Humanized anti-C5A antibody

Abstract

The present invention relates to antibodies that specifically bind to a conformational epitope of human C5a. The present invention particularly relates to humanized anti-C5A antibodies. The antibodies described herein are useful as active agents in pharmaceutical compositions. The antibodies and pharmaceutical compositions are useful, inter alia, for the treatment and prevention of diseases or disorders associated with pathological C5a activity.

Description

Technical Field

[0001] Technical Field The present invention relates to antibodies that specifically bind to the conformational epitopes of human C5a. The present invention relates in particular to humanized anti-C5A antibodies. The antibodies described herein are useful as active agents in pharmaceutical compositions. The antibodies and pharmaceutical compositions are useful, inter alia, for the treatment and prevention of diseases or disorders associated with pathological C5a activity.

Background Art

[0002] Background of the Invention C5a C5a is a 74 amino acid degradation product of its “mother molecule” C5 and represents one endpoint of the complement activation cascade. It can be generated via activation of at least three well-described pathways (alternative, classical and MBL pathways). All pathways converge at the level of C3, form C5 or alternative C5 convertases, leading to cleavage of C5 into C5a and C5b. The latter binds to C6, C7, C8 and multiple C9 molecules, ultimately resulting in pore formation, 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.

[0003] Among the complement activation products, C5a is one of the most potent inflammatory peptides and has a wide range of functions (Guo and Ward 2005). C5a exerts its effects via 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, since few biological responses are seen against the C5a-C5L2 interaction, C5a is thought to exert its biological functions mainly through the C5a-C5aR interaction. However, recent reports have demonstrated signaling involving C5L2 activation as well (Rittirsch et al. 2008).

[0004] C5aR is widely expressed on myeloid cells, including neutrophils, eosinophils, basophils, and monocytes, and on non-myeloid cells in many organs, particularly the lung and liver, indicating the importance of C5a / C5aR signaling. Widespread upregulation of C5aR expression occurs during the onset of sepsis, and blockade of the C5a / C5aR interaction by anti-C5a, or anti-C5aR antibodies, or C5aR antagonists confers a high degree of protection in rodent models of sepsis (Czermak et al. 1999; Huber-Lang et al. 2001; Riedemann et al. 2002).

[0005] 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 breakdown (O2 consumption) in neutrophils and enhances phagocytosis and the release of granular enzymes. C5a has also been found to be a vasodilator. C5a has been shown to be involved in the regulation of cytokine expression from various cell types and to enhance the expression of adhesion molecule expression in neutrophils. High doses of C5a can cause "desensitization" of neutrophil non-specific chemotaxis, thereby causing extensive dysfunction. Many inflammatory diseases, including sepsis, acute lung injury, inflammatory bowel disease, rheumatoid arthritis, etc., are due to the effects of C5a. In an experimental situation of sepsis, exposure of neutrophils to C5a can cause neutrophil dysfunction and paralysis of the signaling pathway, resulting in assembly deficiency of NADPH oxidase, paralysis of the MAPK signaling cascade, severe depression of oxidative breakdown, phagocytosis, and chemotaxis (Guo et al. 2006; Huber-Lang et al. 2002). Apoptosis of thymocytes and delayed neutrophil apoptosis are two important pathogenic events for the development of sepsis, and these are dependent 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 failure (MOF). It has also been found that C5a is due to the activation of the coagulation pathway 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. Since complement activation is an event that occurs during the onset of acute inflammation, C5a may begin to act before the appearance of most inflammatory "cytokine storms". C5a seems to play an important role in regulating and amplifying the ability of the cytokine network and the formation of the systemic inflammatory response syndrome (SIRS).

[0006] In the immunoregulatory network leading to adaptive immunity, C5a affects the crosstalk between dendritic cells (DCs) and γδ T cells, which can lead to the overproduction of inflammatory mediators such as IL-17 (Xu et al. 2010). An important role for C5a has been established and defined in the generation of pathogenic Th17 responses in systemic lupus erythematosus (SLE) (Pawaria et al. 2014). In addition, C5a has been reported to be an important regulatory factor for Treg cells that provides a strong inhibitory effect on Treg proliferation and induction (Strainic et al. 2013). Considering the fact that Tregs and TH17 are important players in autoimmune disease settings, inhibition of C5a signaling is expected to significantly reduce the hyperactive immune state in autoimmune diseases.

[0007] IFX-1 IFX-1 is a chimeric monoclonal IgG4 antibody that specifically binds to the soluble human complement breakdown product C5a. IFX-1 is composed of 1328 amino acids and has a molecular weight of approximately 148,472 daltons. The CDR and FR sequences of IFX-1 are described in Table 3 of WO 2015 / 140304 A1, which is also disclosed as US 2017 / 0137499 A1. The contents of WO 2015 / 140304 A1 and US 2017 / 0137499 A1 are hereby incorporated by reference in their entirety.

[0008] IFX-1 is expressed as a recombinant protein in the mammalian CHO cell line and is finally formulated in phosphate-buffered saline aqueous solution (PBS + 0.05% polysorbate 80) for intravenous administration. The binding of this antibody to human C5a facilitates a very effective blockade of C5a-induced biological effects by abrogating the binding of C5a to its corresponding cell surface receptor and the reaction with the receptor.

[0009] Various non-clinical tests, which can be classified into in vitro / ex vivo tests (using IFX-1) and in vivo tests including GLP toxicology tests in cynomolgus monkeys, were conducted to evaluate the pharmacological and toxicological aspects of IFX-1. The non-clinical tests and studies performed did not show any toxicological or safety concerns with respect to IFX-1. The human Phase I trials showed that safety laboratory parameters, vital signs and ECG parameters did not show any clinically relevant time- or dose-dependent changes.

[0010] In vitro analysis of IFX-1 demonstrated strong binding ability to soluble human C5a and high blocking activity of C5a-induced biological effects, such as lysozyme release from human neutrophils or upregulation of CD11b in neutrophils in human whole blood. One IFX-1 antibody reaches the ability to neutralize the effect of 2 molecules of C5a with almost 100% efficiency in an experimental in vitro situation. Clinical trials with IFX-1 are ongoing to test its clinical efficacy in several inflammatory diseases.

[0011] The technical problem underlying the present invention Antibodies that specifically bind to the C5a portion of C5 but not to the C5b portion have been described in the prior art (Klos et al. (1998) J. Immunol. Meth. 111: 241-252; WO 01 / 15731; WO 03 / 015819). Previously generated anti-C5a antibodies have shown only moderate blocking activity against the biological effects induced by C5a. As a result, prior art anti-C5a antibodies were unable to achieve complete blockade of C5a-induced biological effects or had to be used supra-stoichiometrically to achieve moderately high blockade of C5a activity. The inventors have succeeded in producing two monoclonal anti-C5a antibodies (designated INab308 and INab708) that exhibit strong blocking activity against C5a-induced biological effects even when used at stoichiometric amounts, i.e., 0.5 moles of bivalent antibody per mole of C5a (see WO 2011 / 063980 A1, the content of which is incorporated herein by reference). Based on the monoclonal antibody INab308, the inventors have developed a chimeric antibody, IFX-1, that exhibits the same strong blocking activity (see WO 2015 / 140304 A1, the content of which is incorporated herein by reference).

[0012] However, the two monoclonal antibodies INab308 and INab708 specifically described in WO 2011 / 063980 A1 are murine antibodies. The antibody IFX-1 described in WO 2015 / 140304 A1 is a chimeric antibody. As a result, these antibodies can induce unwanted immunological responses when administered to humans.

[0013] Therefore, in view of the intended clinical use in patients, there remained a need in the prior art for anti-C5a antibodies that are more closely similar to human antibodies but still exhibit excellent blockade of C5a activity.

[0014] In this study, the inventors successfully produced a humanized anti-C5a antibody that has significantly improved human-likeness compared to the antibodies described in WO 2011 / 063980 A1 and WO 2015 / 140304 A1, and still maintains the advantageous properties of the antibodies described in WO 2011 / 063980 A1 and WO 2015 / 140304 A1, that is, it shows a similarly high blocking activity against C5a-induced biological effects without affecting the biological activity of C5b.

[0015] The above overview does not necessarily describe all the problems solved by the present invention.

Summary of the Invention

[0016] Summary of the Invention In a first aspect, the present invention relates to an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH domain has the amino acid sequence according to SEQ ID NO: 10

Chemical formula

Chemical formula

Chemical formula

[0017] In a second aspect, the present invention An antibody or an antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain has the amino acid sequence according to SEQ ID NO: 17 (QVQLVQSGX 9 E X 11 KKPGASVKX 20 SCKASGYSFT TFWMDWVX 38 QA PGQGLEWX 48 GR IDPSDSESRL DQX 63 FKDRX 68 TX 70 TVDKSTSTVY MX 82 LSSX 86 X 87 SED X 91 AVYYCARGN DGYYGFAYWG QGTLVTVSS), where X 9 is A or P, X 11 is L or V, X 20 is I or V, X 38 is K or R, X 48is I or M, and X 63 is K or R, and X 68 is A or V, and X 70 is L or M, and X 82 is E or Q, and X 86 is L or P, and X 87 is R or T, and X 91 is S or T, or an amino acid sequence according to SEQ ID NO: 17 having one, two or three amino acid substitutions, wherein the amino acid sequence having one, two or three amino acid substitutions comprises the CDR1H, CDR2H, CDR3H sequences of SEQ ID NOs: 20 to 22 respectively comprises, consists essentially of, or consists of, and the VL domain is SEQ ID NO: 18 (DIX 3 X 4 TQSPX 9 S LX 12 ASVGDRVTITCKASQSVDYDGD SYMKWYQQKPGKAPKLLIYAASNLQS GX 62 PSRFSGSGSGTDFTLTISSLQX 84 an amino acid sequence according to SEQ ID NO: 18 (EDFATYYCQQSNEDPYTFGQGTKLEI), and X 3 is V or Q, and X 4 is L or M, and X 9 is A or S, and X 12 is A or S, and X 62 is I or V, and X 84 is E or P, or an amino acid sequence according to SEQ ID NO: 18 having one, two, three, four, five, six or seven amino acid substitutions, wherein the amino acid sequence having one, two, three, four, five, six or seven amino acid substitutions comprises the CDR1L, CDR2L, CDR3L sequences of SEQ ID NOs: 23 to 25 respectively comprises, consists essentially of, or consists of relates to an antibody or an antigen-binding fragment thereof.

[0018] In a third aspect, the invention relates to an antibody according to the first aspect or an antigen-binding fragment thereof or an antibody according to the second aspect or an antigen-binding fragment thereof, and further comprises one or more pharmaceutically acceptable carriers, diluents, excipients, extenders, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives,[ a pharmaceutical composition.[

[0019] In a fourth aspect, the invention relates to an antibody according to the first aspect or an antigen-binding fragment thereof or an antibody according to the second aspect or an antigen-binding fragment thereof for use in medicine.[

[0020] In a fifth aspect, the invention relates to an antibody according to the first aspect or an antigen-binding fragment thereof or an antibody according to the second aspect or an antigen-binding fragment thereof for use in the treatment or prevention of a disease or disorder associated with pathological C5a activity.[

[0021] The summary of this invention does not necessarily describe all features of the invention. Other aspects will become apparent from the following detailed description.[ BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1

[0023]

Figure 2

[0024]

Figure 3

[0025] DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Preferably, the terms used herein are defined as described 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 to be interpreted 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.

[0027] Several documents (e.g., patents, patent applications, scientific publications, manufacturer's manuals, instructions, GenBank accession number sequence deposits, etc.) are cited throughout the text of the present specification. In the present specification, it should not be construed as an admission that the present invention has no right prior to such description for reasons of prior inventions. Some of the documents cited in the present specification are characterized as "incorporated by reference". If there is a conflict between the definition or teaching of such incorporated document and the definition or teaching cited in the present specification, the text of the present specification shall prevail.

[0028] Sequences: All sequences mentioned in the present specification are set forth in the accompanying Sequence Listing, which is hereby incorporated by reference in its entirety and forms a part of the present specification.

[0029] As used herein, "human C5a" refers to the following 74 - amino acid peptide:

Chemical formula

[0030] The amino acid sequence of human C5 can be found under accession number UniProtKB P01031 (CO5_HUMAN). The terms "human C5a" and "hC5a" are used interchangeably herein.

[0031] As used herein, a first compound (e.g., an antibody) has a dissociation constant K for a second compound of 1 μM or less, 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, 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, even more preferably 10 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. d When it has this, it is considered to "bind" to a second compound (e.g., an antigen, e.g., a target protein).

[0032] As used in the present invention, the term "binding" preferably relates to specific binding. "Specific binding" means that a binding moiety (e.g., an antibody) binds more strongly to a target, e.g., an epitope, that is specific compared to binding to another target. The binding moiety binds more strongly to a first target compared to a second target when it binds to the first target and has a dissociation constant (K d ) lower than the dissociation constant for the second target. Preferably, the dissociation constant (K d ) for the target to which the binding moiety specifically binds is at least 10-fold lower, preferably at least 20-fold lower, more preferably at least 50-fold lower, even more preferably at least 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (K d ) for the target to which the binding moiety does not specifically bind.

[0033] As used herein, "K dThe term "(measured typically in "mol / L" and sometimes abbreviated as "M")" is intended to refer to the dissociation equilibrium constant of a specific interaction between a binding moiety (e.g., an antibody or a fragment thereof) and a target molecule (e.g., an antigen or an epitope thereof).

[0034] For determining the binding affinity of a compound, i.e., the dissociation constant K d The methods for determining 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 K d is determined at 20 °C, 25 °C, 30 °C, or 37 °C. Unless specifically indicated otherwise, the K d values described in the present specification are determined by SPR at 20 °C.

[0035] An "epitope", also known as an antigenic determinant, is a part of a macromolecule that is recognized by the immune system, specifically by an antibody, a B cell, or a T cell. As used in the present specification, an "epitope" is a part of a macromolecule that can bind to a compound (e.g., an antibody or an antigen-binding fragment thereof) described in the present specification. In this context, the term "binding" preferably relates to specific binding. An epitope usually consists of chemically active surface groups of a molecule, e.g., amino acids or sugar side chains, and usually has 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 latter but not the former is lost in the presence of a denaturing solvent.

[0036] As used herein, "conformational epitope" refers to an epitope of a linear polymer (e.g., a polypeptide) that is formed by the three-dimensional structure of the polymer. In the context of the present application, a "conformational epitope" is a "discontinuous epitope", i.e., a conformational epitope on a polymer (e.g., a polypeptide) formed from at least two separate regions in the primary sequence of the polymer (e.g., the amino acid sequence of a polypeptide). In other words, when an epitope consists of at least two distinct regions in the primary sequence that are simultaneously bound by an antibody (or an antigen-binding fragment thereof) of the present invention, and these at least two distinct regions are interrupted by one or more regions in the primary sequence to which the antibody (or an antigen-binding fragment thereof) of the present invention does not bind, the epitope is considered to be a "conformational epitope" in the context of the present invention. Preferably, such a "conformational epitope" is present on a polypeptide, and in the primary sequence, the two distinct regions are two distinct amino acid sequences that are bound by an antibody (or an antigen-binding fragment thereof) of the present invention, and these at least two distinct amino acid sequences are interrupted by one or more amino acid sequences in the primary sequence to which the antibody (or an antigen-binding fragment thereof) of the present invention does not bind. Preferably, the interrupting amino acid sequence is an adjacent amino acid sequence containing two or more amino acids to which the antibody (or an antigen-binding fragment thereof) does not bind. The at least two distinct amino acid sequences that are bound by an antibody (or an antigen-binding fragment thereof) of the present invention are not particularly limited with respect to their length. Such distinct amino acid sequences may consist of only one amino acid as long as the total number of amino acids within the at least two distinct amino acid sequences is large enough to result in specific binding between the antibody (or an antigen-binding fragment thereof) and the conformational epitope.

[0037] A "paratope" is a part of an antibody that binds to an epitope. In the context of the present invention, a "paratope" is a part of an anti-C5a antibody (or an antigen-binding fragment thereof) described in the present specification that binds to an epitope.

[0038] 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 the antibodies 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 consists of a heavy-chain variable region (designated as VH or V H as omitted herein) and a heavy-chain constant region. Each light chain consists of a light-chain variable region (designated as VL or V L as omitted herein) and a light-chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant regions of the antibody can 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 (C1q) of the classical complement system.

[0039] 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 included within the term "antigen-binding portion" of an antibody are: (i) the Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) the F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) the Fd fragment consisting of the VH and CH1 domains; (iv) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) the dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining regions (CDRs), and (vii) combinations of two or more isolated CDRs which may be linked by a synthetic linker if desired. 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 enables them, using recombinant methods, to be made as 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 included within the term "antigen-binding fragment" of an antibody. Further examples include binding domain immunoglobulin fusion proteins 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.The 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 skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Further examples of "antigen-binding fragments" are so-called microantibodies 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 C.J. 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 comprise two or more CDR regions fused to each other, preferably by cognate framework regions. Cognate V. H V linked by FR2 H CDR1 and V L Such small antibody mimetics that comprise CDR3 are 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).

[0040] Accordingly, as used herein, the term "antibody or antigen-binding fragment thereof" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen. In a broad sense, the term "antibody or antigen-binding fragment thereof" includes any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY). However, the preferred immunoglobulin molecule of the present invention is of the IgG type. Within the IgG type, the preferred immunoglobulin molecule of the present invention can be of any class or subclass (e.g., IgG1; IgG2, preferably IgG2a and IgG2b; IgG3; or IgG4).

[0041] Antibodies and antigen-binding fragments thereof that can be used in the present invention can be of any animal origin, including birds and mammals. Thus, the antibodies or their fragments can be 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. For many applications, it is particularly preferred that the antibody be of human or mouse origin. Antibodies suitable for use in the present invention include chimeric molecules in which the antibody constant region from one species, e.g., human, is combined with an antigen-binding site from another species, e.g., mouse. In a highly preferred embodiment of the present invention, the antibodies and antigen-binding fragments thereof include humanized molecules in which the antigen-binding site of an antibody from a non-human species (e.g., mouse) is combined with constant and framework regions of human origin.

[0042] As exemplified in the present specification, the antibodies of the invention can be obtained directly from hybridomas expressing the antibodies or can be cloned and recombinantly expressed in host cells (e.g., CHO cells, or lymphocyte cells). Further examples of host cells are microorganisms such as Escherichia coli and fungi such as yeast. Alternatively, they can be recombinantly produced in transgenic non-human animals or plants.

[0043] The term "chimeric antibody" refers to an antibody in which one part of the amino acid sequence of each of the heavy and light chains is derived from a particular species or is homologous to the corresponding sequence in an antibody belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequences in another species or class. Generally, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one species of mammal, while the constant portions are homologous to the sequences of antibodies derived from another species. One distinct advantage of such a chimeric form is that, for example, in combination with constant regions derived from human cell preparations, the variable regions can conveniently be derived from currently known sources using readily available B cells or hybridomas from non-human host organisms. The variable regions have the advantage of ease of preparation and specificity that is not affected by the source, while the constant regions, being human, are less likely to elicit an immune response from a human subject when the antibody is injected than constant regions from non-human sources. However, the definition is not limited to this particular example.

[0044] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain or only complementarity-determining regions (CDRs) grafted onto an appropriate framework region in a variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, modified to more closely resemble a human immunoglobulin. Some forms of humanized antibodies conserve all CDR sequences (e.g., humanized mouse antibodies that include all six CDRs from a mouse antibody). Other forms have one or more CDRs that have been changed relative to the parent antibody.

[0045] Various methods for humanizing antibodies are known to those of skill 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 the national stage 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.

[0046] As used herein, "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). The human antibodies of the invention include, for example, antibodies isolated from a human immunoglobulin library or transgenic animals expressing one or more human immunoglobulins and not expressing endogenous immunoglobulins, as described in U.S. Patent No. 5,939,598 to Kucherlapati & Jakobovits.

[0047] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, monoclonal antibodies are produced by hybridomas containing B cells obtained from a non-human animal, such as a mouse, that have been fused to immortalized cells.

[0048] As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, created, or isolated by recombinant means, e.g., (a) antibodies isolated from transgenic or transchromosomal 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 to other DNA sequences.

[0049] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells that express an antibody, e.g., CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi including yeast cells.

[0050] As used herein, "heterologous antibody" is defined with respect to the transgenic organism that produces such antibody. This term corresponds to what is found in an organism that does not consist of a transgenic organism, and generally refers to an antibody having an amino acid sequence derived from a species other than the transgenic organism or a nucleic acid sequence encoding the same.

[0051] As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain with a mouse light chain is a heterohybrid antibody.

[0052] Accordingly, "antibodies and antigen-binding fragments thereof" generally include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (particularly 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. U.S.A. 90(14), 6444-6448), nanobodies (also known as single-domain antibodies), anti-idiotype (anti-Id) antibodies (e.g., including anti-Id antibodies against the antibodies of the present invention), and epitope-binding fragments of any of the above.

[0053] The antibodies described in this specification are preferably isolated. As used herein, an "isolated antibody" is one that is substantially free of other antibodies having different antigen specificities; for example, an isolated antibody that specifically binds to C5a is intended to refer to an antibody that is substantially free of antibodies that specifically bind to antigens other than C5a. However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human C5a may have cross-reactivity with other related antigens, such as antigens from other species (e.g., C5a species homologs, such as rat C5a). Additionally, an isolated antibody may be substantially free of other cellular materials and / or chemical substances. As used herein, a "combination of isolated antibodies" refers to antibodies having different specificities that are combined in a defined composition.

[0054] When applied to an object, as used herein, the term "natural" refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from a natural source and that has not been intentionally modified by a human in the laboratory is natural.

[0055] "Conservative substitutions" may be made, for example, based on the similarity of the relevant amino acid residues in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. Amino acids can be grouped into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0056] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed within the same group of the six standard amino acid groups described above. For example, the replacement of Asp with Glu retains one negative charge in the polypeptide so modified. Additionally, glycine and proline may be substituted for each other based on their ability to disrupt an α - helix. Some preferred conservative substitutions within the six groups described above are those that are exchanged within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code, and recombinant and synthetic DNA techniques, one of ordinary skill in the art can readily construct DNA encoding conservative amino acid variants.

[0057] As used herein, "non - conservative substitution" or "non - conservative amino acid exchange" is defined as the replacement of an amino acid with another amino acid listed in a different group from groups (1) through (6) of the six standard amino acid groups described above.

[0058] The similarity of nucleotide and amino acid sequences, i.e., the percent of sequence identity, can be determined via sequence alignment. Such alignment can be carried out with several algorithms known in the art, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), hmmalign (HMMER package) or the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) or the CLUSTALW2 algorithm (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

[0059] The grade of sequence identity (sequence match) may be calculated, for example, using BLAST, BLAT or BlastZ (or BlastX). Similar algorithms are incorporated in the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Protein searches by BLAST are performed, for example, by the BLASTP program available at the website: http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome The preferred algorithm parameters used are the default parameters as set on the designated website: Expect threshold = 10, word size = 3, max matches in a query range = 0, matrix = BLOSUM62, gap costs = Existence: 11 Extension: 1, compositional adjustments = conditional compositional score matrix adjustment together with the database of non-redundant protein sequences (nr).

[0060] For the purpose of obtaining gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The sequence matching analysis may be complemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov random fields.

[0061] When percentages of sequence identity are referred to in this application, these percentages are calculated with respect to the full length of the indicated reference sequence, unless otherwise specifically indicated. For example, a description such as "an amino acid sequence having at least 80% sequence identity to SEQ ID NO: XYZ" means that the percentage of sequence identity is calculated with respect to the full length of SEQ ID NO: XYZ.

[0062] As used herein, "biological activity" refers to any activity that a polypeptide may exhibit, including but not limited to: enzyme activity; binding activity to another compound (e.g., binding to another polypeptide, particularly binding to a receptor, or binding to a nucleic acid); inhibitory activity (e.g., enzyme inhibitory activity); activating activity (e.g., enzyme activating activity); or toxic effects. With respect to variants and derivatives of a polypeptide, it is not necessary that the variant or derivative exhibit such activity to the same extent as the parent polypeptide. A variant is considered a variant within the context of this application when it exhibits an activity related to that of the parent polypeptide to an extent of at least 10% (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) of the activity of the parent polypeptide. Similarly, a derivative is considered a derivative within the context of this application when it exhibits a biological activity related to that of the parent polypeptide to an extent of at least 10% of the activity of the parent polypeptide. "Biological activity" particularly relevant in the context of the present invention is the binding activity to the conformational epitopes of human C5a formed by the amino acid sequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3). Preferably, "biological activity" relevant in the context of the present invention is the binding activity to the conformational epitopes of human C5a formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and HKDMQ (SEQ ID NO: 5). Even more preferably, "biological activity" relevant in the context of the present invention is the binding activity to the conformational epitopes of human C5a formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and KDM. Assays for determining binding activity are known to those skilled in the art and include ELISA and surface plasmon resonance assays.

[0063] As used herein, "patient" means any mammal or bird that can benefit from treatment with the anti-C5a antibodies described herein. Preferably, "patient" is selected from the group consisting of laboratory animals (e.g., mice or rats), domestic animals (e.g., including guinea pigs, rabbits, chickens, turkeys, pigs, sheep, goats, camels, cows, horses, donkeys, cats, or dogs), or primates including monkeys (e.g., African green monkeys, chimpanzees, bonobos, gorillas) and humans. It is particularly preferred that the "patient" is a human. The terms "patient" and "subject to be treated" (or, for that matter, "subject") are used interchangeably herein.

[0064] As used herein, "treating", "treatment of", 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 development of the symptomatic characteristics of the disorder being treated; (c) inhibiting the worsening of the symptomatic characteristics of the disorder being treated; (d) limiting or preventing recurrence of the disorder in a patient who has previously had the disorder; and (e) limiting or preventing recurrence of symptoms in a patient who has previously been symptomatic for the disorder.

[0065] As used herein, "preventing", "prevention of", "prevent", or "prevention" of a disease or disorder means preventing the disorder from occurring in a subject for a period of time. For example, when an antibody of the invention (or an antigen-binding fragment thereof) is administered to a subject for the purpose of preventing a disease or disorder, the disease or disorder is prevented from occurring at least on the day of administration and preferably for at least one day after administration (e.g., from 1 to 30 days; or from 2 to 28 days; or from 3 to 21 days; or from 4 to 14 days; or from 5 to 10 days).

[0066] As used herein, "administering" includes in vivo administration as well as ex vivo direct administration to a tissue, such as an intravenous graft.

[0067] The "pharmaceutical composition" according to the present invention may be present in the form of a composition in which different active ingredients and diluents and / or carriers are mixed with each other, or may take the form of a combination preparation in which the active ingredients are present in a partially or completely different form. Examples of such combinations or combination preparations are multi-part kits.

[0068] "Effective amount" is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary depending on factors such as the nature of the agent, the route of administration, the size and species of the subject receiving the therapeutic agent, and the purpose of administration. In each individual case, the effective amount can be determined empirically by one of ordinary skill in the art using methods established in the art.

[0069] Unless the context dictates otherwise, the term "active agent" refers to the antibodies of the present invention and the antigen-binding fragments of the present invention. The terms "active agent" and "therapeutic agent" are used interchangeably herein.

[0070] As used herein, the term "adjuvant therapy" refers to a combination therapy in which at least two different agents are administered to a patient. These at least two different drugs can be formulated into a single pharmaceutical composition containing both drugs. Alternatively, each drug can be formulated into a separate pharmaceutical composition and the pharmaceutical compositions can be administered to the patient separately (e.g., at different times and / or by different routes of administration). In this latter alternative, the (at least) two different drugs can be provided in a multi-part kit.

[0071] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0072] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline and oils including those of animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, saline is a preferred carrier. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions can also optionally contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories using conventional binders and carriers, such as triglycerides. The compounds of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from free amino groups, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed from free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington: The Science and Practice of Pharmacy" 22 ndIt is described in the 2012 edition, Loyd V. Allen Jr. et al. (eds.), Pharmaceutical Press. Such compositions contain a therapeutically effective amount of the compound, preferably in a purified form, together with a suitable amount of carrier, so as to provide a form for proper administration to a patient. The formulation should be suitable for the mode of administration.

[0073] Methods generally known and practiced in the fields of molecular biology, cell biology, protein chemistry, and antibody technology are fully described in the continuously updated publications "Molecular Cloning: A Laboratory Manual", (Sambrook et al., Cold Spring Harbor); "Current Protocols in Molecular Biology" (F. M. Ausubel et al. Eds., Wiley & Sons); "Current Protocols in Protein Science" (J. E. Colligan et al. eds., Wiley & Sons); "Current Protocols in Cell Biology" (J. S. Bonifacino et al., Wiley & Sons) and "Current Protocols in Immunology" (J. E. Colligan et al., Eds., Wiley & Sons). Known techniques regarding cell culture and media are described in "Large Scale Mammalian Cell Culture (D. Hu et al., Curr. Opin. Biotechnol. 8:148-153, 1997); "Serum free Media" (K. Kitano, Biotechnol. 17:73-106, 1991); and "Suspension Culture of Mammalian Cells" (J.R. Birch et al. Bioprocess Technol. 10:251-270, 1990).

[0074] Aspects of the present invention The present invention is further described herein. 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 one or more aspects unless expressly indicated to the contrary. In particular, any feature shown to be preferred or advantageous may be combined with any other one or more features shown to be preferred or advantageous.

[0075] In a first aspect, the present invention is an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH domain has the amino acid sequence according to SEQ ID NO: 10

Chemical formula

Chemical formula

[0076] In a preferred embodiment of the first aspect, an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, or even more preferably at least 99%) sequence identity with SEQ ID NO: 10 contains at least 4 (preferably at least 5, more preferably at least 6, more preferably 7) of the following 7 amino acids at the designated positions: - V at amino acid position 5, - E at amino acid position 10, - K at amino acid position 12, - K at amino acid position 13, - A at amino acid position 16, - A at amino acid position 40, or - T at amino acid position 76.

[0077] In a preferred embodiment of the first aspect, an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, or even more preferably at least 99%) sequence identity with SEQ ID NO: 16 contains at least 8 (preferably at least 9, more preferably at least 10, more preferably at least 11, even more preferably at least 12, even more preferably at least 13, even more preferably at least 14, more preferably 15) of the following 15 amino acids at the designated positions: - A at amino acid position 13, - V at amino acid position 15, - D at amino acid position 17, - V at amino acid position 19, - T at amino acid position 22, - K at amino acid position 46, - A at amino acid position 47, - S at amino acid position 64, - T at amino acid position 78, - S at amino acid position 80, - S at amino acid position 81, - L at amino acid position 82, - Q at amino acid position 83, - F at amino acid position 87, or - Q at amino acid position 104.

[0078] In a preferred embodiment of the first aspect, an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, or more preferably at least 99%) sequence identity with SEQ ID NO: 10 contains at least 4 (preferably at least 5, more preferably at least 6, more preferably 7) of the following 7 amino acids at the specified positions: - V at amino acid position 5, - E at amino acid position 10, - K at amino acid position 12, - K at amino acid position 13, - A at amino acid position 16, - A at amino acid position 40, or - T at amino acid position 76, And, optionally, contains 1, 2, or 3 amino acid substitutions, preferably conservative substitutions, located at amino acid positions 1 to 4 or 6 to 9 of SEQ ID NO: 10; And An amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, or most preferably at least 99%) sequence identity with SEQ ID NO: 16 contains at least 8 (preferably at least 9, more preferably at least 10, more preferably at least 11, even more preferably at least 12, even more preferably at least 13, even more preferably at least 14, most preferably 15) of the following 15 amino acids at the designated positions: - A at amino acid position 13, - V at amino acid position 15, - D at amino acid position 17, - V at amino acid position 19, - T at amino acid position 22, - K at amino acid position 46, - A at amino acid position 47, - S at amino acid position 64, - T at amino acid position 78, - S at amino acid position 80, - S at amino acid position 81, - L at amino acid position 82, - Q at amino acid position 83, - F at amino acid position 87, or - Q at amino acid position 104 And optionally contains 1, 2, or 3 amino acid substitutions, preferably conservative substitutions, located at amino acid positions 1 to 10 of SEQ ID NO: 16.

[0079] In a second aspect, the present invention is An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein The VH domain is SEQ ID NO: 17 (QVQLVQSGX 9 E X 11 KKPGASVKX 20SCKASGYSFT TFWMDWVX 38 QA PGQGLEWX 48 GR IDPSDSESRL DQX 63 FKDRX 68 TX 70 TVDKSTSTVY MX 82 LSSX 86 X 87 SED X 91 An amino acid sequence according to AVYYCARGN DGYYGFAYWG QGTLVTVSS), where X 9 is A or P, and X 11 is L or V, and X 20 is I or V, and X 38 is K or R, and X 48 is I or M, and X 63 is K or R, and X 68 is A or V, and X 70 is L or M, and X 82 is E or Q, and X 86 is L or P, and X 87 is R or T, and X 91 is S or T, or An amino acid sequence according to SEQ ID NO: 17 having one, two or three amino acid substitutions, wherein the amino acid sequence having one, two or three amino acid substitutions comprises the CDR1H, CDR2H, CDR3H sequences of SEQ ID NOs: 20 to 22 respectively comprises, consists essentially of, or consists of, and the VL domain is SEQ ID NO: 18 (DIX 3 X 4 TQSPX 9 S LX 12 ASVGDRVT ITCKASQSVD YDGDSYMKWY QQKPGKAPKL LIYAASNLQS GX 62 PSRFSGSG SGTDFTLTIS SLQX 84 An amino acid sequence according to EDFATY YCQQSNEDPY TFGQGTKLEI K), where X 3 is V or Q, and X 4is L or M, and X 9 is A or S, and X 12 is A or S, and X 62 is I or V, and X 84 is E or P, or an amino acid sequence according to SEQ ID NO: 18 having one, two, three, four, five, six, or seven amino acid substitutions, wherein the amino acid sequence having one, two, three, four, five, six, or seven amino acid substitutions comprises amino acid sequences of CDR1L, CDR2L, and CDR3L of SEQ ID NOs: 23 to 25, respectively comprising, consisting essentially of, or consisting of relates to an antibody or an antigen-binding fragment thereof.

[0080] In an embodiment of the first or second aspect, the VH domain comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of VH1 to VH5 (SEQ ID NOs: 7 to 11); and / or the VL domain comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of Vκ1 to Vκ4 (SEQ ID NOs: 13 to 16).

[0081] In an embodiment of the first or second aspect, the antibody or an antigen-binding fragment thereof a) a VH domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 10 (VH4) and a VL domain comprising, consisting essentially of, or consisting of the amino acid sequence according to SEQ ID NO: 16 (Vκ4), or b) a VH domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 11 (VH5) and a VL domain comprising, consisting essentially of, or consisting of the amino acid sequence according to SEQ ID NO: 15 (Vκ3) comprises.

[0082] In an embodiment of the first or second aspect, the antibody is a humanized antibody.

[0083] In an embodiment of the first or second aspect, the antibody or its antigen-binding fragment further comprises a constant domain. In some embodiments of the first or second aspect, the constant domain comprises, consists essentially of, or consists of the amino acid sequence according to SEQ ID NO: 19 (IgG4 WT constant), The amino acid sequence according to SEQ ID NO: 19 optionally comprises the following amino acid exchanges: - Amino acid exchange S108P; - Amino acid exchanges T130Q and M308L; - Amino acid exchanges M132Y, S134T, and T136E including one or more of.

[0084] In an embodiment of the first or second aspect, the antigen-binding fragment of the antibody is selected from the group consisting of Fab fragment, Fab' fragment, F(ab')2 fragment, Fd fragment, Fv fragment, disulfide-linked Fv (dsFv), single-domain antibody and single-chain Fv (scFv) antibody.

[0085] In an embodiment of the first or second aspect, the antibody or its antigen-binding fragment has the following properties: - The antibody or its antigen-binding fragment has a binding constant to C5a at a K d value of 10 nM or less (e.g., 9 nM or less; 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less); - The antibody or its antigen-binding fragment exhibits at least 75% blocking activity against the biological effect induced by one molecule of C5a; - The antibody or its antigen-binding fragment does not inhibit CH50 activity in human plasma; - The antibody or its antigen-binding fragment can reduce Escherichia coli-induced IL-8 production in human whole blood; - The antibody or its antigen-binding fragment has reduced immunogenicity compared to IFX-1, showing one or more of.

[0086] Methods for determining relative immunogenicity are shown in the following examples, namely an ADA screening assay. The abbreviation ADA refers to anti-drug antibodies ( a nti- d rug- a ntibodies).

[0087] In aspects of the first or second aspect, the antibody or antigen-binding fragment thereof binds to a conformational epitope formed by the amino acid sequences NDETCEQRA (SEQ ID NO: 2) and SHKDMQL (SEQ ID NO: 3) of C5a, and the antibody or antigen-binding fragment thereof binds to at least one amino acid within the amino acid sequence according to SEQ ID NO: 2 and at least one amino acid within the amino acid sequence according to SEQ ID NO: 3.

[0088] In a further aspect of the first or second aspect, the antibody or antigen-binding fragment thereof binds to a conformational epitope formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and HKDMQ (SEQ ID NO: 5) of C5a, and the antibody or antigen-binding fragment thereof binds to at least one amino acid within the amino acid sequence according to SEQ ID NO: 4 and at least one amino acid within the amino acid sequence according to SEQ ID NO: 5.

[0089] In a further aspect of the first or second aspect, the antibody or antigen-binding fragment thereof binds to a conformational epitope formed by the amino acid sequences DETCEQR (SEQ ID NO: 4) and KDM of C5a, and the antibody or antigen-binding fragment thereof binds to at least one amino acid within the amino acid sequence according to SEQ ID NO: 4 and at least one amino acid within the amino acid sequence according to KDM.

[0090] In a third aspect, the present invention comprises an antibody or antigen-binding fragment thereof according to the first aspect or an antibody or antigen-binding fragment thereof according to the second aspect, and Further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, extenders, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives, relates to a pharmaceutical composition.

[0091] In a fourth aspect, the invention relates to an antibody or an antigen-binding fragment thereof according to the first aspect or an antibody or an antigen-binding fragment thereof according to the second aspect for use in medicine.

[0092] In a fifth aspect, the invention relates to an antibody or an antigen-binding fragment thereof according to the first aspect or an antibody or an antigen-binding fragment thereof according to the second aspect for use in the treatment or prevention of a disease or disorder associated with pathological C5a activity.

[0093] In an embodiment of the fifth aspect, the disease or disorder is - an autoimmune disease, - an inflammatory disease, an autoinflammatory disorder, or a related condition, - a cardiovascular or cerebrovascular disorder, - a bacterial or viral infection, - a neurodegenerative disorder or related disease, and - cancer or a precancerous condition selected from the group consisting of.

[0094] In some embodiments of the fifth aspect, the viral infection is HIV or AIDS. The present disclosure relates to, for example, the following. [Item 1] An antibody or an antigen-binding fragment thereof comprising a heavy-chain variable domain (VH) and a light-chain variable domain (VL), wherein the VH domain has the amino acid sequence according to SEQ ID NO: 10, or

Chemical formula

Chem.

Example

[0095] Example The following examples are presented to provide a complete disclosure and description of the methods of making and using the compounds, compositions, and methods of the present invention to those skilled in the art and are not intended to limit the scope that the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to the numerical values used, but some experimental errors and deviations should be taken into account. Unless otherwise noted, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0096] Example 1: Preparation of a fully humanized anti-C5A antibody (IFX-2 clone) derived from IFX-1 This example describes an example of constructing a series of fully humanized antibodies using the Composite Human Antibody technology with the V-region gene sequence encoding the chimeric antibody IFX-1 (VH0 / Vκ0). The designed heavy and light chain variable region genes were cloned into vectors encoding the human IgG4 heavy chain constant domain (with the S108P amino acid exchange, see SEQ ID NO: 19) and the human kappa light chain constant domain, respectively. In other publications, the S108P amino acid exchange is sometimes referred to as the S241P amino acid exchange (using the Kabat numbering of full-length human IgG4). The chimeric and humanized antibodies were transiently expressed in HEK EBNA cells and protein A purification was performed. TM

[0097] Synthetic human antibody Design of variable region sequences TM

[0098] The structural model of the V region of the IFX-1 antibody was created using Swiss PDB and analyzed to identify important "constraining" amino acids in the V region that may be essential for the binding properties of the antibody. Most residues, including many framework residues, contained within the CDRs were considered important. The VH and Vκ sequences of IFX-1 contain typical framework residues and the CDR1, 2, and 3 motifs are equivalent to those of many mouse antibodies.

[0099] From the above analysis, it was considered that the composite human sequence of IFX-1 has a wide range of alternative residues outside the CDRs but a narrow menu of possible residues within the CDR sequences. Preliminary analysis has shown that corresponding sequence segments from several human antibodies can be combined to create CDRs similar or identical to those of the mouse sequences. For regions outside and adjacent to the CDRs, a wide selection of human sequence segments was identified as possible components of the novel humanized V region. CD4+ T cell epitope avoidance ​​Based on structural analysis, a large preliminary set of array segments that can be used to create IFX-1 humanized variants was selected, and iTope TM technology (Perry et al New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs R D 9 (6): 385-396) was used, and the TCED of known antibody sequence-related T cell epitopes TM (Bryson et al Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1):1-8) was used for analysis. Array segments identified as significant non-human germline binding factors to human MHC class II or those that recorded significant hits against TCED TM were discarded. This resulted in a reduced set of segments, and these combinations were analyzed again as described above to ensure that they did not contain T cell epitopes where junctions between segments could occur. The selected array segments were assembled into a complete V region sequence lacking significant T cell epitopes. Next, five heavy chain (VH1 to VH5) and four light chain (Vκ1 to Vκ4) sequences were selected for gene synthesis and expression in mammalian cells. The amino acid sequences of VH1 to VH5 are shown in the sequence listing as SEQ ID NOs: 7 to 11, respectively. The amino acid sequences of Vκ1 to Vκ4 are shown in the sequence listing as SEQ ID NOs: 13 to 16, respectively.

[0100] Construction of chimeric antibodies and humanized variants The VH and Vκ sequences of IFX-1 (VH0 (SEQ ID NO: 6) and Vκ0 (SEQ ID NO: 12)) and their humanized variants were synthesized adjacent to restriction enzyme sites for cloning into Abzena's pANT expression vector system for human IgG4 (S241P) heavy and kappa light chains, respectively. The VH region was cloned between Mlu I and Hind III restriction enzyme recognition sites, and the Vκ region was cloned between BssH II and BamH I restriction enzyme recognition sites. All constructs were confirmed by sequencing.

[0101] Expression and purification of antibodies Chimeric IFX-1 (VH0 / Vκ0), two control antibodies (VH0 / Vκ1, VH1 / Vκ0), and combinations of synthetic IgG4 (S241P) VH and Vκ chains (23 combinations in total, Table 1) were transiently transfected into HEK EBNA adherent cells (LGC Standards, Teddington, UK) using the PEI transfection method and incubated for 7 days after transfection. The antibody titers in the supernatants were determined by ELISA, and it was observed that variants containing VH1 consistently showed low expression when compared to other variants (data not shown). Table 1. Chimeric (VH0 / Vκ0), two control humanized variants (VH1 / Vκ0 and VH0 / Vκ1), and 20 IFX-1 synthetic human antibodies TM Summary table of antibodies generated by transient transfection (crossed boxes) containing variants.

Table 1

[0102] Antibodies (excluding VH1 variants with poor expression) were purified from cell culture supernatants using a Protein A Sepharose column (GE Healthcare, Little Chalfont, UK), buffer exchanged into 1x PBS pH 7.2, and the extinction coefficient (Ec (0.1%) ) based on the predicted amino acid sequence was used to calculate OD 280nmQuantified by. SDS-PAGE was used to analyze the antibodies by adding 1 μg of each antibody onto the gel, and bands corresponding to the typical antibody profiles were observed (data not shown).

[0103] Example 2: Comparison of different IFX-2 clones against the parental antibody IFX-1 Materials and Reagents For CD11b assay: o ACD, Sigma Aldrich (Taufkirchen, Germany), Cat. No. C3821-50ML o Reagents for flow cytometer (all from BD Bioscience, NJ, USA) ■ FACS Flow Sheat Fluid, Cat. No. 342003 ■ FACS Shutdown solution, Cat. No. 334224 ■ FACS Clean solution, Cat. No. 340345 ■ Rat anti-mouse CD11b:FITC, Cat. No. 553310, 0.5 mg / mL ■ 10× Lysis solution, BD Bioscience (NJ, USA), Cat. No. 349202 → Diluted 1:10 with AnalaR water o HBSS, Life Technologies GmbH (Darmstadt, Germany), Cat. No. 14025-050 o FBS, Invitrogen (CA, USA), Cat. No. 10099133 → Heat-inactivated: 56°C, 30 min o Sodium azide, Merck (Darmstadt, Germany), Cat. No. 1.06688.0250 o Recombinant human C5a (rhC5a), Sigma Aldrich (Taufkirchen, Germany), Cat. No. C5788-.1MG, expressed in E. coli, purity: ~95%, dissolved in sterile AnalaR water o Staining buffer (SB-buffer): 1% heat-inactivated FBS + 0.1% sodium azide in 1×PBS o Human blood from healthy donors containing 12% ACD (used immediately)

[0104] For C5a-based PK ELISA: o AnalaR water, VWR International (Darmstadt, Germany), Cat. No. 102923C o Recombinant human C5a (rhC5a), expressed in E. coli and dissolved in sterile AnalaR water, Hycult Biotech (Uden, The Netherlands), Cat. No. HC2101 o rhC5a desarginine derivative (rhC5aDArg), expressed in E. coli and dissolved in sterile AnalaR water, Hycult Biotech (Uden, The Netherlands), Cat. No. HC2102 o IFX-1, anti-human C5a antibody used as a control, InflaRx (Jena, Germany), 10 mg / mL in PBS + 0.05% Tween80 o Mouse anti-human IgG4:HRP, AbD Serotec (Puchheim, Germany), Cat. No. MCA2098P, 1 mg / mL o Na2CO3, Sigma-Aldrich (Taufkirchen, Germany), Cat. No. 71350 (Sigma, WI, USA, Cat. No. 31432) o NaHCO3, VWR International (Darmstadt, Germany), Cat. No. L1730 o NaN3, VWR International, (Darmstadt, Germany), Cat. No. 1.06688.0100 o PBS powder, Sigma-Aldrich (Taufkirchen, Germany), Cat. No. P5368-10PAK → 1 package dissolved in 1000 mL of deionized water o Tween 20, Sigma-Aldrich, (Taufkirchen, Germany), Cat. No. P1379-25ML o BSA, Sigma-Aldrich (Taufkirchen, Germany), Cat. No. A7030-100G o Coating buffer: 1.59 g of Na2CO3 + 2.93 g of NaHCO3 + 0.2 g of NaN3 in 1000 mL of AnalaR water, pH 9.6 o Washing buffer: 1x PBS + 0.05% Tween 20 o Assay diluent: 3% BSA in 1×PBS + 0.05% Tween 20 (3% BSA / PBST) o TMB substrate (Substrate Solution C), BioLegend / Biozol (Heidelberg, Germany), Cat. No. BLD-78105 o Stop solution: Sulfuric acid (Sigma-Aldrich, Cat. No. 258105-100ML) diluted 1:10 with AnalaR water

[0105] For plasma hemolytic activity (CH50): o AnalaR water, VWR International (Darmstadt, Germany), Cat. No. 102923C o Complement CH50 assay, Haemoscan (Groningen, Netherlands), Cat. No. K002 o Pooled healthy human plasma, huPP-013, prepared in-house

[0106] Method rhC5a-based ELISA For ELISA, 100 μL of rhC5a (1 μg / mL) was coated onto a 96-well ELISA plate overnight at 4 °C. The plate was washed (5×) and blocked with 200 μL of blocking buffer for 1 h at 37 °C. After the washing step (5×), IFX clones at 2-fold serial dilutions from 250 ng / mL - 3.91 ng / mL were added to the C5a-coated wells. After incubation at 37 °C for 2 h and the washing step (5×), 0.04 μg / mL of HRP-labeled anti-human IgG4 antibody was applied at 37 °C for 1 h to detect the bound IFX clones. For color development, 100 μL of TMB substrate was added after washing (5×) and incubated at RT for 5 min. The color reaction was stopped with 100 μL of stop solution. Absorbance readings at 450 nm were taken within 30 min after the color reaction using a plate reader. Subtraction of the blank was done with the raw data.

[0107] Hemolytic activity of plasma (CH50) The total hemolytic complement titer (CH50) is a conventional method for determining the activation of the classical complement pathway. Briefly, sheep red blood cells (sRBC) were prepared from fresh sheep whole blood by centrifugation and sensitized with anti-sRBC antibody. Plasma samples from healthy volunteers containing IFX clones were serially diluted and incubated with the sensitized sRBC at 37 °C for 30 min. After incubation, the mixture was centrifuged and the degree of hemolysis was quantified by measuring the absorbance of the hemoglobin released into the supernatant at 450 nm. The amount of complement activity was determined by testing the ability of various dilutions of the test plasma sample to lyse the sensitized sRBC.

[0108] CD11b potency assay Human whole blood was stimulated with 16.7 nM rhC5a. To test the blocking activity of IFX-1 and IFX-2 clones against rhC5a-induced CD11b upregulation, the antibodies were diluted to the final concentration at a 1:1 Ag:Ab ratio. Blood incubated with buffer only (HBSS control) was used as the non-stimulated condition to evaluate baseline CD11b expression. Blood with IFX antibody alone was used to exclude the inhibitory or stimulatory effects of the antibody on human blood. The mixed samples were incubated at 37 °C for 20 minutes to induce C5a-mediated CD11b upregulation. Then, FITC-conjugated anti-mouse CD11b antibody was further incubated with the samples on ice for 30 minutes to stain CD11b on the cell surface of granulocytes. The fluorescence signal was captured through a flow cytometer.

[0109] Statistical analysis Graphs and statistical analyses were performed with (登録商標) GraphPad PRISM V7.05.

[0110] Results The IFX-2 clone bound sufficiently equally to C5a compared to IFX-1. To characterize the binding of the IFX-2 clone to C5a, an rhC5a-based ELISA platform was used. 12×IFX-2 and 1×IFX-1 (VH0Vk0) clones in the range of 3.91 ng / mL - 250 ng / mL were applied as samples to 96-well plates coated with 1 μg / mL rhC5a. Next, the C5a-captured IFX clones were detected with 0.04 μg / mL of HRP-conjugated anti-human IgG4 mAb. The more the IFX antibody binds to C5a, the stronger the chemiluminescence signal appears. As shown in Figure 1, all the tested antibodies bound to the coated rhC5a with equivalent dose-dependence in the applied concentration range.

[0111] EC of the IFX clones for C5a binding 50To quantify, a similar ELISA-based assay was performed by ABZENA. In the range of 0 - 100 ng / mL, all IFX clones showed very similar binding behavior to C5a. Compared to the parental molecule IFX1, the relative EC 50 of all IFX2 clones was within a two-fold range and the median was 1.04 (Table 2). Table 2. Relative EC of IFX-2 to IFX-1 (VH0Vk0) regarding C5a binding affinity 50

Table 2

[0112] The IFX-2 antibody was shown not to inhibit the formation of the membrane attack complex (MAC). The parental IFX-1 (VH0Vk0) is known to neutralize C5a without interfering with C5 cleavage to C5a and C5b. C5b is the starting material of the membrane attack complex C5b-9, the end product of the classical complement pathway, which forms pores in the membrane. The total hemolytic complement titer (CH50) was used to indicate the activation of the classical complement pathway via MAC-mediated hemolysis of sensitized sheep red blood cells (SRBC). Pooled human plasma with an initial concentration of 100 μg / mL of each IFX clone (12×IFX-2 and 1×IFX-1 (VH0Vk0)) was serially diluted 4, 8, 16, 32, 64, and 128-fold and incubated with the erythrocyte suspension. The degree of hemolysis was quantified by measuring the absorbance of hemoglobin released into the supernatant at 450 nm. Table 3. CH50 (plasma dilution factor) of each test IFX clone

Table 3

[0113] As shown in Figure 2 and Table 3, in order to achieve 50% hemolysis (CH50) that was completely comparable, a 70 - 77 - fold (median 74 - fold) dilution of the IFX - 2 clone or a 74 - fold dilution of the parental molecule IFX - 1 was required. From this, it was demonstrated that the mutation of the IFX - 2 clone did not impair the activation of the classical complement pathway.

[0114] The IFX - 2 antibody effectively blocked rhC5a - driven up - regulation of CD11b Up - regulation of CD11b on the surface of human granulocytes was detectable within minutes after the addition of rhC5a to human whole blood. CD11b levels were measured using a fluorescently labeled anti - CD11b antibody and shown as mean fluorescence intensity (MFI). Blocking of rhC5a - driven up - regulation of CD11b was evaluated with 12×IFX - 2 antibody compared to IFX - 1 in two CD11b potency assays. In both assays, 16.7 nM rhC5a was used as the stimulus, inducing 3.65 - fold and 5.89 - fold up - regulation of CD11b expression, respectively. All IFX clones were tested at a 1:1 molar ratio for blocking of CD11b up - regulation. The blocking activity of the IFX clones was calculated according to the change in fluorescence intensity for each rhC5a - stimulated condition. The activity of the parental molecule IFX - 1 was set at 100%. At a 1:1 rhC5a:IFX clone ratio, the IFX - 2 clone showed a relative blocking activity of 94% - 104% (see Figure 3). The median of the relative blocking activity of the IFX - 2 clone was 100.68%.

[0115] Example 3: ADA analysis in non-human primates after treatment with IFX-1 / IFX-2 Materials and Reagents Table 4. Reagents and Their Suppliers

Table 4

[0116] Methods Anti-drug antibodies are determined in a two-step approach. Serum samples are screened for anti-drug antibodies by an ADA screening ELISA. A fixed assay cut-off point based on optical density (OD) is 0.102 (by confirmation of ADA screening ELISA using human plasma for detection of IFX-1 ADA), and it should result in 9% false positive samples. Therefore, samples that are positive in the ADA screening ELISA should be analyzed by an ADA confirmation ELISA to confirm or reject the results and determine the ADA concentration when applicable.

[0117] The ADA screening ELISA is a bridging ELISA that uses the drug IFX-1 (anti-human C5a antibody) as the capture antibody (unlabeled) and the detection antibody (biotin-labeled). The anti-drug antibodies used for the creation of the calibration (CAL) curve and spiking of quality control (QC) samples were purified from rabbit serum after immunization of rabbits with purified IFX-1. Since the anti-drug antibodies have two binding sites, they can bind (bridge) to both the capture IFX-1 and the labeled IFX-1 used for detection. All plasma samples screened as ADA positive are re-evaluated by an ADA confirmation ELISA based on the ADA screening ELISA setup. Additional free unlabeled drug (IFX-1) is added to the samples to compete with the possible binding of anti-drug antibodies to the IFX-1 captured on the plate. If available anti-drug antibodies are present, they will bind to the added free unlabeled IFX-1 and will not be detected. When drug-specific ADA inhibition is observed and the screening results are confirmed, the sample is considered to be positive confirmed.

[0118] The ADA screening ELISA and the ADA confirmation ELISA were performed as described below: Briefly, 100 μl / well of capture antibody (IFX-1, 0.5 μg / ml) is incubated overnight at 5 ± 3 °C on a high-binding plate. After the washing step, 200 μl of blocking buffer is dispensed into each well and the plate is incubated at 37 °C ± 2 °C for 2 hours. After another washing step, 100 μl / well of CAL sample, QC sample and sample of interest (test sample) are dispensed into the wells and incubated at 37 °C ± 2 °C for 2 hours. After the washing step, 100 μL of detection antibody (biotinylated IFX-1, 0.08 μg / ml) is dispensed into each well and incubated at 37 °C ± 2 °C for 60 minutes. Then the plate is washed and 100 μl of diluted avidin-HRP (1:3000) is added to each well and incubated at 37 °C ± 2 °C for 30 minutes. After the final washing step, 100 μl / well of TMB (substrate solution) is added, incubated for 5 minutes at room temperature in the dark, and then 100 μl / well of dilute sulfuric acid is added to stop the reaction. The color development intensity is analyzed at 450 nm with a Tecan Infinite M200 reader using magellan TM 6.5 software.

[0119] Plasma samples with unknown ADA concentrations are diluted 1:2 with CrossDown buffer before analysis by ADA screening ELISA. For re-evaluation in the ADA confirmation ELISA, samples are diluted 1:2 once with CrossDown buffer and once with drug-spiked CrossDown buffer (IFX-1 concentration 100 μg / ml).

[0120] Samples with OD values less than 0.102 in the ADA screening assay are classified as "negative". Samples with signal blocking of 50% or more in the ADA confirmation assay are classified as "positive confirmed". The results are shown in Table 5.

[0121] Results IFX-2 treated animals did not develop detectable ADA for 8 weeks after dosing. Table 5. Overview of ADA screening and confirmation ELISA

Table 5

[0122] Sequence Listing Free Text Information

Table 6

Claims

Claim 1 An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), a) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 8 (VH2), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 15 (Vκ3); b) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 8 (VH2), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 16 (Vκ4); c) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 9 (VH3), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 14 (Vκ2); d) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 9 (VH3), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 16 (Vκ4); e) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 10 (VH4), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 13 (Vκ1); f) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 10 (VH4), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 14 (Vκ2); g) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 10 (VH4), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 15 (Vκ3); h) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 10 (VH4), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 16 (Vκ4); i) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 11 (VH5), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 13 (Vκ1); j) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 11 (VH5), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 14 (Vκ2); k) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 11 (VH5), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 15 (Vk3); or l) the VH domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 11 (VH5), and the VL domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 16 (Vk4); The antibody or antigen-binding fragment thereof has a binding constant to human C5a at a K ｄ value of 10 nM or less, and human C5a has the amino acid sequence shown in SEQ ID NO: 1 An antibody or an antigen-binding fragment thereof. **Claim 2** The antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof a) a VH domain comprising, or consisting of, the amino acid sequence according to SEQ ID NO: 10 (VH4) and a VL domain comprising, or consisting of, the amino acid sequence according to SEQ ID NO: 16 (Vk4), or b) a VH domain comprising, or consisting of, the amino acid sequence according to SEQ ID NO: 11 (VH5) and a VL domain comprising, or consisting of, the amino acid sequence according to SEQ ID NO: 15 (Vk3) An antibody or an antigen-binding fragment thereof comprising. **Claim 3** The antibody or an antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody is a humanized antibody. **Claim 4** The antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a constant domain, wherein the constant domain comprises, or consists of, the amino acid sequence according to SEQ ID NO: 19 (IgG4 WT constant), The amino acid sequence according to SEQ ID NO: 19 optionally has the following amino acid exchanges: - Amino acid exchange S108P; - Amino acid exchanges T130Q and M308L; - Amino acid exchanges M132Y, S134T, and T136E An antibody or an antigen-binding fragment thereof comprising one or more of. **Claim 5** The antigen-binding fragment of the antibody is selected from the group consisting of Fab fragment, Fab' fragment, F(ab') ２ fragment, Fd fragment, Fv fragment, disulfide-linked Fv (dsFv), single-domain antibody, and single-chain Fv (scFv) antibody, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. **Claim 6** The antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or the antigen-binding fragment thereof has the following characteristics: - The antibody or the antigen-binding fragment thereof exhibits at least 75% blocking activity against the biological effect induced by one molecule of C5a; - The antibody or the antigen-binding fragment thereof does not inhibit CH50 activity in human plasma; - The antibody or the antigen-binding fragment thereof has reduced immunogenicity compared to IFX-1. An antibody or an antigen-binding fragment thereof exhibiting one or more of. **Claim 7** comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, extenders, binders, lubricants, glidants, disintegrants, adsorbents, and / or preservatives, a pharmaceutical composition. **Claim 8** The pharmaceutical composition according to claim 7, for use in medicine. **Claim 9** The pharmaceutical composition according to claim 7, for use in the treatment or prevention of a disease or disorder associated with pathological C5a activity. **Claim 10** wherein the disease or disorder is - an autoimmune disease, - an inflammatory disease, autoinflammatory disorder, or related condition, - a cardiovascular or cerebrovascular disorder, - a bacterial or viral infection, - a neurodegenerative disorder or related disease, and - cancer or a pre-cancerous state The pharmaceutical composition according to claim 9, selected from the group consisting of.

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