MX2013013911A - Therapeutic antibodies. - Google Patents

Abstract

The present invention concerns human antibodies recognising the human C5a receptor. By binding to C5aR the antibodies inhibit C5a signalling, whereby the pro- inflammatory signal is inhibited. Based on the role of C5a and its receptor in stimulation of inflammation the invention further relates to therapeutic use of said human anti-C5aR antibodies and in particular in relation to treatment of immunological disorders.

Description

THERAPEUTIC ANTIBODIES Field of the Invention The invention relates to the field of therapeutic antibodies.

Background of the Invention The proteolysis of each of the C3-C5 complement proteins gives rise to amino-terminal cationic fragments with signaling molecules called anaphylatoxins. The most powerful of these, C5a, produces the widest responses. By considering the components of the inflammatory response such as marginalization and leukocyte infiltration, the release of proteolytic enzymes bound to granules, the production of active oxygen and nitrogen-derived radicals, changes in blood flow and capillary leak, along with the ability to contracting from smooth muscle, the C5a molecule is the "complete" inflammatory mediator. At sub-nanomolar to nanomolar levels, the C5a molecule causes the chemotaxis of all myeloid lineages (neutrophils, eosinophils and basophils, macrophages and monocytes), and causes vascular permeability that is markedly enhanced by prostaglandins and circulating leukocytes. Higher nanomolar concentrations cause degranulation and activation of NADPH oxidase. This amplitude of bioactivity contrasts with other Ref. 244814 mediators of inflammation. C5a is involved in the pathogenesis of several disorders including rheumatoid arthritis, psoriasis, sepsis, reperfusion injury and respiratory distress syndrome in adults (Gerard and Gerard, 1994, Murdoch and Finn, 2000).

The activities of C5a are mediated by the binding of C5a to its receptor (C5aR). C5aR belongs to the family of seven transmembrane G protein-coupled receptors. C5aR is a high affinity receptor for C5a, with a Kd of ~ 1 nM, and is found in a number of different cell types including leukocytes. The number of receptors per cell is extremely high, of up to 200,000 sites per leukocyte. The biological activation of the receptor occurs in the interval that saturates the junction.

The structure of C5aR conforms to the family of transmembrane receptors of seven domains, with the extracellular N-tertninal end followed by seven transmembrane helices connected by interhelical domains alternating as intracellular and extracellular loops, and ending with a C-terminal domain intracellular C5aR contains an extended N-terminal extracellular domain. This large N-terminal domain is typical of G-protein coupled receptors that bind peptides that include the families of IL-8 and fMet-Leu-Phe (FMLP) receptors.

Inhibition of C5a responses with C5aR antagonists reduce the acute inflammatory response mediated by C5a without affecting other components of the complement.

For this purpose, antagonists of the C5aR peptide and anti-C5a receptor antibodies have been previously described (Watanabe et al, 1995, Pellas et al, 1998, Konteatis et al, 1994, Kaneko et al, 1995, Morgan et al., 1993). For example, WO 95/00164 describes antibodies directed against an N-terminal peptide (residues 9-29) of C5aR. WO 03/062278 also describes antibodies directed against C5aR. Three of these mouse antibodies were designated 7F3, 6C12 and 12D4. It has been shown that these antibodies have excellent properties, such as being very effective in blocking C5a binding to its receptor, stopping C5a directed migration of neutrophils in vivo, and preventing inflammation in animal models. To control chronic diseases, it may be necessary to administer the antibody on successive occasions for months or years. However, a drawback of the administration of mouse antibodies is that the human immune system can generate its own antibodies directed against the mouse antibody (the HAMA response). The HAMA response can neutralize mouse antibodies by rapidly clearing them from the blood, and thus prevent the mouse antibody from binding to its target. To avoid the development of a A HAMA response, a strategy that has been adopted is to "humanize" the mouse antibody by replacing so many "foreign" residues in non-epitope regions with human sequences.

A major problem with humanization procedures has been a loss of affinity for the antigen (Jones et al., 1986), in some cases as much as 10 times or more, especially when the antigen is a protein (Verhoeyen et al., 1988 ). The loss of any affinity is, of course, highly undesirable. At least, this means that more of the humanized antibody will have to be injected into the patient, at a higher cost and a higher risk of adverse effects. Even more critically, an antibody with reduced affinity may have poorer biological functions, such as complement lysis, antibody-dependent cellular cytotoxicity, or virus neutralization. Although faced with these difficulties, the successful humanization of human anti-C5aR antibodies has been described in WO 2009/103113.

A number of strategies have been developed over the years to further minimize the risk of any undesired secondary reaction to the administration of antibodies to patients, which includes reducing the likelihood of antibody formation. against drugs in patients by the generation of "totally" human antibodies.

Even today the identification of antibodies suitable for therapeutic applications is a challenging task. Therefore, alternative and / or improved C5aR antagonists that can be used in diagnostic and / or therapeutic methods are of great interest.

Summary of the Invention The present invention relates to anti-C5aR antibodies and their use for diagnostic and / or therapeutic methods. The inventors have identified a series of antibodies that bind to human C5aR that are in several aspects functionally superior to the anti-C5aR antibodies described above.

As demonstrated herein, the inventors have identified a series of human antibodies that bind to human C5aR (hC5aR) and can displace the binding of hC5a to hC5aR and inhibit neutrophil migration mediated by hC5a. In addition, the inventors have successfully converted non-human residues present in the framework region of one of these anti-hC5aR antibodies to human germline residues without affecting the potency of the antibody.

In addition, by altering the Fe region, the inventors have established an anti-hC5aR antibody that does not induce phagocytosis, ADCC or CDC in vitro. The details of the invention will be apparent from the description of the illustrative modalities.

One aspect of the invention relates to an antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following sequence groups, SEQ ID 1 , 2 and 3, SEQ ID 9, 10 and 11, SEQ ID 17, 18 and 19, SEQ ID 25, 26 and 27 or variants of each of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

One aspect of the invention relates to an antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following groups of sequences, SEQ ID 5 , 6 and 7, SEQ ID 13, 14 and 15, SEQ ID 21, 22 and 23, SEQ ID 29, 30 and 31, or variants of each of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

One aspect of the invention relates to a human antibody that specifically binds to hC5aR, wherein that antibody preferably binds to the second extracellular loop of hC5aR.

One aspect of the invention relates to a human antibody that binds specifically to hC5aR, wherein the Fe region of antibody has been modified in comparison to reference sequences of IgG1, IgG2, IgG4 IgG4 / G2 that reduce the ability of antibodies to induce phagocytosis, ADCC and / or CDC through the interaction of the Fcgamma receptor (FcyR). In a particular embodiment, the Fe region of antibody is IgGl and in another particular embodiment, the Fe region comprises one or more of the following groups of point mutations.

I) N297Q and / or II) L234A and L235E and / or III) G236R and L328R and / or IV) N297Q, L234A and L235E and / or V) N297Q, L234A, L235E, and G237A and / or VI) L234A, L235E, G237A, A330 and P331S In a further aspect, the invention relates to the use of the antibodies according to the invention for the treatment of an immunological disease or disorder.

In a further aspect, the invention relates to a method for the treatment of a disease or disorder comprising administering to a subject in need thereof a therapeutic amount of an antibody as described herein.

In another aspect, the present invention provides a method for treating or preventing a disorder in a subject, the The method comprises administering to the subject an antibody of the invention. In one embodiment, the disorder is an immunopathological disorder such as an autoimmune disease.

Additional aspects and embodiments of the invention will be apparent from the present description which includes illustrative modalities. From the description it follows that the invention has provided new therapeutic antibodies with various benefits and advantages as characterized in this document.

Brief Description of the Figures Figure 1 shows alignments of the variable regions of a selection of monoclonal antibodies isolated and characterized in the application.

Figures 2A-2B show the binding specificity of a selection of antibodies to mouse and human C5aR chimeras. The binding of 32F3A6, 35F12A2 and 35F32A3 to mouse and human C5aR chimeras compared to the binding of Ref. Ab Q. Chimeric receptors are shown schematically. Regions derived from C5aR of human and mouse are shown with a thin line and a thick line, respectively.

Figure 3 shows alignments of the variable regions of an antibody with the variable heavy and light sequences of human germline antibody closest to each other. "/" indicates a break in the sequence, such as between segments V, D or J.

Figure 4 shows clinical scores for three treatment groups that received a single loading dose (arrow) of 0.5, 0.5 or 110 mg / kg i.p. 5 days after the established inflammation in the K / BxN-hC5aR-KO / KI serum transfer model, followed by 9 daily doses of 0.25, 0.5 or 2 mg / kg, respectively, with error bars representing + D.E. The controls received IgGl 3G12.

Figure 5 shows expression of the C5a protein in synovial fluid in patients with psoriatic arthritis and osteoarthritis (controls). The level of C5a was significantly elevated in the group of patients with psoriatic arthritis (p = 0.001, Mann-hitney).

Figures 6A-6D show the semi-quantitative analysis of C5aR protein expression in Crohn's disease and ulcerative colitis. The expression of the C5aR protein was investigated by immunohistochemistry and analyzed by the Kruskal-Walis test with Dunn's multiple comparison after the test on GraphPad Prism 5, and P < 0.05 was considered significant. * P < 0.05; ** P < 0.01; *** P0.001.

Detailed description of the invention Definitions Unless otherwise indicated, the recombinant protein, cell culture and immunological techniques used in the present invention are methods standards, well known to those skilled in the art. These techniques are described and explained in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John ile and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (compilers), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (compilers), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (compilers) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ( 1988), and JE Coligan et al. (compilers) Current Protocols in Immunology, John Wiley and Sons (including all updates to date).

As used herein, "C5a receptor", "C5aR", "C5aRI" or "human C5aR" and variations thereof refer to receptor 1 of component 5 of human complement which is also known in the art as the C5a anaphylatoxin receptor and CD88 antigen. C5aR belongs to the family of seven receptors coupled to transmembrane G protein, and binds to C5a (Gerard and Gerard, 1991). An example of the amino acid sequence of a human C5aR is provided in SEQ ID NO: 41, however, as will be considered by the person skilled in the art there will be natural allelic variants of this molecule, which are also encompassed by the term "C5aR". The various domains of human C5aR are defined as follows: amino acids 1-37: N-terminal extracellular domain, amino acids 38-61: transmembrane domain, amino acids 62-71: intracellular domain, amino acids 72-94: transmembrane domain, amino acids 95-110: extracellular domain - extracellular loop 1, amino acids 111-132: transmembrane domain, amino acids 133-149: intracellular domain, amino acids 150-174: transmembrane domain, amino acids 175-206: extracellular domain - extracellular loop 2, amino acids 207-227: transmembrane domain, amino acids 228-242: intracellular domain, amino acids 243-264: transmembrane domain, amino acids 265-283: extracellular domain - extracellular loop 3, amino acids 284-307: transmembrane domain, amino acids 308-350: C-terminal intracellular domain.

The term "treatment", as used herein, is refers to the medical therapy of any human being or other animal subject that needs it. It is estimated that this subject has undergone physical examination by a doctor or veterinarian, who has given a provisional or definitive diagnosis, which would indicate that the use of that specific treatment is beneficial for the health of that human being or another animal subject. The timing and purpose of this treatment may vary from one individual to another, according to the current health situation of the subject. Therefore, this treatment can be prophylactic, palliative, symptomatic and / or curative. In terms of the present invention, prophylactic, palliative, symptomatic and / or curative treatments may represent separate aspects of the invention.

In relation to medical treatment, by the term "subject", as used herein, is meant any animal, in particular mammals, such as humans, horses, cows, cats and dogs, and where appropriate may be used interchangeably with the term "patient". Preferably, the subject is a human being. As used herein, the terms "treatment", "treating" or "treatment" and variations thereof include the administration of a therapeutically effective amount of an antibody of the invention sufficient to reduce or eliminate at least one symptom of the disorder. .

As used herein, the terms "preventive", "preventive" or "preventive" or variations thereof refer to the protection of a subject against the development of at least one symptom of a disease, or to the reduction of the severity of a symptom of a disorder.

As used herein, the term "cell exposure" refers to providing the antibody in such a manner that it is capable of contacting / binding human C5aR whenever C5aR is present in the cell.

The term "50 percent effective concentration" (abbreviated as "EC50") represents the concentration of an antibody of the invention that is required for 50 percent of a given effect of the molecule to which the antibody is directed (v. gr., the inhibition / displacement of the binding of human C5a to human C5aR). It will be understood by a person skilled in the art that a lower EC50 value corresponds to a more potent antibody.

As used herein, the term "inhibition" refers to a significant reduction, and possibly completely null, of the defined activity. Preferably, the defined activity is reduced or inhibited by at least 50 percent, most preferably at least 75 percent and even most preferably at least 90 percent.

Throughout this description, the word "comprise", or variations such as "comprises" or "comprising", shall be understood to imply the inclusion of an element, integer or indicated step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step or group of elements, integers or steps.

In one embodiment, a molecule consists essentially of the defined sequence.

In another embodiment, a molecule consists of the defined sequence.

In one embodiment, the molecule such as an antibody or DNA sequence is an isolated molecule. The term "isolated antibody" refers to an antibody that has been separated and / or recovered from another / other component (s) of its natural environment and / or purified from a mixture of the components in its natural environment.

The term "antibody", as referred to herein, includes complete antibodies and any antigen-binding fragments (ie, "antigen-binding portion") or single chains thereof. Full-length antibodies (or whole antibodies) comprise four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a region heavy chain constant (CH). Each light chain comprises a variable region of light chain (VL) and a constant region of the light chain (CL). The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The constant region of the light chain is composed of a domain, CL. The VH and VL regions can further be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).

Each VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminal to the carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the antibodies can mediate the binding of immunoglobuin to host tissues or factors, including several cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

As used herein, the term "antibody" is used to describe complete antibodies and any antigen-binding fragments (ie, "antigen-binding portion") or individual chains thereof that specifically bind to their corresponding antigen. Examples of antigen-binding fragments include Fab, Fab ', F (ab) 2, F (ab') 2, F (ab) S, Fv (typically the VL and VH domains of a single arm of an antibody), Single chain Fv (scFv; see, eg, Bird et al, Science 1988; 242: 42S-426; and Huston et al PNAS 1988; 85: 5879-5883), dsFv, Fd fragments (typically the VH and CHI domain) , and (typically a VH domain); dAb domains VH, VL, VHH, and V-NAR; monovalent molecules comprising a single VH chain and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa organisms (see, for example, 111 et al, Protein Eng. 1997; 10: 949-57); Camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, wherein the isolated CDRs or antigen-binding residues or polypeptides may be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 2S: 1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201.

The term "complementarity determining region" ("CDR") or "hypervariable region", when used herein, refers to the amino acid residues of a antibody that are responsible for antigen binding. The CDRs are generally composed of amino acid residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; (Kabat et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and / or those residues of a "hypervariable loop" (residues 26- 32 (Ll), 50-52 (L2) and 91-96 (L3) in the variable domain of the light chain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the domain variable of the heavy chain; Chothia and Lesk, J. Mol. Biol 1987; 196: 901-917). Typically, the numbering of the amino acid residues in this region is carried out by the method described in Kabat et al., Cited above. Phrases such as "Kabat position", "Kabat residue" and "in accordance with Kabat" in this document refer to this numbering system of the variable domains of heavy chain or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or more amino acids corresponding to a shortening of, or insertion in, a frame (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residues 52a, 52b and 52c in accordance with Kabat) after residue 52 of COR H2 and inserted residues (eg, residues 82a, 82b, and 82c, etc., in accordance with Kabat) after residue 82 of heavy chain FR. The Kabat numbering of residues can be determined for a given antibody by aligning in regions of homology of the antibody sequence with a "standard" Kabat numbered sequence.

The term "framework region" or "FR" residues refers to those VH or VL amino acid residues that are not within the CDRs, as defined herein.

The fragment crystallizable region ("Fe region" / "Fe domain") of an antibody is the "tail" region of an antibody that interacts with cell surface receptors called Fe receptors, as well as some proteins of the cell system. complement.

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells of a mouse that has been immunized with the desired antigen. Human monoclonal antibodies can be obtained from transgenic animals (e.g., mice or other suitable species) that encode human antibodies.

Alternatively, recombinant monoclonal antibodies can be made using technologies, known as cloning repertoire or phage display / deployment of yeasts Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, instead of mice.

The term "humanized antibody", as used herein, refers to a human / non-human chimeric antibody that contains sequences, typically at least the minimal complementarity determining regions (CDR sequences) derived from an immunoglobulin sequence of non-human germline. A humanized antibody is, therefore, a human immunoglobulin (receptor antibody) in which the residues of a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as from a mouse , rat, rabbit or non-human primate, which have the desired specificity, affinity and capacity.

Humanized antibodies comprising at least CDR regions not derived from human germline sequences and which may also be referred to as "chimeric antibody" if the antibody light and heavy chain genes have been constructed, typically by genetic engineering, from Variable region and immunoglobulin constant genes that originate from different species. For example, variable segments of the genes of a mouse monoclonal antibody can bind to human constant segments.

The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. It is noted that such antibodies can, however, comprise amino acid residues that are not found in the human germline sequences due to mutations that occur due to maturation in vivo or in vitro. In addition, if the antibody contains a constant region, the constant region is also derived primarily from human germline immunoglobulin sequences. The human antibodies of the invention can, however, include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). On the other hand, the term "human antibody", as used herein, is not intended to include antibodies or alternative antigen-binding regions in which the CDR sequences are derived from the germline of another mammalian species, such as a mouse and have been subsequently grafted onto human frame sequences (see antibody humanized above). The human antibody can be a human monoclonal antibody. The human monoclonal antibody can be produced by a hybridoma including a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Human antibodies can also be isolated from libraries of sequences constructed in the selections of human germline sequences, further diversified with natural and synthetic sequence diversity. Human antibodies can be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus. The sequence of the human antibody can be identified, which allows the production of the antibody by recombinant methods.

In addition, humanized, human and fully human antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance. .

The term "antibody derivatives" refers to any modified form of the antibody, such as an antibody conjugate and the other agent or antibody.

The term "antigen" refers to the molecular entity used for immunization of an immunocompetent vertebrate to produce an antibody that recognizes the antigen. In this document, the term "antigen" is used more broadly and is generally intended to include target molecules that are specifically recognized by the antibody, which therefore include fragments or mimetics of the molecule used in the immunization process to raise the antibody or the molecules used for selective determination after immunization and also used for the selective determination of molecules in the cases in which the antibodies are obtained by alternative methods, such as the selective determination of phage display.

The term "epitope", as used herein, is defined in the context of a molecular interaction between an "antigen-binding polypeptide," such as an antibody, and its corresponding "antigen." In general, "epitope" refers to the area or region in an antigen to which it binds specifically to an antibody, i.e., the area or region in physical contact with the antibody. A protein epitope can comprise amino acid residues in the antigen that are directly involved in binding to the antibody (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues. of antigen binding that are effectively blocked by Ab (in other words, the amino acid residue is within the "solvent surface"). "and / or the" fingerprint "of the antibody is excluded.) A given antigen may comprise a number of different epitopes, which may include, without limitation, the antigenic determinants of linear peptides, conformational antigenic determinants consisting of one or more non-contiguous amino acids , located close to each other in the native conformation (mature), and post-translational antigenic determinants that consist, either in whole or in part, of molecular structures covalently linked to the antigen, such as carbohydrate groups.

From the fact that the descriptions and definitions of the epitopes, depending on the method of epitope mapping used, are obtained in different levels of detail, it follows that the comparison of the epitopes for different Abs in the same Ag is similarly It can be carried out in different levels of detail.

The terms "joining"; "specifically binding" and "binding specificity" are used herein to describe the selectivity of an antibody or an antigen-binding fragment thereof.

The antibodies according to the invention can bind specifically to C5aR, indicating that the antibody has a significantly lower affinity for other antigens, where significantly lower can be such as affinity at least 2 times lower, or 5 times lower or times lower The antibody can be even more species-specific, such as the antibody that is specifically bound to human C5aR, but not mouse C5aR with high affinity.

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, e.g., an antibody, or fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules, e.g., an antibody, or fragment thereof, and an antigen, through a monovalent interaction can be quantified by determining the dissociation constant (KD). In turn, KD can be determined by measuring the kinetics of complex formation and dissociation, e.g., by the SPR method. The rate constants corresponding to the association and dissociation of a monovalent complex are known as the association rate constant ka (or kaso) and the dissociation rate constant K¿ (kd So), respectively. KD is related to ka and kd through the equation KD = ka / ka.

In addition, "affinity" refers to the strength of the binding between a single binding site of a molecule (e.g., an antibody) and a ligand (e.g., an antigen). The affinity of a molecule X for a ligand Y is represented by the dissociation constant (Kd), which is the concentration of Y which is required to occupy the combining sites of half of the X molecules present in a solution. A lower indica indicates a stronger or higher affinity interaction, and a lower concentration of ligand is needed to occupy the sites. Similarly, the specificity of an interaction can be assessed by determining and comparing the KD value for the interaction of interest, such as a specific interaction between an antibody and an antigen, with the KD value of an interaction not of interest.

Typically, the KD for the antibody with respect to the target will be 2 times, preferably 5 times, most preferably 10 times less than KD with respect to the other, non-target molecule, such as unrelated material or accompanying material in the environment or control. Most preferably, the KD will be 50 times less, such as 100 times less, or 200 times less, even very preferably 500 times less, such as 1,000 times less, or 10,000 times less.

The value of this dissociation constant can be determined directly by well-known methods, and can be calculated even for complex mixtures by methods such as those, for example, set forth in Caceci et al (Byte 9: 340-362, 1984). For example, the KD can be established by using the union test of a double filter nitrocellulose filter such as that described by Wong and Lohman (Proc Nati, Acad Sci USA 90, 5428-5432, 1993). Other standard tests for evaluating the binding capacity of ligands such as antibodies to targets are known in the art - including, for example, ELISA, Western blots, RIA, and flow cytometric analysis. The binding kinetics and binding affinity of the antibody can also be evaluated by standard tests known in the art, such as SPR.

A competitive binding test can be carried out in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody. The concentration at which a 50% inhibition occurs is known as the Ki. Under ideal conditions, Ki is equivalent to KD. The Ki value will never be lower than the KD, so the Ki measurement can conveniently be substituted to provide an upper limit for KD.

As will be appreciated by the person skilled in the art, "avidity" refers to the overall resistance of the interaction between two molecules, such as an antibody and antigen. Avidity depends as much on the affinity as on the valence of the interactions.

Other tests to determine the functionality of a given antibody may include cell-based testing which are specific for the given antigen and the effect of antibody binding.

The term "identity", as known in the art, refers to a relationship between the sequences of two or more polypeptides, as determined by comparison of the sequences. In the art, "identity" also means the degree of sequence relationship between polypeptides, as determined by the number of matches between sets of two or more amino acid residues.

"Identity" measures the percent of identical matches between the smallest of two or more sequences with spaces alignments (if any) directed by a particular mathematical model or computer program (ie, "algorithms"). The identity of related polypeptides can be easily calculated by known methods. These methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M. , ed. , Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed. , Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds. , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

The preferred methods for determining identity are designed to provide the best match between the tested sequences. Methods for determining identity are described in publicly available computer programs. Preferred computer program procedures for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucí Acid Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin , Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, Md. 20894, Altschul et al., Cited above). Smith Waterman's well-known algorithm can also be used to determine identity.

For example, by using the GAP computer algorithm (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptides for which the percent sequence identity is to be determined are aligned for the optimal match of their respective amino acids (the "matched slice", as determined by the algorithm). A sanction for opening space (which is calculate as 3 times, the average diagonal; the "average diagonal" is the average of the diagonal of the comparison matrix that is used; the "diagonal" is the score or number assigned to each perfect amino acid matched by the particular comparison matrix) and a penalty for extension of space (which is normally. {fraction (1/10).} times the penalty for opening of space), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used together with the algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978) for the comparison matrix of PAM 250, Henikoff et al, Proc Nati Acad Sci USA 89, 10915 -10919 (1992) for the BLOSUM comparison matrix 62) is also used by the algorithm.

Preferred parameters for a peptide sequence comparison include the following: Algorithm: Needleman et al., J. Mol. Biol. 48, 443-453 (1970); Comparison matrix: BLOSUM 62 by Henikoff et al, PNAS USA. 89, 10915-10919 (1992); Penalty for space: 12, Penalty for length of space: 4, Threshold of similarity: 0.

The GAP program is useful with the above parameters. The aforementioned parameters are the default parameters for peptide comparisons (along with no sanction for the final spaces) using the GAP algorithm.

A "conservative amino acid substitution" may involve a substitution of an amino acid residue with another residue, such that there is little or no effect on the polarity or charge of the amino acid residue in that position. This is illustrated by the following amino acid groups, so substitutions of an amino acid with a different amino acid in the same group is considered a conservative substitution: Hydrophilic: Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr. Aliphatics: Val, lie, Leu, Met. Basics: Lys, Arg, His. Aromatics: Phe, Tyr, Trp. On the other hand, any residue can be frequently substituted with alanine.

In addition, if desired, non-natural amino acids or chemical amino acid analogs may be introduced as a substitution or addition in the antibody and / or immunoglobulin chain of the present invention. These amino acids include, but are not limited to, the D isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino acid. amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citralline, homocitrulin, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, design amino acids such as beta-methyl-amino acids, Ca-methyl-amino acids, Na-methyl-amino acids and amino acid analogs in general.

Mutants of the amino acid sequence of the antibody and / or immunoglobulin chain of the present invention can be prepared by introducing appropriate changes of nucleotides into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. These mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construction, provided that the final polypeptide product possesses the desired characteristics. Mutant (altered) polypeptides can be prepared by the use of any technique known in the art. For example, a polynucleotide of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutant" strain such as E. coli XL-I red (Stratagene) and propagating the transformed bacteria for a number adequate generations. The products derived from the mutated / altered DNA can be easily determined selectively by using the techniques described in this document to determine if they have a receptor binding and / or inhibitory activity.

In the design of mutants of the amino acid sequence, the location of the mutation site and the nature of the mutation will depend on the characteristic (s) to be modified. The sites for the mutation can be modified individually or in series, eg, by (1) substituting first with conservative amino acid choices and then with more radical selections according to the results achieved, (2) deleting the target residue, or (3) Insert other residues adjacent to the localized site.

Amino acid sequence deletions generally range from about 1 to 15 residues, most preferably from about 1 to 10 residues and typically from about 1 to 5 contiguous residues.

The inventors have identified several aspects of relevance for the functionality and effectiveness of biological therapeutics and particular antibodies, and the main area of the present invention is antibodies for the treatment of inflammatory diseases by inhibiting the binding of C5a to C5aR.

One aspect of the invention relates to one or more of a series of antibodies that are characterized by their functionality and / or the amino acid sequence of the CDRs, the variable region of the heavy chains and light chains and / or the sequence of the Fe domain.

In one embodiment, the antibody is an antibody of full length, which includes the standard antibody domains and regions.

In one embodiment, the antibody is an antibody fragment, those fragments can be obtained by the use of conventional recombinant or protein engineering techniques. Antibody fragments of the invention can be made by truncation, eg, by removal of one or more amino acids from the N- and / or C-terminus of a polypeptide. The fragments can also be generated by one or more internal deletions. An antibody of the invention can be, or can comprise, a fragment of any of the antibodies on which this invention is based. An antibody of the invention may be, or may comprise, an antigen binding portion of one of these antibodies, or variants thereof. For example, the antibody of the invention can be a Fab fragment of one of these antibodies or variants thereof, or it can be a single chain antibody, obtained from one of these antibodies, or a variant thereof.

The antibodies of the invention can be of different species, including mammalian species such as mouse, rat, rabbit, pig or non-human primate. The antibody can be a rodent antibody and more particularly a mouse antibody. Alternatively, the antibody can be from a non-mammalian species, such as chicken. The antibody can also be a humanized antibody or a human antibody.

An antibody of the invention may have the ability to compete with another antibody of the invention for binding to C5aR as described herein. These cross-competition antibodies can be identified based on their ability to cross-compete with a known antibody of the invention in standard binding assays. That cross-competition may suggest that the two antibodies bind to identical, overlapping or similar epitopes.

Human antibodies As described in the examples herein, the inventors have identified a series of anti-C5aR antibodies derived from transgenic mice including germline loci of human immunoglobulin. The antibodies are isolated as hybridoma monoclonal antibodies and the binding characteristics are evaluated. As mentioned, C5aR is a transmembrane GPCR of seven domains and it is not possible to produce a soluble form that retains the native conformation. In order to produce human antibodies that block the binding of hC5a to hC5aR, transgenic mice were immunized with expressing cells native hC5aR. However, the blocking antibodies were very difficult to obtain and the inventors carried out 32 fusions before a human antibody having the desired blocking properties was identified. From 35 fusions and selective determination of more than 100,000 hybridoma supernatants a total of 11 blocking antibodies was obtained.

In addition, due to the nature of hC5aR, it was not possible to determine the affinity of the antibodies by standard Biacore analysis, and therefore, tests based on functional hC5aR-dependent readings were established, from which the IC50 and EC50 values were determined. determined as described in example 2 and example 7.

In one aspect, the invention relates to a human antibody that binds C5aR and it is further preferred that the antibody binds specifically to hC5aR, such that binding to hC5aR is stronger than binding to C5aR of other species, such as, in particular, mouse C5aR. In one embodiment, it is preferred that the antibody binds to the second extracellular loop of C5aR and most preferably the 2nd loop of human C5aR. In one embodiment, the antibody binds to the second extracellular loop of human C5aR, but not the second extracellular loop of murine C5aR. In other embodiments, the antibody according to the invention can bind to the second extracellular loop of C5aR in the native conformation only.

The functionality of an anti-C5aR antibody is dependent on the ability of that antibody to inhibit or significantly reduce the binding of C5a to C5aR.

In one embodiment, the invention relates to a human antibody that binds C5aR or an antibody as described herein by sequence definition (see below) wherein that antibody is capable of inhibiting or significantly reducing the binding of C5a to C5aR. This can be determined by a displacement test (SPA) as described in example 2 in this document, from which the IC 50 values can be determined. As is evident from Table 1, the 11 antibodies isolated and described have an IC50 concentration below 50 nM. In a further embodiment of the invention, the antibody is capable of shifting hC5a in a SPA test, with an IC50 below 50 nM, such as below 40 nM, such as below 30 nM, such as below of 20 nM, such as below 10 nM, such as below 5 nM or even below 4 nM, or with IC50 below 3 nM or even below 2.5 nM or 2.0 nM.

In other tests, the ability of anti-C5aR antibodies to inhibit the C5a-dependent migration of human neutrophils was evaluated and some of the human antibodies identified turned out to be inhibitors. more potent neutrophil migration mediated by C5a than a C5aR antibody previously described (Q of WO 2009/103113). In one embodiment, the invention therefore relates to an antibody as described herein by definition of the sequence (see below) or a human antibody that binds to C5aR, wherein that antibody is capable of significantly inhibiting the migration of human neutrophils. In one embodiment, the antibody inhibits migration of less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% compared to the level of migration observed in the presence of 10 nM of C5a and without anti-C5aR antibody. In that embodiment, migration is measured after 30 minutes in the presence of 10 nM of C5a and the antibody compared to the level of migration observed after 30 minutes in the presence of 10 nM of C5a and without antibody. Alternatively, the ability of the antibody to inhibit neutrophil migration can be expressed by the use of IC50 values based on the same configuration. In that embodiment, the IC 50 is below 2.5 ig / ml, such as by deb or 2.5 μg / ml, such as below 10.5 μg / ml, such as below 10.2 μg / ml or even below 1.0 μg / ml.

As an alternative to the standard Biacore analysis, the functionality of the hC5aR antibodies can be determined by a competitive binding test on the neutrophils as described in example 7. This functionality is known as antibody affinity as measured by the competition ligand binding test, but it could also be considered the measure of the avidity of the interaction. The invention in one embodiment refers to an antibody as described herein by definition of the sequence (see below) or a human antibody that binds to C5aR, wherein the affinity or avidity of the antibody as measured by the Competitor ligand binding test in neutrophils is below 0.80 nM, 0.70 nM, 0.60 nM, such as below 0.50 nM, 0.45 nM, 0.40 nM or 0.35 nM.

An additional option for the characterization of the antibodies was explored by the use of a calcium flux test, which measures the ability of an antibody to inhibit the activation of neutrophils induced by C5a ex vivo, in the same manner described in example 7. In a further embodiment, the invention relates to an antibody as described herein by sequence definition (see below) or a human antibody that binds to C5aR, wherein the IC50 as determined in a blood flow test calcium is below 7.0 g / ml, such as below 5.0 μg / ml, such as below 2.5 μ9 / p? 1.

Additional ex vivo tests can be used to determine the ability of an antibody to inhibit or neutralize neutrophil maturation induced by C5a based on side effects, such as the expression of CDllb and CD62L. CDllb and CD62L are the maturation markers of neutrophils as they are up-regulated and down-regulated, respectively, under activation by the interaction of C5a / C5aR.

The effect was determined in an up-regulation CDII test. In one embodiment, the invention relates to an antibody as described herein by sequence definition (see below) or a human antibody that binds to C5aR, wherein the IC50 as determined in an up-regulation test of CDllb is below 3.5 μg / ml, such as 3.0 μg / ml, such as below 2.5 μg / ml, such as below 2.0 g / ml or such as 1.5 g / ml or even below 1.0 g. / ml.

Likewise, the effect of the antibody was determined in a down-regulation test of CD62L. In one embodiment, the invention relates to an antibody as described herein by sequence definition (see below) or a human antibody that binds to C5aR, wherein the IC50 as determined in a down-regulation test of CD62L is below 0.8 μg / ml, such as below 1.5 μg / ml, such as below 1.2 μg / ml or even below 1.0 μg / ml.

Four monoclonal antibodies were selected for sequencing, to determine the sequence of the variable regions and, in particular, CDR sequences. A sequence alignment is presented in Figure 1 and the sequences are also included in the attached sequence listing.

The sequence listing includes the following sequences in relation to isolated antibodies: SEQ ID 1-3: Vh 35F32A3 CDR 1-3 SEQ ID 4: Vh 35F32A3 SEQ ID 5-7: VI 35F32A3 CDR 1-3 SEQ ID 8: VI 35F32A3 Similarly, SEQ ID 9-16 describes 32F3A6 Similarly, SEQ ID 17-24 describes 35F12A2 Similarly, SEQ ID 25-32 describes 35F24A3 Antibodies defined by variable regions or CDR sequences An antibody according to the invention can therefore be defined based on the CDR sequences, the sequences of the variable regions of the heavy and light chains and minor modifications thereto which can be made by the person skilled in the art without alter the functionality of the antibody. This includes substitutions, deletions or insertions of amino acids of one or more, such as one, two or three amino acid residues within each of the CDR sequences.

In one aspect, the invention relates to an antibody that binds to C5aR defined by the sequence of the CDR regions, wherein the variable region of the heavy chain of that antibody comprises CDR1, CDR2 and CDR3 sequences selected from the following groups : a) SEQ ID 1, 2 and 3, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y b) SEQ ID 9, 10 and 11, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y c) SEQ IDs 17, 18 and 19, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y d) SEQ IDs 25, 26 and 27, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue.

In one embodiment, the invention relates to an antibody that binds to C5aR, defined by the sequence of the CDR regions, wherein the variable region of the heavy chain of that antibody comprises sequences of CDR1, CDR2 and CDR3; wherein the CDR1 sequence comprises SEQ ID 1, 9, 17, 25 or one of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with an amino acid residue different; Y wherein the CDR2 sequence comprises SEQ ID 2, , 18, 26 or one of the sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue; Y wherein the CDR3 sequence comprises SEQ ID 3, 11, 19, 27 or one of the sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

In one embodiment, the invention relates to an antibody that binds to C5aR defined by the sequence of the CDR regions, wherein the variable region of the light chain of that antibody comprises CDR1, CDR2 and CDR3 sequences selected from the following groups a) SEQ ID 5, 6 and 7, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y b) SEQ ID 13, 14 and 15, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y c) SEQ ID 21, 22 and 23, wherein none, one, two or three of those sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue; Y d) SEQ ID 29, 30 and 31, where none, one, two or three of these sequences comprise 1, 2 or 3 amino acid (s) substituted with a different amino acid residue.

In one aspect, the invention relates to an antibody that binds to C5aR, defined by the sequence of the CDR regions, wherein the variable region of the light chain of that antibody comprises CDR1, CDR2 and CDR3; wherein the CDR1 sequence comprises SEQ ID 5, 13, 21, 29 or one of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue; Y wherein the CDR2 sequence comprises SEQ ID 6, 14, 22, 30 or one of the sequence wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue; Y wherein the CDR3 sequence comprises SEQ ID 7, 15, 23, 31 or one of the sequence wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

In one embodiment, the invention relates to an antibody wherein the CDRs of the variable region of the heavy chain comprise SEQ ID 1, 2 and 3 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or amino acid insertion (s) and wherein the CDRs of the variable light chain comprise SEQ ID 5, 6 and 7 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or insertion (s) ) of amino acids.

In one embodiment, the invention relates to an antibody wherein the CDRs of the variable region of the heavy chain comprise SEQ ID 9, 10 and 11 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or amino acid insertion (s) and wherein the CDRs of the variable light chain comprise SEQ ID 13, 14 and 15 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or insertion (s) ) of amino acids.

In one embodiment, the invention relates to an antibody wherein the CDRs of the variable region of the heavy chain comprise SEQ IDs 17, 18 and 19 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or amino acid insertion (s) and wherein the CDRs of the variable light chain comprise SEQ ID 21, 22 and 23 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or insertion (s) ) of amino acids.

In one embodiment, the invention relates to an antibody wherein the CDRs of the variable region of the heavy chain comprise SEQ ID 25, 26 and 27 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or amino acid insertion (s) and wherein the CDRs of the variable light chain comprise SEQ ID 29, 30 and 31 or the sequence with 1, 2 or 3 substitution (s), deletion (s) and / or insertion (s) ) of amino acids.

One embodiment of the invention therefore is refers to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following groups of sequences, SEQ ID 1, 2 and 3, SEQ ID 9, 10 and 11, SEQ ID 17, 18 and 19, SEQ ID 25, 26 and 27 or those sequences with up to two substitution, deletion and / or insertion per sequence and wherein the variable region of the light chain of that The antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said CDR sequences comprise one of the following groups of sequences: SEQ ID 5, 6 and 7, SEQ ID 13, 14 and 15, SEQ ID 21, 22 and 23, SEQ ID 29, 30 and 31 or those sequences with up to two substitution, deletion and / or insertion per sequence.

One embodiment of the invention therefore relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following groups of sequences , SEQ ID 1, 2 and 3, SEQ ID 9, 10 and 11, SEQ ID 17, 18 and 19, SEQ ID 25, 26 and 27 or those sequences with up to one substitution, deletion and / or insertion per sequence and where the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following groups of sequences: SEQ ID 5, 6 and 7, SEQ ID 13, 14 and 15 , SEQ ID 21, 22 and 23, SEQ ID 29, 30 and 31 or those sequences with up to one substitution, deletion and / or insertion per sequence.

One embodiment of the invention therefore relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise one of the following groups of sequences, SEQ ID 1, 2 and 3, SEQ ID 9, 10 and 11, SEQ ID 17, 18 and 19, SEQ ID 25, 26 and 27, and wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein CDR sequences comprise one of the following sequence groups, SEQ ID 5, 6 and 7, SEQ ID 13, 14 and 15, SEQ ID 21, 22 and 23, SEQ ID 29, 30 and 31.

In one embodiment, the invention relates to an antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 4, 12, 20 or 28 .

In one embodiment, the invention relates to an antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 4, 12, 20 or 28 .

One aspect of the invention relates to an antibody wherein the variable region of the light chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO 8, 16, 24 or 32.

In one embodiment, the invention relates to an antibody wherein the variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 8, 16, 24 or 32 .

One embodiment of the invention therefore relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 4, 12, 20 or 28 and / or wherein the variable region of the light chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 8, 16, 24 or 32.

In one embodiment, the invention relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 4 and / or where the variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 8.

In one embodiment, the invention relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 12 and / or where the variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 16.

One embodiment of the invention refers to both to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 20 and / or wherein the variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 24.

One embodiment of the invention therefore relates to an antibody, wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 28 and / or wherein the variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 32.

In one embodiment, the invention relates to an antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 39 and / or wherein the The variable region of the light chain of that antibody comprises a sequence of at least 96, 97, 98 or 99% identical to SEQ ID NO: 40.

In one embodiment, the invention is an antibody wherein the variable region of the heavy chain of that antibody is identified by SEQ ID NO 39 and / or wherein the variable region of the light chain of that antibody is identified by SEQ ID NO: 40 During the maturation of antibodies can spontaneous mutations occur in the framework region, as described herein in Examples 6 and 7, the variable region of one of the isolated monoclonal antibodies was compared to human antibody germline sequences to identify the line sequence human germline closest to both the heavy chain and the variable light chain. In order to minimize the risk of immune reaction, it was therefore decided to further optimize the antibodies by introducing point mutations in the framework region to interpret an antibody with the human germline sequence in the regions of framework, as can be seen from the experiments, this did not influence the functionality of the antibody.

In one embodiment, the invention relates to an antibody defined by sequence identity with a variable region of a reference antibody as described hereinabove, wherein the variable region of the heavy chain and / or light chain of that antibody comprises one or more mutations in the framework region. According to the invention, it may be attractive to introduce one or more mutations to increase the identity with the closest human germline sequence, although other mutations may also be considered. In one embodiment, that mutation (s) is / are the conservative mutation (s).

Antibodies defined by Fe region The Fe region allows the antibodies to activate the immune system and the antibodies can be designed to include modifications within the Fe region, typically to alter one or more of their functional properties, such as serum half-life, complement fixation, Fe receptor binding, protein stability and / or antigen-dependent cellular cytotoxicity, or lack of it. In addition, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be bound to the antibody) or can be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. One aspect of the invention relates to an antibody that binds C5aR or an antibody as described herein by definition of the sequence (see below), wherein the Fe region has a reduced or null binding affinity to one. or more FcyRs.

In one embodiment, the invention relates to an antibody that binds C5aR, preferably human C5aR, as described above or an antibody as described herein by definition of the sequence (see below), wherein the Fe region it has reduced binding affinity to one or more FcyRs.

In one embodiment, the antibody of the invention displays reduced binding affinity to one or more FcyRs compared to Fe reference sequences of IgG1, IgG2, IgG2 / 4 or IgG4 as defined by SEQ ID NO: 33, 34, 35 and 36, respectively. Since the specific amino acid residues may be responsible for the interaction of FcyRs and effects meditated herein, it may be advantageous to apply an antibody wherein those specific amino acid residues of the Fe region have been replaced by a different amino acid.

In one embodiment, the Fe region includes one or more point mutations compared to the reference sequences of IgG1, IgG2, IgG4 / G2 or IgG4Fc as defined by SEQ ID 33, 34, 35 and 36, respectively, by reducing the affinity to one or more Fcy receptors or complement components.

In order to evaluate the result of the introduction of point mutations in the Fe region, the effector functions of a series of anti-C5aR antibodies were evaluated as described in example 4. A phagocytosis test was established to measure the role of the Fe region in the ability of anti-hC5aR antibodies to induce phagocytosis of neutrophils (expressing hC5aR) by human monocytes. As can be seen in Table 2, several variants of Fe decrease the level of phagocytosis induced by anti-C5aR antibodies in the test described.

In one embodiment, the antibody according to the invention does not significantly induce neutrophil phagocytosis in vitro, which means that the level of phagocytosis is not significantly above the background, measured in the absence of an anti-C5aR antibody. In one embodiment, the antibody does not give rise to any detectable induction of phagocytosis. The test for evaluating the level of phagocytosis can be performed by the use of human neutrophils as described in example 4.

In the alternative tests, the ability of anti-hC5aR antibodies to induce ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity) was evaluated. The tests were established in order to test the ability of the Fe variants to mediate cell depletion through ADCC or CDC-dependent mechanisms, and it is assumed to be able to simulate activities in an in vivo situation.

The tests apply cells expressing hC5aR as target cells and effector cells (PMBCs depleted of monocytes) or serum containing complement to elicit the responses as described in example 4.

In one embodiment, the antibody according to the invention does not induce ADCC significantly, which means that the ADCC level is not significantly above the background, measured in the absence of an anti-C5aR antibody. In one embodiment, the antibody does not give rise to any detectable induction of ADCC, ie, the ADCC level is not above the background.

In one embodiment, the antibody according to the invention does not significantly induce CDC. In one embodiment, the antibody does not give rise to any detectable induction of CDC, i.e., the CDC level is not above background.

In one embodiment, the antibody according to the invention comprises an Fe region, wherein the sequence has been modified to alter the function or functions of the effector cell. The modification of the sequence of Fe can be obtained by point mutations in the amino acid sequence. The Fe region of the heavy chain may be a chimeric sequence of IgG1, IgG2, IgG4 or IgG2 / 4. The reference sequences are defined in the sequence listing as follows; IgG1 by SEQ ID NO: 33, IgG2 by SEQ ID NO: 34, IgG2 / 4 by SEQ ID NO: 35 and IgG4 by SEQ ID NO: 36.

In one embodiment, the Fe region is an IgG1 (SEQ ID NO: 33), IgG2 (SEQ ID NO: 34), IgG2 / 4 (SEQ ID NO: 35), or IgG4 (SEQ ID NO: 36), with one or more of the following point mutations to. E233P b L234A s or V234A F234L or F234V c. L235E or L235A d. G236R or G236A and. G237A F. S239D g. S254W h. N297Q i. L328R j. A330 k. P331S 1. I332E The difference between the variants of Fe lies in their ability to interact with FcyRs or components of the complement system as described above. The sequence differences in the Fe region further affect the structure and flexibility of the antibody, which can also affect the function of the antibody. As described in Example 5 and Table 3, the inventors further demonstrate that anti-hC5aR antibodies wherein the Fe region is of the IgGl type with or without additional point mutations are more potent inhibitors of the effects mediated by hC5aR than the corresponding antibodies with the Fe region of the IgG4 type. Accordingly, one embodiment of the invention refers to any of the antibodies defined herein with an Fe region of the IgGI isotype or at least one Fe-hinge region of the IgGl isotype.

In one embodiment, the IgGl Fe region comprises from 1 to 10 amino acid substitutions compared to the IgGl Fe reference sequence as defined in SEQ ID NO.33. It is preferred that the Fe regions comprise fewer mutations, such as 1 to 8 amino acid substitutions within AA 231 to 240, or such as 1 to 5 amino acid substitutions within AA 328 to 334. The amino acid substitutions are preferably selected from substitutions that reduce the ability of the antibody to induce significantly phagocytosis of neutrophils, ADCC and / or CDC in vitro as described above.

In one embodiment, the antibody Fe region is an IgGl comprising one or more of the following point mutations: a) N297Q and / or b) L234A and / or c) L235E or L235A and / or d) G236R or G236A and / or e) G237A and / or f) L328R and / or g) A330 and / or h) P331S.

In one embodiment, the antibody Fe region is an IgGl comprising one or more of the following groups of Point mutations: N297Q and / or L234A and L235E and / or L234A and G236R and / or L235E and G236R and / or L234A, L235E and G236R and / or f) G236R and L328R and / or g) N297Q, L234A and L235E and / or h) N297Q, L234A, L235E and G236R and / or N297Q, L234A, L235E and G237A and / or j) L234A, L235E, G237A, A330S and P331S. k) N297Q, L234A, L235E, G237A, A330S and P331S.

In one embodiment, the antibody Fe region is an IgGl comprising one or more of the following groups of point mutations: a) N297Q and / or b) L234A and L235E and / or c) G236R and L328R and / or d) N297Q, L234A and L235E and / or e) N297Q, L234A, L235E and G237A and / or f) L234A, L235E, G237A, A330S and P331S.

It is clear to the person skilled in the art that point mutations within the framework region of both heavy and light chains can be introduced on the basis of standard criteria for the substitution of residues of amino acids is within the scope of the invention. Functional tests as described herein can be used to confirm that these mutations do not influence the functionality of the antibody.

As is clear from the foregoing, the binding specificity of the identified antibodies is provided by the variable regions or CDRs, and it is clear that different types of antibodies possessing a similar antigenic binding region are encompassed by the invention.

In one embodiment of the invention, the antibody is a full-length antibody. In one embodiment of the invention, the antibody is an antibody fragment or a single chain antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a human, mouse, rat, rabbit, pig or non-human primate antibody. In a modality, the antibody is a mouse or human antibody. In one embodiment, the antibody is a human antibody. In one embodiment, the antibody is a humanized antibody. As described in the definition part of the application, a humanized antibody includes at least CDR regions not derived from the human germline sequence. As is more apparent from the foregoing, a human antibody may comprise one or more point mutations compared to the germline sequence, but in general the sequence is considered to be it must be at least in the framework region or the Fe region of at least 95% identity with the human germline sequences.

Pharmaceutical formulations The present invention further includes pharmaceutical compositions / formulations, comprising a pharmaceutically acceptable carrier and a polypeptide or antibody according to the invention, as well as kits comprising those compositions.

The antibody according to the invention may in one aspect of the invention be formulated in a pharmaceutical composition. That pharmaceutical composition can be prepared based on general knowledge in the field, such as in the Pharmacopoeia or Remington.

In one embodiment, the pharmaceutical composition according to the invention comprises an antibody as described herein in combination with a pharmaceutically acceptable carrier. The formulation may be in the form of an aqueous formulation or a dry formulation that is reconstituted in water or an aqueous pH regulator composition prior to administration.

A pharmaceutical composition of antibodies according to the invention may comprise a salt and / or pH regulator, such as the compositions described in WO2011 / 104381.

In another embodiment, the pharmaceutical composition of antibodies according to the invention may be suitable for multiple uses, such as the compositions described in O2011 / 147921 Treatment method An aspect of the invention relates to a method of treating or preventing a disorder in a subject, the method comprising administering to a subject in need thereof a therapeutic amount of an antibody as described herein. As described in previous publications such as WO 2009/103113, anti-C5aR antibodies are usable / suitable for the treatment of various diseases and disorders. One embodiment of the invention therefore relates to a method for the treatment of an immunological disease or disorder, in particular, an inflammatory disease. Example 8 in this document further supports this by demonstrating the functionality of an anti-C5aR antibody according to the invention in a mouse arthritis model. Examples 9 to 11 demonstrate the upregulation of C5aR in tissue samples of psoriatic arthritis, patients with Crohn's disease and ulcerative colitis. In addition, it is demonstrated that an anti-C5aR antibody can inhibit PMN cell migration induced by the synovial fluid of patients with psoriatic arthritis.

A method of treatment may be intended to cure a disease or disorder, but in relation to some diseases, including immunological and inflammatory diseases such as a disease or chronic disorder, relief of one or more symptoms is also considered a treatment, which may be be a significant improvement for the subject even if only partial relief of the symptoms is obtained or the effect is only temporary or partial The method according to the invention includes the treatment of one or more diseases including, but not limited to, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE). in English), lupus nephritis, type I diabetes, Grave's disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC, for its acronyms), irritable bowel syndrome, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atóptic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, nephritis autoimmune, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA-associated vascul, uve, scleroderma, bullous pemphigoid, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's chorea, cystic fibrosis, gout, age-related macular degeneration, allergy, asthma and other autoimmune diseases that are the result of acute or chronic inflammation. In a further embodiment, the disease or disorder is an acute or chronic inflammation, wherein the disorder can be an auto-immune disease. In one embodiment, the disorder is rheumatoid arthr (RA), psoriatic arthr, systemic lupus erythematosus (SLE), lupus nephr, inflammatory bowel disease (IBD) that includes Crohn's disease (CD) or ulcerative col (UC) syndrome or of the irritable bowel. In other modals, the disorder is RA or SLE. In addn to chronic diseases, anti-C5aR antibodies may be relevant in relation to acute indications, such as transplantation, ischemia / reperfusion injury (eg, acute myocardial infarction, cerebrovascular accident), sepsis (v.gr ., SIRS, MODS, ALI), atherosclerosis and intracerebral hemorrhage (ICH).

In a further aspect, the invention relates to an antibody, antibody or antibody composition isolated as described herein, for the treatment of a disease or disorder. In another modality Further, that antibody, antibody or isolated antibody composition is for the treatment of one or more of the diseases and disorders described hereinbefore in connection with a method of treatment.

One aspect of the invention relates to the use of an antibody, antibody or antibody composition isolated as described herein, for the preparation of a drug for the treatment of a disease or disorder, wherein the disease or disorder may be as described above in this document in relation to a treatment method.

Administration mode An antibody of the invention can be administered parenterally, such as intravenously, such as intramuscularly, as well as subcutaneously. Alternatively, an antibody of the invention can be administered through a non-parenteral route, such as per-orally or topically. An antibody of the invention can be administered prophylactically. In a preferred embodiment, the antibody is administered intravenously or subcutaneously.

The dose and time of administration will most likely depend on several factors, including the disease / disorder or symptoms of concern, as well as the subject matter in question. In general, it is expected that the antibody will administer at a dose of 0.010 mg / kg up to 4-6 mg / kg. Also, the dosage regime of the antibody will also depend on the individual subject and disease state of that subject, but it is desirable in accordance with the invention to use a treatment wherein the antibody (or antibody composition) is administered to the subject once a week. or every 2 weeks or even at lower intervals, such as once a month.

An antibody of the invention can be administered on demand, ie the antibody can be administered based on the experience of the patients, e.g., when particular symptoms occur or when the amount of particular biomarkers reaches a predefined level.

Specific combination treatments The antibodies of the invention can be co-administered with one or more other other therapeutic agents or formulations. The other agent can be an agent that improves the effects of the antibodies of the invention. The other agent may aim to treat other symptoms or conditions of the patient. For example, the other agent can be an analgesic, an immunosuppressant or an anti-inflammatory agent. The other agent may be another monoclonal antibody, such as those described in international patent applications WO 2008/022390 and WO 2009/103113 The combined administration of two or more agents is You can achieve it in a number of different ways. In one embodiment, the antibody and the other agent can be administered concomitantly in a single composition. In another embodiment, the antibody and the other agent can be administered in separate compositions as part of a combination therapy. For example, the modulator can be administered before, after or simultaneously with the other agent.

The antibodies according to the present invention can be administered together with other drugs (e.g., methotrexate, dexamethasone and prednisone) and / or other biological drugs. In one embodiment according to the invention, an antibody can be co-administered with one or more therapeutic agent (s) selected from class M01C of the ATC code of antirheumatic drugs and L04 of the ATC code of immunosuppressants as described in the document. WO2009 / 103113 including, but not limited to, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, D-penicillamine, gold salts (sodium aurothiomalate, auranofm), leflunomide, methotrexate, minocycline, sulfasalazine and cyclophosphamide, glucocorticosteroids, mycophenolic acid or mycophenolate and tacrolimus and in separate form one or more of Plaquenil, Azulfidine and methotrexate, dexamethasone and / or prednisone.

In another example, the antibodies of the present invention can also be used in combination with other antibodies (e.g., in combination with antibodies that bind to chemokine receptors, including, but not limited to, CCR2 and CCR3) or with anti-TNF or other anti-inflammatory agents or with existing blood plasma products, such as commercially available gamma globulin and immunoglobulin products used in prophylactic or therapeutic treatments. The antibodies of the present invention can be used as separately administered compositions given together with antibiotics and / or antimicrobial agents.

Accordingly, the antibodies can be administered in combination with agents such as agents that are already in use in autoimmunity, including, but not limited to, immunomodulators, such as IFN-beta, Orencia ™ (CTLA4-lg), Humira ™ (anti-TNF), Cimzia ™ (anti-TNF, PEG Fab), Tysabri ™ (a4-integrin MAB), Simponi ™, Rituxan / MabThera ™, Actemra / RoActemra ™, Kineret ™, Raptiva, Ustekimumab, anti- non-steroidal inflammatory drugs (NSAIDS), such as Aspirin ™, etc., ibuprofen ™, corticosteroids, disease-modifying antirheumatic drugs (DMARDS) such as Plaquenil ™, Azulfidine ™, methotrexate ™, etc., Copaxone ™ (glatirimer acetate), Gilneya ™ (fingolimod), antibiotics + such as Flagyl ™, Cipro ™, topical drugs that include topical corticosteroids (applied to the skin), vitamin D analogue creams (Dovonex ™), topical retinoids (Tazorac ™), humectants, topical immunomodulators (tacrolimus and pimecrolimus), coal tar, anthralin, and others, and also light therapy such as PUVA, UVB and CellCept ™ (mycophenolate mofetil) can be combine with treatment by the use of antibodies according to the invention.

It may be that the subject to be treated is already being treated with one or more other drugs in case the antibody of the invention can be added to that treatment regimen.

Method for the preparation of antibodies An antibody can be prepared by several methods known in the art, mainly based on any of the hybridoma clones for the production of the antibody or the expression of the antibody in a recombinant host, wherein the latter is described in WO2010 / 000864. Based on knowledge in the art, a nucleotide sequence encoding a desired antibody chain can be constructed and used for the recombinant expression of an antibody, wherein the heavy and light chain can be expressed from one or two polynucleotides. separated.

The present invention, in a further aspect, relates to one or more isolated polynucleotides that encode a polypeptide sequence of an antibody chain of an antibody described herein.

A further embodiment refers to a host cell comprising one or more polynucleotides that encode a polypeptide sequence of an antibody chain of an antibody described herein.

The invention further relates to a method for producing an antibody according to the invention, which comprises culturing a host cell described above under conditions that support the expression of one or more polypeptide (s) sequences of an antibody chain. The process may further include that the antibody chains are encoded by two separate open reading frames in a contiguous polynucleotide and, optionally, that the antibody is recovered from that host cell culture.

The present invention, without being limited to the present, can be described by the following modalities.

Modalities 1. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein the sequence of CDR1 comprises SEQ ID 1 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 sequence comprises SEQ ID 2 or that sequence with 1, 2 or 3 substitutions, deletions or amino acid insertions (s) and / or wherein said CDR3 sequence comprises SEQ ID 3 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 2. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ ID 1, 2 and 3, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue. 3. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ ID 1, 2 and 34. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein the sequence of CDR1 comprises SEQ ID 5 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 sequence comprises SEQ ID 6 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 sequence comprises SEQ ID 7 or that sequence with 1, 2 or 3 substitutions, deletions or amino acid inserts (s).

. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CD 1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ IDs 5, 6 and 7, or variants of those sequences wherein 1, 2 or 3 amino acids are substituted with a different amino acid residue. 6. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ IDs 5, 6 and 7. 7. An antibody wherein the variable region of the heavy chain is defined as in any of modes 1, 2 or 3 and wherein the variable region of the light chain is defined as in any of the modes 4, 5 or 6. 8. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein the CDR1 sequence comprises SEQ ID 9 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said sequence of CDR2 comprises SEQ ID 10 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 sequence comprises SEQ ID 11 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 9. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ ID 9, 10 and 11 or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ ID 9, 10 and 11. 11. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said CDR1 sequence comprises SEQ ID 13 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 comprises the sequence SEQ ID 14 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 comprises the sequence SEQ ID 15 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 12. An antibody where the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ IDs 13, 14 and 15, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue. 13. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ IDs 13, 14 and 15. 14. An antibody wherein the variable region of the heavy chain is defined as in any of the modes 8, 9 or 10 and wherein the variable region of the light chain is defined as in any of the modes 11, 12 or 13.

. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein the sequence of CDR1 comprises SEQ ID 17 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said sequence of CDR2 comprises SEQ ID 18 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 sequence comprises SEQ ID 19 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 16. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ IDs 17, 18 and 19, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue. 17. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ IDs 17, 18 and 19. 18. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said CDR1 sequence comprises SEQ ID 21 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 comprises the sequence SEQ ID 22 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 comprises the sequence SEQ ID 23 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 19. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ ID 21, 22 and 23, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue.

. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ ID 21, 22 and 23. 21. An antibody wherein the variable region of the heavy chain is defined as in any of the modes 15, 16 or 17 and wherein the variable region of the light chain is defined as in any of the modes 18, 19 or 20. 22. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein the sequence of CDR1 comprises SEQ ID 25 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said sequence of CDR2 comprises SEQ ID 26 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 sequence comprises SEQ ID 27 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 23. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ ID 25, 26 and 27, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a different amino acid residue. 24. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ IDs 25, 26 and 27.

. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said sequence of CDR1 comprises SEQ ID 29 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 comprises the sequence SEQ ID 30 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 comprises the sequence SEQ ID 31 or that sequence with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s). 26. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences comprise SEQ IDs 29, 30 and 31, or variants of those sequences wherein 1, 2 or 3 amino acid (s) are substituted with a residue of different amino acid. 27. An antibody wherein the variable region of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein those CDR sequences are identical to SEQ IDs 29, 30 and 31. 28. An antibody wherein the variable region of the heavy chain is defined as in any of the modes 22, 23 or 24 and wherein the variable region of the light chain is defined as in any of the modes 25, 26 or 27. 29. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 4, 12, 20 or 28.

. The antibody according to embodiment 29, wherein the variable region of the heavy chain of that antibody comprises one or more mutations in the framework region. 31. The antibody according to mode 29, where that mutation (s) are conservative mutations. 32. The antibody conforms to mode 29, wherein that mutation (s) increases identity with the closest human germline sequence. 33. The antibody according to embodiment 32, wherein the variable region of the heavy chain of that antibody is identified by SEQ ID NO: 39. 34. An antibody where the variable region of the The light chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 8, 16, 24 or 32.

. The antibody according to the modality 34, wherein the variable region of the light chain of that antibody comprises one or more mutations in the framework region. 36. The antibody according to the modality , where that mutation (s) are conservative mutations. 37. The antibody according to mode 35, wherein said mutation (s) increases the identity with the closest human germline sequence. 38. The antibody according to mode 34, wherein the variable region of the light chain of that antibody is identified by SEQ ID NO: 40. 39. An antibody wherein the variable region of the heavy chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 4, 12, 20 or 28 and wherein the variable region of the chain light of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 8, 16, 24 or 32. 40. The antibody according to embodiment 39, wherein the sequence of those variable regions of the heavy chain has at least 96%, such as 97%, such as 98% or such as 99% identity with SEQ ID NO: 4, 12, 20 or 28 and where the sequence of that variable region of light chain it has at least 96%, such as 97%, such as 98% or such as 99% identity with SEQ ID NO: 8, 16, 24 or 32. 41. The antibody according to embodiment 39 or 40, wherein the variable region of the heavy chain of that antibody comprises one or more mutations in the framework region and / or wherein the variable region of the light chain of that antibody comprises a or more mutations in the framework region. 42. The antibody according to mode 41, wherein that mutation (s) are conservative mutations. 43. The antibody according to mode 41, wherein the mutation (s) increase the identity with the nearest human germline sequence. 44. The antibody according to embodiment 41, wherein the variable region of the heavy chain of that antibody is identified by SEQ ID NO 39 and / or wherein the variable region of the light chain of that antibody is identified by SEQ ID NO: 40 45. The antibody according to any of the foregoing embodiments, wherein said antibody binds C5aR. 46. The antibody according to any of the above embodiments, wherein said antibody is a full length antibody or an antibody fragment or a single chain antibody. 47. The antibody according to any of the above embodiments, wherein said antibody is a monoclonal antibody. 48. The antibody according to any of the above embodiments, wherein said antibody is a human, mouse, rat, rabbit, pig or non-human primate antibody. 49. The antibody according to any of the above embodiments, wherein said antibody is a mouse or human antibody. 50. The antibody according to any of the above embodiments, wherein said antibody is a human antibody or a humanized antibody. 51. The antibody according to any of the above embodiments, wherein said antibody is a human antibody. 52. A human antibody binding to C5aR. 53. The antibody according to any of the above embodiments, wherein said antibody binds to human C5aR. 54. The antibody according to any of the above embodiments, wherein said antibody binds to the second extracellular loop of C5aR. 55. The antibody according to any of the above embodiments, wherein said antibody binds to the second extracellular loop of human C5aR. 56. The antibody according to any of the above embodiments, wherein said antibody binds to human C5aR but not to murine C5aR. 57. The antibody according to any of the above embodiments, wherein said antibody binds to the second extracellular loop of human C5aR, but not to the second extracellular loop of murine C5aR. 58. The antibody according to any of the above embodiments, wherein said antibody binds to the second extracellular loop of human C5aR only in the native conformation. 59. The antibody according to any of the above embodiments, wherein the antibody inhibits or significantly reduces the binding of C5a to human C5aR. 60. The antibody according to any of the above embodiments, wherein the antibody is capable of displacing C5a in a SPA test, with an IC50 below 10 nM or below 5 nM or preferably below 3 nM. 61. The antibody according to any of the above embodiments, wherein the antibody significantly inhibits the migration of human neutrophils in vitro. 62. The antibody in accordance with any of the above modalities, wherein the antibody reduces migration to less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, or less than 10%, when measured after 30 minutes in the presence of 10 nM of C5a compared to the level of migration observed after 30 minutes in the presence of 10 μM of C5a and without antibody or where the IC50 in the same configuration is below 2.5 ug / ml, such as below 2.5 μg / ml, such as less than 1.5 ug / ml, such as less than 1 g 0.2 μg / ml or even below 1.0 ug / ml. 63. The antibody according to any of the foregoing embodiments, wherein the affinity of the antibody as measured by the competing ligand binding test in neutrophils is below 0.80 nM, such as below 0.50 nM or 0.35 nM. . 64. The antibody according to any of the foregoing embodiments, wherein the antibody neutralizes neutrophil activation induced by C5a ex vivo with an IC50 as determined in a calcium flux test below 7.0 ug / ml, such as below of 5.0 ug / ml, such as below 2.5 ug / ml. 65. The antibody according to any of the above embodiments, wherein the antibody inhibits neutrophil maturation induced by C5a ex vivo with to. an IC50 as determined in a test of up-regulation of CDllb below 3.5 μg / ml, such as below 2.5 μg / ml, such as less than 1.5 pg / ml or even below 1.0 μg / ml or b. an IC50 as determined in a down-regulation test of CD62L below 1.8 μg / ml, such as below 1.5 μg / ml, such as below 1.2 μg / ml or even below 1.0 μg / ml. 66. An antibody that binds to C5aR, wherein the Fe region has decreased affinity / reduced binding to one or more FCY receptors compared to Fe reference sequences of IgG1, IgG2, IgG4 or IgG4 / G2 as defined by SEQ ID NO: 33, 34, 35 and 36, respectively. 67. The antibody according to any of the foregoing embodiments, wherein the Fe region includes one or more point mutations in comparison with reference sequences Fe of IgG1, IgG2, IgG4 or IgG4 / G2 as defined by SEQ ID NO: 33, 34, 35 and 36, respectively, which reduces affinity to one or more Fcy receptors. 68. The antibody according to any of the above embodiments, wherein the antibody does not significantly induce phagocytosis of neutrophils in vitro. 69. The antibody according to any of the above embodiments, wherein the antibody does not induce ADCC significantly in vitro. 70. The antibody in accordance with any of the above embodiments, wherein the antibody does not significantly induce CDC in vitro. 71. The antibody according to any of the above embodiments, wherein the Fe region is IgGl (SEQ ID NO: 33), IgG2 (SEQ ID NO: 34), IgG2 / 4 (SEQ ID NO: 35), or IgG4 (SEQ. ID NO: 36), with one or more of the following point mutations to. E233P b. L234A or V234A F234L or F234V c. L235E or L235A d. G236R or G236A and. G237A F. N297Q g. L328R h. A330S i. P331S 72. The antibody according to any of the above embodiments, wherein the Fe region is IgG1 or an IgG1 mutant. 73. The antibody according to any of the foregoing embodiments, wherein the Fe region is an IgG1 Fe mutant comprising from 1 to 10 amino acid substitutions as compared to the IgG1 Fe reference as defined in SEQ ID NO: 33 74. The antibody in accordance with any of the above embodiments, wherein the Fe region is an IgG1 Fe mutant comprising from 1 to 8 amino acid substitutions at AA 231 to 240 wherein the IgG1 Fe reference sequence is as defined in SEQ ID NO: 33 . 75. The antibody according to any of the above embodiments, wherein the Fe region is an IgG1 Fe mutant comprising from 1 to 5 amino acid substitutions at AA 328 to 334 wherein the IgG1 Fe reference sequence is as defined in SEQ ID NO: 33. 76. The antibody according to any of the above embodiments wherein the antibody Fe region is IgGl, with one or more of the following groups of point mutations to. N297Q and / or b. L234A and L235E and / or c. G236R and L328R and / or d. N297Q, L234A and L235E and / or and. N297Q, L234A, L235E and G237A and / or F. L234A, L235E, G237A, A330S and P331S 77. The antibody according to any of the above embodiments 52-76, wherein said antibody is a full length antibody or an antibody fragment or a single chain antibody. 78. The antibody according to any of the above embodiments, wherein said antibody is a monoclonal antibody. 79. The antibody according to any of the above embodiments, wherein said antibody is a human, mouse, rat, rabbit, pig or non-human primate antibody. 80. The antibody according to any of the above embodiments, wherein said antibody is a mouse or human antibody. 81. The antibody according to any of the above embodiments, wherein said antibody is a human antibody or a humanized antibody. 82. The antibody according to any of the above embodiments, wherein said antibody is a human antibody. 83. An antibody according to any of the foregoing modalities, for the treatment of an immunological disease or disorder. 84. The antibody according to the modality 83, wherein the disorder is an inflammatory disease. 85. The antibody according to the modality 83, wherein the disorder is an acute or chronic inflammation. 86. The antibody according to mode 83, wherein the disorder is an autoimmune disease. 87. The antibody according to any of the modes 83-86, wherein the antibody is administered intravenously or subcutaneously. 88. The antibody according to any of the above embodiments 83-87, wherein the antibody is administered in doses of 0.010 mg / kg up to 6 mg / kg. 89. The antibody according to any of the above embodiments 83-88, wherein the antibody is administered once a week or every 2 weeks. 90. The antibody according to any of the above embodiments 83-89, wherein the antibody is administered in combination with another drug. 91. The antibody according to any of the above embodiments 83-90, wherein the disease or disorder is rheumatoid arthritis (RA), psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC) or irritable bowel syndrome. 92. The antibody according to any of the above embodiments 83-91, wherein the patient is being treated with another drug such as methotrexate. 93. A method for treating or preventing a disorder in a subject, the method comprises administering to a subject in need thereof a therapeutic amount of an antibody in accordance with any of embodiments 1 to 82. 94. The method of compliance with modality 93, wherein the disorder is an immunological disease or disorder. 95. The method according to mode 93 or 94, wherein the antibody is administered intravenously or subcutaneously. 96. The method according to any of embodiments 93-95, wherein the antibody is administered in doses of 0.010 mg / kg up to 6 mg / kg. 97. The method according to any of the embodiments 93-96, wherein the antibody is administered once a week or every 2 weeks. 98. The method according to any of embodiments 93-97, wherein the antibody is administered in combination with at least one other drug. 99. The method according to any of embodiments 93-98, wherein the disorder is an immunopathological disorder such as an autoimmune disease. 100. The method according to any of embodiments 93-99, wherein the subject is a patient suffering from rheumatoid arthritis (RA), psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease (IBD), disease of Crohn's disease (CD), ulcerative colitis (UC) or irritable bowel syndrome. 101. The method of compliance with any of the 93-101 modalities, where the patient is being treated with another drug. 102. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 82 optionally in combination with a pharmaceutically acceptable carrier. 103. The pharmaceutical composition according to the embodiment 102, in the form of an aqueous formulation or a dry formulation which is reconstituted in water / an aqueous pH regulator before administration. 104 An isolated polynucleotide encoding a polypeptide sequence (s) of an antibody according to any one of embodiments 1 to 82. 105. A host cell comprising one or more polynucleotides conforming to embodiment 104. 106. A method for producing an antibody according to any of embodiments 1 to 82, which comprises culturing a host cell according to mode 105 under conditions that support the expression of one or more polypeptide sequences of that antibody. 107. The method according to embodiment 106, wherein the heavy chain and the light chain are encoded by two separate open reading frames in a contiguous polynucleotide. 108. The procedure according to modality 106 or 107, which also includes recovering that antibody from the culture of host cells. 109. The use of an antibody according to any of embodiments 1 to 82, for the manufacture of a drug. 110. The use of an antibody according to any of embodiments 1 to 82, for the manufacture of a drug for the treatment of an immunological disease or disorder such as rheumatoid arthritis (RA), psoriatic arthritis, systemic lupus erythematosus (SLE), nephritis for lupus, inflammatory bowel disease (IBD) or irritable bowel syndrome.

And emplos Example 1: Generation of human anti-hC5aR antibodies Immunization and selective determination In general, the production of antibodies against GPCR is difficult since the soluble protein that has the conformation of the correct native protein is very difficult, if not possible, to produce. Traditionally, cells that over-express GPCRs have been used for immunization, but the resulting antibody responses tend to be very nonspecific which makes it difficult to identify antibodies that have the desired profile, ie be able to block the binding to ligand and GPCR signaling. In fact, the inventors have found that it is very difficult to develop human anti-hC5aR antibodies that could block the binding of C5a to C5aR, and a series of immunization strategies were applied before these antibodies were identified.

HuMAb mice (Medarex) were immunized with mouse Ll2 cells (a mouse B cell lymphoma line) with high expression of human C5aR (-80,000 copies per cell) (Lee et al Nat. Biotechnol, 2006, 10: 1279-1284) and the splenocytes from the immunized mice were used for cell fusions by the use of standard procedures. Due to the lack of soluble hC5aR, the supernatants could not be evaluated in a standard ELISA test, and therefore a cell-based binding test was established. The supernatants of the obtained hybridomas were tested for binding to a transfected rat cell line (RBL) which stably expressed a high number (-1,000,000 copies per cell) of native hC5aR by FACS analysis as described in WO2008 / 022390. In general, hybridoma supernatants were incubated with a mixture of untransfected cells (labeled with CellTracker) and cells transfected with hC5aR, or neutrophils from mice inactivated / activated for hC5aR (KOKI) (document O 2005/060739), and incubated with goat anti-human IgG APC conjugated with F (ab ') 2 (IgG-APC). Supernatants that bound to hC5aR- cells were identified transfected but not to non-transfected cells, and hybridomas producing anti-hC5aR were subcloned and tested for binding to human neutrophils and neutrophils derived from bone marrow isolated from KOKI mice (data not shown). Anti-hC5aR antibodies were purified from hybridoma supernatants by using protein A sepharose and standard protocols.

Example 2: Identification and characterization of anti-hC5aR antibodies As mentioned, the process of obtaining human anti-hC5aR antibodies is problematic, and 32 fusions had to be performed before a hC5a / hC5aR blocking antibody was identified. From 35 mergers and selective determination of more than 100, 000 supernatants of hybridomas only 11 clones were identified that could block the binding of hC5a to hC5aR. The tests applied in the characterization of antibodies are described below. The reference antibody (Ref. Ab Q) is described in WO2009 / 103113. In addition, other tests suitable for determining the affinity and functionality in a calcium flux test and up-regulation of CDllb are described in example 7.

Displacement Test A scintillation proximity test (SPA) was applied in order to determine the potency of anti-hC5aR antibodies to displace the binding of hC5a to hC5aR. A detailed description of the SPA is provided in the patent of E.U.A. 4568649 and protocols provided by the manufacturer (Amershara Biosciences). Briefly, receptor-carrying membrane fragments purified from the binding of RBL-hC5aR cells to scintillating microparticles coated with wheat germ agglutinin (WGA). After the addition of the radiolabelled hC5a tracer (125I), binding to the receptors will result in the emission of light from the particles. Specific to the SPA principle, only the radioisotope and particles in close proximity to one another will emit light, that is, only radiolabeled hC5a bound to a receptor is close enough to a WGA particle to produce light. The amount of light emitted is therefore an expression of the amount of 125 I-hC5a bound to receptor. The test is a competition test, where unlabelled anti-hC5aR / hC5a competes with the tracer on binding to the receptors. In the test, a fixed amount of C5a labeled with 125I is added to WGA particles and the C5aR receptors resulting in the emission of a certain amount of light measured as counts per minute (cpm). If unlabeled C5a or anti-C5aR is added, binding them to the receptors herein will cause a lower cpm due to displacement of 125 I of C5a. % Of Displacement was calculated as follows: s ~ S mx - 100% So - S max S: Sample SmSx: Non-specific union. Measurement by adding hC5a not labeled in an amount sufficient to replace the 125i-hC5a specifically bound.

S0: Maximum union. No hC5a is added without labeling.

The IC50 value is defined as the concentration that displaces 50% of C5a. The cpm remained constant between the experiments, so the IC50 values are relative as the tracking decades over time. The potency (IC50) of the human anti-hC5aR antibodies to displace 125I-hC5a was determined and the data are presented in Table 1.

Neutrophil migration test (chemotaxis) The potency of the antibodies to inhibit the neutrophil migration dependent on hC5a (or mC5a) was analyzed in a Boyden chamber. Neutrophils isolated from human or animal blood were stained with calcein and added to the upper compartment of the Boyden chamber and mixed with the antibodies. hC5a or mC5a is applied to the lower compartment of the Boyden chamber and acts as a chemoattractant for neutrophils. The ability of neutrophils to migrate to the lower chamber is determined by count the number of neutrophils stained with calcein that pass through a FluoroBlok membrane of 3 or 5 pm.

Human PMN (polymorphonuclear leukocytes, -granulocytes) were obtained from human blood samples obtained in flasks containing EDTA. The blood cells were separated by centrifugation of blood (4 parts) through a gradient of Ficoll-Paque PLUS (GE Health Care) (3 parts) for 30 min (400 x g) at room temperature. The PMN-containing layer was suspended in PBS (phosphate-buffered saline) containing dextran-500 (Sigma) for 1 hr to remove contaminating erythrocytes. The supernatant was centrifuged for 5 min (250 x g) at room temperature and the remaining erythrocytes were lysed osmotically by using 0.2% NaCl for 55 sec. The solution was made isotonic by 1.2% NaCl + PBS and centrifuged at 250 x g for 5 min, before the osmotic lysis was repeated. After centrifugation, the PMN were resuspended in the reaction mixture (RM): HBSS (Gibco Cat. No. 14175) contained 137 mM NaCl, 5.3 mm KC1, 0.33 mm Na2HP04, 4 mM NaHCO3, 0.44 mM KH2P04, glucose 5 mM, supplemented with MgSO4 · 7H20 0.4 mM, MgCl2, 0.5 mM, 0.5 mM CaCl2, 20 mM HEPES. The cell density was determined by NucleoCounter (Chemometec). The PMN suspension contained > 95% of neutrophils as evaluated by microscopy of samples stained with Giemsa.

PMN load: calcein, AM, (Fluka) was dissolved in DMSO (dimethyl sulfoxide) and diluted 1000X in RM with cells (2xl06 cells per ml) to give a concentration of 10 μ ?. The suspension was incubated for 30 min in the incubator at 37 ° C and then washed 3 times with RM to remove the excess calcein. Finally, the cells were resuspended in MRI (4 × 10 6 cells / ml).

Migration was evaluated by the Boyden chamber technique using FluoroBlok® 3 and pore size, 96 wells (Cat. No. 351 161 BD Falcon (VWR)). The upper chamber i.e., the inserts containing the FluoroBlok membrane, was coated with human fibrinogen (Cat. No. F3879-1 G, Sigma) in 1 mg / ml of PBS at 37 ° C for 2 hours. After washing, the membranes were blocked with a solution containing 2% bovine serum albumin (BSA) in PBS. After another MRI wash, 105 PMN loaded with calcein with or without the hC5aR-antibodies were added to each well and placed in the receptor plate (lower chamber) containing the control solution or the hC5a chemoattractant hC5a (Sigma, C5788 ). Each group comprised at least 6 wells. Subsequently, the plate was measured at 485/538 nm, 37 ° C every 5 min for 60 min in a plate reader (SpectraMax, Molecular Devices, or Fluoroscan, Thermo Labsystems.). The value at 30 minutes in relative fluorescence units was used as a measure of migration.

Curve adjustment. The ability of the antibodies to inhibit migration was expressed by IC50 as determined with the use of GraphPad Prism 5 (GraphPad Software, Inc).

Table 1 includes the data from the displacement test and the chemotaxis test that tested the capacity of human mAbs of 10 pg / ml to inhibit human neutrophil migration dependent on hC5a (10 nM). The value obtained in the absence of antibody was set at 100. The data was compiled from 3 donors. The average values are included in table 1. The three mAbs 32F3A6, 35F12A2 and 35F32A3 showed the strongest power in both tests, which was equal to or slightly higher than the potency of a control antibody Ref. Ab is Q described in the document WO2009 / 103113.

Table 1. Functional characteristics of anti-hC5aR antibodies Characterization of CDR sequences anti-hC5aR mAb The variable regions of the anti-hC5aR Abs 35F32A3, 32F3A6, 35F12A2 and 35F24A3 were cloned in recombinant form and the nucleotide and amino acid sequences were characterized by the use of standard methods. The amino acid sequences are included in Figure 1 and the sequence listing annexed.

Characterization of binding specificity Chimeric human-mouse C5aR constructs were used to determine the binding region of C5aR. The chimeric receptors were transiently expressed in HEK cells and the binding of the individual antibodies was determined by FACS as previously described in document O 2008/022390 except for the cell line change. The binding of 32F3A6, 35F32A3 and 35F12A2 was dependent on the human sequence of the extracellular loop 2, whereas the human N-terminus was dispensable (Figures 2A-2B).

Example 3. Generation of Fe variants The four subclasses of human IgG (IgG1, IgG2, IgG3 and IgG4) share more than 95% homology in the Fe regions, but show important differences in the hinge region. The Fe region mediates effector functions, such as cell-mediated cytotoxicity antibody-dependent (ADCC) and complement-dependent cytotoxicity (CDC, for its acronym in English). In ADCC, the Fe region of an antibody binds to Fe activation receptors (FCYRS) on the surface of immune effector cells, such as natural killer cells and monocytes, which leads to phagocytosis or lysis of target cells . In the CDC, the Fe region binds to complement at a different site of the FcyR binding sites, and the antibodies kill the target cells by triggering the complement cascade on the cell surface. The different isoforms of IgG exert different levels of increasing effector functions in the order IgG4 <; IgG2 < IgGl < IgG3. A number of IgG Fe variants, comprising the entire variable region of Ref. Ab Q, were generated by site-directed mutagenesis through the use of the QuickChange® Site-directed Mutagenesis Kit (Cat. No. 200518, Stratagene) and it was characterized as described in example 4.

Example 4: Characterization of the effector functions of the Fe variants Affinity of union of variants of Fe to FcgRs The affinity of the Fe variants towards FcyRs was determined by plasmon resonance measurements of surface (SPR), which were performed on a BIAcore T100 instrument by using a CM5 sensor chip (GE). The Fe variants were immobilized on the flow cells by the use of amine coupling chemistry. For the measurement of kinetic SPR, the affinity of FcyR to the Fe variants, HIS-FCYRS were used as analytes and injected into flow cells in pH regulator HBS-EP. The high affinity receptor FcyRI was injected with a flow rate of 40 μm / min, a contact time of 180 seconds, and a dissociation time of 300 seconds. The other FcyRs were injected with a flow rate of 50 μm / min, a contact time of 30 seconds, and a dissociation time of 120 seconds. Chip surfaces were regenerated with a solution containing 10 mM NaOH and 500 mM NaCl. The affinities (Kd values) are listed in table 2 ?.

Table 2A. Summary of the results obtained from the analysis of the affinity of Fe variants towards FcyRs (Kd in M). (- = no union, 0 = no change in the union, nda = Kd not calculated due to very weak union). (1) a Fe IgG2 / lgG4 variant comprising the CH1 region and the lower hinge region of IgG2, and the CH2-CH3 moiety of IgG4; (2) IgG4 mutant that includes the S228P point mutation.

Phagocytosis test In order to identify Fe variants with reduced or nullified capacities to mediate phagocytosis of neutrophils, a phagocytosis test was established in vitro. The phagocytosis test described below involves the labeling of human neutrophils isolated from human peripheral blood (the target cell for phagocytosis) with a fluorescent dye, CMFDA, and the addition thereof to a culture of human monocytes, also isolated from human peripheral blood. Neutrophils labeled with CMFDA are pre-coated with test mAb (or PBS), and after incubation with human monocytes, the number of CD14 / CMFDA positive double monocytes is determined by FACS. The results of several Fe variants are presented in Table 2B.

All antibodies tested include the variable regions of the Q antibody described in WO2009 / 103113 and used as Ref. Ab previously.

Both monocytes and macrophages were found to be able to mediate neutrophil-dependent phagocytosis of antibodies, and phagocytosis tests were established using both cell types. The results were qualitatively similar in both tests, but since the macrophage test was more variable, the analysis was carried out mainly through the use of monocytes.

Preparation of human monocytes Human monocytes and lymphocytes were isolated from peripheral venous blood collected from healthy human volunteers in tubes containing EDTA as an anticoagulant (K2E, BD Biosciences, Cat. No. 367525) by the use of Percoll gradient centrifugation. 100 ml of blood usually gave ~ 8 - 20 x 107 peripheral blood mononuclear cells (CMSP, for its acronym in English). At least 3 volumes of dPBS were added to the isolated cells which were then centrifuged at 100 x g for 10 min at room temperature (RT). After discarding the supernatant of the lymphocyte / monocyte layer, it was resuspended in the same volume of a 50:50 mixture of dPBS: Culture medium as the previous step and centrifuged again at 100 × g for 10 min at RT. The supernatant was discarded and the lymphocyte / monocyte layer was resuspended in 1-2 x 10 6 cells / ml in culture medium. The resuspended cells were seeded in 6-well tissue culture plates (Corning, Cost Cat. No. 3516) in 2 ml / well with 4 x 106 cells / well and incubated for 2 hours at 37 ° C in 5% C02. The non-adherent cells (lymphocytes and dead cells) were removed by aspiration and the adhered cells (monocytes) were washed four times in 1 ml of culture medium (RPMI 1640 (Invitrogen-Gibco, Cat. No. 11875) + 19% of FCS (GIBCO-Invitrogen, Cat. No. 16000) inactivated with heat at 56 ° C for 30 min + 25 mM Hepes (Invitrogen-Gibco, Cat. No. 15630) + 1% penicillin / streptomycin (Invitrogen-Gibco, Cat. No. 15070)) with gentle agitation before aspiration of the washing medium. After washing, the monocytes derived from the blood were added with 1 ml of fresh culture medium to each well. The cells were scraped from a well and suspended in culture medium in order to estimate the number of monocytes per water well .

Preparation of human neutrophils Human neutrophils were isolated from peripheral venous blood obtained from healthy volunteers) by using Percoll gradient centrifugation and stained with CellTracker ™ Green (5-chloromethyl-fluorescein diacetate, CMFDA). 100 ml of blood usually gave -10-20 x 107 neutrophils. Staining was performed by dissolving CellTracker ™ Green CMFDA in DMSO at 10 mM final concentration. Neutrophils were resuspended in 1 x 107 cells / ml in dPBS and CMFDA was added to a final concentration of 2 μ? . The cells and the dye were incubated for 15 min at 37 ° C. The excess dye was removed by washing the cells 3 times with 10 ml of DPBS (by centrifugation at 300 x g for 5 min at room temperature). A cell count was performed after the last wash step. Neutrophils labeled with CMFDA were resuspended in 2 x 10+ cells / ml in dPBS and incubated with the antibody (final concentration of 0.001, 0.01, 0.1, 1, 10 or 100 g / ml) or PBS (for control without antibody ). In some tests (as indicated) the neutrophil incubation step + Ab also contained 4 mg / ml of human IgG. The cells plus the antibodies were incubated for 30 min at 37 ° C. The neutrophils were washed twice with dPBS after centrifugation at 300x g for 5 minutes at RT and resuspended in culture medium at 1 x 10 7 cells / ml.

Analysis of FACS Neutrophils labeled with CMFDA, pre-coated with the antibody (prepared as described above) were added to the monocytes (as described above) at the desired concentration in 1 ml of culture medium. The total volume in each well of the 6-well plate was 2 ml. In some tests (as indicated) the culture medium also contained 4 mg / ml of human IgG. A ratio of 5:01 (neutrophils: monocytes) was used in general. If the number of adherent monocytes was less than 4 x 105 per well and then 2 x 106 neutrophils was added (ie, the ratio of neutrophils: monocytes exceeded 05:01). If the number of monocytes exceeded 4 x 105 per well then five times that number of neutrophils was added to maintain the ratio of neutrophils: monocytes to 05:01. The cultures were incubated for 1 hour at 37 ° C in an incubator with 5% C02.

After incubation, the medium was aspirated to remove non-adherent and non-ingested neutrophils. Adherent monocytes were washed (with gentle centrifugation) three times with 1 ml / well of culture medium. Monocytes were harvested in 15 ml tubes by scraping the cells in well culture medium with cell scrapers (Corning, Cat. No. CP3010). The cells were centrifuged at 300x g for 5 min at room temperature and the supernatant was eliminated. The cell pellet was resuspended in 160 μ? 1% (w / v) of paraformaldehyde in PBS to fix before the FACS.

The samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Neutrophils labeled with CMFDA were identified and measured in FL-1 (fluorescence channel 1) by the use of fluorescein isothiocyanate (FITC), and monocytes were identified by staining a single sample of monocytes with labeled anti-CD14 with phycoerythrin, which was measured in FL-2 (fluorescence channel 2). A monocyte gate was defined by using the SSC profile (forward scatter) vs. SSC (lateral dispersion) of the single monocyte sample and was extended (along the axes of FSC and SSC) to include monocytes whose size had increased during incubation. This gate excluded the profile region of FSC vs. SSC containing neutrophils as defined in the FSC profile vs. SSC in a single sample of neutrophils. The degree of phagocytosis was calculated to be the percentage of FL-1 + monocytes seen at the total monocyte gate.

The background level of non-specific phagocytosis was the percentage of FL-1 + ves monocytes in a sample containing neutrophils labeled with CMFDA not coated with antibody ("No Ab" sample). The background was subtracted from each sample with Ab before data (% FL-l + ve monocytes vs concentration of Ab) were introduced into a prism (v4.0c, GraphPad Software Inc) for graphic representation. The data were subjected to non-linear regression by using the sigmoidal dose-response (variable slope), ie 4-parameter logistic equation, in order to determine the EC50 values in their case.

The data is presented in Table 2B. "-" Represents phagocytosis not detectable and "+" to "++++" represents lower to higher level of phagocytosis, measured in the tests.

Tests for ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity) The following in vitro tests were established in order to test the ability of Fe variants to mediate cell depletion through ADCC or CDC-dependent mechanisms.

Target cells In these tests, the target cells were Ramos E2 clones expressing hC5aR or human neutrophils. The E2 clones expressing hC5aR were developed by stable transfection of Ramos clone E2 cells with a mammalian expression vector encoding hC5aR by the use of standard procedures. The resulting cell line expresses high levels of human C5aR (5-7 times higher than in human neutrophils) and CD20. Human neutrophils were obtained as described above in relation to the phagocytosis test.

The target cells were stained with the fluorescent cell membrane dye, PKH-26. The required number of target cells (5 x 10 4 / sample / well x 4) was diluted to 15 ml in dPBS and centrifuged at 1, 200 rpm for 5 min at RT. The cells were then resuspended in 2 μ? of PKH-26 (100 μl of solution per 1 x 10s target cells). The labeling was allowed to proceed at room temperature for exactly 3 minutes before an equal volume of heat-inactivated FCS was added (or human serum inactivated by heat (Millipore)) to stop the labeling reaction. After exactly 1 min, RPMI was added to a total volume of 15 ml. The cells were centrifuged as before and resuspended at 2 x 10 6 cells / ml in test medium. For coating with aliquots of antibodies (25 μm ie 5 × 10 4) of target cells labeled with PKH-26 were placed in the wells of a 96-well U-shaped plate containing 25 μm. of 200 mg / ml of antibody diluted in test medium (final concentration of 100 μ9 / t? 1) and incubated at 37 ° C in 5% C02 for 30 min.

Effector cells The effector cells were PMBCs depleted in monocytes from healthy donors. The PMBCs were obtained as described earlier. Resuspended cells (lymphocytes / monocytes) were seeded in 6-well tissue culture plates (Corning) in 2 ml / well with ~ 4 x 10 6 cells / well or T75 flasks (Corning) at 20 ml per flask and incubated for 2 hours at 37 ° C in 5% C02. The non-adherent cells (including lymphocytes and NK cells) were removed by aspiration and centrifuged at 100 g for 10 min at room temperature. The cells were resuspended in 20 ml of medium containing 100 ng / ml of sterile recombinant human IL-2 solution to increase the number of lymphocytes and natural killer cells. The cells were incubated overnight at 37 ° C in 5% C02. The next day the cells were centrifuged at 1400 rpm for 10 min at room temperature, then resuspended in test medium at 2.5 x 10 7 cells / ml for use as effector cells in the ADCC test.

ADCC test After labeling of the target cells with PKH-26 and the coating with antibody 100 μ? of effector cells or 100 μ? Test medium (control, only target cell) was added directly to 50 μ? of target cells. The samples were incubated for another 3 hours at 37 ° C in 5% C02. Samples were transferred to 1.2 ml microtiter FACS tubes containing 10 μ? of 10 μ? of viability dye To-Pro-3 (TP-3) for a final concentration of -625 nM and the samples were analyzed by FACS. In a graph of FSC vs. SSC, all cells except the waste were separated. The separated cells were analyzed in FL-2 vs. FSC and FL-2 positive cells (ie, PKH-26 target labeled cells) were separated. The FACS data was analyzed using the FlowJo software (Tree Star, Inc. v6.3.4).

Specific ADCC was calculated by subtracting the average% of TP3 + ve Objectives Only '(A) from the average% TP3 + ve + Obj ective + Effectors' (B) from the corresponding samples after subtracting the average% from TP3 + ve vNo Ab Only '(C) and the average% of TP3 + ve' There are no Objectives of Ab + Effectors' (D) respectively, that is, Specific ADCC = (B-D) - (A-C) or = (B-A) - (D-C) Equation 1 The results presented in Table 2B, were obtained by the use of depleted monocytes, human PBMC stimulated by IL-2, predominantly NK cells but including B cells, T cells and dendritic cells, as the population of effector cells. The target cells were a Ramos E2 transfected cell line that expressed both hC5aR and CD20 that allowed the use of the anti-CD20 antibody rituximab as the positive control. The results vary from "+++", which is indicative of a variant Fe that induces ADCC with the same potency as rituximab, "+ / -", which is indicative for a variant of Fe for which a high degree of donor variation was observed and "-" which is indicative of a Fe variant for which no significant induction of ADCC was detected. A variant Fe that mediates increased ADCC (IgGl_S239D, I332E) (Chu SY, Vostiar I, Karki S et al, Mol Immunol, 2008, 45 (15): 3926-3933) was included as a positive control for the test.

CDC test The Fe variants were also analyzed for their potency to induce CDC. The experimental approach was essentially as described for the ADCC test, except that the effector cells were replaced by human serum.

Target cells, such as Ramos E2 cells (2 x 106 cells / ml) in medium with 3% rabbit complement serum were mixed with an equal volume of 2x antibody solution (200 mg ml), containing 3% serum of rabbit complement in a 96-well U-bottom tissue culture plate. A duplicate set of wells contained 25 μ? of Ramos E2 cells (2 x 106 cells / ml) mixed with 25 μ? of 2x antibody solution (200 mg ml) in medium without complement. A set of 3 wells contained 25 μ? of E2 Ramos cells (2 x 106 cells / ml), in addition to 25 μ? of test medium ("non-ab 'samples) in 3% rabbit complement serum Another set of 3 wells contained 25 μ? of E2 Ramos cells (2 x 106 cells / ml), in addition to 25 μ? of test medium ("no ab 'samples) without complement." Prior to incubation, 100 μl of test medium, with or without 3% rabbit complement serum where appropriate, was added to each well. they were incubated for 3 hours at 37 ° C in 5% C02.

Determination of the viability of the target cells Fluorescent viability dye To-Pro-3 (Molecular Probes) was added to each sample immediately before analysis by flow cytometry. The final concentration of To-Pro-3 in each tube was -62.5 nM. To-Pro-3 positive cells (TP3 +) were defined as non-viable or lysed.

Flow cytometry and data analysis The samples were analyzed in a FACSCalibur (BD Biosciences) and the data obtained were analyzed by using FlowJo software (v6.3.4, TreeStar Inc.). In the scatter plot of FSC vs. SSC, the separation of all cells except the residues, 5,000 events of target cells were collected for each sample. A histogram of the separate target cells was created in the FL-4 channel that showed the level of absorption of To-Pro-3 by the cells. The number of TP3 + cells (ie non-viable cells) in each sample was determined - these are defined as cells to the right of the main peak - and this number was expressed as a percentage of the total target cells in the sample.

Samples, tested in triplicate, could be classified into one of 4 categories, A, B, C and D as shown in the lower category overview.

Category overview Categories of samples analyzed For each sample containing the antibody, the reactions were carried out with or without complement. Samples containing antibody without complement gave the background level lysis of antibodies. Samples of categories B and D gave nonspecific background lysis in the absence of either antibody or both antibody and complement.

The percentage of TP3 + non-viable target cells (% lysis) was calculated for each sample with 3% complement and for each sample without complement. For each antibody and the 'no Ab' control, data from the triplicate samples were averaged.

To calculate the specific activity of CDC for each antibody, the specific lysis of antibody in the absence of complement (% average lysis in the samples ?? ' ) was subtracted from the% lysis of the Ab sample with complement (deCi2 / 3 'samples), before subtracting the non-specific background lysis. Non-specific background lysis was the lysis of the 'no ab' samples with complement (% of average lysis in the samples of "D") less lysis in the samples' no ab 'without complement (% of average lysis in the samples' ? '). The variant Fe that mediates the increase in CDC (IgGl_S254W) (WO08030564) was included as a positive control for the test.

Specific CDC (¾ lysis) = (C-A) - (D-B) [or = (C-D) - (A-B)] Equation 2 The statistical analysis was carried out in GraphPad Prism (v4.0) to determine if the differences between any of the groups were significant. The specific CDC activity of each sample of the 4 tests was entered into a spreadsheet according to antibody use. The groups were compared using a parametric test: unidirectional analysis of variance (ANOVA) followed by Tukey's multiple post comparison test.

The results are included in Table 2B. The results vary from "+++", which is indicative of a Fe variant that induce CDC with equal potency as the mutant IgGl S254W. "+/-", which is indicative of a variant of Fe for which a high degree of variation was observed and # - "which is indicative of a variant of Fe for which it is not detected no significant induction of CDC.

Table 2B. Activity of Fe variants in cell-based effector function tests Summary of the results obtained from the analysis of Fe variants in phagocytosis, ADCC and CDC tests. (- = no effector function; + = effector function). (1) a Fe IgG2 / IgG4 variant comprising the CH1 region and the lower hinge region of IgG2, and the remaining CH2-CH3 of IgG4; (2) S228P mutation introduced into the Fe region of IgG4 to eliminate the formation of semi-antibodies.

Example 5: Characterization of the potency of Fe variants of anti-hC5aR antibodies In order to test if the mutations in the region of Fe affect the potency of the antibodies to inhibit the binding of hC5a to hC5aR and neutrophil migration mediated by hC5a, respectively; the different Fe variants were tested in the displacement and migration tests described above. The neutrophil migration test was performed as described above, except that the mouse PMNs used were isolated from hC5aR-K0 / KI mice (deactivated for mC5a / activated receptor for human C5aR, WO 2005 060 739). The cells were obtained in the following manner. Bone marrow PMN were isolated from the femurs and tibias of two hC5aR-K0 / KI mice. The marrow cells were flushed from the bones by the use of PBS before the cell suspension was filtered through a cell filter (BD Falcon, 352350; 70 micron nylon mesh) in a 50 ml tube and centrifuged (10 min, 1600 rpm). The cells were resuspended in medium and carefully placed in layers on top of 3 ml of Ficoll-Paque PLUS (GE Healthcare) in a sterile 15 ml tube. After centrifugation, for 20 minutes at 600xg at room temperature, the neutrophil / erythrocyte pellet was isolated. The erythrocytes were used by using lysis pH regulator (Sigma, R7757, 8.3 g / L ammonium chloride in 10 mM Tris-HCl, pH 7.5) for 1 min. After two rounds of centrifugation and washing the pellet cells were resuspended in the reaction mixture. The suspension contained > 95% of neutrophils as evaluated by microscopy of samples stained with Giemsa. The variable region of the antibodies tested was identical to the variable region of Ref. Ab Q. The data are shown in table 3.

A significant difference in potency was observed to inhibit the binding of hC5a to hC5aR, for the Fe variants in the SPA analysis (Table 3, column 1). An IgGl version of Ref. Ab Q was analyzed together with additional variants of IgG Fe, and the data showed that IgGl Fe variants generally inhibited the binding of hC5a more potently than both the IgG4 and IgG2 / IgG4 variants. Fe. An F (ab ') 2 fragment from Ref. Ab Q was also included in the analysis and was found to inhibit the binding of hC5a to the same extent as Ref. Ab Q (IgG4) full-length. These findings indicated that the hinge region is important for the ability of antibodies to inhibit the binding of hC5a and this idea was supported by the fact that F (ab ') 2 fragments were able to inhibit the migration of neutrophils to the same degree that Ref. Ab Q (Table 3). It was also found that the IgG1 variants were more potent in inhibiting the migration of neutrophils than IgG comprising hinge regions of IgG2 or IgG4 (Table 3, column 2).

The greatest power of versions of Ref. Ab of IgGl on the IgG4 versions could be related to the increase in avidity due to the greater flexibility of the hinge region of IgG. To investigate this, the union of the versions of Ref. Ab of IgG1 and IgG4 to human neutrophils was analyzed by FACS. The data showed that the IgG1 version bound neutrophils with greater avidity than the IgG version. No data shown Taken together, these findings support that the increased flexibility in the hinge regions of IgGl contribute to the increased binding to hC5aR, which leads to an increase in potency.

Table 3. Effect of Fe variants on the binding of hC5a (SPA) and migration of neutrophils to hC5a (+ = low activity, ++ = average activity, +++ / + high).

Example 6. Generation and characterization of "fully" human anti-hC5aR antibodies From the analyzes described in Example 2, the 32F3A6 antibody was selected for further studies. During the recombinant cloning of this antibody, seven mutations were identified in the framework region of VH that differs from the human germline sequences, while no frame mutations were found in the LC (Figure 3). Mutations were found by aligning the VH and VL sequences of 32F3A6 to all available human germline sequences.

In order to make the antibody even more human-like, the seven point mutations in the VH region of 32F3A6 were mutated back to the human germline residues, and grafted into the IgGI Fe region comprising the five mutations L234A_L235E_G237A_A330S_P331S that were shown to cancel the induction of phagocytosis, ADCC and CDC as described above. The compound is known as 32F3A6 GL. The potency of the mutated antibody was again compared to the original antibody and no difference in potency was observed to inhibit the binding of hC5a to hC5aR (tested in SPA) or in the potency to inhibit neutrophil migration mediated by hC5a between 32F3A6 or 32F3A6 GL (data not shown).

The ability of the fully human antibodies described above to induce phagocytosis of neutrophils, ADCC or CDC was evaluated as described in example 4 and the results are summarized in table 4.

The results relative to the specific ADCC included in Table 4 were obtained by the use of human PBMCs depleted in monocytes such as the effector cell, and human neutrophils as the target cell.

Table 4. Fe-mediated cellular effector functions of anti-C5aR antibodies. "-" Represents no detectable effector function and "+" to "+++" represents low to high level of effector functions as measured in the tests described in example 4. ND (not determined).

As noted above, the 5 point mutations in the IgGl Fe region abolish phagocytosis, ADCC and CDC compared to the Fe region of wild-type IgGl.

Example 7. Additional characterization of human anti-hC5aR antibody (32F3A6 GL) To further elucidate the functionality of the identified antibodies and to determine affinity and potency, additional tests were performed by using one of the anti-C5aR antibodies compared to Ref AB Q. Affinity was determined by ligand binding test of competition in human neutrophils. This functionality is known as antibody affinity as measured by the competition ligand binding test, but the measurement of the avidity of the interaction could also be considered. The ex vivo tests measure the ability of the antibodies to neutralize the actions mediated by C5a in in vitro establishment. The potency tests measure the flux neutralization of Ca induced by C5a, upregulation of CDII receptor and down-regulation of CD62L, respectively, in human neutrophils. The data obtained for 32F3A6GL are given in Table 5.

Affinity measurements Isolation of neutrophils from fresh human blood The blood was diluted 1: 1 with PBS + 2% FBS and layered on Ficoll-Paque PLUS (GE Healthcare # 17-1440-03) in a ratio of 3 parts of Ficoll and 4 blood parts (15 ml of Ficoll and 20 ml of blood in a 50 ml tube) and, subsequently, stratified by centrifugation at 400 xg for 30 minutes at room temperature. By aspiration, the intermediate PBMC band was gently removed. The stratified granulocytes in the packed red blood cells were aspirated with a plastic Pasteur pipette. Granulocytes and red blood cells were transferred and pelleted in a new 50 ml tube. The pellet was diluted to 40 ml with 1 x PBS and 10 ml of a solution of DEXTRAN 500 (Sigma, 31392) at 4% in PBS (ratio 1: 5) was added and gently mixed by inversion. After 20-30 minutes, the obtained granulocyte rich supernatant was transferred to a new tube and centrifuged at 250 x g for 5 minutes at room temperature. The contaminating red blood cells were removed by osmotic lysis upon resuspending. the cell pellet in 7.5 ml 0.2% NaCl and mixed gently for 55-60 seconds. Subsequently, 17.5 ml of 1.2% NaCl was added and then diluted to 50 ml with PBS and centrifuged at 250 x g for 5 min. This step was repeated once. The cell pellets were subsequently resuspended in 1 ml of reaction mixture (dPBS / RPMI). Viability and cell count were monitored by using NucleoCounter®.

Neutrophil competition ligand binding test Human neutrophils were purified, washed and resuspended in binding pH buffer (50 mM HEPES, pH 7.5, 1 mM CaCl 2, 5 mM MgCl 2 and 0.5% bovine serum albumin (Fraction V IgG free) ) at ~ 5 x 106 cells / ml. For each sample, 40 μ? of cell suspension (1 x 10 5 cells / well) were seeded in a 96-well V-shaped plate (Greiner, Cat. # 651101). Competency studies were conducted by using 12 concentrations of unlabeled ligand competition in semi-log dilutions that started with 1 μ? as the "highest concentration." 40 μ? of antibodies were added when considering a final test volume of 120 μ ?. 40 μ? of radioligand [12 I] -hC5a (Perkin Elmer, Cat. No. NEX250) were added to all samples, except background control.The final concentration of radioligand in the test was 1 nM and the final volume was 120 μ? . All samples were made in triplicate and incubated for 4 hours at 4 ° C. The cells were then harvested by centrifugation at 1200 rpm, at 4 ° C for 2 min and washed three times in 100 μ? of washing pH regulator (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 150 mM NaCl and 0.5% bovine serum albumin (Fraction V IgG free)). Finally, the cells were resuspended in 30 μ? of washing pH regulator and were transferred to an OptiPlate (Perkin Elmer, Cat. No. 6005290) and 150 μ? of MicroScint 20 (Perkin Elmer, Cat. No. 6013621) were added to each well. The plates were covered, mixed well in a counter Counter calibrated with delay of 1 hr. The total amount of radioligand added to the test was determined on a separate plate. The number of counts in each sample was expressed as normalized values in percent where 100% is the maximum level of counts where 1 nM of [125I] -hC5a and no cold antibody was added, and 0% is the non-specific binding determined in presence of 1 μ? of hC5a cold. The data were analyzed by non-linear regression using PRISM (GraphPad).

Calcium flow test Staining of human neutrophils with Fluo-04 AM cell dye The neutrophils were centrifuged and washed in PBS and then resuspended at 1 x 10 7 cells / ml in cell dye and incubated at room temperature for 40 min in the dark. The cells were centrifuged and washed (to remove excess dye), centrifuged again and resuspended at 2 x 10 6 cells / ml in pH buffer of the cells. The cells (0.5 ml) were divided into aliquots in non-sterile glass FACS tubes - one tube for each sample - stored at room temperature and used within two hours. Each sample used 1 x 106 neutrophils.

Proof The calcium flow test was carried out in the following manner. Briefly, 1 x 106 neutrophils loaded with Fluo-4 AM in 0.5 ml of pH regulator cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences) with separated neutrophils using the FSC of the x-axis. SSC of the y axis. The FL-1 channel (FITC) was used to measure the fluorescence of neutrophils after the addition of various reagents for the tube (e.g., antibodies, C5a, ionomycin - dissolved at a final concentration lOx in pH regulator of cells instead of I-GB or C-MGB). The fluorescence of the sample was measured continuously with a mean fluorescence intensity value (MFI) acquired every 1 second. These data were saved in a CellQuest file (BD Biosciences) and they were transferred to Excel (Microsoft) and Prisma (v4.0c, GraphPad Software Inc.) for further processing and analysis. The order of addition of reagents to neutrophils and incubation times varied according to the type of test performed.

Neutralization test of C5a A 3-fold serial dilution of 10x antibodies, with concentrations ranging from 1,000 μg / μl to 1.37 g / ml, was prepared. Neutrophils loaded with Fluo-4 AM (1 x 106 in 0.5 ml of pH regulator cells) were incubated with 50 μ? of lOx antibody solution (final concentration of Ab in the tube: 100-0.137 ug / ml) for 10 min at room temperature. The more antibody cells were analyzed by FACS for -60 sec to establish baseline fluorescence. Then, 50 μ? from C5a to 10 nM to give a final concentration of ~ 1 nM and the fluorescence measurement continued for others -60 sec. If the antibody blocked the release of Ca2 + induced by C5a, there was no peak fluorescence. If the antibody did not neutralize C5a, then there was an increase in fluorescence. Finally, 50 μ9 of 1 μ9 / 1 1 of ionomycin was added to a final concentration of 0.1 μg / ml and the fluorescence measurement was continued for another -60 sec to ensure that the cells were still sensitive.

Up-regulation of CDII receiver Configuration test The following configuration was designed to determine the ability of the identified antibodies to neutralize neutrophil activation induced by C5a by measuring changes in CDII expression.

Anti-C5aR and isotype control antibody were diluted in PBS at 2x final concentration in a series of 3-fold dilutions (from 600 to 0.003387 9 p1) and 50 μ? supplied in duplicate in wells of 96-well U-bottom plates. An aliquot of 50 μl of heparinized whole blood was added to each well. Four well control sets (in duplicate) contained 50 μ? of PBS plus 50 μ? of blood only. The plates were incubated for 20 min at 37 ° C in an incubator with 5% C02. To activate neutrophils, 50 μ? of human C5a, final concentration 10 or 100 nM as specified, was added to the wells containing Ab and a set of control wells without antibody. PBS (50 μ?) Was added to a second set of control wells without antibody. Myristate-phorbol acetate (PMA), final concentration of 5 μg / ml, was added to a third set of control wells without antibody. The plates were incubated again for 20 min at 37 ° C in an incubator with 5% C02. Finally 50 μ? of a mixture of anti-CDII-PE (BD Biosciences, Cat. No. 555388) diluted 1/50 in PBS (final concentration 1/200) was added to all wells (except for the 4th control set of 2 wells without Ab and without C5a or PMA - these samples provided the baseline IFM values) . The plates were incubated again for 20 min at 37 ° C in an incubator with 5% C02, then centrifuged for 3 min at 2000 rpm to pellet the blood cells. The supernatant (150 μ?) Was removed and the pellets resuspended in 200 μ? of Lysis FACS lx solution to lyse the red blood cells. After 5 min at room temperature, the plates were centrifuged again, 200-225 μ? of supernatant was removed and the pellets were resuspended in 160 μ? of Lysis FACS solution lx. The cells were transferred to microtitre tubes for analysis by flow cytometry.

FACS and data analysis The FACSCalibur flow cytometer (BD Biosciences) was configured with set compensation parameters for the FL-2 channels. Samples were separated to exclude dead cells and debris. Neutrophils were identified as high FSC and SSC and were separated. The mean fluorescence intensity (MFI) of the separated neutrophils in the FL-2 channel (CDllb-PE) was calculated.

The results were expressed as a percentage of the expression of maximal CDllb with the background subtracted. The expression of maximal CDllb (CDllbMáx) was the average MFI of neutrophils incubated with C5a but without Ab. The expression of minimal (background) CDllb (CDllbMin) was the average MFI of neutrophils incubated without C5a and without Ab. The formula used to calculate the% of the maximum CDII expression for each sample was: |¾ M xmues a = (FIsample- MFIMín) / (MFlMáx- MFIMín) x 100 The data were entered in GraphPad Prism (v4.0) and adjusted to the sigmoidal dose-response curve (variable slope), that is, the 4-parameter logistic equation by using non-linear regression to calculate the EC50.

Descending regulation receiver of CD62L Configuration test The following set was designed to determine the ability of the identified antibodies to neutralize neutrophil activation induced by C5a by measuring changes in CD62L expression.

The CDIIb test above was adapted for the detection of CD62L by the use of a conjugated antibody that recognizes CD62L (BD Biosciences, Cat. No. 559772). The specific experimental details for CD62L are given below.

FACS and data analysis The flow cytometer FACSCalibur (BD Biosciences) was configured with compensation parameters established for the FL-4 channels. Samples were separated to exclude dead cells and debris. Neutrophils were identified as high FSC and SSC and were separated. The mean fluorescence intensity (MFI) of the neutrophils separated in the FL-3 channel (CD62L-APC) was calculated.

The results were expressed as a percentage of maximal CD62L expression with the background subtracted. The expression of maximal CD62L (CD62LMax) was the average MFI of neutrophils incubated with C5a but without Ab. The expression of minimal CD62L (background) (CD62Lmin) was the average MFI of neutrophils incubated without C5a and without Ab. The formula used to calculate the% of the maximum CD62L expression for each sample was: % Max sample = (MFIsample-MFIMin) / (MFIMáx- MFIMín) x 100 The data were entered in GraphPad Prism (v4.0) and adjusted to the sigmoidal dose-response curve (variable slope), that is, the 4-parameter logistic equation by using non-linear regression to calculate the EC50.

The results of the previous tests are summarized in Table 5 below.

Table 5. Data obtained in the affinity test, Calcium flow test, CDII and CD6 test The data confirmed that 32F3A6 GL inhibits the action of C5a in a dose-dependent manner. Inhibition of the release of Ca2 + neutrophils increased with increasing concentrations of 32F3A6 GL and with a higher efficiency than Ref. Ab Q, as seen by the lower IC50 value.

Similarly, 32F3A6 GL is also more efficient than Ref. Ab Q in the regulation test of CDllb and CD62L which displays a high power of 4-5 times that of Ref. Ab Q.

The additional test of 32F3A6 GL in the neutrophil migration test (chemotaxis) also showed dose dependence with an IC 50 of 1.0 μg / ml.

Example 8. Arthritis model of mice in vivo The in vivo effect was tested in a K / BxN model in hC5aR KO / KI mice (WO 2009/103113 and Lee et al, Nat Biotechnol 2006 Oct; 24 (10): 1279-84). K / BxN mice spontaneously develop an autoimmune type disease mediated by circulating Ab against GPI (self-antigen glucose 6-phosphate isomerase). Serum from K / BxN arthritic mice induces disease in other strains of mouse with many characteristics of human RA markings that includes chronic progressive disease with destruction of the joint.

Animals Transgenic mice KO / KI for human C5aR (C57BL / 6, H-2b, human C5aR + / + / mouse C5aR - / -, strain abbreviation: H5Rtg) aged 8-27 weeks.

Serum with K / BxN To produce serum for the experiments, male KRNtg mice were crossed with female NOD mice. The offspring Fl (8-10 weeks of age) carrying the KRN transgene, which developed inflamed joints, were sacrificed and blood was collected by cardiac puncture. After 2 hours of incubation at 37 ° C the serum was collected and centrifuged for 10 min at 4000 rpm. The serum of several mice was put in stock, aliquots were formed and stored at -80 ° C. All mice were injected with the same batch of serum with K / BxN.

Induction and scoring of arthritis Inflammatory arthritis was induced in H5Rtg recipient mice by injection of 150 μ? of serum with K / BxN i.p. both day 0 and day 2. The progress of the disease was monitored daily by measuring paw size and determining a clinical score based on the degree of inflammation in the paws front and back and the ankle joints. The change in the average size of the paw from day 0 was calculated as follows. The thickness (in mm) of the ankles on each of the hind legs was measured daily by the use of a caliper. The average of the one or two readings of each of the hind legs was the size of the average daily paw (PS). The average paw size on day 0 was subtracted from the average paw size every day to give the average change in paw size (AAPS) for each day of the experiment. A clinical score was calculated for each leg of each mouse based on the scoring system shown in Table 6. The 4-leg score was added to give the total clinical score (CS) for each mouse on each day of the experiment.

Table 6. Clinical scoring system of arthritis To determine which mice entered the treatment phase on day 5, an "RA score" was calculated for each mouse by multiplying the clinical score by changing the size of the paw from day 0 (in mm. they had a score of > 0.7 were introduced to the treatment phase of the study.

Therapeutic treatment with 32F3A6 GL After the onset of the disease (day 0), the KO / KI mice for hC5aR were given a loading dose of 32F3A6 GL on day 5 and then 9 daily doses. The loading doses were 10, 1.5 and 0.5 mg / kg and the daily doses of 2, 0.5 and 0.25 mg / kg. Clinical scores (mean +/- D.E.) for each treatment group are shown in Figure 4. Treatment with NNC0215-0384 produced a dose-dependent reduction in inflammation compared to mice treated with an irrelevant control antibody. A similar effect was observed based on changes in average leg size (not shown).

Example 9. Expression level of C5a in patients with psoriasic arthritis C5a was measured in synovial fluid samples from 11 patients with psoriatic arthritis and 12 patients with osteoarthrosis as controls. The protocol of a commercial C5a ELISA kit was followed (BD OptEIA ™, Human C5a ELISA Kit II (BD Biosciences, Cat. No. 557965)). The data are provided in Figure 5 and are summarized in Table 7 below. The level of C5a was significantly elevated in the group of patients with psoriatic arthritis (p = 0.001; Mann-Whitney) indicating that C5a may be a driver of inflammation Synovial in psoriatic arthritis.

Table 7. Levels of detection of C5a in synovial fluid of controls and patients with psoriatic arthritis Example 10. Expression of C5aR in the synovial membrane of patients with psoriatic arthritis Tissue microarray preparations (TMAs) containing formalin-fixed and paraffin-embedded synovial membrane biopsies from patients with psoriatic arthritis (PsA) (n = 9), and within normal limits (n = 5) were obtained from Biochain Institute Inc./BioCat GmbH, Heidelberg, Germany. A sample of PsA from the collaboration with Dr. Bliddal (Frederiksberg Hospital, Denmark) and Dr. S e (Gentofte Hospital, Denmark). All human materials were obtained with the informed consent of the donors / close relatives, and the approval of the relevant local ethical committees BioCat Ge, personal communication; Cambridge Biosciences, provider information: Tissue Supply Network (www.bioscience, co.uk). The sample of Drs. Bliddal / S e was obtained under the ethical permit No. H-4-2009-117. The following antibodies were used: mouse monoclonal anti-C5aR (R & D Systems, MAB3648 clone 347214 (IgG2a)). Isotype-specific control of mouse IgG2a (Dako, X0943, clone DAK-G05). Anti-mouse conjugate with biotin Jackson ImmunoReseach (715-065-150).

Immunohistochemistry was performed as follows. Sections were deparaffinized in xylene and rehydrated in decreasing concentrations of alcohols. The recovery of the antigen was carried out in Tris-EGTA pH buffer (10 mM, 0.5 mM), pH 9.0 in a microwave oven for 15 min. The endogenous peroxidase activity was blocked with 3% H202, and the endogenous biotin was blocked by incubation with avidin and biotin blocking solutions for 10 min, respectively, according to the manufacturer. Non-specific binding was blocked by incubation with TBS containing 3% skim milk, 7% donkey serum, 3% human serum, and 3.2 mg / ml poly-L-lysine (PLL) for 30 min. The primary and secondary antibodies were diluted in a Tris pH buffer containing 0.5% skim milk, 7% donkey serum and 3% human serum, and the incubation was carried out overnight at 4 ° C, and 60 min. at room temperature, respectively. The first step of amplification was performed by incubation with the Vectastain ABC peroxidase kit, diluted in pH buffer Tris-HCl 0.1 M (pH 7.5) containing 0.5% of Du Pont blocking reagent (TNB) for 30 min, followed by a second amplification step with incubation in biotinylated tiramide for 6 minutes. The final amplification performed by additional incubation with the Vectastain ABC peroxidase kit, diluted as described above for 30 min. The chromogenic reaction was achieved with diaminobenzidine. The nuclei were counterstained with hematoxylin and the sections were rehydrated, rinsed in xylene and mounted with Eukitt. The evaluation of the TMAs for the expression of C5aR protein in RA, OA and the normal synovial membrane was carried out blinded to the observer. An Olympus BX51 microscope equipped with a DP70 digital camera (Olympus Denmark A / S, Ballerup, Denmark) was used for the evaluation of the sections.

Results C5aR-immunopositive cells were found intermixed in the lymphoid aggregates in the synovial sub-coat layer in 8 out of 10 psoriatic arthritis patients and in the stroma of 10 of the 10 patients with psoriatic arthritis. The controls did not show any C5aR staining in these synovial compartments (0/5). C5aR-immunopositive synoviocytes were detected in the cells of the coating layer in controls 4 of 5, as well as in 10 of 10 patients with psoriatic arthritis. The results are summarized in table 8 below.

Table 8. Detection of C5aR + cells in the normal synovial membrane and the synovial membrane of patients with psoriatic arthritis. The value of P (Fisher's exact test) for the difference between the C5aR expression in patients with psoriatic arthritis compared to normal synovial membrane: 0.007 (lymphoid aggregates) and 0.0003 (stroma).

Example 11. Inhibition of neutrophil migration induced by synovial fluid of patients with psoriatic arthritis by anti-C5aR Migration test of neutrophil granulocytes (chemotaxis) The potency of antibodies to inhibit hC5a - dependent migration of. Human neutrophil granulocytes (human PMNs (polymorphonuclear leukocytes)) were analyzed in a Boyden chamber test by using BD FluoroBlok 96-well multiple insertion systems.

Human PMN were obtained from human blood samples collected in vials containing EDTA. The blood cells were separated by centrifugation from the blood (4 parts) through one of a gradient of Ficoll-Paque PLUS (GE Health Care) (3 parts) for 30 min (400 x g) at room temperature. The PMN-containing layer was suspended in PBS (phosphate buffered saline) containing dextran-500 (Sigma) for 1 hr to remove the polluting erythrocytes. The supernatant was centrifuged for 5 min (250 x g) at room temperature and the remaining erythrocytes were lysed osmotically by using 0.2% NaCl for 55 sec. The solution was made isotonic by 1.2% NaCl + PBS and centrifuged at 250 x g for 5 min, before the osmotic lysis was repeated. After centrifugation, the PMN were resuspended in the reaction mixture (RM): HBSS (Gibco Ca. No. 14175) contained 137 mM NaCl, 5.3 mm KC1, 0.33 mm Na2HP04, 4 mM NaHCO3, 0.44 mM KH2P04, glucose 5 mM, supplemented with 0.4 M MgS0 | 7H20, 0.5 mM MgCl2, 0.5 mM CaCl2, 20 mM HEPES. The cell density was determined by NucleoCounter (Chemometec). The PMN suspension contained > 95% of neutrophils as evaluated by microscopy of samples stained with Giemsa.

Load of PMN: Calcein, AM, (Fluka) was dissolved in DMSO (dimethyl sulfoxide) and was diluted 1000X in RM with cells (2xl06 cells per ml) to give a concentration of 10 μ ?. The suspension was incubated for 30 min in the incubator at 37 ° C and then washed 3 times with RM to remove the excess calcein. Finally, the cells were resuspended in MRI (4 × 10 6 cells / ml).

Human synovial fluid (SF) was obtained from 2 patients with psoriatic arthritis by knee puncture. After removal of cells by centrifugation, the samples were frozen and stored at -80 ° C. For the migration experiments, the samples were thawed and diluted by the use of 2X RM containing 0.2% EDTA.

The migration was evaluated by the Boyden chamber technique through the use of FluoroBlok® 3 μt? pore size, 96 wells (Cat. No. 351161. BD Falcon (VWR)). The upper chamber i.e., the inserts containing the FluoroBlok membrane, was coated with human fibrinogen (Cat. No. F3879-1G, Sigma) in 1 mg / ml of PBS at 37 ° C for 2 hours. After washing, the membranes were blocked with a solution containing 2% bovine serum albumin (BSA), in PBS. After another wash with the use of RM, 105 PMN loaded with calcein with or without the hC5aR-antibodies (100 mg mi) were added to each well and placed in the receptor plate (lower chamber) containing the control solution or of the chemoattractant solution (hC5a (Sigma, or synovial fluid samples)). Each group comprised 4-6 wells. The quantification of cell migration is achieved by measuring the fluorescence of the cells in the lower chamber. Since the FluoroBlok membrane effectively blocks the passage of light at 490-700 nm, the fluorescence of cells that have not entered the lower chamber is not detected at 485/530 nm. The plate was read at excitation / emission wavelengths of 485 / 538nm, 37 ° C every 5 min for 60 min in the fluorescence plate reader with lower reading capabilities (SpectraMax, Molecular Devices, or Fluoroscan, Thermo Labsystems).

The migration was evaluated by fluorescence values in 60 min expressed as relative fluorescence values. In Table 9, migration in the presence of isotype antibodies is set at 100% and the ability of the anti-C5aR antibody to inhibit migration is calculated. The migration was clearly attenuated by the hC5aR antibody. The migration caused by 10 nM of hC5a was inhibited 83%. The values for the three SF samples were: 15%, 70% and 48%. The results demonstrate that the C5aR-antibody inhibited the chemoattractant effect of SF from patients with psoriatic arthritis.

Table 9. Migration of PMN in response to hC5a or synovial fluid from three patients with psoriatic arthritis and inhibition of this document by hC5aR antibodies (Ref Q antibodies). All values are normalized to detected migration when incubated with the isotype antibody.

Example 12. C5aR expressing in the intestine of patients with Crohn's disease and ulcerative colitis Samples of intestinal tissues within the normal limits (n = 14) of patients with ulcerative colitis (n = 21) and Crohn's disease (n = 25) were obtained from Cambridge Bioscience (Cambridge, R.U.). All human materials were obtained with the informed consent of the donors / close relatives, and the approval of the relevant local ethical committees Cambridge Biosciences, Supplier information: Tissue Supply Network (www.bioscience, co.uk). The antibodies used and the immunohistochemistry protocol as described in example 9.

Semi-quantitative score The immunopositive C5aR cells (C5aR +) were semi-quantitatively qualified as follows: The mucosa-associated lymphoid compartments were individually graded: Mucosa (M): lymphocyte intraepithelial compartment (IEL) (surface epithelium), lamina propria, and epithelium associated with the follicle (FAE, for its acronym in English ). Submucosa (SM): isolated lymphoid follicles (solitary) (ILF), Peyer's patches (ileus) / colonic IEL (colon) and isolated infiltrating lymphocytes. External muscle (ME): IEL and isolated infiltrating lymphocytes. Each compartment was rated on a scale of 0-4: 0, no; 1, few; 2 moderate; 3, many; and 4 abundant numbers of C5aR + cells. A cumulative score was calculated for each intestinal layer (M, SM, ME) and in total (M + SM + ME) for the entire intestine. Maximum rating: M = 12, SM = 12, ME = 8 and for the entire intestine 32. The semi-quantitative score of the immunohistochemical data for the expression of C5aR protein was analyzed by the Kruskal-Walis test with post-Dunn multiple comparison test on GraphPad Prism 5. P < 0.05 was considered significant.

Results Positive C5aR neutrophils and myeloid-like cells were found in the intraepithelial lymphocyte compartment, in the associated follicular epithelium, and as solitary cells in the mucosal lamina propria in 23 of the 25 patients with CD, 19 of the 21 patients with UC and in 7 out of 14 normal intestinal samples (P values (Fisher's exact test) 0.005 and 0.015, respectively). In addition, no positive C5aR cells were found in the Peyer's plaques / colonic lymphoid follicles; isolated lymphoid follicles (solitary) and, as solitary cells of the submucosa in 21 of the 25 patients with CD and 18 of the 21 patients with UC compared to 7 of 14 normal intestinal specimens (P values (Fisher's exact test) 0.03 and not significant, respectively). Finally, positive C5aR cells were found with infiltration of the external muscle of patients with CD and UC, as well as in the normal intestine. The results are presented in figures 6A-6D and summarized in table 10. Based on Semi-quantitative analysis, it was found that C5aR is expressed significantly higher in the intestine of patients with CD (P <0.01) and UC (P <0.05) compared to normal bowel throughout the intestinal wall, v. gr. , the accumulated score in the three intestinal layers (mucosa, submucosa, and external muscle).

Table 10. Summary of the expression of C5aR in the intestine of patients with Crohn's disease and ulcerative colitis compared to the normal intestine.

Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it is understood that the annexed modalities intend to cover all the modifications and changes that fall within the true essence of the invention.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An antibody that binds to C5aR, characterized in that the variable on of the heavy chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said CDR1 sequence comprises SEQ ID 1, 9, 17 or 25 or any of those sequences with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 sequence comprises SEQ ID 2 , 10, 18 or 26 or any of those sequences with 1, 2 or 3 substitutions, deletions or amino acid insertions (s) and / or wherein said CDR3 sequence comprises SEQ ID 3, 11, 19 or 27 or any of those sequences with 1, 2 or 3 (s) substitutions, deletions or insertions of amino acid (s).

2. An antibody that binds to C5aR, characterized in that the variable on of the light chain of that antibody comprises a sequence of CDR1, CDR2 and CDR3, wherein said CDR1 sequence comprises SEQ ID 5, 13, 21 or 29 or any of those sequences with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR2 sequence comprises SEQ ID 6 , 14, 22 or 30 or any of those sequences with 1, 2 or 3 substitutions, deletions or insertions of amino acid (s) and / or wherein said CDR3 sequence comprises SEQ ID 7, 15, 23 or 31 or any of those sequences with 1, 2 or 3 (s) amino acid substitutions, deletions or insertions (s)

3. The antibody according to any of claims 1 or 2, characterized in that the antibody is selected from: to. an antibody wherein the CDRs of the variable on of the heavy chain comprise SEQ ID 1, 2 and 3 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s) and in wherein the CDRs of the variable light chain comprise SEQ ID 5, 6 and 7 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s), b. an antibody wherein the CDRs of the variable on of the heavy chain comprise SEQ ID 9, 10 and 11 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s) and wherein the CDRs of the variable light chain comprise SEQ ID 13, 14 and 15 or that amino acid sequence with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s), c. an antibody wherein the CDRs of the variable on of the heavy chain comprise SEQ ID 17,18 and 19 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s) and in where the CDRs of the variable light chain comprising SEQ ID 21, 22 and 23 or those amino acid sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s), d. an antibody wherein the CDRs of the variable on of the heavy chain comprise SEQ IDs 25, 26 and 27 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s) and in wherein the CDRs of the variable light chain comprising SEQ ID 29, 30 and 31 or those sequences with 1 substitution (s), deletion (s) and / or insertion (s) of amino acid (s).

4. The antibody according to any of the preceding claims, characterized in that the variable on of the heavy chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 4, 12, 20 or 28 and / or wherein the variable on of the light chain of that antibody comprises a sequence of at least 80, 85, 90 or 94% identical to SEQ ID NO: 8, 16, 24 or 32.

5. The antibody according to any of the preceding claims, characterized in that it is a human antibody.

6. A human antibody characterized in that it binds to the second extracellular loop of C5aR.

7. The antibody according to any of the preceding claims, characterized in that it binds to human C5aR and preferably to the second extracellular loop of human C5aR.

8. The antibody according to any of the preceding claims, characterized in that the affinity of the antibody as measured by the competing ligand binding test in neutrophils is below 0.80 nM.

9. The antibody according to any of the preceding claims, characterized in that it inhibits or significantly reduces the binding of C5a to C5aR.

10. The antibody according to any of the preceding claims, characterized in that it significantly inhibits the migration of human neutrophils in vitro.

11. An antibody that binds to human C5aR, characterized in that the Fe region has reduced the binding affinity to one or more Fcy receptors compared to the Ig reference sequences of IgG1, IgG2, IgG4 or IgG4 / G2 as defined by SEQ ID NO: 33, 34, 35 and 36, respectively.

12. The antibody according to any of the preceding claims, characterized in that it does not significantly induce ADCC, CDC and / or neutrophil phagocytosis in vitro.

13. The antibody in accordance with any of the preceding claims, characterized in that the Fe region is IgGl (SEQ ID NO: 33), IgG2 (SEQ ID NO: 34), IgG2 / 4 (SEQ ID NO: 35), or IgG4 (SEQ ID NO: 36), with one or more of the following point mutations to. E233P b L234A or V234A or F234L or F234V c. L235E or L235A d. G236R O G236A and. G237A F. N297Q g. L328R h. A330S i. P331S.

14. An antibody according to any of the preceding claims for use in treatment.

15. An antibody according to any one of the preceding claims, for use in the treatment of an immunological disease or disorder, such as rheumatoid arthritis (RA), psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease ( IBD), or irritable bowel syndrome.