RU2823245C2 - ANTIBODIES TARGETING C5aR - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to an isolated antibody or its antigen-binding fragment, which specifically bind to the C5a human complement receptor C5aR. Also disclosed is an isolated nucleic acid molecule coding said antibody; an expression vector and a host cell containing said nucleic acid molecule; pharmaceutical composition containing said antibody. Disclosed is a method of producing said antibody.

EFFECT: invention makes it possible to effectively treat diseases associated with C5aR.

18 cl, 18 ex, 23 tbl, 11 dwg

Description

Field of technology to which the invention relates

The present invention relates to antibodies that interact with C5aR, in particular with human C5aR. The present invention also relates to nucleic acid-based compositions, vector-based compositions and host cells capable of expressing said antibodies, pharmaceutical compositions containing said antibodies, and methods of using said antibodies for the treatment of certain diseases and/or for diagnostic purposes.

BACKGROUND OF THE INVENTION

Anaphylatoxin C5a chemotactic receptor 1 (C5aR) (also known as CD88) is a G protein-coupled receptor (GPCR) belonging to the rhodopsin family and is one of two high-affinity receptors for C5a ligand that is produced in serum blood as one of the main effector components of the response involving the complement system. Under physiological conditions, C5a acts as a chemotactic agent for inflammatory cells, stimulates their respiratory burst as well as the release of cytokines and chemokines, and functions to increase vascular permeability.

C5aR appears to be widely expressed by different cell types. The highest levels of C5aR expression have been described for neutrophils. Low to moderate expression levels have been shown for macrophages/monocytes, dendritic cells, mast cells, eosinophils, pulmonary vascular smooth muscle cells, astrocytes, microglia, osteoblasts, osteoclasts, epithelial and endothelial cells (Monk PN et al., Br J Pharmacol. 2007 , 152: 429-448; Wetsel RA, Immunol Lett. 1995, 44: 183-187). Low levels of expression were detected in human T cells (Nataf S, J Immunol. 1999, 162: 4018-4023).

Cellular responses to C5a are tightly controlled by ligand-induced internalization of the receptor. C5aR is described to be internalized rapidly and in a dose-dependent manner when treated with C5a, and up to 90% of the receptors are recycled back to the cell surface. Thus, high expression levels of C5aR coupled with its high switching rate may be limiting in terms of efficacy due to target-mediated drug distribution (TMDD) effects.

The interaction of C5aR with C5a has been described in many different diseases; most are involved in inflammatory and autoimmune diseases (Morgan BP et al., Nat Rev Drag Discov. 2015, 14: 857-877; Hawksworth OA et al., Mol Immunol. 2017, 89: 36-43). Some initial studies have been performed to identify the underlying mechanisms by which C5a stimulates tumor growth (Markiewski MM et al., Nat Immunol. 2008, 9: 1225-1235; Corrales L. et al, J Immunol. 2012, 189: 4674-4683 ; Cho MS et al., Cell Rep.2014, 6: 1085-1095). Most evidence indicates that C5a enhances cancer cell proliferation, intratumoral angiogenesis, and increases tumor invasiveness and metastasis. More recent evidence also points to a role for C5a/C5aR in creating immunosuppressive environments in the context of solid tumors (Sayegh ET et al., Cancer Med. 2014, 3: 747-758; Darling VR et al., Expert Rev Clin Immunol. 2015, 11 : 255-263; Markiewski MM et al., Cancer Res. 2009, 69: 6367-6370), which leads to increased growth of the primary tumor by suppressing antitumor responses (for example, increased recruitment of myeloid cells expressing C5aR, such as myeloid suppressor cells origin (MDSC) or M2 macrophages). From these data, it can be concluded that strategies for combining with established anticancer agents, such as immune checkpoint protein inhibitors, to enhance a subject's immune response by reducing the immunosuppressive microenvironment are the focus of research and clinical development (Wang Y, et al: Cancer Discov. 2016, 6: 1022-1035).

To date, only one specific complement-based therapeutic has been approved that targets the upstream C5a molecule, namely C5. The humanized anti-C5 mAb eculizumab is able to bind C5, preventing its cleavage and the formation of the C5a and C5b proteins, as well as the subsequent formation of MAC. Various other therapeutic monoclonal antibodies specific for C5, such as ALXN1210 or LFG316, are in clinical evaluation (Hawksworth OA et al., Mol Immunol. 2017, 89: 36-43). However, since the increased risk of infection is a major concern in the treatment of chronic C5 induction, specific targeting of downstream molecules while preventing the biological activities of other complement components is a clear advantage. Accordingly, a number of antagonistic C5a-specific monoclonal antibodies are in development.

Directly targeting C5aR has several advantages over targeting C5 or C5a, respectively. First, inhibiting the receptor alone will preserve MAC activity, thereby reducing the potential risk of infections. Second, blockade of C5aR allows C5a to continue interacting with its second receptor, C5L2. Since C5L2 has been reported to have anti-inflammatory effects, maintaining an effective C5L2 signaling pathway may result in increased efficacy or reduced dosage requirements. Third, direct targeting of C5aR may provide pharmacodynamic advantages over inhibition of soluble C5a due to its small molecular weight and high switching rate. In general, there is great interest in the development of C5aR inhibitors, such as aptamers, peptides, and non-peptide small molecules, which are undergoing preclinical and clinical trials.

The prior art has described the neutralization of polyclonal antiserum or monoclonal antibodies directed against the N-terminal extracellular region of human C5aR and capable of interfering with the C5aR-C5a interaction (see, for example, Morgan et al., The Journal of Immunology, Vol 151, 377-388 . No. 1, July 1, 1993; Oppermann et al., The Journal of Immunology, Vol 151, 3785-3794, No. 7, October 1, 1993),

However, these C5aR-specific antibodies are not suitable for clinical development and therapeutic use in humans, particularly due to their animal origin (making them immunogenic in human patients), clonality, and/or lack of cross-reactivity in relevant animal species.

Clinical development of therapeutic antibodies targeting C5aR has been considered. However, clinical development of the C5aR-specific antagonist antibody neutrazomab, a humanized IgG4 mAb, was stopped in phase II clinical trials due to issues with immune cell depletion and immunogenicity (Daniluk S et al., Annals of the Rheumatic Diseases. 2014, 73: 684-685). Because C5aR appears to be constitutively expressed in a variety of cell types, it is important that the antagonist antibody does not cause any depletion of the target cells.

To overcome the limitations of neutrazomab, a second generation of C5aR-specific antibodies was generated, namely NNC0215-0384 (US 2013/0295116 (NOVO NORDISK); clone 32F3A6GL). This antibody is a human IgG1 antibody derived from transgenic mice and is currently in clinical development as IPH5401 in the field of oncology (Olivier Demaria et al., Innate Pharma 2017. Poster #B184. CRI-CIMT-EATI-AACR Mainz) . IPH5401 carries a silenced Fc region of human IgG1 to eliminate the ability of the antibody to induce effector function. This antibody is referred to herein as RefMAB #1.

Brief description of the invention

The present invention provides new antibodies and antibody fragments.

The antibodies and antibody fragments disclosed herein can specifically bind human C5aR and preferably cross-react with cynomolgus C5aR. Therefore, in some embodiments, the disclosed antibodies are specific for human C5aR and cynomolgus C5aR. In some other embodiments, the disclosed antibodies or antibody fragments bind to the N-terminal extracellular region of human and cynomolgus C5aR.

This is in contrast to the prior art antibody IPH5401 mentioned above, which binds to the second extracellular loop of the human C5aR, resulting in a lack of binding to the cynomolgus C5aR commonly used in toxicology studies of the relevant species.

In addition, the present inventors found that the C5aR-specific antibodies claimed herein were not only significantly more effective in neutralizing pathophysiological concentrations of C5a compared to IPH5401, but also demonstrated increased effectiveness over time in suppressing C5-mediated neutrophil activation in vitro.

Accordingly, in some embodiments, the disclosed antibodies are capable of effectively inhibiting C5a-induced C5aR activity in vitro, particularly at pathophysiological concentrations of C5a. The disclosed antibodies or antibody fragments are also capable of suppressing C5a-induced leukocyte activation in vitro, as determined by their ability to suppress C5a-induced up-regulation of CD11b in granulocytes and/or monocytes. In some embodiments, the disclosed antibodies or antibody fragments inhibit human C5a-induced CD11b expression in human granulocytes at an IC ₅₀ concentration of 42 nM in the presence of 150 nM human C5a in vitro. In some other embodiments, the disclosed antibodies are capable of exhibiting increased potency in suppressing C5a-induced CD11b up-regulation in granulocytes and/or monocytes after an extended period of incubation. The disclosed antibodies may also be effective in inhibiting C5a-induced neutrophil migration.

In general, the present invention provides new antibodies that are superior to C5aR specific antibodies known in the art. In particular, the antibodies of the present invention are human antibodies with high binding affinity to human C5aR, which preferentially cross-react with cynomolgus C5aR and have favorable functional and safety properties that have never been previously observed. These properties make the antibodies of the present invention highly desirable for therapeutic applications such as the prevention and/or treatment of inflammatory and autoimmune diseases, as well as cancer.

The present invention provides isolated antibodies or antibody fragments that specifically bind to human C5aR, containing CDR regions according to Table 1 or Table 2 herein. The present invention also provides isolated antibodies or antibody fragments specific for human C5aR comprising a heavy chain variable region (VH) and a light chain variable region (VL) containing amino acid sequences according to Table 1 or Table 2 herein. The present invention also provides isolated antibodies or antibody fragments specific for C5aR, comprising a heavy chain (HC) and a light chain (LC), containing amino acid sequences according to Table 1 or Table 2 herein.

The isolated antibodies of the present invention have virtually no effector function in vitro. Such effector function may include ADCP, ADCC or CDC. In addition, the isolated antibody or antibody fragments of the present invention contain one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S and P331S, numbered according to the EU index. In particular, the isolated antibodies or antibody fragments of the present invention contain a variant of the Fc region of human IgG1, which contains the following amino acid substitutions: L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

The present invention also provides isolated antibodies or antibody fragments of the present invention for use in medicine.

The present invention also provides methods for treating a subject suffering from a disease, such as an inflammatory or autoimmune disease or cancer, by administering to said subject an effective amount of antibodies or antibody fragments of the present invention. Preferably, said subject is human.

The present invention also provides pharmaceutical compositions containing isolated antibodies or antibody fragments of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides nucleic acid compositions encoding isolated antibodies or antibody fragments of the present invention. The present invention also provides vector-based compositions including nucleic acid-based compositions encoding isolated antibodies or antibody fragments of the present invention. The present invention also provides host cells containing vector-based compositions or nucleic acid-based compositions encoding isolated antibodies or antibody fragments of the present invention.

The present invention also provides methods for treating a subject suffering from a disease, such as an inflammatory disease, an autoimmune disease, or cancer, by administering to the subject an effective amount of isolated antibodies or antibody fragments of the present invention. Preferably, said subject is human.

The present invention also provides pharmaceutical compositions containing isolated antibodies or antibody fragments of the present invention and a pharmaceutically acceptable carrier.

The claimed antibodies or antibody fragments are applicable. Moreover, the claimed method is applicable for the identification of such antibodies or antibody fragments.

The claimed antibodies or antibody fragments are useful for modifying the biological activity of human C5aR. In particular, the claimed antibodies or antibody fragments are intended for therapeutic use, such as the treatment of an inflammatory or autoimmune disease or cancer.

Brief description of graphic materials

Figure 1. Cellular binding of MAB #1, MAB #2, RefMAB #1 and the negative isotype control MOR03207 to human and cynomolgus C5aR determined by FACS. A. Dose-dependent binding to human C5aR overexpressed on CHO Flp-In cells. B. Dose-dependent binding to cynomolgus C5aR overexpressed on CHO Flp-In cells. C. Average dose-dependent binding to purified human neutrophils obtained from whole blood from three different donors. D. Average dose-dependent binding to purified cynomolgus monkey neutrophils obtained from the whole blood of three different monkeys.

Figure 2. Cellular binding of MAB #1, MAB #2, RefMAB #1 and the negative isotype control MOR03207 to human, cynomolgus and rodent C5aR, as well as GPCR-related C5aR, human C5L2, human C3aR, human FPR1 and human ChemR23 , determined by FACS, at an IgG concentration of 600 nM.

Figure 3. ELISA binding of MAB No. 1 to two natural variants of human C5aR, as well as to mouse C5aR expressed on virus-like particles (VLPs).

Figure 4. PathHunter β-arrestin assay from DiscoveRx. Neutralization of β-arrestin recruitment induced by human C5a. A. Log dose response curves for increasing concentrations of recombinant human C5a in the absence or presence of MAB #1 (50 nM) , MAB #2 (50 nM) and RefMAB #1 (50 nM). B. Percent inhibition was calculated for three increasing concentrations of human C5a (1.2 nM, 11 nM and 100 nM, respectively) with a final IgG concentration of 50 nM.

Figure 5. Suppression of human C5a-induced CD11b up-regulation in human granulocytes at normal and pathophysiological concentrations of C5a. A+B. Comparison of MAB #1, MAB #2 and RefMAB #1 in whole blood CD11b assay. Logarithmic suppression versus dose curves are shown. Human granulocytes were gated as target cells. The IgG concentration required to achieve 50% suppression of CD11b up-regulation (IC ₅₀ concentration) was calculated as a quantifiable readout. The IC ₅₀ concentrations for each IgG are shown below on the x-axis. A. Logarithmic dose response curves for increasing concentrations of human IgG and 15 nM C5a. B. Logarithmic dose response curves for increasing concentrations of IgG and 150 nM human C5a.

Figure 6. Suppression of human C5a-induced CD11b up-regulation in human granulocytes and monocytes determined over an extended period of incubation time. Comparison of MAB #1 and RefMAB #1 in the CD11b whole blood assay after IgG incubation with target cells for either 20 minutes or 300 minutes. IgG was added in serial dilutions and incubated for either 20 minutes or 300 minutes, followed by stimulation with using 15 nM human C5a. Either granulocytes (Figures 6A and B) or monocytes (Figures 6C and D) were gated as target cells. CD11b levels were determined. Logarithmic suppression versus dose curves are shown. Data are expressed as % suppression of CD11b up-regulation at 15 nM human C5a. The results for MAB #1 are shown in Figures 6A and C, and the results for RefMAB #1 are shown in Figures 6B and D.

Figure 7. Suppression of human C5a-induced migration of human neutrophils. MAB #1 and the negative control MOR03207 were tested at two concentrations of IgG (100 nM and 600 nM, respectively) in the presence of 10 nM human C5a. Shown are the average values from three independent assays performed at 3 different time points (15 min, 25 min, 35 min). Neutrophils were obtained from 3 different human donors. The percentage of inhibition was calculated based on neutrophil migration in the absence of antibody.

Figure 8. ADCC and ADCP reporter bioassay using Promega. Comparison of MAB #1 and a monoclonal control IgG to C5aR carrying either the wild-type (unsilenced) human IgG1 Fc region or a variant (silenced) Fc region identical to the Fc region of MAB #1. In addition, the isotype control antibody MOR03207 was included with either the variant or the Fc region of wild-type human IgG1. Assays were performed according to the supplier's instructions using either engineered Jurkat cells expressing FcγRIIa_H to mimic the ADCP pathway, or Jurkat cells expressing FcγRIIIa high affinity variant V158 to mimic the ADCC pathway, and CHO cells expressing C5aR. A. ADCP reporter bioassay results at 10 μg/mL IgG concentration. B. ADCC reporter bioassay results at 10 μg/mL IgG concentration. Data are presented as the average fluorescence signal above background.

Figure 9. Group mean pharmacokinetic profile for MAB #1 in Han Wistar rats after a single intravenous administration of 10 mg/kg IgG. Data are presented as mean values (IgG concentration over time) (standard deviation (S) (N=3).

Figure 10. Results of profiling a panel of proteins (3P) for MAB No. 1 and MAB No. 2. The numbers for each antibody represent the resulting binding signal of each antibody and test protein compared to the binding of the negative isotype control antibody MOR03207. Antibodies were tested at concentrations of 10 nM and 100 nM, respectively.

Figure 11. Cytokine release by in vitro matured monocyte-derived macrophages M1 and M2. The levels of IL-10 and IL-12 were determined by ELISA after treatment with MAB No. 1 and incubation with C5a overnight.

Detailed Description of the Invention

The present invention relates to a series of human antibodies that recognize human C5aR.

Definitions

The term "C5aR" refers to a protein known as chemotactic anaphylatoxin receptor 1 C5a or CD88.

Human C5aR (Uniprot: P21730⏐1-350) (herein referred to as the “D/K variant”) is characterized by its amino acid sequence

Two natural missense mutations of human C5aR have been described ((http://www.uniprot.org/uniprot/P21730): Reference SNP (refSNP) Cluster Report: rs4467185 (MAF: 0.03) and Cluster Report: rs11880097 (MAF: 0.03). One mutation is within the N-terminal extracellular region of human C5aR (position 2 in SEQ ID NO: 1), resulting in a D to N substitution.

A human C5aR protein that contains both naturally occurring missense mutations in its sequence (also referred to herein as an “N/N variant”) is characterized by its amino acid sequence

C5aR of cynomolgus macaque (Macaca fascicularis) characterized by amino acid sequence

Mouse (Mus musculus) C5aR characterized by amino acid sequence

Rat (Rattus norvegicus) C5aR characterized by amino acid sequence

The term "C5a" refers to the protein known as human complement component C5a.

Human C5a (Uniprot: P01031⏐678-751) is characterized by its amino acid sequence

The term "C5L2" refers to a protein known as anaphylatoxin C5a chemotactic receptor 2.

Human C5L2 characterized by amino acid sequence

The term "C3aR" refers to a protein known as the anaphylatoxin C3a chemotactic receptor.

Human C3aR is characterized by its amino acid sequence

The term "FPR1" refers to a protein known as the fMet-Leu-Phe receptor.

Human FPR1 characterized by amino acid sequence

The term "ChemR23" refers to a protein known as chemokine-like receptor 1.

Human ChemR23 characterized by amino acid sequence

As used herein, the term “antibody” refers to a protein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds that interacts with an antigen. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CHI, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies and chimeric antibodies. Antibodies can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

As used herein, the phrase “antibody fragment” refers to one or more portions of an antibody that retain the ability to specifically interact with (eg, by binding, steric mismatch, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, the Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of VH and CH1 domains; Fv fragment, consisting of the VL and VH domains of one arm of the antibody; dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and a dedicated complementarity determining region (CDR). Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined recombinantly using a synthetic linker that allows them to form a single protein chain in which the VL and VH regions are joined in pairs to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antibody fragment". These antibody fragments are prepared using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be included in single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136) . Antibody fragments can be grafted onto polypeptide backbones such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes monobodies based on a fibronectin polypeptide). Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and US patent No. 5641870).

As used herein, the terms “human antibody” or “human antibody fragment” are an antibody or antibody fragment containing variable regions in which both the framework and CDR regions are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (eg XenoMouse), provided that the system produces antibodies containing variable regions in which both the framework and CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, then the constant region is also derived from such sequences. Sources of human origin include, for example, human germline sequences or mutant versions of human germline sequences or antibodies containing consensus framework sequences obtained from analysis of human framework sequences, for example, as described in Knappik et al., (2000) J Mol Biol 296: 57-86).

The structures and locations of immunoglobulin variable domains, such as CDRs, can be determined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of the Kabat and Chothia numbering schemes (see, for example, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Lazikani et al., (1997) J. Bio. 273: 927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit, NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948.

As used herein, the terms “humanized antibody” or “humanized antibody fragment” are defined as an antibody molecule that contains antibody constant regions derived from sequences of human origin, and wherein the antibody variable regions and portions thereof or CDRs alone are derived from another species. For example, a humanized antibody may contain grafted CDRs, wherein the variable domain CDRs are of non-human origin, while one or more variable domain backbones are of human origin, and the constant domain (if any) is of human origin.

The terms “chimeric antibody” or “chimeric antibody fragment” are defined herein as an antibody molecule that contains antibody constant regions derived from or corresponding to sequences found in one species and antibody variable regions derived from another species. Preferably, the antibody constant regions are derived from or correspond to sequences found in humans, and the antibody variable regions (e.g., VH, VL, CDR, or FR regions) are derived from sequences found in a non-human animal, e.g. mouse, rat, rabbit or hamster.

The term "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having a different antigenic specificity. Moreover, the isolated antibody or antibody fragment may contain substantially no other cellular material and/or chemicals. Thus, in some aspects, the antibodies provided are isolated antibodies that have been separated from antibodies with other specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of the target may, however, be cross-reactive with other related antigens, for example from other species (eg homologue species).

As used herein, the term “recombinant antibody” includes all antibodies that are produced, expressed, created or isolated by means not existing in nature. For example, antibodies isolated from a host cell transformed to express an antibody, antibodies selected and isolated from a recombinant combinatorial library of human antibodies, and antibodies produced, expressed, generated or isolated by any other means that involve splicing all or part of a human immunoglobulin gene , sequences with other DNA sequences or antibodies isolated from an animal (eg mouse) that are transgenic or transchromosomal with respect to human immunoglobulin genes or a hybridoma derived from them. Preferably, such recombinant antibodies contain variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are are sequences that, because they are derived from and related to human germline VH and VL sequences, may not naturally occur within the human germline antibody repertoire in vivo. The recombinant antibody may be a monoclonal antibody. In one embodiment, the antibodies and antibody fragment disclosed herein are isolated from the HuCAL library (Rothe et al, J. Mol. Biol. (2008) 376, 1182-1200).

As used herein, an antibody “binds specifically to,” “specifically binds to,” is “specific for,” or “specifically recognizes” an antigen, such as human C5aR, if such antibody is capable of distinguishing such antigen and one or more reference antigens , since binding specificity is not an absolute, but a relative property. For example, a standard ELISA assay can be performed. Scoring can be achieved by standard stain development (eg secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in certain wells is assessed by optical density, for example at 450 nm. A typical background (=negative reaction) may correspond to 0.1 OD; a typical positive reaction may correspond to 1 OD. This means that the difference between positive and negative reactions can be more than 10-fold. Typically, the determination of binding specificity is carried out using not a single reference antigen, but a set of approximately three to five unrelated antigens, such as milk powder proteins, BSA, transferrin, etc.

As used herein, the term “affinity” refers to the strength of interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target at many sites through weak non-covalent forces; the more interactions, the higher the affinity.

As used herein, the term "K _D " refers to the dissociation constant, which is derived from the ratio of k _off and K _on (ie k _off /k _on ) and is expressed as molar concentration (M). K _D values for antigen binding fragments, such as, for example, monoclonal antibodies, can be determined using methods well known in the art. Methods for determining the K _D of an antigen binding fragment, such as, for example, a monoclonal antibody, are SET (solution equilibrium titration) or surface plasmon resonance using a biosensor system such as the Biacore® system. In the present invention, the antibody specific for C5aR typically has a dissociation rate constant (K _D ) (k _off /k _on ) of less than 5×10 ^-2 M, less than 10 ^-2 M, less than 5× 10 ^-3 M, less than 10 ^-3 M, less than 5×10 ^-4 M, less than 10 ^-4 M, less than 5×10 ^-5 M, less than 10 ^-5 M, less than 5×10 ^{- 6} M, less than 10 ^-6 M, less than 5×10 ^-7 M, less than 10 ^-7 M, less than 5×10 ^-8 M, less than 10 ^-8 M, less than 5×10 ^-9 M , less than 10 ^-9 M, less than 5×10 ^-10 M, less than 10 ^-10 M, less than 5×10 ^-11 M, less than 10 ^-11 M, less than 5×10 ^-12 M, less less than 10 ^-12 M, less than 5×10 ^-13 M, less than 10 ^-13 M, less than 5×10 ^-14 M, less than 10 ^-14 M, less than 5×10 ^-15 M or less than 10 ^-15 M or less.

The term "epitope" includes any protein region that is specifically recognized by an antibody or antibody fragment or otherwise interacts with a molecule. Typically, epitopes are chemically active surface groups of molecules such as amino acids or the side chains of carbohydrates or sugars, and can usually have specific three-dimensional structure characteristics as well as specific charge characteristics. As one skilled in the art will appreciate, virtually anything to which an antibody is capable of specifically binding can be an epitope.

The "compositions" of the present invention can be used for therapeutic or prophylactic applications. The present invention, therefore, relates to a pharmaceutical composition containing an antibody or antibody fragment disclosed herein and a pharmaceutically acceptable carrier or excipient therefor. In a related aspect, the present invention provides a method for treating cancer. Such a method involves the steps of administering to a subject in need thereof an effective amount of a pharmaceutical composition that contains an antibody or antibody fragment described herein.

The present invention relates to therapeutic methods comprising administering a therapeutically effective amount of an antibody or antibody fragment disclosed herein to a subject in need of such treatment. As used herein, the expressions “therapeutically effective amount” or “effective amount” refer to the amount of anti-C5aR antibody required to induce the desired biological response. In accordance with the present invention, a therapeutically effective amount is the amount of anti-C5aR antibody needed to treat and/or prevent a disease.

“Administered” or “administration” includes, without limitation, the delivery of a drug by an injectable form, such as, for example, by the intravenous, intramuscular, intradermal, subcutaneous, or transmucosal route, such as a nasal spray or inhalation aerosol or ingestible inside a solution, capsule or tablet. Preferred administration is via an injection form.

As used herein, the terms “treating,” “treating,” or “providing treatment,” and the like, refer to a clinical intervention in an attempt to change the natural course of a disease in a subject being treated, which may be performed either prophylactically or during clinical pathology. Desired effects of treatment include, but are not limited to, prevention of onset or recurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or mitigation of disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments of the present invention are used to delay the development of a disease or to slow the progression of a disease.

The term "effector function" refers to those biological activities inherent in the Fc region of an antibody that vary depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP); negative regulation of cell surface receptors (eg B cell receptor) and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies binding to Fc receptors (FcRs) present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages) allow those cytotoxic effector cells to specifically bind to the antigen-bearing target cell, and then the cytotoxic cells destroy the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) of the present invention that bind to their cognate antigen.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to the mechanism of elimination of antibody-coated target cells through internalization by phagocytic cells such as macrophages or dendritic cells.

"Prevent" or "prevention" refers to reducing the risk of acquiring or developing a disease (i.e., preventing the development of at least one of the clinical symptoms of a disease in a subject who may be exposed to a disease-causing factor or has a predisposition to the disease before the onset of the disease) . "Prevention" also refers to methods that are intended to prevent the onset of a disease or symptoms thereof or that delay the onset of a disease or symptoms thereof.

As used herein, the terms "subject" or "species" refer to any mammal, including rodents such as mice or rats, and primates such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatto) or humans ( Homo sapiens). Preferably the subject is a primate, most preferably a human.

As used herein, unless the context otherwise requires, the words “comprise”, “have” and “include” and their corresponding variations such as “comprises”, “comprising”, “has”, “having”, “includes” and “ "including" shall be understood to mean the inclusion of a specified element or whole number or group of elements or whole numbers, but not the exclusion of any other element or whole number or group of elements or whole numbers.

As used herein, the terms “engineered” or “modified” include the manipulation of nucleic acids or polypeptides through synthetic techniques (eg, recombinant techniques, in vitro peptide synthesis, enzymatic or chemical peptide coupling, or some combination of these techniques). Preferably, the antibodies or antibody fragments of the present invention are designed or modified to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, safety, and the like.

As used herein, the term “variant” refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitutions, insertions, or deletions.

As used herein, the term “amino acid mutation” includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification can be carried out provided that the final construct has the desired characteristics, for example reduced binding to the Fc receptor. Deletions and insertions of amino acid sequences include N- and/or C-terminal deletions and insertions of amino acid residues. Specific amino acid mutations are amino acid substitutions. Amino acid substitutions involve replacing twenty standard amino acids with non-naturally occurring amino acids or derivatives of naturally occurring amino acids. Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering the side chain group of an amino acid residue through methods other than genetic engineering, such as chemical modification, may also be applicable. Various designations may be used herein to refer to the same amino acid mutation. For example, a change from glycine at position 327 of the Fc region to alanine may be designated 237A, G337, G337A, or Gly329Ala.

As used herein, the term "EC ₅₀ " refers to the concentration of an antibody, or antibody fragment, or ligand that induces a response in an assay that is the average of baseline and maximum. Therefore, it represents the concentration of antibody or ligand at which an effect of 50% of the maximum is observed.

As used herein, the term "IC ₅₀ " refers to the concentration of an antibody or antibody fragment that inhibits the response in an assay that is the average of the maximum response and the baseline. It represents the concentration of antibody that reduces this response by 50%.

The terms "suppression" or "suppress" or "reduce" or "reduce" or "neutralize" or "neutralize" refer to the reduction or termination of any phenotypic characteristic (such as binding or biological activity or function) or to a reduction or cessation of occurrence, degree or likelihood of that characteristic. “Suppression”, “reduction” or “neutralization” do not need to be complete, but what is important is that they can be identified using suitable analysis. In some embodiments, the terms “reduce” or “suppress” or “neutralize” mean the ability to cause a reduction of 20% or more. In another embodiment, the terms “reduce” or “suppress” or “neutralize” mean the ability to cause a reduction of 50% or more. In yet another embodiment, the terms "reduce" or "suppress" or "neutralize" mean the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

As used herein, the term "antagonistic" antibody refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes the biological activity or function or any other phenotypic characteristic of the target antigen. A “wild type” protein is a version or variant of a protein that occurs in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of a naturally occurring protein. Due to allotypic differences, a wild-type protein may have more than one amino acid sequence. For example, there are several allotypes of naturally occurring heavy chain constant regions of human IGg1 (see, for example, Jeffries et al. (2009) mAbs 1:1).

The term "Fc region" is used to refer to the C-terminal region of the heavy chain of an immunoglobulin. The Fc region of an immunoglobulin usually contains two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the IgG heavy chain Fc region may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the C terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

Embodiments

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region containing the amino acid sequence under SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence under SEQ ID NO: 28, the HCDR3 region containing the amino acid sequence under SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence under SEQ ID NO: 32, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

b) the HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 39, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 39, the HCDR3 region under SEQ ID NO: 40, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region containing the amino acid sequence under SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence under SEQ ID NO: 31, the HCDR3 region containing the amino acid sequence under SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence under SEQ ID NO: 32, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

b) the HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 41, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region containing the amino acid sequence under SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence under SEQ ID NO: 28, the HCDR3 region containing the amino acid sequence under SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence under SEQ ID NO: 32, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

b) the HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 31, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

c) the HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 39, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

d) the HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 41, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 28 NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 39 NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 31 NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 41 NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR comprising the Kabat 6 CDRs of any of the antibodies disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR comprising the 6 Chothia-defined CDRs of any of the antibodies disclosed in Table 1 or Table 2.

In one embodiment of the present invention, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of the present invention, the isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In another embodiment of the present invention, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment of the present invention, the isolated antibody or antibody fragment is of the IgG isotype. In one embodiment of the present invention, the isolated antibody or antibody fragment is of the IgG1 class. In another embodiment of the present invention, the isolated antibody or antibody fragment does not induce substantially effector function in vitro.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, and further comprise VH of SEQ ID NO: 35, or VL of SEQ ID NO: 36, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, and further comprise VH of SEQ ID NO: 35, or VL of SEQ ID NO: 36, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprise VH of SEQ ID NO: 42, or VL of SEQ ID NO: 43, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprises VH of SEQ ID NO: 42, or VL of SEQ ID NO: 43.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36 or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the antibody or antibody fragment comprises VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the antibody or antibody fragment comprises the HC of SEQ ID NO: 37 and the LC of SEQ ID NO: 38.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the antibody or antibody fragment comprises the HC of SEQ ID NO: 44 and the LC of SEQ ID NO: 45.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR comprising a variable heavy chain (VH) and a variable light chain (VL) of any of the antibodies disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR comprising a heavy chain (VH) and a light chain (LC) of any of the antibodies disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 28 NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 35, or VL of SEQ ID NO: 36

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 39 NO: 40, region LCDR1 of SEQ ID NO: 32, region LCDR2 of SEQ ID NO: 33, and region LCDR3 of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42, or VL of SEQ ID NO: 43 .

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 31 NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 35, or VL of SEQ ID NO: 36 .

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 41 NO: 40, region LCDR1 of SEQ ID NO: 32, region LCDR2 of SEQ ID NO: 33, and region LCDR3 of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42, or VL of SEQ ID NO: 43 .

In one embodiment of the present invention, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of the present invention, the isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In another embodiment of the present invention, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or SEQ ID NO: 42 and with VL of SEQ ID NO: 36 or SEQ ID NO: 43.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or VH and VL that are characterized by at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are characterized by at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or SEQ ID NO: 44 and with the LC of SEQ ID NO:38 or SEQ ID NO:45.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises an HC of SEQ ID NO: 37 and an LC of SEQ ID NO: 38, or an HC and LC that are characterized by at least 80%, at least 85%, at least 90%, or at least 95% identity with HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment comprises an HC of SEQ ID NO: 44 and an LC of SEQ ID NO: 45, or an HC and LC that are characterized by at least 80%, at least 85%, at least 90%, or at least 95% identity with HC of SEQ ID NO: 44 and LC of SEQ ID NO: 45.

In one embodiment of the present invention, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of the present invention, the isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In another embodiment of the present invention, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human or humanized antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human antibody or antibody fragment.

In one embodiment, the isolated human C5aR-specific antibody or antibody fragment of the present invention is a recombinant or synthetic antibody or antibody fragment. In a further embodiment, an isolated antibody or antibody fragment specific for C5aR in accordance with the present invention is an isolated recombinant monoclonal antibody or antibody fragment. In a further embodiment, an isolated antibody or antibody fragment specific for C5aR in accordance with the present invention is an isolated recombinant human monoclonal antibody or antibody fragment.

In one embodiment, the isolated antibody or antibody fragment specific for C5aR in accordance with the present invention is of the IgG isotype. In another embodiment, said isolated antibody or antibody fragment is of the IgG1 class. In another embodiment, said isolated antibody or antibody fragment is of the human IgG1 class.

Nucleic acids

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody or antibody fragment comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In another embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a heavy chain sequence and a light chain sequence of an isolated antibody or antibody fragment specific for human C5aR, wherein the nucleic acid sequence or plurality nucleic acid sequences contain

a) the HCDR1 region under SEQ ID NO: 46, the HCDR2 region under SEQ ID NO: 47, the HCDR3 region under SEQ ID NO: 48, the LCDR1 region under SEQ ID NO: 51, the LCDR2 region under SEQ ID NO: 52 and the LCDR3 region under SEQ ID NO: 53, or

b) the HCDR1 region under SEQ ID NO: 49, the HCDR2 region under SEQ ID NO: 50, the HCDR3 region under SEQ ID NO: 48, the LCDR1 region under SEQ ID NO: 51, the LCDR2 region under SEQ ID NO: 52 and the LCDR3 region under SEQ ID NO: 53, or

c) the HCDR1 region under SEQ ID NO: 58, the HCDR2 region under SEQ ID NO: 59, the HCDR3 region under SEQ ID NO: 60, the LCDR1 region under SEQ ID NO: 63, the LCDR2 region under SEQ ID NO: 64 and the LCDR3 region under SEQ ID NO: 64 SEQ ID NO: 65, or

d) the HCDR1 region under SEQ ID NO: 61, the HCDR2 region under SEQ ID NO: 62, the HCDR3 region under SEQ ID NO: 60, the LCDR1 region under SEQ ID NO: 63, the LCDR2 region under SEQ ID NO: 64 and the LCDR3 region under SEQ ID NO: 64 SEQ ID NO: 65.

In another embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a heavy chain sequence and a light chain sequence of an isolated antibody or antibody fragment specific for human C5aR, wherein the nucleic acid sequence or plurality nucleic acid sequences contain

a) the HCDR1 region under SEQ ID NO: 70, the HCDR2 region under SEQ ID NO: 71, the HCDR3 region under SEQ ID NO: 72, the LCDR1 region under SEQ ID NO: 75, the LCDR2 region under SEQ ID NO: 76 and the LCDR3 region under SEQ ID NO: 77, or

b) the HCDR1 region of SEQ ID NO: 73, the HCDR2 region of SEQ ID NO: 74, the HCDR3 region of SEQ ID NO: 72, the LCDR1 region of SEQ ID NO: 75, the LCDR2 region of SEQ ID NO: 76, and the LCDR3 region of SEQ ID NO: 77, or

c) the HCDR1 region under SEQ ID NO: 82, the HCDR2 region under SEQ ID NO: 83, the HCDR3 region under SEQ ID NO: 84, the LCDR1 region under SEQ ID NO: 87, the LCDR2 region under SEQ ID NO: 88 and the LCDR3 region under SEQ ID NO: 88 SEQ ID NO: 89, or

d) the HCDR1 region under SEQ ID NO: 85, the HCDR2 region under SEQ ID NO: 86, the HCDR3 region under SEQ ID NO: 84, the LCDR1 region under SEQ ID NO: 87, the LCDR2 region under SEQ ID NO: 88 and the LCDR3 region under SEQ ID NO: 88 SEQ ID NO: 89.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises a VH under SEQ ID NO: 54 and VL under SEQ ID NO: 55 or VH and VL, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with VH of SEQ ID NO: 54 and/or VL of SEQ ID NO: 55.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises a VH under SEQ ID NO: 66 and VL under SEQ ID NO: 67 or VH and VL, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with VH of SEQ ID NO: 66 and/or VL of SEQ ID NO: 67.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises a VH under SEQ ID NO: 78 and VL under SEQ ID NO: 79 or VH and VL, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with VH of SEQ ID NO: 78 and/or VL of SEQ ID NO: 79.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises a VH under SEQ ID NO: 90 and VL under SEQ ID NO: 91 or VH and VL, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with VH of SEQ ID NO: 90 and/or VL of SEQ ID NO: 91.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 56 and LC under SEQ ID NO: 57 or HC and LC, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with HC under SEQ ID NO: 56 and/or LC under SEQ ID NO: 57.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 68 and LC under SEQ ID NO: 69 or HC and LC, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with HC under SEQ ID NO: 68 and/or LC under SEQ ID NO: 69.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 80 and LC under SEQ ID NO: 81 or HC and LC, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with HC under SEQ ID NO: 80 and/or LC under SEQ ID NO: 81.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 92 and LC under SEQ ID NO: 93 or HC and LC, which are characterized by at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identity with HC under SEQ ID NO: 92 and/or LC under SEQ ID NO: 93.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 56 and LC under SEQ ID NO: 57.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 68 and LC under SEQ ID NO: 69.

In one embodiment, the present invention relates to a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein said nucleic acid sequence or plurality of nucleic acid sequences comprises an HC under SEQ ID NO: 80 and LC under SEQ ID NO: 81.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for human C5aR disclosed herein, wherein said nucleic acid sequence or plurality of nucleic acid sequences acids contain HC under SEQ ID NO: 92 and LC under SEQ ID NO: 93.

In another embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or fragment specific for human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises VH and VL of any from the antibodies disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR encoded by any of the nucleic acid sequences or plurality of nucleic acid sequences disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any of the isolated antibodies or antibody fragments disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any of the isolated antibodies or antibody fragments specific for human C5aR in accordance with the present invention.

In another embodiment, the present invention provides a nucleic acid composition comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises HC and LC any of the antibodies or antibody fragments disclosed in Table 1 or Table 2.

In one embodiment, said nucleic acid composition and/or said nucleic acid sequence and/or a plurality of nucleic acid sequences are isolated.

Vectors

In one embodiment, the present invention provides a vector-based composition containing a vector or a plurality of vectors containing a nucleic acid-based composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention.

In one embodiment, the present invention provides a vector-based composition containing a vector or a plurality of vectors containing a nucleic acid-based composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding any of the isolated antibodies or antibody fragments specific for human C5aR, disclosed in Table 1 or Table 2.

In one embodiment, the present invention provides a vector-based composition comprising a vector or a plurality of vectors containing a nucleic acid sequence or a plurality of nucleic acid sequences disclosed in Table 1 or Table 2.

In one embodiment, said vector-based composition and/or vector and/or plurality of vectors are dedicated.

Host cells

In one embodiment, the present invention provides a host cell containing a vector-based composition containing a vector or a plurality of vectors containing a nucleic acid-based composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for Human C5aR according to the present invention.

In one embodiment, the present invention relates to a host cell containing a vector-based composition containing a vector or a plurality of vectors containing a nucleic acid-based composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to regarding C5aR, disclosed in Table 1 or Table 2.

In one embodiment, a host cell in accordance with the present invention is capable of expressing an isolated antibody or antibody fragment specific for human C5aR encoded by a vector composition or a nucleic acid composition.

In a further embodiment, the host cell is an isolated host cell. In a further embodiment, said host cell is a mammalian cell. In one embodiment, said mammalian cell is a human cell. In another embodiment, said mammalian cell is a CHO cell. In one embodiment, said cell is a HEK cell. In another embodiment, said cell is a PERC.6 cell. In one embodiment, said cell is an HKB11 cell.

One skilled in the art will appreciate that a nucleic acid sequence or multiple nucleic acid sequences encoding the heavy and/or light chain of an antibody or antibody fragment of the present invention can be cloned into different vectors or into the same vector.

Vectors can be introduced into appropriate host cells, such as prokaryotic (eg bacterial) or eukaryotic (eg yeast or mammalian) cells, by methods known in the art (see, for example, "Current Protocol in Molecular Biology", Ausubel et al. al. (eds.), Greene Publishing Assoc. and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those skilled in the art, and selection of a suitable cloning vector is a matter of choice. The gene may be under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as “control” elements) such that the nucleic acid sequence encoding the desired protein is transcribed into RNA in the transformed host cell vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. When expressed in host cells, antibodies or antibody fragments of the present invention are obtained. These steps can be performed in a variety of ways known to one skilled in the art. In general, such steps typically involve transformation or transfection of a suitable host cell with a nucleic acid composition, or vector composition, or infectious particle that encodes an antibody or antibody fragments. In addition, such steps typically include culturing said host cells under conditions suitable for proliferation of said host cells, and a culturing step under conditions suitable for producing (expressing, synthesizing) the encoded antibody or antibody fragment. Cultivation of host cells under conditions suitable for proliferation or expression is typically carried out in the presence of a medium containing components suitable for cell growth or induction of expression. In specific embodiments, methods for producing antibodies or antibody fragments of the present invention further include the step of isolating and purifying the resulting antibody or antibody fragment from host cells or media. If the expression system secretes the protein into the growth medium, then the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of suitable growth conditions and extraction methods is within the competence of those skilled in the art. The antibody or antibody fragment of the present invention can then be purified by a variety of techniques known to one of ordinary skill in the art.

In one embodiment, the present invention provides a method for producing an isolated antibody or antibody fragment specific for human C5aR, any of the antibodies disclosed in Table 1 or Table 2. In one embodiment, there is provided a method for producing an isolated antibody or antibody fragment in accordance with the present the invention, where the method includes culturing a host cell containing a vector-based composition containing a vector or a plurality of vectors containing a nucleic acid-based composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment in accordance with the present invention, under conditions suitable for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or the culture medium of the host cell. The antibody or antibody fragment isolated as described herein can be purified by techniques known in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The methods used to purify a particular antibody or antibody fragment will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For purification by affinity chromatography, an antibody, ligand, receptor, or antigen to which the antibody or antibody fragment binds can be used. For example, a Protein A or Protein G matrix may be used to purify an antibody or antibody fragment of the present invention by affinity chromatography. The purity of the antibody or antibody fragment may be determined by any of a variety of well known analytical techniques, including gel electrophoresis, liquid chromatography high pressure, etc.

Specificity

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR disclosed in Table 1 or Table 2. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention is specific for human C5aR.

In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention is specific for the human C5aR encoded by the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In one embodiment, an isolated antibody or antibody fragment in accordance with with the present invention are specific for a polypeptide containing the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention is specific for a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.

In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention specifically binds to the extracellular region of human C5aR. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention specifically binds to the N-terminal extracellular region of human C5aR. In one embodiment, an isolated antibody or antibody fragment of the present invention specifically binds to the N-terminal extracellular region of human C5aR, wherein the N-terminal extracellular region comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the isolated antibody or antibody fragment in accordance with the present invention specifically binds to the N-terminus of human C5aR containing the amino acid sequence of SEQ ID NO: 13. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: : 13. In one embodiment, an isolated antibody or antibody fragment according to the present invention specifically binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 13.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, the isolated human C5aR-specific antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment specific for human C5aR is a recombinant antibody or antibody fragment. In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is an isolated recombinant human monoclonal antibody or antibody fragment.

Species cross-reactivity

In additional embodiments, an isolated antibody or antibody fragment of the present invention cross-reacts with cynomolgus monkey C5aR. In another embodiment, an isolated antibody or antibody fragment according to the present invention is specific for human C5aR and cynomolgus C5aR.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment binds via cross-reactivity to cynomolgus C5aR. In one embodiment, an isolated antibody or antibody fragment of the present invention specifically binds to the extracellular region of human C5aR and cynomolgus C5aR. In one embodiment, an isolated antibody or antibody fragment of the present invention binds to the N-terminal extracellular region of human C5aR and cynomolgus C5aR. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention binds to the N-terminal extracellular region of human C5aR and cynomolgus C5aR, wherein the N-terminal extracellular region of C5aR contains the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

In yet another embodiment, an isolated antibody or antibody fragment in accordance with the present invention does not bind to a rodent C5aR, such as a mouse or rat C5aR.

In one embodiment, an antibody or antibody fragment of the present invention is coupled to a peptide comprising the amino acid sequence of SEQ ID NO: 13 and/or SEQ ID NO: 14.

In one embodiment, an antibody or antibody fragment of the present invention is coupled to a peptide consisting of the amino acid sequence of SEQ ID NO: 13 and/or SEQ ID NO: 14.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR and cynomolgus C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

NS and LC, which are characterized by at least at least 80%, at least 85%, at least 90% or at least 95%

identical to HC under SEQ ID NO: 37 or 44 and to LC under SEQ ID NO: 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Monovalent affinity for C5aR peptides

In further embodiments, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment has a monovalent affinity for a human C5aR peptide comprising SEQ ID NO: 13, with a K _D of 100 nM or less, for example, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or 1 nM or less.

In one embodiment, said monovalent affinity is determined in IgG format. In certain embodiments, said monovalent affinity is determined by surface plasmon resonance (SPR), as described herein in Example 4.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34. In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment. Apparent affinity (bivalent) for C5aR peptides

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for the human C5aR peptide comprising SEQ ID NO: 13, with a K _D of 1 nM or less, for example, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, or 1 pM or less.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for the cynomolgus C5aR peptide comprising SEQ ID NO: 14, with a K _D of 300 nM or less, for example, 250 nM or less, 200 nM or less, 150 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or 1 nM or less.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for a human C5aR peptide comprising SEQ ID NO: 13, with a K _D of 0.3 nM or less, and with respect to the cynomolgus C5aR peptide containing SEQ ID NO: 14, with a K _D of 150 nM or less.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for a human C5aR peptide comprising SEQ ID NO: 13 with a K _D of 0.05 nM or less and for a cynomolgus C5aR peptide comprising SEQ ID NO: 14, with a K _D of 80 nM or less.

In certain embodiments, said apparent affinity is determined in IgG format. In one embodiment, said apparent affinity is determined by biolayer interferometry (BLI), as described herein in Example 5.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Apparent affinity (bivalent) for full-length C5aR

In further embodiments, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for human C5aR with a K _D of 10 nM or less, e.g., 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

In embodiments, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for cynomolgus C5aR with a K _D 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 1 nM or less.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody has an apparent affinity for human C5aR with a K _D of 0.5 nM or less and for cynomolgus C5aR with a K _D of 5 nM or less.

In one embodiment, said human C5aR contains the amino acid sequence of SEQ ID NO: 1. In one embodiment, said human C5aR contains the amino acid sequence of SEQ ID NO: 2. In one embodiment, said cynomolgus C5aR contains the amino acid sequence of SEQ ID NO : 3.

In certain embodiments, said apparent affinity is determined in IgG format. In embodiments, said human C5aR or cynomolgus C5aR is expressed on cells. In embodiments, said human C5aR or cynomolgus C5aR is expressed on engineered full-length human CHO cells. In embodiments, said CHO cells are Flp-In CHO cells.

In certain embodiments, said apparent affinity is determined as described herein in Example 6.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HCDR1 region under SEQ ID NO: 27, HCDR2 region under SEQ ID NO: 28,

HCDR3 region under SEQ ID NO: 29, LCDR1 region under SEQ ID NO: 32,

the LCDR2 region of SEQ ID NO: 33 and the LCDR3 region of SEQ ID NO: 34, or

b) HCDR1 region under SEQ ID NO: 30, HCDR2 region under SEQ ID NO: 31,

HCDR3 region under SEQ ID NO: 29, LCDR1 region under SEQ ID NO: 32,

the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

c) HCDR1 region under SEQ ID NO: 27, HCDR2 region under SEQ ID NO: 39,

HCDR3 region under SEQ ID NO: 40, LCDR1 region under SEQ ID NO: 32,

the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

d) HCDR1 region under SEQ ID NO: 30, HCDR2 region under SEQ ID NO: 41,

HCDR3 region under SEQ ID NO: 40, LCDR1 region under SEQ ID NO: 32,

the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Apparent EU ₅₀ in relation to full-length C5aR

In further embodiments, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody binds to human C5aR with an EC50 concentration of 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less , or 0.1 nM or less.

In embodiments, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said isolated antibody binds to cynomolgus C5aR at an EC50 concentration of 20 nM or less, 10 nM or less, 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 1 nM or less.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody binds to human C5aR and cynomolgus C5aR at an EC ₅₀ K _D concentration of 10 nM or less.

In one embodiment, said human C5aR contains the amino acid sequence of SEQ ID NO: 1. In one embodiment, said human C5aR contains the amino acid sequence of SEQ ID NO: 2. In one embodiment, said cynomolgus C5aR contains the amino acid sequence of SEQ ID NO : 3.

In certain embodiments, said EC ₅₀ concentration is determined in IgG format. In embodiments, said human C5aR or cynomolgus C5aR is expressed on cells. In embodiments, said human C5aR or cynomolgus C5aR is expressed on engineered full-length human CHO cells. In embodiments, said CHO cells are Flp-In CHO cells. In certain embodiments, said human C5aR or cynomolgus C5aR is expressed on neutrophils. In embodiments, said human C5aR is expressed on human neutrophils. In embodiments, said cynomolgus C5aR is expressed on cynomolgus neutrophils. In certain embodiments, said neutrophils are obtained from whole blood. In certain embodiments, said EC50 concentration is determined as described herein in Example 7.

In additional embodiments, the isolated human C5aR-specific antibody or antibody fragment binds substantially not to a C5aR-related antigen selected from the group consisting of human C5L2, human ChemR23, human FPR1, and human C3aR. In certain embodiments, said isolated human C5aR-specific antibody or antibody fragment binds substantially not to a C5aR-related antigen selected from the group consisting of human C5L2, human ChemR23, human FPR1, and human C3aR at an IgG concentration of 300 nM. In one embodiment, said binding is determined as described in Example 7.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 37 or 44 and with VL of SEQ ID NO: : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein said antibody or antibody fragment binds to human C5aR comprising SEQ ID NO: 1 and/or SEQ ID NO: 2, at an EC50 concentration, being 5 nM or less, for example, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less.

In certain embodiments, said human C5aR is presented on virus-like particles. In certain embodiments, said human C5aR is expressed as a fusion protein. In certain embodiments, said human C5aR is fused to a GAG protein. In embodiments, said fusion protein comprises the amino acid sequence disclosed in Table 8. In certain embodiments, said binding is determined by ELISA as described in Example 8.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ Ш NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ Ш NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ Ш NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Functionality - C5A-induced activation of C5aR

In general, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention can be used to prevent or inhibit the interaction between human C5aR and human C5a, thereby preventing, inhibiting, neutralizing or reducing signaling pathways that are mediated by C5aR , and/or modulating biological pathways and mechanisms in which C5aR is involved.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody or antibody fragment specifically disrupts C5aR-mediated signal transduction.

In a further embodiment of the present invention, an isolated antibody or antibody fragment specific for human C5aR specifically disrupts the interaction of C5a with C5aR expressed on cells. In yet another further embodiment, said isolated antibody or antibody fragment is capable of specifically disrupting C5a-induced signal transduction mediated by C5aR.

Methods for analyzing the functional activity of a C5aR-specific antibody may use binding assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and other methods that are known in the art (see Hampton, R. et al. (1990; Serological Methods a Laboratory Manual, APS Press, St. Paul, MN) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216)). Alternatively, assays can test the ability of an isolated antibody or antibody fragment of the present invention to elicit a biological response as a result of binding to C5aR either in vivo or in vitro. Such assays are described in the examples disclosed herein. Other suitable assays will be known to those skilled in the art.

In certain embodiments, an isolated antibody or antibody fragment of the present invention antagonizes human C5aR activity. In one embodiment, the isolated antibody or antibody fragment neutralizes human C5aR activity. In one embodiment, an isolated antibody or antibody fragment of the present invention inhibits human C5aR activity. In one embodiment, an isolated antibody or antibody fragment of the present invention inhibits human C5aR signaling. In one embodiment, said human C5aR activity or human C5aR signaling is induced by C5a. In one embodiment, said human C5aR activity or human C5aR signaling is induced through the interaction of human C5a with human C5aR. In one embodiment, said C5aR activity or C5aR signaling is induced by binding of human C5a to human C5aR. In one embodiment, said C5aR is expressed on cells. In one embodiment, said human C5aR activity or human C5aR signaling is inhibited in vitro and/or ex vivo and/or in vivo.

C5A-induced β-arrestin recruitment

The ability of the isolated human C5aR-specific antibody or antibody fragment of the present invention to inhibit C5a-induced C5aR activation was tested in a β-arrestin recruitment assay as described in Example 14 and revealed that both antibodies were even more effective antagonists of C5aR activity compared to the prior art antibody RefMAB No. 1, especially at high pathophysiological concentrations of C5a.

Therefore, in one embodiment of the present invention, an isolated antibody or antibody fragment specific for human C5aR inhibits the ability of C5a to induce C5aR activity. In one embodiment, said C5a-induced C5aR activity is determined in a β-arrestin recruitment assay as described in Example 14. In one embodiment, said C5a-induced C5aR activity is determined in vitro.

In one such embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody or antibody fragment inhibits the ability of human C5a to induce human C5aR-mediated β-arrestin recruitment. In one embodiment, said isolated antibody or antibody fragment inhibits human C5a-induced recruitment of β arrestin. In one embodiment, said isolated antibody or antibody fragment inhibits human C5aR-mediated recruitment of β-arrestin.

In a further embodiment of the present invention, an isolated antibody or antibody fragment specific for human C5aR inhibits human C5a-induced interaction of human C5aR with β-arrestin. In one such embodiment, said human C5a-induced interaction of human C5aR with β-arrestin and/or such human C5a-induced beta-arrestin recruitment are measured using beta-galactose enzyme fragment complementation. In one embodiment, said human C5a-induced human C5aR interaction with β-arrestin and/or such human C5a-induced β-arrestin recruitment are determined as described in Example 14. In one such embodiment, said human C5a-induced human C5aR interaction with β-arrestin and/or such human C5a-induced β-arrestin recruitment is tested at an IgG concentration of 50 nM.

The ability of an isolated antibody or antibody fragment according to the present invention to inhibit human C5a-induced human C5aR activity, e.g., inhibiting human C5a-induced C5aR β-arrestin interaction and/or human C5a-induced human C5aR-mediated β-recruitment, can be determined by constructing dependence curves dose response for increasing human C5a concentrations and fixed IgG concentrations and by calculating the corresponding EC50 concentrations.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody or antibody fragment increases the EC ₅₀ concentration determined for human C5a in a β-arrestin recruitment assay by at least 5-fold or more , for example, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times or at least 13 times, at an IgG concentration of 50 nM, compared to the EC50 concentration determined in the absence of the specified isolated antibody or antibody fragment.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody or antibody fragment increases the EC ₅₀ concentration determined for human C5a in a β-arrestin recruitment assay at an IgG concentration of 50 nM. about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, or about 13-fold compared to the _EC50 concentration determined in the absence of said isolated antibody or antibody fragment.

In one embodiment, the present invention provides an antibody or antibody fragment specific for human C5aR, wherein said human C5a is present at a concentration that is at least 5 times or greater, such as at least 6 times or greater, at least at least 7 times or more, at least 8 times or more, at least 9 times or more, at least 10 times or more, at least 11 times or more, at least 12 times or more, or at least 13 times or more to induce the same human C5aR activity in a β-arrestin recruitment assay at a 50 nM IgG concentration compared to a human C5a concentration in the absence of said antibody or antibody fragment.

In one embodiment, said β-arrestin recruitment assay is performed as described in Example 14. In one embodiment, said β-arrestin recruitment assay is performed in vitro.

Alternatively, the ability of an isolated C5aR-specific antibody or antibody fragment according to the present invention to inhibit C5a-induced C5aR-mediated β-arrestin recruitment can be determined by calculating the % inhibition for various concentrations of human C5a.

In one such embodiment, an isolated antibody or antibody fragment specific for C5aR in accordance with the present invention inhibits human C5a-induced β-arrestin mediated recruitment by at least 50%, by at least 55%, by at least 60%, at least 70%, at least 80%, or at least 90% at 50 nM IgG concentration and in the presence of 1.2 nM or 11 nM human C5a compared to the level of induced human C5a human C5aR-mediated β-arrestin recruitment in the presence of 1.2 nM or 11 nM human C5a and in the absence of the indicated antibody or antibody fragment.

In one such embodiment, the isolated antibody or antibody fragments specific for C5aR in accordance with the present invention inhibit human C5a-induced beta-arrestin mediated recruitment by at least 25%, for example by at least 30%, by at least 35%, at least 40%, at least 45% or at least 50% at an IgG concentration of 50 nM and in the presence of 1.2 nM or 11 nM human C5a compared to the induced level Human C5a-mediated human C5aR recruitment of β-arrestin in the presence of 100 nM human C5a and in the absence of the indicated antibody or antibody fragment.

In one embodiment, said β-arrestin recruitment assay is performed as described in Example 14. In one embodiment, said β-arrestin recruitment assay is performed in vitro.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

C5A-induced positive regulation of CD11b expression

C5a, as an effective activator of human neutrophils and monocytes, induces positive regulation of the cell surface antigen CD11b in such cells. Thus, the ability of an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention to suppress C5a-induced activation of granulocytes and/or monocytes can be assessed by determining the expression levels of CD11b in such cells.

The ability of isolated antibodies or antibody fragments according to the present invention to inhibit CD11b expression in granulocytes and/or monocytes can be determined by constructing dose response curves for increasing concentrations of IgG and a fixed concentration of human C5a and calculating the corresponding IC50 concentrations. Alternatively, the ability of isolated antibodies or antibody fragments according to the present invention to suppress CD11b expression in granulocytes and/or C5a monocytes can be determined by calculating the % suppression of CD11b expression for various IgG concentrations.

In one such embodiment, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention inhibits human C5a-induced CD11b expression in human granulocytes and/or human monocytes with an IC ₅₀ concentration of 30 nM or less, 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, or 5 nM or less, in the presence of 15 nM human C5a.

In another embodiment, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention suppresses human C5a-induced CD11b expression in human granulocytes and/or human monocytes by at least 70%, by at least 75%, by at least 80%, at least 85%, or at least 90% in the presence of 15 nM human C5a and 600 nM IgG concentration compared to the level of CD11b expression in the presence of 15 nM human C5a and in the absence of these antibody or antibody fragment.

In a further embodiment, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention inhibits human C5a-induced CD11b expression in human granulocytes with an IC50 concentration of 150 nM or less, 125 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, or 40 nM or less in the presence of 150 nM human C5a.

In one embodiment, an isolated antibody or antibody fragment specific for human C5aR according to the present invention inhibits human C5a-induced CD11b expression in human granulocytes with an IC ₅₀ concentration of 42 nM in the presence of 150 nM human C5a.

In a further embodiment, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention suppresses human C5a-induced CD11b expression in human granulocytes by at least 65%, at least 70%, at least 75%, by at least 80% or at least 90% in the presence of 150 nM human C5a and at an IgG concentration of 600 nM, compared with the level of CD11b expression in the presence of 150 nM human C5a and in the absence of the specified antibody or antibody fragment.

In one embodiment, isolated antibody or antibody fragments specific for human C5aR in accordance with the present invention suppress C5a-induced CD11b expression in human granulocytes by at least 45%, at least 50%, at least 55%, at least at least 60% or at least 65% in the presence of 150 nM human C5a and at an IgG concentration of 100 nM compared to the expression level of CD11b in the presence of 150 nM human C5a and in the absence of said antibody or antibody fragment.

In one embodiment, said determination of CD11b expression is performed as described in Example 15. In one embodiment, said determination of CD11b expression is performed in vitro and/or ex vivo.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

In addition, the inhibitory activity of the isolated antibody or antibody fragment according to the present invention against human C5a-induced CD11b expression was further analyzed over a long period of time, which means that the antibodies were pre-incubated with granulocytes and/or monocytes present in whole blood for for varying periods of time (eg 300 minutes vs. 20 minutes) before adding human C5a. The experiment demonstrated that the isolated antibodies of the present invention are even more effective at antagonistizing C5aR activity over a longer period of time, for example a longer period of incubation time, in particular compared to the prior art RefMAB#1 antibody.

Accordingly, the ability of an isolated antibody or antibody fragment according to the present invention to suppress human C5a-induced CD11b expression in granulocytes and/or monocytes over an extended period of time can be determined by constructing dose response curves for increasing concentrations of IgG and a fixed concentration of human C5a and by calculating the corresponding EC50 values after incubation of the indicated isolated antibodies with the indicated granulocytes and/or monocytes for various periods of incubation time.

As shown in Figures 6A and C, the isolated antibodies or antibody fragments of the present invention showed a clear shift in the determined dose response curve towards lower IgG concentration values determined after 300 minutes of incubation compared to the dose response curve determined after 20 minutes incubation. RefMAB#l did not show an increase in effectiveness over time (see Figures 6B and D).

Thus, in one embodiment, an isolated antibody or antibody fragment according to the present invention is more effective in inhibiting C5a-induced CD11b expression in human granulocytes and/or human monocytes after an extended period of incubation with the cells.

In one such embodiment, an isolated antibody or antibody fragment according to the present invention inhibits human C5a-induced CD11b expression in human granulocytes and/or human monocytes by at least 2-fold, at least 4-fold, at least 5-fold, or at least 6 times lower IC ₅₀ concentration determined after an extended incubation time period of 50 minutes, 100 minutes, 150 minutes, 200 minutes, 250 minutes or 300 minutes compared to the IC ₅₀ concentration determined after an incubation time period of 20 minutes.

In one embodiment, an isolated antibody or antibody fragment according to the present invention inhibits human C5a-induced CD11b expression in human granulocytes and/or human monocytes at approximately 5-fold lower IC ₅₀ concentration, determined after an extended incubation time of 300 minutes, compared to the IC ₅₀ concentration determined after an incubation time of 20 minutes.

In one embodiment, an isolated antibody or antibody fragment according to the present invention inhibits human C5a-induced CD11b expression in human granulocytes and/or human monocytes by at least 3-fold, at least 4-fold, at least 5-fold, at least at least 10 times, at least 15 times or at least 19 times the lower IC ₅₀ concentration determined after an extended incubation time of 300 minutes compared to the corresponding IC ₅₀ concentration of RefMAB#l.

In one embodiment, an isolated antibody or antibody fragment according to the present invention inhibits human C5a-induced CD11b expression in human granulocytes and/or human monocytes with an IC ₅₀ concentration of 3 nM or less, 2.5 nM or less, 2 nM or less , 1.5 nM or less, or 1 nM or less, after an extended period of incubation with the indicated cells of 300 minutes.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

C5A-induced neutrophil migration

The following assays evaluated the ability of the isolated human C5aR-specific antibody or antibody fragment of the present invention to inhibit human C5a-induced human neutrophil migration and found that MAB#1 was effective in inhibiting C5-induced neutrophil migration in vitro.

Thus, in one embodiment of the present invention, said isolated human C5aR-specific antibody or antibody fragment of the present invention inhibits human C5a-induced migration of human neutrophils in vitro.

In a further embodiment, said isolated antibody or antibody fragment inhibits human C5a-induced human neutrophil migration by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least at least 70%, at least 75%, or at least 80% compared to the level of migration in the presence of 10 nM human C5a and in the absence of said isolated antibody or antibody fragment in vitro.

In one embodiment, said migration of human neutrophils is determined after 15 minutes, after 25 minutes and/or after 35 minutes. In certain embodiments, said isolated antibody or antibody fragment in accordance with the present invention is tested at an IgG concentration of 100 nM and/or 600 nM.

In one embodiment, said isolated antibody or antibody fragment of the present invention inhibits human C5a-induced human neutrophil migration by at least 25% after 35 minutes and at an IgG concentration of 100 nM, compared to the level of migration after 35 minutes in the presence of 10 nM human C5a and in the absence of said antibody or antibody fragment.

In one embodiment, said isolated antibody or antibody fragment of the present invention inhibits human C5a-induced migration of human neutrophils by at least 60% after 25 minutes and at an IgG concentration of 100 nM and/or 600 nM, compared to the level of migration after 25 minutes in the presence of 10 nM human C5a and in the absence of the indicated antibody or antibody fragment.

In one embodiment, the isolated antibody or antibody fragment of the present invention inhibits human C5a-induced human neutrophil migration by at least 40% after 35 minutes at a 600 nM IgG concentration, compared to the level of migration after 35 minutes in the presence of 10 nM C5a human and in the absence of said antibody.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Effector function

The Fc region of an immunoglobulin generally provides favorable pharmacokinetic properties of antibodies, such as a prolonged serum half-life and the ability to induce effector function through binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors can also lead to undesirable activation of certain cell surface receptors, leading to undesirable release of cytokines and serious side effects when administered systemically.

Accordingly, for certain therapeutic situations it is desirable to reduce or eliminate the normal binding of a wild-type antibody Fc region, such as a wild-type IgG Fc region, to one, more, or all Fc receptors and/or binding to a complement component such as Clq. to reduce or eliminate the ability of an antibody to induce effector function. For example, it may be desirable to reduce or eliminate binding of the Fc region of an antibody to one, more, or all Fcγ receptors, such as FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa.

The effector function may include, without limitation, one or more of the following: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, NK cell binding, binding macrophages, monocyte binding, polymorphonuclear cell binding, direct apoptosis-inducing signal transduction, target-bound antibody cross-linking, dendritic cell maturation or T cell priming.

Reduced or eliminated Fc region binding to the Fc receptor and/or Clq is typically achieved by mutation of a wild-type Fc region, such as the IgG1 Fc region, more specifically the human IgG1 Fc region, resulting in a variant Fc region or an engineered Fc region of said wild-type Fc region, for example a human IgG1 Fc region variant. Substitutions that result in reduced binding may be beneficial. To reduce or eliminate the binding properties of the Fc region to the Fc receptor, non-conservative amino acid substitutions are preferred, i.e. replacement of one amino acid with another amino acid characterized by different structural and/or chemical properties.

Accordingly, in one embodiment, an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention comprises a variant Fc region characterized by reduced or eliminated binding to the Fc receptor and/or Clq compared to the wild-type Fc region. type. In one such embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains an Fc region variant that reduces or eliminates the ability of the antibody to induce effector function. In a further embodiment, an isolated antibody or antibody fragment in accordance with the present invention substantially does not induce effector function.

In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present invention substantially does not induce ADCC and/or CDC and/or ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present invention does not induce ADCC or ADCP in vitro.

In one embodiment, the Fc region variant of an isolated antibody or antibody fragment in accordance with the present invention contains one or more amino acid substitutions that reduce or eliminate the binding of the Fc region variant to one or more Fc receptors and/or C1q compared to Fc -wild type region. In one embodiment, a variant Fc region of an isolated antibody or antibody fragment in accordance with the present invention contains one or more amino acid substitutions that reduce or eliminate the ability of the antibody to induce effector function compared to the wild-type Fc region.

In a specific embodiment, one or more amino acid substitutions may reduce the binding affinity of the Fc region variant for one or more Fc receptors and/or C1q by at least 2-fold, at least 5-fold, at least 10-fold, at least at least 20-fold or even at least 50-fold compared to the wild type Fc region. In alternative embodiments, one or more amino acid substitutions may reduce the ability of an isolated antibody or antibody fragment according to the present invention to induce effector function by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or even at least 50-fold compared to the wild-type Fc region.

In one embodiment, the Fc region variant of an isolated antibody or antibody fragment in accordance with the present invention substantially does not bind to one or more Fc receptors and/or C1q. In one embodiment, the Fc region variant of an antibody of the present invention substantially eliminates the ability of said antibody to induce effector function. In one embodiment, an antibody or antibody fragment of the present invention induces substantially no effector function. In one embodiment, said effector function is ADCC and/or ADCP and/or CDC. In one embodiment, an antibody or antibody fragment of the present invention substantially does not induce effector function, which means that the level of effector function induced is not significantly above background measured in the absence of the antibody.

In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is a Psureceptor. In one embodiment, the Fc receptor is human FcγRIIIa, FcγRI, FcγRIIa and/or FcγRIIb.

In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC and ADCP. In a specific embodiment, the effector function is ADCC, CDC and ADCP. In a more specific embodiment, the effector function is ADCC and ADCP.

In one embodiment, the wild-type Fc region is an IgG1 Fc region. In one embodiment, the wild-type Fc region is a human IgG1 Fc region. In one embodiment, the wild-type Fc region is a human IgG1 Fc region. In one embodiment, the wild-type Fc region is a human IgG1 Fc region comprising the amino acid sequence

PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention comprises a variant human IgG1 Fc region that contains one or more amino acid substitutions relative to the wild-type human IgG1 Fc region. In one embodiment, one or more amino acid substitutions reduce or eliminate the binding of the variant Fc region to the Fc receptor and/or C1q, and/or reduce the ability of the antibody to induce effector function compared to the wild-type Fc region.

In one embodiment, a variant Fc region of a human IgG1 antibody or antibody fragment in accordance with the present invention contains an amino acid substitution at one or more positions selected from the group consisting of 234, 235, 237, 330 and 331, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence SEQIDNO: 11.

In one embodiment, said variant human IgG1 Fc region contains an amino acid substitution at one or more positions selected from the group consisting of L234, L235 and G237, numbered according to the EU index, compared to a wild-type IgG1 Fc region containing an amino acid sequence under SEQ ID NO: 11.

In one embodiment, the variant human IgG1 Fc region contains amino acid substitutions at one or more positions selected from the group consisting of L234A, L235E, G237A, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence under SEQIDNO: 11.

In one embodiment, the variant human IgG1 Fc region contains amino acid substitutions L234A and L235E, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the variant human IgG1 Fc region contains amino acid substitutions L234A, L235E and G237A, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

In one embodiment, a variant human IgG1 Fc region contains an amino acid substitution at one or more positions selected from the group consisting of AD3O and P331, numbered according to the EU index, compared to a wild-type IgG1 Fc region comprising the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the variant human IgG1 Fc region contains amino acid substitutions at one or more positions selected from the group consisting of A330S and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the variant human IgG1 Fc region contains the amino acid substitutions A330S and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the variant human IgG1 Fc region contains amino acid substitutions L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

In one embodiment, an antibody or antibody fragment in accordance with the present invention comprises a variant human IgG1 Fc region that contains one or more amino acid substitutions relative to a wild-type human IgG1 Fc region comprising the sequence of SEQ ID NO: 11 that reduce or eliminate the binding affinity of the Fc region variant to one or more Fc receptors and/or C1q, and/or reduce the ability of said antibody to induce effector function, where one or more amino acid substitutions are L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

In one embodiment, the isolated antibody or antibody fragment of the present invention is of the human IgG1 class. In one embodiment, the isolated antibody or antibody fragment of the present invention is a human IgG1 variant. In one embodiment, an isolated antibody or antibody fragment according to the present invention substantially does not induce effector function in vitro. In one embodiment, the human IgG1 Fc region variant does not bind substantially to one or more Fc receptors and/or C1q. In one such embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains an amino acid substitution selected from the group consisting of L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, compared to the Fc region of wild-type IgG1 , containing the amino acid sequence under SEQIDNO: 11.

In one embodiment, the isolated antibody or antibody fragment in accordance with the present invention is of the human IgG1 class, containing the amino acid substitutions L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains amino acid substitutions L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains the amino acid substitution L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, compared to the Fc region of wild-type IgG1 containing the amino acid sequence of SEQ ID NO : eleven.

In one such embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains the amino acid substitutions L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, and substantially does not induce effector function in vitro. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC and ADCP.

In one such embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains the amino acid substitution L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, compared to the wild-type human IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11, and practically does not bind to one or more Fc receptors and/or C1q in vitro.

In one embodiment, the isolated antibody or antibody fragment of the present invention is of the human IgG1 class. In one embodiment, the isolated antibody or antibody fragment of the present invention substantially does not induce effector function in vitro. In one embodiment, an isolated antibody or antibody fragment in accordance with the present invention contains one or more amino acid substitutions selected from the group consisting of L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

The binding of an antibody to Fc receptors by its Fc region can be easily determined, for example by ELISA or surface plasmon resonance (SPR) using standard measurement equipment such as the Biacore® device (GE Healthcare), and Fc receptors can be obtained through recombinant expression. Alternatively, the binding affinity of Fc regions can be assessed using cell lines known to express specific Fc receptors, such as NK cells expressing the FcγIIIa receptor. The effector function of an antibody can be measured by methods known in the art. Suitable in vitro assays for assessing the ADCC activity of a molecule of interest are described, for example, in WO2012130831. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). A CDC assay can be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). C1q binding assays (such as ELISA) can be performed to determine whether an antibody is capable of binding C1q and therefore has CDC activity (see, for example, WO 2006/029879). In vitro methods for assessing Fc receptor binding or for assessing immune effector function are described herein in Examples 10-13.

Fusion proteins

An isolated antibody or antibody fragment in accordance with the present invention may or may not be fused to one or more other amino acid residues, polypeptides or fragments. Such a fusion protein can be produced by any suitable route, including genetic or chemical approaches. These linked fragments may contain secretory or leader sequences, sequences that facilitate detection, expression, separation or purification, or sequences that impart increased stability to the protein, for example during recombinant production. Non-limiting examples of potential fragments include beta-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragment, secretion signal peptide, antibody or antibody fragment, toxin, reporter enzyme, fragment capable of binding a metal ion such as a polyhistidine tag, tag, suitable for detection and/or purification, a homo- or heteroassociation domain, a fragment that increases protein solubility, or a fragment that contains an enzymatic cleavage site.

Accordingly, an isolated antibody or antibody fragment in accordance with the present invention may optionally contain one or more fragments for binding to other targets or target proteins of interest. It should be appreciated that such additional fragments may or may not provide additional functionality to the antibody and may modify or not to modify the properties of the isolated antibody or antibody fragment in accordance with the present invention. Diagnostic Application

In one embodiment, the present invention provides for the use of an isolated antibody or antibody fragment specific for human C5aR in accordance with the present invention for the diagnosis of a disease. In one embodiment, the present invention provides the use of an antibody or antibody fragment in accordance with the present invention to detect C5aR, in particular human C5aR and/or cynomolgus C5aR. In one embodiment, the present invention provides a method for detecting C5aR in a subject or sample, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment specific for human C5aR of the present invention. In one embodiment, the present invention provides a method for diagnosing a disease in a subject, comprising the step of contacting said subject or sample with an isolated antibody or antibody fragment in accordance with the present invention.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Therapeutic methods

The isolated antibody or antibody fragment in accordance with the present invention can be used in therapeutic methods. An antibody or antibody fragment according to the present invention can be used to treat a disease such as cancer, an autoimmune disease, or an inflammatory disease.

In one embodiment, the present invention provides a method for treating a disease.

In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for treating a disease. In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for use in treating a disease. In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for use in treating a disease in a subject in need thereof.

In one embodiment, the present invention provides for the use of an isolated antibody or antibody fragment in accordance with the present invention for the manufacture of a medicinal product. In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for use as a drug. In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for use in medicine. In one embodiment, the present invention provides an isolated antibody or antibody fragment in accordance with the present invention for use as a drug for treating a subject in need thereof.

In one embodiment, the disease is associated with the undesirable presence of C5aR, in particular human C5aR. In one embodiment, the disease is associated with the undesirable presence of C5a, in particular human C5a.

In one embodiment, the disease being treated is a proliferative disease. In a specific embodiment, the disease is cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, colorectal cancer, stomach cancer, prostate cancer, blood cancer, sarcoma, skin cancer, squamous cell carcinoma, bone cancer, melanoma, renal cell carcinoma and kidney cancer.

In one embodiment, the disease being treated is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory disease include rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary heart disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo , graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA-associated vasculitis, uveitis, scleroderma, bullous pemphigoid, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's chorea, cystic fibrosis, gout, age-related macular degeneration, allergies, asthma, antiphospholipid syndrome (APS), atherosclerosis, C3 glomerulopathy and IgA nephropathy, ischemia/reperfusion injury, peritonitis, sepsis and other autoimmune diseases that result from acute or chronic inflammation.

In one embodiment, the present invention provides an isolated antibody or antibody fragment specific for human C5aR, in accordance with the present invention, for use in a method of treating a subject having a disease, comprising administering to the subject a therapeutically effective amount of the antibody or antibody fragment in accordance with the present invention.

In one embodiment, the method further includes administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more specifically a human. For use in therapeutic methods, the isolated antibody or antibody fragment in accordance with the present invention can be formulated, dosed and administered in accordance with good medical practice.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) HCDR1 region under SEQ ID NO: 30, HCDR2 region under SEQ ID NO: 41, HCDR3 region under SEQ ID NO: 40, LCDR1 region under SEQ ID NO: 32, LCDR2 region under SEQ ID NO: 33 and LCDR3 under SEQ ID NO: 33 ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 37 or 44 and with the LC of SEQ ID NO : 38 or 45.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Pharmaceutical compositions

In one embodiment, the present invention provides a pharmaceutical composition comprising an isolated antibody or antibody fragment in accordance with the present invention and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical compositions may further contain at least one other pharmaceutically active compound. The pharmaceutical composition in accordance with the present invention can be used in the diagnosis, prevention and/or treatment of diseases associated with the undesirable presence of C5aR, in particular human C5aR. In particular, the present invention provides pharmaceutical compositions containing an antibody or antibody fragment according to the present invention that are suitable for prophylactic, therapeutic and/or diagnostic use in a mammal, more particularly in a human.

In general, an antibody or antibody fragment according to the present invention may be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present invention and at least one pharmaceutically acceptable a carrier or excipient and optionally one or more additional pharmaceutically active compounds. Such a formulation may be suitable for oral, parenteral, topical or inhalation administration. Accordingly, a pharmaceutical composition containing at least one antibody or antibody fragment according to the present invention may be administered parenterally, for example intravenously, or intramuscularly, or subcutaneously. Alternatively, the antibody of the present invention may be administered by a route other than parenteral, such as orally or topically. In a preferred embodiment, a pharmaceutical composition containing an antibody or antibody fragment according to the present invention is administered intravenously or subcutaneously.

In particular, an antibody or antibody fragment in accordance with the present invention can be used in combination with one or more pharmaceutically active compounds that are or can be used for the prevention and/or treatment of diseases in which the target antigen of interest is involved, resulting in what a synergistic effect may or may not be obtained. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

In one embodiment, the present invention provides a pharmaceutical composition containing an antibody or antibody fragment in accordance with the present invention, for use in the prevention and/or treatment of a disease associated with the undesirable presence of C5aR. In one embodiment, the present invention provides a pharmaceutical composition containing an antibody or antibody fragment according to the present invention for use as a drug. In one embodiment, the present invention provides a pharmaceutical composition containing an antibody or antibody fragment in accordance with the present invention, for use in the prevention and/or treatment of an autoimmune disease and/or an inflammatory disease and/or cancer.

In one embodiment, the present invention provides a method of treating an autoimmune disease and/or an inflammatory disease and/or a cancer in a subject in need thereof using a pharmaceutical composition containing an antibody or antibody fragment in accordance with the present invention.

Further provided is a method of producing an antibody or antibody fragment in accordance with the present invention in a form suitable for administration in vivo, the method comprising (a) producing the antibody or antibody fragment by a method in accordance with the present invention and (b) formulating said antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, wherein the antibody or antibody fragment is formulated for in vivo administration. Pharmaceutical compositions in accordance with the present invention contain a therapeutically effective amount of one or more antibodies or antibody fragments in accordance with the present invention, dissolved in a pharmaceutically acceptable carrier or excipient.

In one embodiment, said isolated human C5aR-specific antibody or antibody fragment in accordance with the present invention comprises

a) the HCDR1 region under SEQ ID NO: 27, the HCDR2 region under SEQ ID NO: 28, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

b) the HCDR1 region under SEQ ID NO: 30, the HCDR2 region under SEQ ID NO: 31, the HCDR3 region under SEQ ID NO: 29, the LCDR1 region under SEQ ID NO: 32, the LCDR2 region under SEQ ID NO: 33 and the LCDR3 region under SEQ ID NO: 34, or

c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or

d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) VH under SEQ ID NO: 35 and VL under SEQ ID NO: 36, or

b) VH under SEQ ID NO: 42 and VL under SEQ ID NO: 43, or

VH and VL that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and with VL of SEQ ID NO: : 36 or 43.

In one embodiment, said isolated antibody or antibody fragment specific for human C5aR comprises

a) HC under SEQ ID NO: 37 and LC under SEQ ID NO: 38, or

b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45, or

HC and LC that have at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 37 or 44 and with VL of SEQ ID NO: : 38 or 45. In one embodiment, said isolated antibody or antibody fragment specific for human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, said isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

Antibody sequences

Demo examples

Example 1: Antigen Generation and Quality Control

Amino acid sequences of C5aR and C5aR-related GPCRs from various species were obtained from publicly available sources (e.g., Uniprot), verified, and obtained in-house or purchased from third parties.

Synthetic peptides

Linear peptides covering the N-terminal extracellular region of human C5aR were used as antigens for initial panning and screening. The peptides were chemically synthesized with a biotin tag (JPT), purified by RP-HPLC, and delivered as lyophilized material. Lyophilized peptides were stored at -80°C. Alternatively, the peptides were conjugated to transferrin or bovine serum albumin (BSA).

Linear peptides containing the N-terminal region of human and cynomolgus macaque C5aR were used as antigens for subsequent binding studies. Peptides were chemically synthesized with a biotin tag (Genscript), purified by RP-HPLC, and delivered as lyophilized material. Lyophilized peptides were stored at -80°C. For recovery, the peptides were dissolved in the required volume of PBS and stored at -80°C.

Recombinant proteins

Protein C1q

C1q protein purified from pooled normal human plasma was purchased from Complement Technology, Inc. (cat. no. A099).

Human protein C5a

Recombinant human C5a was either purchased from R&D Systems (cat. no. 2037-C5) or prepared locally.

For in situ production, DNA encoding human C5a amino acids (Uniprot: P01031 | Lys679 - Arg751) was cloned into the expression vector pET21a (Novagen) in frame with the N-terminal signal sequence ompA followed by the sequence encoding maltose binding protein (MBP) , FXa cleavage site and GS linker.

Human C5a (hC5a) was expressed in E. coli BL21 (DE3) cells (Novagen) as a maltose binding protein (MBP) fusion protein with an N-terminal tag. Protein expression was induced by the addition of IPTG and the cultures were further cultured for 20-23 hours. Cells were collected by centrifugation and the pellet was resuspended in lysis buffer (PBS buffer plus 2 mM MgCl ₂ , 20 U/ml benzonase (Roche) and 1 tablet/50 ml cOmplete, non-EDTA protease inhibitor cocktail tablets (Roche)). Cells were destroyed either by chemical lysis or by high pressure homogenization. The resulting suspension was centrifuged and the supernatant was subjected to sterilizing filtration for further purification steps.

The hC5a-MBP fusion protein was purified by dextrin-Sepharose affinity chromatography using an MBP-Trap column (GE-Healthcare) and optionally fine-purified by cation exchange chromatography using a Hi-Trap SP FF column (GE LifeSciences). Purified MBP fusion products were buffer exchanged using PD10 columns (GE Healthcare) with FXa digestion buffer (20 mM Tris/HCl, pH 8.0; 100 mM NaCl; 2 mM CaCl ₂ ). The hC5a protein was released from maltose binding protein by adding factor Xa (1:100 (w/w)) and incubating overnight in a rotary shaker at room temperature. Released hC5a was purified by cation exchange chromatography using a Hi-Trap SP FF column (GE LifeSciences). All affinity chromatography steps were performed using a preparative chromatography system Avant 25.

Buffer exchange to PBS was performed using PD 10 columns (GE Healthcare). Samples were subjected to sterilization filtration and hC5a concentrations were determined using UV spectrophotometry. The purity and integrity of the samples were analyzed by SDS-PAGE under denaturing, reducing or non-reducing conditions, SEC-HPLC and mass spectrometry.

Fc gamma receptors (FcγR) and FcRn receptors

DNA encoding the extracellular region of human FcγRI, human FcγRIIa (131H), human FcγRIIa (131R), human FcγRIIb, human FcγRIIIa (158F) and human FcγRIIIa (158V) was cloned in frame with the N-terminal Vκ leader sequence and the C-terminal 6x His tag into the pMAX expression vector, which is a modified pcDNA3.1-based expression vector (Thermo Fisher).

DNA encoding the extracellular region of the p51 large subunit of human, cynomolgus, mouse or rat FcRn was cloned in frame with an N-terminal Vκ leader sequence and a C-terminal AVI-6xHis tag into the expression vector pMAX, which is a modified expression vector on based on pcDNA3.1 (Thermo Fisher). In addition, DNA encoding the human, cynomolgus, mouse or rat small subunit p14 FcRn protein (=identical to beta-2 microglobulin) was cloned into a second open reading frame in frame with the N-terminal Vκ leader sequence. The amino acid sequences of the resulting receptors are briefly described in Tables 6 and 7.

The HEK293-6E cell line was developed by the National Research Council of Canada (NRC). Cells were maintained in Freestyle F17 medium (Thermo Scientific) in a humidified CO2 incubator at 37°C and 6% _CO2 . HKB11 (parent clone: US Patent No. 6136599, J. Biomed. Sci. 2002; 9:631-638) is a human hybrid cell line derived from the fusion of HEK293 human embryonic kidney cells and 2B8 Burkitt lymphoma cells. HKB11 #52 cells were maintained in MAC1.0 medium containing 1% FCS in a humidified CO ₂ incubator at 37°C and 6% CO ₂ .

HKB11 #52 or HEK293-6E cells were transiently transfected one day after plating using a commercially available transfection reagent according to the manufacturer's instructions. The cells were cultured for 3 days and the conditioned cell culture supernatant was collected by centrifugation followed by sterilizing filtration (0.22 μm). Stably transfected pools of HKB11 #52 were generated by transfecting cells followed by selection with 800 μg/ml G418 (Thermo Scientific). Expression of antigens in stable pools was carried out within 4 days after seeding. Conditioned cell culture supernatants were collected by centrifugation followed by sterilizing filtration (0.22 μm).

The corresponding proteins were purified by IMAC using Protino Ni-NTA columns (Macherey-Nagel). All chromatography steps were performed using chromatography systems (GE Healthcare). Samples were buffer exchanged to D-PBS using PD10 columns (GE Healthcare). In some cases, the fine purification step of preparative SEC was performed in D-PBS using a Superdex 200 column (GE Healthcare).

Biotinylation of FcRn heterodimers was performed by in vitro biotinylation using the BirA kit (Avidity) followed by preparative SEC using a Superdex 200 column (GE Healthcare).

Sample quality was analyzed by SDS-PAGE under denaturing, reducing or non-reducing conditions, streptavidin shift assay, HP-SEC and DLS.

Virus-like particles (VLP)

VLPs stably expressing one of two natural human C5aR variants (D/K variant or N/N variant) as well as mouse C5aR were generated in situ as described in WO 2015/193143. All cloning experiments were performed using standard techniques. Antigens of interest were cloned into the appropriate two-vector system for expression in mammalian cells. In this system, one vector expresses GAG and the other vector expresses a GPCR-GAG fusion protein. Expression of these vectors is under the control of the CMV promoter. Host cells were then transfected with the two vectors. The generated constructs were produced as fusion proteins in which the antigen of interest was fused at the N-terminus to a GAG protein (HV1B1 (Uni-Prot ID: P03347)). Protein expression and VLP production were carried out under standard conditions in suspension cultures. The host cells used in these experiments were HKB11 cells (ATCC; CRL-12568) and HEK cells (Life Technologies). Three days after transfection, VLP-containing supernatants were collected and purified using standard procedures (including precipitation and ion exchange chromatography). The isolated proteins were subjected to SDS-PAGE chromatography. The supernatants were analyzed with a commercial anti-GAG antibody or a commercial anti-C5aR antibody and found that co-expression of GAG and a GPCR-GAG fusion protein resulted in high levels of GAG and GPCR-GAG expression in the VLPs. This confirmed that C5aR antigens were efficiently incorporated into VLPs and that C5aR antigens were detectable by antibodies. The amino acid sequences of the resulting fusion proteins are summarized in Table 8.

Cell lines

CHO Flp-In cells were generated stably expressing full-length human C5aR, cynomolgus C5aR, mouse C5aR, rat C5aR, and C5aR-related GPCRs, human C5L2, human C3aR, human FPR1, and human ChemR23. To create CHO Flp-In cells, various vector constructs were synthesized by in situ genetic engineering and cell transfection was performed according to the methodological developments (Thermofischer/Invitrogen). All constructs contained an N-terminal V5/His tag. Commercially available secondary antibodies - anti-His (e.g., Dianova, catalog no. DIA-910) or anti-V5 (e.g., AbD Serotec, catalog no. MCA2285GA) were used to confirm expression of the corresponding GPCR on the surface of the cell line, even in the absence of a commercially available specific working antibody to GPCR.

Example 2: Generation of Human C5aR-Specific Antibodies from the HuCAL PLATINUM ^® Library

For antibody generation, the HuCAL Platinum ^® library was used to select antibodies with specificity for human C5aR. The HuCAL PLATINUM ^® library is a phagemid library based on the HuCAL concept (Knappik et al., (2000) J Mol Biol 296:57-86) and uses CysDisplay ^® technology to display Fabs on the surface of phages (Lohning et al. , WO 2001/05950).

To identify antibodies specific for human C5aR, panning strategies were implemented using human and cynomolgus C5aR antigenic material to select for antibodies characterized by cross-species cross-reactivity. Each panning operation performed involved at least 3 separate rounds of selection for different C5aR antigens (either soluble recombinant antigens or antigens overexpressed on cells).

Although the overall homology of 90% between cynomolgus and human C5aRs is quite high, the extracellular domains share only 75% protein sequence identity. Indeed, identifying antibodies that are cross-reactive against cynomolgus C5aR has proven challenging because the precursor antibodies MAB#1 and MAB#2 were among the few candidates demonstrating cell-specific binding to human C5aR expressed on cells. cross-reactivity with cynomolgus C5aR and lack of binding to other related GPCRs.

Panning operations in a bead-based solution were carried out on peptides representing the N-terminus (NT) of human C5aR, which led to the identification of precursor antibodies MAB#1 and MAB#2, which have specificity for human and cynomolgus C5aR and are capable of binding to full-length human C5aR expressed on cells.

This clone was subjected to two sequential affinity maturation panning operations using CHO Flp-In cells engineered to overexpress either human C5aR or cynomolgus C5aR to further increase affinity and specificity for human and cynomolgus C5aR. In addition, antibodies have been engineered to further enhance specificity, remove potential post-translational modification sites (PTM motifs), and for germline targeting purposes.

At this last stage of the design process, MAB#1 and MAB#2 were identified as potential therapeutic candidates. Because both candidates MAB#1 and MAB#2 are derived from the same precursors, they have similar amino acid sequences and in vitro characteristics.

These two antibodies are further described in the examples below.

Example 3. Preparation of antibodies specific for human C5aR

Both MAB#1 and MAB#2 are of the human IgG1f isotype, but their Fc region is engineered to eliminate the ability of antibodies to mediate immune effector function. The Fc region contains 5 amino acid substitutions compared to the wild-type human IgG1 Fc region, namely L234A, L235E, G237A, A330S and P331S (h_IgG1f_AEASS), numbered according to the EU index.

The antibodies consist of a VH3-15 heavy chain backbone and a lambda 1 light chain backbone.

Transient Antibody Production - HKB11 Enables Improved Microscale Production

Eukaryotic HKB11#52 cells were transiently transfected with mammalian expression vectors encoding the heavy and light chains of MAB#1 or MAB#2 (human IgG1_AEA88), respectively. Cell culture supernatants were collected 7 days after transfection and subjected to protein A affinity chromatography (MabSelect SuRe | GE Healthcare) using a liquid handling unit. Samples were left in neutralized elution buffer (NaPS: 137 mM sodium phosphate, 81 mM NaCl, pH 7). Samples were subjected to sterilizing filtration (pore size 0.2 μm). Protein concentrations were determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denaturing, reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer). UHP-SEC was performed to analyze IgG preparations in the native state.

Obtaining antibodies in a transient mode - obtaining on a research scale in SHO

CHO3-E7 cells were transiently transfected with a mammalian expression vector encoding the heavy and light chains of MAB#1 or MAB#2 (human IgG1_AEASS), respectively. Cell culture supernatants were collected 6 days posttransfection and subjected to standard protein A affinity chromatography (MabSelect SuRe | GE Healthcare). Unless otherwise stated, buffer exchange was performed with 1x Dulbecco's PBS (pH 7.2 | Invitrogen) and samples were subjected to sterilization filtration (pore size 0.2 μm). Protein concentrations were determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denaturing, reducing, and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer). UHP-SEC was performed to analyze IgG preparations in the native state.

Transient mode acquisition results

Product quality data (SEC monomer content) and productivity for MAB#1 and MAB#2 are summarized in Table 9. Overall, acceptable monomer content (>95%) and yield (>55 mg/L in HKB11 cells). The volumetric yields obtained from transient production in CHO3E-7 at research scale were also in the expected range.

Creating stable HKB11 pools

A two-vector co-transfection system was used to generate pools of HKB11#52 stably expressing MAB#1 or MAB#2.

To facilitate pool selection, these vectors contain zeocin and neomycin resistance cassettes. The two vectors were transiently co-transfected into actively dividing HKB11#52 cells in a 1:1 ratio. Selection one day after transfection was started by adding 160 μg/ml zeocin and 800 μg/ml geneticin to the cell suspension. During selection, cell number and viability initially decreased. 20 days after transfection, the cells began to recover. After reaching ~80% viability, the size of the stable pools was increased to the desired volume depending on the quantity needed. Cell culture supernatants, when prepared in a batchwise manner, were collected on day 6 after seeding. Large-scale purification of MAVNo. 1 and MAV No. 2

Purification of MAB#1 and MAB#2 from HKB11#52 stable pool cell culture supernatants using protein A affinity chromatography (MabSelect SURE | GE Healthcare) was performed using 100 mM citrate, 150 mM NaCl, pH 3.5, in quality of the elution buffer. After incubation at pH 3.5 for 60 minutes, samples were neutralized. Buffer exchange was performed with 150 mM histidine, pH 6.0, and the samples were subjected to sterilization filtration (pore size 0.2 μm). Protein concentrations were determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denaturing, reducing, and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer). UHP-SEC was performed to analyze IgG preparations in the native state.

Large scale acquisition results

As summarized in Table 10, the preparation of MAB#1 and MAB#2 provided favorable yields, purity and integrity.

Control antibodies

Various control antibodies were prepared and included in experiments for comparison purposes.

RefMAB№1 - reference antibody

Nucleotide sequences encoding the VH and VL regions from the human C5aR-specific antibody "IPH5401" were taken from US patent application US 2013/0295116 (NOVO NORDISK - clone 32F3A6GL). Nucleotide sequences were genetically synthesized as linear DNA fragments with appropriate flanking regions (eg, suitable restriction enzyme recognition sites, linker sequences) either in-house or from an external supplier. The DNA fragments were cloned into suitable mammalian IgG expression vectors encoding the human IgG1 AEASS heavy and light chains as described above using standard molecular biology techniques. RefMAB#1 was obtained in transient mode as described above. The amino acid sequences of the heavy and light chains of RefMAB#1 are presented in Table 3.

Additional control antibodies

An in-house negative isotype control antibody with specificity for hen's egg lysozyme (MOR03207) as well as an in-house positive control antibody (anti-C5aR) with specificity for the N-terminus of human C5aR were generated transiently as described above, in format either human IgG1 or human IgG1_AEASS.

Determination of binding properties of MAB#1 and MAB#2

Example 4: Determination of the monovalent binding affinity of MAB#1 and MAB#2 for N-terminal C5aR peptides using SPR

Way

Determination of K _D using the IgG capture model was performed at 25°C on a Biacore T200 device (Biacore, GE Healthcare). Approximately 500 relative units of IgG diluted with HBS-EP+, pH 7.4, were captured on a CM5 chip (Biacore, GE Healthcare) onto which anti-human Fc antibody (GE Healthcare) was immobilized using standard EDC-NHS amine coupling chemistry . Reference flow cell 1 was only activated and deactivated. Kinetic measurements were performed using 6 different concentrations of human or cynomolgus_NT-bio C5aR peptide (SEQ ID NO: 13 or SEQ ID NO: 14, respectively) (2-fold serial dilution, 2000 to 62.5 nM) using HBS -EP+ (GE Healthcare) as mobile phase buffer (injection time 300 s; dissociation time 600 s; flow rate 30 μl/min). After each cycle, the sensor chip was regenerated to remove bound peptide/antibody complex using 3×20 second injections of 3 mM MgCl ₂ . For double comparison with the standard, a blank injection of mobile phase buffer was used. All sensorgrams were fitted using Biacore T200 Evaluation 3.1 software (Biacore, GE Healthcare) to determine the rate constants k _on and k _off , which were used to calculate K _D . The raw data were fitted using a 1:1 binding model, with R _max parameters set locally and RI set to 0.

results

The results are summarized in Table 11. Both antibodies showed similar binding to the human C5aR peptide, with K _D values in the lower end of the double-digit nanomolar range and binding to the cynomolgus C5aR peptide being weaker.

Example 5: Determination of Apparent Affinity (Bivalent) of MAB#1 and MAB#2 for N-Terminal C5aR Peptides Using Octet

Way

Determination of apparent _KD using the C5aR_NT-bio peptide capture model was performed at 27°C on an Octet HTX device ( , Pall Life Sciences). Approximately 0.03 nM of peptide, diluted in PBS, pH 7.4, was loaded onto the sensors ( , Pall Life Sciences) with streptavidin (SA). Kinetic parameter measurements were performed using 7 different concentrations of IgG (3-fold serial dilution, from 200 to 0.27 nM for human C5aR peptide_NT-bio (SEQ ID NO: 13) and from 1000 to 1.4 nM for macaque C5aR peptide crabeater_NT-bio (SEQ ID NO: 14) in Octet buffer (PBS, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA) with an association time of 480 s and time dissociation 900 s. After each dissociation step, the sensors were regenerated to remove bound antibody (3x30 with Gly/HCl, pH 1.5). All sensorgrams were fitted using Octet 10.0 data analysis software (). ) to determine the apparent rate constant k _on and the approximated rate constant k _off , which were used to calculate the apparent K _D (using a 1:1 binding model).

results

The results are summarized in Table 12. Both antibodies exhibited high levels of binding to human C5aR peptide in bivalent format with apparent K _D values in the two- to three-digit picomolar range. Binding to the cynomolgus monkey C5aR peptide was approximately 1000-fold weaker. The observed weaker binding to the cynomolgus monkey C5aR peptide arose primarily due to the high rate constants k _off . In terms of apparent affinity values for human C5aR_NT, MAB#2 exhibited approximately 10-fold weaker binding affinity compared to MAB#1 ( _KD : 290 pM vs. 20 pM).

Example 6 Determination of Apparent Affinity (Bivalent) of MAB#1 for Full Length C5aR Expressed on Cells Using KinExA

Because C5aR belongs to the GPCR family, it is very difficult to create a recombinant full-length antigen material that can be used for SPR measurements. Therefore, measurements were performed using CHO Flp-m cells stably expressing human C5aR or cynomolgus C5aR and KinExA.

Way

Determination of apparent _KD on C5aR-expressing cells was performed at room temperature. using a KinExA 3200 device (Sapidyne Instruments). PBS (Gibco) supplemented with BSA (1 mg/ml) and 0.02% (v/v) NaN ₃ was used as the assay buffer. MAB#1 (concentration: 2 nM and 30 pM) was used as the analyte, and Flip-In CHO hC5aR_V5/His cells (final concentration: 3 million cells/ml and 1 million cells/ml, 2-fold serial dilution) or cells Flip-In CHO cyC5aR_V5/His (final concentration: 25 million cells/ml and 6 million cells/ml, 2-fold serial dilution) as titrant and equilibrated overnight at RT. in a top drive shaker. After equilibration, samples were centrifuged and supernatants were used for analysis. The free concentration of MAB#1 was determined using polymethyl methacrylate (PMMA) beads coated with MabSSL (GE Healthcare), and anti-human Fab2 antibody Alexa Fluor 647 (500 ng/ml) was used for detection. Apparent _KD was obtained using the KinExA software and using “n-curve analysis”, which allows all given curves to be fitted simultaneously to a single _KD value.

results

The results are summarized in Table 13. MAB#1 exhibited high levels of binding to full-length human C5aR with an apparent K _D value of 130 pM. Binding to full-length cynomolgus C5aR was approximately 20-fold weaker, with an apparent K _D value of approximately 3 nM. Similar results regarding binding to cynomolgus C5aR were previously observed in Octet and Biacore measurements (see Example 4 and Example 5).

Example 7. Binding of MAB#1 and MAB#2 to full-length C5aR expressed on CHO Flp-In cells and whole blood-derived neutrophils (FACS analysis)

Cellular binding to CHO Flp-In cell lines stably expressing various full-length C5aR antigens and related GPCRs, as well as binding to purified human or cynomolgus neutrophils endogenously expressing human or cynomolgus C5aR, were examined by FACS. Crab-eating macaque neutrophils were obtained from cynomolgus macaque whole blood from three different animals (LPT Hamburg).

Methods

C5aR-CHO Flp-In cell lines were blocked and IgG was added either in serial dilutions or at a single (high) concentration of 300 nM. To detect IgG binding, R-phycoerythrin (R-PE)-conjugated human IgG antibody was added and fluorescence was measured using a FACS array device or a Novocyte device.

Neutrophils were purified from EDTA whole blood samples using MACSexpress Neutrophil Isolation Mixture from Miltenyi Biotec. Briefly, using this kit, cells were isolated using magnetic labeling and negative magnetic separation. After removal of red blood cells, cells were stained by adding serial dilutions of IgG followed by the addition of Alexa Fluor 647-conjugated detection antibodies specific for the human P(ab)2 fragment. Measurements were performed on a FACS array device. FACS data were assessed using FlowJo, entered into GraphPad Prism (v4.0) and fitted to a sigmoidal dose response curve using nonlinear regression to calculate EC50.

results

The results are summarized in Table 14 and Figures 1 and 2. Figure 1A and C depicts the binding of MAB#1, MAB#2 and RefMAB#1 to human and cynomolgus C5aR present on engineered CHO cells expressing the corresponding full-length receptor. Overall, highly comparable human C5aR binding curves with nearly identical EC ₅₀ concentrations were determined for MAB#1, MAB#2 and RefMAB#1. Similar results were obtained with cynomolgus monkey C5aR for MAB#1 and MAB#2. As expected, RefMAB11 did not exhibit binding to C5aR C5aR expressed on CHO cells.

Binding to cynomolgus and human C5aR was also confirmed using purified neutrophils derived from human or cynomolgus whole blood (Figure 1B and D). Again, highly comparable binding curves with nearly identical EC ₅₀ values on human and cynomolgus neutrophils were observed for MAB#1. Interestingly, MAB#2 showed an overall lower signal compared to background levels on cynomolgus monkey neutrophils compared to MAB#1. This result was not observed in a binding assay with cynomolgus C5aR expressed on CHO cells. The absence of binding to cynomolgus macaque neutrophils was confirmed for RefMAB№l

For rodent C5aR, cross-reactivity with rat and mouse C5aR was not expected due to low sequence homology (66% overall identity). Indeed, neither MAB#1 nor MAB#2 showed any significant binding to rat or mouse C5aR expressed on CHO-Flp cells when tested at 300 nM IgG concentration (Figure 2). To exclude cross-reactivity with any other member of the C5aR subfamily, binding to full-length human C5L2, ChemR23, FPR1, and C3aR expressed on CHO Flp-In cells was also determined by FACS (compared to untransfected parental CHO Flp-In cells). As shown in Figure 2, even at 300 nM IgG concentration, there was no significant level of cellular binding of MAB#1 and MAB#2 to any of the C5aR-related GPCRs or to parental CHO cells.

Together, both MAB#1 and MAB#2 showed specific binding to human and cynomolgus C5aR.

Example 8. Binding of MAB#1 and MAB#2 to Full Length Human C5aR Presented on Virus-Like Particles (VLPs) - Link to Natural Human C5aR Variants

Two naturally occurring human C5aR variants have been reported (SEQ ID NO: 1; variant D/K and SEQ ID NO: 2; variant N/N).

To compare the binding of MAB#1 and Mab#2 to the two natural variants, VLPs were created expressing one of the two C5aR variants as described in Example 1. VLPs expressing mouse C5aR were included as a negative control, since MAB#1 and Mab#2 cross do not react with mouse C5aR.

Way

To assess binding, the resulting VLPs were plated overnight and, after the blocking step, IgG titers were added the next day. IgG binding to the coated antigen was detected using alkaline phosphatase-conjugated anti-human IgG2 antibody and AttoPhos as substrate.

results

The results are summarized in Table 15 and shown in Figure 3. Both MAB#1 and MAB#2 exhibited comparable titration curves against both naturally occurring human C5aR variants with equal _EC50 concentrations. As expected, no binding to mouse C5aR was detected. Based on these data, a high level of binding affinity of MAB#1 and MAB#2 to human C5aR in vivo could be expected, regardless of the natural variant present on the respective target cells.

Example 9. Profiling a panel of proteins (PP)

Potential non-specific off-target binding for MAB#1 was determined in the overall ZR assay.

Way

Protein panel profiling was essentially performed as described by Frese et al. (mAbs 5:2, 279-287; March/April 2013). 32 different proteins and controls were coated on two 384-well standard MSD plates at a concentration of 1.0 μg/ml at 4°C overnight. The coating solution was discarded and the plates were blocked with 50 μl of 3% (w/v) BSA in PBS for one hour at RT. on a microtiter plate shaker (-500 rpm) followed by three wash steps with 50 μl wash buffer (PBS with 0.05% (v/v) Tween 20). IgG samples were diluted to 100 nM and 10 nM assay buffer (PBS with 0.5% (w/v) BSA, 0.05% (v/v) Tween 20). Isotype control antibody MOR03207 (IgG1f_AEASS) and assay buffer were used as controls. Samples and controls were added at 30 μl/well and incubated for three hours at RT. on a shaker for microtiter plates. The plates were washed three times and 30 μl of detection antibody (ECL-labeled anti-human Fab antibody) was added per well and incubated for one hour on a microtiter plate shaker (~500 rpm). After washing the MSD plate and adding 35 μl/well of MSD T reading buffer with surfactant, the electro-chemiluminescence signal was detected using a SECTOR Imager S600 (Meso Scale Diagnostics).

For evaluation, the binding signals of the antibody sample to a specific protein were divided by the corresponding binding signals of the reference antibody MOR03207 to obtain the binding ratio (BR). The cumulative binding ratio (CBR) of all proteins except controls (25 in total) was then calculated: a CBR of up to 150 indicates IgG with no detectable nonspecific binding. Values above 150 indicate IgG with increased levels of nonspecific binding compared to the reference antibody MOR03207.

results

The results for MAB#1 and MAB#2 are shown in Figure 10. Overall, no critical nonspecific binding was detected to any of the proteins tested. Very low (MAB#2) and low (MAB#1) levels of binding to bovine transferrin were observed. Binding to bovine transferrin was confirmed in an Octet-based binding assay, but binding to rat and cynomolgus monkey transferrin was not detected (data not shown). Thus, the observed binding to bovine transferrin was considered non-critical.

Final antibody format and safety

C5aR is expressed on various immune cells such as leukocytes, neutrophils and lymphocytes, and human IgG1 Fc-mediated depletion of such cells must be prevented to avoid unwanted side effects. Accordingly, the final C5aR IgG antibody format must be silenced for the ability to induce any effector function during a therapeutic intervention.

Both MAB#1 and MAB#2 contain five amino acid substitutions in the Fc region of human IgG1f, namely L234A, L235E, G237A, A330S and P331S (hlgG1f_AEASS, EU numbering) to eliminate antibody-induced effector function. The clinical safety of this format in the context of anti-C5aR antibody therapy has been described in the prior art (Wagner F et al. Annals of the Rheumatic Diseases. 2014; 73: 499. doi: 10.1136/annrheumdis-2014-eular.2156.)

The lack of ability of MAB#1 and MAB#2 to induce effector function was confirmed in various assays, such as Fcγ receptor or C1q binding studies, as well as in vitro ADCC and ADCP assays disclosed below.

Example 10. Binding of MAB#1 and MAB#2 to the FcRn receptor using Octet

Way

Determination of apparent K _D for immobilized neonatal Fc receptor (FcRn) from different species was performed at pH 6.0 and 7.2 at 27°C using an Octet NTX device ( , Pall Life Sciences). Capture 0.5 nM biotinylated human, cynomolgus, mouse, and rat FcRn onto streptavidin (SA)-coated sensors ( , Pall Life Sciences). Kinetic measurements were performed using 8 different concentrations of IgG (3-fold serial dilution, 1000-0.46 nM) in Octet buffer (PBS, 0.05% (v/v), Tween-20, 0.1% (w/v) BSA) with an association time of 240 s and a dissociation time of 180 s. After each cycle, the sensor was regenerated to remove the bound peptide/antibody complex (2 × 30 s in HBS-EP+, pH 8.0). All sensorgrams were fitted using data analysis software 10.0 ( , Pall Life Sciences) to determine apparent affinity and the data were fit to a steady state model.

results

The results are summarized in Table 16. MAB#1 and MAB#2 exhibited apparent binding affinities for different species FcRn in the expected range of affinity values (comparable to isotype control antibody MOR03207 IgG1f), and the physiological binding behavior for binding to human FcRn could be confirmed for both IgG molecules, i.e. no binding was detected at neutral pH (7.2). Therefore, the 5 mutations introduced into the Fc region of the antibodies did not have a negative effect on human FcRn binding.

Example 11. Binding of MAB#1 and MAB#2 to human Fcy receptors using Octet

Way

Determination of K _D using the IgG capture model was performed at 27°C using Octet ( , Pall Life Sciences). Capture 2.0 nM IgG diluted in Octet assay buffer (PBS, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA) onto Protein A-coated sensors ( , Pall Life Sciences). Kinetic measurements were performed using 7 concentrations of Fc-gamma receptors (2-fold serial dilution) in assay buffer. After each cycle, the sensors were regenerated to remove the bound peptide/antibody complex (2 × 30 s in 10 mM Gly/HCl, pH 1.5). All sensorograms were fitted using data analysis software 10.0 ( , Pall Life Sciences) to determine the rate constants k _on and k _off , which were used to calculate K _D . The raw data were fitted using a 1:1 linkage model with R _max set locally.

results

The results are summarized in Table 17. No or very weak binding of MAB#1 and MAB#2 to any of the Fcγ receptors tested could be detected and the introduced mutations in the Fc region of human IgG1 were confirmed to be effective in abolishing Fcγ receptor binding.

Example 12: Binding of MAB#1 and MAB#2 to C1q Using Octet

Way

Determination of apparent (bivalent) K _D using the IgG capture model was performed at 27°C using Octet ( Pall Life Sciences). Capture 2.0 nM IgG diluted in Octet buffer with anti-human kappa/lambda CHI Fab (BAC) immobilized on streptavidin (SA)-coated sensors ( , Pall Life Sciences). Kinetic measurements were performed using 8 different concentrations of C1q (3-fold serial dilution, 500–0.69 nM) in Octet assay buffer (see above) with an association time of 240 s and a dissociation time of 240 s. After each cycle, the sensors were regenerated to remove the bound peptide/antibody complex (2 × 50 mM NaOH, 1 × 10 mM Gly/HCl, pH 1.5, 30 s each). All sensorograms were fitted using data analysis software 9 ( , Pall Life Sciences) to determine apparent affinity. The data were fitted to a steady state model.

results

The results are summarized in Table 18. As expected, no binding of MAB#1 and MAB#2 to C1q (isolated from pooled human plasma) was observed and the introduced mutations in the Fc region of human IgG1 were confirmed to be effective in eliminating C1q binding.

Example 13. Activity of ADCC and ADCP in vitro MAB#1

Methods

ADCC and ADCP activities for MAB#1 were tested using ADCC and ADCP reporter bioassays using Promega according to the manufacturer's instructions (cat. no. G7017 and catalog no. G988A, respectively). The kits use engineered Jurkat cells as effector cells. The cells either stably express the FcyRIIIa receptor variant V158 (high affinity) for ADCC and the FcγRIIa_H receptors for ADCP, and the NFAT response element driving firefly luciferase expression. CHO Flp-In cells expressing human C5aR were used as target cells. Binding of effector cells to the target through an antibody bridge (eg, through MAB#1 or MAB#2) triggers a cascade of events in the NFAT pathway, leading to expression of the firefly luciferase protein. The enzymatic reaction produces luminescence, the level of which is proportional to the concentration of luciferase, which directly correlates with ADCC or ADCP activity. ADCC or ADCP activity was analyzed for MAB#1 by plotting the average signal against background values.

Because the wild-type version of human IgG1f was not available for MAB#1, wild-type IgG1f as well as the Fc-silenced version of human IgG1f_AEASS, derived in place of the human control antibody to C5aR, were included as positive controls. In addition, the Fc-silenced version (hIgG1_AEASS) and the wild-type version (hIgG1f) of the isotopic control antibody MOR03207 were included as negative controls.

results

The results are summarized in Table 19 and shown in Figure 8A (ADCP) and B (ADCC) for an IgG concentration of 10 μg/ml. MAB#1 did not induce FcγRIIIa or FcγRIIa_H activation of the NFAT pathway in engineered effector cells in the presence of C5aR-overexpressing CHO cells, similar to the Fc-silenced version of the anti-C5aR control antibody and the Fc-silenced version of MOR03207. A wild-type (unsilenced) version of a C5aR-specific control antibody clearly induced luciferase production in engineered Jurkat cells in the presence of C5aR-expressing CHO cells.

Overall, the experiment clearly confirmed that introduced mutations into the Fc region of wild-type human IgG1 are effective in preventing ADCC and ADCP activity.

Functional characteristics of MAV No. 1 and MAV No. 2

The neutralizing activities of MAB#1 and MAB#2 were analyzed in different in vitro assays that monitored C5a-induced C5aR activation.

Since elevated levels of C5a have been described in pathophysiological conditions, the ability of a C5aR antagonist antibody to neutralize high concentrations of C5a that may be present locally at the site of disease is expected to provide a beneficial therapeutic effect in vivo. Therefore, in vitro experiments were also performed to reflect such pathological conditions in vivo.

Example 14 PathHunter® β-Arrestin Assay (DiscoveRx)

Methods

The PathHunter® β-arrestin assay from DiscoveRx was performed according to the manufacturer's instructions. Briefly, human C5a-induced human C5aR activity was measured by detecting the interaction of β-arrestin with activated C5aR using fragment complementation of the β-galactosidase enzyme.

β-Arrestin recruitment was induced using recombinant human C5a, and enzyme activity was measured using chemiluminescent detection reagents from DiscoverRx. Chinese hamster ovary (CHO) cells expressing an engineered version of human C5aR were seeded overnight, supplemented with serial dilutions of human C5a (R&D Systems), and incubated at 37°C and 5% CO2 for 1.5 hours (titration curves generated in the absence of antagonist). In parallel, human C5a titration curves were determined in the presence of C5aR-specific antibodies (fixed IgG concentration of 50 nM). To do this, IgG was added to the cells before stimulation with human C5a and incubated for 1 hour at 37°C and 5% CO ₂ . The results were expressed in relative luminescence units. Human C5a titration curves in the presence and absence of antagonistic IgG were generated using GraphPad Prism.

For the β-arrestin analysis, shifts of the human C5a dose response curves toward higher doses (ie, horizontally to the right of the dose axis) were compared for MAB#1, MAB#2, and RefMAB#1 (Figure 4A). In addition, % inhibition at three increasing concentrations of C5a (1.2 nM, 11 nM, and 100 nM) was calculated and compared (Figure 4B).

results

The results of the β-arrestin assays are shown in Figures 4A and 4B and are summarized in Table 20 and Table 21. In the absence of antibody, a dose response curve was generated for human C5a with an average EC ₅₀ concentration of 2.9 nM (Figure 4A).

Addition of MAB#1 or MAB#2 to human C5a at a final IgG concentration of 50 nM resulted in a significant shift in the dose response curve of more than 12-fold toward higher doses of human C5a. More specifically, in the presence of 50 nM MAB#1 or MAB#2, a dose response curve was obtained for human C5a with an average _EC50 concentration of 37 nM (Figure 4A, Table 20). In other words, the presence of MAB#1 or MAB#2 significantly reduces the ability of human C5 to induce C5aR activation or, alternatively, in the presence of MAB#1 or MAB#2 human C5 must be added to at least a 12-fold higher concentration to induce the same activity of C5aR compared to its activity in the absence of the antibody.

This observation was also reflected when calculating the % inhibition at three increasing concentrations of C5a (Figure 4B). At 1.2 nM human C5a, all three antibodies tested, MAB#1, MAB#2, and RefMAB#1, showed comparable suppression of C5a-induced C5aR activity by more than 80% at an IgG concentration of 50 nM. However, when an approximately 10-fold higher concentration of human C5a (11 nM) was used to activate C5aR, RefMABIII was only able to neutralize approximately 50% of C5a-induced C5aR activity. This is in sharp contrast to MAB#1 and MAB#2, which were still able to neutralize up to 80% of C5a-induced C5aR activity.

This effect was even more pronounced when a 100-fold higher concentration of human C5a (100 nM) was used to activate C5aR. In this case, virtually no neutralizing activity was detected in RefMAB#1, whereas MAB#1 and MAB#2 were still able to neutralize approximately 40% and 30% of C5a-induced human C5aR activity, respectively.

Therefore, both MAB#1 and MAB#2 are effective in neutralizing pathophysiological concentrations of C5a in vitro and are significantly more effective compared to RefMAB#1.

Example 15. Suppression of neutrophil and monocyte activation using MAB#1 and MAB#2 - CD11b assay

The neutralization efficiency of MAB#1 and MAB#2 was further determined in a whole blood CD11b functional assay, which represents a more physiological model. CD11b binds to CD18 to form the Mac-1 integrin complex, which serves as a multiligand receptor. CD11b is constitutively expressed on the surface of >50% of peripheral blood leukocytes; upon activation of leukocytes, its expression increases due to the fusion of CD11b-containing secretory granules with the cell membrane. Thus, CD11b expression is widely used as a marker of leukocyte activation both in vivo and in vitro.

C5a, as an effective activator of human neutrophils and monocytes, induces positive regulation of the CD11b surface antigen. Therefore, the ability of MAB#1 and MAB#2 to prevent C5a-induced activation of granulocytes and monocytes was examined by assessing CD11b levels in granulocytes and monocytes obtained from whole blood. Experiments were essentially performed as described in US patent application US2013/0295116 (NOVO NORDISK).

Way

In the first assay, heparinized whole blood was mixed with IgG (in serial dilutions) and incubated for 20 minutes at 37°C, 5% CO ₂ . Human C5a was added at a standard concentration of 15 nM and incubated for 20 minutes at 37°C, 5% CO ₂ . Anti-CD11b-PE antibody or isotype control antibody MOR03207 was added and the plate was incubated for 20 minutes at 37°C, 5% CO ₂ . Finally, red cell lysis buffer was added and incubated at room temperature for 15 minutes in the dark, and the cells were washed and resuspended in lysis buffer.

In the second experimental setup, human C5a was added at the more clinically relevant pathophysiological concentration of 150 nM without modifying the remaining assays as described above.

To examine MAB1 receptor retention time, the assay was further adapted by extending the incubation time of heparinized whole blood with IgG from 20 minutes to 300 minutes.

Fluorescence levels were measured using a FACS or Novocyte array device. Samples were gated to exclude dead cells and debris. Monocytes and granulocytes were identified according to their FSC and SSC profiles and gated. The median fluorescence intensity (MFI) of gated granulocytes and/or monocytes in the CD11b-PE (Yellow-A) channel was calculated. Results were expressed as percent suppression (% suppression). The maximum level of CD11b expression (MFI _Max ) was the average MFI of cells incubated with C5a but without IgG. The minimum (background) level of CD11b expression (MFI _Min ) was the average MFI of cells incubated without C5a and without IgG. The formula used to calculate % suppression for each sample was as follows:

Data were assessed using FlowJo, entered into GraphPad Prism (v4.0) and fitted to a sigmoidal dose response curve using nonlinear regression to calculate IC ₅₀ .

Results for MAB#1 using 15 nM C5a ligand

Results for MAB#1 from CD11b whole blood assays are summarized in Table 22. For both gated cell populations (monocytes and granulocytes), IC ₅₀ values were determined in the single digit nM range with maximum suppression of nearly 88% for granulocytes and 83% for monocytes.

Results for MAB#1, MAB#2 and RefMAB#l using 15 nM versus 150 nM C5a ligand

In addition to adding 15 nM human C5a to stimulate granulocytes (as described above), a tenfold higher concentration (150 nM) of ligand was used to mimic a more pathophysiological ligand concentration.

Again, MAB#1, MAB#2, and RefMAB#l were dose titrated and suppression curves were generated using GraphPad Prism using the nonlinear regression function. The results were quantified as “antagonist concentration required to induce 50% inhibition.”

The results of this whole blood CD11b assay are shown in Figure 5A and B.

Figure 5A shows the neutralizing activity of MAB#1, MAB#2 and RefMAB#1 at 15 nM C5a, and Figure 5B shows the corresponding neutralizing activity at 150 nM C5a. As quantifiable readouts, the IgG concentration to achieve 50% suppression of CD11b up-regulation was calculated and the results are shown below the x-axis in both figures.

In general, similar observations as for the β-arrestin assay (Example 14) were made using increasing concentrations of C5a in the whole blood CD11b assay, gating granulocytes as the primary target cells. To block the effects of 15 nM human C5a to 50%, an IgG concentration of 12 nM was required for MAB#1 and RefMAB#1, whereas for MAB#2 an IgG concentration of 17 nM was sufficient. However, significantly different amounts of antagonists were required to block 50% of the CD11b up-regulation induced by a 10-fold higher concentration of C5a (150 nM). While for MAB#1 an IgG concentration of 42 nM was now sufficient to suppress 50% of C5a-induced C5aR activation, a 7-fold higher concentration of RefMAB#1 (291 nM) was required to achieve the same blocking effect. For MAB#2, an IgG concentration of 114 nM was sufficient.

Therefore, it was possible to perform the same ranking of the 3 IgGs in terms of potency as already shown in the b-arrestin assay (MAB#1 > MAB#2 > RefMAB#1), and both MAB#1 and MAB#2 showed high potency in neutralizing pathophysiological concentrations of C5a in vitro and were significantly more effective compared to RefMAB№l.

Results for MAB#1 - effect on receptor retention time

Seow et al. (Seow V et al., Sci Rep.2016; 6: 24575) reported that C5a production is localized to the cell membrane and can be very high for short but repeated periods. Thus, it has been suggested that a C5aR antagonist with a long retention time may be useful in systems with fast and transient signaling. It was also concluded that increased receptor retention time measured in vitro may also influence the duration of action and degree of efficacy in vivo.

To compare the receptor retention times for the two antibodies in a similar formulation published by Seow et al., logarithmic dose-repression curves were evaluated for 20 minutes and 300 minutes of incubation of IgG with granulocytes and monocytes present in whole blood as described above.

The results of this experimental setup of whole blood CD11b assay are shown in Figure 6A - D. Surprisingly, MAB#1 observed a significant increase in neutralizing activity over a long period of incubation time. As shown in Figure 6A and C, for both granulocyte and monocyte populations, the estimated IC ₅₀ concentration for MAB#1 decreased over time by approximately 6-fold (IC ₅₀ values are summarized in Table 23). For RefMAB#l, no shift or decrease was observed in the suppression curves after 300 minutes (Figure 6B and D).

Overall, the combination of effective neutralization of pathophysiological C5a levels and increased efficacy over time is expected to be beneficial in vivo.

Example 16. C5a-induced neutrophil migration

C5a-induced chemotaxis of purified neutrophils was analyzed. The experiment was essentially performed as described in US patent application US2013/0295116.

Methods

Neutrophils were isolated and purified from the whole blood of three different human donors using the MACSxpress Human Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, Cat. No. 130-104-434) and the MACSxpress Human Red Blood Cell Depletion Kit (Miltenyi Biotec, Cat. No. 130-104-434). according to catalog 130-098-196).

The effectiveness of IgG in inhibiting hC5a-dependent neutrophil migration was analyzed using the Boyden chamber technique using 3.0 μm pore size FluoroBlok® 96-well plates. The pore membrane of the Boyden chamber was coated with 1 mg/ml human fibrinogen for 2 hours at 37°C. After washing, the membranes were blocked with a solution containing 2% bovine serum albumin (BSA) for 1 hour at 37°C. Purified neutrophils were then stained with calcein and 10 ⁵ stained cells were added to the upper compartment of the Boyden chamber with and without antagonistic IgG (100 and 600 nM). hC5a (R&D Systems, 10 nM) was added to the lower compartment of the Boyden chamber. The plate was measured at 485/538 nm at 37°C every 5 minutes for 60 minutes in a plate reader (Tecan M1000Pro). The ability of neutrophils to migrate into the lower chamber was determined by measuring calcein-stained neutrophils passing through a FluoroBlok membrane. Results were expressed as migration kinetic curves (5-60 minutes) as well as percent inhibition calculated at selected time points (15, 25 and 35 minutes).

The formula used to calculate % suppression was:

results

The average percent inhibition of neutrophil migration at selected time points at the two IgG concentrations tested was calculated from 3 independent experiments using neutrophils from three different human donors.

As shown in Figure 7, after 15 minutes of migration, almost complete inhibition of C5a-induced neutrophil migration was achieved for MAB#1. After 35 min of migration and the highest IgG concentration tested (600 nM), MAB#1 still showed >50% inhibition. The negative isotype control antibody MOR03207 did not demonstrate an inhibitory effect on neutrophil migration.

Example 17 C5a-induced cytokine release by macrophages

Macrophages release pro-inflammatory or anti-inflammatory cytokines upon activation depending on their polarization. Therefore, we assessed whether blockade of the interaction of C5a/C5aR with MAB1 affects C5a-induced cytokine release by Ml and M2 macrophages. Cytokine ELISA was performed to detect the production of anti-inflammatory IL-10 by M2 macrophages and pro-inflammatory IL-12 by Ml macrophages.

Methods

Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy human donors by density gradient centrifugation with Biocoll separation solution and SepMate tubes (Stemcell). Monocytes were isolated from PBMCs using a CD 14+ selection kit (Miltenyi Biotech) and cultured in RPMI supplemented with 10% FCS and 1x GlutaMax in 96-well plates. Monocytes seeded overnight were polarized for 24 hours at 37°C to Ml macrophages using LPS (20 ng/ml) and IFNγ (50 ng/ml) and preincubated with MAB#1 (30 nM) for 30 minutes followed by the addition of C5a (15 nM) for another 24 hours. M1 macrophages stimulated with C5a in the absence of MAB#1 and untreated controls were included. After 24 hours, cell supernatants were analyzed using IL-12/IL23 DuoSet ELISA (R&D Systems) according to the manufacturer's instructions. To generate M2 macrophages, monocytes were predifferentiated into macrophages by culturing for 5 days in RPML10% FCS supplemented with M-CSF and adding M-CSF, IL-4, IL-13, and IL-6 (40 ng/ml each) to day 5. C5a (15 nM)+/- MAB#1 (30 nM) was added daily from days 5 to 9. On day 9, cell supernatants were analyzed using IL-10 DuoSet ELISA (R&D Systems) according to the manufacturer's instructions.

results

Although incubation with C5a resulted in decreased IL-12 production by Ml macrophages, no reduction in IL-12 levels was observed after pretreatment with MAB#1 compared to untreated controls. On the other hand, treatment with MAB#1 resulted in suppression of C5a-induced IL-10 production in M2 macrophages (see Figure 11).

In conclusion, MAB#1 effectively suppressed C5a-induced production of anti-inflammatory IL-10 by M2 macrophages and restored the production of proinflammatory IL-12 by Ml macrophages, thereby demonstrating the mechanism of action of MAB#1 in vitro.

Pharmacokinetics

Example 18 Pharmacokinetics

The pharmacokinetic profile of MAB#1 was assessed in male Han-Wistar rats (n=3 animals) after a single intravenous (i.v.) administration of 10 mg/kg IgG.

Methods

Plasma samples were collected from each animal via retroorbital sinus or mandibular vein puncture at the following time points: predose, 0.083, 1, 3, 8, 24, 48, 72, 96, 168, 240, 336, and 504 hours postdose .

Concentrations of free, bioactive MAB#1 in rat plasma were determined using an MSD-based ligand binding assay. Briefly, biotinylated human C5aR N-terminal peptide was coated onto the surface of a 96-well streptavidin-coated MSD plate. Bound analyte was detected using a drug-specific anti-idiotypic ECL-labeled antibody. The pharmacokinetic properties of MAB1 were assessed using non-compartmental analysis (NCA) based on plasma free drug concentrations. results

Mean plasma concentrations over time are shown in FIG. 9. The average maximum concentrations of MAB#1 in blood plasma after a single iv injection were observed 5 minutes (i.e. 0.083 hours) after administration ( _Tmax ) in all three animals (i.e. the first time point of sampling after administration) . The mean volume of distribution (Vz) of 106 ml/kg was between plasma volume and extracellular volume (Davies et al., 1993). The average terminal half-life after IV administration was determined after 9.0 days, the average total clearance was determined at 0.341 ml/h/kg.

Overall, MAB#1 exhibited a typical pharmacokinetic profile of human IgG1 antibody in rat plasma without cross-reactivity with rodent C5aR. No evidence of anti-drug antibody (ADA)-mediated clearance could be detected.

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LIST OF SEQUENCES

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130 135 140

Gln Asn Phe Arg Gly Ala Gly Leu Ala Trp Ile Ala Cys Ala Val Ala

145 150 155 160

Trp Gly Leu Ala Leu Leu Leu Thr Ile Pro Ser Phe Leu Tyr Arg Ala

165 170 175

Val Arg Gln Glu Glu Tyr Ser Pro Lys Val Leu Cys Gly Val Asp Tyr

180 185 190

Asn Asn Asp Thr Arg Arg Glu Arg Ala Val Ala Ile Val Arg Leu Val

195 200 205

Leu Gly Phe Leu Trp Pro Leu Leu Thr Leu Met Ile Cys Tyr Thr Phe

210 215 220

Leu Leu Leu Arg Thr Trp Ser Arg Arg Ala Thr Arg Ser Thr Lys Thr

225 230 235 240

Leu Lys Val Val Val Ala Val Val Ala Ser Phe Phe Ile Phe Trp Leu

245 250 255

Pro Tyr Gln Val Thr Gly Thr Met Met Ser Phe Leu Arg Pro Ser Ser

260 265 270

Pro Thr Tyr Leu Gln Leu Lys Lys Leu Asp Ser Leu Ser Ile Ser Phe

275 280 285

Ala Tyr Ile Asn Cys Cys Ile Asn Pro Val Ile Tyr Val Val Ala Gly

290 295 300

Gln Gly Phe Gln Gly Arg Leu Arg Lys Ser Leu Pro Ser Leu Leu Arg

305 310 315 320

Asn Val Leu Thr Glu Glu Ser Val Val Arg Glu Ser Lys Ser Phe Thr

325 330 335

Arg Ser Thr Val Asp Thr Met Thr Glu Lys Thr Gln Ala Val

340 345 350

<210> 4

<211> 351

<212> PROTEIN

<213> Mus musculus

<400> 4

Met Asp Pro Ile Asp Asn Ser Ser Phe Glu Ile Asn Tyr Asp His Tyr

1 5 10 15

Gly Thr Met Ala Pro Asn Ile Pro Ala Asp Gly Ile His Leu Pro Lys

20 25 30

Arg Gln Pro Gly Asp Val Ala Ala Leu Ile Ile Tyr Ser Val Val Phe

35 40 45

Leu Val Gly Val Pro Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe

50 55 60

Glu Ala Arg Arg Ala Val Asn Ala Ile Trp Phe Leu Asn Leu Ala Val

65 70 75 80

Ala Asp Leu Leu Ser Cys Leu Ala Leu Pro Val Leu Phe Thr Thr Val

85 90 95

Leu Asn His Asn Tyr Trp Tyr Phe Asp Ala Thr Ala Cys Ile Val Leu

100 105 110

Pro Ser Leu Ile Leu Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala

115 120 125

Thr Ile Ser Ala Asp Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys

130 135 140

Gln Lys Val Arg Gly Thr Gly Leu Ala Trp Met Ala Cys Gly Val Ala

145 150 155 160

Trp Val Leu Ala Leu Leu Leu Thr Ile Pro Ser Phe Val Tyr Arg Glu

165 170 175

Ala Tyr Lys Asp Phe Tyr Ser Glu His Thr Val Cys Gly Ile Asn Tyr

180 185 190

Gly Gly Gly Ser Phe Pro Lys Glu Lys Ala Val Ala Ile Leu Arg Leu

195 200 205

Met Val Gly Phe Val Leu Pro Leu Leu Thr Leu Asn Ile Cys Tyr Thr

210 215 220

Phe Leu Leu Leu Arg Thr Trp Ser Arg Lys Ala Thr Arg Ser Thr Lys

225 230 235 240

Thr Leu Lys Val Val Met Ala Val Val Ile Cys Phe Phe Ile Phe Trp

245 250 255

Leu Pro Tyr Gln Val Thr Gly Val Met Ile Ala Trp Leu Pro Pro Ser

260 265 270

Ser Pro Thr Leu Lys Arg Val Glu Lys Leu Asn Ser Leu Cys Val Ser

275 280 285

Leu Ala Tyr Ile Asn Cys Cys Val Asn Pro Ile Ile Tyr Val Met Ala

290 295 300

Gly Gln Gly Phe His Gly Arg Leu Leu Arg Ser Leu Pro Ser Ile Ile

305 310 315 320

Arg Asn Ala Leu Ser Glu Asp Ser Val Gly Arg Asp Ser Lys Thr Phe

325 330 335

Thr Pro Ser Thr Thr Asp Thr Ser Thr Arg Lys Ser Gln Ala Val

340 345 350

<210> 5

<211> 352

<212> PROTEIN

<213> Rattus norvegicus

<400> 5

Met Asp Pro Ile Ser Asn Asp Ser Ser Glu Ile Thr Tyr Asp Tyr Ser

1 5 10 15

Asp Gly Thr Pro Asn Pro Asp Met Pro Ala Asp Gly Val Tyr Ile Pro

20 25 30

Lys Met Glu Pro Gly Asp Ile Ala Ala Leu Ile Ile Tyr Leu Ala Val

35 40 45

Phe Leu Val Gly Val Thr Gly Asn Ala Leu Val Val Trp Val Thr Ala

50 55 60

Phe Glu Ala Lys Arg Thr Val Asn Ala Ile Trp Phe Leu Asn Leu Ala

65 70 75 80

Val Ala Asp Leu Leu Ser Cys Leu Ala Leu Pro Ile Leu Phe Thr Ser

85 90 95

Ile Val Lys His Asn His Trp Pro Phe Gly Asp Gln Ala Cys Ile Val

100 105 110

Leu Pro Ser Leu Ile Leu Leu Asn Met Tyr Ser Ser Ile Leu Leu Leu

115 120 125

Ala Thr Ile Ser Ala Asp Arg Phe Leu Leu Val Phe Lys Pro Ile Trp

130 135 140

Cys Gln Lys Phe Arg Arg Pro Gly Leu Ala Trp Met Ala Cys Gly Val

145 150 155 160

Thr Trp Val Leu Ala Leu Leu Leu Thr Ile Pro Ser Phe Val Phe Arg

165 170 175

Arg Ile His Lys Asp Pro Tyr Ser Asp Ser Ile Leu Cys Asn Ile Asp

180 185 190

Tyr Ser Lys Gly Pro Phe Phe Ile Glu Lys Ala Ile Ala Ile Leu Arg

195 200 205

Leu Met Val Gly Phe Val Leu Pro Leu Leu Thr Leu Asn Ile Cys Tyr

210 215 220

Thr Phe Leu Leu Ile Arg Thr Trp Ser Arg Lys Ala Thr Arg Ser Thr

225 230 235 240

Lys Thr Leu Lys Val Val Met Ala Val Val Thr Cys Phe Phe Val Phe

245 250 255

Trp Leu Pro Tyr Gln Val Thr Gly Val Ile Leu Ala Trp Leu Pro Arg

260 265 270

Ser Ser Ser Thr Phe Gln Ser Val Glu Arg Leu Asn Ser Leu Cys Val

275 280 285

Ser Leu Ala Tyr Ile Asn Cys Cys Val Asn Pro Ile Ile Tyr Val Met

290 295 300

Ala Gly Gln Gly Phe His Gly Arg Leu Arg Arg Ser Leu Pro Ser Ile

305 310 315 320

Ile Arg Asn Val Leu Ser Glu Asp Ser Leu Gly Arg Asp Ser Lys Ser

325 330 335

Phe Thr Arg Ser Thr Met Asp Thr Ser Thr Gln Lys Ser Gln Ala Val

340 345 350

<210> 6

<211> 74

<212> PROTEIN

<213> Homo sapiens

<400> 6

Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser

1 5 10 15

Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys Val Asn Asn Asp Glu

20 25 30

Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile

35 40 45

Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn

50 55 60

Ile Ser His Lys Asp Met Gln Leu Gly Arg

65 70

<210> 7

<211> 337

<212> PROTEIN

<213> Homo sapiens

<400> 7

Met Gly Asn Asp Ser Val Ser Tyr Glu Tyr Gly Asp Tyr Ser Asp Leu

1 5 10 15

Ser Asp Arg Pro Val Asp Cys Leu Asp Gly Ala Cys Leu Ala Ile Asp

20 25 30

Pro Leu Arg Val Ala Pro Leu Pro Leu Tyr Ala Ala Ile Phe Leu Val

35 40 45

Gly Val Pro Gly Asn Ala Met Val Ala Trp Val Ala Gly Lys Val Ala

50 55 60

Arg Arg Arg Val Gly Ala Thr Trp Leu Leu His Leu Ala Val Ala Asp

65 70 75 80

Leu Leu Cys Cys Leu Ser Leu Pro Ile Leu Ala Val Pro Ile Ala Arg

85 90 95

Gly Gly His Trp Pro Tyr Gly Ala Val Gly Cys Arg Ala Leu Pro Ser

100 105 110

Ile Ile Leu Leu Thr Met Tyr Ala Ser Val Leu Leu Leu Ala Ala Leu

115 120 125

Ser Ala Asp Leu Cys Phe Leu Ala Leu Gly Pro Ala Trp Trp Ser Thr

130 135 140

Val Gln Arg Ala Cys Gly Val Gln Val Ala Cys Gly Ala Ala Trp Thr

145 150 155 160

Leu Ala Leu Leu Leu Thr Val Pro Ser Ala Ile Tyr Arg Arg Leu His

165 170 175

Gln Glu His Phe Pro Ala Arg Leu Gln Cys Val Val Asp Tyr Gly Gly

180 185 190

Ser Ser Ser Thr Glu Asn Ala Val Thr Ala Ile Arg Phe Leu Phe Gly

195 200 205

Phe Leu Gly Pro Leu Val Ala Val Ala Ser Cys His Ser Ala Leu Leu

210 215 220

Cys Trp Ala Ala Arg Arg Cys Arg Pro Leu Gly Thr Ala Ile Val Val

225 230 235 240

Gly Phe Phe Val Cys Trp Ala Pro Tyr His Leu Leu Gly Leu Val Leu

245 250 255

Thr Val Ala Ala Pro Asn Ser Ala Leu Leu Ala Arg Ala Leu Arg Ala

260 265 270

Glu Pro Leu Ile Val Gly Leu Ala Leu Ala His Ser Cys Leu Asn Pro

275 280 285

Met Leu Phe Leu Tyr Phe Gly Arg Ala Gln Leu Arg Arg Ser Leu Pro

290 295 300

Ala Ala Cys His Trp Ala Leu Arg Glu Ser Gln Gly Gln Asp Glu Ser

305 310 315 320

Val Asp Ser Lys Lys Ser Thr Ser His Asp Leu Val Ser Glu Met Glu

325 330 335

Val

<210> 8

<211> 482

<212> PROTEIN

<213> Homo sapiens

<400> 8

Met Ala Ser Phe Ser Ala Glu Thr Asn Ser Thr Asp Leu Leu Ser Gln

1 5 10 15

Pro Trp Asn Glu Pro Pro Val Ile Leu Ser Met Val Ile Leu Ser Leu

20 25 30

Thr Phe Leu Leu Gly Leu Pro Gly Asn Gly Leu Val Leu Trp Val Ala

35 40 45

Gly Leu Lys Met Gln Arg Thr Val Asn Thr Ile Trp Phe Leu His Leu

50 55 60

Thr Leu Ala Asp Leu Leu Cys Cys Leu Ser Leu Pro Phe Ser Leu Ala

65 70 75 80

His Leu Ala Leu Gln Gly Gln Trp Pro Tyr Gly Arg Phe Leu Cys Lys

85 90 95

Leu Ile Pro Ser Ile Ile Val Leu Asn Met Phe Ala Ser Val Phe Leu

100 105 110

Leu Thr Ala Ile Ser Leu Asp Arg Cys Leu Val Val Phe Lys Pro Ile

115 120 125

Trp Cys Gln Asn His Arg Asn Val Gly Met Ala Cys Ser Ile Cys Gly

130 135 140

Cys Ile Trp Val Val Ala Cys Val Met Cys Ile Pro Val Phe Val Tyr

145 150 155 160

Arg Glu Ile Phe Thr Thr Asp Asn His Asn Arg Cys Gly Tyr Lys Phe

165 170 175

Gly Leu Ser Ser Ser Leu Asp Tyr Pro Asp Phe Tyr Gly Asp Pro Leu

180 185 190

Glu Asn Arg Ser Leu Glu Asn Ile Val Gln Pro Pro Gly Glu Met Asn

195 200 205

Asp Arg Leu Asp Pro Ser Ser Phe Gln Thr Asn Asp His Pro Trp Thr

210 215 220

Val Pro Thr Val Phe Gln Pro Gln Thr Phe Gln Arg Pro Ser Ala Asp

225 230 235 240

Ser Leu Pro Arg Gly Ser Ala Arg Leu Thr Ser Gln Asn Leu Tyr Ser

245 250 255

Asn Val Phe Lys Pro Ala Asp Val Val Ser Pro Lys Ile Pro Ser Gly

260 265 270

Phe Pro Ile Glu Asp His Glu Thr Ser Pro Leu Asp Asn Ser Asp Ala

275 280 285

Phe Leu Ser Thr His Leu Lys Leu Phe Pro Ser Ala Ser Ser Asn Ser

290 295 300

Phe Tyr Glu Ser Glu Leu Pro Gln Gly Phe Gln Asp Tyr Tyr Asn Leu

305 310 315 320

Gly Gln Phe Thr Asp Asp Asp Gln Val Pro Thr Pro Leu Val Ala Ile

325 330 335

Thr Ile Thr Arg Leu Val Val Gly Phe Leu Leu Pro Ser Val Ile Met

340 345 350

Ile Ala Cys Tyr Ser Phe Ile Val Phe Arg Met Gln Arg Gly Arg Phe

355 360 365

Ala Lys Ser Gln Ser Lys Thr Phe Arg Val Ala Val Val Val Val Ala

370 375 380

Val Phe Leu Val Cys Trp Thr Pro Tyr His Ile Phe Gly Val Leu Ser

385 390 395 400

Leu Leu Thr Asp Pro Glu Thr Pro Leu Gly Lys Thr Leu Met Ser Trp

405 410 415

Asp His Val Cys Ile Ala Leu Ala Ser Ala Asn Ser Cys Phe Asn Pro

420 425 430

Phe Leu Tyr Ala Leu Leu Gly Lys Asp Phe Arg Lys Lys Ala Arg Gln

435 440 445

Ser Ile Gln Gly Ile Leu Glu Ala Ala Phe Ser Glu Glu Leu Thr Arg

450 455 460

Ser Thr His Cys Pro Ser Asn Asn Val Ile Ser Glu Arg Asn Ser Thr

465 470 475 480

Thr Val

<210> 9

<211> 350

<212> PROTEIN

<213> Homo sapiens

<400> 9

Met Glu Thr Asn Ser Ser Leu Pro Thr Asn Ile Ser Gly Gly Thr Pro

1 5 10 15

Ala Val Ser Ala Gly Tyr Leu Phe Leu Asp Ile Ile Thr Tyr Leu Val

20 25 30

Phe Ala Val Thr Phe Val Leu Gly Val Leu Gly Asn Gly Leu Val Ile

35 40 45

Trp Val Ala Gly Phe Arg Met Thr His Thr Val Thr Thr Ile Ser Tyr

50 55 60

Leu Asn Leu Ala Val Ala Asp Phe Cys Phe Thr Ser Thr Leu Pro Phe

65 70 75 80

Phe Met Val Arg Lys Ala Met Gly Gly His Trp Pro Phe Gly Trp Phe

85 90 95

Leu Cys Lys Phe Leu Phe Thr Ile Val Asp Ile Asn Leu Phe Gly Ser

100 105 110

Val Phe Leu Ile Ala Leu Ile Ala Leu Asp Arg Cys Val Cys Val Leu

115 120 125

His Pro Val Trp Thr Gln Asn His Arg Thr Val Ser Leu Ala Lys Lys

130 135 140

Val Ile Ile Gly Pro Trp Val Met Ala Leu Leu Leu Thr Leu Pro Val

145 150 155 160

Ile Ile Arg Val Thr Thr Val Pro Gly Lys Thr Gly Thr Val Ala Cys

165 170 175

Thr Phe Asn Phe Ser Pro Trp Thr Asn Asp Pro Lys Glu Arg Ile Asn

180 185 190

Val Ala Val Ala Met Leu Thr Val Arg Gly Ile Ile Arg Phe Ile Ile

195 200 205

Gly Phe Ser Ala Pro Met Ser Ile Val Ala Val Ser Tyr Gly Leu Ile

210 215 220

Ala Thr Lys Ile His Lys Gln Gly Leu Ile Lys Ser Ser Pro Pro Leu

225 230 235 240

Arg Val Leu Ser Phe Val Ala Ala Ala Phe Phe Leu Cys Trp Ser Pro

245 250 255

Tyr Gln Val Val Ala Leu Ile Ala Thr Val Arg Ile Arg Glu Leu Leu

260 265 270

Gln Gly Met Tyr Lys Glu Ile Gly Ile Ala Val Asp Val Thr Ser Ala

275 280 285

Leu Ala Phe Phe Asn Ser Cys Leu Asn Pro Met Leu Tyr Val Phe Met

290 295 300

Gly Gln Asp Phe Arg Glu Arg Leu Ile His Ala Leu Pro Ala Ser Leu

305 310 315 320

Glu Arg Ala Leu Thr Glu Asp Ser Thr Gln Thr Ser Asp Thr Ala Thr

325 330 335

Asn Ser Thr Leu Pro Ser Ala Glu Val Ala Leu Gln Ala Lys

340 345 350

<210> 10

<211> 373

<212> PROTEIN

<213> Homo sapiens

<400> 10

Met Arg Met Glu Asp Glu Asp Tyr Asn Thr Ser Ile Ser Tyr Gly Asp

1 5 10 15

Glu Tyr Pro Asp Tyr Leu Asp Ser Ile Val Val Leu Glu Asp Leu Ser

20 25 30

Pro Leu Glu Ala Arg Val Thr Arg Ile Phe Leu Val Val Val Tyr Ser

35 40 45

Ile Val Cys Phe Leu Gly Ile Leu Gly Asn Gly Leu Val Ile Ile Ile

50 55 60

Ala Thr Phe Lys Met Lys Lys Thr Val Asn Met Val Trp Phe Leu Asn

65 70 75 80

Leu Ala Val Ala Asp Phe Leu Phe Asn Val Phe Leu Pro Ile His Ile

85 90 95

Thr Tyr Ala Ala Met Asp Tyr His Trp Val Phe Gly Thr Ala Met Cys

100 105 110

Lys Ile Ser Asn Phe Leu Leu Ile His Asn Met Phe Thr Ser Val Phe

115 120 125

Leu Leu Thr Ile Ile Ser Ser Asp Arg Cys Ile Ser Val Leu Leu Pro

130 135 140

Val Trp Ser Gln Asn His Arg Ser Val Arg Leu Ala Tyr Met Ala Cys

145 150 155 160

Met Val Ile Trp Val Leu Ala Phe Phe Leu Ser Ser Pro Ser Leu Val

165 170 175

Phe Arg Asp Thr Ala Asn Leu His Gly Lys Ile Ser Cys Phe Asn Asn

180 185 190

Phe Ser Leu Ser Thr Pro Gly Ser Ser Ser Trp Pro Thr His Ser Gln

195 200 205

Met Asp Pro Val Gly Tyr Ser Arg His Met Val Val Thr Val Thr Arg

210 215 220

Phe Leu Cys Gly Phe Leu Val Pro Val Leu Ile Ile Thr Ala Cys Tyr

225 230 235 240

Leu Thr Ile Val Cys Lys Leu His Arg Asn Arg Leu Ala Lys Thr Lys

245 250 255

Lys Pro Phe Lys Ile Ile Val Thr Ile Ile Ile Thr Phe Phe Leu Cys

260 265 270

Trp Cys Pro Tyr His Thr Leu Asn Leu Leu Glu Leu His His Thr Ala

275 280 285

Met Pro Gly Ser Val Phe Ser Leu Gly Leu Pro Leu Ala Thr Ala Leu

290 295 300

Ala Ile Ala Asn Ser Cys Met Asn Pro Ile Leu Tyr Val Phe Met Gly

305 310 315 320

Gln Asp Phe Lys Lys Phe Lys Val Ala Leu Phe Ser Arg Leu Val Asn

325 330 335

Ala Leu Ser Glu Asp Thr Gly His Ser Ser Tyr Pro Ser His Arg Ser

340 345 350

Phe Thr Lys Met Ser Ser Met Asn Glu Arg Thr Ser Met Asn Glu Arg

355 360 365

Glu Thr Gly Met Leu

370

<210> 11

<211> 216

<212> PROTEIN

<213> Homo sapiens

<400> 11

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

1 5 10 15

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

20 25 30

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

35 40 45

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

50 55 60

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

65 70 75 80

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

85 90 95

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

100 105 110

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

115 120 125

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

130 135 140

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

145 150 155 160

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

165 170 175

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

180 185 190

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

195 200 205

Ser Leu Ser Leu Ser Pro Gly Lys

210 215

<210> 12

<211> 31

<212> PROTEIN

<213> Homo sapiens

<400> 12

Met Asp Ser Phe Asn Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp

1 5 10 15

Lys Asp Thr Leu Asp Leu Asn Thr Pro Val Asp Lys Thr Ser Asn

20 25 30

<210> 13

<211> 37

<212> PROTEIN

<213> Homo sapiens

<400> 13

Met Asp Ser Phe Asn Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp

1 5 10 15

Lys Asp Thr Leu Asp Leu Asn Thr Pro Val Asp Lys Thr Ser Asn Thr

20 25 30

Leu Arg Val Pro Asp

35

<210> 14

<211> 37

<212> PROTEIN

<213> Macaca fascicularis

<400> 14

Met Asp Pro Phe Ser Ser Thr Thr Leu Asp Tyr Glu His Tyr Asp Gly

1 5 10 15

Lys Asn Val Leu Asp Ser Asp Thr Pro Val Asp Lys Thr Ser Asn Thr

20 25 30

Leu Arg Val Pro Asp

35

<210> 15

<211> 398

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 15

Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser

1 5 10 15

Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro

20 25 30

Gln Gln Tyr Leu Ser Tyr Asn Ser Leu Arg Gly Glu Ala Glu Pro Cys

35 40 45

Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu

50 55 60

Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys

65 70 75 80

Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys

85 90 95

Glu Leu Gly Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu

100 105 110

Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly

115 120 125

Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln

130 135 140

Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro

145 150 155 160

His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp

165 170 175

Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Ser Ser Pro Gly

180 185 190

Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu

195 200 205

Gln Leu Arg Phe Leu Arg Asn Gly Leu Ala Ala Gly Thr Gly Gln Gly

210 215 220

Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu

225 230 235 240

Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His

245 250 255

Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Ser Pro Ala Lys

260 265 270

Ser Ser Val Asn Ser Arg Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys

275 280 285

Ile Glu Trp His Glu His His His His His Ile Gln Arg Thr Pro

290 295 300

Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn

305 310 315 320

Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val

325 330 335

Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp

340 345 350

Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu

355 360 365

Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val

370 375 380

Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

385 390 395

<210> 16

<211> 398

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 16

Ala Glu Ser His Leu Ser Leu Leu Tyr His Leu Thr Ala Val Ser Ser

1 5 10 15

Pro Ala Pro Gly Thr Pro Ala Phe Trp Val Ser Gly Trp Leu Gly Pro

20 25 30

Gln Gln Tyr Leu Ser Tyr Asp Ser Leu Arg Gly Gln Ala Glu Pro Cys

35 40 45

Gly Ala Trp Val Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu

50 55 60

Thr Thr Asp Leu Arg Ile Lys Glu Lys Leu Phe Leu Glu Ala Phe Lys

65 70 75 80

Ala Leu Gly Gly Lys Gly Pro Tyr Thr Leu Gln Gly Leu Leu Gly Cys

85 90 95

Glu Leu Ser Pro Asp Asn Thr Ser Val Pro Thr Ala Lys Phe Ala Leu

100 105 110

Asn Gly Glu Glu Phe Met Asn Phe Asp Leu Lys Gln Gly Thr Trp Gly

115 120 125

Gly Asp Trp Pro Glu Ala Leu Ala Ile Ser Gln Arg Trp Gln Gln Gln

130 135 140

Asp Lys Ala Ala Asn Lys Glu Leu Thr Phe Leu Leu Phe Ser Cys Pro

145 150 155 160

His Arg Leu Arg Glu His Leu Glu Arg Gly Arg Gly Asn Leu Glu Trp

165 170 175

Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Gly Asn Pro Gly

180 185 190

Phe Ser Val Leu Thr Cys Ser Ala Phe Ser Phe Tyr Pro Pro Glu Leu

195 200 205

Gln Leu Arg Phe Leu Arg Asn Gly Met Ala Ala Gly Thr Gly Gln Gly

210 215 220

Asp Phe Gly Pro Asn Ser Asp Gly Ser Phe His Ala Ser Ser Ser Leu

225 230 235 240

Thr Val Lys Ser Gly Asp Glu His His Tyr Cys Cys Ile Val Gln His

245 250 255

Ala Gly Leu Ala Gln Pro Leu Arg Val Glu Leu Glu Thr Pro Ala Lys

260 265 270

Ser Ser Val Asn Ser Arg Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys

275 280 285

Ile Glu Trp His Glu His His His His His Ile Gln Arg Thr Pro

290 295 300

Lys Ile Gln Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys Pro Asn

305 310 315 320

Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val

325 330 335

Asp Leu Leu Lys Asn Gly Glu Lys Met Gly Lys Val Glu His Ser Asp

340 345 350

Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu

355 360 365

Phe Thr Pro Asn Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val

370 375 380

Thr Leu Ser Gly Pro Arg Thr Val Lys Trp Asp Arg Asp Met

385 390 395

<210> 17

<211> 400

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 17

Ser Glu Thr Arg Pro Pro Leu Met Tyr His Leu Thr Ala Val Ser Asn

1 5 10 15

Pro Ser Thr Gly Leu Pro Ser Phe Trp Ala Thr Gly Trp Leu Gly Pro

20 25 30

Gln Gln Tyr Leu Thr Tyr Asn Ser Leu Arg Gln Glu Ala Asp Pro Cys

35 40 45

Gly Ala Trp Met Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu

50 55 60

Thr Thr Asp Leu Lys Ser Lys Glu Gln Leu Phe Leu Glu Ala Leu Lys

65 70 75 80

Thr Leu Glu Lys Ile Leu Asn Gly Thr Tyr Thr Leu Gln Gly Leu Leu

85 90 95

Gly Cys Glu Leu Ala Ser Asp Asn Ser Ser Val Pro Thr Ala Val Phe

100 105 110

Ala Leu Asn Gly Glu Glu Phe Met Lys Phe Asn Pro Arg Ile Gly Asn

115 120 125

Trp Thr Gly Glu Trp Pro Glu Thr Glu Ile Val Ala Asn Leu Trp Met

130 135 140

Lys Gln Pro Asp Ala Ala Arg Lys Glu Ser Glu Phe Leu Leu Asn Ser

145 150 155 160

Cys Pro Glu Arg Leu Leu Gly His Leu Glu Arg Gly Arg Arg Asn Leu

165 170 175

Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Gly Asn

180 185 190

Ser Gly Ser Ser Val Leu Thr Cys Ala Ala Phe Ser Phe Tyr Pro Pro

195 200 205

Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly Leu Ala Ser Gly Ser Gly

210 215 220

Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly Ser Phe His Ala Trp Ser

225 230 235 240

Leu Leu Glu Val Lys Arg Gly Asp Glu His His Tyr Gln Cys Gln Val

245 250 255

Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val Asp Leu Asp Ser Ser

260 265 270

Ala Arg Ser Ser Val Asn Ser Arg Gly Leu Asn Asp Ile Phe Glu Ala

275 280 285

Gln Lys Ile Glu Trp His Glu His His His His His Ile Gln Lys

290 295 300

Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys

305 310 315 320

Pro Asn Ile Leu Asn Cys Tyr Val Thr Gln Phe His Pro Pro His Ile

325 330 335

Glu Ile Gln Met Leu Lys Asn Gly Lys Lys Ile Pro Lys Val Glu Met

340 345 350

Ser Asp Met Ser Phe Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His

355 360 365

Thr Glu Phe Thr Pro Thr Glu Thr Asp Thr Tyr Ala Cys Arg Val Lys

370 375 380

His Asp Ser Met Ala Glu Pro Lys Thr Val Tyr Trp Asp Arg Asp Met

385 390 395 400

<210> 18

<211> 274

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 18

Gln Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val

1 5 10 15

Ser Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His

20 25 30

Leu Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr

35 40 45

Gln Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp

50 55 60

Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro

65 70 75 80

Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser

85 90 95

Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp

100 105 110

Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala

115 120 125

Phe Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn

130 135 140

Ile Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg

145 150 155 160

Tyr Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala

165 170 175

Pro Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu

180 185 190

Val Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu

195 200 205

Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg

210 215 220

Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser

225 230 235 240

Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys

245 250 255

Arg Ser Pro Glu Leu Glu Leu Gln Val Asn Ser Arg His His His His

260 265 270

His His

<210> 19

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 19

Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

1 5 10 15

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

20 25 30

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

35 40 45

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

50 55 60

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

65 70 75 80

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

85 90 95

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

100 105 110

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

115 120 125

Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

130 135 140

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

145 150 155 160

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

165 170 175

Val Asn Ser Arg His His His His His

180 185

<210> 20

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 20

Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp

1 5 10 15

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala

20 25 30

Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

35 40 45

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

50 55 60

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

65 70 75 80

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

85 90 95

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser

100 105 110

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

115 120 125

Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala

130 135 140

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

145 150 155 160

Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser

165 170 175

Val Asn Ser Arg His His His His His

180 185

<210> 21

<211> 187

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 21

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

1 5 10 15

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

20 25 30

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

35 40 45

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

50 55 60

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

65 70 75 80

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

85 90 95

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

100 105 110

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

115 120 125

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

130 135 140

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe

145 150 155 160

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

165 170 175

Gly Val Asn Ser Arg His His His His His

180 185

<210> 22

<211> 187

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 22

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

1 5 10 15

Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

20 25 30

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

35 40 45

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

50 55 60

Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

65 70 75 80

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

85 90 95

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

100 105 110

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

115 120 125

Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro

130 135 140

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

145 150 155 160

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

165 170 175

Gly Val Asn Ser Arg His His His His His

180 185

<210> 23

<211> 186

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 23

Thr Pro Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Gln Trp

1 5 10 15

Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Arg Gly Thr

20 25 30

His Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu

35 40 45

Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn

50 55 60

Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp

65 70 75 80

Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr Pro

85 90 95

His Leu Glu Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys His Ser

100 105 110

Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys

115 120 125

Ser Lys Lys Phe Ser Arg Ser Asp Pro Asn Phe Ser Ile Pro Gln Ala

130 135 140

Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr

145 150 155 160

Thr Leu Tyr Ser Ser Lys Pro Val Thr Ile Thr Val Gln Ala Pro Ser

165 170 175

Asp Asn Ser Arg His His His His His

180 185

<210> 24

<211> 907

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 24

Asp Gly Ser His His His His His Gly Thr Met Asp Ser Phe Asn

1 5 10 15

Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp Lys Asp Thr Leu Asp

20 25 30

Leu Asn Thr Pro Val Asp Lys Thr Ser Asn Thr Leu Arg Val Pro Asp

35 40 45

Ile Leu Ala Leu Val Ile Phe Ala Val Val Phe Leu Val Gly Val Leu

50 55 60

Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe Glu Ala Lys Arg Thr

65 70 75 80

Ile Asn Ala Ile Trp Phe Leu Asn Leu Ala Val Ala Asp Phe Leu Ser

85 90 95

Cys Leu Ala Leu Pro Ile Leu Phe Thr Ser Ile Val Gln His His His

100 105 110

Trp Pro Phe Gly Gly Ala Ala Cys Ser Ile Leu Pro Ser Leu Ile Leu

115 120 125

Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala Thr Ile Ser Ala Asp

130 135 140

Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys Gln Asn Phe Arg Gly

145 150 155 160

Ala Gly Leu Ala Trp Ile Ala Cys Ala Val Ala Trp Gly Leu Ala Leu

165 170 175

Leu Leu Thr Ile Pro Ser Phe Leu Tyr Arg Val Val Arg Glu Glu Tyr

180 185 190

Phe Pro Pro Lys Val Leu Cys Gly Val Asp Tyr Ser His Asp Lys Arg

195 200 205

Arg Glu Arg Ala Val Ala Ile Val Arg Leu Val Leu Gly Phe Leu Trp

210 215 220

Pro Leu Leu Thr Leu Thr Ile Cys Tyr Thr Phe Ile Leu Leu Arg Thr

225 230 235 240

Trp Ser Arg Arg Ala Thr Arg Ser Thr Lys Thr Leu Lys Val Val Val

245 250 255

Ala Val Val Ala Ser Phe Phe Ile Phe Trp Leu Pro Tyr Gln Val Thr

260 265 270

Gly Ile Met Met Ser Phe Leu Glu Pro Ser Ser Pro Thr Phe Leu Leu

275 280 285

Leu Lys Lys Leu Asp Ser Leu Cys Val Ser Phe Ala Tyr Ile Asn Cys

290 295 300

Cys Ile Asn Pro Ile Ile Tyr Val Val Ala Gly Gln Gly Phe Gln Gly

305 310 315 320

Arg Leu Arg Lys Ser Leu Pro Ser Leu Leu Arg Asn Val Leu Thr Glu

325 330 335

Glu Ser Val Val Arg Glu Ser Lys Ser Phe Thr Arg Ser Thr Val Asp

340 345 350

Thr Met Ala Gln Lys Thr Gln Ala Val Asp Ile Asp Tyr Lys Asp Asp

355 360 365

Asp Asp Lys Ile Glu Gly Arg Met Asp Gly Ala Arg Ala Ser Val Leu

370 375 380

Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys Ile Arg Leu Arg Pro Gly

385 390 395 400

Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val Trp Ala Ser Arg Glu

405 410 415

Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly

420 425 430

Cys Arg Gln Ile Leu Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly Ser

435 440 445

Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val

450 455 460

His Gln Arg Ile Glu Ile Lys Asp Thr Lys Glu Ala Leu Asp Lys Ile

465 470 475 480

Glu Glu Glu Gln Asn Lys Ser Lys Lys Lys Ala Gln Gln Ala Ala Ala

485 490 495

Asp Thr Gly His Ser Ser Gln Val Ser Gln Asn Tyr Pro Ile Val Gln

500 505 510

Asn Ile Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu

515 520 525

Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val

530 535 540

Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu

545 550 555 560

Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met

565 570 575

Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His

580 585 590

Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg

595 600 605

Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly

610 615 620

Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg

625 630 635 640

Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr

645 650 655

Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr

660 665 670

Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln Glu

675 680 685

Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro

690 695 700

Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu

705 710 715 720

Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala

725 730 735

Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Asn Thr Ala Thr Ile

740 745 750

Met Met Gln Arg Gly Asn Phe Arg Asn Gln Arg Lys Met Val Lys Cys

755 760 765

Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys Arg Ala Pro

770 775 780

Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln Met Lys

785 790 795 800

Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys Ile Trp Pro Ser

805 810 815

Tyr Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser Arg Pro Glu Pro Thr

820 825 830

Ala Pro Pro Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu

835 840 845

Glu Ser Phe Arg Ser Gly Val Glu Thr Thr Thr Pro Pro Gln Lys Gln

850 855 860

Glu Pro Ile Asp Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser Leu

865 870 875 880

Phe Gly Asn Asp Pro Ser Ser Gln Val Asn Ser Arg Gly Leu Asn Asp

885 890 895

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

900 905

<210> 25

<211> 874

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 25

Asp Gly Ser His His His His His Gly Thr Met Asn Ser Phe Asn

1 5 10 15

Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp Lys Asp Thr Leu Asp

20 25 30

Leu Asn Thr Pro Val Asp Lys Thr Ser Asn Thr Leu Arg Val Pro Asp

35 40 45

Ile Leu Ala Leu Val Ile Phe Ala Val Val Phe Leu Val Gly Val Leu

50 55 60

Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe Glu Ala Lys Arg Thr

65 70 75 80

Ile Asn Ala Ile Trp Phe Leu Asn Leu Ala Val Ala Asp Phe Leu Ser

85 90 95

Cys Leu Ala Leu Pro Ile Leu Phe Thr Ser Ile Val Gln His His His

100 105 110

Trp Pro Phe Gly Gly Ala Ala Cys Ser Ile Leu Pro Ser Leu Ile Leu

115 120 125

Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala Thr Ile Ser Ala Asp

130 135 140

Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys Gln Asn Phe Arg Gly

145 150 155 160

Ala Gly Leu Ala Trp Ile Ala Cys Ala Val Ala Trp Gly Leu Ala Leu

165 170 175

Leu Leu Thr Ile Pro Ser Phe Leu Tyr Arg Val Val Arg Glu Glu Tyr

180 185 190

Phe Pro Pro Lys Val Leu Cys Gly Val Asp Tyr Ser His Asp Lys Arg

195 200 205

Arg Glu Arg Ala Val Ala Ile Val Arg Leu Val Leu Gly Phe Leu Trp

210 215 220

Pro Leu Leu Thr Leu Thr Ile Cys Tyr Thr Phe Ile Leu Leu Arg Thr

225 230 235 240

Trp Ser Arg Arg Ala Thr Arg Ser Thr Lys Thr Leu Lys Val Val Val

245 250 255

Ala Val Val Ala Ser Phe Phe Ile Phe Trp Leu Pro Tyr Gln Val Thr

260 265 270

Gly Ile Met Met Ser Phe Leu Glu Pro Ser Ser Pro Thr Phe Leu Leu

275 280 285

Leu Asn Lys Leu Asp Ser Leu Cys Val Ser Phe Ala Tyr Ile Asn Cys

290 295 300

Cys Ile Asn Pro Ile Ile Tyr Val Val Ala Gly Gln Gly Phe Gln Gly

305 310 315 320

Arg Leu Arg Lys Ser Leu Pro Ser Leu Leu Arg Asn Val Leu Thr Glu

325 330 335

Glu Ser Val Val Arg Glu Ser Lys Ser Phe Thr Arg Ser Thr Val Asp

340 345 350

Thr Met Ala Gln Lys Thr Gln Ala Val Asp Ile Gly Ala Arg Ala Ser

355 360 365

Val Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys Ile Arg Leu Arg

370 375 380

Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val Trp Ala Ser

385 390 395 400

Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser

405 410 415

Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu Gln Pro Ser Leu Gln Thr

420 425 430

Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr

435 440 445

Cys Val His Gln Arg Ile Glu Ile Lys Asp Thr Lys Glu Ala Leu Asp

450 455 460

Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Lys Ala Gln Gln Ala

465 470 475 480

Ala Ala Asp Thr Gly His Ser Ser Gln Val Ser Gln Asn Tyr Pro Ile

485 490 495

Val Gln Asn Ile Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg

500 505 510

Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro

515 520 525

Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln

530 535 540

Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met

545 550 555 560

Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg

565 570 575

Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu

580 585 590

Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln

595 600 605

Ile Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr

610 615 620

Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser

625 630 635 640

Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg

645 650 655

Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser

660 665 670

Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala

675 680 685

Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr

690 695 700

Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His

705 710 715 720

Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Asn Thr Ala

725 730 735

Thr Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Arg Lys Met Val

740 745 750

Lys Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys Arg

755 760 765

Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln

770 775 780

Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys Ile Trp

785 790 795 800

Pro Ser Tyr Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser Arg Pro Glu

805 810 815

Pro Thr Ala Pro Pro Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro

820 825 830

Pro Glu Glu Ser Phe Arg Ser Gly Val Glu Thr Thr Thr Pro Pro Gln

835 840 845

Lys Gln Glu Pro Ile Asp Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg

850 855 860

Ser Leu Phe Gly Asn Asp Pro Ser Ser Gln

865 870

<210> 26

<211> 908

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 26

Asp Gly Ser His His His His His Gly Thr Met Asp Pro Ile Asp

1 5 10 15

Asn Ser Ser Phe Glu Ile Asn Tyr Asp His Tyr Gly Thr Met Ala Pro

20 25 30

Asn Ile Pro Ala Asp Gly Ile His Leu Pro Lys Arg Gln Pro Gly Asp

35 40 45

Val Ala Ala Leu Ile Ile Tyr Ser Val Val Phe Leu Val Gly Val Pro

50 55 60

Gly Asn Ala Leu Val Val Trp Val Thr Ala Phe Glu Ala Arg Arg Ala

65 70 75 80

Val Asn Ala Ile Trp Phe Leu Asn Leu Ala Val Ala Asp Leu Leu Ser

85 90 95

Cys Leu Ala Leu Pro Val Leu Phe Thr Thr Val Leu Asn His Asn Tyr

100 105 110

Trp Tyr Phe Asp Ala Thr Ala Cys Ile Val Leu Pro Ser Leu Ile Leu

115 120 125

Leu Asn Met Tyr Ala Ser Ile Leu Leu Leu Ala Thr Ile Ser Ala Asp

130 135 140

Arg Phe Leu Leu Val Phe Lys Pro Ile Trp Cys Gln Lys Val Arg Gly

145 150 155 160

Thr Gly Leu Ala Trp Met Ala Cys Gly Val Ala Trp Val Leu Ala Leu

165 170 175

Leu Leu Thr Ile Pro Ser Phe Val Tyr Arg Glu Ala Tyr Lys Asp Phe

180 185 190

Tyr Ser Glu His Thr Val Cys Gly Ile Asn Tyr Gly Gly Gly Ser Phe

195 200 205

Pro Lys Glu Lys Ala Val Ala Ile Leu Arg Leu Met Val Gly Phe Val

210 215 220

Leu Pro Leu Leu Thr Leu Asn Ile Cys Tyr Thr Phe Leu Leu Leu Arg

225 230 235 240

Thr Trp Ser Arg Lys Ala Thr Arg Ser Thr Lys Thr Leu Lys Val Val

245 250 255

Met Ala Val Val Ile Cys Phe Phe Ile Phe Trp Leu Pro Tyr Gln Val

260 265 270

Thr Gly Val Met Ile Ala Trp Leu Pro Pro Ser Ser Pro Thr Leu Lys

275 280 285

Arg Val Glu Lys Leu Asn Ser Leu Cys Val Ser Leu Ala Tyr Ile Asn

290 295 300

Cys Cys Val Asn Pro Ile Ile Tyr Val Met Ala Gly Gln Gly Phe His

305 310 315 320

Gly Arg Leu Leu Arg Ser Leu Pro Ser Ile Ile Arg Asn Ala Leu Ser

325 330 335

Glu Asp Ser Val Gly Arg Asp Ser Lys Thr Phe Thr Pro Ser Thr Thr

340 345 350

Asp Thr Ser Thr Arg Lys Ser Gln Ala Val Asp Ile Asp Tyr Lys Asp

355 360 365

Asp Asp Asp Lys Ile Glu Gly Arg Met Asp Gly Ala Arg Ala Ser Val

370 375 380

Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys Ile Arg Leu Arg Pro

385 390 395 400

Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val Trp Ala Ser Arg

405 410 415

Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser Glu

420 425 430

Gly Cys Arg Gln Ile Leu Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly

435 440 445

Ser Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys

450 455 460

Val His Gln Arg Ile Glu Ile Lys Asp Thr Lys Glu Ala Leu Asp Lys

465 470 475 480

Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Lys Ala Gln Gln Ala Ala

485 490 495

Ala Asp Thr Gly His Ser Ser Gln Val Ser Gln Asn Tyr Pro Ile Val

500 505 510

Gln Asn Ile Gln Gly Gln Met Val His Gln Ala Ile Ser Pro Arg Thr

515 520 525

Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu

530 535 540

Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp

545 550 555 560

Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln

565 570 575

Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val

580 585 590

His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro

595 600 605

Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile

610 615 620

Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys

625 630 635 640

Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro

645 650 655

Thr Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Arg Asp

660 665 670

Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ser Gln

675 680 685

Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn

690 695 700

Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu

705 710 715 720

Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys

725 730 735

Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Asn Thr Ala Thr

740 745 750

Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Arg Lys Met Val Lys

755 760 765

Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala Arg Asn Cys Arg Ala

770 775 780

Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln Met

785 790 795 800

Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu Gly Lys Ile Trp Pro

805 810 815

Ser Tyr Lys Gly Arg Pro Gly Asn Phe Leu Gln Ser Arg Pro Glu Pro

820 825 830

Thr Ala Pro Pro Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro

835 840 845

Glu Glu Ser Phe Arg Ser Gly Val Glu Thr Thr Thr Pro Pro Gln Lys

850 855 860

Gln Glu Pro Ile Asp Lys Glu Leu Tyr Pro Leu Thr Ser Leu Arg Ser

865 870 875 880

Leu Phe Gly Asn Asp Pro Ser Ser Gln Val Asn Ser Arg Gly Leu Asn

885 890 895

Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

900 905

<210> 27

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 27

Ser Tyr Ala Met His

15

<210> 28

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 28

Arg Ile Lys Ser Lys Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala His

1 5 10 15

Val Lys Gly

<210> 29

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 29

Val Ser Phe Ser Thr Phe Asp Val

15

<210> 30

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 30

Gly Phe Thr Phe Ser Ser Tyr

15

<210> 31

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 31

Lys Ser Lys Ala Gln Gly Gly Thr

15

<210> 32

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 32

Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr Tyr Val Ser

1 5 10

<210> 33

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 33

Arg Asn Asn Gln Arg Pro Ser

15

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 34

Asp Ser Trp Asp His Ser Ser Met Asn Val

1 5 10

<210> 35

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

His Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Ser Phe Ser Thr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 36

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 36

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Val Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Trp Asp His Ser Ser

85 90 95

Met Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

<210> 37

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

His Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Ser Phe Ser Thr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 38

<211> 215

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 38

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Val Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Trp Asp His Ser Ser

85 90 95

Met Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 39

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 39

Arg Ile Lys Ser Val Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala His

1 5 10 15

Val Lys Gly

<210> 40

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 40

Val Ser His Ser Thr Phe Asp Val

15

<210> 41

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 41

Lys Ser Val Ala Gln Gly Gly Thr

15

<210> 42

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Val Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

His Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Ser His Ser Thr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 43

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 43

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Val Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Trp Asp His Ser Ser

85 90 95

Met Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

<210> 44

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Val Ala Gln Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

His Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Val Ser His Ser Thr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 45

<211> 215

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 45

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Val Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asp Ser Trp Asp His Ser Ser

85 90 95

Met Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 46

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 46

agctatgcga tgcac

15

<210> 47

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 47

cgtatcaaat ccaaagccca gggcggtacg accgactacg cggcgcacgt gaaaggc

57

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 48

gtttctttct ccactttcga tgtt

24

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 49

ggatttacct tcagcagcta t

21

<210> 50

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 50

aaatccaaag cccagggcgg tacg

24

<210> 51

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 51

agcggcagct cctccaatat tggtagctat tacgtgagc

39

<210> 52

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 52

cgtaataatc aacgtcctag c

21

<210> 53

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 53

gacagctggg atcacagctc catgaatgtt

thirty

<210> 54

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 54

gaggtgcaat tggtggaaag cggcggtggc ctggtgaaac caggcggcag cctgcgcctg

60

agctgcgccg cctccggatt taccttcagc agctatgcga tgcactgggt gcgccaggcc

120

ccgggcaaag gtctcgaatg ggtgggtcgt atcaaatcca aagcccaggg cggtacgacc

180

gactacgcgg cgcacgtgaa aggccgcttt accattagcc gcgatgattc gaaaaacacc

240

ctgtatctgc aaatgaacag cctgaaaacc gaagatacgg ccgtgtatta ttgcgcgcgt

300

gtttctttct ccactttcga tgtttggggc caaggcaccc tggtgactgt ctcgagc

357

<210> 55

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 55

cagagcgtgc tgacccagcc tcctagcgtg agcggtgcac cgggccagcg cgtgaccatt

60

agctgtagcg gcagctcctc caatattggt agctattacg tgagctggta tcagcagctg

120

ccgggcacgg cgccgaaagt tctgatctat cgtaataatc aacgtcctag cggcgtgccg

180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa

240

gcagaagatg aagcggatta ttactgcgac agctgggatc acagctccat gaatgttttt

300

ggcggcggta ccaagctgac cgtgctgggc cag

333

<210> 56

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 56

gaggtgcaat tggtggaaag cggcggtggc ctggtgaaac caggcggcag cctgcgcctg

60

agctgcgccg cctccggatt taccttcagc agctatgcga tgcactgggt gcgccaggcc

120

ccgggcaaag gtctcgaatg ggtgggtcgt atcaaatcca aagcccaggg cggtacgacc

180

gactacgcgg cgcacgtgaa aggccgcttt accattagcc gcgatgattc gaaaaacacc

240

ctgtatctgc aaatgaacag cctgaaaacc gaagatacgg ccgtgtatta ttgcgcgcgt

300

gtttctttct ccactttcga tgtttggggc caaggcaccc tggtgactgt ctcgagcgcg

360

tcgaccaaag gccccagcgt gttccctctg gcccccagca gcaagagcac ctctggcgga

420

acagccgccc tgggctgcct ggtcaaggac tacttccccg agcccgtgac cgtgtcctgg

480

aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctcca gagcagcggc

540

ctgtacagcc tgagcagcgt cgtgaccgtg cccagcagca gcctgggcac ccagacctac

600

atctgcaacg tgaaccacaa gcccagcaac acaaaggtgg acaagcgggt ggaacccaag

660

agctgcgaca agacccacac ctgtcccccc tgccctgccc ctgaagcgga gggagccccc

720

tccgtgttcc tgttcccccc aaagcctaag gacaccctga tgatcagccg gacccccgaa

780

gtgacctgcg tggtggtgga cgtgtcccac gaggaccctg aagtgaagtt taattggtac

840

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gtacaacagc

900

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag

960

tacaagtgca aggtgtccaa caaggccctg ccttcctcca tcgagaaaac catcagcaag

1020

gccaaaggcc agccccgcga gccccaggtg tacacactgc cccctagccg ggaagagatg

1080

accaagaacc aggtgtccct gacctgcctc gtgaagggct tctaccccag cgacattgcc

1140

gtggaatggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

1200

gacagcgacg gctcattctt cctgtacagc aagctgaccg tggacaagag ccggtggcag

1260

cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

1320

aagtccctga gcctgagccc cggcaag

1347

<210> 57

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 57

cagagcgtgc tgacccagcc tcctagcgtg agcggtgcac cgggccagcg cgtgaccatt

60

agctgtagcg gcagctcctc caatattggt agctattacg tgagctggta tcagcagctg

120

ccgggcacgg cgccgaaagt tctgatctat cgtaataatc aacgtcctag cggcgtgccg

180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa

240

gcagaagatg aagcggatta ttactgcgac agctgggatc acagctccat gaatgttttt

300

ggcggcggta ccaagctgac cgtgctgggc cagcccaaag ccgcccctag cgtgaccctg

360

ttccccccct cgagtgagga actccaggcc aacaaggcca ccctcgtgtg cctgatcagc

420

gacttctacc ctggcgccgt gaccgtggcc tggaaggccg atagcagccc tgtgaaggcc

480

ggcgtggaaa ccaccacccc cagcaagcag agcaacaaca aatacgccgc cagcagctac

540

ctgagcctga cccccgagca gtggaagtcc cacagatcct acagctgcca ggtcacacac

600

gagggcagca ccgtggaaaa gaccgtggcc cccaccgagt gcagc

645

<210> 58

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 58

agctatgcga tgcac

15

<210> 59

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 59

cgtatcaaat ccgtggccca gggcggtacg accgactacg cggcgcacgt gaaaggc

57

<210> 60

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 60

gtttctcatt ccactttcga tgtt

24

<210> 61

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 61

ggatttacct tcagcagcta t

21

<210> 62

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 62

aaatccgtgg cccagggcgg tacg

24

<210> 63

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 63

agcggcagct cctccaatat tggtagctat tacgtgagc

39

<210> 64

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 64

cgtaataatc aacgtcctag c

21

<210> 65

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 65

gacagctggg atcacagctc catgaatgtt

thirty

<210> 66

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 66

gaggtgcaat tggtggaaag cggcggtggc ctggtgaaac caggcggcag cctgcgcctg

60

agctgcgccg cctccggatt taccttcagc agctatgcga tgcactgggt gcgccaggcc

120

ccgggcaaag gtctcgaatg ggtgggtcgt atcaaatccg tggcccaggg cggtacgacc

180

gactacgcgg cgcacgtgaa aggccgcttt accattagcc gcgatgattc gaaaaacacc

240

ctgtatctgc aaatgaacag cctgaaaacc gaagatacgg ccgtgtatta ttgcgcgcgt

300

gtttctcatt ccactttcga tgtttggggc caaggcaccc tggtgactgt ctcgagc

357

<210> 67

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 67

cagagcgtgc tgacccagcc tcctagcgtg agcggtgcac cgggccagcg cgtgaccatt

60

agctgtagcg gcagctcctc caatattggt agctattacg tgagctggta tcagcagctg

120

ccgggcacgg cgccgaaagt tctgatctat cgtaataatc aacgtcctag cggcgtgccg

180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa

240

gcagaagatg aagcggatta ttactgcgac agctgggatc acagctccat gaatgttttt

300

ggcggcggta ccaagctgac cgtgctgggc cag

333

<210> 68

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 68

gaggtgcaat tggtggaaag cggcggtggc ctggtgaaac caggcggcag cctgcgcctg

60

agctgcgccg cctccggatt taccttcagc agctatgcga tgcactgggt gcgccaggcc

120

ccgggcaaag gtctcgaatg ggtgggtcgt atcaaatccg tggcccaggg cggtacgacc

180

gactacgcgg cgcacgtgaa aggccgcttt accattagcc gcgatgattc gaaaaacacc

240

ctgtatctgc aaatgaacag cctgaaaacc gaagatacgg ccgtgtatta ttgcgcgcgt

300

gtttctcatt ccactttcga tgtttggggc caaggcaccc tggtgactgt ctcgagcgcg

360

tcgaccaaag gccccagcgt gttccctctg gcccccagca gcaagagcac ctctggcgga

420

acagccgccc tgggctgcct ggtcaaggac tacttccccg agcccgtgac cgtgtcctgg

480

aactctggcg ccctgaccag cggcgtgcac acctttccag ccgtgctcca gagcagcggc

540

ctgtacagcc tgagcagcgt cgtgaccgtg cccagcagca gcctgggcac ccagacctac

600

atctgcaacg tgaaccacaa gcccagcaac acaaaggtgg acaagcgggt ggaacccaag

660

agctgcgaca agacccacac ctgtcccccc tgccctgccc ctgaagcgga gggagccccc

720

tccgtgttcc tgttcccccc aaagcctaag gacaccctga tgatcagccg gacccccgaa

780

gtgacctgcg tggtggtgga cgtgtcccac gaggaccctg aagtgaagtt taattggtac

840

gtggacggcg tggaagtgca caacgccaag accaagccca gagaggaaca gtacaacagc

900

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag

960

tacaagtgca aggtgtccaa caaggccctg ccttcctcca tcgagaaaac catcagcaag

1020

gccaaaggcc agccccgcga gccccaggtg tacacactgc cccctagccg ggaagagatg

1080

accaagaacc aggtgtccct gacctgcctc gtgaagggct tctaccccag cgacattgcc

1140

gtggaatggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

1200

gacagcgacg gctcattctt cctgtacagc aagctgaccg tggacaagag ccggtggcag

1260

cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

1320

aagtccctga gcctgagccc cggcaag

1347

<210> 69

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 69

cagagcgtgc tgacccagcc tcctagcgtg agcggtgcac cgggccagcg cgtgaccatt

60

agctgtagcg gcagctcctc caatattggt agctattacg tgagctggta tcagcagctg

120

ccgggcacgg cgccgaaagt tctgatctat cgtaataatc aacgtcctag cggcgtgccg

180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa

240

gcagaagatg aagcggatta ttactgcgac agctgggatc acagctccat gaatgttttt

300

ggcggcggta ccaagctgac cgtgctgggc cagcccaaag ccgcccctag cgtgaccctg

360

ttccccccct cgagtgagga actccaggcc aacaaggcca ccctcgtgtg cctgatcagc

420

gacttctacc ctggcgccgt gaccgtggcc tggaaggccg atagcagccc tgtgaaggcc

480

ggcgtggaaa ccaccacccc cagcaagcag agcaacaaca aatacgccgc cagcagctac

540

ctgagcctga cccccgagca gtggaagtcc cacagatcct acagctgcca ggtcacacac

600

gagggcagca ccgtggaaaa gaccgtggcc cccaccgagt gcagc

645

<210> 70

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 70

agctacgcta tgcac

15

<210> 71

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 71

cggatcaaga gcaaggctca aggcggcacc accgattacg ccgctcatgt gaagggc

57

<210> 72

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 72

gtgtccttct ccaccttcga tgtg

24

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 73

ggcttcacct tctccagcta c

21

<210> 74

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 74

aagagcaagg ctcaaggcgg cacc

24

<210> 75

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 75

tccggctcct cctccaacat cggctcctac tacgtgtcc

39

<210> 76

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 76

cggaacaacc agcggccttc t

21

<210> 77

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 77

gactcttggg accactcctc catgaacgtg

thirty

<210> 78

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 78

gaagtgcagc tggtggaatc tggcggcgga cttgtgaaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgcta tgcactgggt ccgacaggcc

120

cctggcaaag gattggagtg ggtcggacgg atcaagagca aggctcaagg cggcaccacc

180

gattacgccg ctcatgtgaa gggcagattc accatctctc gggacgactc caagaacacc

240

ctgtacctgc agatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcgccaga

300

gtgtccttct ccaccttcga tgtgtggggc cagggcacac tggttacagt ctcgagc

357

<210> 79

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 79

cagtccgtgc tgacccagcc tccttctgtt tctggtgctc ctggccagag agtgaccatc

60

tcttgctccg gctcctcctc caacatcggc tcctactacg tgtcctggta tcagcagctg

120

cctggcaccg ctcctaaggt gctgatctac cggaacaacc agcggccttc tggcgtgccc

180

gatagattct ccggctctaa gtctggcacc tctgccagcc tggctatcac tggactgcag

240

gctgaggacg aggccgacta ctactgcgac tcttgggacc actcctccat gaacgtgttc

300

ggcggaggta ccaagctgac cgtgctggga cag

333

<210> 80

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 80

gaagtgcagc tggtggaatc tggcggcgga cttgtgaaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgcta tgcactgggt ccgacaggcc

120

cctggcaaag gattggagtg ggtcggacgg atcaagagca aggctcaagg cggcaccacc

180

gattacgccg ctcatgtgaa gggcagattc accatctctc gggacgactc caagaacacc

240

ctgtacctgc agatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcgccaga

300

gtgtccttct ccaccttcga tgtgtggggc cagggcacac tggttacagt ctcgagcgcc

360

tccaccaaag gaccctctgt gtttcctctg gctccctcca gcaagtctac ctctggtgga

420

acagctgccc tgggctgcct ggtcaaggat tactttcctg agcctgtgac cgtgtcctgg

480

aactctggcg ctctgacatc tggcgtgcac acctttccag ctgtgctgca gtcctctggc

540

ctgtacagcc tgtcctctgt cgtgaccgtg ccttctagct ctctgggcac ccagacctac

600

atctgcaatg tgaaccacaa gccttccaac accaaggtgg acaagagagt ggaacccaag

660

tcctgcgaca agacccacac ctgtcctcca tgtcctgctc cagaagctga gggcgctcct

720

tccgtgttcc tgtttcctcc aaagcctaag gacaccctga tgatctctcg gacccctgaa

780

gtgacctgcg tggtggtgga tgtgtctcac gaggacccag aagtgaagtt caattggtac

840

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc

900

acctacagag tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag

960

tacaagtgca aggtgtccaa caaggccctg ccttccagca tcgaaaagac catctccaag

1020

gccaagggcc agcctaggga accccaggtt tacaccctgc ctccaagccg ggaagagatg

1080

accaagaacc aggtgtccct gacctgcctc gtgaagggct tctacccttc cgatatcgcc

1140

gtggaatggg agagcaatgg ccagcctgag aacaactaca agacaacccc tcctgtgctg

1200

gactccgacg gctcattctt cctgtactcc aagctgacag tggacaagtc cagatggcag

1260

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaatca ctacacacag

1320

aagtccctgt ctctgtcccc tggcaag

1347

<210> 81

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 81

cagtccgtgc tgacccagcc tccttctgtt tctggtgctc ctggccagag agtgaccatc

60

tcttgctccg gctcctcctc caacatcggc tcctactacg tgtcctggta tcagcagctg

120

cctggcaccg ctcctaaggt gctgatctac cggaacaacc agcggccttc tggcgtgccc

180

gatagattct ccggctctaa gtctggcacc tctgccagcc tggctatcac tggactgcag

240

gctgaggacg aggccgacta ctactgcgac tcttgggacc actcctccat gaacgtgttc

300

ggcggaggta ccaagctgac cgtgctggga cagcctaagg ctgccccttc cgtgacactg

360

ttccctccat cctctgagga actgcaggcc aacaaggcta ccctcgtgtg cctgatctcc

420

gacttttacc ctggcgctgt gaccgtggcc tggaaggctg atagttctcc tgtgaaggcc

480

ggcgtggaaa ccaccacacc ttccaagcag tccaacaaca aatacgccgc ctcctcctac

540

ctgtctctga cccctgaaca gtggaagtcc caccggtcct acagctgcca agtgacccat

600

gagggctcca ccgtggaaaa gaccgtggct cctaccgagt gctct

645

<210> 82

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 82

agctacgcta tgcac

15

<210> 83

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 83

cggatcaaga gcgttgccca aggcggcacc accgattacg ctgctcatgt gaagggc

57

<210> 84

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 84

gtgtcccact ctaccttcga tgtg

24

<210> 85

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 85

ggcttcacct tctccagcta c

21

<210> 86

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 86

aagagcgttg cccaaggcgg cacc

24

<210> 87

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 87

tccggctcct cctccaacat cggctcctac tacgtgtcc

39

<210> 88

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 88

cggaacaacc agcggccttc t

21

<210> 89

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 89

gactcttggg accactcctc catgaacgtg

thirty

<210> 90

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 90

gaagtgcagc tggtggaatc tggcggcgga cttgtgaaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgcta tgcactgggt ccgacaggcc

120

cctggcaaag gattggagtg ggtcggacgg atcaagagcg ttgcccaagg cggcaccacc

180

gattacgctg ctcatgtgaa gggcagattc accatcagcc gggacgactc caagaacacc

240

ctgtacctgc agatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcgccaga

300

gtgtcccact ctaccttcga tgtgtggggc cagggcacac tggttacagt ctcgagc

357

<210> 91

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 91

cagtccgtgc tgacccagcc tccttctgtt tctggtgctc ctggccagag agtgaccatc

60

tcttgctccg gctcctcctc caacatcggc tcctactacg tgtcctggta tcagcagctg

120

cctggcaccg ctcctaaggt gctgatctac cggaacaacc agcggccttc tggcgtgccc

180

gatagattct ccggctctaa gtctggcacc tctgccagcc tggctatcac tggactgcag

240

gctgaggacg aggccgacta ctactgcgac tcttgggacc actcctccat gaacgtgttc

300

ggcggaggta ccaagctgac cgtgctggga cag

333

<210> 92

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 92

gaagtgcagc tggtggaatc tggcggcgga cttgtgaaac ctggcggctc tctgagactg

60

tcttgtgccg cttccggctt caccttctcc agctacgcta tgcactgggt ccgacaggcc

120

cctggcaaag gattggagtg ggtcggacgg atcaagagcg ttgcccaagg cggcaccacc

180

gattacgctg ctcatgtgaa gggcagattc accatcagcc gggacgactc caagaacacc

240

ctgtacctgc agatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcgccaga

300

gtgtcccact ctaccttcga tgtgtggggc cagggcacac tggttacagt ctcgagcgcc

360

tccaccaaag gaccctctgt gtttcctctg gctccctcca gcaagtctac ctctggtgga

420

acagctgccc tgggctgcct ggtcaaggat tactttcctg agcctgtgac cgtgtcctgg

480

aactctggcg ctctgacatc tggcgtgcac acctttccag ctgtgctgca gtcctctggc

540

ctgtacagcc tgtcctctgt cgtgaccgtg ccttctagct ctctgggcac ccagacctac

600

atctgcaatg tgaaccacaa gccttccaac accaaggtgg acaagagagt ggaacccaag

660

tcctgcgaca agacccacac ctgtcctcca tgtcctgctc cagaagctga gggcgctcct

720

tccgtgttcc tgtttcctcc aaagcctaag gacaccctga tgatctctcg gacccctgaa

780

gtgacctgcg tggtggtgga tgtgtctcac gaggacccag aagtgaagtt caattggtac

840

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc

900

acctacagag tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag

960

tacaagtgca aggtgtccaa caaggccctg ccttccagca tcgaaaagac catctccaag

1020

gccaagggcc agcctaggga accccaggtt tacaccctgc ctccaagccg ggaagagatg

1080

accaagaacc aggtgtccct gacctgcctc gtgaagggct tctacccttc cgatatcgcc

1140

gtggaatggg agagcaatgg ccagcctgag aacaactaca agacaacccc tcctgtgctg

1200

gactccgacg gctcattctt cctgtactcc aagctgacag tggacaagtc cagatggcag

1260

cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaatca ctacacacag

1320

aagtccctgt ctctgtcccc tggcaag

1347

<210> 93

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polynucleotide"

<400> 93

cagtccgtgc tgacccagcc tccttctgtt tctggtgctc ctggccagag agtgaccatc

60

tcttgctccg gctcctcctc caacatcggc tcctactacg tgtcctggta tcagcagctg

120

cctggcaccg ctcctaaggt gctgatctac cggaacaacc agcggccttc tggcgtgccc

180

gatagattct ccggctctaa gtctggcacc tctgccagcc tggctatcac tggactgcag

240

gctgaggacg aggccgacta ctactgcgac tcttgggacc actcctccat gaacgtgttc

300

ggcggaggta ccaagctgac cgtgctggga cagcctaagg ctgccccttc cgtgacactg

360

ttccctccat cctctgagga actgcaggcc aacaaggcta ccctcgtgtg cctgatctcc

420

gacttttacc ctggcgctgt gaccgtggcc tggaaggctg atagttctcc tgtgaaggcc

480

ggcgtggaaa ccaccacacc ttccaagcag tccaacaaca aatacgccgc ctcctcctac

540

ctgtctctga cccctgaaca gtggaagtcc caccggtcct acagctgcca agtgacccat

600

gagggctcca ccgtggaaaa gaccgtggct cctaccgagt gctct

645

<210> 94

<211> 454

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asp Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Tyr Tyr Tyr Ala Ser Gly Ser Tyr Tyr Lys Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 95

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 95

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Arg

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 96

<211> 400

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 96

Ala Glu Pro Arg Leu Pro Leu Met Tyr His Leu Ala Ala Val Ser Asp

1 5 10 15

Leu Ser Thr Gly Leu Pro Ser Phe Trp Ala Thr Gly Trp Leu Gly Ala

20 25 30

Gln Gln Tyr Leu Thr Tyr Asn Asn Leu Arg Gln Glu Ala Asp Pro Cys

35 40 45

Gly Ala Trp Ile Trp Glu Asn Gln Val Ser Trp Tyr Trp Glu Lys Glu

50 55 60

Thr Thr Asp Leu Lys Ser Lys Glu Gln Leu Phe Leu Glu Ala Ile Arg

65 70 75 80

Thr Leu Glu Asn Gln Ile Asn Gly Thr Phe Thr Leu Gln Gly Leu Leu

85 90 95

Gly Cys Glu Leu Ala Pro Asp Asn Ser Ser Leu Pro Thr Ala Val Phe

100 105 110

Ala Leu Asn Gly Glu Glu Phe Met Arg Phe Asn Pro Arg Thr Gly Asn

115 120 125

Trp Ser Gly Glu Trp Pro Glu Thr Asp Ile Val Gly Asn Leu Trp Met

130 135 140

Lys Gln Pro Glu Ala Ala Arg Lys Glu Ser Glu Phe Leu Leu Thr Ser

145 150 155 160

Cys Pro Glu Arg Leu Leu Gly His Leu Glu Arg Gly Arg Gln Asn Leu

165 170 175

Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys Ala Arg Pro Gly Asn

180 185 190

Ser Gly Ser Ser Val Leu Thr Cys Ala Ala Phe Ser Phe Tyr Pro Pro

195 200 205

Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly Leu Ala Ser Gly Ser Gly

210 215 220

Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly Ser Phe His Ala Trp Ser

225 230 235 240

Leu Leu Glu Val Lys Arg Gly Asp Glu His His Tyr Gln Cys Gln Val

245 250 255

Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val Asp Leu Asp Ser Pro

260 265 270

Ala Arg Ser Ser Val Asn Ser Arg Gly Leu Asn Asp Ile Phe Glu Ala

275 280 285

Gln Lys Ile Glu Trp His Glu His His His His His Ile Gln Lys

290 295 300

Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro Glu Asn Gly Lys

305 310 315 320

Pro Asn Phe Leu Asn Cys Tyr Val Ser Gln Phe His Pro Pro Gln Ile

325 330 335

Glu Ile Glu Leu Leu Lys Asn Gly Lys Lys Ile Pro Asn Ile Glu Met

340 345 350

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Ile Leu Ala His

355 360 365

Thr Glu Phe Thr Pro Thr Glu Thr Asp Val Tyr Ala Cys Arg Val Lys

370 375 380

His Val Thr Leu Lys Glu Pro Lys Thr Val Thr Trp Asp Arg Asp Met

385 390 395 400

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Claims (29)

1. Isolated antibody or antibody fragment containing a) heavy chain complementarity determining region (HCDR1) under SEQ ID NO: 27, HCDR2 under SEQ ID NO: 28, HCDR3 under SEQ ID NO: 29, light chain complementarity determining region (LCDR1) under SEQ ID NO: 32, LCDR2 under SEQ ID NO: 33 and LCDR3 under SEQ ID NO: 34, or b) HCDR1 under SEQ ID NO: 27, HCDR2 under SEQ ID NO: 39, HCDR3 under SEQ ID NO: 40, LCDR1 under SEQ ID NO: 32, LCDR2 under SEQ ID NO: 33 and LCDR3 under SEQ ID NO: 34; wherein the isolated antibody or antibody fragment specifically binds to the human complement receptor C5a (C5aR). 2. An isolated antibody or antibody fragment according to claim 1, where said antibody or antibody fragment contains: a) a heavy chain variable region (VH) of SEQ ID NO: 35 and a light chain variable region (VL) of SEQ ID NO: 36, or b) VH under SEQ ID NO: 42 and VL under SEQ ID NO 43. 3. An isolated antibody or antibody fragment according to claim 1 or 2, where said antibody or antibody fragment contains: a) heavy chain (HC) of SEQ ID NO: 37 and light chain (LC) of SEQ ID NO: 38 or b) HC under SEQ ID NO: 44 and LC under SEQ ID NO: 45. 4. An isolated antibody or antibody fragment according to any of the previous paragraphs, wherein said antibody is of the human IgG1 class. 5. An isolated antibody or antibody fragment according to any of the previous paragraphs, wherein said antibody substantially does not induce effector function in vitro. 6. An isolated antibody or antibody fragment according to any of the previous paragraphs, where the specified antibody contains amino acid substitutions L234A, L235E, G237A, A330S and P331S, numbered according to the EU index. 7. The isolated antibody or antibody fragment of any one of the previous paragraphs, wherein said isolated antibody or antibody fragment is specific for human C5aR and cross-reacts with cynomolgus macaque C5aR. 8. The isolated antibody or antibody fragment of any one of the preceding claims, wherein the isolated antibody or antibody fragment inhibits human C5a-induced CD11b expression in human granulocytes at an IC50 concentration of 42 nM in the presence of 150 nM human C5a in vitro. 9. The isolated antibody or antibody fragment of any one of the preceding claims, wherein said isolated antibody or antibody fragment is a monoclonal antibody. 10. The isolated antibody or antibody fragment of any one of the preceding claims, wherein the isolated antibody or antibody fragment is a human or chimeric antibody. 11. Use of an isolated antibody or antibody fragment according to any of the preceding claims for the treatment of a disease associated with the undesirable presence of C5aR. 12. A nucleic acid molecule encoding an isolated antibody or antibody fragment according to any one of claims. 1-10. 13. An expression vector containing a nucleic acid molecule according to claim 12. 14. Host cell for obtaining an isolated antibody or antibody fragment according to any one of paragraphs. 1-10, containing the vector according to claim 13 or a nucleic acid molecule according to claim 12. 15. Pharmaceutical composition for the treatment of a disease associated with the undesirable presence of C5aR, containing an effective amount of an isolated antibody or antibody fragment according to claims. 1-10 and a pharmaceutically acceptable carrier or excipient. 16. A method for producing an isolated antibody or antibody fragment according to any one of claims 1 to 10, including: (a) obtaining a host cell according to claim 14 under conditions suitable for expression of the antibody or antibody fragment; And (b) isolating the antibody or antibody fragment. 17. The production method according to claim 16, further comprising purifying the isolated antibody or antibody fragment. 18. The production method according to claim 17, where the purification is carried out by high performance liquid chromatography (HPLC), ion exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography and/or gel permeation chromatography (SEC).

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