TR201816428T4 - Therapeutic antibodies. - Google Patents

Abstract

The present invention relates to human antibodies that recognize the human C5a receptor. By binding to C5aR, antibodies inhibit C5a signaling, thereby inhibiting the pro-inflammatory signal. Based on the role of C5a and its receptor in inducing inflammation, the invention further relates to the therapeutic use of said human anti-C5aR antibodies, and in particular for the treatment of immunological disorders.

Description

DESCRIPTION THERAPEUTIC ANTIBODIES TECHNICAL FIELD The invention relates to the field of therapeutic antibodies.

BACKGROUND OF THE INVENTION The proteolysis of each of the CIS-CS complement proteins as anaphylatoxins cause amino-terminal cationic fragments with so-called signaling molecules It is possible. The strongest of these, the COA, provides the broadest responses. Your inflammatory response margination and infiltration of leukocytes, release of granule-bound proteolytic enzymes, production of activated oxygen and nitrogen-derived radicals, combined with smooth muscle contraction Considering its components, such as changes in blood flow and capillary leakage, The C5a molecule is the "complete" pro-inflammatory mediator. Nanomolar-sub-nanomolar levels, the C5a molecule is present in all myeloid lineages (neutrophils, eosinophils, and basophils, macrophages and monocytes) provide chemotaxis and produce prostaglandins and Vascular permeability significantly enhanced by circulating Iukocytes causes. Higher nanomolar concentrations, NADPH oxidazine It provides degranulation and activation. This breadth of bioactivity conflicts with inflammatory mediators. C5a, rheumatoid arthritis, psoriasis, sepsis, including reperfusion injury and adult respiratory distress syndrome It is involved in the pathogenesis of various disorders (Gerard and Gerard, 1994; Murdoch and The activities of C5al are mediated by the binding of C5a to its receptor (C5aR). CSAR, It belongs to seven families of transmembrane G-protein-coupled receptors. CöaR is ~1 nM It is a high-affinity receptor for Kd and CSa and a number of cells including leukocytes located in different cell types. Number of receptors per cell, 200,000 per leukocyte It is extremely high until you get it. Biological activation of the receptor, binding occurs over the saturating range.

The structure of the CSaR consists of the extracellular N-terminus, alternating as intracellular and extracellular loops, and a seven transmembranes connected by inter-helix domains terminating with an intracellular C-terminal domain Following the helix, it belongs to seven families of transmembrane receptors. it is compatible. CSaR contains an extended N-terminal extracellular domain. This large N-terminal area It is a feature of G-protein coupled receptors.

Inhibition of CSa responses by C5aR antagonists inhibits other complement components. It reduces acute inflammatory responses mediated through C5a without affecting This For this purpose, C5aR peptide antagonists and anti-C5a receptor antibodies have been previously described. Three of the antibodies were named 7F3, 6C12 and 12D4. These antibodies, CSa being very effective in blocking the binding of neutrophils to the C5a-direction receptor in vitro excellent as inhibiting migration and inhibiting inflammation in animal models. properties have been shown. To control chronic diseases, It may be necessary to administer the antibody in successive situations over months or years.

One drawback of administering mouse antibodies, however, is that the human immune system the ability to produce its own antibodies against the antibody (HAMA response). HAMA response mouse antibodies, by quickly clearing them from the blood, thereby reaching the target of the mouse antibody It can neutralize it by preventing it from binding. To prevent a HAMA response One strategy that has been adopted is that the mouse antibody replicates at non-epitope binding sites. is the "humanization" of many "alien" remains by replacing them with human sequences.

A fundamental problem with humanization procedures is that some of the antigens are a protein. for antigen, which may be 10-fold or more in samples (Verhoeyen et al., 1988). there was a loss of affinity (Jones et al., 1986). Any loss of affinity, of course, It is highly undesirable. At the very least, the humanized antibody is more would have to be injected at higher cost and with greater risk of adverse effects. means. More critically, an antibody with reduced affinity complement lysis, antibody-dependent cellular cytotoxicity, or virus neutralization. may have weaker biological functions. Although these difficulties Successful humanization of anti-human CSaR antibodies has been The risk of any undesirable side effects resulting from the administration of antibodies to patients to further minimize it in patients with the production of "fully" human antibodies. over the years, including reducing the likelihood of the formation of anti-drug antibodies Multiple strategies have been developed.

Even today, identifying antibodies suitable for therapeutic applications is a challenging one. is duty. Therefore, an alternative that can be used in diagnostic and/or therapeutic methods. and/or advanced CSaR antagonists remain of considerable interest.

BRIEF DESCRIPTION OF THE INVENTION The present invention is associated with anti-CSaR antibodies that bind to cycle 2 of human C6aR and relates to their use in the treatment of an immunological disease or disorder. Promise The variable region of the heavy chain of the antibody of interest is at least 90% identical to SEQ ID NO: 12 contains a sequence and a CDR1, a CDR2, and a CDR3 containing the sequences of SEO ID 9, 10, and includes. The variable region of the light chain of the antibody of interest has at least SEQ ID NO: 16 and a CDR3. The antibody has an Fc hinge region of the IgG1 isotype.

The authors of the invention, bind human CöaR in many ways, which have already been described. A number of antibodies have been identified that are functionally superior to anti-C5aR antibodies.

As set forth herein, the inventors of human CSaR (hCSaR) binding and inhibit hCSa-mediated neutrophil migration, which can alter hCSa binding to hCSaR A number of human antibodies have been identified that can In addition, the inventors of this anti- Non-human residues present in the framework region of one of the hCSaR antibodies. successfully to human germline remnants in influencing the potency of the antibody. converted.

Moreover, the inventors can modify the Fc region to induce phagocytosis, ADCC or CDC. produced an in vitro non-inducing anti-hCöaR antibody. Details of the invention, precedent will be understood from the description of the applications.

In one embodiment, the anti-C5aR antibody is a human antibody that specifically binds hCSaR. wherein said antibody preferentially binds the 2nd extracellular cycle of hCSaR.

In one embodiment, the anti-CSaR antibody specifically binds hCSaR and its antibody Fc region, phagocytosis of the antibody via Fcgamma receptor (FcγR) interaction, ADCC and/or lgGi, IgG2, lgG4, and IgG4/G2 reference that reduce the ability to induce CDC modified compared to the series. In a particular embodiment, the antibody Fc region is IgG1. and in other particular embodiment, the Fc region may contain one of the following point mutations or includes more In another aspect, the invention provides that antibodies according to the invention are associated with an immunological disease or related to its use for the treatment of ailment.

In another aspect, the invention is for the treatment of a disease or ailment, for a needy containing the administration of a therapeutic amount of the antibody as described herein to the subject. relates to an antibody of the invention for use in the method.

In another aspect, the present invention involves treating or treating a condition in a subject. provides an antibody of the invention for use in a method for inhibiting the method comprises administering the antibody of the invention to the subject. In one embodiment, the discomfort is a It is an immunopathological disorder like an autoimmune disease.

Other aspects and applications of the invention, including precedent will be clear from the description. The invention has many benefits and It will be seen from the description that it has provided new therapeutic antibodies with advantage.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a selection of monoclonal antibodies isolated and characterized in practice. shows the alignment of the variable regions.

Figure 2 Binding of a selection of antibodies to mouse and human C5aR chimeras binding to the chimeric human/mouse C5aR. Chimeric receptors are semantically is shown. Regions derived from human and mouse C5aR, respectively, with a thin line and is indicated by a bold line.

Figure 3 shows a strain with variable heavy and light sequences of the nearest germ-line human antibody. shows the alignments of the regions variable from the antibody. “”/, V, D or J It shows a "break" in the sequence as between its segments.

Figure 4. 5 from inflammation induced in the K/BxN-hC5aR-KO/Kl serum transfer model given a single loading dose (arrow) of 0.5, 1.5, or 10mg/kg i.p. Clinical manifestations of the three treatment groups given 9 daily doses of 0.25, 0.5, or 2 mg/kg, respectively. scores, error bars represent ±SD. Controls received IgG1 3G12.

Figure 5. C5a in synovial fluids in Psoriatic Arthritis and Osteoarthritis patients (controls) protein expression. CSa level in the psoriatic arthritis patient group (p = 0.001; Mann- Whitney) increased significantly.

Figure 6. Half-expression of C5aR protein in Crohn's disease and ulcerative colitis quantitative analysis. C5aR protein expression was examined by immunohistochemistry and With Dunn's multiple comparison post-test in GraphPad Prism 5 with KruskaI-Wallis test analyzed and P<0.05 was evaluated as significant. *P< 0.05; **P<0.01; DEFINITIONS Unless otherwise stated, the recombinant protein used in the present invention is cell culture and immunological techniques are standard procedures known to those skilled in the art. this type techniques in the literature, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and U.S. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996) and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, all including updates), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988) and J.E. Coligan et al. (editors) Current Protocols Immunology, John Wiley and Sons (to date all are described and explained in sources such as

As used herein, "C5a receptor", "C5aR", "C5aRI" or "human CSaR" and variations of these are also known in the art as C5a anaphylatoxin receptor and CD88 antigen. known human complement receptor 5 refers to receptor 1. C5aR, seven It belongs to the transmembrane G-protein-coupled receptor family and binds C5a (Gerard and Gerard, 1991). An example of the amino acid sequence of a human C5aR is in SEQ ID NO: 41 provided, however, a person skilled in the art would recognize that this molecule is designated by the term "C5aR". will notice that there are naturally occurring allelic variants covered as well. Human The various domains of the C5aR are as follows: amino acids 1 - 37: extracellular domain N-terminus, amino acids 38 - 61: transmembrane domain, amino acids 62 - 71: intracellular space, 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 space, amino acids 150 - 174: transmembrane domain, amino acids 175 - 206: extracellular domain - extracellular cycle 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: intracellular domain - C-terminus.

As used herein, the term "treatment" refers to any person or other person who needs it. animal refers to the medical therapy of the subject. the subject in question, that the specific treatment is beneficial to the health of the human or other animal subject in question a medical doctor who has made an uncertain or specific diagnosis that may indicate that A physical examination by a veterinarian is expected. The treatment in question The timing and purpose of it varies from one individual to another according to the subject's state of health. it can change. Thus, the treatment in question is prophylactic, palliative, symptomatic and/or can be curative. In the sense of the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the invention.

With regard to medical treatment, the term "subject" as used herein means any animal, especially mammals, such as humans, horses, cows, cats and dogs and, where appropriate, interchangeably with the term "patient" can be used. Preferably, the subject is a human. As used herein, "treatment", sufficient to reduce or eliminate at least one symptom of the disorder contains the administration of a therapeutically effective amount of

As used herein, the terms "do not hinder", "obstruct" or "obstruct" or variations of these cause a subject to develop at least one symptom of a disease. refers to protection against or the reduction of the severity of a symptom of a disorder is doing.

As used herein, the term "cell exposure" means that the antibody, C5aR able to contact/bind to human C5aR provided it is present on the cell It means to be provided in a way. antibody has a specific effect of the molecule targeted by the antibody (for example, human Inhibition/displacement of C5atsin binding to human CSaR) percent It represents the required concentration for 50. A person skilled in the art A lower E050 value corresponds to a stronger antibody. will be understood.

As used herein, the term "inhibit" refers to a significant effect of the activity described. denotes reduction and possibly inhibition. Preferably, defined activity of at least 50 percent, more preferably at least 75 percent, and even more preferably at least at least one percent. 90 is reduced or inhibited.

Throughout this specification, the word "comprises" or the words "contains" or "contains" variations of a specified item, integer, or step, or of an item, integer, or the inclusion of sets of steps only includes any other item, integer, or step or element, meaning that integers or groups of steps are not excluded. will be understood.

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

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

In one embodiment, the molecule, such as an antibody or DNA sequence, is an isolated molecule. “Isolated The term “antibody” is a term that has been separated and/or recycled from another/other component(s) of its natural environment. a purified from a mixture of components that have been acquired and/or in its natural environment. stands for antibody. antigen-binding fragments (ie, the “antigen-binding portion”) or single Chains includes. Full-length antibodies (or full antibodies) are four polypeptide chains with disulfide bonds. It consists of two heavy (H) chains and two light (L) chains linked together. Each heavy chain is a heavy It consists of a chain variable region (VH) and a heavy chain constant region (CH). Each one light chain from a light chain variable region (VL) and one light chain constant region (CL). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. occurs. The variable regions of the heavy and light chains are a binding site that interacts with the antigen. includes. Each light chain (abbreviated here as LCVR or VL) is a light chain. It consists of a variable region and a light chain constant region. light chain fixed region consists of a field, CL. VH and VL regions, complementarity determination as more conserved framework regions (FRs) called CDRs. further into regions of hypervariability distributed among the named regions. fissile.

Each VH and VL are three amino-terminus-to-carboxy-terminus arranged in the following order. It consists of CDR and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Constant regions of antibodies, immunoglobulin, various cells of the immune system (for example, effector cells) and the first component of the classical complement system (Clq) It may mediate its binding to host tissues or factors, including

As used herein, the term "antibody" includes complete antibodies and their corresponding Any antigen-binding fragment that specifically binds to the antigen (i.e. “antigen- binder part") or to describe single Chains. Antigen binder Examples of fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)S, Fv (typically a VL and VH domains of a single arm of the antibody), single-Chain Fv (scFv; see eg Bird et al., Science , dst, Fd (typically a VH and CHI domain) and dAb (typically a VH domain) fragments; VH, VL, VhH and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minichors, diachors, trichores, tetrachors, and kappa cores (see, for example, III et al. or a functional antibody fragment of antigen-binding residues or polypeptides one or more of which it can be associated or linked together to form CDR or a functional paratope is included. Various types of antibody fragments, for example, or examined. amino acid residues of an antibody responsible for antigen binding. means. CDRs are usually in the light-chain variable domain, and Sequences of Proteins NIH Publication No. 91-3242) and/or 0 residues are a part of the light chain variable domain. 96 (L3) and in the heavy-chain variable area enumeration of amino acid residues in the region is described in Kabat et al., above. carried out by the given method. such as "Kabat location", "Kabat residue" and "According to Kabat" expressions here for heavy chain variable domains or light chain variable domains represents the numbering system. Using the coarse numbering system, the actual linear amino acid sequence of a peptide, variable alanine a framework (FR), or Fewer or additional amino acids corresponding to a shortening or addition to its CDR may contain. For example, from residue 52 of a heavy chain variable domain CDR H2 then amino acid adducts (residues 52a, 52b and 520 according to Kabat) and heavy chain FR residues added after residue 82 (for example, residues 82a, 82b and, according to Kabat, 820 etc.). Kabat enumeration of residues for a particular antibody with a "standard" Kabat numbering sequence in the homology regions of the sequence can be determined by alignment. acid residues refer to those not included in the CDRs.

The fragment crystallizable region of an antibody (“Fc region”/“/Fc domain”) of an antibody, Cell surface receptors called Fc receptors as well as complement It is the “tail” region that interacts with some of the proteins of the system.

Monoclonal antibodies typically target myeloma cells with the desired antigen. It is made by fusing with spleen cells from an immunized mouse. Human monoclonal antibodies from transgenic animals encoding human antibodies (for example, mice or other suitable species). Alternatively, recombinant monoclonal antibodies, repertoire cloning or phage display/yeast display It can be done by including the technologies expressed as recombinant antibody engineering, using viruses or yeast rather than mice to create antibodies As used herein, the term "humanized antibody" refers to a non-human germline Sequences derived from the immunoglobulin sequence are usually at least minimally a human/non-human containing complementarity-determination regions (CDR sequences) represents a chimeric antibody. A humanized antibody thus allows the recipient to have a Remains from the hypervariable region may be from a mouse, rat, rabbit, or human- from a hypervariable region of a non-human species such as a non-primate (donor antibody) with residues of desired specificity, affinity and capacity It is a human immunoglobulin (receiver antibody) that has been modified.

Contain at least CDR regions not derived from human germline sequences humanized antibodies also contain antibody light and heavy chain genes, typically genetic variable and constant region of immunoglobulin from different species It may also be referred to as a “chimeric antibody” if it is constructed from genes. variable segments of genes from mouse monoclonal antibody to human constant segments can be combined.

As used herein, the term "human antibody" refers to both the framework and CDR regions. have variable regions from which they are derived from human germline immunoglobulin sequences It is intended to contain antibodies. However, in vivo or in vitro maturation of such antibodies in human germline sequences due to mutations caused by It is recorded that it contains amino acid residues that do not exist. Moreover, the antibody is a constant region, the constant region is also primarily composed of human germline immunoglobulin. derived from strings. Human antibodies of the invention nevertheless, human germline immunoglobulin May contain residues of amino acids that are not encoded by sequences (for example, random in vitro or mutations introduced by site-specific mutagenesis or in vivo somatic mutation).

On the other hand, the term "human antibody" as used herein means that CDR sequences are a derived from the germline of another mammalian species, such as the mouse, and subsequently human antibodies on which framework sequences are grafted (see humanized antibody above) or alternative antigenic binding sites. human antibody may be a human monoclonal antibody. This type of human monoclonal antibody is a human heavy chain transgene fused to the immortalized cell and a light from a transgenic non-human animal with a genome containing the chain transgene, for example, by a hybridoma containing a B cell from a transgenic mouse. can be produced. Human antibodies are also built on selections of human germline sequences. It is also one of the series libraries, which are more diversified with natural and synthetic series diversity. can be isolated. Human antibodies are released after in vitro immunization of human lymphocytes. The sequence may be defined to allow the antibody to be produced by recombinant methods.

Furthermore, humanized, human, and fully human antibodies can be found in either the recipient antibody or the donor antibody. may contain residues not found in the antibody. These modifications improve antibody performance. It is made to refine it even more. conjugate and another agent or antibody. refers to the molecular entity used for the immunization of the vertebrate. Here The term antigen is used more broadly and is usually associated with an antibody. contain specifically recognized target molecules so that the molecule, the antibody contain fragments or imitations used in the immunization process for amplification or such molecules used for screenings on immunization, as well as where antibodies are obtained by alternative methods such as phage display scanning It is intended to include molecules used for screening.

The term "epitope" as used herein refers to an "antigen-binding agent" such as an antibody. a molecular interaction between the polypeptide and its corresponding "antigen" defined in context. Generally, the "epitope" is an antibody on an antigen. the area or region to which it specifically binds, that is, the area in physical contact with the antibody, or represents the region. A protein epitope in the antigen that binds directly to the antibody amino acid (also called the immunodominant component of the epitope) residues and the antigen effectively blocked by Ab (i.e., amino acid residue is within the "solvent-free surface" and/or "footprint" of the antibody) other amino acids not directly involved in binding, such as amino acid residues may contain residues. A particular antigen is an antigen which may include without limitation the sequence may contain different epitopes; linear peptide antigenic markers, native (mature) structural structure consisting of one or more non-contiguous amino acids located close together antigenic markers; and wholly or partially antigen, such as carbohydrate groups. Equivalently, post-translational antigenic composed of attached molecular structures determinants.

Depending on the epitope mapping method used, descriptions of epitopes and Based on the fact that their definitions are obtained at different levels of detail, comparison of epitopes for different Abs on the same Ag with different levels of detail can be carried out in a similar way. or to describe the selectivity of an antigen-binding fragment thereof is used.

Antibodies according to the invention can bind specifically to CSaR, which means that the antibody can bind to other shows that it has a significantly lower affinity for antigens, here significantly lower at least 2 times lower or 5 times lower or times lower affinity. The antibody is also species-specific, eg human CSaR. Antibody that binds specifically but not with high affinity to mouse C5aR it could be. as a measure of the strength of a non-equivalent interaction between an antigen is used. The term "binding affinity" refers to univalent interactions (intrinsic activity) used to describe. Two molecules, for example, an antibody or a binding via a single value interaction between fragment and an antigen Its affinity can be quantified by determining the dissociation constant (KD). Therefore, KD, by measuring the kinetics of complex formation and dissociation, for example, SPR method can be determined. In response to the merger and dissociation of a monovalent complex the incoming rate constants, the association rate constant ka (or blood) and the dissociation rate constant kd, respectively (or koir). Ko relates to ka and kd through the equation KD = kd/ka.

Also, "affinity" is a combination of a molecule (for example, an antibody) with a single binding site. relates to the strength of the binding between the ligand (eg, an antigen). of a molecule (X) its affinity for a ligand (Y) is the coupling of half of the X molecules present in a solution represented by the dissociation constant (Kd), which is the concentration of Y required to occupy is done. A smaller Kd will result in a stronger or higher affinity interaction. and to occupy areas with a lower concentration of ligandin. is necessary. Similarly, the specificity of an interaction is the difference between an antibody and an antigen. by determining the KD value for the interaction of interest, such as a specific interaction, and can be determined by comparison with the KD value for an interaction that is not the subject of

Typically, for the antibody, KD relative to the target, unrelated material or media, or control 2-fold, preferably 5-fold, KD relative to other, non-target molecule, such as the accompanying material times, more preferably 10 times less. More preferably, K0 is 50-fold less, eg 100-fold less or 200-fold less; even more preferably 500-fold less, eg It would be 1,000-fold less or 10,000-fold less.

The value of this dissociation constant can be determined directly by well-known methods and, for example, can be calculated even for mixtures. For example, KD, Wong & Lohman (Proc. Natl. Acad. Sci. can be generated using the binding determination. As antibodies are towards targets known, for example ELISAs, Western blots, RIAs and flow cytometry analysis are included. The binding kinetics and binding affinity of the antibody have also been demonstrated in techniques such as SPR. It can also be determined by known standard assays.

The binding of the antibody to the target means that the target is transferred to another target like any other antibody. executable. The concentration at which 50% inhibition occurs is known as Ki.

Under ideal conditions, Ki is equivalent to KD. However, the value of Ki will not be less than KD, thus the measurement of Ki is conveniently substituted to provide an upper bound for KD. can be done.

As the person skilled in the art will understand, "avidity" is two molecules such as an antibody and an antigen. relates to the overall strength of the interaction between Avidity depends on both affinity and interactions. depends on its worth.

To determine the functionality of a particular antibody, other assays, specific antigen and Cell-based assay specific for antibody binding may be included.

As is known in the art, the term "identity", as determined by comparing sequences, refers to a relationship between the sequences of two or more polypeptides. in technique, The degree of sequence relationship between polypeptides as determined by the number of matches also means. "Identity" with the smaller of two or more sequences handled by a particular mathematical model or computer program (that is, "algorithms") measures the percentage of identical matches between the spacing alignments received (if any). Relating to The identity of the polypeptides can be easily calculated by known methods. This type of method Without being limited to these, 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. @, 1073 (1988). The preferred methods for determining identity are the most common of the sequences tested. Designed to be the perfect match. Methods for determining identity, ring described in open computer programs. The identity between the two series GAP (Devereux et al., Nucl. acid. pic. 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 21_5, 403- 410 (1990)) is included in the GCG program suite. BLASTX program 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., above) is open to the public. The well-known Smith Waterman algorithm for determining identity is also can be used. For example, the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.) to determine the percent sequence identity. the polypeptide is aligned (by the algorithm) for optimal matching of the amino acids of interest. “matched range”) as determined. One gap penalty (3 times the average diagonal) is calculated as; "mean crossover" comparison matrix being used is the mean of the cross; “cross” is defined by the particular comparison matrix for each is the score or number assigned to the perfect amino acid match) and (usually spaces) a space extension penalty {fraction (1/10)} times the opening penalty plus PAM 250 or a comparison matrix such as BLOSUM 62 is used with the algorithm. algorithm A standard comparison matrix is also used by (PAM 250 comparison matrix see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, supp.3 (1978); For the BLOSUM 62 comparison matrix, see Henikoff et al., Proc. Natl. Acad.

Preferred parameters for a peptide sequence comparison include: Gap Length Penalty: 4, Similarity Threshold: 0.

The GAP program is useful with the above parameters. The above mentioned parameters are Peptide using the GAP algorithm (with no penalty for end-gaps) are the default parameters for comparisons.

A "conservative amino acid substitution" means that one amino acid residue with another, 0 little or no effect on the polarity or charge of the amino acid residue at position may contain a substitute where it is not. with the following amino acid groups When exemplified, substitutions of an amino acid with a different amino acid in the same group are conservative. considered a substitution: Hydrophilic: Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr.

Aliphatic: Val, Ile, Leu, Met. Basic: Lys, Arg, His. Aromatic: Phe, Tyr, Trp. Moreover, any residue may be frequently substituted by alanine.

Also, if desired, unnatural amino acids or chemical amino acid analogs, a substitution into the antibody and/or immunoglobulin chain of the present invention, or can be added additionally. These types of amino acids are commonly, but not limited to, D-isomers of amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-aminobutyric acid, 3 - amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citraline, homocitrulline, cysteic acid, t-butiglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta- design amino acids such as alanine, fluoro-amino acids, beta-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.

Amino acid sequence of the antibody and/or immunoglobulin chain of the present invention mutants, where appropriate nucleotide changes are incorporated into a nucleic acid of the present invention. or by in vitro synthesis of the desired polypeptide. this type mutants, for example, deletions, insertions, or insertions of residues in the amino acid sequence. substitutions are included. A combination of deletion, insertion, and substitution, the final polypeptide to reach the final structure, provided that the product has the desired characteristics. can be done. Mutant (modified) polypeptides can be made by any technique known in the art. can be prepared using For example, a polynucleotide of the invention is subject to in vitro mutagenesis. can be held. For such in vitro mutagenesis techniques, insert the polynucleotide into a suitable vector. subcloning the vector into a "mutator" strain such as E. coli XL-I red (Strategene). transformation and propagation of the transformed bacteria for production in appropriate numbers are included. Products derived from mutated/altered DNA, receptor- here to determine whether they have binding and/or -inhibitory activity. can be easily scanned using the techniques described.

When designing amino acid sequence mutants, the location of the mutation site and the mutation's its structure will depend on the characteristic(s) to be modified. for mutation domains individually or in series, for example, (1) conservative amino acid first choices, and then more radical choices depending on the results achieved. substitution, (2) deletion of target residue, or (3) other can be modified by inserting residues.

Amino acid sequence deletions are generally 1 to 15 residues, more preferably about 1 to 10 residues. residue and typically ranges from 1 to 5 adjacent residues.

EXPLANATION Functionality and efficacy of biological therapeutics and certain antibodies has described various aspects of the relationship for Antibodies for the treatment of inflammatory diseases by preventing their binding.

One aspect of the disclosure is heavy, with their functionality and/or the amino acid sequence of the CDRs. with the variable region of chains and light chains and/or the sequence of the FC domain relates to one or more characterized series of antibodies.

In one embodiment, the antibody is a full-length image containing standard antibody domains and regions. is an antibody.

In one embodiment, the antibody is an antibody fragment such fragments can be obtained using recombinant or protein processing techniques. Invention antibody by pruning fragments of, for example, one or more amino acids into a polypeptide N and/or It can be done by removing it from the C-terminal ends. Fragments also include one or more It can also be produced with an internal eraser. An antibody of the invention is one of the antibodies on which this invention is based. may contain or be a fragment of any of them. An antibody of the invention, this antibodies or variants thereof may have an antigen-binding moiety or may contain. For example, the antibody of the invention is one of these antibodies or variants thereof. may be a Fab fragment or from one of these antibodies or a variant thereof may be a derived single-chain antibody.

The antibodies described herein can be used in mice, rats, rabbits, pigs, or non-human primates. may be of different species, including mammalian species such as The antibody is a rodent antibody, and more particularly a mouse antibody. Alternatively, the antibody, such as chicken may be of a non-mammalian species. The antibody is also a humanized antibody or human Could be antibodies.

An antibody is combined with an antibody of the invention for binding to C5aR* as described herein. can compete. Such cross-competing antibodies their ability to cross-compete with a known antibody of the invention in binding assays. can be defined based on This type of cross-competition is when two antibodies are identical, overlapping or may reveal binding to similar epitopes.

human antibodies As described in the examples herein, the inventors of the human immunoglobulin germ anti-CSaR antibodies derived from transgenic mice, including defined a series. Antibodies are isolated as monoclonal hybridoma antibodies and The binding characteristics are evaluated. As mentioned, CSaR is a seven-transmembrane It is GPCR and produces a soluble form that retains the native conformation. It is not possible. Human antibodies that block hCöa binding to hCSaR Transgenic mice were cultured with native hCSaR-expressing cells for breeding. immunized. However, blocking antibodies were very difficult to obtain and desired. Prior to the identification of a human antibody with blocking properties, the inventors A total of 11 blocking antibodies were obtained from the screening of the filtrate.

Also, due to the nature of hC5aR, the affinity of the antibodies was determined by standard Biacore analysis. It was not possible to determine the ICSO and E050 values in Example 2 and functional hCSaR-dependent reads, determined as described in Example 7. based appointments were made.

In one aspect, the invention relates to a human antibody that binds C5aR and that binding to hC5aR will be more potent than other strains, especially mouse C5aR, without binding to CSaR. More preferably, it specifically binds hC5aR. Antibody human C5aR It connects to the 2nd loop. In one embodiment, the antibody is used in cell 2 of human CSaR. It binds to the 2nd extracellular loop of the murine CSaR but not to the 2nd extracellular loop.

In other embodiments, the antibody according to the invention only enters the 2nd extracellular cycle of CSaR. can be bound in the native conformation.

The functionality of an anti-C6aR antibody depends on its ability to link C5a to C5aRS. It depends on its ability to significantly inhibit or reduce its binding.

In one embodiment, the antibody of the invention significantly inhibits C5a binding to C5aR. may or may not be reduced. This is ICSO, as described in Example 2 herein. It can be determined by a displacement assay (SPA) from which the values can be determined. From Table 1 As it turned out, the 11 antibodies isolated and described had an IC 50 below 50nM. has concentration. In another embodiment of the invention, antibody hC5a is used in a SPA. in determination, below 50nM, for example below 40nM, for example below 30nM, for example Below 20nM eg below 10nM eg below 5nM or even 4nMl with an IC50 below or below 3nM or even an ICSO below 2.5nM or 2.0nM can be replaced with .

In other assays, anti-C5aR antibodies inhibit C5a-dependent migration of human neutrophils. The ability to inhibit was evaluated and some of the human antibodies identified were mediated neutrophil migration from a previously described CSaR antibody (WO The antibody of the invention can significantly inhibit the migration of human neutrophils. A in practice, in the presence of antibody migration, 10nM CSa and zero anti-C5aR antibody less than 50%, less than 40%, less than 30% compared to the observed level of migration less, less than 20%, or less than 10%. This type of in practice, migration is 10 nM C5a and after 30 min in the presence of antibody, 10 nM C5a and zero It is measured against the level of migration observed after 30 minutes in the presence of antibodies.

Alternatively, the antibody's ability to inhibit neutrophil migration may be altered based on the same setting. below ug/ml, eg below 2.5 ug/ml, eg below 1.5 ug/ml, eg It is below 1.2 ug/ml or even below 1.0 ug/ml.

As an alternative to the standard Biacore analysis, the functionality of hCSaR antibodies By a competition binding assay on neutrophils as described in Example 7. can be determined. This functionality, as measured by the competitive ligand binding assay. expressed as the affinity of the antibody, but also, the avidity of the interaction It can also be considered as a measurement. In one embodiment, the antibody acts on neutrophils. its affinity or avidity as measured by the competition ligand binding assay is 0.80nM, is below.

Another option for characterizing antibodies is similarly described in Example 7. inhibiting CSa-induced neutrophil activation by an antibody ex vivo It was discovered using a calcium-flow assay that measures ability. In another application, the ICSO of the antibody is below 7.0ug/ml as determined by a calcium-flow assay, eg below 50ug/ml, eg below 2.5ug/ml.

Additional ex vivo assays show secondary effects of an antibody, such as expression of CD11b and CD62L. ability to inhibit or neutralize C5a-induced neutrophil maturation based on can be used to determine Activation of CD11b and CD62L, Coa/CSaR interaction maturation of neutrophils as the neutrophils are arranged up and down respectively. are markers.

The effect in a CD11b upregulation assay was determined. In one embodiment, a CDi1b ICSO, as determined in the upregulation assay, below 3.5ug/ml, eg Below 3.0ug/ml, eg below 2.5ug/ml, eg below 2.0ug/ml, or eg below 1.5ug/ml or even below 1.0ug/ml.

Similarly, the effect of the antibody in a CD62L downregulation assay was determined. A In practice, ICSO 1.8 as determined in a CD62L downregulation assay below µg/ml, eg below 1.5 µg/ml, eg below 1.2 µg/ml or even It is below 1.0 ug/ml.

Sequencing for sequencing of variable regions and particularly CDR sequences Four monoclonal antibodies were selected for An alignment of the arrays is shown in Figure 1. are presented, and the sequences are likewise included in the accompanying sequence list.

The sequence list includes the following sequences for isolated antibodies: SEQ ID 4: Vh 35F32A3 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 identified by variable regions or CDR sequences An antibody according to the present disclosure thus has CDR sequences, heavy and light chains. sequences of the variable regions and antibody detection by the person skilled in the art. based on minor modifications that can be performed without changing the functionality definable. This includes one, two, or three amino acids in each of the CDR sequences. amino acid substitutions, deletions, or additions of one or more are included.

In one aspect, the disclosure binds the CSaR defined by the sequence of CDR regions. associated with an antibody, wherein the variable region of the heavy chain of said antibody is Contains the CDR1, CDR2 and CDR3 sequences selected from the following groups: a) SEO IDs 1, 2, and 3, where none, one, two or three of the threads in question, contains 1, 2 or 3 amino acid(s) substituted by a different amino acid residue; and b) SEQ IDs 9, 10 and 11 where none, one, two or three of said sequences, contains 1, 2 or 3 amino acid(s) substituted by a different amino acid residue; and c) SEQ IDs 17, 18 and 19 where none, one, two or three of said sequences, contains 1, 2 or 3 amino acid(s) substituted by a different amino acid residue; and d) SEQ IDs 25, 26 and 27 where none, one, two or three of said sequences, contains 1, 2, or 3 amino acid(s) substituted by a different amino acid residue.

In one aspect, the disclosure binds C5aR, which is defined by the sequence of CDR regions. associated with an antibody, wherein the variable region of the heavy chain of said antibody is includes the sequences CDR1, CDR2, and CDR3; wherein said CDR1 sequence is SEQ ID 1, 9, 17, 25 or one of said sequences wherein 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue; and where said CDR2 sequence SEO ID 2, 10, 18, 26 or one of said sequences wherein 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue; and wherein said CDR3 sequence is SEQ ID 3, 11, 19, 27 or one of said sequences contains, where 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue.

In one aspect, the disclosure binds C5aR, defined by the sequence of CDR regions. associated with an antibody, wherein the variable region of the light chain of said antibody is Contains the CDR1, CDR2 and CDR3 sequences selected from the following groups a) SEQ IDs 5, 6 and 7 where none, one, two or three of said sequences, contains 1, 2 or 3 amino acid(s) substituted by a different amino acid residue; and b) SEQ ID 13, 14, and 15 wherein neither, one, two, or 1, 2, or 3 amino acid(s), three of which are substituted by a different amino acid residue c) SEO IDs 21, 22 and 23, where none, one, two or 1, 2, or 3 amino acid(s), three of which are substituted by a different amino acid residue d) SEQ IDs 29, 30, and 31, wherein neither, one, two, or 1, 2, or 3 amino acid(s), three of which are substituted by a different amino acid residue In one aspect, the disclosure binds C5aR, defined by the sequence of CDR regions. associated with an antibody, wherein the variable region of the light chain of said antibody is includes the sequences CDR1, CDR2, and CDR3; wherein said CDR1 sequence is SEQ ID 5, 13, 21, 29 or one of said sequences wherein 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue; and where said CDR2 sequence SEO ID 6, 14, 22, 30 or one of said sequences wherein 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue; and wherein said CDR3 sequence is SEQ ID 7, 15, 23, 31 or one of said sequences contains, where 1, 2 or 3 amino acid(s) are substituted by a different amino acid residue.

In one aspect, the explanation is that the CDRs of the variable region of the heavy chain are SEQ ID 1, 2 and 3 or 1, 2, or 3 amino acid substitution(s), deletion(s), and/or the string in question with the extension(s) it contains and the variable light chain SEQ ID 5, 6 and 7 or 1, 2 or 3 amino acid substitutions, deletions and/or said sequence having the appendix relates to an antibody it contains.

In one aspect, the disclosure is that the CDRs of the variable region of the heavy chain SEQ ID 9, 10 and 11 or 1, 2, or 3 amino acid substitution(s), deletion(s), and/or the string in question with the extension(s) it contains and the variable light chain to SEQ ID 13, 14 and 15 or 1, 2 or 3 amino acid substitution(s) of their CDRIs, The array in question with its deletion(s) and/or its extension(s) contains relates to an antibody.

In one aspect, the explanation is that the CDRs of the variable region of the heavy chain SEQ ID 17, 18 and 19 or 1, 2, or 3 amino acid substitution(s), deletion(s), and/or The string in question with its plugin(s) contains and is the variable light chain substitutions of CDRs for SEQ ID 21, 22 and 23 or 1, 2 or 3 amino acid substitutions), The array in question with its deletion(s) and/or its extension(s) contains relates to an antibody.

In one aspect, the explanation is that the CDRs of the variable region of the heavy chain SEQ ID 25, 26 and 27 or 1, 2, or 3 amino acid substitution(s), deletion(s), and/or The string in question with its plugin(s) contains and is the variable light chain SEQ ID 29, 30, and 31 of CDRs, or to 1, 2, or 3 amino acid substitution(s), The sequence in question with its deletion(s) and/or its attachment(s) contains a related to the antibody.

In one aspect, the disclosure thus relates to an antibody, wherein said antibody is heavy. The variable region of the chain contains a CDR1, a CDR2, and a CDR3 sequence. said CDR sequences comprise one of the following sequence groups; SEQ ID 1, 2 and 3, SEO ID 9, said sequences with substitution, deletion and/or insertion, and The variable region of the light chain of the antibody contains a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences comprise one of the following sequence groups; SEQ ID 5, 6 and 7, said sequences with as many substitutions, deletions and/or insertions.

In one aspect, the disclosure thus relates to an antibody, wherein said antibody is heavy. The variable region of the chain contains a CDR1, a CDR2, and a CDR3 sequence. said CDR sequences comprise one of the following sequence groups; SEQ ID 1, 2 and 3, SEQ ID 9, said sequences having deletion and/or insertion, and wherein said antibody The variable region of the light chain contains a CDR1, a CDR2, and a CDR3 sequence, where said CDR sequences comprise one of the following sequence groups; SEO ID 5, 6 and 7, SEO said sequences with substitution, deletion and/or insertion.

In one aspect, the disclosure thus relates to an antibody, wherein said antibody is heavy. The variable region of the chain contains a CDR1, a CDR2, and a CDR3 sequence. said CDR sequences comprise one of the following sequence groups; SEQ ID 1, 2 and 3, SEQ ID 9, The variable region of the light chain contains a CDR1, a CDR2, and a CDR3 sequence, where said CDR sequences comprise one of the following sequence groups; SEQ ID 5, 6 and 7, SEQ One aspect of the disclosure is that the variable region of the heavy chain of the antibody in question is related to the antibody.

In one aspect, application of the disclosure of the invention is that the antibody in question is heavy. It relates to an antibody in which it contains an identical sequence of 99.

One aspect of the disclosure is that the variable region of the light chain of the antibody in question is related to the antibody.

In one aspect, the disclosure is that the variable region of the light chain of the antibody of interest is related to the antibody.

One embodiment of the disclosure is thus wherein the heavy chain of the antibody in question contains a sequence and/or wherein the light chain of the antibody in question is variable It is related to an antibody it contains.

In one aspect, the disclosure is that the variable region of the heavy chain of said antibody is contains a sequence that is at least 96, 97, 98 or 99 percent identical to SEO ID NO: 4 and/or mentioned here at least 96.97% of the variable region of the light chain of the subject antibody to SEO ID NO 8, It relates to an antibody containing a 98 or 99 identical sequence.

In one aspect, the variable region of the heavy chain of the antibody of the invention is closest to SEQ 1D NO: 12. contains at least 96, 97, 98, or 99 percent identical sequences and the light chain of the antibody of the invention In one aspect, the disclosure is that the variable region of the heavy chain of said antibody is at least 96% of the variable region of the light chain of the antibody in question, to SEO ID NO 24, 97, 98 or 99 relates to an antibody containing an identical sequence.

In one aspect, the disclosure is that the variable region of the heavy chain of said antibody is the variable region of the light chain of the subject antibody, at least 96.97% to SEO ID NO 32, It relates to an antibody containing a 98 or 99 identical sequence.

In one embodiment, the variable region of the heavy chain of the antibody of the invention, SEO ID NO 39*a contains at least 96, 97, 98, or 99 percent identical sequences and is part of the light chain of the antibody of the invention. In one embodiment, the antibody of the invention is where the heavy chain of said antibody that the variable region is defined by SEO ID NO 39 and that in which the variable region of the antibody's light chain is defined by SEQ lD NO 40 is an antibody.

During maturation of antibodies, as described herein in Examples 6 and 7 Spontaneous mutations may occur in the framework region, isolated monoclonal The variable region of one of the antibodies is the closest human germ for both the heavy and light chain. The human antibody was compared with the germline sequences to identify the lineage sequence. This Therefore, in order to minimize the risk of immunological reactions, antibodies should be framework for interpretation by an antibody with the human germline sequence in It was decided to further optimize it by adding point mutations in the As can be seen from the experiments, this does not affect the functionality of the antibody. it does not affect. In one aspect, the disclosure is a reference as described above herein. an antibody identified by sequence identity to a variable region of the antibody, wherein the variable region of the heavy chain and/or light chain of said antibody contains one or more mutations in the framework region. although other Although mutations may also be considered, identity to the nearest human germline sequence is It may be tempting to add one or more mutations to increase This mutation(s) may be conservative mutation(s).

Antibodies defined by the Fc region The Fc region allows antibodies to activate the immune system, and antibodies typically as serum half-life, complement fixation, Fc-receptor binding, protein stability and/or antigen-dependent cellular cytotoxicity or lack thereof. Fc region for modification of one or more of its functional properties can be processed to include modifications in it. Also, an antibody of the invention is chemically can be modified (for example, one or more chemical segments to the antibody) may be added) or to alter its glycosylation, again one or more of the antibody It can be modified to change more functional features.

In one embodiment, the antibody of the invention includes an Fc region, where the Fc region is an Fc region. or more has reduced or impaired binding affinity to FcγR.

In one embodiment, the antibody of the invention is detected by SEQ ID NOs 33, 34, 35 and 36, respectively. one or more of the identified IgGf, IgG2, IgG2/4 or IgG4 Fc reference sequences. exhibits reduced binding affinity to excess FcγR. Specific amino acid residues FC may be responsible for the interaction of FcyRs and its mediated effects. where such specific amino acid residues of the region are substituted with a different amino acid. It may be advantageous to administer an antibody with

In one embodiment, the FC region in question was identified by SEO LD 33, 34, 35 and 36, respectively. Compared to IgG1, IgG2, IgG4/G2 or IgG4Fc reference sequences as defined, a reducing affinity for one or more Fcv receptors or complement components It contains one or more point mutations in the figure.

Evaluation of the consequences of introducing point mutations in the FC region effector functions for a number of anti-CSaR antibodies, as described in Example 4. evaluated in the following. FC region, anti-hCSaR antibodies, (hCöaR-expressing ability of neutrophils to induce phagocytosis by human monocytes A phagocytosis assay was created to quantify its role. As can be seen in Table 2, Various Fc variants were induced by anti-C5aR antibodies in the described assay. reduces the level of phagocytosis.

In one embodiment, the antibody according to the invention significantly inhibits phagocytosis of neutrophils in vitro. does not induce phagocytosis, that is, the level of phagocytosis is measured in the absence of an anti-C5aR antibody. not significantly above the background. In one embodiment, the antibody phagocytosis does not cause any detectable induction. phagocytosis level The assay for evaluation was performed using human neutrophils as described in Example 4. can be realized.

In alternative assays, anti-hCSaR antibodies can be used to detect ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity) inducing ability were evaluated.

The assays show that Fc variants are cell-mediated via ADCC or CDC dependent mechanisms. was created to test its ability to mediate It was assumed that he could imitate the activities.

Assays, target cells and effector to provide responses as described in Example 4. hCSAR-expressing cells as cells (monocyte-depleted PBMCs), or administers serums containing complement.

In one embodiment, the antibody according to the invention does not significantly induce ADCC, ie ADCC level significantly above background as measured in the absence of an anti-CSaR antibody. not on. In one embodiment, any detectable antibody ADCC induced, that is, the ADCC level is not above background.

In one embodiment, the antibody according to the invention does not significantly induce CDC. A in practice, the antibody does not cause any detectable induction of CDC, that is, the CDC level is not above the background.

In one embodiment, the antibody according to the invention includes an Fc region where the sequence is the effector modified to alter cell function or functions. FC Modification of the sequence can be achieved by point mutations in the amino acid sequence. Heavy The chain Fc region may be IgG1, I962, I964 or an IgGZ/4 chimeric sequence. Reference sequences are defined in the sequence list as follows; IgG1 by SEQ ID NO: 33 IgG2 by SEQ ID NO: 34, IgG2/4 by SEQ ID NO: 35 and SEO ID NO: 36 by IgG4.

In one embodiment, the Fc region has one or more of the following point mutations. an IgG1 (SEO ID NO: 33), IgG2 (SEQ ID NO: 34), IgG or c. L235E or L235A D. G236R or G236A g. 8254W The difference between F0 variants is with FcγRs or complement as described above. takes place in their ability to interact with the components of the system. Sequence differences in the FC region It also affects the structure and flexibility of the antibody, which in turn also affects antibody function. may affect. As described in Example 5 and Table 3, the inventors also The Fc region is of the IgG1 type with or without additional point mutations. anti-hC5aR antibodies, hCSaR-mediated effects, have an IgG4-type Fc region proved to be more potent inhibitors than the corresponding antibodies.

Thus, antibodies of the invention have an Fc hinge region of the IgG1 isotype.

In one embodiment, the IgG1 Fc region is SEQ 1D NO. IgG1 Fc as defined in 33 contains 1 to 10 amino acid substitutions compared to the reference sequence. Fc regions, AA to 231 It is preferred that it contains fewer mutations, such as acid substitution. Preferably, amino acid substitutions phagocytosis of neutrophils, ADCC and/or CDC as described above. It is selected from substitutions that significantly reduce the ability to induce in vitro.

In one embodiment, the antibody Fc region contains 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) A3308 and/or In one embodiment, the antibody FC region represents one of the following groups of point mutations. is an IgG1 containing one or more of: a) N297Q and/or b) L234A and L235E and/or o) L234A and GZ36R and/or d) L235E and G236R and/or f) G236R and L328R and/or g) N297Q, L234A and L235E and/or In one embodiment, the antibody Fc region represents one of the following groups of point mutations. is an lgGl containing one or more of: a) N297Q and/or b) L234A and L235E and/or c) G236R and L328R and/or d) N297Q, L234A and L235E and/or For the person skilled in the art, both heavy and light chains within the framework region based on standard criteria for substitution of point mutations, amino acid residues obviously can be added. Functional assays as described herein can be used to confirm that the mutations do not affect the functionality of the antibody.

As will be understood from above, the binding specificity of the identified antibodies, their CDR or by variable domains and have a similar antigenic binding site. It is clear that different types of antibodies having antibodies 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 It is a monoclonal antibody. In one embodiment, the antibody is a human antibody. Description of the application As described in the section on, a humanized antibody is at least human germline Contains CDRs that are not derived from the sequence. As can be understood from the above, a human The antibody may contain one or more point mutations compared to the germline sequence. but in general, human from at least the frame region or Fc region of the sequence It is thought that it should have at least 95% identity to the germline sequences.

PHARMACEUTICAL FORMULATIONS The present invention also provides a pharmacologically acceptable carrier and, according to the invention, a pharmaceutical compositions/formulations containing polypeptide or antibody, as well as this It also includes kits containing the compositions.

The antibody according to the invention is formulated as a pharmaceutical composition in one aspect of the invention. can be done. Such a pharmaceutical composition, as in Pharmacopeia or Remington can be prepared based on general knowledge in the art.

In one embodiment, the pharmaceutical composition according to the invention is a pharmacologically acceptable in combination with the carrier, an antibody as described herein.

The formulation is an aqueous formulation or a buffer in water or an aqueous buffer prior to administration. may be in the form of a reconstituted dry formulation in its composition. Like the compositions, it may contain a salt and/or buffer. may be suitable for multiple uses, such as the compositions described.

Treatment method One aspect of the invention is a method for the treatment or prevention of a condition in a subject. relates to an antibody of the invention for use in the method, comprising administering to the subject a therapeutic amount of an antibody as described herein. It is useful/suitable for the treatment of diseases and ailments. Thus, you find a administration of an immunological disease or condition, especially an inflammatory It relates to an antibody of the invention for use in the treatment of disease. Example 8 here This demonstrates the functionality of an anti-CSaR antibody according to the invention in a mouse model of arthritis. supporting it with proof. Examples 9-11, psoriatic arthritis, Crohn's and ulcerative colitis revealed upregulation of CSaR in tissue samples from patients puts it. In addition, an anti-C5aR antibody was obtained from patients with psoriatic arthritis. It has also been revealed that PMNs induced by synovial fluid can inhibit cell migration. put r.

A method of treatment may be aimed at curing a disease or condition, but immunological and inflammatory diseases such as a chronic disease or condition Only partial symptoms may be associated with some diseases, including significant for the subject, even if relief is achieved or the effect is only temporary or partial. Relief of one or more symptoms may also be a worthwhile improvement. can be seen as a treatment.

The antibody of the invention includes, but is not limited to, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic Iupus erythematosus (SLE), Iupus nephritis, type I diabetes, Grave disease, Inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, autoimmune thyroid, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, patch-borne host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA- associated vasculitis, uveitis, scleroderma, bullous pemphigoid, Alzheimer's Disease, amyotrophic lateral sclerosis, Huntington's disease, cystic fibrosis, gout, age-related macular degeneration, allergy, asthma, and other autoimmune diseases that are the result of acute or chronic inflammation It can be used in the treatment of one or more diseases, including diseases.

In another embodiment, the disease or disorder is an acute or chronic inflammation, the ailment here is an auto-immune disease. Discomfort rheumatoid arthritis in an application (RA), psoriatic arthritis, systemic Iupus erythematosus (SLE), Iupus nephritis, inflammatory bowel disease (IBD), Crohn's disease (CD) or ulcerative colitis (UC) or irritable bowel syndrome. In other applications, the disorder is RA or SLE. Chronic anti-CSaR antibodies, organ transplant, ischemia/reperfusion injury, except for diseases (eg, acute myocardial infarction, stroke), sepsis (eg, SIRS; MODS; ALI), associated with acute indications such as atherosclerosis and intracerebral hemorrhage (ICH) may be related.

In another aspect, the invention is for the treatment of a disease or disorder as described herein. An antibody, such as, relates to an isolated antibody or antibody composition. In other application said antibody, isolated antibody or antibody composition, wherein the above treatment one or more of the diseases and conditions described in connection with the method is for treatment.

One aspect of the invention is the use of an antibody, an isolated antibody, or of the antibody composition, a medicine for the treatment of a disease or condition relating to its use for the preparation of may be as described above in connection with a treatment modality.

GIVEN PATH An antibody of the invention can be administered parenterally, eg intravenously, eg intramuscularly. may be given subcutaneously, for example. Alternatively, an antibody of the invention, per- It may be administered via a non-parenteral route such as orally or topically. find it An antibody may be given prophylactically. In a preferred embodiment, the antibody is administered intravenously. given orally or subcutaneously.

Dosage and timing of verisin is often determined by the disease/condition of concern or depending on a variety of factors, including symptoms as well as the subject in question. will be. In general, doses of 0.010 mg/kg to 4-6 mg/kg of antibody are is expected to be given. Similarly, the dosage regimen of the antibody also depends on the individual subject and will also depend on the disease state of the subject in question, but according to the invention, the antibody (or antibody composition) to the subject once a week or every 2 weeks or less It may be desirable to administer a treatment that is given at intervals, eg monthly.

An antibody of the invention can be delivered upon request, ie the antibody is based on the patient's experience. for example, when certain symptoms occur or the amount of certain biomarkers can be given when it reaches a predefined level.

SPECIFIC COMBINATION TREATMENTS Antibodies of the invention in combination with one or more other therapeutic agents or formulations can be given. The other agent may be an agent that potentiates the effects of the antibodies of the invention. Other The purpose of the agent may be to treat the patient's other symptoms or conditions. For example, the other agent is an analgesic, an immunosuppressant, or an anti-inflammatory agent. The combined administration of two or more agents can be achieved in a number of different ways. A In practice, the antibody and other agent may be administered together in a single composition. Another In practice, the antibody and other agent are combined in separate compositions as part of a combined therapy. can be given. For example, the modulator can be used before, after, or simultaneously with the other agent. can be given.

Antibodies according to the present invention are associated with other drugs (eg, methotrexate, dexamethasone and prednisone) and/or other biologic drugs. According to the invention a provided that it does not remain, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, D-penicillamine, gold salts (sodium aurothiomalate, auranofm), Ieflunomide, methotrexate, minocycline, sulfasalazine and cyclophosphamide, glucocorticosteroids, mycophenolic acid or mycophenolate and tacrolimus and in separate administration Plakenil, Azulfidine and Methotrexatin, anti-inflammatory drugs, including one or more of dexamethasone and/or prednisone ATC code of rheumatic drugs from MO1C class and ATC code of immunosuppressants It may be administered with one or more therapeutic agent(s) selected from L04.

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

Antibodies, therefore, in autoimmunity including but not limited to in use, IFN-beta, OrenciaTM (CTLA4-Ig), HumiraTM (anti-TNF), CimziaTM (anti- TNF, PEG Fab), TysabriTM (α4-integrin mAb), SimponiTM, Rituxan/MabTheraTM, immune modulators such as Actemra/RoActemraTM, KineretTM, Raptiva, Ustekimumab; Non-steroidal anti-inflammatory drugs (NSAIDs) such as AsprinT'V', IbuprofenT'V' etc. Corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs) eg PlaquenilT'V', AzulfidineTM, MethotrexateTM, etc., CopaxoneTM (glatirimer acetate), Antibiotics such as GilneyaTM (fingolimod), FlagleM, CiproTM, topical corticosteroids, D vitamin analog creams (DovonexTM), topical retinoids (TazoracTM), moisturizers, topical immunomodulators (tacrolimus and pimecrolimus), coal tar anthralin and with agents such as Topical (applied to the skin) drugs and other It can be given in combination and additionally, PUVA, UVB and CellCeptTM Light therapy, such as (mycophenolate mofetil), is also combined with treatment using antibodies according to the invention. can be combined.

The subject is currently being treated with one or more other drug(s) In that case, the antibody of the invention can be added to said treatment regimen.

Antibody preparation method An antibody is applied to hybridoma clones or clones known in the art primarily for antibody production. various methods based on expression of the antibody in a recombinant host a nucleotide sequence encoding a desired antibody chain based on the information can be configured and the heavy and light chain of an antibody is made of one or two separate polynucleotides. It can be used for recombinant expression in which it is expressed.

In another aspect, the present disclosure provides that an antibody described herein is an antibody chain. relates to one or more isolated polynucleotide(s) encoding a polypeptide sequence.

Another aspect is a polypeptide of an antibody chain of an antibody described herein. A host containing one or more isolated polynucleotide(s) encoding sequence(s) relates to the cell.

The invention also uses a host as described above to produce an antibody according to the invention. one or more polypeptide sequence(s) of an antibody chain It relates to a process comprising culturing it under conditions that favor its expression.

The process also allows two separate open reads of the antibody chains on an adjacent polynucleotide. encoded by the framework and optionally the antibody to the host in question. may include recovery from cell culture.

The current description also includes the following aspects. 1. An antibody, wherein the variable region of the heavy chain of said antibody is a CDR1 contains a CDR2 and a CDR3 sequence, where the CDR1 sequence in question is SEO ID 1 or 1, 2 or 3 amino acid(s) substituted, deleted, or added sequence of interest and/or wherein said CDR2 sequence SEO ID 2 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The subject CDR3 sequence is SEO ID 3 or substituted, deleted, or replaced by 1, 2, or 3 amino acids. contains the array in question with the appendix. 2. An antibody, wherein the variable region of the heavy chain of said antibody is a CDR1 contains a CDR2 and a CDR3 sequence. where the CDR sequences in question are SEO ID 1, 2 and 3 or a different sequence of said sequences, 1, 2 or 3 amino acid(s) includes variants in which it is substituted by amino acid residue. 3. Where the variable region of the heavy chain of the antibody in question is a CDR1, a An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEO ID 1, 2 and 3. 4. An antibody wherein the variable region of the light chain of said antibody is a CDR1 contains a CDR2 and a CDR3 sequence, where the CDR1 sequence in question is SEO ID or the sequence of interest substituted, deleted, or added with 1, 2, or 3 amino acid(s) and/or wherein said CDR2 sequence SEO ID 6 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The subject CDR3 sequence SEO ID 7 or 1, 2 or 3 amino acid(s) is substituted, deleted, or contains the array in question with the appendix.

. An antibody wherein the variable region of the light chain of said antibody is a CDR1 contains a CDR2 and a CDR3 sequence, wherein said CDR sequences are SEO ID , 6 and 7 or a different sequence of said sequences, 1, 2 or 3 amino acid(s) includes variants in which it is substituted by amino acid residue. 6. Here, a CDR1 of the variable region of the light chain of the antibody in question An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEO ID 5, 6 or 7. 7. Here in any of 1, 2 or 3 of the variable region of the heavy chain as defined and where the variable region of the light chain is 4, 5 or It is an antibody as defined in any of 6. 8. An antibody, wherein the variable region of the heavy chain of said antibody is a CDR1 contains a CDR2 and a CDR3 sequence, where the CDR1 sequence in question is SEO ID 9 or 1, 2 or 3 amino acid(s) substituted, deleted or added to the sequence of interest. and/or wherein said CDR2 sequence is SEQ ID 10 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein the subject CDR3 sequence SEQ ID 11 or the 1, 2 or 3 amino acid(s) is substituted, deleted, or contains the array in question with the appendix. 9. An antibody wherein the variable region of the heavy chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR sequences are SEQ ID 9, 10 or 11 or a different sequence of said sequences, 1, 2 or 3 amino acid(s) includes variants in which it is substituted by amino acid residue.

wherein the variable region of the heavy chain of the antibody in question is a CDR1, a An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences 11. An antibody wherein the variable region of the light chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR1 sequence is SEQ ID 13 or 1, 2 or 3 amino acid(s) substituted, deleted or added the sequence of interest includes and/or wherein said CDR2 sequence is SEQ ID 14 or substituted by 1, 2 or 3 amino acid(s), contains said sequence deleted or inserted, and/or wherein said CDR3 sequence SEQ ID 15 or 1, 2 or 3 amino acid substitutes, deletions or additions in question contains the array. 12. An antibody, wherein the variable region of the light chain of said antibody includes a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are SEQ includes variants in which it is substituted by an amino acid residue. 13. Here, a CDR1 of the variable region of the light chain of the antibody in question An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEQ ID 13, 14 and 15. 14. Here in any of 8, 9 or 10 of the variable region of the heavy chain as defined and where the variable region of the light chain is 11, 12 or It is an antibody as defined in any of 137.

. An antibody wherein the variable region of the heavy chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR1 sequence is SEQ ID 17 or 1, 2 or 3 amino acid(s) substituted, deleted or added sequence of interest and/or wherein said CDR2 sequence is SEQ ID 18 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The CDR3 sequence in question is SEQ ID 19 or substituted, deleted, or replaced with 1, 2, or 3 amino acid(s). contains the array in question with the appendix. 16. An antibody, wherein the variable region of the heavy chain of said antibody includes a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are SEQ includes variants in which it is substituted by an amino acid residue. 17. Where the variable region of the heavy chain of the antibody in question is a CDR1, a An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEQ ID 17, 18 and 19”. 18. An antibody wherein the variable region of the light chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR1 sequence is SEQ 1D 21 or 1, 2 or 3 amino acid(s) substituted, deleted or added the sequence of interest and/or wherein said CDR2 sequence is SEQ ID 22 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The CDR3 sequence in question is SEQ ID 23 or substituted, deleted, or replaced with 1, 2, or 3 amino acid(s). contains the array in question with the appendix. 19. An antibody, wherein the variable region of the light chain of said antibody includes a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are SEQ includes variants in which it is substituted by an amino acid residue.

. Here, a CDR1 of the variable region of the light chain of the antibody in question An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEQ ID 21, 22 and 23. 21. Here in any of the 15, 16 or 17 of the variable region of the heavy chain as defined and where the variable region of the light chain is 18, 19 or as defined in any. 22. An antibody wherein the variable region of the heavy chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR1 sequence is SEQ ID or the sequence of interest substituted, deleted, or added with 1, 2, or 3 amino acid(s) and/or wherein said CDR2 sequence is SEQ ID 26 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The CDR3 sequence in question is SEQ ID 27 or substituted, deleted, or replaced with 1, 2, or 3 amino acid(s). contains the array in question with the appendix. 23. An antibody, wherein the variable region of the heavy chain of said antibody includes a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are SEQ lD 25, 26 and 27 or different sequences of said sequences, 1, 2 or 3 amino acid(s) includes variants in which it is substituted by an amino acid residue. 24. Where the variable region of the heavy chain of the antibody in question is a CDR1, a An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences It is identical to SEQ ID 25, 26 and 27.

. An antibody wherein the variable region of the light chain of said antibody is a CDR1 includes a CDR2 and a CDR3 sequence, wherein said CDR1 sequence is SEQ ID 29 or 1, 2 or 3 amino acid(s) substituted, deleted or added the sequence of interest. and/or wherein said CDR2 sequence is SEQ ID 30 or 1, 2 or 3 amino acid(s) contains the sequence in question substituted, deleted or added, and/or referred to herein The CDR3 sequence in question is SEQ ID 31 or substituted, deleted, or replaced with 1, 2, or 3 amino acid(s). contains the array in question with the appendix. 26. An antibody wherein the variable region of the light chain of said antibody includes a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are SEQ includes variants in which it is substituted by an amino acid residue. 27. Where the variable region of the light chain of the antibody in question is a CDR1, a An antibody comprising CDR2 and a CDR3 sequence, wherein said CDR sequences SEQ ID 29, 30 and 31.8 are identical. 28. Here in any of 2, 23 or 24 of the variable region of the heavy chain as defined and wherein the variable region of the light chain is 25, 26 or It is an antibody as defined in any of the 27. 29. Herein, the variable region of the heavy chain of the antibody in question is SEQ ID NO: 4, . 29, wherein the heavy chain of said antibody is variable. region contains one or more mutations in the framework region. 31. The antibody according to 29.3, wherein said mutation(s) are conservatively are mutations. 32. The antibody according to 298, wherein said mutation(s) are the closest human germ Increases the identity to the line array. 33. The antibody of 32, wherein the heavy chain of said antibody is variable. region is defined by SEQ ID NO 39. 34. Here is the SEO ID NO of the variable region of the light chain of the antibody in question: is an antibody. . 34, wherein the light chain of said antibody is variable. region contains one or more mutations in the framework region. 36. The antibody of 35, wherein said mutation(s) are conservatively are mutations. 37. The antibody according to 357, wherein said mutation(s) are the closest human germ Increases the identity to the line array. 38. The antibody of 34, wherein the light chain of said antibody is variable. region is defined by SEQ ID NO 40. 39. Herein, the variable region of the heavy chain of the antibody in question is SEQ ID NO: 4, the variable region of the light chain of the subject antibody, SEO ID NO: 8, 16, 24 or 40. The antibody of 39, wherein said heavy chain variable regions for example, it has 99% identity, where the light chain in question is variable. 41. The antibody of 39 or 40, wherein the heavy chain of said antibody the variable region contains one or more mutations in the framework region and/or wherein the variable region of the light chain of said antibody is in the framework region contains one or more mutations. 42. The antibody of 41, wherein said mutation(s) are conservatively are mutations. 43. The antibody of 41, wherein said mutation(s) is the nearest human germ Increases the identity to the line array. 44. The antibody of 41, wherein the heavy chain of said antibody is variable region is defined by SEQ ID NO 39 and/or where the antibody in question The variable region of the light chain is defined by SEQ ID NO 40. 45. The antibody of any one of 1 to 44, wherein said the antibody binds to CSaR. 46. The antibody of any of 1 to 45, wherein said the antibody is a full length antibody or an antibody fragment or a single Chain antibody. 47. The antibody of any of 1 to 46, wherein the The antibody is a monoclonal antibody. 48. The antibody of any of 1 to 47, wherein antibody a human, mouse. rat, rabbit, pig or non-human is an antibody. 49. The antibody of any of 1 to 48, wherein the the antibody is a mouse or human antibody. 50. The antibody of any of 1 to 49, wherein the the antibody is a human antibody or a humanized antibody. 51. The antibody of any of 1-50, wherein 52. It is a human antibody that binds C5aR. 53. The antibody of any of 1 to 52, wherein the the antibody binds the human CSaR. 54. The antibody of any one of 1-53, wherein the the antibody binds the 2nd extracellular loop of C5aR. 55. The antibody of any one of 1-54, wherein the the antibody binds the 2nd extracellular cycle of the human CSaR. 56. The antibody of any of 1 to 55. promise here The antibody binds the human C5aR but not the murine CSaR. primate 57. The antibody of any one of 1-56, wherein said The antibody binds the 2nd extracellular loop of the human C5aR but not the 2nd one of the murine CSaR. 58. The antibody of any one of 1-57, wherein said antibody 2 extracellular cycle of human C5aR only in native conformation 59. The antibody of any of 1 to 58, wherein the antibody is C5a Significantly inhibits or reduces its binding to human C5aR. 60. The antibody of any one of 1-59, wherein the antibody is a SPA determination of C5a below 10 nM or below 5 nM or preferably above 3 nM. It can be replaced with an IC50 under it. 61. The antibody of any of 1-60, wherein the antibody is human Significantly inhibits the in vitro migration of neutrophils. 62. The antibody of any one of 1-61, wherein antibody migration is After 30 minutes in the presence of nM C5a, 30 minutes in the presence of 10 nM C5a and zero antibodies less than 50%, less than 40% as measured against the level of migration observed after IC50 at setting below 2.5 ug/ml, eg below 2.5 ug/ml, eg 1.5 It is below ug/ml, eg below 1.2 ug/ml or even below 1.0 ug/ml. 63. The antibody of any one of 1-62, wherein the antibody is affinity of 0.80 as measured in the competition ligand binding assay on neutrophils Below nM, for example, below 0.50 nM or below 0.35 nM. 64. The antibody of any one of 1-63. where antibody C5a induced neutrophil activation ex vivo, as determined in a calcium-flow assay Below 7.0 µg/ml, eg below 5.0 µg/ml, eg below 2.5 µg/ml 65. The antibody of any of 1 to 64, wherein the antibody is CSa inhibits induced neutrophil maturation ex vivo as follows a. of 35 µg/ml as determined in a CD11b upregulation assay. below, eg below 2.5 pg/ml, eg below 1.5 ug/ml, or even with an IC50 below 1.0 pg/ml or b. of 1.8 pg/ml as determined in a CD62L downregulation assay. below, eg below 1.5 pg/ml, eg below 1.2 pg/ml or even with an ICSO below 1.0 pg/ml. 66. An antibody-binding CSaR, wherein the FC region is one or more of Fcy receptor as defined by SEQ ID NOs 33, 34, 35 and 36, respectively. decreased compared to lgG1, lgG2, lgG4, or IgG4/G2 Fc reference sequences. It has affinity/reduced binding. 67. The antibody of any one of 1-66, wherein the FC region is Compared to the lgG4 or lgG4/82 FC reference sequences, one or more Fcy one or more point mutations that reduce affinity for its receptor 68. The antibody of any one of 1-67, wherein the antibody is does not significantly induce phagocytosis of neutrophils in vitro. 69. The antibody of any one of 1-68, wherein the antibody inhibits ADCC. does not induce significantly in vitro. 70. The antibody of any of 1 to 70, wherein the antibody inhibits CDC does not induce significantly in vitro. 71. The antibody of any one of 1 to 68, wherein the Fc region is LGGi (SEQ ID) with one or more of the following point mutations NO: 33), IgG2 (SEQ ID NO: 34), IgG or IgG4 (SEO ID c. L235E or L235A D. G236R or G236A 72. The antibody of any of 1-71, wherein the Fc region is IgG1 or an IgG1 mutant. 73. The antibody of any of 1-72, wherein the Fc region is the SEQ It is an IgG1 Fc mutant containing the substitution. 74. The antibody of any one of 1-73, wherein the Fc region is AA It is an IgG1 Fc mutant containing 1 to 8 amino acid substitutions from 231 to 240, where IgG1 FC reference sequence SEQ ID NO. It is as defined in 33. 75. The antibody of any one of 1-74, wherein the Fc region is AA It is an IgG1 FC mutant containing 1 to 5 amino acid substitutions from 328 to 334, where [961 Fc reference sequence SEQ ID NO. It is as defined in 33. 76. The antibody of any one of 1-75. where antibody Fc region has one or more of the following groups of point mutations is lgG1 a. N297Q and/or b. L234A and L235E and/or c. G236R and L328R and/or D. N297Q, L234A and L235E and/or 77. The antibody of any one of 52-76, wherein said the antibody is a full length antibody or an antibody fragment or a single chain antibody. 78. The antibody of any one of 1-77, wherein said The antibody is a monoclonal antibody. 79. The antibody of any one of 1-78, wherein said antibody to a human, mouse, rat, rabbit, pig, or non-human primate is an antibody. 80. The antibody of any of 1 to 79. at issue here the antibody is a mouse or human antibody. 81. The antibody of any one of 1-80, wherein said the antibody is a human antibody or a humanized antibody. 82. The antibody of any of 1 to 81, wherein said antibody is a human antibody 83. The antibody of any of 1 to 82, an Immunological disease or for the treatment of discomfort. 84. The antibody of 83, wherein the disorder is an inflammatory disease. 85. The antibody of 83, wherein the disorder is an acute or chronic inflammation. 86. The antibody of 83, wherein the disorder is an auto-immune disease. 87. The antibody of any of 83-86, wherein the antibody given intravenously or subcutaneously. It is given in doses up to 6 mg/kg to 6 mg/kg. 89. The antibody of any of 83-88, wherein the antibody is given once or every 2 weeks. 90. The antibody of any one of 83-89, wherein the antibody is another given in combination with medication. 91. The antibody of any of 83-90, wherein disease or discomfort rheumatoid arthritis (RA), psoriatic arthritis, systemic Iupus erythematosus (SLE), colitis (UC) or irritable bowel syndrome. 92. The antibody of any of 83-91, wherein the patient, It is treated with another drug such as methotrexate. 93. A method for treating or preventing a condition in a subject according to a subject needing the method, any of 1 to 82 comprising the administration of a therapeutic amount of an antibody. 94. The method of 93, wherein the disorder is an immunological disease or discomfort r. 95. The method of 93 or 94, wherein the antibody is administered intravenously or subcutaneously. given as. It is given in doses up to 6 mg/kg to 6 mg/kg. 97. The method according to any one of 93-96, wherein the antibody is given once or every 2 weeks. 98. The method according to any one of 93-97, wherein the antibody is It is given in combination with another drug. 99. The method according to any of those 93-98 where discomfort. a It is an immunopathological disorder like an autoimmune disease. 100. The method according to any one of 93-99, wherein the subject is rheumatoid arthritis (RA), psoriatic arthritis, systemic Iupus erythematosus (SLE), Iupus nephritis, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), or He is a patient with irritable bowel syndrome. 101. The method according to any of 93-101, wherein the patient is a is treated with medication. 102. Optional combination with a pharmacologically acceptable carrier a pharmaceutical product containing an antibody according to any of 1 to 82 is the composition. 103. The pharmaceutical composition according to 102, an aqueous formulation or administration a dry formulation reconstituted in water/aqueous buffer before is in the form. 104. A polypeptide of an antibody according to any of 1 to 82 is an isolated polynucleotide encoding the sequence(s). 105. A host cell comprising one or more polynucleotides according to 104. 106. To produce an antibody according to any of 1 to 82, 105,e one or more polypeptides of a host cell, said antibody culturing the sequence(s) under conditions that favor expression containing a noun. 107. The process according to 106, wherein the heavy chain and light chain are a contiguous polynucleotide It is encoded by two separate open reading frames on it. 108. The process according to 106 or 107, furthermore, that the antibody in question is a host cell includes recovery from culture. 109. An antibody according to any one of 1 to 82, a medicinal drug use for its production. 110. An antibody according to any one of 1 to 82, rheumatoid arthritis (RA), psoriatic arthritis, systemic Iupus erythematosus (SLE), Iupus nephritis, Inflammatory bowel an immunological disease such as irritable bowel syndrome (IBD) or It is used for the manufacture of a medicinal drug for the treatment of ailment. EXAMPLES Example 1: Generation of human anti-hC5aR antibodies Immunization and scanning Raising antibodies against GPCRs in general, correct native protein It is very difficult, if not impossible, to produce a soluble protein with the same conformation it is difficult as it is. Traditionally, cells overexpressing GPCRs used for immunization, but the antibody responses generated are not quite specific. tends to have the desired profile, i.e. Iigand binding and GPCR. This complicates the identification of antibodies that can block the signaling In addition, the inventors of human anti-hCSaR, which can block CÖa binding to CSaR found the development of antibodies quite challenging, and these antibodies A number of immunization strategies have been implemented before they were identified.

HumAb mice (Medarex) have high human C5aR expression (~80,000 per cell). copy) were immunized with L1,2-mouse cells (a mouse B-cell Ienfoma line) (Lee et al. used for cell fusions using procedures. Deprivation of soluble hCSaR Because of this, the supernatants could not be screened in a standard ELISA assay and therefore a cell based binding determination was established. The obtained hybridoma filtrates, (~1,000,000 copies per cell) a transfected rat expressing native hCSaR tested for binding to the cell line (RBL). In general, hybridoma supernatants, untransfected cells (labeled with CeIITracker) and hCSaR-transfected cells or from hCSaR knock-out/knock-in (KOKI) mice with neutrophils (WO 2 goat anti-human Supernatants that did not bind to untreated cells were identified and produced anti-hCSaR. hybridomas were subcloned and bone isolated from human neutrophils and KOKI mice. Tested for binding to marrow-derived neutrophils (data not shown). Anti-HCSaR antibodies, protein A sepharose, and hybridoma using standard protocols. was purified from the filtrate. Example 2: Identification and characterization of anti-hCSaR antibodies As noted, the process of obtaining human anti-hCSaR antibodies was problematic and performing the fusion before a hC5a/hCSaR blocking antibody is identified needed. From 35 fusions and screening of more than 100,000 hybridoma supernatants, Only 11 clones were identified that were able to block hCöa binding to hC5aR. your antibodies The determinations applied in the characterization are described below. Reference antibody for determination of affinity and functionality in the assay and upregulation of CD11b Other adapted assays are described in Example 7.

Displacement Determination The displacement force of anti-hCSaR antibodies to hC5aR binding to hCöa A Scintillation Proximity Assay (SPA) was applied to determine of SPA a detailed specification is in US Patent 4568649 and the manufacturer (Amersham Biosciences) provided by the protocols provided by Briefly, from RBL-hCSaR cells purified receptor-carrier membrane fragments coated with rosette agglutinin (WGA) binds to luminescent microparticles. Addition of radiolabelled hCSa (125I) tracer The binding of the receptors will then result in the emission of light from the particles.

Specific to the SPA-principle, only radio isotope and particles will emit light. That is, radiolabeled hC5a bound to only one receptor, light close enough to a WGA-particle to produce Thus, the amount of light emitted is is an expression of the amount of receptor-bound 125I-hC5a. Detection, anti-hCSaR/unlabeled It is a competition assay in which hC5a competes with the tracer for binding to receptors.

In the assay, a fixed amount of 125I-labeled C5a is attached to WGA-particles and C6aR receptors. is added, which means that a certain amount of light measured in counts per minute (cpm) results in its spread. If unlabeled CSa or anti-CSaR is added, it binding to receptors, 125! To a lower cpm due to displacement of C5a will cause. The displacement % was calculated as follows: Q: Sample Smax: Non-specific binding. To replace specifically bound 125l-hC5a was measured by adding an amount of unlabeled hC5a sufficient for

So: Maximum binding. No unlabeled hC5a was added. were kept constant between experiments, so that ICSO values increased over time. proportional to their tens. 125I-hCSa displacement strength of human anti-hC5aR antibodies (ICSO) was determined and the data are presented in table 1.

Determination of Neutrophil Migration (Chemotaxis) The ability of antibodies to inhibit hCSa (or mC5a)-dependent neutrophil migration is a Boyden analyzed in the reservoir. Neutrophils isolated from human or animal blood are treated with calcein. stained and added to the upper chamber of the Boyden chamber and mixed with antibodies. hCSa or mCSa is added to the lower chamber of the Boyden chamber and is chemoattractant for neutrophils. functions. The ability of neutrophils to migrate into the lower chamber is a 3 or 5 pm fluoroblock. by counting the number of calcein-stained neutrophils crossing the membrane determines.

Human PMNs (PolyMorphoNuclear leukocytes; granulocytes), EDTA-containing vials obtained from human blood drawn samples. blood cells (4 parts) canine 30 for minutes ( separated by centrifugation over a gradient (3 parts). PMN-containing layer, dextran for 1 hour to destroy contaminating erythrocytes. suspended in PBS (phosphate buffered saline). filtrate 5 minutes centrifuged at room temperature (250 x 9) for 55 h and the remaining erythrocytes osmotically for a period of time using 02% NaCl. The solution should be rinsed before repeating the osmotic lysis. After centrifugation, PMNs were resuspended in the reaction mixture (RM): CaCl2 0.5mM, make up with HEPES 20mM. Cell density NucleoCounter (Chemometec) was determined. PMN suspension, Giemsa-stained samples contained >95% neutrophils as assessed by microscopy.

Loading PMNs: Calcein, AM, (Fluka) dissolved in DMSO (Dimethyl sulfoxide) and RM containing cells (2x106 cells per ml) to provide a concentration of 10 pM It was diluted 1000X in The suspension was incubated at 37°C in an incubator for 30 minutes. and then washed 3 times with RM to remove excess Calcein. Finally cells were resuspended in RM (4x10 6 cells/ml).

Migration, FIuoroBlok® 3pm pore size evaluated using the Boyden chamber technique. Upper chamber, aka Fluoroblock membrane-containing chambers in 1 mg/ml PBS at 37°C for 2 hours. coated with fibrinogen (cat no F3879-1 G, Sigma). Membranes after washing, PBS It was blocked with a solution containing 2% bovine serum albumin (BSA) in it. RM After another wash using the hC5aR-antibody or antibody Added '105 Calcein-loaded PMNs without control solution or chemoattractant The receiver containing hCSa (Sigma, C5788) was placed in the plate (lower chamber). Each group is It consisted of at least 6 eyes. Then, plate one plate every 5 minutes for 60 minutes. in the reader (SpectraMax, Molecular devices or Fluoroscan, Thermo Labsystems.) was used as a measurement.

Curve fabrication. The ability of antibodies to inhibit migration, GraphPad Prism 5 (GraphPad Software, Table 1 shows the hCSa-dependent (10 nM) migration of human neutrophils of 10ug/ml human mAb. from the displacement assay and the chemotaxis assay, which test the ability to inhibit contains the received data. The value obtained in the absence of antibody was set to 100.

Data were collected from 3 donors. Average values are given in table 1. three mAbs the control antibody Ref. Equal to or slightly higher than the power of Ab Q showed his mighty strength.

Ab hC5a displacement Migration of human neutrophils (in the absence of antibodies) (SPA) IC50 (nM) % compared to migration).

Ab hCSa displacement Migration of human neutrophils (in the absence of antibodies) (SPA) ICSO (nM) % as compared to migration). 32F3A6 1.90 2 35F3A1 10.7 38 35F6A1 22.9 ND hC5a 4.1 100 Table 1. Functional characteristics of anti-hCSaR antibodies Characterization of anti-hCSaR mAb CDR sequences were cloned recombinantly and their nucleotide and amino acid sequences were determined by standard methods. was characterized using The amino acid sequences are in Figure 1 and the accompanying sequence list. is located.

Characterization of binding specificity Human-mouse CSaR chimeric constructs for determination of the binding site of C5aR used. Chimeric receptors were transiently expressed in HEK cells and binding of individual antibodies, except for cell line change, previously Binding of 35F12A2 is bound to the human sequence of extracellular loop 2 while the human N-end was insignificant (Figure 2). Example 3. Generation of Fc variants More than 95% in Fc regions of the four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) share homogeneity, but differ greatly in the hinge region. fc region, Antibody-Independent Cell-Mediated Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity It mediates effector functions such as (CDC). In ADCC, the Fc region of an antibody is activator on the surface of immune effector cells such as natural killer cells and monocytes It binds to Fc receptors (FCyRs), which causes phagocytosis or phagocytosis of targeted cells. binds at one site and antibodies bind targeted cells to complement on the cell surface. It kills by triggering the cascade. Various IgG isoforms, IgG4 < IgG2 < IgGl < IgG3 exerts different levels of effector functions, increasing during Full Ref Ab A series of IgG Fc variants containing the variable region of Q, QuickChange® Domain-Directed By site-directed mutagenesis using the Mutagenesis Kit (Cat no. 200518, Stratagene) was produced and characterized as described in Example 4. Example 4: Characterization of effector functions of Fc variants Binding affinity of Fc variants to Fch The affinity of Fc variants for FcγRs can be determined using a CMS sensor chip (GE). BIAcore T determined by measurements. Fc variants flow using amine-coupling chemistry. immobilized on cells. Measuring the affinity of FcγR to Fc variants For kinetic SPR, His-FovRs were used as analytes and flow in HPS-EP buffer. injected into the cells. The high-affinity receptor FcγRI has a flow of 40pI/min. It injects quickly, with a contact time of 180 seconds and a separation time of 300 seconds. was done. Other FcyRs have a flow rate of 50uI/minute, a contact time of 30 seconds and a 120 It was injected with a separation time of one second. Chip surfaces, 10mM NaOH and 500mM It was reconstituted with a solution containing NaCl. Affinities (Kd values) Table It is listed in 2A.

Antibody Fc region L234A, L235E, G237A, A33OS N297Q, L234A, L235E, G237A N297Q, L234A, L235E G236R, L328R L234A, L235E N297Q N297Q N297Q V234A, G236A reference N297Q, F234L, L235A N297Q, E233P, F234V, L235A E233P, F234V, L235A N297Q 0.36E- 6.64E- 0.38E- 0.13E- FCvRIIA (131 R) 0.95E-6 2.79E-6 FCvRIIA (131H) 0.64E-6 0.22E-6 1.03E-6 FcvRIIB 2.10E-6 0.42E-6 1.54E-6 .34E-6 1.83E-6 FCvRHI 0.33E-6 (158V) 0.96E-6 1.50E-6 Table 2A. From analysis of the affinity (Kd as M) of Fc variants to FcγRs summary of results. (- = zero binding; 0 = zero change in binding; rida = weak Kd not calculated due to binding). (1) Lower hinge region of CH1 and IgG2 an IgGZ/IgG4 Fc variant containing and the remaining CH2-CH3 of IgG4; (2) 8228P dot IgG4 mutant containing the mutation.

Determination of phagocytosis reduced or impaired capacities to mediate phagocytosis of neutrophils An in vitro phagocytosis assay was established to identify Fc variants with

The phagocytosis assay described below was obtained from peripheral human blood (the target for phagocytosis). cell) labeling isolated human neutrophils with a fluorescent dye, CMFDA. and a culture thereof, of human monocytes, also isolated from human peripheral blood. includes the addition. CMFDA-labeled neutrophils are pre-coated with test mAbts (or PBS) and Number of CDM/CMFDA double positive monocytes after incubation with human monocytes It is determined by FACS. Results from various Fc variants are in Table ZB is offered. contains variable regions of the Q antibody used.

Mediate antibody-dependent phagocytosis of both monocytes and macrophages, neutrophils and phagocytosis assays using both cell types were generated.

Results were qualitatively similar in both assays, but macrophage assay was more variable. analysis was performed using monocytes as the basis.

Preparation of human monocytes Human monocytes and lymphocytes were isolated using Percoll gradient centrifugation. in tubes containing EDTA as coagulant (K2E. BD Biosciences, Cat. No. 367525) isolated from peripheral venous blood from healthy human volunteers. 100 ml of snow it provided ~8 - 20x overall. isolated At least 3 volumes of dPBS were added to the cells and the cells were then at 100X g for 10 min. centrifuged at room temperature (RT) for a period of time. After removal of the filtrate resuspended in the mixture and again at 100X g for 10 minutes at RT. centrifuged. The filtrate was discarded and the lymphocyte/monocyte layer 1 - 2 X 106 in Culture Media resuspended at cells/ml. Resuspended cells 4 x 10 6 cell/well to 2 ml/well in 6-well cell culture plates (Coming, Costar Cat. No. 3516) and incubated for 2 hours at 37°C, 5% CO2. non-adhesive cells (lymphocytes and dead cells) were destroyed by aspiration and adherent cells (monocytes) four times 1 ml of Culture Media (RPMI 1640 (Invitrogen-GIBCO, Cat. No.

Floor. no. + 1% Pen/Strep (Invitrogen-GIBCO, Cat. No. 15070)) before aspiration of the wash media gently swirled. 1 ml fresh after washing of blood-derived monocytes Culture Medium was added to each eye. Cells were scraped from one eye and monocytes per eye. suspended in Culture Media to calculate the number of

Preparation of human neutrophils Human neutrophils, percoll gradient from peripheral venous blood from healthy volunteers was isolated using centrifugation and CellTrackerT'V1 Green (5-chloromethylfluorocein) Staining was performed to a final 10 mM in CeIITrackerTM Green CMFDAhin DMSO. carried out by solubilization to concentration. Neutrophils 1 x 107 in dPBS cells/ml and CMFDA for a final concentration of 2 pM. added. Cells and dye were incubated for 15 minutes at 37°C. too much paint, cells with 10 ml dPBS 3 times (by centrifugation at 300X 9 for 5 minutes at RT) removed by washing. A cell count was performed after the last wash step.

CMFDA-labeled-neutrophils were resuspended in dPBS at 2 x 10 6 cells/ml and for antibody control). In some assays (as shown), neutrophil+Ab The incubation step also contained 4 mg/ml of human IgG. Cells plus antibody 30 min was incubated at 37°C for a period of time. Neutrophils at 300X for 5 minutes at RT After centrifugation, it was washed twice with dPBS and in Culture Media at 1 x 10 7 cells/ml. resuspended.

FACS analysis CMFDA-labeled pre-coated with antibody (prepared as described above) neutrophils (as described above) in 1 ml of Culture Medium concentration was added to monocytes. Total volume in each well of the G-well plate It was 2 ml. In some assays (as shown), the culture medium also contained 4 mg/ml of human IgG. contained. Generally, a ratio of 5:1 (neutrophilsmonocytes) was used. Sticky If the number of monocytes was less than 4 x 105 per well, 2 x 106 neutrophils were added (i.e. neutrophilsmonocyte ratio exceeded 5:1). If the number of monocytes exceeds 4 x 105 per well, this In this case, five times that neutrophil count was added to keep the neutrophilsmonocyte ratio at 5:1.

Cultures were incubated for 1 hour at 37°C in a 5% CO 2 incubator.

After incubation, to destroy non-adherent and undigested neutrophils medium was aspirated. Adherent monocytes (by gentle swirling) three times 1 ml/well Culture Washed mediocrely. Monocytes, Cell Scrapers from wells of cells in Culture Media (Coming, Cat. No. CP3010). Cells 300X It was centrifuged at 9 for 5 minutes at RT and the filtrate was destroyed. Cell pellet, FACS Reconstitute in 160 µl of 1% (w/v) paraformaldehyde in PBS to fix before was suspended.

Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences).

Neutrophils labeled with CMFDA were identified and Fluorescein isothiocyanate (FITC) monocytes were measured in FL-1 (Florisima channel 1) using identified only by staining with anti-CD14 labeled Phycoerythrin as the sample and FL-2ide (Florisima channel 2) was measured. increased in size during incubation for the inclusion of monocytes, only the sample monocyte must be added to the FSC (Forward diffuser) A monocyte gate was identified using the opposite SSC (Side-distributor) profile and (FSC and along the SSC axes). This gate is only accessible to the FSC in a sample neutrophil. SSC versus FSCS containing neutrophils as defined in the anti-SSC profile excluded the region of his profile. Degree of phagocytosis, FL-1+ve in total monocyte gate Calculated as the percentage of monocytes. Background size of non-specific phagocytosis not coated with antibody (“Zero Ab” sample) FL-1+Ve monocytes in a sample containing CMFDA-labeled neutrophils. was the percentage. Background with Ab from each sample, data (FL-1+ and Ab vs. Monocytes) % concentration) into a Prism program (v4.0c, GraphPad Software) for graphing. 3-form dose-response (variable slope) i.e. 4-parameter logistic were subjected to non-linear regression using the equation.

The data are presented in Table ZB. “-“ represents no detectable phagocytosis and “+” to “++++” represent low to high levels of phagocytosis as measured in assays. represents.

A_DCC (in Mkor dependent cellular sltotoxil) and C_DC (complement dependent cytotoxicity) The following in vitro assays refer to cell depletion of Fc variants as ADCC or CDC. to test the ability to mediate through dependent mechanisms. created.

target cells In these assays, target cells were either hC5aR or human expressing Ramos clone E2. were neutrophils. hCSaR expressing Ramos clone E2, Ramos clone E2 cells, using standard procedures with a mammalian expression vector encoding hCSaR. Developed by stably transfecting High levels of the resulting cell line express human CSaR (5-7 times higher than human neutrophils) and CD20.

Human neutrophils were obtained as described above for phagocytosis assay.

Target cells were stained with the fluorescent cell membrane dye, PKH-26. the required number of centrifuged at RT for 10 min. The cells were then 2 µM PKH-26 (each 1 x 106 resuspended in 100 µl of solution for the target cell. room of labeling Equal temperature for about 3 minutes to stop the labeling reaction. one volume of heat-inactivated FCS (or heat-inactivated human serum) (Millipore)) was allowed to continue before being added. Exactly 1 minute later RPMI was added for a volume of about 15 ml. Cells were centrifuged as above and It was resuspended in assay medium at 2 x 10 6 cells/ml. antibody of PKH-26 Target cells for plating with segments (25 µl ie 5 x 104) in 25 µl Containing 200 tig/ml antibody diluted in medium (final concentration 100ug/ml) dispensed into the wells of a sterile 96-well U-bottom plate and in 5% CO 2 at 37°C. minutes incubated.

effector cells Effector cells were monocyte-depleted PMBCs from healthy donors. PMBCs obtained as described above. Resuspended cells (lymphocyte/monocytes) eu-well tissue culture as ~4 x 106 cells/well 2 ml/well plates (Coming) or 20 mL per vial into T75 vials (Coming) inserted and incubated in 5% 002% at 37°C for 2 hours. (Lymphocytes and NK non-adherent cells (including cells) were removed by aspiration and 100X The gl was centrifuged for 10 minutes at RT. Cells, lymphocytes and natural killer 20 containing 100 ng/ml recombinant human IL-2 to increase the number of cells mL was resuspended in medium. Cells 5% (within 302) at 37° overnight incubated. The next day, cells were centrifuged at 1,400 rpm for 10 minutes at RT. and 2.5 x 10 7 for later use as effector cells in ADCC assay It was resuspended in Assay medium at cells/ml.

ADCC determination After labeling target cells with PKH-26 and coating with antibodies, 100 pl effector cell or 100 µl Detection medium (control, target cell only) directly 50 ul was added to target cells. Samples were placed in 5% CO2 at 37°C for an additional 3 hours. incubated. Samples were prepared with 10 µl of 10 µM To-Pro- Transferred to 1.2 ml microtiter FACS tubes containing 3 viability dyes (TP-3) and samples were analyzed by FACS. In an FSC vs. SSC drawing, all but the residual cells were gated. Passed cells were analyzed on FSC against FL-2 and FL- 2 positive cells (ie PKH-26 labeled target cells) were passaged. FACS data, FlowJo software (Tree Star, Inc. v6.3.4).

Specific ADCC, mean TP3+And 'Target Only, % of (A), mean TP3+Ve, respectively After subtracting % of 'Targets+Effectors' (D), the corresponding samples' Calculated by subtracting the mean TP3+And from % of 'Targets+Effectors' (B); well: Specific ADCC = (B - D) - (A - C) or = (B - A) - (D - C) Equation 1 The results presented in Table 2B are monocyte-depleted, IL-depleted as the effector cell population. 2-stimulated human PMBCls, but including B-cells, T-cells and dendritic cells obtained using predominantly NK cells. target cells, anti- The CD20 antibody allows both rituximab to be used as a positive control. The hC5aR was a transfected cell line Ramos E2 that expressed both CD20. Results, For an Fc variant that induces ADCC with equal potency with Rituximab, “+++” is a high “+/-” indicative for an Fc variant in which the degree of donor variation is observed, and as an indicator of Fc variant in which no notable induction of ADCC was detected. was included as a positive control for

CDC determination Fc variants were also analyzed for their CDC inducing strength. Experimental The setup was essentially ADCC, except that the effector cells were replaced with human serum. was the same as described for the determination. cells/ml) contain an equal amount of 3% Tavsan Complement Serum. in a 96-well U-bottom tissue culture plate with 2x antibody solution (200 µg/ml). mixed up. Two of the eyes, 25 to 2x antibody in medium without complement he did. 3-well batch 25 µl Ramos E2 cells (2 x 10 6 cells/ml) plus 3% Rabbit It contained 25 µl of assay medium ('zero Ab` samples) in Complement Serum. 3 eyes another batch 25 µl of Ramos E2 cells (2 x 10 6 cells/ml) plus 25 without complement pl contained assay medium ('zero Ab' samples). Before incubation, as appropriate With or without 3% Rabbit Complement Serum, 100 µl assay media per each added to the eye. Samples were incubated in 5% 002% at 37°C for 3 hours.

Determination of target cell viability Fluorisir viability dye To-Pr from analysis by flow cytometry added to each sample just before. Final test of To-Pr0-3 in each tube concentration ~ cells, non-viable or Flow cytometry & data analysis Samples were analyzed on a FACSCaIbur (BD Biosciences) and the data acquired Analyzed using FlowJo software (v6.3.4, TreeStar Inc.). SSC vs. FSC in the diffuser plot, gating in all cells except residual, 5,000 for each sample target cell event was collected. Gated target cells in the FL-4 channel created a histogram showing the level of To-Pr0-3 uptake. Each the number of TP3+ cells (ie non-viable cells) in the sample was determined - these were identified as cells to the right of the main peak - and this number expressed as a percentage of the total target cell. Samples assigned in triplicate can be found in the Category overview below. can be classified into one of 4 categories as A, B, C and D as shown.

Category overview Target cells were incubated with: 100 pglml Ab Zero Ab Zero Complement A B Rabbit complement serum C D Categories of analyzed samples For each sample containing the antibody, the reactions were performed with or without complement. carried out without Samples containing antibody without complement provided a specific level of background lysis. Samples in categories B & D, either antibody or non-specific background lysis in the absence of both antibody and complement provided.

Percentage of TP3+ non-viable target cells (% of Isis). Each containing 3% complement calculated for one sample and for each uncomplemented sample. Each antibody and To calculate the specific CDC activity for each antibody, complement In the absence of antibody-specific Iizis (“A, mean % Iizis in their samples), non-specific Before removing the background lysis, the complemented Ab sample ('Ci/zis' samples) were subtracted from Iizis %. Non-specific background trace, uncomplemented *zero Less than lysis in 'Ab' samples (mean % Iizis in 'B' samples) Iizi in complemented 'zero Ab' samples (mean % lysis in 'D' samples). included as positive control.

Specific CDC (% of Iizis = (C- A) (D - B) [or = (C - D) - (A - B)] Equation 2 Determining whether the differences between any of the groups are significant Statistical analysis was performed in the GraphPad Prism (v4.0) program. your 4 appointments The specific CDC activity of each sample from the whole is relative to the antibody used. entered on a separate page. Groups were compared using a parametric test: variance Tukey's multiple comparison post test after one-way analysis (ANOVA).

The results are given in Table ZBy. The results equate CDC with the IgG1 8254W mutant. “+++” indicative for a vigor-inducing FC variant, a high donor “+/-” indicative for an FC variant where the degree of variation is observed, and no as an indicator of Fc variant in which no notable induction of CDC was detected. as it changes.

N297Q_L234A_L235E - ++ - L234A_L235E + +++ - Antibody Fc region N297Q lgG1 reference 8239D_I332E 8254W N297Q N297Q V234A_6236A N297Q_F234L_L235A E233P_F234V_L235A N297Q L235A F234L_L235A Phagocytosis Table 28. Activity of FC variants in cell-based effector function assays.

Obtained from analysis of Fc variants in phagocytosis, ADCC and CDC assays. summary of results. (- = zero effector function; + = effector function). (1) CH1 and An IgG2/IgG4 FC variant containing the bottom hinge region of IgG2 and the remaining CH2- CH3; (2) to the IgG4 Fc region to abolish the formation of semi-antibodies added 8228P mutation. Example 5: Characterization of strength of anti-hC5aR antibody Fc variants Mutations in the FC region result in antibodies binding to hCSaR and hCSa, respectively. To test whether it affects the potency of inhibiting hC5a-mediated neutrophil migration For this purpose, different Fc variants were used in the displacement and migration assays described above. tested. Neutrophil Migration Assay from hC5aR-KO/KI mice of PMNs used It was performed as described above, except with PMNs. cells water was obtained in sequence. Bone marrow PMNs were obtained from two hCSaR-KO/KI mice in the femur and isolated from their tibia. Marrow cells from bones, cell suspension from a Cell removed and centrifuged (10 min, 1600 rpm) using PBS before filtering.

Cells were resuspended in medium and carefully inserted into a sterile 15 ml tube. layered on 3 ml of FicoII-Paque PLUS (GE Healthcare). chamber at 600X g After centrifugation at temperature for 20 minutes, the neutrophil/erythrocyte pellet is isolated.

Erythrocytes, Lyse Buffer (Sigma, R7757; 10 mM Tris-HCl pH 7.5) for 1 minute It is lysed using 8.3g/L ammonium chloride in it. Two rounds of centrifugation and washing The cell pellet is then resuspended in the Reaction Mix. Suspension, Contain >95% neutrophils as assessed by microscopy of Giemsa-stained samples he did. Variable region of tested antibodies, Ref. To the variable region of Ab Q was identical. Data are provided in Table 3. A significant difference in the inhibitory potency of hC5a binding to hC5aR* observed for Fc variants in the analysis (Table 3, column 1). Ref. Ab Q is an IgG1 version was analyzed with additional IgG Fc variants and data were analyzed for IgG1 Fc variants. variants generally bind to hCSa, both IgG4 and IgG2/IgG4 Fc. showed that it inhibited more strongly than its variants. Ref. One of Ab Q The F(ab')2 fragment was also included in the analysis and binds hC5a to full length Ref. Ab Q (IgG4) was found to inhibit at the same level. These findings indicate that the hinge region, demonstrated that antibodies are important for the ability to inhibit hC5a binding, and this notion supports neutrophil migration of F(ab')2 fragments Ref. Inhibits at the same level as Ab O supported by the fact that he could (Table 3). In addition, IgG1 variants are associated with neutrophil than IgGs containing IgG2 or !984 hinge regions. was found to be strong (Table 3, column 2).

Ref. Higher potency of IgG1 versions of Ab than IgG4 versions, IgG This may be related to increased avidity due to increased flexibility of the hinge region. this much to review, Ref. Binding of IgG1 and IgG4 versions of Ab to human neutrophils analyzed by FACS. Data are from IgG1 version to neutrophils, IgG4 version to neutrophils. revealed that it binds with higher avidity. Data are not shown.

Taken together, these findings suggest that increased flexibility in the IgG1 hinge regions, contributes to increased binding to hC5aR resulting in increased potency supports.

The antibody Fc region of human neutrophils involved in hCSa inhibition of displacement migration (SPA) Ref. as F(ab)2. Ab Q++++ Table 3. Binding of Fc variants to hCSa (SPA) and neutrophil migration towards hC5a effect on it. (+ = low activity, ++= medium activity, +++/+ high). Example 6. Generation of “fully” human anti-hCSaR antibodies and land characterization. From the analyzes described in Example 2, the 32F3A6 antibody was selected for further studies. This During recombinant cloning of the antibody, the human germline in the VH framework region Seven mutations were identified that differed from their sequences, while no frames were identified in the LC. mutation was not found (Figure 3). Mutations are present in all of the VH and VL sequences from 32F3A6l. found by alignment to existing human germline sequences.

To make the antibody more human-like, the VH region of 32F3A6 seven point mutations were back mutated into human germline remnants and Five have been shown to impair phagocytosis, ADCC, and CDC as described above. was noble. The compound is expressed as 32F3A6 GL.

The strength of the back-mutated antibody was compared to the specific antibodies and 32F3A6 or 32F3A6 GL, inhibiting hCSa binding to hCSaR (in SPA determined) or the potency to inhibit hC5a-mediated neutrophil migration. no difference observed (data not shown).

The fully human antibodies described above do not prevent neutrophil phagocytosis, ADCC, or The ability to induce CDC was evaluated as described in example 4, and the results were in Table 1. It is summarized in 4.

The results for specific ADCC in Table 4 include monocytes as effector cells. obtained using depleted human PBMCs and human neutrophils as target cells. Compound Phagocytosis Specific ADCC Specific CDC PBS as control - ND - variable region FC region 3G12 (unlinked antigen) lgG1 - + - 32F3A6 GL IgG1AEASS - + - 32F3A6 GL lgG1 + +++ ND Sigma lgG4 ND + ND Table 4. Fc-mediated cellular effector functions of anti-CSaR antibodies. "-" none represents detectable effector function and ”+” to “+++” are described in example 4. low to high levels of effector functions as measured in assays represents. ND (Not determined). As previously observed, the 5-point mutation in the IgGllin Fc region is wild-type IgG1 disrupts phagocytosis, ADCC and CDC compared to the Fc region. Example 7. Further characterization of human anti-hCSaR antibody (32F To further clarify the functionality of the identified antibodies and with affinity using one of the anti-CSaR antibodies compared to Ref Ab Q to determine potency. Additional assignments were made. Affinity for competition ligand binding on human neutrophils determined by appointment. This functionality, as measured by the competitive ligand binding assay. expressed as the affinity of the antibody, but also the avidity of the interaction. It can also be considered as a measurement. Ex-vivo assays show antibodies to C5a at in-vitro settings. measures the ability to neutralize mediated actions. Strength determinations on human neutrophils, CSa-induced Ca-flux, CD11b receptor upregulation and CD26L, respectively. Measures down regulation. The data obtained for the 32F3A6GL are given in Table 5.

Afini measurements Isolation of neutrophils from fresh human blood Blood was diluted 1:1 with PBS + 2% FBS and 3 parts Ficoll and 4 parts blood (a 50 ml tube 15 ml of Ficoll and 20 ml of blood) in Ficoll-Paque PLUS (GE Healthcare #17- stratified by centrifugation. The intermediate PBMC band was gently removed by aspiration.

Granulocytes layered on packed red cells a plastic Pasteur was aspirated with a pipette. Granulocytes and red cells were transferred to a new 50 ml tube and pelleted. The pellet was diluted to 40 ml with 1x PBS and 10 ml of 4% DEXTRAN 500 in PBS (sigma, 31392) solution (ratio 1:5) was added and mixed gently by inversion. 20- The granulocyte-rich filtrate obtained after 1 minute was transferred to a new tube. and inverted at 250 x 9 for 5 min at RT. contaminating red cells, by resuspending the cell pellet in 7.5 ml of 02% NaCl and for 55-60 seconds It was removed by osmotic lysis with gentle mixing for a period of time. Subsequently, 17.5 ml of 12% NaCl was added and then diluted to 50 ml with PBS and run down at 250 x 9 for 5 minutes. translated. This step was repeated once. Cell pellets followed by 1 ml reaction resuspended in the mixture (dPBS/RPMI). Viability and cell count.

Monitored using NucleoCounter®.

Determination of competitive ligand binding on neutrophils.

Human neutrophils were purified, washed, and bound in binding buffer (50 x 106 cells/ml). mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2 and 0.5% bovine serum albumin (FractionV was resuspended in lg G-free. 40 µl for each sample cell suspension (1 x 10 5 cells/well) in a 96-well V-shaped plate (Greiner, Cat# 651101) was planted in. Competition studies, with 1 pM as the highest concentration Competition unlabeled Iigandin 12 concentration in half-log dilutions starting from was made using 40 µl of antibody was added considering a final assay volume of 120 µl. 40 pl radioligand [, excluding background control added to all samples. The final concentration of the radioligand in the assay was 1 nM and the final volume was 120 µL. All samples were run in triplicate and at 4°C for 4 hours. incubated. Cells were then incubated at 1200 rpm for 2 minutes at 4°C. collected by centrifugation and three times 100 µl wash buffer (50 mM HEPES; pH 7.5, 1mM CaClz, 5 mM MgClz, 150 mM NaCl and 0.5% bovine serum albumin (FractionV lg G not include)) washed in. Finally, cells were resuspended in 30 µl wash buffer. pl MicroScint 20 (Perkin Elmer, Cat. No. 6013621) was added. The plates were covered, thoroughly mixed and counted with a calibrated Top Counter with a delay of 1 hour. designation The total amount of added radioligand was determined in a separate plate. in each sample The number of counts was expressed as percent normalized values and was 1 nM [125I]- 100% max count with hC5a added and no cold antibody added level, and 0.1% is non-specific binding determined in the presence of 1 µM cold hC5a.

Data were analyzed by nonlinear regression using PRISM (GraphPad).

Calcium-flow determination Staining of human neutrophils with Fluo-4 AM cell dye Neutrophils were centrifuged and washed in PBS, then Cell at 1 x 10 7 cells/ml It was resuspended in its dye and left in the dark at room temperature for 40 minutes. incubated. Cells were centrifuged and washed (to remove excess dye), re-centrifuged and resuspended in Cell Buffer at 2 x 10 6 cells/ml was done. Cells (0.5 ml) - one tube per sample - non-sterile glass It was segmented into FACS tubes, stored at room temperature, and within two hours. used. Each sample used 1 X 106 neutrophils.

Calcium flow determination was performed as follows. Briefly, in 0.5 ml Cell Buffer 1 x 106 neutrophils loaded with FIuo-4 AM, x-axis FSC vs. y-axis SSC A FACSCaIibur flow cytometry (BD) using neutrophils gated using Biosciences) was analyzed. The FL-1 (FITC) channel allows various reagents to be injected into the tube. (eg, antibodies, C5a, ionomycin - 10x final rather than 1-MBG or C-MGB concentration dissolved in Cell Buffer) followed by neutrophil was used to measure fluorisimia. Sample fluorescence continuously. every 1 It was measured by a mean fluorescence density (MFI) value obtained every second. This data was recorded in a CellQuest (BD Biosciences) file and was used for further processing and analysis. Exported to Excel (Microsoft) and Prism (v4.00, GraphPad Software Inc.).

The order of addition of markers to neutrophils and incubation times, assay performed changed according to the type.

C5a neutralization determination serial dilution was prepared. FIu0-4 AM loaded neutrophils (1 x in 0.5 ml Cell Buffer) ug/ml) was incubated for 10 minutes at room temperature. Cells plus antibody, reference line was analyzed by FACS for ~60 seconds to establish fluorescence. More 50 µl of 10 nM C5a was then added to give a final concentration of ~1 nM and florisima measurement continued for another ~60 seconds. Antibody CSa-induced Jazz*- If it blocked its release, there was no florisima spayki. If the antibody does not neutralize C5a, this In one case, there was a spike in Florida. Finally, a final concentration of 0.1 ug/ml 50 µl of tug/ml ionomycin was added and the cells were still responsive. Fluorescence measurement continued for another ~60 seconds to ensure that

CD11b receptor upregulation Assignment setup The following setup describes CSa-induced neutrophil activation of the identified antibodies. neutralizing ability by measuring changes in CD11b expression designed to determine.

Anti-CSaR and isotype control antibody at a 3-fold serial dilution (from 600”) in PBS dispersed in the eyes of the bottom plates. A 50 µl aliquot of fully heparinized canine into each well added. A set of four control eyes (in pairs) contained only 50 µl of PBS plus 50 µl of blood. he did. Plates were incubated for 20 minutes at 37°C in a 5% CO 2 incubator.

50 µl human C5a to activate neutrophils, 10 or 100 nM final as indicated concentration, to wells containing Ab and a group of antibody-free control eyes. added. PBS (50 µl) was added to a second set of antibody-free control eyes. 5 ug/ml final concentration of phorbol myristate acetate (PMA) antibody-free third control eye added to the group. Plates were again placed in a 5% CO 2 incubator at 37°C for 20 minutes. incubated. Finally, 50 µl diluted 1/50 in PBS (final concentration Except for group 4 of 2 control wells without C5a or PMA - these samples were baseline MFl values) were added to all wells. Plates were again kept for 20 minutes. It was incubated in a 5% CO 2 incubator at 37°C and then for 3 minutes. It was centrifuged at 2,000 rpm to pellet the blood cells. The filtrate (150 pl) was removed and pellets Isolation of red blood cells in 200 pl 1x FACS Lysis Solution resuspended for. Plates again for 5 minutes at room temperature. resuspended in solution. Cells for analysis by flow cytometry transferred to microtiter tubes.

FACS and data analysis FACSCalibur flow cytometry (BD Biosciences) generated for FL-2 channels adjusted with balancing parameters. Samples, excluding dead cells and residues gated to be released. Neutrophils were defined as having high FSC and SSC and passed. Mean fluorescence of gated neutrophils in the FL-2 (CD11b-PE) channel density (MFI) was calculated.

The results are a percentage of maximum CD11b expression with background subtracted. expressed as. Maximum expression of CD11b (MaxCD11b) incubated with C5a The mean MFI of neutrophils with no Ab was obtained. Minimum (background) CD11b expression (MinC11b) incubated without CSa and Ab was the mean MFI of neutrophils. Maximum CD11b expression for each sample The data were entered into the GraphPad Prism (v4.0) program and used to calculate the EC50. s-shaped dose-response curve (variable slope) using non-linear regression that is, it was fitted to the 4-parameter logistic equation.

CD62L reregulator down edit Assignment setup The following setup describes CSa-induced neutrophil activation of the identified antibodies. determination of neutralization ability by measuring changes from CD62L expression was designed for. adapted for the detection of CD62L using an antibody. Experimental specific to CD62L details are given below.

FACS and data analysis FACSCalibur flow cytometry (BD Biosciences) generated for FL-4 channels adjusted with balancing parameters. Samples, excluding dead cells and residues gated to be released. Neutrophils were defined as having high FSC and SSC and passed. The mean of gated neutrophils in the FL-4 (CD62L-APC) channel fluorescence density (MFI) was calculated.

The results are a percentage of maximum CD62L expression with background subtracted. expressed as. Maximum expression of CD62L (MaxCD62L) without C5a and Ab was the mean MF1 of neutrophils without Minimal (background) CD62L expression (MinC62L) was the mean MFI of neutrophils incubated with C5a but without Ab.

To calculate % of maximum CD62L expression for each sample The formula used is as follows: UI:: Man,... ...5. = lliilFI.;_.,-;,.:. - IlilFIr...) ijMFl`ti - MFIU..) x 100 The data was entered into the GraphPad Prism (v4.0) program and used to calculate the ECSO. s-shaped dose-response curve (variable slope) using non-linear regression that is, it was fitted to the 4-parameter logistic equation.

The results from the above determinations are summarized in Table 5 below.

Ref. Ab 0.84 nM 7.3 ug/ml 3.6 tig/ml 1.9 ug/ml Table 5. Affinity determination, Calcium-flow assay, CD11b and CD62L determination data obtained.

The data show that 32F3A6 GL inhibits the action of C5a in a dose-dependent manner. has confirmed. Inhibition on neutrophil Ca2+ release increased 32F3A6 GL Ref. from Ab Q increased with greater effectiveness.

Similarly, 32F3A6 GL also in the CD11b and CD62L regulation assay, Ref. EU Exhibiting a strength 4-5 times higher than Q, Ref. More effective than Ab Q. Further testing in 32F Determination. a 1.0 ug/ml It showed dose dependence with lC50. Example 8. In vivo mouse model of arthritis As a result, GPI (auto-antigen glucose 6-phosphate isomerase) is mediated by circulating Ab. develops an autoimmune-like disease. Serum from arthritic K/BXN mice, of human RA, including chronic progressive disease with joint destruction induces disease in other strains of mice with many of the hallmarks.

animals Human CSaR KO/K1 transgenic mice (C57BL/6; H-2b; human C5aR+/+/mouse CSaR-/-; sus abbreviation: H5Rtg) from 8 to 27 weeks.

K/BxN serum To generate serum for experiments, KRth male mice were paired with non-NOD mice. crossed. F1 offspring carrying the KRN transgene, developing inflamed joints (8-10 weekly) was killed and blood was drawn by cardiac puncture. After 2 hours of incubation at 37°C and Serum was collected after centrifugation at 4,000 rpm for 1 minute. More than one serum from mice was pooled, segmented and stored at -80°C. same for all mice K/BxN serum was injected from the batch.

Arthritis injury and wnlamg An inflammatory arthritis in recipient H5Rtg mice, 150 µl of K/BXN on both days 0 and 2. serum i.p. induced by injection. Disease progression paw daily size of the fore and hind paws and wrist joints monitored by determining a clinical score based on the degree of inflammation. Average from day 0 The change in claw size was calculated in the figure below. Each of the hind paws The thickness (in mm) of one wrist was measured daily using a caliper.

Average daily paw of one or two readings from each of the hindpaws was the size (PS). Average claw size on day 0, claw for each day of experiment average daily claw to provide average change in size (APS) subtracted from size. A clinical score for each paw of each mouse is in Table 6. calculated based on the scoring system shown. score from 4 claws, to provide the total clinical score (CS) for each mouse on each day of the experiment. gathered.

Score View 0 normal joints 1 mild/moderate swelling of the wrist and/or one swollen finger 2 swollen wrists or two or more swollen fingers Score View Severe swelling along all directions of 3 paws or all five toes Table 6. Arthritis clinical scoring system To determine which mice entered the treatment phase on day 5, one for each mouse was required. calculated by multiplying. Overall only mice with an RA score of >0.7 entered the treatment phase of the study. Therapeutic treatment with 3_2F3A6 GL After disease onset (day 0), KO/KI hCSaR mice are 32F3A6 at day 5 A loading dose of GL was given followed by 9 daily doses. Loading doses 10, clinical scores (mean +/- SD) are shown in Figure 4. treatment with NNC0215-0384, dose-dependent in inflammation compared to mice treated with an unrelated control antibody provided a reduction. A similar effect is based on changes in average claw size. observed (not shown). Example 9. C5a expression level in patients with psoriatic arthritis C5a, synovial from 11 Psoriatic Arthritis and 12 Osteoarthritis patients as controls measured in liquid samples. A commercial 053 ELISA kit (BD OptEIATM, Human C5a ELISA Kit II (BD Biosciences; cat. No. 557965» protocol followed. Data Figure Site are given and summarized in Table 7 below. C5a level psoriatic arthritis increased significantly in the group of patients (p = 0.001; Mann-Whitney), which It shows that C5a is a factor of synovial inflammation in psoriatic arthritis.

Controls (Osteoarthritis Psoriatic arthritis patients) Table 7. Synovial fluid C5a from control and psoriatic arthritis patients detection levels. Example 10. C5aR expression in synovium from Psoriatic Arthritis patients Formalin-fixed and paraffin-embedded synovial specimens from PsA patients (n=9) tissue microarray (TMA) containing biopsies and within normal nerves (n=5) Slides are available from Biochain Institute Inc./BioCat GmbH, Heidelberg, Germany. was done. A sample of PSA was obtained from Dr. Bliddal (Frederiksberg Hospital, Denmark) and Dr.

Provided in cooperation with Sße (Gentofte Hospital, Denmark). All human materials with the informed consent of donors/close relatives and relevant local ethics committees. BioCat Ge, personal communication; Cambridge BioSciences, Supplier information: Tissue Supply Obtained with the approval of Network (www.bioscience.co.uk). Doctors The sample from BIIddal/Soe was obtained under ethical clearance H-4-2009-117.

The following antibodies were used: Mouse monoclonal anti-human C5aR (R&D Systems, MAB3648 clone . Mouse IgG2a isotype-specific control (Dako, X0943, clone Immunohistochemistry was performed as follows. Paraffin of sections in xylene removed and rehydrated for increasing alcohol concentrations. Antigen recovery Tris- In EGTA buffer (10mM; pH 9.0, 15 min in a microwave oven period was carried out. Endogenous peroxidase activity was blocked with 3% H2O2 and endogenous biotin, according to the manufacturer's instructions, for 10 minutes, respectively, Avidin and It was blocked by incubation with biotin. Non-specific attachment, 3% skim milk, 7% donkey 30 minutes with TBS containing serum, 3% human serum and blocked by incubation. Primary and secondary antibodies, 0.5% skim milk, 7% donkey and in a Tris buffer containing 3% human serum and incubation, were carried out at 4°C overnight and at room temperature for 60 minutes, respectively.

First strengthening step, 0.1 M containing % 30 with Vectastain ABC peroxidase kit diluted in Tris-HCl buffer (pH 7.5) minutes and then biotinylated for 6 minutes. A second step of reinforcement was performed with tyramide. Final retrofit, described above An additional 30 minutes with the Vectastatin ABC peroxidase kit diluted as done by incubation. The chromogenic reaction was obtained with diaminobenzidine. Nuclei were counterstained with hematoxylin and sections were rehydrated in xylene. cleared and merged with Eukitt. C5aR in RA, OA and normal synovium of TMAs Evaluation for protein expression was performed in an observer-blind manner.

An Olympus BX51 equipped with a DP7O digital camera for the evaluation of sections microscope (Olympus Denmark A/S; Ballerup, Denmark) was used.

Results CöaR-immunopositive cells were found in the synovial subset of 8 out of 10 patients with psoriatic arthritis. lymph in the lining layer and stroma of 10 of 10 patients with psoriatic arthritis aggregates were found to be mixed with each other. Controls do not have any of these synovial compartments. It did not exhibit C5aR staining (0/5). CSaR-immunopositive synoviocytes of 5 controls In lining layer cells in 4 and 10 out of 10 patients with psoriatic arthritis detected. The results are summarized in Table 8 below.

Synovial compartment Normal Psoriatic arthritis patients In lymphoid aggregates in synovial sub-lining tissue 0/5 8/10 Infiltrating C5aR+ cells C5aR+ cells in the stroma 0/5 10/10 CSaR+ synoviocytes in the primer layer 4/5 10/10 Table 8. CSaR+ cells in normal synovium and from patients with psoriatic arthritis detection in the taken synovium. Those with psoriatic arthritis compared to normal synovium P-value for difference between C5aR expression in patients (Fischer's exact test): 0.007 (lymphoid aggregates) and 0.0003 (stroma). Example 11. Induced by synovial fluid from patients with psoriatic arthritis inhibition of neutrophil migration by anti-C5aR.

Determination of Neutrophil G_ranulocyte Remover (Chemotgxis) Antibodies are found in human neutrophil granulocytes (human PMNs (PolyMorphoNuclear analyzed in a Boyden chamber assay using add-on systems.

Human PMNs are obtained from human blood samples drawn into vials containing EDTA. obtained. Blood cells canine (4 parts) a FicoII-Paque PLUS (GE Health Care) over a gradient (3 parts) for 30 minutes (400 x 9) at room temperature separated by centrifugation. PMN-containing layer, containing dextran-5OO (Sigma) Destroy contaminating erythrocytes for 1 hour in PBS (phosphate buffered saline) was suspended for The filtrate was at room temperature for 5 minutes (250 x 9). centrifuged and the remaining erythrocytes osmotically in 0.2% NaCl for 55 seconds. Iized using . The solution was made isotonic with 1.2% NaCl + PBS and osmotic It was centrifuged at 250 x increments for 5 minutes before the lysis was repeated. without centrifugation The PMNs were then resuspended in the reaction mixture (RM): HBSS (cat no. Complete with HEPES 20mM. Cell density with NucleoCounter (Chemometec) determined. PMN suspension by microscopy of Giemsa-stained samples contained >95% neutrophils as evaluated.

Loading PMNs: Calcein, AM, (Fluka) dissolved in DMSO (Dimethyl sulfoxide) and in RM containing (2X106 cells per ml) to provide a concentration of 10 µM 1000X diluted. The suspension was incubated at 37°C in an incubator for 30 minutes and then washed 3 times with RM to remove excess Calcein. Finally, cells It was resuspended in RM (4x10 6 cells/ml).

Human synovial fluid (SF) was obtained by puncturing the knee from 2 psoriatic arthritis patients.

After the cells were destroyed by centrifugation, the samples were frozen at -80°C and was stored. For migration experiments, the samples were thawed and RM containing 0.2% EDTA. was diluted 2X using

Migration, FluoroBlok® 3um pore size evaluated using the Boyden chamber technique. Upper chamber, aka Fluoroblock membrane-containing chambers in 1 mg/ml PBS at 37°C for 2 hours. coated with fibrinogen (cat no F3879-1 G, Sigma). Membranes after washing, PBS It was blocked with a solution containing 2% bovine serum albumin (BSA) in it. RM After another wash using 105 Calcein-loaded PMNs were added with or without antibodies and control solution or To the recipient plate containing the chemoattractant (hCSa (Sigma or synovial fluid samples)) (lower chamber) is placed. Each group consisted of 4-6 eyes. Quantification of cell migration, sub obtained by measuring the fluorisimia of cells in the well. FIuoroBlock membrane Since it effectively blocks the passage of light at 490-700 nm, it enters the lower chamber. Fluorescence from wells that did not penetrate is not detected at 485/530 nm. Plate 60 minutes every 5 minutes at 37°C at excitation/emission wavelengths of 485/538nm, in a florisima plate reader with sub-reading capability (SpectraMax, Molecular devices or Fluoroscan, Thermo Labsystems) were read.

Migration is measured by fluorescence values at 60 minutes, expressed as relative fluorescence values. was appointed. Table food, Migration is set to 100% in the presence of Isotype antibody and anti-C5aR The ability of the antibody to inhibit migration is calculated. Migration is evident in the hCSaR antibody. reduced by. Migration by 10 nM hCSa was 83% inhibited. three SF The values for the sample were: 15%, 70% and 48%. The results show that C5aR-antibody, revealed that SF taken from psoriatic arthritis patients inhibited the chemoattractive effect. has put. hC5a (10 nM SF sample SF sample SF sample CSa) donor 1 donor 1 donor 1 anode Anti-CSaR 17 85 30 52 Table 9. PMNs to hCSa or synovial fluid from three psoriatic arthritis patients its migration in response and its inhibition by the hC5aR antibody (Ref antibody Q).

All values were normalized to migration detected when incubated with the isotype antibody. Example 12. Intestine from patients with Crohn's Disease and ulcerative colitis Expressing CSaR Normal limits (n=14) from patients with ulcerative colitis (n=21) and Crohn's disease (n=25) intestinal tissue samples in Cambridge Bioscience (Cambridge, UK) obtained from the company. All human material from donors or close relatives with the informed consent of the relevant local ethics committees, BioCat Ge, personal communication; Cambridge BioSciences, Supplier information: Tissue Supply Network (www. bioscience. co.uk) was obtained with the approval of institutions. Antibodies used and immunohistochemistry the protocol is as described in Example 9.

semi-quantitative scoring C5aR immunopositive (CSaR*) cells were scored semi-quantitatively as follows: Mucosa-associated Ienfoid compartments scored individually: Mucosa (M): intraepithelial (FAE). Submucosa (SM): isolated (single) lymph follicles (ILF), Peyer's patches (ileum)/colonic infiltrative lymphocytes. Each split 0-4: 0, none; 1, several; 2, medium; 3, many and 4, plenty scored on a scale with the number of C5aR+ cells. An accumulated score per for the intestinal layer (M, SM, ME) and for the whole intestine in total (M+SM+ME) calculated. Maximum score: M=12, SM=12, ME=8 and 32 for the whole bowel. C5aR semi-quantitative scoring of immunohistochemical data for protein expression, With Dunn's multiple comparison post-test in GraphPad Prism 5 with KruskaI-Wallis test analyzed. P<0.05 was evaluated as significant.

Results C5aR positive neutrophils and myeloid-like cells, CD" in 23 of 25 patients with UC Intraepithelial lymphocytes in 19 of 21 patients and 7 of 14 normal bowel samples compartment, in the follicle-associated epithelium and as solitary cells in the lamina of the mucosa propria (P-values (Fischer complete test), 0.005 and 0.015, respectively). Additional Peyer patches/colonic-lymph compared to the sample of normal bowel in the follicles; as solitary cells in isolated lymphatic follicles and submucosa found (P-values (Fischer exact test), 0.03 and not significant, respectively). Finally, C5aR positive cells were obtained from CD and UC patients, as well as from normal intestine. found in the infiltrative muscularis externa. The results are presented in Figure and Table. It is summarized in 10. Based on semi-quantitative analysis, C5aR Compared to the normal intestine in its wall, for example, the three intestinal layers (mucosa, lower mucosa and muscularis externa) accumulated score, CD (P<0.01) and UC Significantly higher expression in the gut from patients with (P<0.05) was found.

CSaR expression in Diagnosis Mucosa Submucosa Muscularis Externa Normal 7/14 7/14 8/14 Table 10. Patients with Crohn's disease and ulcerative colitis compared to normal bowel Summary of C5aR expression in the ingested gut.

SERIES LIST <110> Novo Nordisk A/S <120> THERAPEUTIC ANTIBODIES <160> 41 <170> Patented version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 1 <210> 2 <211> 16 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 2 Vai With Trp Phe Asp Wear With Asn Lys Tyr Tyr Gly Asp Ser Vai Lys 1 5 10 15 <210>3 <211>13 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 3 Thr Ty: Phe Gly Pro Gly Thr Thr Glu Phe Phe Gln His <210>4 <211> 122 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 4 <210>5 <211> 11 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 5 Arg Ala Ser Gln Set Val Ser Ser Tyr Leu Ala <210> 6 <211> 7 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 6 <210> 7 <211> 8 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 7 Gln Gln Arg Ser Asn Trp Pro Thr <210> 8 <211> 107 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 8 <210>9 <211> 5 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 9 <210> 10 <211> 15 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 10 Ala Ile Asp Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Vai Lys <210> 11 <211> 16 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 11 Asp Tyr Tyr Tyr Tyr Ala Set GLy Ser Ty: Tyr Lys Ala Phe With Asp 1 5 10 15 <210> 12 <211> 124 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 12 Giu Val Gln Leu Vai Gin Sex Wear Gly Wear Leu Val His Pro Wear Gly 1S 10 15 Ser Leu Arq Leu Ser Cys Ala GLy Ser Giy Phe Thr Phe Ser Ser Tyr 25 30 Val Hat Hi: Trp Val Arg Gln Ala Pro Gly Lys Gly Liu Glu Trp Val 40 45 Set Aia 11e Asp Thr Gly Gly Gly Thr Ty: Tyr Ala Asp Ser vai Lys 50 55 60 Gly Arg Phe Thr With Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 HP Gin Met Asn Ser Lan Arg Ala GLu Asp Met Ala Vai Ty: Tyr Cys A1& 85 90 95 Arg Asp Tyr Tyr Tyr Ty: Wear Aia Sex Ser Ty: Ty: Lys Ala Phe Asp 100 105 110 115 120 <210> 13 <211> 12 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 13 Arg Ala Ser Gin Ser Val Ser Ser Arg Ty: Len Ala <210> 14 <211> 7 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 14 <210> 15 <211> 8 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 15 Gln Gln Tyr Gly Ser Pro Leu Thr <210> 16 <211> 108 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 16 Glu Ile Vai Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly <210> 17 <211> 5 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 17 <210> 18 <211> 16 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 18 Val Iie Trp Ty:: Wear Asp With Ann Lys Ty: Tyr Ala Asp Ser Vai Lya <210> 19 <211> 13 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 19 Thr Tyr Tyr Thr Ser Giy Ser Ser Lys His Phe Gin Pro <210> 20 <211> 122 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 20 <210> 21 <211> 11 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 21 Arg Ala Ser Gln Se: Val Ser Ser Tyr Leu Ser <210> 22 <211> 7 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 22 <210> 23 <211> 8 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 23 (fln Gln Arg Ser 151511 Trp Pro Thr <210> 24 <211> 107 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 24 Sin Arg Ala Thr LouLou Ser Trp Ty: GlnTyr Asp Ala Ser: AsnSer Gly Ser Gly ThrGlu Asp Phe Ala vaiphe Gly Pro Gly Thr <210> 25 <211> 5 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 25 <210> 26 <211> 15 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 26 Ala Phe Ser Ser Asp Gly Ty: Thr Phe Ty: Pro Asp Ser Leu Lys 1 5 10 IS <210> 27 <211> 12 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 27 His Ala Asp Tyr Ala Asu Ty: Pro Val Met Asp Tyr <210> 28 <211> 120 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 28 Glu Val Lya Leu Vai Glu Se: Gly GLy Gly Leu Ser Lou Lys Lan Ser Cys So: Ala Ser Gly Phe Asp Mit Sir Trp Vai Arg Gln Thr Pro Glu Lys Ala Aia Pbe Ser Ser Asp Wear Ty: Thr Phe Ty: Gly Arg ?he Thr With Ser Arg Asp Asn Ala Arg 65 70 75 Gin Hat Ser Ser Leu Giy Ser Giu Asp Thr Ala Arg Ris Ala Asp Tyr Aia Asn Ty: Pro Vai Met 100 105 Gly Thr Ser Val Thr Val Ser Ser ii5 120 <210> 29 <211> 11 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 29 Arg Ala Ser Gln Wear With Ser Ser Trp Leu Ala <210> 30 <211> 7 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 30 <210> 31 <211> 9 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 31 Gln Gln Tyr Asn Ser Tyr Pro Arg Thr <210> 32 <211> 108 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 32 <210> 33 <211> 330 <212> PRT <213> Homo sapiens <400> 33 <210> 34 <211> 326 011.: <212> PRT <213> Homo Sapiens <400> 34 Ala Ser TR): <210> 35 <21 1 > 326 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 35 Ala Se:: Thz <210> 36 <211> 327 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 36 225 230 305 310 <210> 37 <211> 120 <212> PRT <213> Homo sapiens <400> 37 <210> 38 <211> 105 <212> PRT <213> Homo Sapiens <400> 38 <210> 39 <211> 124 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 39 Sar Ldu Arq Lûu Ser Cys Ala Ala Wear Wear Wear Leu Vai Gin Pro Wear Wear Ser Gly Ph & Thr Ph. Ser Sor Tyr Ala Thr Gly Lys Gly Leu Glu Trp Val <210> 40 <211> 108 <212> PRT <213> Artificial Array <220> <223> Synthetic array <400> 40 <210> 41 <211> 350 <212> PRT <213> Homo sapiens <400> 41

Claims (1)

CLAIM An antibody that binds to the second extracellular loop of the human CSaR, characterized in that the heavy chain of said antibody contains a CDR1, a CDR2, and a CDR3 sequence, wherein said CDR sequences are a CDR1, a CDR2, and a CDR3 variable region of the SEO chain. wherein said CDR sequences include SEQ ID 13, 14 and name sequences, wherein the variable region of the heavy chain of said antibody contains a sequence that is at least 90% identical to SEQ ID NO: 12, wherein the variable region of the light chain of said antibody Contains a sequence that is at least 90% identical to SEQ ID NO: 16, where the antibody has an Fc hinge region of the IgG1 isotype. The antibody of claim 1, wherein the variable region of the heavy chain of said antibody contains a sequence that is at least 94% identical to SEQ ID NO: 12, wherein the variable region of the light chain of said antibody is at least %9 to SEQ ID NO: 16. It contains 94 identical sequences. An antibody according to any one of the preceding claims, characterized in that i. the variable region of the heavy chain of said antibody contains a sequence that is at least 96, 97, 98, or 99 percent identical to SEQ ID NO: 12; and ii. the light chain of said antibody contains a sequence that is at least 96, 97, 98, or 99 percent identical to SEQ ID NO: 16. The antibody according to claim 3, characterized by i. the variable region of the heavy chain of said antibody contains the sequence identified by SEQ ID NO: 12; and ii. wherein the light chain of said antibody contains the sequence defined by SEQ ID NO: 16. An antibody according to claim 1 or 2, characterized by i. the variable region of the heavy chain of said antibody contains a sequence that is at least 96, 97, 98, or 99 percent identical to SEQ ID NO: 39; and ii. the light chain of said antibody contains a sequence that is at least 96, 97, 98, or 99 percent identical to SEQ ID NO: 40. The antibody according to claim 5, characterized by i. the variable region of the heavy chain of said antibody contains the sequence defined by SEQ ID NO: 39; and ii. wherein the light chain of said antibody contains the sequence defined by SEQ ID NO: 40. An antibody according to any preceding claim, characterized in that the affinity of the antibody is less than 0.80 nM as measured by the competitive ligand binding assay on neutrophils. An antibody according to any preceding claim, characterized in that the antibody significantly inhibits or reduces C5a binding to CSaR at less than 10 nM in a Scintillation Proximity Assay (SPA). . An antibody according to any preceding claim, characterized in that the antibody significantly inhibits in vitro migration of Human neutrophils. An antibody according to any preceding claim, characterized in that the antibody reduces migration in the presence of 10 nM CSa and zero antibody to less than 20% compared to the observed migration level. An antibody according to any preceding claim, characterized in that the antibody has an FC region of the IgG1 isotype. The antibody of any preceding claim, characterized in that the FC region has reduced binding affinity to one or more Fcγ receptors compared to the IgGi reference sequences as defined by SEQ ID NO 33. An antibody according to any preceding claim, characterized in that the antibody does not significantly induce ADCC, CDC and/or phagocytosis of neutrophils in vitro. The antibody according to any preceding claim, characterized in that the Fc region is an IgG1 FC mutant containing 1 to 5 amino acid substitutions from AA 328 to 334, wherein the IgG1 Fc reference sequence is as defined in SEQ ID NO 33. The antibody of any preceding claim, characterized in that the Fc region has 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. The antibody according to any of the preceding claims N297Q, L234A and L235E and/or wherein the FC region is IgG1 (SEQ ID NO: 33) with the following point mutations: The antibody according to any one of the preceding claims, characterized in that the antibody is a human antibody. An antibody according to any one of the preceding claims, characterized in that it is for medical use. An antibody according to any preceding claim, characterized 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.