 2011/063980 A1 are incorporated herein by reference in their entirety.

As used herein, a "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. 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 by somatic mutation in vivo). Human antibodies of the invention include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins, e.g., as described in U.S. Patent No. 5,939,598 by Kucherlapati & Jakobovits.

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal, e.g., a mouse, fused to an immortalized cell.

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

As used herein, the term "transfectoma" includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a "heterologous antibody" is defined with respect to the transgenic organism producing such an antibody. The term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence that corresponds to one found in an organism that is not a transgenic organism and generally originates from a species other than the transgenic organism.

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

Accordingly, suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, xenogeneic, heterohybrid, chimeric, humanized (especially CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, fragments produced by a Fab expression library, Fd, Fv, disulfide-linked Fv (dsFv), single chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to 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 antigens other than C5a). However, an isolated antibody that specifically binds to an epitope, isoform or variant of human C5a may have cross-reactivity to other related antigens, such as antigens from other species (e.g., C5a species homologs, e.g., rat C5a). Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one aspect of the present invention, an "isolated" monoclonal antibody combination relates to antibodies that have different specificities and are combined in a defined composition.

As used herein, when applied to an object, the term "naturally occurring" refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence is naturally occurring if it exists in an organism (including viruses) that can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory.

As used herein, the term "nucleic acid aptamer" refers to a nucleic acid molecule that has been engineered through an iterative process of in vitro selection or SELEX (Systematic Exploration of Ligands by Exponential Expansion) to bind to a target molecule (for illustration, see Brody E.N. and Gold L. (2000), Aptamers as therapeutic and diagnostic agents. J. Biotechnol. 74(1):5-13). Nucleic acid aptamers can be DNA or RNA molecules. Aptamers can include modifications, such as modified nucleotides, such as 2'-fluorine-substituted pyrimidines, and/or one or more nucleotides having an L-ribose unit (or L-deoxyribose) instead of the 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 loop mutagenesis) to specifically bind to a target molecule. Typically, such antibody-like proteins contain at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint significantly increases the binding affinity of the antibody-like protein to a level comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small, globular protein. Antibody-like proteins include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins (designed ankyrin repeat proteins), phynomers, Kunitz domain peptides, and monobodies (for illustration, see Binz H.K. et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10):1257-1268). Antibody-like proteins can be obtained from large libraries of mutants, e.g., panned from large phage display libraries, and isolated similarly to conventional antibodies. Antibody-like binding proteins can also be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins are sometimes referred to as "peptide aptamers" or "antibody mimetics."

As used herein, a "peptide mimetic" is a small protein-like chain designed to mimic a peptide. Peptide mimetics generally result from the modification of existing peptides to alter the properties of the molecule. For example, they may result from modifications to change the stability or biological activity of the molecule. This can have a role in the development of drug-like compounds from existing peptides. These modifications include changes to peptides that do not occur in nature (e.g., altered backbones and the 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 2 kDa or less, preferably 1 kDa or less. The term "small molecule" refers in particular to a molecule that is not an oligopeptide or oligonucleotide.

In the context of the present invention, the general expression "A competes with B for binding to C" (e.g., in the expression "the antibody or antigen-binding fragment thereof competes with one of the antibodies shown under (a) for binding to C5a") is used to define the binding properties of the compounds listed in position A. Compound A binds to C and compound B also binds to C, but compounds A and B cannot bind to C simultaneously; i.e., A and B bind to the same epitope (or at least to overlapping epitopes) on C. Such competition in binding can be determined by competitive ELISA or surface plasmon resonance (SPR)-based techniques or any of the other techniques mentioned above in the context of determining binding affinity. Unless otherwise stated, the competitive binding properties of the compounds are determined by ELISA at 20° C. using equimolar concentrations of the two competing compounds.

As used herein, "skin, neutrophilic, inflammatory disease" refers to any disease associated with inflammation of the skin and neutrophil infiltration into the skin (e.g., into the epidermis) in an individual suffering from the disease. The term "skin, neutrophilic, inflammatory diseases" refers in particular to hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa); Sweet's syndrome (SS); subcorneal pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutigenti (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's syndrome.

As used herein, the term "HS-related disorders" includes, but is not limited to, pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa); Sweet's syndrome (SS); and subcorneal pustular purulence (SPD).

IFX-1 (alternative name: CaCP29; InflaRx GmbH, Germany) is an antibody that specifically binds to C5a. The CDR and FR sequences of IFX-1 are described in WO 2015/140304 A1 (Table 3), the contents of which are hereby incorporated by reference in their entirety.

INab708 (InflaRx GmbH, Germany) is another antibody that specifically binds to C5a. The CDR and FR sequences of INab708 are also described in WO 2015/140304 A1 (Table 3), the contents of which are incorporated by reference in their entirety.

MEDI-7814 (MedImmune) is a recombinant humanized anti-C5a antibody. The crystal structure of human C5a in complex with MEDI7814 is available in the RCSB Protein Data Bank (DOI: 10.2210/pdb4uu9/pdb) under 4UU9.

ALXN-1007 (Alexion) is a humanized anti-C5a antibody.

NOX-D21 (Noxxon) is a PEGylated mixed L-RNA/DNA-aptamer (Spiegelmer™) with the sequence 40 kDa PEG-aminohexyl-GCG AUG (dU) GG UGG UGA AGG GUU GUU GGG (dU) GU CGA CGC A(dC)GC (SEQ ID NO:34 ⁾ . NOX-D21 targets C5a (Hyzewicz J, Tanihata J, Kuraoka M, Nitahara-Kasahara Y, Beylier T, Ruegg UT, Vater A, and Takeda S. 2017. Low-Intensity Training and the C5a Complement Antagonist NOX-D21 Rescue the mdx Phenotype through Modulation of Inflammation. Am. J. Pathol., 187(5):1147-1161; Published electronically ahead of print: March 18, 2017).

Eculizumab (alternative names: Soliris ^™ , 5G1-1; h5G1.1; Alexion Pharmaceuticals) is a recombinant humanized monoclonal IgG2/4 kappa antibody produced by mouse myeloma cell culture and purified by standard bioprocessing techniques. Eculizumab specifically binds human C5. Eculizumab contains human constant regions from human IgG2 and IgG4 sequences and mouse complementarity determining regions grafted onto human framework light and heavy chain variable regions. Eculizumab is composed of two 448 amino acid heavy chains and two 214 amino acid light chains, with a molecular weight of approximately 148 kDa. The heavy and light chains of eculizumab are described, for example, in WO 2016/061066 A1 as SEQ ID NO: 1 and SEQ ID NO: 34, respectively. Nucleic acids encoding the heavy and light chains of eculizumab are described, for example, in U.S. Patent No. 6,355,245.

ALXN1210 (alternative name: BNJ441; Alexion Pharmaceuticals) is an anti-C5 antibody. The heavy and light chains of ALXN1210 are described in WO 2016/209956 A1 as SEQ ID NOs: 14 and 11, respectively.

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

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

Coversin ^™ (alternative names: EV 576; PAS-coversin; rEV 576; Tissue targeted Coversin ^™ - Akari; Akari Therapeutics, Evolutec) is a recombinant protein molecule (16.7 kDa) derived from saliva molecules from Ornithodoros moubata ticks that aids in parasite feeding without eliciting a host immunological response. The amino acid sequence of the EV576 protein (i.e. Coversin) as well as its encoding nucleotide sequence are shown in Figure 2 of WO 2008/029167. Coversin ^™ binds to C5.

RA101495 (Ra Pharma) is a macrocyclic synthetic peptide inhibitor of C5 (Ricardo A, Arata M, DeMarco S, Dhamnaskar K, Hammer R, Fridkis-Hareli M, Rajagopal V, Seyb K, Tang G-Q, Tobe S and Treco D. 2015. Preclinical Evaluation of RA101495, a Potent Cyclic Peptide Inhibitor of C5 for the Treatment of Paroxysmal Nocturnal Hemoglobinuria. Blood 126:939).

Zimura® (alternative names: anti-C5 aptamer; ARC-187; ARC-1905; Avacincaptad pegol sodium; OphthoTech Corporation, Archemix Corporation) is a pegylated RNA aptamer that inhibits complement factor C5. The nucleotide sequence of ARC1905 (i.e. Zimura) is shown, for example, in WO 2005/079363 A2 as SEQ ID NO:67, and its structure is shown in FIG. 22 of WO 2005/079363 A2.

AMY-201 (Amyndas Pharmaceuticals) is an engineered form of factor H that directly links the regulatory and surface recognition domains; it is thus a type of mini-FH molecule.

Mirococept (alternative names: APT070 and APT 070C; inventor: Adprotech; developer: Inflazyme Pharmaceuticals) consists of the first three short consensus domains of human complement receptor 1, produced in recombinant bacteria and modified with a membrane-targeting amphipathic peptide based on the natural membrane-bound myristoyl electrostatic switch peptide (Souza DG, Esser D, Bradford R, Vieira AT, and Teixeira MM. 2005. APT070 (Mirococept), a membrane-localised complement inhibitor, inhibits inflammatory responses that follow intestinal ischaemia and reperfusion injury. Br J Pharmacol 145(8):1027-1034).

BikacioMab (Novelmed) is an F(ab) ₂ fragment of an anti-factor Bb antibody designated NM001. Antibody NM001 is produced by hybridoma cell line 1D3 deposited under ATCC Accession Number PTA-8543.

Lampalizumab (alternative names: anti-Factor D Fab; FCFD4514S; RG7417; TNX-234; Inventor: Tanox, Developer: Genentech) is a humanized anti-Factor D Fab fragment that inhibits Factor D and the alternative complement pathway through binding to an exosite on Factor D.

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

Avacopan (also known by the name CCX168; Chemocentryx) has the formula I:
Avacopan is a small molecule (MW=581.66 g/mol) having the structure according to the 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 C5aR. In the context of the present invention, the term "avacopan" refers to the compound according to formula I as well as to the physiologically acceptable salts thereof.

Compounds similar to Avacopan that are also suitable for carrying out the present invention are described in International Patent Applications WO 2010/075257 A1 and WO 2011/163640 A1, the contents of which are incorporated herein by reference in their entirety. Thus, in some embodiments, inhibitors of C5a activity are represented by formula II
[Wherein,
^C1 is selected from the group consisting of aryl and heteroaryl, where the heteroaryl group has 1-3 heteroatoms as ring members selected from N, O and S; said aryl and heteroaryl groups are optionally substituted with 1 to 3 ^R1 substituents;
^C2 is selected from the group consisting of aryl and heteroaryl, the heteroaryl group having 1-3 heteroatoms as ring members selected from N, O and S; said aryl and heteroaryl groups are optionally substituted with 1 to 3 ^R2 substituents;
C ³ is selected from the group consisting of C _1-8 alkyl or heteroalkyl, C _3-8 cycloalkyl, C _3-8 cycloalkyl-C _1-4 alkyl, aryl, aryl-C _1-4 alkyl, heteroaryl, heteroaryl-C _1-4 alkyl, heterocycloalkyl or heterocycloalkyl-C _1-4 alkyl, where the heterocycloalkyl group or moiety has 1-3 heteroatoms selected from N, O and S, and the heteroaryl group has 1-3 heteroatoms as ring members selected from N, O and S, and each C ³ is optionally substituted with 1-3 R ³ substituents;
each R ¹ is independently selected from the group consisting of halogen, -CN, -R ^c , -CO ₂ R ^a , -CONR ^a R ^b , -C(O)R ^a , -OC(O)NR ^a R ^b , -NR ^b C(O)R ^a , -NR ^b C(O)2R ^c , -NR ^a -C(O)NR ^a R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a R ^b , -OR ^a , and -S(O) ₂ NR ^a R ^b ; each R ^a and R ^b is independently selected from the group consisting of hydrogen, C _1-8 alkyl, and C _1-8 haloalkyl, or when bonded to the same nitrogen atom, may be bonded to the nitrogen atom to form a 5- or 6-membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S, optionally substituted with 1 or 2 oxo; each R ^c is independently selected from the group consisting of C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and the aliphatic and cyclic portions of R ^a , R ^b and R ^c are optionally further substituted with 1 to 3 halogen, hydroxy, methyl, amino, alkylamino and dialkylamino groups; optionally when two R ¹ substituents are adjacent atoms, they are bonded to form a fused 5- or 6-membered carbocyclic or heterocyclic ring;
each R ² is independently selected from the group consisting of halogen, -CN, -NO ₂ , -R ^f , -CO ₂ R ^d , -CONR ^d R ^e , -C(O)R ^d , -OC(O)NR ^d R ^e , -NR ^e C(O)R ^d , -NR ^e C(O) ₂ R ^f , -NR ^d C(O)NR ^d R ^e , -NR ^d C(O)NR ^d R ^e , -NR ^d R ^e , -OR ^d , and -S(O) ₂ NR ^d R ^e ; each R ^d and R ^e are independently hydrogen, C _1-8 alkyl, and C _1-8 haloalkyl, or when bonded to the same nitrogen atom, may be bonded to the nitrogen atom to form a 5- or 6-membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S, optionally substituted with 1 or 2 oxo; each R is independently selected from the group consisting of C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl, the aliphatic and cyclic portions of R ^d , R ^e and R ^f optionally further substituted with 1 to 3 halogen, hydroxy, methyl, amino, alkylamino and dialkylamino groups, optionally bonded to form a 5- or 6-membered ring when two R ² groups are adjacent atoms;
Each R ³ is independently halogen, —CN, —R ⁱ , —CO ₂ R ^g , —CONR ^g R ^h , —C(O)R ^g , —C(O)R ⁱ , —OC(O)NR ^g R ^h , —NR ^h C(O)R ^g , —NR ^h CO ₂ R ⁱ , —NR ^g C(O)NR ^g R ^h , —NR ^g R ^h , —OR ^g , —OR ^j , —S(O) ₂ NR ^g R ^h , —X ⁴ -R ^j , —NH-X ⁴ -R ^j , —O-X ⁴ -R ^j , —X ⁴ -NR ^g R ^h , —X ⁴ -NHR ^j , —X ⁴ -CONR ^g R ^{h is} selected from the group consisting of -X ⁴ -NR ^h C(O)R ^g , -X ⁴ -CO ₂ R ^g , -O-X ⁴ -CO ₂ R ^g , -NH-X ⁴ -CO ₂ R ^g , -X ⁴ -NR ^h CO ₂ R ⁱ , -O-X ⁴ -NR ^h CO ₂ R ⁱ , -NHR ^j and -NHCH ₂ R ^j , wherein X ⁴ is C _1-4 alkylene; each R ^g and R ^h is independently hydrogen, C _1-8 alkyl or heteroalkyl, C _3-6 cycloalkyl and C _1-8 haloalkyl, or when attached to the same nitrogen atom, may be attached to the nitrogen atom to form a 4-, 5- or 6-membered ring having 0 to 2 additional heteroatoms as ring members selected from N, O or S, optionally substituted with 1 or 2 oxo; each R ⁱ is independently selected from the group consisting of C _1-8 alkyl or heteroalkyl, C _1-8 haloalkyl, C _3-6 cycloalkyl, heterocycloalkyl, aryl and heteroaryl; each R ^j is selected from the group consisting of C _3-6 cycloalkyl, imidazolyl, pyrimidinyl, pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and S,S-dioxo-tetrahydrothiopyranyl, and the aliphatic and cyclic portions of R ^g , R ^h , R ⁱ and R ^j are optionally selected from 1 to 3 halogen, methyl, CF ₃ , hydroxy, C _1-4 alkoxy, C _1-4 alkoxy-C further substituted with -C(O)O- _Ci-4 alkyl, -C(O)O-Ci _-8 alkyl, amino, alkylamino and dialkylamino groups, optionally joined to form a 5- or 6-membered ring when two ^R3 groups are adjacent atoms; and X is hydrogen or _CH3 .
and pharma- ceutically acceptable salts, hydrates and rotomers thereof.

Compounds similar to Avacopan, but with improved solubility profiles, are described in WO 2017/176620 A2, the contents of which are incorporated herein by reference in their entirety. Thus, in some other embodiments, inhibitors of C5a activity are represented by the following formula III:
[Wherein,
R ¹ is selected from the group consisting of H, -O-CH ₂ -O-P(O)OR ^a OR ^b , -O-C(O)-C _1-6 alkylene-L ² -X ¹ , O-P(O)OR ^a OR ^b , and -O-C(O)-A ¹ -(C _1-3 alkylene) _n -C _4-7 heterocyclyl, wherein the C _4-7 heterocyclyl is optionally substituted with 1 to 6 R ^c groups;
A ¹ is selected from the group consisting of C _6-10 aryl, C _3-10 cycloalkyl, C _5-10 heteroaryl, and C _5-10 heterocyclyl, each of which is optionally substituted with 1 to 5 R ^x , which may be the same or different;
n=0 or 1;
L ² is independently selected from the group consisting of a bond, -O-C(O)-C _1-6 alkylene-, and -NR ^d -C(O)-C _1-6 alkylene-;
X ¹ is independently selected from the group consisting of -NR ^e R ^f , -P(O)OR ^a OR ^b , -OP(O)OR ^a OR ^b , and -CO ₂ H;
R ² is selected from the group consisting of H, -L ³ -C _1-6 alkylene-L ⁴ -X ² , -L ³ -(C _1-6 alkylene) _m -A ² -X ² , -P(O)OR ^a OC(O)-C _1-6 alkyl, -P(O)OR ^a NR ^g R ^h and -P(O)OR ^a OR ^b ;
^L3 is independently selected from the group consisting of -C(O)-O-, and -C(O)-;
L ⁴ is independently selected from the group consisting of a bond, —O—C(O)—C _2-6 alkenylene-, —O—C(O)—C _1-6 alkylene-, and —NR ^d —C(O)—C _1-6 alkylene-, wherein the C _1-6 alkylene in —NR ^d —C(O)—C _1-6 alkylene- and —O—C(O)—C _1-6 alkylene- is optionally substituted with NR ^e R ^f ;
^X2 is independently selected from _the group consisting of ^{-NRkRl ,} -P(O) ^{ORaORb ,} -OP(O) ^ORaORb , and ^-CO2H ;
m=0 or 1;
^A2 is selected from the group consisting of _C6-10 aryl, _C3-10 cycloalkyl, _C5-10 heteroaryl, and _C5-10 heterocyclyl, each of which is optionally substituted with 1 to 5 ^Rx , which may be the same or different;
R ³ is H or -L ⁵ -P(O)OR ^a OR ^b , where L ⁵ is independently selected from the group consisting of a bond and -CH ₂ -O-;
each R ^x is independently selected from the group consisting of halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 heteroalkyl, CN, NR ^y R ^z , SR ^y , and OR ^y ;
each R ^c is independently selected from the group consisting of halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 heteroalkyl, CN, NR ^y R ^z , SR ^y , and OR ^y ;
each R ^a , R ^b , R ^d , R ^e , R ^f , R ^g , R ^k , R ^l , R ^y and R ^z is independently selected from the group consisting of H and C _1-6 alkyl;
each R ^h is independently selected from the group consisting of H and C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with 1 to 5 substituents independently selected from CO ₂ H, NR ⁱ R ^j , C _6-10 aryl, C _3-10 cycloalkyl, C _5-10 heteroaryl and C _5-10 heterocyclyl, and each R ⁱ and R ^j is independently H or C _1-6 alkyl;
Two of R ¹ , R ² and R ³ are H, and one of R ¹ , R ² and R ³ is other than H.
or a pharma- ceutically acceptable salt thereof.

PMX-53 is a potent antagonist of C5aR (CD88). It is a cyclic peptide composed of six amino acids with the following sequence: Ac-Phe-cyclization (Orn-Pro-D-Cha-Trp-Arg) with a lactam bridge between Orn-2 and Arg-6. Because PMX-53 contains at least one D-amino acid (i.e. D-Cha), it is not included in the sequence listing included in this application. PMX-53 is commercially available from bio-techne GmbH (Wiesbaden-Nordenstadt, Germany), Cat. No. 5473.

Compounds similar to PMX-53 that are also suitable for practicing the present invention are described in International Patent Applications WO 99/00406 A1, WO 03/033528 A1, and WO 2008/009062 A1, which are incorporated herein by reference in their entirety. Thus, in some embodiments, inhibitors of C5a activity are represented by formula IV
[Wherein,
A is H, alkyl, aryl, NH ₂ , NH-alkyl, N(alkyl) ₂ , NH-aryl, NH-acyl, NH-benzoyl, NHSO ₃ , NHSO ₂ -alkyl, NHSO ₂ -aryl, OH, O-alkyl, or O-aryl;
B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the side chain of a D- or L-amino acid, but not the side chain of glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L-homotryptophan, L-tyrosine, or L-homotyrosine;
C is the side chain of a D-, L-, or homo-amino acid, but is not the side chain of isoleucine, phenylalanine, or cyclohexylalanine;
D is the side chain of a neutral D-amino acid, but not the side chain of glycine or D-alanine, a bulky planar side chain, or a bulky charged side chain;
E is a bulky substituent but is not 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 a bioisostere thereof; and ^X1 is -( _CH2 ) _nNH- or ( _CH2 ) _nS- (where n is an integer from 1 to 4); -( _CH2 ) _2O- ; -( _CH2 ) _3O ; -( _CH2 ) _3- ; -( _CH2 ) _4- , -CH2-COCHRNH-; or _{-CH2 -} CHCOCHRNH- (where R is the side chain of any common or uncommon amino acid).
is a cyclic peptide or peptidomimetic compound of the formula:

In this context, the term "common amino acids" refers to the 20 proteinogenic amino acids defined by the standard genetic code. The term "uncommon amino acids" includes, but is not limited to, D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids other than phenylalanine, tyrosine and tryptophan, ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline, canavanine, norleucine, delta-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine, and α,α-disubstituted amino acids.

Specific antagonists of C5aR (CD88) suitable for carrying out the present invention include PMX95, PMX218, PMX200, PMX273, PMX205, and PMX201, which are described in WO 2008/009062 A1.

Clone S5/1 is a monoclonal antibody that recognizes the human receptor for C5a (CD88). Clone S5/1 was raised against a synthetic peptide containing the N-terminal domain (Met1-Asn31) of C5aR. The antibody has been shown to inhibit binding of C5a to its receptor. It is commercially available through Hycult Biotech (Uden, The Netherlands), Cat. No. HM2094.

Clone 7H110 is a monoclonal mouse antibody that recognizes the human receptor for C5a (CD88). It is commercially available through Biomol GmbH (Hamburg, Germany); Cat. No. C2439-60N.

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

As used herein, "treat," "treating," or "treatment" of a disease or disorder means achieving one or more of the following: (a) reducing the severity and/or duration of the disorder; (b) limiting or preventing the onset of symptoms characteristic of the disorder being treated; (c) inhibiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting or preventing the recurrence of the disorder in a patient who previously had the disorder; and (e) limiting or preventing the recurrence of symptoms in a patient who was previously symptomatic for the disorder.

As used herein, "prevent," "preventing," "prevention," or "prophylaxis" of a disease or disorder means preventing the disorder from occurring in a subject.

An "effective amount" is an amount of a therapeutic agent sufficient to accomplish its intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of the administration. The effective amount in each individual case can be empirically determined by one of ordinary skill in the art using methods established in the art.

"Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

Aspects of the present invention The present invention is now further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect defined below may be combined with any other aspect or aspects, unless expressly 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 provides a compound for use in treating a skin, neutrophilic, inflammatory disease in a subject, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa) ; Sweet's syndrome (SS); Subcorneal pustulosis (SPD); Epidermolysis bullosa acquisita, erythema elevatus diutentiata (EED); Neutrophilic panniculitis; Bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; Rheumatic neutrophilic dermatosis; Familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's syndrome.

In a second aspect, the present invention provides a method for the treatment of a skin, neutrophilic, inflammatory disease in a subject, comprising administering to a subject in need thereof a therapeutic amount of a compound, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne) The present invention relates to a method for treating a skin disease selected from the group consisting of: ne, PG and hidradenitis suppurativa; Sweet's syndrome (SS); subkeratotic pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutentiata (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's 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 a skin, neutrophilic, inflammatory disease, the compound being an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease being selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa ulcer); Sweet's syndrome (SS); subcorneal pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutentiata (EED); neutrophilic panniculitis; bowel-associated dermatosis-arthritis syndrome (BADAS); SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome; rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler's syndrome.

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is
- 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 the transcription of the C5 gene; by blocking the translation of C5 mRNA; by increasing the degradation of C5 mRNA; by increasing the degradation of C5 protein; or by preventing the secretion of C5 from the liver);
- inhibiting the cleavage of C5 into C5a and C5b (e.g., by inhibiting the C5 convertase or by binding to the cleavage site on C5, thereby blocking cleavage);
- reducing the concentration of C5a (e.g., by increasing the degradation of the C5a protein);
- inhibits the binding between C5a and the C5a receptor (e.g., by binding to C5a or by binding to the C5a receptor);
- decreasing the concentration of C5a receptor (e.g. by inhibiting transcription of the C5a receptor gene; by blocking translation of C5a receptor mRNA; by increasing the degradation of C5a receptor mRNA; by increasing the degradation of C5a receptor protein); and/or - inhibiting the activity of C5a receptor.

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is selected from the group consisting of a protein ligand (as defined above); an oligonucleotide; and a small molecule (as defined above). An oligonucleotide that acts as an inhibitor of C5a activity can achieve an 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 a nucleic acid aptamer).

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is a protein ligand that specifically binds to C5 protein, or C5a protein, or C5a receptor protein.
(i) an antibody (e.g., an anti-C5 antibody, an anti-C5a antibody, an anti-C5aR antibody, or an anti-C5L2 antibody);
(ii) an antigen-binding fragment of an antibody;
(iii) antibody-like proteins,
(iv) inhibitory mutants of C5a;
(v) inhibitory mutants of the C5a receptor (e.g., decoy receptors);
(vi) proteins acting on the complement pathway (e.g., Coversin); and (vii) peptides (e.g., RA101495 (Ra Pharma, Cambridge, Mass.); PMX-53 (bio-techne GmbH (Wiesbaden-Nordenstadt, Germany)).
is selected from the group consisting of:

In some embodiments of any aspect of the present 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 sequences NDETCEQRA (SEQ ID NO:2) and SHKDMQL (SEQ ID NO:3) of human C5a. Binding to the conformation formed by the amino acid sequences according to SEQ ID NO:2 and 3 means that the protein ligand or oligonucleotide binds to at least one amino acid in the amino acid sequence according to SEQ ID NO:2 and at least one amino acid in the amino acid sequence according to SEQ ID NO:3. 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 present invention, the protein ligand or oligonucleotide, preferably the 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 present 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 at least one amino acid within the amino acid sequence KDM. SEQ ID NO:5 corresponds to amino acids 67-71 of human C5a; the sequence KDM corresponds to amino acids 68-70 of human C5a.

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

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

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

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

In some embodiments of any aspect of the present invention, the protein ligand or oligonucleotide, preferably the protein ligand, that specifically binds to 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, and even more preferably at least 95% blocking activity against the biological effect induced by one molecule of C5a, particularly human C5a. These specific blocking activities refer to those embodiments in which the binding moiety comprises a single paratope that binds to C5a, preferably human C5a. In embodiments in which 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, and more preferably at least 95% blocking activity against the biological effect induced by C5a. The preferred biological effect that is blocked is C5a-induced lysozyme release from human whole blood cells. Assays for determining this C5a-induced lysozyme release and its blocking are described, for example, in WO 2011/063980 A1 and corresponding US national stage application US 2012/0231008 A1.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:
(i) the heavy chain CDR3 sequence shown in SEQ ID NO:6; or (ii) the heavy chain CDR3 sequence shown in SEQ ID NO:7;
and the heavy chain CDR3 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:
(iii) the light chain CDR3 sequence shown in SEQ ID NO:8; or (iv) the light chain CDR3 sequence shown in SEQ ID NO:9;
and the light chain CDR3 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising:
(i) the heavy chain CDR3 sequence shown in SEQ ID NO:6 and the light chain CDR3 sequence shown in SEQ ID NO:8; or (ii) the heavy chain CDR3 sequence shown in SEQ ID NO:7 and the light chain CDR3 sequence shown in SEQ ID NO:9;
wherein the heavy chain CDR3 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions; and the light chain CDR3 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof having at least one of the following sequences:
(v) a heavy chain CDR2 sequence according to SEQ ID NO:10;
(vi) a heavy chain CDR2 sequence according to SEQ ID NO:11;
(vii) a light chain CDR2 sequence according to SEQ ID NO:12;
(viii) a light chain CDR2 sequence according to SEQ ID NO:13;
(ix) a heavy chain CDR1 sequence according to SEQ ID NO:14;
(x) a heavy chain CDR1 sequence according to SEQ ID NO: 15;
(xi) a light chain CDR1 sequence according to SEQ ID NO: 16; or (xii) a light chain CDR1 sequence according to SEQ ID NO: 17;
wherein the heavy chain CDR2 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions;
the light chain CDR2 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
The heavy chain CDR1 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions; and the light chain CDR1 sequence optionally comprises one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.

In a particular embodiment, the total number of these desired changes listed above in each one of the amino acid sequences according to 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, i.e., the total number of exchanges, deletions, and additions in each sequence, is 1 or 2.

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

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof and comprises one of the sets A through H of heavy chain CDR3, heavy chain CDR2, and heavy chain CDR1 sequences listed in Table 1 below:
Each heavy chain CDR3 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
Each heavy chain CDR2 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions; and each heavy chain CDR1 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.
Table 1: Set of heavy chain CDR sequences suitable for use in the antibodies or fragments thereof of the present invention.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof and comprises one of the following sets I to IV of light chain CDR3, light chain CDR2, and light chain CDR1 sequences listed in Table 2:
Each light chain CDR3 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
Each light chain CDR2 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions; and each light chain CDR1 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.
Table 2: Sets of light chain CDR sequences suitable for use in the antibodies or fragments thereof of the present 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 set including SEQ ID NO:13 should be redundant with the set including SEQ ID NO:12. Therefore, the table lists only four sets of light chain CDR sequences.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof and comprises 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 following combinations of sets: A-I, A-II, A-III, A-IV, B-I, B-II, B-III, B-IV, C-I, C-II, C-III, C-IV, D-I, D-II, D-III, D-IV, E-I, E-II, E-III, E-IV, F-I, F-II, F-III, F-IV, G-I, G-II, G-III, G-IV, H-I, H-II, H-III, or H-IV (wherein the combination A-I and H-IV is especially preferred);
Each heavy chain CDR3 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
Each heavy chain CDR2 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
Each heavy chain CDR1 sequence optionally contains one, two, or three amino acid exchanges, preferably conservative amino acid exchanges, one, two, or three amino acid deletions, and/or one, two, or three amino acid additions;
Each light chain CDR3 sequence optionally contains one, two or three amino acid exchanges, particularly conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions;
Each light chain CDR2 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions; and each light chain CDR1 sequence optionally contains one, two or three amino acid exchanges, preferably conservative amino acid exchanges, one, two or three amino acid deletions, and/or one, two or three amino acid additions.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof and comprises a VH domain that comprises, consists essentially of, or consists of (i) the VH domain of IFX-1 or (ii) the VH domain of INab708.

The FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 sequences defining the VH domains of IFX-1 and INab708 are shown in Table 3 below.

In some embodiments of any aspect of the present invention, the protein ligand is an antibody or antigen-binding fragment thereof and comprises a VL domain that comprises, consists essentially of, or consists of (i) the VL domain of IFX-1 or (ii) the VL domain of INab708.

The FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 sequences defining the VL domains of IFX-1 and INab708 are shown in Table 3 below.
Table 3: CDR and FR sequences of antibodies IFX-1 and INab708 (Chothia classification mode)

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

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity reduces expression of C5 protein or C5a receptor protein. In further embodiments, 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 present invention, the C5a receptor is C5aR and/or C5L2. In preferred embodiments of any aspect of the present invention, the C5a receptor is C5aR (also known as CD88 or C5aR1).

In some embodiments of any aspect of the present invention, the inhibitor of C5a activity is
(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 that competes with one of the antibodies shown under (a) for binding to C5a;
(c) eculizumab, ALXN1210, ALXN5500, or LFG316, or an antigen-binding fragment thereof;
(d) an antibody or antigen-binding fragment thereof that competes with one of the antibodies set forth under (c) for binding to C5;
(e) Coversin or RA101495;
(f) an antibody or antigen-binding fragment thereof or a protein or macrocyclic peptide that competes with one of the proteins or peptides set out under (e) for binding to C5;
(g) Zimura;
(h) an antibody or antigen-binding fragment or aptamer thereof that competes with Zimura for binding to C5;
(i) AMY-201 or Mirococept;
(j) an antibody or antigen-binding fragment or protein that competes with one of the proteins set out under (i) for binding to C3b;
(k) Bikaciomab;
(l) an antibody or antigen-binding fragment thereof that competes with Bikaciomab for binding to Factor B;
(m) Lampalizumab;
(n) an antibody or antigen-binding fragment thereof that 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 that competes with avacopan or PMX-53 for binding to C5aR;
(r) clone S5/1 or clone 7H110, or an antigen-binding fragment thereof; and (s) an antibody or an antigen-binding fragment thereof that competes with one of the antibodies set forth under (r) for binding to C5aR.

In some embodiments of any aspect of the present invention, the skin, neutrophilic, inflammatory disease is
- autoinflammatory diseases (more precisely: cutaneous, neutrophilic, autoinflammatory diseases); or - autoimmune diseases with cutaneous inflammation (more precisely: cutaneous, autoimmune diseases with neutrophilic inflammation).
It is.

In some embodiments of any aspect of the present invention, the skin, neutrophilic, inflammatory disease is an autoinflammatory disease selected from the group consisting of hidradenitis suppurativa (HS); pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa); Sweet's syndrome (SS); subcorneal pustulosis (SPD); epidermolysis bullosa acquisita, erythema elevatum diutinum (EED); neutrophilic panniculitis; gut-associated dermatosis-arthritis syndrome (BADAS); and SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome.

In some embodiments of any aspect of the present invention, the skin, neutrophilic, inflammatory disease is HS or an HS-related disease selected from the group consisting of pyoderma gangrenosum (PG); PAPA (septic arthritis, PG and acne); PASH (PG, acne and hidradenitis suppurativa); PAPASH (septic arthritis, acne, PG and hidradenitis suppurativa); Sweet's syndrome (SS); and subcorneal pustulosis (SPD).

In some embodiments of any aspect of the present invention, the skin, neutrophilic, inflammatory disease is an autoimmune disease with skin inflammation selected from the group consisting of rheumatic neutrophilic dermatosis; familial Mediterranean fever, cryopyrin-associated disorder, gout, and Schnitzler syndrome.

In some embodiments of the first or third aspect of the present invention, the compound is administered in a dose of 800 mg once a week or in a dose of 800 mg twice a week.
- the inhibitor of C5a activity is a compound which specifically binds to C5a (preferably selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007, NOX-D21, and antigen-binding fragments thereof; more preferably, the inhibitor of C5a activity is selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007, and antigen-binding fragments thereof; even more preferably, the inhibitor of C5a activity is selected from the group consisting of IFX-1 and antigen-binding fragments thereof; more preferably, the inhibitor of C5a activity is IFX-1); and - the skin, neutrophilic, inflammatory disease is hidradenitis suppurativa (HS); and - the compound is administered at a dose of 800 mg once a week or at a dose of 800 mg twice a week.

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

In some embodiments of the second aspect of the present invention, the compound is administered in a dose of 800 mg once per week or in a dose of 800 mg twice per week.
- the inhibitor of C5a activity is a compound which specifically binds to C5a (preferably selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007, NOX-D21, and antigen-binding fragments thereof; more preferably, the inhibitor of C5a activity is selected from the group consisting of IFX-1, INab708, MEDI-7814, ALXN-1007, and antigen-binding fragments thereof; even more preferably, the inhibitor of C5a activity is selected from the group consisting of IFX-1 and antigen-binding fragments thereof; more preferably, the inhibitor of C5a activity is IFX-1); and - the skin, neutrophilic, inflammatory disease is hidradenitis suppurativa (HS); and - the compound is administered at a dose of 800 mg once a week or at a dose of 800 mg twice a week.

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

Pharmaceutical Compositions and Modes of Administration In the practice of any aspect of the present invention, the compounds (e.g., inhibitors of C5a activity described herein) or pharmaceutical compositions comprising the compounds may be administered to a patient by any route established in the art that provides sufficient levels of the compounds in the patient. They may be administered systemically or locally. Such administration may be parenteral, mucosal, e.g., oral, nasal, rectal, vaginal, sublingual, submucosal, transdermal, or by inhalation. Preferably, administration is parenteral, e.g., via intravenous or intraperitoneal injection, including, but not limited to, intraarterial, intramuscular, intradermal, and subcutaneous administration. When the compounds (e.g., inhibitors of C5a activity described herein) or pharmaceutical compositions comprising the compounds are administered locally, they may be injected directly into the organ or tissue to be treated.

Pharmaceutical compositions adapted for oral administration may be provided as capsules or tablets; powders or granules; solutions, syrups or suspensions (in aqueous or non-aqueous liquids); edible foams or whips; or emulsions. Tablets or hard gelatin capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharinate, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may contain vegetable oils, waxes, fats, semisolid or liquid polyols, etc. Solutions and syrups may contain water, polyols and sugar.

Active agents intended for oral administration may be coated with or mixed with a material that delays degradation and/or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be used). Thus, sustained release of the active agent can be achieved over several hours, and when necessary, the active agent can be protected from being destroyed in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of the active agent at a particular gastrointestinal location by specific pH or enzymatic conditions.

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

Pharmaceutical compositions adapted for nasal administration may include a solid carrier, such as a powder (preferably having a particle size in the range of 20 to 500 microns). Powders can be administered in the manner in which one takes a snuff, i.e. by rapid inhalation through the nose from a container of the powder held close to the nose. Alternatively, compositions employed for nasal administration may include a liquid carrier, such as a nasal spray or nasal drops. These compositions may include an aqueous or oily solution of the active ingredient. Compositions for administration by inhalation may be delivered in specially adapted devices, including, but not limited to, pressurized aerosols, nebulizers or inhalers, which may be constructed to provide a predetermined dose of the active ingredient. Pharmaceutical compositions may also be administered to the lungs via the nasal passages.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable 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 adapted 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 requiring only the addition of a sterile liquid carrier for injection, for example sterile saline, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In a preferred embodiment, the compounds described herein (e.g., inhibitors of C5a activity described herein) are formulated according to routine procedures as pharmaceutical compositions adapted for intravenous administration to humans. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the compositions may also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of injection. Generally, the ingredients are provided either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachette indicating the quantity of active agent. When the composition is administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampule of sterile saline may be provided so that the ingredients can be mixed prior to administration.

In other embodiments, for example, a compound (e.g., an inhibitor of C5a activity described herein) or a pharmaceutical composition including the compound can be delivered in a controlled release system. For example, the compound can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can 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 other embodiments, the compounds can be delivered in vesicles, in particular liposomes (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., 353-365; WO 91/04014; U.S. 4,704,355). In other embodiments, polymeric materials can 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 other embodiments, the controlled release system can be placed in proximity of the therapeutic target, i.e., the target cell, tissue, or organ, thus requiring only a fraction of the systemic dose (see, e.g., Goodson (1984) 115-138 in Medical Applications of Controlled Release, vol. 2). Other controlled release systems are described in the review by Langer (1990, Science 249: 1527-1533).

In certain embodiments, 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 locally to the area requiring treatment. This may be accomplished, for example, but not limited to, by local infusion during surgery, e.g., with a wound dressing after surgery, topical application, injection, by means of a catheter, by means of a suppository, or by means of an implant, which may be a porous, non-porous, or gelatinous material, including a membrane, such as a silastic membrane, or a fiber.

Selection of a preferred effective dose will be determined by the skilled artisan based on consideration of several factors known to those skilled in the art. Such factors include the particular form of the pharmaceutical composition, e.g., polypeptide or vector, and its pharmacokinetic parameters, e.g., bioavailability, metabolism, half-life, etc., and are established during the normal development process commonly used in obtaining regulatory approval for pharmaceutical compounds. Additional factors in considering dosage include the condition or disease to be prevented and/or treated or the benefit to be achieved in normal individuals, the patient's weight, the route of administration, whether administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of the administered pharmaceutical agent. Thus, the exact dose should be determined by standard clinical techniques, e.g., depending on the condition and immune status of the individual patient, according to the judgment of the practitioner and each patient's circumstances.

EXAMPLES The following examples are provided to further illustrate the present invention, but the present invention is not limited thereto, and the following examples merely demonstrate the feasibility of the present invention based on the above description.

1. Methods 1.1 Preparation of Zymosan A Stock Solution and Zymosan A Activated Plasma (ZAP)
Zymosan A was dissolved at 2 mg/ml in 50 ml sterile saline and boiled at 100° C. for 1 hour. After centrifugation, the supernatant was discarded and the pellet was resuspended in 50 ml sterile saline. After a second centrifugation step, the pellet was resuspended in 5 ml sterile saline to obtain a 20 mg/ml stock solution. The stock solution was aliquoted and stored at −20° C. until use. To activate the plasma, Zymosan A stock solution and 100 μl plasma were mixed and incubated at 37° C. for 30 minutes. After incubation, the tubes were centrifuged and the supernatant was aliquoted and stored at −20° C. until use.

1.2 CD11b Assay Using rhC5a or ZAP as Stimulants
Human whole blood was stimulated with rhC5a or ZAP. To test the blocking activity of IFX-1 and irrelevant control IgG4 against rhC5a, 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 reach final Ab/Ag molar ratios of approximately 4:1/3:1/2:1/1:1/0.5:1. Blood with buffer alone served as an unstimulated control to assess baseline CD11b expression. Blood with antibody alone was used to determine the effect of antibody on CD11b expression under unstimulated conditions. The complete mixture (Ab/Ag/blood) was incubated at 37° C. for 20 minutes to assess C5a-induced upregulation of CD11b. After addition of anti-mouse CD11b:FITC, samples were incubated on ice for 30 minutes to minimize background staining. Granulocytes were gated and the mean fluorescence intensity (MFI) of FITC-labeled (CD11b expressing) granulocytes was examined by flow cytometer.

1.3 CD11b Assay Using rhC5a or Zymosan A in Whole Blood
Human blood was stimulated with rhC5a or zymosan A and the complete mixture (Ab/Ag/blood) was incubated for 20 min at 37°C to stimulate C5a-induced upregulation of CD11b. After incubation, 2 μl of anti-mouse CD11b:FITC or isotype:FITC control was added and samples were incubated on ice for 30 min to minimize background staining. After lysis, cells were analyzed using a flow cytometer. Granulocytes were gated on FSC/SSC dot-plots and the mean fluorescence intensity (MFI) of FITC-labeled (CD11b expressing) granulocytes was examined for the entire sample set.

1.4 Cytokine IL-8 ELISA
Human IL-8 ELISA was performed as recommended in the instruction manual under "Assay procedure" (eBioscience Inc., San Diego, CA). Briefly, coating was performed with 100 μl 1× capture antibody overnight at 4° C. Plates were blocked with 200 μl 1× assay diluent for 1 h at RT. Standard stock solutions were diluted to the desired concentrations in 1× assay diluent and then diluted 6 times serially 1:2. Sample supernatants were diluted in 1× assay diluent as required. According to the "Assay procedure", 100 μl of standard and sample dilutions were added to the coated plate and incubated for 1 h at RT, followed by incubation with 100 μl 1× detection antibody (RT, 1 h) and 100 μl 1× avidin-HRP (RT, 30 min). Color development was performed with 100 μl TMB substrate solution for 10 min at RT in the dark and terminated with 100 μl stop solution. Absorbance was read within 30 minutes using a plate reader at 450 nm. Zero standard values (blanks) were subtracted from all standards and samples. Cytokine concentrations of samples were 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 at a final concentration of 0.5 μg/mL on the ELISA plate. After blocking with assay diluent (1× PBS with 0.05% Tween 20 and 2% heat-inactivated FBS), calibration samples (recombinant human C5a, Sigma, Taufkirchen, Germany) diluted in assay diluent and samples were incubated for 90 min at room temperature. Mouse anti-human C5/C5a antibody clone 561 (Hycult Biotech, Uden, The Netherlands) diluted to 2 μg/mL in assay diluent was applied as the primary detection antibody for a 60 min incubation at room temperature, followed by a 30 min incubation with a secondary horseradish peroxidase-labeled antibody (goat anti-mouse IgG2a polyclonal antibody, SouthernBiotech, Birmingham, USA) diluted to 0.05 μg/mL in assay diluent. Color development was performed with tetramethylbenzidine substrate solution (TMB, Biozol, Eching, Germany) and stopped with 3.7 N sulfuric acid. OD was read as absorbance at 450 nm on a Tecan Infinite® 200 reader equipped with a Tecan Magellan ^™ (Tecan Group, Maennedorf, Switzerland). The in-house developed C5a ELISA was validated according to the EMA guidelines for bioanalytical method validation.

Intra- and interassay precision tested at five different concentrations showed coefficients of variation (CV) of 0.65% to 4.96% and 1.50% to 4.88% for 6 and 18 replicates, respectively. Recovery analysis of spiked recombinant human C5a in buffer resulted in a recovery of 86.98 ± 1.20% (mean ± SD) at the lower limit of quantification and 91.50 ± 3.29% at the upper limit of quantification. No cross-reactivity was detected for C3, C3a and C4, and <0.01% cross-reactivity was detected for C5b-6. Human IgG4 antibodies did not interfere with the assay. The mean C5a level in citrated plasma from 20 human volunteers is 17.08 ng/mL ± 6.96 ng/mL, ranging from 7.52 ng/mL to 30.17 ng/mL.

1.6 Measurement of complement activation products
Concentrations of complement activation products C3a, C5a and membrane attack complex C5b-9 were measured by ELISA. C3a ELISA (BD OptEIA ^™ Human C3a ELISA Kit, BD Bioscience, Germany) was performed according to the manufacturer's instructions. C5b-9 concentrations were determined using an InflaRx-validated C5b-9 ELISA based on the BD OptEIA ^™ Human C5b-9 ELISA Set (BD Bioscience). C5a concentrations were measured using the InflaRx-established and validated C5a ELISA.

1.7 Statistical analysis
All results were presented as mean ± standard deviation. Statistical differences between groups were calculated by one-way analysis of variance with Tukey's multiple comparison test or by Student's t-test for two groups after ^{baseline correction. A p-value of 0.05 was used in the calculations to determine whether there was a significant difference between any two groups. Graphing and statistical analysis were performed with GraphPad PRISM®} V6.05 (CA, USA).

2. Preclinical related data 2.1 Neutrophil activation by C5a and the blocking effect of IFX-1
Neutrophil activation was evaluated using CD11b levels in neutrophils, since CD11b upregulation is a sensitive feature for neutrophil activation. In this study, a human whole blood model was used to evaluate the blocking activity of IFX-1 against recombinant human C5a (rhC5a). Human whole blood was incubated with buffer, antibody alone, rhC5a alone, or a combination of different antibody concentrations and rhC5a. After incubation, cells were stained with anti-mouse CD11b:FITC and analyzed by flow cytometry, where CD11b MFI is a check for activation levels of blood neutrophils. As shown in Figure 1, recombinant human C5a strongly stimulates CD11b upregulation in human neutrophils. This effect can be completely blocked in the presence of the anti-human C5a antibody IFX-1. This inhibition was strong and specific, and an irrelevant human IgG4 antibody showed no blocking activity.

Zymosan-activated plasma (ZAP) was used to stimulate blood neutrophils as a source of endogenous C5a (eC5a). The amount of eC5a in ZAP was measured using a commercially available C5a ELISA Kit. The data presented here (Figure 2) indicate that eC5a in ZAP induced CD11b upregulation at levels comparable to rhC5a. The presence of IFX-1 significantly reduced CD11b expression in human neutrophils, even at an Ab:Ag molar ratio of 0.5:1. The total blocking activity of IFX-1 against ZAP-induced CD11b upregulation ranged from 100% to 82%, depending on the Ab:Ag ratio. Despite the presence of high levels of eC3a and other complement activation products in ZAP, IFX-1 can specifically block CD11b upregulation up to 100%. Therefore, it can be concluded that eC5a is the sole driving force behind neutrophil activation upon ZAP stimulation and that IFX-1 can completely block it.

2.2 C5a blockade attenuates zymosan-induced inflammatory responses in human whole blood
Zymosan A, as an active fungal wall component, can induce a strong inflammatory response in human whole blood, characterized by neutrophil activation with elevated cytokine and chemokine levels. In this study, human whole blood was spiked with zymosan A in the presence or absence of IFX-1, and CD11b expression in blood neutrophils was measured by flow cytometry analysis. As shown in Figure 3, CD11b in blood neutrophils was strongly upregulated in human whole blood in the presence of zymosan. CD11b upregulation by zymosan stimulation could be inhibited by 79%-93% depending on the concentration of IFX-1 added. As a positive control, CD11b upregulation stimulated by rhC5a was 100% blocked by IFX-1. Therefore, it is confirmed that CD11b upregulation in blood neutrophils upon zymosan A stimulation is mainly caused by eC5a. In addition, it can be concluded that eC5a, when generated in whole blood by zymosan A, initially binds to IFX-1, thereby blocking its access to its natural receptor.

In the same experimental setting, IL-8 levels were measured and used to evaluate the inflammatory response. IL-8 concentrations after stimulation with various doses of zymosan A ranged from 458 pg/ml to 3218 pg/ml in the absence of IFX-1. As shown in Figure 4, the presence of IFX-1 significantly reduced IL-8 production upon stimulation with various concentrations of zymosan A, with a maximum reduction rate of 54% observed. Thus, in an inflammatory whole blood environment, zymosan-induced inflammatory responses are highly dependent on the presence of C5a.

3. Clinically relevant data 3.1 Data obtained from clinical samples 3.1.1 Complement activation in HS patients
A total of 54 patients with HS and 14 healthy controls were enrolled in the study. Patients were followed up in the Outpatient Department of Infectious Diseases and Immunology at the ATTIKON University Hospital, Greece. The study was approved by the Ethics Committee of the hospital. Written informed consent was provided by all patients. The diagnosis of HS was based on the following criteria: a) early onset after puberty; b) presence of subcutaneous nodules in areas of skin rich in apocrine glands; and c) a compatible history of recurrent drainage of pus from the affected area.

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

However, it was unclear from the above results which one of C3a, C5a or C5-9b or other complement activation products was the most promising target for this novel therapeutic strategy, and whether targeting only one of these factors would be sufficient, or whether two or more factors involved in complement activation should be targeted.

3.1.2 Blockade of CD11b upregulation in blood neutrophils induced by HS plasma
To determine the role of C5a in HS plasma samples in neutrophil activation, HS plasma samples with high levels of C5a were selected and evaluated by using a human whole blood model. As shown in Figure 6, in contrast to control plasma samples with low C5a levels (Ctrl 008 and Ctrl 012), HS plasma samples with high levels of C5a (Pat.088 and Pat.092) strongly upregulated CD11b expression in blood neutrophils. Recombinant human C5a was used as a positive control, while plasma from a healthy individual was used as a negative control. CD11b upregulation induced by HS plasma could be 100% inhibited by IFX-1, indicating that C5a is the most important activating factor in HS plasma to initiate neutrophil activation. From these novel results, we conclude that blocking C5a is sufficient to achieve a strong inhibition of neutrophil activation in HS patients.

3.2 Data from Clinical Trials 3.2.1 Study Design
An open-label Phase II study was conducted in 11 patients with moderate to severe hidradenitis suppurativa at the Department of Internal Medicine, ATTIKON University Hospital, Greece.

The primary objective of the study was to investigate the safety and tolerability of IFX-1 administered over 8 weeks. Secondary objectives of the study were to evaluate the pharmacokinetics and pharmacodynamics of IFX-1, and to generate preliminary data of the efficacy of IFX-1 on clinical endpoints to generate further hypotheses (e.g., HiSCR, DLQI, VAS for disease status, VAS for pain, HS-PGA, modified Sartorius Score). Enrolled patients were treated with 800 mg IFX-1 twice in the first week and once weekly thereafter for a total of 8 weeks of treatment; i.e., IFX-1 was administered in 9 intravenous doses of 800 mg IFX-1 on days 1, 4, 8, 15, 22, 29, 36, 43, and 50. All patients were followed for an additional 12 weeks.

Screening inclusion criteria:
1. Male or female patient > 18 years of age 2. Written informed consent 3. Diagnosis of HS for at least 1 year 4. HS lesions in at least 2 different anatomical regions, one of which is Hurley Stage II or III 5. Total AN (abscesses and nodules) number > 3
6. Patients with either primary or secondary failure of biological treatment or who are not suitable for other biological treatments. Note: Primary failure is defined as at least 12 weeks of treatment with a biological compound without effect, secondary failure is defined as achieving an initial response after at least 12 weeks of treatment with a biological compound followed by relapse. 7. Failure of previous antimicrobial treatments.

Screening Exclusion Criteria:
1. Body weight >150 kg or <60 kg; 2. Having a baseline draining fistula count of 30 or more; 3. Planned surgical procedure within the next 24 weeks; 4. Occurrence of HS flare leading to intravenous antibiotic treatment within the past 14 days; 5. Other diseases and conditions that may interfere with the evaluation of the study product, outcome assessment, or satisfactory conduct of the study. a) Active infection; b) Severe congestive heart failure (i.e., NYHA class IV).
c) Depression d) History of systemic lupus erythematosus or rheumatoid arthritis e) Immunodeficiency disease f) Active hematologic or solid malignancies g) Patients must not have other active skin diseases or conditions (e.g., bacterial, fungal, or viral infections) that may preclude evaluation for HS 6. One of the following abnormal laboratory results a) White blood cell count < 2,500/mm ³
b) Neutrophil count <1000/ ^mm
c) Serum creatinine >3x upper limit of normal (UNL)
d) Total bilirubin > 2 x UNL
e) Alanine aminotransferase (ALAT)>2xUNL
f) positive screening test for Hepatitis B, Hepatitis C, or HIV 1/2; 7. prior administration of a biologic compound in the past 3 months; 8. intake of corticosteroids, defined as daily intake of ≥ 1 mg/kg prednisone or equivalent in the past 3 weeks;
9. Taking immunosuppressants within the past 30 days (e.g., cyclosporine, tacrolimus)
10. General Exclusion Criteria a) Pregnant (a urine pregnancy test should be performed in women of childbearing potential) or breastfeeding women b) Women of childbearing potential (defined as within 2 years since their last menstrual period) who are not willing to practice adequate contraception while participating in the study (e.g., Implanon, injections, oral contraceptives, intrauterine devices, vasectomy partner, abstinence)
c) Participation in an interventional clinical trial within the past 3 months d) Known intravenous drug abuse e) Spouse/partner or relative of an investigational site employee, study staff (e.g., investigator, subinvestigator, or study nurse), or blood relationship to the sponsor

3.2.2 Clinical trial results
IFX-1 is well tolerated by HS patients. No drug-related serious adverse events were reported over the treatment period.

A commonly used efficacy parameter in hidradenitis suppurativa clinical response (HiSCR). HiSCR is defined by three lesion states (defining criteria): abscess (fluid, tender or painful with or without drainage), inflammatory nodule (tender, erythematous, suppurating granulomatous lesion), and draining fistula (sinus tract, with communication to skin surface, draining purulent fluid). The proposed definition of responder to treatment (HiSCR achiever) is (i) at least 50% reduction in AN, (ii) no increase in number of abscesses, and (iii) no increase in number of draining fistulas from baseline. HiSCR has recently been validated as a responsive and clinically meaningful endpoint of inflammatory manifestations of HS (Kimball and others, 2014).

The HiSCR response over an 8-week treatment period was investigated in this study, with 8 of 11 patients who had been treated up to 56 days responding, representing a response rate of 72.7% and a 95% confidence interval of 43% to 91%. To compare these results with historical data, a literature search was performed to find placebo-controlled clinical trials that used HiSCR as an efficacy parameter. Table 4 below summarizes five recently completed trials:
Table 4. Completed clinical trials using HiSCR as an efficacy parameter


¹ Humira EMA evaluation report:
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Assessment_Report_-_Variation/human/000481/WC500195564.pdf
^2. Anakinra trial (Tzanetakou and others, 2016)
³ Press Release XBiotech (http://investors.xbiotech.com/phoenix.zhtml?c=253990&p=irol-newsArticle&ID=2246777)

In total, 179 patients have been treated in the placebo group of these studies with a response rate of 19.0% with a 95% confidence interval of 14% to 25%. As both confidence intervals (e.g., historical placebo patients and patients treated with IFX-1) do not overlap, a significant treatment effect of IFX-1 can be concluded.

Photographic documentation of the affected areas confirmed these findings with significantly reduced inflammation in the skin as evidenced by a visual reduction in inflammatory hyperplasia and redness following treatment.

Thus, anti-C5a represents a potent anti-inflammatory agent in the disease environment of HS. The clinical findings demonstrate that blockade of C5a is highly effective in reducing neutrophil activation, thereby effectively alleviating cutaneous neutrophilic inflammatory disease.

4. Blockade of Activated Complement Factor-Induced CD11B Upregulation in HS Patients via Inhibition of the C5A-C5AR Axis 4.1 Objectives The objective of the following study was to demonstrate blockade of HS patient plasma-induced CD11b upregulation on the surface of neutrophils by anti-human C5a monoclonal antibody IFX-1, anti-human C5a receptor C5aR (CD88) antibodies, and C5aR antagonists and inhibitors.

4.2 Assay Principle Neutrophil accumulation at sites of inflammation depends on the expression of adhesion molecules, including CD11b (also known as integrin alpha M) (Larson and Springer, 1990; Carlos and Harlan, 1990). Upregulation and recruitment of CD11b/CD18 from intracellular pools to the surface of neutrophils is essential for human neutrophil rolling and migration (Smith et al., 1989). Thus, enhanced expression of CD11b/CD18 reflects events that trigger inflammation. Human CD11b assays are performed using flow cytometry to detect FITC-conjugated anti-CD11b antibodies on the surface of neutrophils. Activated complement products, especially elevated endogenous C5a (eC5a), in HS patient plasma samples can strongly upregulate CD11b expression via binding of C5a to its receptor C5aR (CD88) on neutrophils. As a result, blockade of the C5a-C5aR axis is expected to abolish or attenuate CD11b upregulation on the surface of neutrophils.

IFX-1, as the first anti-human C5a monoclonal antibody introduced into clinical development, has been proven to control inflammatory responses that cause tissue and organ damage. The antibody is currently being evaluated in a Phase IIb trial for patients with moderate or severe hidradenitis. It specifically and directly neutralizes the terminal complement anaphylatoxin C5a, blocking its deleterious effects as a key inflammatory mediator in both acute and chronic inflammatory diseases (Klos et al., 2009; Guo and Ward, 2005; Riedemann et al., 2017).

C5a exerts its effects through interaction with the 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 understood that C5a-C5aR signaling is crucial in the pathogenesis of inflammatory outcomes (Ward, 2009). Therefore, targeting C5aR is another strategy to inhibit complement-dependent inflammatory diseases. A series of small molecules derived from the C-terminus of C5a have been developed as C5aR antagonists. Among them, the lead compound, the cyclic hexapeptide PMX-53 (AcF-[OP(D-Cha)WR]) (Finch et al., 1999), was shown to reduce damage in many animal models of inflammation after intravenous, subcutaneous, intraperitoneal, and oral administration (Proctor et al. 2006). With structural similarity to C5a, such antagonists compete with C5a for the C5a receptor in neutrophils (March et al., 2004). In addition, anti-C5aR antibodies could block the binding of C5a to C5aR, thereby reducing the accumulation and activation of myeloid-derived suppressor cells and neutrophils (Markiewski et al., 2008). Two commercially available monoclonal anti-C5aR antibodies, clones S5/1 and 7H110, were tested in this study. They were raised in mice against synthetic peptides containing the N-terminal extracellular domain of C5aR (Met1-Asn31) and recombinant human C5aR (Met1-Val350), respectively, and both antibodies were described as neutralizing antibodies. Avacopan (CCX168) is an orally administered small molecule drug candidate that selectively inhibits the complement C5a receptor (C5aR) and is being developed for inflammatory and autoimmune diseases (Bekker et al., 2008; Jayne et al., 2017).

The inhibitory effects of these blockers targeting the C5a-C5aR axis were monitored via flow cytometry for blockade of CD11b upregulation in neutrophils.

4.3 Experimental Details 4.3.1 Sample
According to the consensus definition and diagnostic criteria of the Hydradenitis Suppurativa Foundation 2009, plasma samples of two HS patients (Pat. 088 & Pat. 092) and two healthy controls (Ctrl 009 & Ctrl 010) were included in this study. The complement system was activated in the pathogenesis of HS, as manifested by elevated levels of C3a, C5a and C5b-9 (see Table 5 below). The C5a levels of patients 088 and 092 (93.77 ng/mL and 70.01 ng/mL, respectively) were significantly higher than those of the healthy controls (21.02 ng/mL and 11.77 ng/mL).
Table 5. Concentrations of complement factors measured in plasma samples of test subjects

4.3.2 Reagents
AnaLaR water, VWR (Darmstadt, Germany), Cat. No. 102923C, NORMAPUR analytical grade, sterile filtered ACD, Sigma Aldrich (Taufkirchen, Germany), Cat. No. C3821-50ML
Reagents for flow cytometers: FACS Flow Sheet Fluid, BD Bioscience (NJ, USA), Cat. No. 342003
FACS Shutdown solution, BD Bioscience (NJ, USA), Cat. No. 334224
FACS Clean solution, BD Bioscience (NJ, USA), Cat. No. 340345
Rat anti-mouse CD11b: FITC, BD Bioscience (NJ, USA) Cat. No. 553310, 0.5 mg/mL
○ 10x FACS Lysing Solution, BD Bioscience (NJ, USA), Cat. No. 349202 → Working solution: 1x FACS Lysing Solution (diluted 1:10 in Analar water)
Staining buffer: 1% heat-inactivated FBS + 0.1% sodium azide in 1x PBS solution. FBS, Thermo Fisher Scientific (Darmstadt, Germany), Cat. No. 10099133. Heat inactivation: 56°C, 30 min. 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. No. HC2101, expressed in E. coli dissolved in sterile AnaLaR water; 0.9% sterile sodium chloride (saline), B. Braun (Melsungen, Germany), Cat. No. 3200950
IFX-1, an anti-human C5a antibody applied as a control, InflaRx (Jena, Germany), 10 mg/mL in PBS + 0.05% Tween 80
PMX-53, bio-techne (Wiesbaden-Nordenstadt, Germany), Cat. No. 5473
Anti-C5aR (CD88) antibody Clone S5/1, Hycult Biotech (Uden, Netherlands), Cat. No. HM2094
Anti-C5aR (CD88) antibody Clone 7H110, biomol (Hamburg, Germany), Cat. No. C2439-60N
-Avacopan, MedKoo Biosciences Inc. (Morrisville, USA), Cat. No. 319575
Human blood from healthy donors containing 12% ACD (for immediate use)
Pooled human plasma (citrated plasma) from Jena University Hospital

4.3.3 Equipment
Flow cytometer (FACS Canto II with DIVA software V6.1.2)

4.3.4 Procedure
a) Human CD11b potency assay (flow cytometry assay)
Two patient plasma samples Pat. 088 and Pat. 092 (5 μL) were incubated with fresh human blood (60 μL, as a source of neutrophils) in the absence or presence of C5a-C5aR axis blockers (anti-C5a antibody IFX-1, anti-C5aR antibody clone S5/1 and clone 7H110, C5aR antagonist PMX-53, and C5aR inhibitor Avacopan; 10 μL) in a total volume of 100 μL. Two control plasma samples (Ctrl 009 and Ctrl 010, 5 μL), prepared according to the procedure applied to the patient samples, served as controls for non-specific activation. Blood with saline only (40 μL) or normal human plasma pool (5 μL huPP + 35 μL saline) served as unstimulated controls to define baseline expression of CD11b. Blood samples with normal human plasma pool and spiked with recombinant human C5a (rhC5a) mimicked stimulating conditions. All samples were incubated at 37°C for 20 min to activate CD11b upregulation. After cooling on ice, 2 μL of FITC-conjugated anti-mouse CD11b antibody was added to the samples. To minimize background signal, the labeled samples were left on ice for an additional 30 min in the dark. Red blood cells were then lysed with 1× FACS Lysing solution for 10 min at room temperature. The remaining cells were washed twice using 2 mL Staining buffer. After centrifugation at 2500 rpm for 3 min, cells were resuspended in 0.5 mL staining buffer and prepared for FACS analysis. A gate was set on the FSC vs. SSC plot to allow analysis of only cells with the size of neutrophils.

The percent blocking activity (BA) of C5a-C5aR axis blocking test substances was calculated using the following formula: where MFI is the mean fluorescence intensity emitted from CD11b-bound FITC in neutrophils.
BA (%) = (MFI _{patient plasma} - MFI _{test substance spiked patient plasma} ) ÷ (MFI _{patient plasma} - MFI _huPP ) x 100

b) Statistical Analysis Graphs were generated with GraphPad Prism 7 (CA, USA).

4.4 Results and Discussion
Expression of integrin CD11b on neutrophils
CD11b expression on neutrophils was evaluated in plasma samples from two healthy blood donors (Ctrl 009 and Ctrl 010) and two diagnosed HS patients (Pat. 088 and Pat. 092). The mean fluorescence intensity (MFI) of the Ctrl- samples was 2156.9 ± 114.3, which is within the range of unstimulated CD11b baseline expression (MFI < 3500). In contrast, a 2.3- to 3.9-fold increase in CD11b expression was induced by either HS patient samples (Pat. 088, Pat. 092) or 20 nM recombinant human C5a (rhC5a) (Figures 8, 9, 10, and 11; Tables 6, 7, and 8). These data suggest that the significantly upregulated CD11b expression was mediated by inflammatory factors in HS patients. Complement activation products, especially C5a, are hypothesized to play a major role in CD11b upregulation (Table 5).
Table 6. Blocking activity of anti-C5aR antibody clones S5/1 and 7H110, and the C5aR antagonist PMX-53, on complement factor-induced CD11b upregulation.

Table 7. Blocking activity of anti-C5a antibodies IFX-1 and C5aR antagonist PMX-53 on complement factor-induced CD11b upregulation

Inhibition of CD11b upregulation by anti-human C5aR monoclonal antibodies, C5aR antagonists and C5aR inhibitors As shown in Figure 8, rhC5a (20 nM) and two HS patient plasma samples with high levels of eC5a (Pat. 088 and Pat. 092) strongly upregulated CD11b expression in blood neutrophils, whereas the presence of a blocking antibody targeting C5aR at a final concentration of 50 nM could significantly attenuate the CD11b expression driven by either rhC5a or HS patient plasma samples. High blocking activity, ranging from 71% to 79%, was achieved by using anti-C5aR antibodies, clone 7H110 and clone S5/1 (Figure 8, Tables 9 and 6). In contrast, the C5aR antagonist PMX-53, a small hexapeptide, was not as effective as the C5aR-specific monoclonal antibody. The blocking activity obtained by the same concentration of PMX-53 (50 nM) was within 50% to 57% (Figure 9A, Tables 9 and 6). To test whether abolition of CD11b upregulation could be achieved via C5aR inhibition, a higher concentration of PMX53 (20 μM) was further evaluated for blocking activity in the same experimental setup. As a result, CD11b upregulation induced by HS patient plasma samples as well as rhC5a was completely abolished in the presence of high levels of PMX-53 (Figure 9B, Tables 9 and 7). Similar data were achieved in the presence of the C5aR inhibitor Avacopan (Figure 11, Tables 8 and 9). Blocking activity in the presence of 100 μM Avacopan was within 77% to 80%, and CD11b upregulation induced by HS patient plasma was completely blocked in the presence of 500 μM Avacopan (FIG. 11, Tables 8 and 9).

These results suggested that the C5a/C5aR axis is primarily involved in CD11b upregulation in blood neutrophils and that C5aR may serve as a possible target for blocking C5a activity.
Table 8. Blocking activity of the C5aR inhibitor Avacopan on complement factor-induced CD11b upregulation

Table 9. Summary blocking activity (%) of all C5a-C5aR axis blocking tested molecules in CD11b upregulation induced by HS patient plasma or rhC5a spiked healthy human plasma

IFX-1, an anti-human C5a monoclonal antibody, completely blocked HS-induced CD11b upregulation. By using the same experimental setup as above, as expected, rhC5a and HS patient plasma samples (Pat. 088 and Pat. 092) strongly activated CD11b expression in blood neutrophils, and the addition of IFX-1 at a concentration as low as 20 nM could completely abolish CD11b upregulation (Figure 10). These results indicate that IFX-1 is more efficient in inhibiting C5a/C5aR-driven inflammatory responses.

4.5 Conclusion Taken together, our results show that a C5a-targeting approach, i.e. application of the anti-C5a monoclonal antibody IFX-1, effectively abolishes C5a-mediated CD11b upregulation. Moreover, anti-C5aR antibodies, C5aR antagonists as well as C5aR inhibitors also showed strong blocking activity under the same experimental conditions. Thus, targeting C5aR with blocking antibodies or antagonists represents an alternative strategy to block the C5a/C5aR axis under inflammatory conditions, e.g. HS.

References

Claims (1)

1. A composition comprising a compound for use in treating a skin, neutrophilic, inflammatory disease in a subject, wherein the compound is an inhibitor of C5a activity, the skin, neutrophilic, inflammatory disease is hidradenitis suppurativa (HS), the inhibitor of C5a activity directly binds to the C5a receptor and inhibits activity of the C5a receptor, and the inhibitor of C5a activity is avacopan .