Antibodies targeting C5aR

Abstract

To provide novel antibodies or antibody fragments specifically binding to human C5aR.SOLUTION: The present invention relates to antibodies or antibody fragments that have combined beneficial properties and are therefore useful for the treatment of inflammatory or autoimmune diseases or cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to C5aR and, in particular, antibodies that interact with human C5aR. This disclosure also relates to nucleic acid compositions, vector compositions and host cells capable of expressing the antibodies, pharmaceutical compositions comprising the antibodies, and the use of the antibodies for the treatment and / or diagnostic purposes of specific diseases. [Background technology]

[0002] C5a anaphylatoxin chemotactic receptor 1 (C5aR) (also known as CD88) is a G protein coupling receptor (GPCR) belonging to the rhodopsin family. It is produced in serum as one of the core effector components of the complement response and is one of two high-affinity receptors for its ligand, C5a. Under physiological conditions, C5a acts as a chemotactic on inflammatory cells, stimulating their respiratory burst, cytokine and chemokine release, and vascular permeability.

[0003] C5aR appears to be widely expressed by various cell types. The maximum C5aR expression level has been described for neutrophils. Low to moderate expression levels have been shown for macrophages / monocytes, dendritic cells, mast cells, eosinophils, pulmonary vascular smooth muscle cells, astrocytes, microglia, osteoblasts, osteoclasts, epithelial and endothelial cells (Non-Patent Literature 1, Non-Patent Literature 2). Low expression levels have been observed in human T cells (Non-Patent Literature 3).

[0004] The cellular response to C5a is tightly regulated by ligand-induced receptor internalization. C5aRs have been described as rapidly and dose-dependently internalizing upon C5a treatment, with up to 90% of the receptor being recirculated back to the cell surface. Therefore, high levels of C5aR expression, coupled with their rapid turnover rate, may be limiting in terms of efficiency due to targeted drug elimination (TMDD) effects.

[0005] The interaction between C5a and C5aR has been described in many different disease settings; most of these are involved in inflammatory and autoimmune diseases ((Non-Patent Document 4), (Non-Patent Document 5)). Several preliminary studies have been conducted to identify the underlying mechanisms by which C5a stimulates tumor growth ((Non-Patent Document 6), (Non-Patent Document 7), (Non-Patent Document 8)). Most of the data suggest that C5a increases cancer cell proliferation, intratumoral angiogenesis, and promotes tumor invasiveness and metastasis. More recent data also suggest a role for C5a / C5aR in creating an immunosuppressive environment in solid tumor settings ((Non-Patent Document 9), (Non-Patent Document 10), (Non-Patent Document 11)), resulting in the promotion of primary tumor growth by inhibiting the antitumor response (e.g., increased recruitment of C5aR-expressing myeloid cells such as myeloid-derived suppressor cells (MDSCs) or M2 macrophages). Based on these findings, combination strategies with known antitumor agents, such as immune checkpoint protein inhibitors, to promote the target immune response by reducing the immunosuppressive microenvironment are a focus of research and clinical development (Non-Patent Literature 12).

[0006] To date, only one specific complement therapy has been approved, targeting the upstream molecule of C5a, namely C5. The humanized anti-C5 mAb eculizumab can bind to C5 and prevent its cleavage and formation of C5a and C5b proteins, and subsequent MAC formation. Various other therapeutic monoclonal C5-specific antibodies, such as ALXN1210 or LFG316, are under clinical evaluation (Non-Patent Literature 5). However, since the increased risk of infection is a major concern associated with chronic C5 treatment, specifically targeting downstream molecules while preventing the biological activity of other complement components is clearly advantageous. Therefore, the development of many antagonistic C5a-specific monoclonal antibodies is underway.

[0007] Direct targeting of C5aRs offers several advantages over targeting C5 or C5a, respectively. Firstly, inhibition of the receptor alone preserves MAC activity, thereby reducing the potential risk of infection. Secondly, C5aR blockade allows for continued interaction with C5a through its secondary receptor, C5L2. Since C5L2 has been reported to have anti-inflammatory effects, maintaining an effective C5L2 signaling pathway may result in improved efficacy or reduced administration requirements. Thirdly, direct C5aR targeting may offer pharmacodynamic advantages over soluble C5a inhibition due to its low molecular weight and high turnover rate. Overall, there is strong interest in the development of C5aR inhibitors, including aptamers, peptides, and non-peptide small molecules, which are being tested in preclinical and clinical trials.

[0008] Neutralizing polyclonal antisera or monoclonal antibodies targeting the N-terminal extracellular region of human C5aR and capable of interfering with C5aR-C5a interactions have been described in the Art (e.g., Non-Patent Document 13, Non-Patent Document 14).

[0009] However, these C5aR-specific antibodies are not suitable for clinical development and therapeutic use in humans, particularly because they are animal-derived (which would make them immunogenic in human patients), clonal, and / or lack cross-reactivity to related animal species.

[0010] Therapeutic antibodies targeting C5aR are being investigated for clinical development. However, clinical development of the antagonist C5aR-specific antibody neutrazumab, a humanized IgG4 mAb, was discontinued in Phase II clinical trials due to problems involving immune cell depletion and immunogenicity (Non-Patent Literature 15). Since C5aR is thought to be constitutively expressed in various cell types, it is important that antagonist antibodies do not induce any depletion of target cells.

[0011] To overcome the shortcomings of neutrazumab, a second generation of C5aR-specific antibodies, namely NNC0215-0384 ((Patent Document 1) (NOVO NORDISK); clone 32F3A6GL), was developed. This antibody is a human IgG1 antibody derived from transgenic mice and is currently undergoing clinical development in the oncology field as IPH5401 (Non-Patent Document 16). IPH5401 possesses a suppressed human IgG1 Fc region to eliminate the antibody's effector function induction ability. This antibody is referred to herein as RefMAB#1. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent Application Publication No. 2013 / 0295116 [Non-patent literature]

[0013] [Non-Patent Document 1] Monk PN et al.,Br J Pharmacol.2007,152:429-448 [Non-Patent Document 2] Wetsel RA, Immunol Lett.1995,44:183-187 [Non-Patent Document 3] Nataf S,J Immunol.1999,162:4018-4023 [Non-Patent Document 4] Morgan BP et al.,Nat Rev Drug Discov.2015,14:857-877 [Non-Patent Document 5] Hawksworth OA et al.,Mol Immunol.2017,89:36-43 [Non-Patent Document 6] Markiewski MM et al.,Nat Immunol.2008,9:1225-1235 [Non-Patent Document 7] Corrales L.et al.,J Immunol.2012,189:4674-4683

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0014] This disclosure provides novel antibodies and antibody fragments. [Means for solving the problem]

[0015] The antibodies and antibody fragments disclosed herein can specifically bind to human C5aR and preferably cross-react with cynomolgus monkey-derived C5aR. Therefore, in some embodiments, the disclosed antibodies are specific to both human and cynomolgus monkey C5aR. In some other embodiments, the disclosed antibodies or antibody fragments bind to the N-terminal extracellular regions of human and cynomolgus monkey C5aR.

[0016] This is in contrast to the prior art antibody IPH5401, referenced above, which binds to the second extracellular loop of human C5aR and consequently lacks binding to cynomolgus monkey C5aR, a species for which appropriate toxicology is commonly used.

[0017] Furthermore, the inventors of this invention have surprisingly found that the C5aR-specific antibody claimed in this invention is significantly more potent than IPH5401 in neutralizing pathophysiological C5a concentrations, and has also demonstrated increased efficacy over the long term in inhibiting C5-mediated activation of neutrophils in vitro.

[0018] Therefore, in some embodiments, the disclosed antibodies can efficiently inhibit C5a-inducible C5aR activity in vitro, particularly at pathophysiological C5a concentrations. The disclosed antibodies or antibody fragments can also inhibit C5a-inducible leukocyte activation in vitro, as determined by their ability to inhibit the C5a-inducible upregulation of CD11b in granulocytes and / or monocytes. In some embodiments, the disclosed antibodies or antibody fragments inhibit 42 nM IC in vitro in the presence of 150 nM human C5a. 50At certain concentrations, the antibody inhibits human C5a-induced CD11b expression in human granulocytes. In some other embodiments, the disclosed antibody may exhibit increased efficacy in inhibiting C5a-induced upregulation of CD11b in granulocytes and / or monocytes after prolonged incubation. The disclosed antibody may also be effective in inhibiting C5a-induced neutrophil migration.

[0019] In summary, this disclosure provides a novel antibody that is superior to known C5aR-specific antibodies from the art. In particular, the antibody of this disclosure is a human antibody that has high affinity binding to human C5aR, which preferably cross-reacts with cynomolgus monkey C5aR and has desirable functional and safety properties that have not been observed previously. These properties make the antibody of this disclosure highly desirable for therapeutic applications, such as for the prevention and / or treatment of inflammatory and autoimmune diseases and cancer.

[0020] This disclosure provides isolated antibodies or antibody fragments that specifically bind to human C5aR, having a CDR region as shown in Table 1 or Table 2 of this specification. This disclosure also provides isolated antibodies or antibody fragments specific to human C5aR, having a variable heavy chain region (VH) and a variable light chain region (VL) containing amino acid sequences as shown in Table 1 or Table 2 of this specification. This disclosure also provides isolated antibodies or antibody fragments specific to C5aR, having a heavy chain (HC) and a light chain (LC) containing amino acid sequences as shown in Table 1 or Table 2 of this specification.

[0021] The isolated antibodies of this disclosure do not substantially induce effector function in vitro. Such effector function may include ADCP, ADCC, or CDC. Furthermore, the isolated antibodies or antibody fragments of this disclosure contain one or more amino acid substitutions selected from the group L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index. In particular, the isolated antibodies or antibody fragments of this disclosure contain a variant human IgG1 Fc region, which includes the following amino acid substitutions: L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index.

[0022] This disclosure also provides isolated antibodies or antibody fragments of this disclosure for use in pharmaceuticals.

[0023] The disclosure also provides a method for treating a subject suffering from an inflammatory or autoimmune disease or cancer, by administering an effective amount of the antibody or antibody fragment of the disclosure to the subject. Preferably, the subject is a human.

[0024] The Disclosure also provides a pharmaceutical composition comprising an isolated antibody or antibody fragment of the Disclosure and a pharmaceutically acceptable carrier.

[0025] The Disclosure also provides nucleic acid compositions encoding the isolated antibody or antibody fragment of the Disclosure. The Disclosure also provides vector compositions comprising the nucleic acid composition encoding the isolated antibody or antibody fragment of the Disclosure. The Disclosure also provides host cells comprising the vector composition or nucleic acid composition encoding the isolated antibody or antibody fragment of the Disclosure.

[0026] The disclosure also provides a method for treating subjects suffering from inflammatory diseases, autoimmune diseases, or cancer by administering an effective amount of the isolated antibody or antibody fragment of the disclosure to subjects suffering from such diseases, preferably human subjects.

[0027] The Disclosure also provides a pharmaceutical composition comprising an isolated antibody or antibody fragment of the Disclosure and a pharmaceutically acceptable carrier.

[0028] The claimed antibody or antibody fragment is useful. Furthermore, the claimed method for identifying such antibody or antibody fragment is useful.

[0029] The claimed use of the antibody or antibody fragment alters the biological activity of human C5aR. In particular, the claimed antibody or antibody fragment is intended for therapeutic uses, such as the treatment of inflammatory or autoimmune diseases or cancer. [Brief explanation of the drawing]

[0030] [Figure 1] Cellular binding of MAB#1, MAB#2, RefMAB#1, and negative isotype control MOR03207 to human and cynomolgus monkey C5aR determined via FACS. A. Dose-response binding to human C5aR overexpressed on Flp-In CHO cells. B. Dose-response binding to cynomolgus monkey C5aR overexpressed on Flp-In CHO cells. C. Average dose-response binding to purified human neutrophils obtained from whole blood of three different donors. D. Average dose-response binding to purified cynomolgus monkey neutrophils obtained from whole blood of three different monkeys. [Figure 2] Cell binding of MAB#1, MAB#2, RefMAB#1, and negative isotype control MOR03207 to human, cynomolgus monkey, and rodent C5aR, as well as to C5aR-related GPCRs human C5L2, human C3aR, human FPR1, and human ChemR23, as determined by FACS at an IgG concentration of 600 nM. [Figure 3] ELISA conjugation of MAB#1 to two native variants of human C5aR, as well as to mouse C5aR expressed on virus-like particles (VLPs). [Figure 4] PathHunter®-β-arrestin assay from DiscoveRx. Neutralization of human C5a-inducible β-arrestin recruitment. A. Log dose-response curves for incremental concentrations of recombinant human C5a in the absence or presence of MAB#1 (50 nM), MAB#2 (50 nM), and RefMAB#1 (50 nM). B. % inhibition was calculated for three incremental concentrations of human C5a (1.2 nM, 11 nM, and 100 nM, respectively) at a final IgG concentration of 50 nM. [Figure 5]Inhibition of human C5a-induced CD11b upregulation in human granulocytes at typical and pathophysiological C5a concentrations. Comparison of MAB#1, MAB#2, and RefMAB#1 in CD11b whole blood assays A+B. Log dose-response curves are shown. Human granulocytes were gated as target cells. As a quantitative reading, the IgG concentration required to achieve 50% inhibition of CD11b upregulation was calculated (IC50 concentration). IC50 concentrations for each IgG are shown below the x-axis. A Log dose-response curve for IgG escalation and 15 nM human C5a. B Log dose-response curve for IgG escalation and 150 nM human C5a. [Figure 6] Inhibition of human C5a-induced CD11b upregulation in human granulocytes and monocytes, determined over extended incubation periods. Comparison of MAB#1 and RefMAB#1 in CD11b whole blood assays after IgG incubation with target cells for either 20 minutes or 300 minutes. IgG was added in serial dilutions and incubated for either 20 minutes or 300 minutes, followed by stimulation with 15 nM human C5a. Either granulocytes (Figure 6A and B) or monocytes (Figure 6C and D) were gated as target cells. CD11b levels were determined. Log dose-inhibition curves are shown. Data are expressed as % inhibition of CD11b upregulation with 15 nM human C5a. Results for MAB#1 are shown in Figures 6A and C, and results for RefMAB#1 are provided in Figures 6B and D. [Figure 7] Inhibition of human C5a-induced human neutrophil migration. MAB#1 and the negative control MOR03207 were tested at two IgG concentrations (100 nM and 600 nM, respectively) in the presence of 10 nM human C5a. Mean values ​​from three independent assay runs at three different time points (15 min, 25 min, and 35 min) are shown. Neutrophils were obtained from three different human donors. Percentage inhibition was calculated based on neutrophil migration in the absence of the antibody. [Figure 8]Promega ADCC and ADCP reporter bioassays. Comparison of MAB#1 and monoclonal anti-C5aR control IgG possessing either the wild-type (unrepressed) human IgG1 Fc region or a mutant (repressed) Fc region identical to the MAB#1 Fc region. Furthermore, isotype control antibody MOR03207 possessing either the mutant or wild-type human IgG1 Fc region was included. Assays were performed according to supplier instructions using modified Jurkat cells expressing FcγRIIa_H to mimic the ADCP pathway, or Jurkat cells expressing FcγRIIIa, a V158 high-affinity mutant to mimic the ADCC pathway, and C5aR-expressing CHO cells. A. Results from the ADCP reporter bioassay at an IgG concentration of 10 μg / ml. B. Results from the ADCC reporter bioassay at an IgG concentration of 10 μg / ml. Data are provided as mean fluorescence signal relative to background. [Figure 9] Group-mean pharmacokinetic profiles of MAB#1 in Han Wistar rats after a single intravenous administration of 10 mg / kg IgG. Data are provided as mean (IgG concentration over time) ± standard deviation (S) (n=3). [Figure 10] Protein panel profiling (3P) results for MAB#1 and MAB#2. The number for each antibody represents the binding signal obtained for each antibody and the tested protein compared to the binding of the isotype-negative control antibody MOR03207. Antibodies were tested at concentrations of 10 nM and 100 nM, respectively. [Figure 11] Cytokine release by monocyte-derived in vitro mature M1 and M2 macrophages. After treatment with MAB#1 and overnight incubation with C5a, IL-10 and IL-12 levels were determined by ELISA. [Modes for carrying out the invention]

[0031] This disclosure relates to a number of human antibodies that recognize human C5aR.

[0032] definition The term "C5aR" refers to the protein known as C5a anaphylatoxin chemotactic receptor 1 or CD88.

[0033] Human C5aR (Uniprot:P21730|1-350) (referred to herein as the "D / K variant") is, MDSFNYTTPDYGHYDDKDTLDLNTPVDKTSNTLRVPDILALVIFAVVFLVGVLGNALVVWVTAFEAKRTINAIWFLNLAVADFLSCLALPILFTSIVQHHHWPFGGAACSILPSLILLNMYASILLLATISADRFLLVFKPIWCQNFRGAGLAWIACAVAWGLALLLTIPSFLYRVVREEYFPPKVLCGVDYSHDKRRERAVAIVRLVLGFLWPLLTLTICYTFILLRTWSRRATRSTKTLKVVVAVVASFFIFWLPYQVTGIMMSFLEPSSPTFLLLKKLDSLCVSFAYINCCINPIIYVVAGQGFQGRLRKSLPSLLRNVLTEESVVRESKSFTRSTVDTMAQKTQAV(Sequence ID 1) It has the following amino acid sequence.

[0034] Two native missense mutations in human C5aR have been described ((http: / / www.uniprot.org / uniprot / P21730): refer SNP (refSNP) cluster report: rs4467185 (MAF: 0.03) and cluster report: rs11880097 (MAF: 0.03). One mutation is located within the N-terminal extracellular region of human C5aR (position 2 of SEQ ID NO: 1), resulting in a D-to-N substitution.

[0035] The human C5aR protein containing both native missense mutations in its sequence (also referred to herein as the “N / N variant”) MNSFNYTTPDYGHYDDKDTLDLNTPVDKTSNTLRVPDILALVIFAVVFLVGVLGNALVVWVTAFEAKRTINAIWFLNLAVADFLSCLALPILFTSIVQHHHWPFGGAACSILPSLILLNMYASILLLATISADRFLLVFKPIWCQNFRGAGLAWIACAVAWGLALLLTIPSFLYRVVREEYFPPKVLCGVDYSHDKRRERAVAIVRLVLGFLWPLLTLTICYTFILLRTWSRRATRSTKTLKVVVAVVASFFIFWLPYQVTGIMMSFLEPSSPTFLLLNKLDSLCVSFAYINCCINPIIYVVAGQGFQGRLRKSLPSLLRNVLTEESVVRESKSFTRSTVDTMAQKTQAV(Sequence ID 2) It has the following amino acid sequence.

[0036] Cynomolgus macaque (Macaca fascicularis) C5aR is, MDPFSSTTLDYEHYDGKNVLDSDTPVDKTSNTLRVPDILALVVFAVVFLVGVLGNALVVWVTAFEVKRTINAIWFLNLAVADFLSCLALPILFTSIVQHHHWPFGGTACRILPSLILLNMYASILLLATISADRFLLVFNPIWCQNFRGAGLAWIACAVAWGLALLLTIPSFLYRAVRQEEYSPKVLCGVDYNNDTRRERAVAIVRLVLGFLWPLLTLMICYTFLLLRTWSRRATRSTKTLKVVVAVVASFFIFWLPYQVTGTMMSFLRPSSPTYLQLKKLDSLSISFAYINCCINPVIYVVAGQGFQGRLRKSLPSLLRNVLTEESVVRESKSFTRSTVDTMTEKTQAV (Sequence ID 3) It has the following amino acid sequence.

[0037] The mouse (Mus musculus) C5aR is, MDPIDNSSFEINYDHYGTMAPNIPADGIHLPKRQPGDVAALIIYSVVFLVGVPGNALVVWVTAFEARRAVNAIWFLNLAVADLLSCLALPVLFTTVLNHNYWYFDATACIVLPSLILLNMYASILLLATISADRFLLVFKPIWCQKVRGTGLAWMACGVAWVLALLLTIPSFVYREAYKDFYSEHTVCGINYGGGSFPKEKAVAILRLMVGFVLPLLTLNICYTFLLLRTWSRKATRSTKTLKVVMAVVICFFIFWLPYQVTGVMIAWLPPSSPTLKRVEKLNSLCVSLAYINCCVNPIIYVMAGQGFHGRLLRSLPSIIRNALSEDSVGRDSKTFTPSTTDTSTRKSQAV(Sequence ID 4) It has the following amino acid sequence.

[0038] Rat (Rattus norvegicus) C5aR is, MDPISNDSSEITYDYSDGTPNPDMPADGVYIPKMEPGDIAALIIYLAVFLVGVTGNALVVWVTAFEAKRTVNAIWFLNLAVADLLSCLALPILFTSIVKHNHWPFGDQACIVLPSLILLNMYSSILLLATISADRFLLVFKPIWCQKFRRPGLAWMACGVTWVLALLLTIPSFVFRRIHKDPYSDSILCNIDYSKGPFFIEKAIAILRLMVGFVLPLLTLNICYTFLLIRTWSRKATRSTKTLKVVMAVVTCFFVFWLPYQVTGVILAWLPRSSSTFQSVERLNSLCVSLAYINCCVNPIIYVMAGQGFHGRLRRSLPSIIRNVLSEDSLGRDSKSFTRSTMDTSTQKSQAV(Sequence ID 5) It has the following amino acid sequence.

[0039] The term "C5a" refers to a protein known as human complement component C5a.

[0040] Human C5a (Uniprot:P01031|678-751) is, TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCVVASQLRANISHKDMQLGR(Sequence ID 6) It has the following amino acid sequence.

[0041] The term "C5L2" refers to a protein known as C5a anaphylatoxin chemotactic receptor 2.

[0042] Human C5L2 is, MGNDSVSYEYGDYSDLSDRPVDCLDGACLAIDPLRVAPLPLYAAIFLVGVPGNAMVAWVAGKVARRRVGATWLLHLAVADLLCCLSLPILAVPIARGGHWPYGAVGCRALPSIILLTMYASVLLLAALSADLCFLALGPAWWSTVQRACGVQVACGAAWTLALLLTVPSAIYRRLHQEHFPARLQCVVDYGGSSSTENAVTAIRFLFGFLGPLVAVASCHSALLCWAARRCRPLGTAIVVGFFVCWAPYHLLGLVLTVAAPNSALLARALRAEPLIVGLALAHSCLNPMLFLYFGRAQLRRSLPAACHWALRESQGQDESVDSKKSTSHDLVSEMEV (Sequence ID 7) It has the following amino acid sequence.

[0043] The term "C3aR" refers to a protein known as the C3a anaphylatoxin chemotactic receptor.

[0044] Human C3aR is, MASFSAETNSTDLLSQPWNEPPVILSMVILSLTFLLGLPGNGLVLWVAGLKMQRTVNTIWFLHLTLADLLCCLSLPFSLAHLALQGQWPYGRFLCKLIPSIIVLNMFASVFLLTAISLDRCL VVFKPIWCQNHRNVGMACSICGCIWVVACVMCIPVFVYREIFTTDNHNRCGYKFGLSSSLDYPDFYGDPLENRSLENIVQPPGEMNDRLDPSSFQTNDHPWTVPTVFQPQTFQRPSADSLPR GSARLTSQNLYSNVFKPADVVSPKIPSGFPIEDHETSPLDNSDAFLSTHLKLFPSASSNSFYESELPQGFQDYYNLGQFTDDDQVPTPLVAITITRLVVGFLLPSVIMIACYSFIVFRMQRGRFAKSQSKTFRVAVVVVAVFLVCWTPYHIFGVLSLLTDPETPLGKTLMSWDHVCIALASANSCFNPFLYALLGKDFRKKARQSIQGILEAAFSEELTRSTHCPSNNVISERNSTTV(Sequence ID 8) It has the following amino acid sequence.

[0045] The term "FPR1" refers to a protein known as the fMet-Leu-Phe receptor.

[0046] Human FPR1 is, METNSSLPTNISGGTPAVSAGYLFLDIITYLVFAVTFVLGVLGNGLVIWVAGFRMTHTVTTISYLNLAVADFCFTSTLPFFMVRKAMGGHWPFGWFLCKFLFTIVDINLFGSVFLIALIALDRCVCVLHPVWTQNHRTVSLAKKVIIGPWVMALLLTLPVIIRVTTVPGKTGTVACTFNFSPWTNDPKERINVAVAMLTVRGIIRFIIGFSAPMSIVAVSYGLIATKIHKQGLIKSSPPLRVLSFVAAAFFLCWSPYQVVALIATVRIRELLQGMYKEIGIAVDVTSALAFFNSCLNPMLYVFMGQDFRERLIHALPASLERALTEDSTQTSDTATNSTLPSAEVALQAK (Sequence ID 9) It has the following amino acid sequence.

[0047] The term "ChemR23" refers to a protein known as chemokine-like receptor 1.

[0048] Human ChemR23 is, MRMEDEDYNTSISYGDEYPDYLDSIVVLEDLSPLEARVTRIFLVVVYSIVCFLGILGNGLVIIIATFKMKKTVNMVWFLNLAVADFLFNVFLPIHITYAAMDYHWVFGTAMCKISNFLLIHNMFTSVFLLTIISSDRCISVLLPVWSQNHRSVRLAYMACMVIWVLAFFLSSPSLVFRDTANLHGKISCFNNFSLSTPGSSSWPTHSQMDPVGYSRHMVVTVTRFLCGFLVPVLIITACYLTIVCKLHRNRLAKTKKPFKIIVTIIITFFLCWCPYHTLNLLELHHTAMPGSVFSLGLPLATALAIANSCMNPILYVFMGQDFKKFKVALFSRLVNALSEDTGHSSYPSHRSFTKMSSMNERTSMNERETGML(Sequence ID 10) It has the following amino acid sequence.

[0049] The term "antibody," as used herein, refers to a protein that interacts with an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further classified into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. The antibody may be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0050] When used herein, the term “antibody fragment” refers to one or more portions of an antibody that possess the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of the antibody; dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and separate complementarity-determining regions (CDRs). Furthermore, although two of the Fv fragment's domains, VL and VH, are encoded by separate genes, they can be linked together using recombinant methods via synthetic linkers that allow the VL and VH regions to pair to form a monovalent single-chain protein (known as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also encompassed within the term "antibody fragment." These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same way as intact antibodies. Antibody fragments can also be incorporated into monodomain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be implanted into polypeptide-based scaffolds such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide monobodies).Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that form an antigen-binding site pair together with a complementary light chain polypeptide (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0051] When used herein, "human antibody" or "human antibody fragment" refers to an antibody and antibody fragment having a variable region in which both the framework and CDR region are derived from human sequences. Human antibodies may also be derived from synthetic libraries or transgenic mice (e.g., Xenomouse), provided that the individual systems produce antibodies having a variable region in which both the framework and CDR region are derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a sequence. Examples of human origins include antibodies containing human germline sequences or mutant versions of human germline sequences, or consensus framework sequences derived from human framework sequence analysis, such as those described in Knappik et al., (2000) J Mol Biol 296:57-86).

[0052] The structure and location of immunoglobulin variable domains, such as CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J.Mol.Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 USD Department of Health and Human Services; Chothia et al., (1987) J.Mol.Bio. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al. See al., (1997) J.Mol.Biol.273:927-948.

[0053] In this specification, "humanized antibody" or "humanized antibody fragment" is defined as an antibody molecule having a constant antibody region derived from a human sequence, with only the variable antibody region or a portion thereof, or the CDR, being of another species origin. For example, a humanized antibody may have a CDR implanted, where the CDR of the variable domain is of non-human origin, while one or more frameworks of the variable domain are of human origin, and the constant domain (if any) is of human origin.

[0054] The terms “chimeric antibody” or “chimeric antibody fragment” are defined herein as an antibody molecule having a constant antibody region derived from or corresponding to a sequence found in one species and a variable antibody region derived from another species. Preferably, the constant antibody region is derived from or corresponds to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR, or FR region) is derived from a sequence found in a non-human animal, such as a mouse, rat, rabbit, or hamster.

[0055] The term "isolated antibody" refers to an antibody or antibody fragment that substantially contains no other antibodies or antibody fragments having different antigen specificities. Furthermore, an isolated antibody or antibody fragment may not substantially contain other cellular material and / or chemical substances. Therefore, in some embodiments, the antibody provided is an isolated antibody, which has been separated from antibodies having different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may cross-reactive to other relevant antigens, such as those from other species (e.g., species homologs).

[0056] The term “recombinant antibody,” as used herein, includes all antibodies prepared, expressed, produced, or isolated by means not naturally occurring. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, produced, or isolated by any other means, including splicing all or part of human immunoglobulin genes, sequences against other DNA sequences, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes with respect to human immunoglobulin genes, or hybridomas prepared therefrom. Preferably, such recombinant antibodies have a variable region in which the framework and CDR region are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis if an animal transgenic to the human Ig sequence is used), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, while being sequences that do not naturally exist in vivo within the human antibody germline repertoire. The recombinant antibody may be a monoclonal antibody. In one embodiment, the antibodies and antibody fragments disclosed herein are isolated from a HuCAL library (Rothe et al., J.Mol.Biol.(2008)376,1182-1200).

[0057] When used herein, such an antibody is able to distinguish such an antigen from one or more reference antigens. Binding specificity is a relative, not absolute, property; therefore, when an antibody "binds specifically to" or "specifically binds to" an antigen, such as human C5aR, it is either "specific to" or "specifically recognizes" it. For example, a standard ELISA assay may be performed. Scoring may be performed by standard color development (e.g., secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). This reaction is scored in a certain number of wells by optical density, e.g., 450 nm. A typical background (= negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. This means that the positive / negative difference can be more than 10 times. Generally, binding specificity is determined by using a series of about 3-5 unrelated antigens, such as powdered milk, BSA, transferrin, etc., rather than a single reference antigen.

[0058] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target at many sites through weak non-covalent forces; the more interactions there are, the stronger the affinity.

[0059] "K D Where used herein, the term "K" refers to the dissociation constant, which is K on k for off Obtained from the ratio (i.e., k off / k on ), expressed as molar concentration (M). For example, K for the antigen-binding site of a monoclonal antibody. D The value can be determined using methods well established in the art, such as the K of the antigen-binding moiety of a monoclonal antibody. DThe method for determination is SET (solution equilibrium titration) or surface plasmon resonance using a biosensor system such as the Biacore® system. In the present disclosure, an antibody specific to C5aR is generally less than 5×10 -2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10 -5 M, less than 5×10 -6 M, less than 10 -6 M, less than 5×10 -7 M, less than 10 -7 M, less than 5×10 -8 M, less than 10 -8 M, less than 5×10 -9 M, less than 10 -9 M, less than 5×10 -10 M, less than 10 -10 M, less than 5×10 -11 M, less than 10 -11 M, less than 5×10 -12 M, less than 10 -12 M, less than 5×0 -13 M, less than 10 -13 M, less than 5×10 -14 M, less than 10 -14 M, less than 5×10 -15 M or less than 10 -15 M or lower dissociation rate constant (K D )(k off / k on ).

[0060] The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or their antibody fragments or otherwise interacts with a molecule. Generally, an epitope is a chemically active surface group of a molecule such as an amino acid or a carbohydrate or a sugar side chain, and can generally have characteristics of a specific three-dimensional structure and specific charge characteristics. As will be recognized by those skilled in the art, anything to which an antibody specifically binds can be an epitope.

[0061] The “compositions” of this disclosure may be used for therapeutic or prophylactic applications. Accordingly, this disclosure includes pharmaceutical compositions comprising an antibody or antibody fragment as disclosed herein and a pharmaceutically acceptable carrier or excipient thereto. In relevant embodiments, this disclosure provides a method for treating cancer. Such a method includes the step of administering an effective amount of a pharmaceutical composition containing an antibody or antibody fragment as described herein to a subject in need thereof.

[0062] This disclosure provides a therapeutic method comprising administering a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject requiring such treatment. “Therapeutically effective amount” or “effective amount,” as used herein, refers to the amount of C5aR antibody required to induce a desired biological response. According to this disclosure, a therapeutically effective amount is the amount of C5aR antibody required to treat and / or prevent a disease.

[0063] "Administered" or "administration" includes, but is not limited to, the delivery of a drug in an injectable form, such as intravenous, intramuscular, intradermal, subcutaneous, or mucosal route, for example, as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule, or tablet. Preferably, administration is by an injectable form.

[0064] As used herein, “treatment,” “treat,” or “treating” refers to a clinical intervention in an attempt to alter the natural course of a disease in the subject being treated, and may be performed for prevention or in any of a series of clinical conditions. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation of the disease state, and improving or enhancing the prognosis. In some embodiments, antibodies or antibody fragments according to this disclosure are used to delay the onset of the disease or to slow the progression of the disease.

[0065] The term "effector function" refers to the biological activity that may be attributable to the Fc region of an antibody, which varies with antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0066] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to antigen-carrying target cells, subsequently killing the target cells cytotoxicly. NK cells, the main cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII.

[0067] "Complement-dependent cell injury" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component (C1q) of the complement system to the antibodies (of the appropriate subclass) of this disclosure, which are bound to their alloantigens.

[0068] "Antibody-dependent phagocytosis" or "ADCP" refers to the mechanism by which antibody-coated target cells are eliminated by internal migration of phagocytic cells, such as macrophages or dendritic cells.

[0069] "Preventing" or "prevention" refers to reducing the risk of acquiring or developing a disease (i.e., preventing at least one clinical symptom of a disease from developing in a person who may be exposed to a disease-causing substance or who is susceptible to developing the disease prior to its onset). "Prevention" also refers to methods aimed at preventing or delaying the onset of a disease or its symptoms.

[0070] When used in connection therewith, "subject" or "species" refers to rodents, such as mice or rats, and any mammals, including primates, such as crab-eating macaques (Macaca fascicularis), rhesus macaques (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0071] Throughout this specification, unless otherwise indicated by the context, the individual variations of “comprise,” “have,” and “include,” and “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are understood to indicate the inclusion of the element or integer, or group of elements or integers, being described, but not the exclusion of any other element or integer, or group of elements or integers.

[0072] The terms "engineered" or "modified," as used herein, include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or by some combination of these techniques). Preferably, antibodies or antibody fragments according to this disclosure are modified or engineered to improve one or more properties such as antigen binding, stability, half-life, effector function, immunogenicity, and safety.

[0073] As used herein, "mutant" refers to a polypeptide that differs from the reference polypeptide due to one or more modifications, such as amino acid substitutions, insertions, or deletions.

[0074] The term "amino acid mutation," as used herein, encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications is possible, as long as the final construct retains the desired characteristics, such as reduced binding to the Fc receptor. Deletions and insertions of amino acid sequences include N and / or C-terminal deletions and insertions of amino acid residues. A specific amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions by non-natural amino acids or by natural amino acid derivatives of 20 standard amino acids. Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic modification, such as chemical modifications, that alter the side chain groups of amino acid residues may also be useful. Various nomenclature may be used herein to indicate the same amino acid mutation. For example, a substitution of glycine to alanine at position 327 in the Fc region may be indicated as 237A, G337, G337A, or Gly329Ala.

[0075] "EC 50Where used herein, the term "baseline" refers to the concentration of an antibody, antibody fragment, or ligand that induces the response in the assay, midway between baseline and maximum. Thus, this corresponds to the antibody or ligand concentration at which 50% of the maximum effect is observed.

[0076] "I C 50 As used herein, the term "maximum response" refers to the concentration of an antibody or antibody fragment that inhibits the response in an assay, midway between the maximum response and the baseline. This corresponds to an antibody concentration at which the given response is reduced by 50%.

[0077] The terms "inhibition," "inhibit," "reduction," "reduce," "neutralization," and "neutralize" refer to a reduction or cessation of any phenotypic feature (such as binding, biological activity, or function) or a decrease or cessation of the incidence, degree, or possibility of such feature. "Inhibition," "reduction," or "neutralization" does not need to be complete, as long as it is detectable using an appropriate assay. In some embodiments, "reduce," "inhibit," or "neutralize" means the ability to cause a reduction of 20% or more. In other embodiments, "reduce," "inhibit," or "neutralize" means the ability to cause a reduction of 50% or more. In another embodiment, “reduce,” “inhibit,” or “neutralize” means the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or more.

[0078] The term "antagonist" antibody, as used herein, refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes any biological activity or function or any other phenotypic feature of the target antigen.

[0079] A "wild-type" protein is the version or variant of a protein that it is found in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of the protein as it occurs in nature. Due to allotypic differences, there can be multiple amino acid sequences for a wild-type protein. For example, there are several allotypes of the natural human IgG1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0080] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally contains two constant domains, the CH2 domain and the CH3 domain. Although the boundary of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is typically defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0081] Embodiments: In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 28, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or b) HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, HCDR2 region containing the amino acid sequence of SEQ ID NO: 39, HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0082] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) HCDR1 region of SEQ ID NO: 27, HCDR2 region of SEQ ID NO: 39, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0083] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 31, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or b) HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, HCDR2 region containing the amino acid sequence of SEQ ID NO: 41, HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0084] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0085] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 28, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or b) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 31, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 29, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or c) The HCDR1 region containing the amino acid sequence of SEQ ID NO: 27, the HCDR2 region containing the amino acid sequence of SEQ ID NO: 39, the HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, the LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, the LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34, or d) HCDR1 region containing the amino acid sequence of SEQ ID NO: 30, HCDR2 region containing the amino acid sequence of SEQ ID NO: 41, HCDR3 region containing the amino acid sequence of SEQ ID NO: 40, LCDR1 region containing the amino acid sequence of SEQ ID NO: 32, LCDR2 region containing the amino acid sequence of SEQ ID NO: 33, and LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0086] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0087] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

[0088] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

[0089] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

[0090] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34.

[0091] In one embodiment, the disclosure refers to a human C5aR-specific isolated antibody or antibody fragment containing six CDRs defined by one of the antibodies disclosed in Table 1 or Table 2.

[0092] In one embodiment, the Disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, comprising six CDRs defined by one of the antibodies disclosed in Table 1 or Table 2.

[0093] In one embodiment of this disclosure, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of this disclosure, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment. In another embodiment of this disclosure, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment of this disclosure, the isolated antibody or antibody fragment is an IgG isotype. In one embodiment of this disclosure, the isolated antibody or antibody fragment is of the IgG1 class. In another embodiment of this disclosure, the isolated antibody or antibody fragment does not substantially induce effector function in vitro.

[0094] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) Including the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further including VH of SEQ ID NO: 35 or VL of SEQ ID NO: 36, or b) Including the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further including VH of SEQ ID NO: 35 or VL of SEQ ID NO: 36, or c) comprising the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42 or VL of SEQ ID NO: 43, or d) comprising the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42 or VL of SEQ ID NO: 43.

[0095] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43 Includes.

[0096] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising SEQ ID NO: 35 VH and SEQ ID NO: 36 VL.

[0097] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment includes VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43.

[0098] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment includes the HC of SEQ ID NO: 37 and the LC of SEQ ID NO: 38.

[0099] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment includes the HC of SEQ ID NO: 44 and the LC of SEQ ID NO: 45.

[0100] In one embodiment, the disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, comprising a variable heavy chain (VH) and a variable light chain (VL) of any one of the antibodies disclosed in Table 1 or Table 2.

[0101] In one embodiment, the disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, comprising the heavy chain (HC) and light chain (LC) of any one of the antibodies disclosed in Table 1 or Table 2.

[0102] In one embodiment, the disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 35 or VL of SEQ ID NO: 36.

[0103] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42 or VL of SEQ ID NO: 43.

[0104] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 35 or VL of SEQ ID NO: 36.

[0105] In one embodiment, the disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, and further comprising VH of SEQ ID NO: 42 or VL of SEQ ID NO: 43.

[0106] In one embodiment of the Disclosure, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of the Disclosure, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment. In another embodiment of the Disclosure, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0107] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or SEQ ID NO: 42 and VL of SEQ ID NO: 36 or SEQ ID NO: 43 Includes.

[0108] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or VH and VL having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36.

[0109] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43.

[0110] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to HCs of SEQ ID NO: 37 or SEQ ID NO: 44 and LCs of SEQ ID NO: 38 or SEQ ID NO: 45 Includes.

[0111] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HC of SEQ ID NO: 37 and the LC of SEQ ID NO: 38, or an HC and LC having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to the HC of SEQ ID NO: 37 and the LC of SEQ ID NO: 38.

[0112] In one embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, the antibody or antibody fragment comprising the HC of SEQ ID NO: 44 and the LC of SEQ ID NO: 45, or the HC and LC having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to the HC of SEQ ID NO: 44 and the LC of SEQ ID NO: 45.

[0113] In one embodiment of this disclosure, the isolated antibody or antibody fragment is a monoclonal antibody or antibody fragment. In one embodiment of this disclosure, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment. In another embodiment of this disclosure, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human or humanized antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human antibody or antibody fragment.

[0114] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR according to the Disclosure is a recombinant or synthetic antibody or antibody fragment. In a further embodiment, the isolated antibody or antibody fragment specific to C5aR according to the Disclosure is an isolated recombinant monoclonal antibody or antibody fragment. In a further embodiment, the isolated antibody or antibody fragment specific to C5aR according to the Disclosure is an isolated recombinant monoclonal human antibody or antibody fragment.

[0115] In one embodiment, the isolated antibody or antibody fragment specific to C5aR according to this disclosure is an IgG isotype. In another embodiment, the isolated antibody or antibody fragment is of the IgG1 class. In yet another embodiment, the isolated antibody or antibody fragment is of the human IgG1 class.

[0116] nucleic acid In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody or antibody fragment is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0117] In another embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a heavy chain sequence and a light chain sequence of an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences are a) The HCDR1 region of sequence number 46, the HCDR2 region of sequence number 47, the HCDR3 region of sequence number 48, the LCDR1 region of sequence number 51, the LCDR2 region of sequence number 52, and the LCDR3 region of sequence number 53, or b) The HCDR1 region of SEQ ID NO: 49, the HCDR2 region of SEQ ID NO: 50, the HCDR3 region of SEQ ID NO: 48, the LCDR1 region of SEQ ID NO: 51, the LCDR2 region of SEQ ID NO: 52, and the LCDR3 region of SEQ ID NO: 53, or c) The HCDR1 region of SEQ ID NO: 58, the HCDR2 region of SEQ ID NO: 59, the HCDR3 region of SEQ ID NO: 60, the LCDR1 region of SEQ ID NO: 63, the LCDR2 region of SEQ ID NO: 64, and the LCDR3 region of SEQ ID NO: 65, or d) HCDR1 region of SEQ ID NO: 61, HCDR2 region of SEQ ID NO: 62, HCDR3 region of SEQ ID NO: 60, LCDR1 region of SEQ ID NO: 63, LCDR2 region of SEQ ID NO: 64, and LCDR3 region of SEQ ID NO: 65 Includes.

[0118] In another embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a heavy chain sequence and a light chain sequence of an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences are a) The HCDR1 region of sequence number 70, the HCDR2 region of sequence number 71, the HCDR3 region of sequence number 72, the LCDR1 region of sequence number 75, the LCDR2 region of sequence number 76, and the LCDR3 region of sequence number 77, or b) The HCDR1 region of sequence number 73, the HCDR2 region of sequence number 74, the HCDR3 region of sequence number 72, the LCDR1 region of sequence number 75, the LCDR2 region of sequence number 76, and the LCDR3 region of sequence number 77, or c) The HCDR1 region of SEQ ID NO: 82, the HCDR2 region of SEQ ID NO: 83, the HCDR3 region of SEQ ID NO: 84, the LCDR1 region of SEQ ID NO: 87, the LCDR2 region of SEQ ID NO: 88, and the LCDR3 region of SEQ ID NO: 89, or d) HCDR1 region of SEQ ID NO: 85, HCDR2 region of SEQ ID NO: 86, HCDR3 region of SEQ ID NO: 84, LCDR1 region of SEQ ID NO: 87, LCDR2 region of SEQ ID NO: 88, and LCDR3 region of SEQ ID NO: 89 Includes.

[0119] In one embodiment, the disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55, or VH and VL having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 54 and / or VL of SEQ ID NO: 55.

[0120] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67, or VH and VL having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 66 and / or VL of SEQ ID NO: 67.

[0121] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises VH of SEQ ID NO: 78 and VL of SEQ ID NO: 79, or VH and VL having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 78 and / or VL of SEQ ID NO: 79.

[0122] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises VH of SEQ ID NO: 90 and VL of SEQ ID NO: 91, or VH and VL having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 90 and / or VL of SEQ ID NO: 91.

[0123] In one embodiment, the disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 56 and the LC of SEQ ID NO: 57, or an HC and LC having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 56 and / or the LC of SEQ ID NO: 57.

[0124] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 68 and the LC of SEQ ID NO: 69, or an HC and LC having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 68 and / or the LC of SEQ ID NO: 69.

[0125] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 80 and the LC of SEQ ID NO: 81, or an HC and LC having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 80 and / or the LC of SEQ ID NO: 81.

[0126] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 92 and the LC of SEQ ID NO: 93, or an HC and LC having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identity with the HC of SEQ ID NO: 92 and / or the LC of SEQ ID NO: 93.

[0127] In one embodiment, the disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 56 and the LC of SEQ ID NO: 57.

[0128] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 68 and the LC of SEQ ID NO: 69.

[0129] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 80 and the LC of SEQ ID NO: 81.

[0130] In one embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific to human C5aR as disclosed herein, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the HC of SEQ ID NO: 92 and the LC of SEQ ID NO: 93.

[0131] In another embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or fragment specific to human C5aR, wherein the nucleic acid sequence or plurality of nucleic acid sequences comprises the VH and VL of any one of the antibodies disclosed in Table 1 or Table 2.

[0132] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment specific to human C5aR encoded by any one of the nucleic acid sequences disclosed in Table 1 or Table 2 or a plurality of nucleic acid sequences.

[0133] In one embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments disclosed in Table 1 or Table 2.

[0134] In one embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding either one of the isolated antibodies or antibody fragments specific to human C5aR according to the present disclosure.

[0135] In another embodiment, the present disclosure refers to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR, wherein the nucleic acid sequence or the plurality of nucleic acid sequences comprises the HC and LC of any one of the antibodies or antibody fragments disclosed in Table 1 or Table 2.

[0136] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or a plurality of nucleic acid sequences are isolated.

[0137] vector In one embodiment, the present disclosure provides a vector or vector composition comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR according to the present disclosure.

[0138] In one embodiment, the present disclosure provides a vector or vector composition comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the isolated antibodies or antibody fragments specific to human C5aR disclosed in Table 1 or Table 2.

[0139] In one embodiment, the present disclosure provides a vector comprising a nucleic acid sequence or a plurality of nucleic acid sequences disclosed in Table 1 or Table 2, or a vector composition comprising a plurality of vectors.

[0140] In one embodiment, the vector composition and / or the vector and / or the plurality of vectors are isolated.

[0141] host cell In one embodiment, the present disclosure provides a host cell comprising a vector or a vector composition comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to human C5aR according to the present disclosure.

[0142] In one embodiment, the present disclosure refers to a host cell comprising a vector or a vector composition comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific to C5aR as disclosed in Table 1 or Table 2.

[0143] In one embodiment, the host cells according to this disclosure can express an isolated antibody or antibody fragment specific to human C5aR encoded by a vector composition or nucleic acid composition.

[0144] In further embodiments, the host cell is an isolated host cell. In further embodiments, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a CHO cell. In one embodiment, the cell is a HEK cell. In another embodiment, the cell is a PERC.6 cell. In one embodiment, the cell is an HKB11 cell.

[0145] Experts will recognize that nucleic acid sequences or multiple nucleic acid sequences encoding the heavy and / or light chains of the antibody or antibody fragment of this disclosure may be cloned into different vectors or into the same vector.

[0146] The vector can be introduced into suitable host cells, such as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells, by methods well known in the art (see, e.g., “Current Protocol in Molecular Biology”, Ausubel et al. (eds.), Greene Publishing Assoc. and John Wiley Interscience, New York, 1989 and 1992). Many cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of choice. The gene can be placed under the control of a promoter, a ribosome binding site (in the case of bacterial expression), and optionally an operator (collectively referred to herein as “control” elements), so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in host cells transformed by a vector containing this expression configuration. The encoding sequence may or may not contain a signal peptide or a leader sequence. Upon expression in host cells, the antibody or antibody fragment of the disclosure is obtained. These steps can be achieved in different ways, as would be known to those skilled in the art. Generally, such steps typically involve transforming or transferring genes into suitable host cells with a nucleic acid composition, vector composition, or infectious particle encoding the antibody or antibody fragment. Furthermore, such steps typically include culturing the host cells under conditions suitable for proliferation (growth) and culturing under conditions suitable for the production (expression, synthesis) of the encoded antibody or antibody fragment. Culturing host cells under conditions suitable for proliferation or expression is generally completed in the presence of a medium containing components suitable for cell proliferation or expression induction. In certain embodiments, the method for producing the antibody or antibody fragment of this disclosure further includes the step of isolating and purifying the antibody or antibody fragment produced from the host cells or medium. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from the cell lysate or recovered from the cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the art.Next, the antibodies or antibody fragments of this disclosure can be purified by many techniques that are known to those skilled in the art.

[0147] In one embodiment, the Disclosure refers to a method for producing an isolated antibody or antibody fragment specific to human C5aR of any of the antibodies disclosed in Table 1 or Table 2. In one embodiment, a method is provided for producing an isolated antibody or antibody fragment according to the Disclosure, the method comprising culturing a host cell containing a vector or a vector composition containing a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibody or antibody fragment according to the Disclosure under conditions suitable for the expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or the culture medium of the host cell. The antibody or antibody fragment isolated as described herein can be purified by techniques known in the art, such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The conditions used to purify a particular antibody or antibody fragment depend in part on factors such as overall charge, hydrophobicity, and hydrophilicity, which will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antibody or antibody fragment binds may be used. For example, a matrix of protein A or protein G may be used for affinity chromatography purification of antibodies or antibody fragments according to this disclosure. The purity of the antibody or antibody fragment can be determined by any of the various well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0148] specificity In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific to human C5aR, as disclosed in Table 1 or Table 2. In one embodiment, the isolated antibody or antibody fragment according to the disclosure is specific to human C5aR.

[0149] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is specific to human C5aR encoded by the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is specific to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is specific to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0150] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to the extracellular region of human C5aR. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to the N-terminal extracellular region of human C5aR. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to the N-terminal extracellular region of human C5aR, which includes the amino acid sequence of SEQ ID NO: 13. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to the N-terminus of human C5aR containing the amino acid sequence of SEQ ID NO: 13. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 13. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure specifically binds to a peptide consisting of the amino acid sequence of SEQ ID NO: 13.

[0151] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0152] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0153] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0154] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a human, humanized, or chimeric antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is an isolated recombinant human monoclonal antibody or antibody fragment.

[0155] Species cross-reactivity In further embodiments, the isolated antibody or antibody fragment according to the Disclosure is cross-reactive to cynomolgus monkey C5aR. In another embodiment, the isolated antibody or antibody fragment according to the Disclosure is specific to human C5aR and cynomolgus monkey C5aR.

[0156] In one embodiment, the disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, which cross-reactively binds to cynomolgus monkey C5aR. In one embodiment, the isolated antibody or antibody fragment according to the disclosure specifically binds to the extracellular regions of human C5aR and cynomolgus monkey C5aR. In one embodiment, the isolated antibody or antibody fragment according to the disclosure binds to the N-terminal extracellular regions of human C5aR and cynomolgus monkey C5aR. In one embodiment, the isolated antibody or antibody fragment according to the disclosure binds to the N-terminal extracellular regions of human C5aR and cynomolgus monkey C5aR, and the N-terminal extracellular region of C5aR includes the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0157] In another embodiment, the isolated antibody or antibody fragment according to this disclosure does not bind to rodent C5aR such as mouse or rat C5aR.

[0158] In one embodiment, the antibody or antibody fragment according to this disclosure is bound to a peptide containing the amino acid sequence of SEQ ID NO: 13 and / or SEQ ID NO: 14.

[0159] In one embodiment, the antibody or antibody fragment according to this disclosure is bound to a peptide consisting of the amino acid sequence of SEQ ID NO: 13 and / or SEQ ID NO: 14.

[0160] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR and cynomolgus monkey C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0161] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0162] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0163] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0164] Monovalent affinity for C5aR peptide In further embodiments, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment has a K content of 100 nM or less, for example, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, or less than 1 nM. D Therefore, it has monovalent affinity for the human C5aR peptide containing SEQ ID NO: 13.

[0165] In one embodiment, the monovalent affinity is determined by the IgG method. In certain embodiments, the monovalent affinity is determined by surface plasmon resonance (SPR) as described in Example 4 herein.

[0166] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0167] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL having at least 80%, at least 85%, at least 90%, or at least 95% identity with VH of SEQ ID NO: 35 or 42 and VL of SEQ ID NO: 36 or 43 Includes.

[0168] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0169] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0170] Apparent affinity (divalent) for C5aR peptide In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, or 1 pM or less, etc., with a K content of 1 nM or less. D It exhibits apparent affinity for the human C5aR peptide, including SEQ ID NO: 13.

[0171] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 300 nM or less, such as 250 nM or less, 200 nM or less, 150 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or 1 nM or less, etc. D It exhibits apparent affinity for the cynomolgus monkey C5aR peptide containing SEQ ID NO: 14.

[0172] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 0.3 nM or less. D For the human C5aR peptide containing SEQ ID NO: 13, and K below 150 nM, D It exhibits apparent affinity for the cynomolgus monkey C5aR peptide containing SEQ ID NO: 14.

[0173] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 0.05 nM or less. D For human C5aR peptides including SEQ ID NO: 13, and K2 below 80 nM D It exhibits apparent affinity for the cynomolgus monkey C5aR peptide containing SEQ ID NO: 14.

[0174] In certain embodiments, the apparent affinity is determined by the IgG method. In one embodiment, the apparent affinity is determined by biolayer interferometry (BLI) as described in Example 5 of this specification.

[0175] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0176] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0177] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0178] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0179] Apparent affinity (divalent) for full-length C5aR In further embodiments, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 10 nM or less, such as 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. D It exhibits apparent affinity for human C5aR.

[0180] In embodiments, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 1 nM or less. D It exhibits apparent affinity for C5aR in cynomolgus macaques.

[0181] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 0.5 nM or less. D In relation to human C5aR, and K below 5 nM, D It exhibits apparent affinity for C5aR in cynomolgus macaques.

[0182] In one embodiment, the human C5aR includes the amino acid sequence of SEQ ID NO: 1. In one embodiment, the human C5aR includes the amino acid sequence of SEQ ID NO: 2. In one embodiment, the cynomolgus monkey C5aR includes the amino acid sequence of SEQ ID NO: 3.

[0183] In certain embodiments, the apparent affinity is determined by the IgG method. In embodiments, the human C5aR or cynomolgus monkey C5aR is expressed on cells. In embodiments, the human C5aR or cynomolgus monkey C5aR is expressed on modified CHO cells expressing full-length human C5aR. In embodiments, the CHO cells are Flp-In CHO cells.

[0184] In certain embodiments, the apparent affinity is determined as described in Example 6 herein.

[0185] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region including the amino acid sequence of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region including the amino acid sequence of SEQ ID NO: 34, or d) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0186] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL having at least at least 80%, at least 85%, at least 90% or at least 95% identity to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 and comprises.

[0187] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of SEQ ID NO: 44 and LC of SEQ ID NO: 45, or HC and LC having at least at least 80%, at least 85%, at least 90% or at least 95% identity to HC of SEQ ID NO: 37 or 44 and to LC of SEQ ID NO: 38 or 45 and comprises.

[0188] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized or chimeric antibody or antibody fragment.

[0189] Apparent EC for full-length C5aR 50 In a further embodiment, the present disclosure refers to an isolated antibody or antibody fragment specific for human C5aR, wherein the isolated antibody binds to human C5aR at an EC 50 concentration of 20 nM or less, 15 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less or 0.1 nM or less.

[0190] In embodiments, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has an EC of 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 1 nM or less. 50 It binds to C5aR in cynomolgus monkeys depending on the concentration.

[0191] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody has a K content of 10 nM or less. D EC 50 It binds to human C5aR and cynomolgus monkey C5aR at certain concentrations.

[0192] In one embodiment, the human C5aR includes the amino acid sequence of SEQ ID NO: 1. In one embodiment, the human C5aR includes the amino acid sequence of SEQ ID NO: 2. In one embodiment, the cynomolgus monkey C5aR includes the amino acid sequence of SEQ ID NO: 3.

[0193] In a certain embodiment, the EC 50 The concentration is determined by the IgG method. In some embodiments, the human C5aR or cynomolgus monkey C5aR is expressed on cells. In some embodiments, the human C5aR or cynomolgus monkey C5aR is expressed on modified CHO cells expressing full-length human C5aR. In some embodiments, the CHO cells are Flp-In CHO cells. In certain embodiments, the human C5aR or cynomolgus monkey C5aR is expressed on neutrophils. In some embodiments, the human C5aR is expressed on human neutrophils. In some embodiments, the cynomolgus monkey C5aR is expressed on cynomolgus monkey neutrophils. In some embodiments, the neutrophils are derived from whole blood. In some embodiments, the EC 50 The concentration is determined as described in Example 7 of this specification.

[0194] In further embodiments, the isolated antibody or antibody fragment specific to human C5aR substantially does not bind to a C5aR-related antigen selected from the group consisting of human C5L2, human ChemR23, human FPR1, and C3aR. In certain embodiments, the isolated antibody or antibody fragment specific to human C5aR substantially does not bind to a C5aR-related antigen selected from the group consisting of human C5L2, human ChemR23, human FPR1, and C3aR at an IgG concentration of 300 nM. In one embodiment, the binding is determined as described in Example 7.

[0195] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region including the amino acid sequence of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region including the amino acid sequence of SEQ ID NO: 34, or d) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0196] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0197] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HC and LC having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to VH of SEQ ID NO: 37 or 44 and VL of SEQ ID NO: 38 or 45 Includes.

[0198] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0199] In one embodiment, this disclosure refers to an isolated antibody or antibody fragment specific to human C5aR, wherein the antibody or antibody fragment has an EC of 5 nM or less, such as 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less. 50 At this concentration, it binds to human C5aR, including SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0200] In certain embodiments, the human C5aR is presented on a virus-like particle. In certain embodiments, the human C5aR is expressed as a fusion protein. In certain embodiments, the human C5aR is fused to a GAG protein. In some embodiments, the fusion protein includes the amino acid sequence disclosed in Table 8. In certain embodiments, the binding is determined by an ELISA as described in Example 8.

[0201] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR is a) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) the HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) the HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34 comprises.

[0202] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL having at least at least 80%, at least 85%, at least 90% or at least 95% identity to VH of SEQ ID NO: 35 or 42 and VL of SEQ ID NO: 36 or 43.

[0203] In one embodiment, the isolated antibody or antibody fragment specific for human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of SEQ ID NO: 44 and LC of SEQ ID NO: 45, or HCs and LCs having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to HCs of sequence number 37 or 44 and LCs of sequence number 38 or 45.

[0204] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0205] Functional C5aR-induced activation of C5A Generally, isolated antibodies or antibody fragments specific to human C5aR according to this disclosure may be used to prevent or inhibit the interaction between human C5aR and human C5a, thereby preventing, inhibiting, neutralizing, or mitigating C5aR-mediated signaling pathways and / or modulating biological pathways and mechanisms in which C5aR is involved.

[0206] In one embodiment, the disclosure relates to an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody or antibody fragment specifically interferes with C5aR-mediated signal transduction.

[0207] In further embodiments of this disclosure, an isolated antibody or antibody fragment specific to human C5aR specifically interferes with the interaction between C5a and C5aR expressed on cells. In even further embodiments, the isolated antibody or antibody fragment can specifically interfere with C5a-induced signal transduction mediated by C5aR.

[0208] Methods for assaying the functional activity of C5aR-specific antibodies may utilize binding assays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence-activated cell sorting (FACS), and other methods well known in the art (see Hampton, R. et al. (1990; Serological Methods a Laboratory Manual, APS Press, St Paul, MN) and Maddox, DE et al. (1983; J.Exp. Med. 158:1211-1216)). Alternatively, assays may be used to test the ability of the isolated antibodies or antibody fragments of this disclosure to induce a biological response as a result of binding to C5aR, either in vivo or in vitro. Such assays are described in the examples disclosed herein. Other suitable assays are known to those skilled in the art.

[0209] In certain embodiments, the isolated antibody or antibody fragment of the Disclosure antagonizes human C5aR activity. In one embodiment, the isolated antibody or antibody fragment neutralizes human C5aR activity. In one embodiment, the isolated antibody or antibody fragment of the Disclosure inhibits human C5aR activity. In one embodiment, the isolated antibody or antibody fragment of the Disclosure inhibits human C5aR signaling. In one embodiment, the human C5aR activity or human C5aR signaling is induced by C5a. In one embodiment, the human C5aR activity or human C5aR signaling is induced by the interaction between human C5a and human C5aR. In one embodiment, the C5aR activity or C5aR signaling is induced by the binding of human C5a to human C5aR. In one embodiment, the C5aR is expressed on a cell. In one embodiment, the human C5aR activity or human C5aR signaling is inhibited in vitro and / or ex vivo and / or in vivo.

[0210] C5A-induced β-arrestin mobilization As described in Example 14, the ability of the human C5aR-specific isolated antibody or antibody fragment according to this disclosure to inhibit C5a-inducible C5aR activation was tested in a β-arrestin recruitment assay. Compared with the prior art antibody RefMAB#1, both antibodies were found to be even more potent antagonists of C5aR activity, particularly at high pathophysiological C5a concentrations.

[0211] Accordingly, in one embodiment of the present disclosure, an isolated antibody or antibody fragment specific to human C5aR inhibits the ability of C5a to induce C5aR activity. In one embodiment, the C5a-inducible C5aR activity is determined in a β-arrestin recruitment assay as described in Example 14. In one embodiment, the C5a-inducible C5aR activity is determined in vitro.

[0212] In one such embodiment, the disclosure provides an isolated antibody or antibody fragment specific to human C5aR, which inhibits the ability of human C5a to induce human C5aR-mediated β-arrestin mobilization. In one embodiment, the isolated antibody or antibody fragment inhibits human C5a-induced β-arrestin mobilization. In one embodiment, the isolated antibody or antibody fragment inhibits human C5aR-mediated β-arrestin mobilization.

[0213] In further embodiments of this disclosure, an isolated antibody or antibody fragment specific to human C5aR inhibits the human C5a-inducible human C5aR interaction with β-arrestin. In one such embodiment, the human C5a-inducible human C5aR interaction with β-arrestin and / or its human C5a-inducible β-arrestin recruitment is measured using enzyme fragment complementation. In one embodiment, the human C5a-inducible human C5aR interaction with β-arrestin and / or its human C5a-inducible β-arrestin recruitment is determined as described in Example 14. In one such embodiment, the human C5a-inducible human C5aR interaction with β-arrestin and / or its human C5a-inducible β-arrestin recruitment is tested at an IgG concentration of 50 nM.

[0214] The ability of the isolated antibodies or antibody fragments according to this disclosure to inhibit human C5a-inducible human C5aR activity, such as inhibiting the human C5a-inducible human C5aR interaction with β-arrestin and / or human C5a-inducible human C5aR-mediated β-mobilization, can be determined by creating dose-response curves for increasing human C5a concentrations and fixed IgG concentrations, and by calculating individual EC50 concentrations.

[0215] In one embodiment, the disclosure provides an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody or antibody fragment is determined in the absence of the isolated antibody or antibody fragment. 50 Compared to other concentrations, the EC determined for human C5a in the β-arrestin mobilization assay at a 50 nM IgG concentration is... 50 Increase the concentration by at least five times, for example, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, at least twelve times, or at least thirteen times.

[0216] In one embodiment, the disclosure provides an isolated antibody or antibody fragment specific to human C5aR, wherein the isolated antibody or antibody fragment is determined in the absence of the isolated antibody or antibody fragment.50 Compared to other concentrations, the EC determined for human C5a in the β-arrestin mobilization assay at a 50 nM IgG concentration is... 50 Increase the concentration by approximately 5, 6, 7, 8, 9, 10, 11, 12, or 13 times.

[0217] In one embodiment, the Disclosure provides an antibody or antibody fragment specific to human C5aR, and in order to induce the same human C5aR activity in a β-arrestin recruitment assay at a 50 nM IgG concentration compared to the concentration of human C5a in the absence of the antibody or antibody fragment, the human C5a must be present at a concentration of at least 5 times, for example, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, or at least 13 times.

[0218] In one embodiment, the β-arrestin recruitment assay is performed as described in Example 14. In one embodiment, the β-arrestin recruitment assay is performed in vitro.

[0219] Alternatively, the ability of a C5aR-specific isolated antibody or antibody fragment according to this disclosure to inhibit C5a-inducible C5aR-mediated β-arrestin mobilization can be determined by calculating the percentage inhibition for different human C5a concentrations.

[0220] In such one embodiment, the C5aR-specific isolated antibody or antibody fragment according to the Disclosure inhibits human C5a-inducible human C5aR-mediated β-arrestin mobilization by at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% in the presence of 1.2 nM or 11 nM human C5a, at a concentration of 50 nM IgG, compared to the levels of human C5a-inducible human C5aR-mediated β-arrestin mobilization in the presence of 1.2 nM or 11 nM human C5a and in the absence of the antibody or antibody fragment.

[0221] In such one embodiment, the C5a-specific isolated antibody or antibody fragment according to the Disclosure inhibits human C5a-inducible human C5aR-mediated β-arrestin mobilization by at least 25%, for example, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at a concentration of 50 nM IgG and in the presence of 1.2 nM or 11 nM human C5a, compared to the level of human C5a-inducible human C5aR-mediated β-arrestin mobilization in the presence of 100 nM human C5a and in the absence of the antibody or antibody fragment, respectively.

[0222] In one embodiment, the β-arrestin recruitment assay is performed as described in Example 14. In one embodiment, the β-arrestin recruitment assay is performed in vitro.

[0223] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 41, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0224] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0225] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0226] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0227] C5A-induced upregulation of CD11b expression C5a acts as a potent activator of human neutrophils and monocytes, inducing the upregulation of the cell surface antigen CD11b in such cells. Therefore, the ability of human C5aR-specific isolated antibodies or antibody fragments according to this disclosure to inhibit C5a-inducible activation of granulocytes and / or monocytes can be assessed by determining CD11b expression levels in such cells.

[0228] The ability of the isolated antibodies or antibody fragments according to this disclosure to inhibit CD11b expression in granulocytes and / or monocytes to create dose-response curves for IgG escalating concentrations and human C5a fixed concentrations, and for individual ICs. 50The concentration may be determined by calculation. Alternatively, the ability of the isolated antibody or antibody fragment according to this disclosure to inhibit CD11b expression in granulocytes and / or monocyte C5a may be determined by calculating the percentage inhibition of CD11b expression for different IgG concentrations.

[0229] In one such embodiment, the isolated antibody or antibody fragment specific to human C5aR according to this disclosure exhibits IC50 ≤ 30 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM, ≤ 10 nM, or ≤ 5 nM in the presence of 15 nM human C5a. 50 At this concentration, it inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes.

[0230] In another embodiment, an isolated antibody or antibody fragment specific to human C5aR according to this disclosure inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes by at least 70%, at least 75%, 80%, at least 85%, or at least 90% in the presence of 15 nM human C5a and 600 nM IgG, compared to CD11b expression levels in the presence of 15 nM human C5a and in the absence of the antibody or antibody fragment.

[0231] In further embodiments, the isolated antibody or antibody fragment specific to human C5aR according to this disclosure exhibits IC50 ≤ 150 nM, ≤ 125 nM, ≤ 100 nM, ≤ 90 nM, ≤ 80 nM, ≤ 70 nM, ≤ 60 nM, ≤ 50 nM, or ≤ 40 nM in the presence of 150 nM human C5a. 50 At certain concentrations, it inhibits human C5a-induced CD11b expression in human granulocytes.

[0232] In one embodiment, the human C5aR-specific isolated antibody or antibody fragment according to this disclosure exhibits a 42 nM IC in the presence of 150 nM human C5a. 50 At certain concentrations, it inhibits human C5a-induced CD11b expression in human granulocytes.

[0233] In further embodiments, the human C5a-specific isolated antibody or antibody fragment according to the present disclosure inhibits human C5a-induced CD11b expression in human granulocytes by at least 65%, at least 70%, at least 75%, at least 80%, or at least 90% in the presence of 150 nM human C5a and 600 nM IgG, compared to the CD11b expression levels in the presence of 150 nM human C5a and in the absence of the antibody or antibody fragment.

[0234] In one embodiment, an isolated antibody or antibody fragment specific to human C5aR according to this disclosure inhibits C5a-induced CD11b expression in human granulocytes by at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% in the presence of 150 nM human C5a and 100 nM IgG, compared to CD11b expression levels in the presence of 150 nM human C5a and in the absence of the antibody or antibody fragment.

[0235] In one embodiment, the determination of CD11b expression is performed as described in Example 15. In one embodiment, the determination of CD11b expression is performed in vitro and / or ex vivo.

[0236] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0237] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0238] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0239] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0240] Furthermore, the inhibitory activity of the isolated antibody or antibody fragment according to this disclosure on human C5a-induced CD11b expression was further analyzed over extended periods, meaning that the antibody was pre-warmed with granulocytes and / or monocytes present in whole blood for varying lengths of time (e.g., 300 minutes vs. 20 minutes) before the addition of human C5a. Surprisingly, these experiments revealed that the isolated antibody according to this disclosure is a more potent antagonist of C5aR activity over longer periods, such as longer warming times, particularly compared to the prior art antibody RefMAB#1.

[0241] Therefore, the ability of the isolated antibodies or antibody fragments according to this disclosure to inhibit human C5a-induced CD11b expression in granulocytes and / or monocytes over a long period of time can be demonstrated by creating dose-response curves for gradual IgG concentrations and fixed human C5a concentrations, and by incubating the isolated antibodies with the granulocytes and / or monocytes over different incubation times, and by observing individual EC 50 It can be determined by calculating the value.

[0242] As shown in Figures 6A and C, the isolated antibodies or antibody fragments of this disclosure showed a clear shift in the determined dose-response curve to lower IgG concentrations when determined after 300 minutes of incubation, compared to the dose-response curve determined after 20 minutes of incubation. Interestingly, RefMAB#1 revealed no increase in potency over time (see Figures 6B and D).

[0243] Therefore, in one embodiment, the isolated antibody or antibody fragment according to the present disclosure is more potent in inhibiting C5a-inducible CD11b expression in human granulocytes and / or human monocytes after a long incubation period with the cells.

[0244] In such one embodiment, the isolated antibody or antibody fragment according to the Disclosure is determined after a 20-minute incubation period. 50When compared to the concentration, the IC50 concentration, when determined after a long incubation period of 50, 100, 150, 200, 250, or 300 minutes, decreases to at least half, at least one-quarter, at least one-fifth, or at least one-sixth of the original IC50 concentration. 50 At this concentration, it inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes.

[0245] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is determined after a 20-minute incubation period. 50 When compared to the concentration, the IC50, when determined after a long incubation period of 300 minutes, had decreased to about one-fifth. 50 At this concentration, it inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes.

[0246] In one embodiment, the isolated antibody or antibody fragment according to this disclosure is the corresponding IC of RefMAB#1 50 When compared to the concentration, the IC250, when determined after a long incubation period of 300 minutes, was reduced to at least one-third, at least one-quarter, at least one-fifth, at least one-tenth, at least one-fifteenth, or at least one-nineteenth. 50 At this concentration, it inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes.

[0247] In one embodiment, the isolated antibody or antibody fragment according to this disclosure undergoes an IC50 of 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, or 1 nM or less after a long incubation period of 300 minutes with the cells. 50 At this concentration, it inhibits human C5a-induced CD11b expression in human granulocytes and / or human monocytes.

[0248] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0249] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0250] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0251] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0252] C5A-induced migration of neutrophils Further assays evaluated the ability of the human C5aR-specific isolated antibody or antibody fragment according to this disclosure to inhibit human C5a-induced migration of human neutrophils, revealing that MAB#1 efficiently inhibited C5a-induced neutrophil migration in vitro.

[0253] Therefore, in one embodiment of the present disclosure, the isolated antibody or antibody fragment specific to human C5aR of the present disclosure inhibits human C5a-inducible migration of human neutrophils in vitro.

[0254] In further embodiments, the isolated antibody or antibody fragment inhibits human C5a-induced migration of human neutrophils by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80% in vitro, compared to migration levels in the presence of 10 nM human C5a and in the absence of the isolated antibody or antibody fragment.

[0255] In one embodiment, the migration of human neutrophils is determined at 15 minutes, 25 minutes, and / or 35 minutes. In certain embodiments, the isolated antibody or antibody fragment according to this disclosure is tested at IgG concentrations of 100 nM and / or 600 nM.

[0256] In one embodiment, the isolated antibody or antibody fragment of the present disclosure inhibits human C5a-induced migration of human neutrophils by at least 25% at a 100 nM IgG concentration at 35 minutes, compared to migration levels at 35 minutes in the presence of 10 nM human C5a and in the absence of the antibody or antibody fragment.

[0257] In one embodiment, the isolated antibody or antibody fragment of the present disclosure inhibits human C5a-induced migration of human neutrophils by at least 60% at IgG concentrations of 100 nM and / or 600 nM after 25 minutes, compared to migration levels after 25 minutes in the presence of 10 nM human C5a and in the absence of the antibody or antibody fragment.

[0258] In one embodiment, the isolated antibody or antibody fragment of the present disclosure inhibits human C5a-induced migration of human neutrophils by at least 40% at a 600 nM IgG concentration after 35 minutes, compared to migration levels after 35 minutes in the presence of 10 nM human C5a and in the absence of the antibody.

[0259] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0260] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0261] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0262] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0263] Effector function The Fc region of immunoglobulins generally confers desirable pharmacokinetic properties to antibodies, such as prolongation of serum half-life, and the ability to induce effector function through binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors can also result in undesirable activation of certain cell surface receptors, which can lead to undesirable cytokine release and serious side effects during systemic administration.

[0264] Therefore, in certain therapeutic situations, it is desirable to reduce or eliminate the normal binding of the wild-type Fc region of an antibody, such as the wild-type IgG Fc region, to one or more Fc receptors and / or to complement components such as C1q, in order to reduce or eliminate the ability of the antibody to induce effector function. For example, it may be desirable to reduce or eliminate the binding of the Fc region of an antibody to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa.

[0265] Effector functions may include, but are not limited to, one or more of the following: complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling including apoptosis, crosslinking of target-binding antibodies, dendritic cell maturation, or T cell priming.

[0266] By mutating a wild-type Fc region, such as the IgG1 Fc region, or more specifically, the human IgG1 Fc region, and as a result obtaining a mutated or modified Fc region, such as a mutant human IgG1 Fc region, a reduction or loss of the Fc region's binding to the Fc receptor and / or C1q is generally achieved. Substitutions that reduce binding may be useful. Non-conservative amino acid substitutions, i.e., substituting one amino acid with another amino acid having a different structure and / or chemical properties, are preferred to reduce or eliminate the binding properties of the Fc region to the Fc receptor.

[0267] Accordingly, in one embodiment, the isolated antibody or antibody fragment specific to human C5aR according to the Disclosure includes a mutant Fc region in which binding to the Fc receptor and / or to C1q is reduced or absent compared to the wild-type Fc region. In such an embodiment, the isolated antibody or antibody fragment according to the Disclosure includes a mutant Fc region that reduces or eliminates the antibody's ability to induce effector function. In a further embodiment, the isolated antibody or antibody fragment according to the Disclosure substantially does not induce effector function.

[0268] In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, the isolated antibody or antibody fragment according to this disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, the isolated antibody or antibody fragment according to this disclosure does not induce ADCC or ADCP in vitro.

[0269] In one embodiment, the mutant Fc region of the isolated antibody or antibody fragment according to the Disclosure comprises one or more amino acid substitutions that reduce or eliminate the binding of the mutant Fc region to one or more Fc receptors and / or C1q compared to the wild-type Fc region. In one embodiment, the mutant Fc region of the isolated antibody or antibody fragment according to the Disclosure comprises one or more amino acid substitutions that reduce or eliminate the antibody's ability to induce effector function compared to the wild-type Fc region.

[0270] In certain embodiments, one or more amino acid substitutions may reduce the binding affinity of the mutant Fc region to one or more Fc receptors and / or C1q to at least half, at least one-fifth, at least one-tenth, at least one-twentieth, or even at least one-fiftieth, compared to the wild-type Fc region. In alternative embodiments, one or more amino acid substitutions may reduce the ability of the isolated antibody or antibody fragment according to this disclosure to induce effector function to at least half, at least one-fifth, at least one-tenth, at least one-twentieth, or even at least one-fiftieth, compared to the wild-type Fc region.

[0271] In one embodiment, the mutant Fc region of the isolated antibody or antibody fragment according to the Disclosure substantially does not bind to one or more Fc receptors and / or C1q. In one embodiment, the mutant Fc region of the antibody according to the Disclosure substantially eliminates the ability of the antibody to induce effector function. In one embodiment, the antibody or antibody fragment according to the Disclosure substantially does not induce effector function. In one embodiment, the effect function is ADCC and / or ADCP and / or CDC. In one embodiment, the antibody or antibody fragment according to the Disclosure substantially does not induce effector function, meaning that when measured in the absence of the antibody, the level of induced effector function does not significantly exceed the background.

[0272] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is human FcγRIIIa, FcγRI, FcγRIIa and / or FcγRIIb.

[0273] In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In a particular embodiment, the effector function is ADCC, CDC, and ADCP. In a more particular embodiment, the effector function is ADCC and ADCP.

[0274] In one embodiment, the wild-type Fc region is the IgG1 Fc region. In one embodiment, the wild-type Fc region is the human IgG1 Fc region. In one embodiment, the wild-type Fc region is the human IgG1 Fc region. In one embodiment, the wild-type Fc region is, This is the human IgG1 Fc region containing the amino acid sequence PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 11).

[0275] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure comprises a mutant human IgG1 Fc region containing one or more amino acid substitutions compared to the wild-type human IgG1 Fc region. In one embodiment, the one or more amino acid substitutions reduce or eliminate the binding of the mutant Fc region to the Fc receptor and / or to C1q, and / or reduce the antibody's ability to induce effector function, compared to the wild-type Fc region.

[0276] In one embodiment, the mutant human IgG1 Fc region of the antibody or antibody fragment according to the present disclosure includes amino acid substitutions at one or more positions selected from the group 234, 235, 237, 330, and 331, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0277] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions at one or more positions selected from the group L234, L235, and G237, which are numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0278] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions at one or more positions selected from the group L234A, L235E, and G237A, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0279] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions L234A and L235E, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0280] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions L234A, L235E, and G237A, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0281] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions at one or more positions selected from the group A330 and P331, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0282] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions at one or more positions selected from the group A330S and P331S, which are numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0283] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions A330S and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0284] In one embodiment, the mutant human IgG1 Fc region includes amino acid substitutions L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0285] In one embodiment, the antibody or antibody fragment according to the present disclosure comprises a mutant human IgG1 Fc region comprising one or more amino acid substitutions that reduce or eliminate the binding affinity of the mutant Fc region to one or more Fc receptors and / or C1q, and / or reduce the effector function-inducing ability of the antibody, compared to a wild-type human IgG1 Fc region comprising the sequence of SEQ ID NO: 11, wherein the one or more amino acid substitutions are L234A, L235E, G237A, A330S and P331S, numbered according to the EU index.

[0286] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is of the human IgG1 class. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is of the mutant human IgG1 class. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure does not substantially induce effector function in vitro. In one embodiment, the mutant human IgG1 Fc region does not substantially bind to one or more Fc receptors and / or C1q. In such an embodiment, the isolated antibody or antibody fragment according to the Disclosure includes amino acid substitutions selected from the group L234A, L235E, G237A, A330S and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0287] In one embodiment, the isolated antibody or antibody fragment according to this disclosure is a human IgG1 class comprising amino acid substitutions L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index.

[0288] In one embodiment, the isolated antibody or antibody fragment according to the present disclosure includes amino acid substitutions L234A, L235E, G237A, A330S, and P331S, which are numbered according to the EU index.

[0289] In one embodiment, the isolated antibody or antibody fragment according to the present disclosure includes amino acid substitutions L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index, compared to the wild-type IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11.

[0290] In one such embodiment, the isolated antibody or antibody fragment according to the Disclosure comprises amino acid substitutions L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index, and does not substantially induce effector function in vitro. In one embodiment, the effector function is one or more selected from the group CDC, ADCC, and ADCP.

[0291] In such one embodiment, the isolated antibody or antibody fragment according to the present disclosure comprises amino acid substitutions L234A, L235E, G237A, A330S, and P331S, numbered according to the EU index, compared to the wild-type human IgG1 Fc region containing the amino acid sequence of SEQ ID NO: 11, and substantially does not bind to one or more Fc receptors and / or C1q in vitro.

[0292] In one embodiment, the isolated antibody or antibody fragment according to the Disclosure is of the human IgG1 class. In one embodiment, the isolated antibody or antibody fragment against the Disclosure does not substantially induce effector function in vitro. In one embodiment, the isolated antibody or antibody fragment according to the Disclosure contains one or more amino acid substitutions selected from the group L234A, L235E, G237A, A330S and P331S, which are numbered according to the EU index.

[0293] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0294] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0295] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0296] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0297] For example, the binding of an antibody to an Fc receptor via its Fc region can be easily determined by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a Biacore® instrument (GE Healthcare), and the Fc receptor can be obtained by recombinant expression. Alternatively, the binding affinity of the Fc region can be evaluated using cell lines known to express a specific Fc receptor, such as NK cells expressing the FcγIIIa receptor. The effector function of the antibody can be measured by methods known in the art. Appropriate in vitro assays for evaluating the ADCC activity of molecules of interest are described, for example, in International Publication No. 2012130831. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of molecules of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). To evaluate complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). C1q binding assays (such as ELISA) may be performed to determine whether an antibody is capable of binding to C1q and therefore possesses CDC activity (see, e.g., International Publication No. 2006 / 029879). In vitro methods for evaluating binding to the Fc receptor or for evaluating immunoeffector function are described in Examples 10-13 herein.

[0298] Fusion protein The isolated antibodies or antibody fragments according to this disclosure may or may not be fused to one or more other amino acid residues, polypeptides, or moieties. Such fusion proteins can be appropriately prepared, including by genetic or chemical approaches. The fused moieties may contain secretion or leader sequences, sequences that facilitate detection, expression, isolation, or purification, or sequences that improve protein stability during recombinant production, for example. Non-limiting examples of possible moieties include beta-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragments, secretory signal peptides, antibodies or antibody fragments, toxins, reporter enzymes, moieties capable of binding to metal ions such as polyhistidine tags, tags suitable for detection and / or purification, homo- or hetero-associating domains, moieties that improve protein solubility, or moieties containing enzymatic cleavage sites.

[0299] Therefore, the isolated antibodies or antibody fragments according to this disclosure may optionally contain one or more moieties for binding to other targets or target proteins of interest. Such further moieties may or may not provide further functionality to the antibody, and may or may not modify the properties of the isolated antibodies or antibody fragments according to this disclosure. Diagnostic applications

[0300] In one embodiment, the Disclosure provides the use of a human C5aR-specific isolated antibody or antibody fragment according to the Disclosure for disease diagnosis. In one embodiment, the Disclosure provides the use of an antibody or antibody fragment according to the Disclosure for the detection of C5aR, particularly human C5aR and / or cynomolgus monkey C5aR. In one embodiment, the Disclosure provides a method for detecting C5aR in a subject or sample, comprising the step of contacting the subject or sample with a human C5aR-specific isolated antibody or antibody fragment according to the Disclosure. In one embodiment, the Disclosure provides a method for diagnosing a disease in a subject, comprising the step of contacting the subject or sample with an isolated antibody or antibody fragment according to the Disclosure.

[0301] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0302] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0303] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0304] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0305] Treatment method The isolated antibodies or antibody fragments provided in this disclosure may be used in therapeutic methods. The antibodies or antibody fragments provided in this disclosure may be used for the treatment of diseases, such as cancer, autoimmune diseases, or inflammatory diseases.

[0306] In one embodiment, the present disclosure provides a method for treating a disease.

[0307] In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for the treatment of a disease. In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for use in the treatment of a disease. In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for use in the treatment of a disease in a subject requiring treatment of the disease.

[0308] In one embodiment, the Disclosure provides the use of an isolated antibody or antibody fragment according to the Disclosure for the manufacture of a pharmaceutical product. In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for use as a pharmaceutical product. In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for use in a pharmaceutical product. In one embodiment, the Disclosure provides an isolated antibody or antibody fragment according to the Disclosure for use as a pharmaceutical product for the treatment of a subject requiring treatment.

[0309] In one embodiment, the disease is associated with the undesirable presence of C5aR, particularly human C5aR. In another embodiment, the disease is associated with the undesirable presence of C5a, particularly human C5a.

[0310] In one embodiment, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, hematological cancer, sarcoma, skin cancer, squamous cell carcinoma, bone cancer, melanoma, renal cell carcinoma, and kidney cancer.

[0311] In one embodiment, the disease to be treated is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory diseases include rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes, Graves' disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, and Sjögren's disease. Examples include nephritis, 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, allergies, asthma, antiphospholipid syndrome (APS), atherosclerosis, C3 glomerulopathy and IgA nephropathy, ischemia / reperfusion injury, peritonitis, sepsis, and other autoimmune diseases resulting from either acute or chronic inflammation.

[0312] In one embodiment, the Disclosure provides a human C5aR-specific isolated antibody or antibody fragment according to the Disclosure for use in a method of treating a diseased subject, which involves administering a therapeutically effective amount of the antibody or antibody fragment according to the Disclosure to the subject.

[0313] In one embodiment, the method further includes administering a therapeutically effective amount of at least one further therapeutic agent to the target. The target requiring treatment is generally a mammal, more specifically a human. For use in therapeutic methods, the isolated antibodies or antibody fragments according to this disclosure are prescribed, taken orally, and administered in a manner consistent with proper medical practice.

[0314] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0315] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0316] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HCs and LCs that are at least 80%, at least 85%, at least 90%, or at least 95% identical to HCs of SEQ ID NO: 37 or 44 and to LCs of SEQ ID NO: 38 or 45. Includes.

[0317] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0318] Pharmaceutical composition In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0319] The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to this disclosure may be used in the diagnosis, prevention and / or treatment of diseases associated with the undesirable presence of C5aR, particularly human C5aR. In particular, this disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to this disclosure that is suitable for prophylactic, therapeutic and / or diagnostic applications in mammals, more specifically in humans.

[0320] Generally, the antibodies or antibody fragments according to the present disclosure may be formulated as a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharmaceutically acceptable carrier or excipient, and optionally one or more further pharmaceutically active compounds. Such formulations may be suitable for oral, parenteral, topical, or inhalation administration. Accordingly, a pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure may be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention may be administered orally or via parenteral routes such as topically. In preferred embodiments, a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

[0321] In particular, the antibodies or antibody fragments provided herein may be used in combination with one or more pharmaceutically active compounds used or that may be used for the prevention and / or treatment of diseases involving a target antigen of interest, and as a result, a synergistic effect may or may not be obtained. Examples of such compounds, as well as routes, methods, and formulations or compositions for administering them, are obvious to clinicians.

[0322] In one embodiment, the Disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the Disclosure for use in the prevention and / or treatment of diseases associated with the undesirable presence of C5aR. In one embodiment, the Disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the Disclosure for use as a pharmacopoeia. In one embodiment, the Disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the Disclosure for use in the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases and / or cancer.

[0323] In one embodiment, the Disclosure provides a method for treating autoimmune diseases and / or inflammatory diseases and / or cancer in subjects requiring treatment of autoimmune diseases and / or inflammatory diseases and / or cancer, using a pharmaceutical composition comprising an antibody or antibody fragment according to the Disclosure.

[0324] A method is further provided for producing an antibody or antibody fragment according to the Disclosure in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody or antibody fragment by the method according to the Disclosure and (b) formulating the antibody or antibody fragment together with at least one pharmaceutically acceptable carrier or excipient, thereby formulating a preparation of the antibody or antibody fragment for in vivo administration. A pharmaceutical composition according to the Disclosure comprises a therapeutically effective amount of one or more antibodies or antibody fragments according to the Disclosure dissolved in a pharmaceutically acceptable carrier or excipient.

[0325] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR according to the Disclosure is a) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 28, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or b) The HCDR1 region of SEQ ID NO: 30, the HCDR2 region of SEQ ID NO: 31, the HCDR3 region of SEQ ID NO: 29, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or c) The HCDR1 region of SEQ ID NO: 27, the HCDR2 region of SEQ ID NO: 39, the HCDR3 region of SEQ ID NO: 40, the LCDR1 region of SEQ ID NO: 32, the LCDR2 region of SEQ ID NO: 33, and the LCDR3 region of SEQ ID NO: 34, or d) HCDR1 region of SEQ ID NO: 30, HCDR2 region of SEQ ID NO: 41, HCDR3 region of SEQ ID NO: 40, LCDR1 region of SEQ ID NO: 32, LCDR2 region of SEQ ID NO: 33, and LCDR3 region of SEQ ID NO: 34 Includes.

[0326] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43, or VH and VL that are at least 80%, at least 85%, at least 90%, or at least 95% identical to VH of SEQ ID NO: 35 or 42 and to VL of SEQ ID NO: 36 or 43 Includes.

[0327] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a) HC of SEQ ID NO: 37 and LC of SEQ ID NO: 38, or b) HC of sequence number 44 and LC of sequence number 45, or HC and LC having at least 80%, at least 85%, at least 90%, or at least 95% identity with respect to VH of SEQ ID NO: 37 or 44 and VL of SEQ ID NO: 38 or 45 Includes.

[0328] In one embodiment, the isolated antibody or antibody fragment specific to human C5aR is a monoclonal antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a recombinant antibody or antibody fragment. In one embodiment, the isolated antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.

[0329] antibody sequence

[0330] [Table 1]

[0331] [Table 2]

[0332] [Table 3]

[0333] [Table 4]

[0334] Table 5

[0335] Table 6

[0336] Table 7

[0337] Table 8

[0338] Table 9

[0339] Table 10

[0340] Table 11

[0341] Table 12

[0342] Table 13

[0343] Table 14

[0344] Table 15 [Examples]

[0345] Example 1: Antigen production and quality control We searched for and validated the amino acid sequences of C5aR and C5aR-related GPCRs from various species using publicly available sources (e.g., Uniprot), and then prepared them in-house or through external vendors.

[0346] Synthetic peptides For initial panning and screening, a linear peptide covering the N-terminal extracellular region of human C5aR was used as the antigen. This peptide was chemically synthesized with a biotin tag (JPT), purified by RP-HPLC, and delivered as a lyophilized material. The lyophilized peptide was stored at -80°C. Alternatively, this peptide was conjugated with transferrin or bovine serum albumin (BSA).

[0347] [Table 16]

[0348] For subsequent binding assays, linear peptides containing the N-terminal regions of human and cynomolgus monkey C5aR were used as antigens. These peptides were chemically synthesized with biotin tags (Genscript), purified by RP-HPLC, and delivered as lyophilized material. The lyophilized peptides were stored at -80°C. For reconstitution, the peptides were dissolved in the desired volume of PBS and stored at -80°C.

[0349] [Table 17]

[0350] Recombinant protein C1q protein The C1q protein, purified from pooled normal human plasma, was purchased from Complement Technology, Inc. (Catalog#A099).

[0351] Human C5a Protein Recombinant human C5a cells were either purchased from R&D Systems (CAT#:2037-C5) or manufactured in-house.

[0352] For in-house production, DNA encoding the amino acids of human C5a (Uniprot:P01031|Lys679-Arg751), along with the N-terminal ompA signal sequence, the subsequent sequence encoding maltose-binding protein (MBP), the FXa cleavage site, and the GS linker, was cloned in-frame into the pET21a expression vector (Novagen).

[0353] Human C5a (hC5a) was expressed as an N-terminally tagged maltose-binding protein (MBP) fusion protein in E. coli BL21 (DE3) cells (Novagen). Protein expression was induced by IPTG addition, and the cultures were further incubated for 20-23 hours. Cells were harvested by centrifugation, and the pellet was resuspended in lysis buffer (PBS buffer + 2 mM MgCl2, 20 U / ml Benzonase (Roche) and 1 tablet / 50 mL cOmplete, EDTA-free protease inhibitor cocktail tablet (Roche)). Cells were destroyed by either chemical lysis or high-pressure homogenization. The resulting suspension was centrifuged, and the supernatant was filtered for further purification.

[0354] The hC5a-MBP fusion protein was purified by dextrin-Sepharose affinity chromatography using an MBP-Trap column (GE-Healthcare), and optionally polished by cation exchange chromatography using a Hi-Trap SP FF column (GE LifeSciences). The purified MBP fusion was buffer-exchanged to FXa-digestion buffer (20mM Tris / HCl pH8.0; 100mM NaCl; 2mM CaCl2) using a PD10 column (GE Healthcare). The hC5a protein was released from the maltose-binding protein by adding factor Xa (1:100 (w / w)), and the mixture was incubated over-temperature in a rotary shaker at room temperature. The released hC5a was purified by cation exchange chromatography using a Hi-Trap SP FF column (GE LifeSciences). All affinity chromatography steps were performed using an AKTA Avant 25-part preparative chromatography system.

[0355] Buffer exchange to PBS was performed using a PD10 column (GE Healthcare). The sample was sterile filtered, and the hC5a concentration was determined by UV spectrophotometric analysis. The purity and integrity of the sample were analyzed by denaturation, reduction, or non-reduction SDS-PAGE, SEC-HPLC, and mass spectrometry.

[0356] Fc gamma receptor (FcγR) and FcRn receptor DNA encoding the extracellular regions of human FcγRI, human FcγRIIa(131H), human FcγRIIa(131R), human FcγRIIb, human FcγRIIIa(158F), and human FcγRIIIa(158V) was cloned in-frame, along with the N-terminal Vκ reader sequence and C-terminal 6xHis- tag, into a pMAX expression vector, a modified expression vector based on pcDNA3.1 (Thermo Fisher).

[0357] DNA encoding the extracellular region of the human, cynomolgus monkey, mouse, or rat FcRn large subunit p51 was cloned in-frame, along with the N-terminal Vκ reader sequence and C-terminal AVI-6xHis- tag, into a pMAX expression vector, a modified expression vector based on pcDNA3.1 (Thermo Fisher). Furthermore, DNA encoding the human, cynomolgus monkey, mouse, or rat FcRn small subunit p14 (= identical to beta-2 microglobulin) protein was cloned into a second open reading frame, along with the N-terminal Vk reader sequence in-frame. The amino acid sequences of the produced receptors are summarized in Tables 6 and 7.

[0358] The HEK293-6E cell line was developed by the National Research Council of Canada (NRC). Cells were maintained in Freestyle F17 medium (Thermo Scientific) at 37°C and 6% CO2 in a humidified CO2 incubator. HKB11 (parent clone: ​​U.S. Patent No. 6,136599; J.Biomed.Sci.2002;9:631-638) is a human hybrid cell line obtained from the fusion of HEK293 human embryonic kidney and 2B8 Burkitt lymphoma cells. HKB11#52 cells were maintained in MAC1.0 medium containing 1% FCS at 37°C and 6% CO2 in a humidified CO2 incubator.

[0359] Following the manufacturer's instructions, transient gene transfer was performed on HKB11#52 or HEK293-6E cells one day after seeding with a commercially available gene transfer reagent. The cells were cultured for three days, and the conditioned cell culture supernatant was collected by centrifugation followed by sterile filtration (0.22 μm). A stable HKB11#52 pool was created by gene transfer into the cells and subsequent selection with 800 μg / mL G418 (Thermo Scientific). Antigen expression was performed from the stable pool four days after seeding. The conditioned cell culture supernatant was collected by centrifugation and followed by sterile filtration (0.22 μm).

[0360] Individual proteins were purified by IMAC using a Protino Ni-NTA column (Macherey-Nagel). All chromatography steps were performed using an AKTA chromatography system (GE Healthcare). Buffer exchange to D-PBS was performed on the samples using a PD10 column (GE Healthcare). In some cases, a preparative SEC step involving polishing in D-PBS was performed using a Superdex 200 column (GE Healthcare).

[0361] Using the BirA Kit (Avidity), biotinylation of FcRn heterodimers was performed by in vitro biotinylation, followed by preparative SEC using a Superdex 200 column (GE Healthcare).

[0362] Sample quality was analyzed by denaturation, reduction, or non-reduction SDS-PAGE, streptavidin-shift assay, HP-SEC, and DLS.

[0363] [Table 18]

[0364] [Table 19]

[0365] Virus-like particles (VLPs) As described in International Publication No. 2015 / 193143, we in-house constructed VLPs that stably express either human C5aR (D / K or N / N variant) or one of two native variants of mouse C5aR. All cloning experiments were performed using standard techniques. The antigen of interest was cloned in two appropriate vector systems for expression in mammalian cells. In these systems, one vector expresses a GAG, and the other expresses a GPCR-GAG fusion protein. Expression in these vectors is under the control of the CMV promoter. Subsequently, gene transfer into host cells was performed using these two vectors. The resulting constructs were fusion proteins in which the antigen of interest was fused to the GAG ​​protein at the N-terminus (HV1B1 (Uni-Prot ID: P03347)). Protein expression and VLP production were performed under standard conditions in suspension culture. The host cells used in this experiment were HKB11 cells (ATCC;CRL-12568) and HEK cells (Life Technologies). Three days after gene transfer, the supernatant containing VLP was collected and purified using standard procedures (including precipitation and ion-exchange chromatography). The isolated proteins were subjected to SDS-PAGE chromatography. The supernatant was examined using commercially available anti-GAG antibodies or commercially available anti-C5aR antibodies, and it was revealed that the co-expression of GAG and GPCR-GAG fusion proteins resulted in increased expression levels of GAG and GPCR-GAG in the VLP. This confirmed that the C5aR antigen was efficiently incorporated into the VLP and that the C5aR antigen was detectable by the antibody. The amino acid sequences of the produced fusion proteins are summarized in Table 8.

[0366] [Table 20]

[0367] [Table 21]

[0368] cell line CHO Flp-In cells were created that stably express full-length human C5aR, cynomolgus monkey C5aR, mouse C5aR, rat C5aR, and C5aR-related GPCRs human C5L2, human C3aR, human FPR1, and human ChemR23. Various vector constructs were synthesized in-house to produce Flp-In CHO cells, and gene transfer into cells was performed according to the instructor's manual (Thermofischer / Invitrogen). All constructs contained an N-terminal V5 / His tag. To confirm the expression of individual GPCRs on the cell line surface, even in the absence of commercially available specific anti-GPCR tool antibodies, commercially available anti-His (e.g., Dianova CAT#DIA-910) or anti-V5 (e.g., AbD Serotec CAT#MCA2285GA) detection antibodies were used.

[0369] Example 2: Production of human C5aR-specific antibodies from the HuCAL PLATINUM® library For antibody production, the HuCAL Platinum® library was used to select antibodies specific to human C5aR. The HuCAL PLATINUM® library is a phagemide library based on the HuCAL concept (Knappik et al., (2000) J Mol Biol 296:57-86), and uses CysDisplay® technology to present Fab on the phage surface (Lohning et al., International Publication No. 2001 / 05950).

[0370] To identify human C5aR-specific antibodies, a panning strategy was performed using human and cynomolgus monkey C5aR antigens to select species-crossreactive antibodies. Each panning strategy consisted of at least three individual selection rounds against various C5aR antigens (either soluble recombinant antigens or those overexpressed on cells).

[0371] Although the overall homology between cynomolgus monkey and human C5aR is quite high at 90%, the extracellular domain has only 75% protein sequence identity. In fact, identifying cynomolgus monkey C5aR cross-reactive antibodies has proven difficult, and the ancestral antibodies MAB#1 and MAB#2 are among several candidates that reveal cell binding specific to human C5aR expressed on cells, cross-reactivity to cynomolgus monkey C5aR, and no binding to other related GPCRs.

[0372] Bead-based solution panning was performed against the peptide corresponding to the N-terminus (NT) of human C5aR, and as a result, ancestral antibodies of MAB#1 and MAB#2 were identified that are specific to human and cynomolgus monkey C5aR and can bind to full-length human C5aR expressed on cells.

[0373] To further improve affinity and specificity for human and cynomolgus monkey C5aR, modified CHO Flp-In cells overexpressing either human or cynomolgus monkey C5aR were used, and these clones were subjected to two consecutive affinity maturation panning cycles. Furthermore, antibody modifications were performed to remove potential post-translational modification sites (PTM motifs) and for germline purposes to further improve specificity.

[0374] In this final stage of the modification process, MAB#1 and MAB#2 were identified as potential therapeutic candidates. Since both candidates MAB#1 and MAB#2 originate from the same ancestor, they share similar amino acid sequences and in vitro characteristics.

[0375] These two antibodies are further described in the examples outlined below.

[0376] Example 3: Production of human C5aR-specific antibodies Both MAB#1 and MAB#2 are human IgG1f isotypes, but have been modified in the Fc region to eliminate the antibody's ability to mediate immunoeffector function. The Fc region contains five amino acid substitutions compared to the wild-type human IgG1 Fc region, namely L234A, L235E, G237A, A330S, and P331S (h_IgG1f_AEASS), which are numbered according to the EU index.

[0377] This antibody consists of a heavy chain framework VH3-15 and an antibody light chain framework Lambda-1.

[0378] Transient antibody production - Advanced Micro-scale production HKB11 Transient gene transfer was performed into eukaryotic HKB11#52 cells using mammalian expression vectors encoding the heavy and light chains of MAB#1 or MAB#2 (human IgG1_AEASS), respectively. Seven days after gene transfer, the cell culture supernatant was collected and subjected to protein A affinity chromatography (MabSelect SuRe│GE Healthcare) using a liquid handling station. The sample remained in neutralized elution buffer (NaPS: 137 mM NaPhosphate, 81 mM NaCl, pH 7). The sample was sterile filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denaturation and reduction conditions using CE-SDS (LabChip GXII│Perkin Elmer). UHP-SEC was performed to analyze the IgG preparation in its native state.

[0379] Transient antibody production - exploratory scale production in CHO Transient gene transfer was performed into CHO3-E7 cells using mammalian expression vectors encoding the heavy and light chains of MAB#1 or MAB#2 (human IgG1_AEASS), respectively. Six days after gene transfer, the cell culture supernatant was collected and subjected to standard protein A affinity chromatography (MabSelect SuRe│GE Healthcare). Unless otherwise specified, the buffer was changed to 1× Dulbecco's PBS (pH 7.2│Invitrogen), and the sample was sterile filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denatured, reduced, and non-reduced conditions using CE-SDS (LabChip GXII│Perkin Elmer). UHP-SEC was performed to analyze IgG preparations in their native state.

[0380] Result: Transient production Table 9 summarizes the data on production quality (SEC monomer content) and the productivity of MAB#1 and MAB#2. Overall, acceptable monomer content (>95%) and yield (>55 mg / L in HKB11 cells) were achieved. The volume yield derived from transient exploratory scale production of CHO3E-7 was also within the expected range.

[0381] [Table 22]

[0382] Creating a stable HKB11 pool Two vector systems were used for simultaneous gene transfer to create an HKB11#52 pool that stably expresses MAB#1 or MAB#2.

[0383] To facilitate pool selection, these vectors contained zeosin and neosin-resistant cassettes. These two vectors were transiently and simultaneously transferred into actively dividing HKB11#52 cells in a 1:1 ratio. One day after transfer, selection was initiated by adding 160 μg / mL zeosin and 800 μg / mL genethecin to the cell suspension. During selection, cell number and viability initially decreased. Twenty days after transfer, cell harvesting was initiated. Once approximately 80% viability was reached, the stable pool was expanded to the desired volume depending on the required amount. Cell culture supernatant from batch-produced cells was collected six days after seeding.

[0384] Large-scale purification of MAB#1 and MAB#2 MAB#1 and MAB#2 were purified from the cell culture supernatant of a stable pool of HKB11#52 via protein A affinity chromatography (MabSelect SURE│GE Healthcare) using 100 mM citrate and 150 mM NaCl pH 3.5 as the elution buffer. After incubation at pH 3.5 for 60 minutes, the samples were neutralized. The buffer was changed to 150 mM histidine pH 6.0, and the samples were sterile filtered (0.2 μm pore size). Protein concentrations were determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed under denatured, reduced, and non-reduced conditions using CE-SDS (LabChip GXII│Perkin Elmer). UHP-SEC was performed to analyze the IgG preparations in their native state.

[0385] Results Large-scale production As summarized in Table 10, the production of MAB#1 and MAB#2 resulted in favorable yield, purity, and integrity.

[0386] [Table 23]

[0387] control antibody Various control antibodies were prepared and included in the experiment for comparative purposes.

[0388] RefMAB#1: Benchmark antibody The nucleotide sequences encoding the VH and VL regions from the human C5aR-specific antibody "IPH5401" were searched for in U.S. Patent Application Publication No. 2013 / 0295116 (NOVO NORDISK-clone 32F3A6GL). The nucleotide sequences were synthesized as linear DNA fragments, either in-house or by an external company, along with appropriate adjacent regions (e.g., appropriate restriction enzyme recognition sites, linker sequences). Using standard molecular biological methods, the DNA fragments were cloned into appropriate mammalian IgG expression vectors encoding the heavy and light chains of human IgG1_AEASS as described above. RefMAB#1 was transiently produced as described above. The heavy and light chain amino acid sequences of RefMAB#1 are shown in Table 3.

[0389] Further control antibodies: An in-house negative isotype control antibody (MOR03207) with specificity to chicken egg lysozyme and an in-house positive control antibody (anti-C5aR antibody) with specificity to the N-terminus of human C5aR were transiently produced using either human IgG1 or human IgG1_AEASS format, as described above.

[0390] Characteristic evaluation of the coupling properties of MAB#1 and MAB#2 Example 4: Determination of monovalent affinity for MAB#1 and MAB#2 to C5aRN terminal peptide using SPR method Using the Biacore T200 instrument (Biacore, GE Healthcare), K12 was captured via IgG capture setting at 25°C. DThe determination was made. Using standard EDC-NHS amine coupling chemistry, approximately 500 RU of IgG, diluted in HBS-EP+, pH 7.4, was captured on a CM5 chip (Biacore, GE Healthcare) immobilized with anti-human Fc antibody (GE Healthcare). Reference flow cell 1 was simply activated and deactivated. Using HBS-EP+ (GE Healthcare) as the running buffer, kinetic measurements were performed using six different human or cynomolgus monkey C5aR_NT biopeptide concentrations (SEQ ID NO: 13 or SEQ ID NO: 14, respectively) (2n serial dilutions, 2000-62.5 nM) (injection time 300 s; dissociation time 600 s; flow rate 30 μL / min). After each cycle, the sensor chip was regenerated to remove the bound peptide / antibody complex with a 3 × 20 s injection of 3 mM MgCl2. A blank injection of running buffer was used for dual reference. on and k off To determine the rate constant, all sensorgrams were fitted using Biacore T200 Evaluation Software 3.1 (Biacore, GE Healthcare), and these were used to determine the K D The following was calculated: The raw data was fitted to a 1:1 joined model, and the parameter R max I set it to local and set RI to 0.

[0391] result The results are summarized in Table 11. Both antibodies have similar binding properties to the human C5aR peptide, and K D The values ​​were in the low two-order-of-a-kind nanomolar range, indicating that the binding to the cynomolgus monkey C5aR peptide was weaker.

[0392] [Table 24]

[0393] Example 5: Determination of apparent affinity (divalent) of MAB#1 and MAB#2 to C5aRN terminal peptide using Octet method Using the Octet HTX instrument (ForteBIO, Pall Life Sciences), apparent K via C5aR_NT-bio peptide capture setting D The determination was performed at 27°C. A peptide of approximately 0.03 nm, diluted in PBS, pH 7.4, was placed on a streptavidin (SA) sensor (ForteBIO, Pall Life Sciences). Seven different IgG concentrations (3-fold serial dilutions, Octet buffer (PBS, 0.05% (v / v) Tween-20, 0.1% (w / v) BSA)) were used, with concentrations ranging from 200 to 0.27 nM for human C5aR_NT-bio peptide (SEQ ID NO: 13) and 1000 to 1.4 nM for cynomolgus monkey C5aR_NT-bio peptide (SEQ ID NO: 14) at 480 s association time and 900 s dissociation time) for kinetic measurements. After each dissociation step, the sensor was regenerated to remove the bound antibody (3 × 30 s Gly / HCl, pH 1.5). on and apparent k off To determine the rate constant, all sensorgrams were fitted using Octet Data Analysis Software 10.0 (ForteBIO), and these were used to determine the apparent K D The result was calculated (using a 1:1 joint model).

[0394] result The results are summarized in Table 12. Both antibodies showed an apparent K in the 2- to 3-order-of-magnitude picomolar range. D We revealed strong binding to human C5aR peptide in a bivalent form at a specific value. Binding to cynomolgus monkey C5aR peptide was approximately 1 / 1000th. The weaker binding observed to cynomolgus monkey C5aR peptide was mainly due to fast k off This is thought to be due to the speed. Regarding the apparent affinity for human C5aR_NT, MAB#2 showed a binding affinity that was about one-tenth of that of MAB#1 (K D (290 pM vs. 20 pM).

[0395] [Table 25]

[0396] Example 6: Determination of the apparent affinity (bivalent) of MAB#1 for the full-length C5aR expressed on cells by using KinExA Since C5aR belongs to the GPCR family, it is very difficult to produce a recombinant full-length antigen that can be used for SPR measurement. Therefore, Flp-In CHO cells stably expressing human C5aR or cynomolgus C5aR and KinExA measurements were performed.

[0397] Method Using a KinExA3200 instrument (Sapidyne Instruments), the apparent K D in C5aR-expressing cells was determined at RT. PBS (Gibco) supplemented with BSA (1 mg / mL) and 0.02% (v / v) NaN3 was used as the assay buffer. MAB#1 (concentrations: 2 nM and 30 pM) was used as the analyte and, as titrants, Flip-In CHO hC5aR_V5 / His cells (final concentrations 3 Mio cells / mL and 1 Mio cells / mL, 2n serial dilutions) or Flip-In CHO cyC5aR_V5 / His cells (final concentrations 25 Mio cells / ml and 6 Mio cells / ml, 2n serial dilutions) were used and equilibrated overnight at RT in an overhead shaker. After equilibration, the samples were centrifuged and the supernatants were used for analysis. Polymethylmethacrylate (PMMA) beads coated with MabSSL (GE Healthcare) were used to determine the free concentration of MAB#1 and anti-human Fab2 Alexa Fluor 647 (500 ng / mL) was used for detection. Using KinExA software, the apparent K D was obtained by "n-curve analysis" that simultaneously fits all of a certain curve to a single K D value.

[0398] [[ID=​​​​We revealed strong binding to full-length human C5aR at a specific value. Binding to full-length cynomolgus monkey C5aR was approximately 3 nM with an apparent K D The value is estimated to be approximately 20 × weak. Similar findings regarding binding to cynomolgus monkey C5aR have been previously observed in Octet and Biacore measurements (see Examples 4 and 5).

[0399] [Table 26]

[0400] Example 7: Binding of MAB#1 and MAB#2 to full-length C5aR expressed on Flp-In CHO cells and whole blood-derived neutrophils (FACS analysis) Cells that bind to CHO Flp-In cell lines stably expressing various full-length C5aR antigens and related GPCRs, as well as cells that bind to purified human or cynomolgus monkey neutrophils endogenously expressing human or cynomolgus monkey C5aR, were investigated via FACS. Cynomolgus monkey neutrophils were obtained from whole blood of cynomolgus monkeys from three different animals (LPT Hamburg).

[0401] method C5aR-CHO Flp-In cell lines were blocked, and IgG was added either in serial dilutions or as a single (high) concentration of 300 nM. For detection of IgG binding, R-phycoerythrin (R-PE)-complexed anti-human IgG (Fc-gamma fragment specific) 2 antibody was added, and fluorescence was measured using a FACS array or Novocyte instrument.

[0402] Neutrophils were purified from EDTA whole blood samples using MACSexpress Neutrophil Isolation Cocktail from Miltenyi Biotec. Briefly, cells were isolated using this kit by magnetic labeling and negative magnetic separation. After erythrocyte removal, these cells were stained by serial dilution IgG followed by detection antibodies specific to the Alexa Fluor 647-complexed anti-human F(ab)2 fragment. Measurements were performed using a FACS array instrument. FACS data were evaluated using FlowJo and entered into GraphPad Prism (v4.0), where a sigmoid dose-response curve was fitted using nonlinear regression, and EC50 was calculated.

[0403] result The results are summarized in Table 14 and Figures 1 and 2. Figures 1A and 1C show the binding of MAB#1, MAB#2, and RefMAB#1 to human and cynomolgus monkey C5aR present on modified CHO cells expressing individual full-length receptors. Overall, EC 50 Remarkably similar binding curves for human C5aR at nearly identical concentrations were determined for MAB#1, MAB#2, and RefMAB#1. Similar results were obtained for cynomolgus monkey C5aR for MAB#1 and MAB#2. As expected, RefMAB#1 demonstrated no binding to cynomolgus monkey C5aR expressed on CHO cells.

[0404] Binding to cynomolgus monkey and human C5aR was also confirmed using purified neutrophils obtained from human or cynomolgus monkey whole blood (Figures 1B and D). Again, EC binding in human and cynomolgus monkey neutrophils was confirmed. 50 Extremely similar binding curves with nearly identical values ​​were observed for MAB#1. Interestingly, MAB#2 showed a generally lower signal relative to background levels in cynomolgus monkey neutrophils compared to MAB#1. This finding was not observed when analyzing binding to cynomolgus monkey C5aR expressed on CHO cells. For RefMAB#1, a lack of binding to cynomolgus monkey neutrophils was confirmed.

[0405] Due to the low sequence homology (66% overall identity) to rodent C5aR, non-cross-reactivity to rat and mouse C5aR was expected. Indeed, when tested at a 300 nM IgG concentration, neither MAB#1 nor MAB#2 showed any significant binding to rat or mouse C5aR expressed on CHO-Flp cells (Figure 2). To rule out cross-reactivity to all other members of the C5aR subfamily, binding to full-length human C5L2, ChemR23, FPR1, and C3aR expressed on CHO Flp-In cells was also determined via FACS (compared to non-gene-transferred parental CHO Flp-In cells). As shown in Figure 2, even at a 300 nM IgG concentration, no significant cell binding of MAB#1 and MAB#2 to any C5aR-related GPCR or to parental CHO cells was detectable.

[0406] In summary, both MAB#1 and MAB#2 demonstrated specific binding to human and cynomolgus monkey C5aR.

[0407] [Table 27]

[0408] Example 8: Binding of MAB#1 and MAB#2 to full-length human C5aR presented on virus-like particles (VLPs) - Binding to native variants of human C5aR We report two natural variants of human C5aR (SEQ ID NO: 1; D / K variant and SEQ ID NO: 2; N / N variant).

[0409] To compare the binding of MAB#1 and Mab#2 to two native mutants, VLPs expressing one of these two C5aR mutants were prepared as described in Example 1. Since MAB#1 and Mab#2 do not cross-react to mouse C5aR, a VLP expressing mouse C5aR was included as a negative control.

[0410] method For the evaluation of binding, the prepared VLPs were coated overnight, and after the blocking treatment step, IgG titrants were added the next day. Binding of IgG to the coated antigen was detected using an alkaline phosphatase-conjugated anti-human IgG2 antibody and AttoPhos as the substrate.

[0411] Results The results are summarized in Table 15 and shown in Figure 3. Both MAB#1 and MAB#2 revealed equivalent titration curves for both native variants of human C5aR, and the EC 50 concentrations were equal. As expected, binding to mouse C5aR was not detected. Based on these data, high-affinity binding of MAB#1 and MAB#2 to human C5aR can be predicted in vivo, independent of the native variants present on individual target cells.

[0412] [Table 28]

[0413] Example 9: Protein Panel Profiling (3P) In a common 3P assay, potential non-specific off-target binding to MAB#1 was determined.

[0414] Method: Protein panel profiling was performed primarily as described by Frese et al. (mAbs 5:2, 279-287; March / April 2013). Thirty-two different proteins and controls were coated at a concentration of 1.0 μg / mL on two 384-well MSD standard plates overnight at 4°C. The coating solution was discarded, and the plates were blocked with 50 μL of 3% (w / v) BSA in PBS for 1 hour on a microtiter plate shaker (approximately 500 rpm) at RT, followed by three washes with 50 μL of wash buffer (PBS containing 0.05% (v / v) Tween20). IgG samples were diluted to 100 nM and 10 nM in assay buffer (PBS containing 0.5% (w / v) BSA and 0.05% (v / v) Tween20). As controls, the isotype control antibody MOR03207 (IgG1f_AEASS) and assay buffer were used. 30 μL of sample and control were added to each well and incubated on a microtiter plate shaker at RT for 3 hours. The plate was washed three times, 30 μL of detection antibody (ECL-labeled anti-human Fab) was added to each well, and incubated on a microtiter plate shaker for 1 hour (approximately 500 rpm). After washing the MSD plate and adding 35 μL / well of MSD read buffer T containing surfactant, the electro-chemiluminescence signal was detected using a SECTOR Imager S600 instrument (Meso Scale Diagnostics).

[0415] For evaluation, the binding signal of an antibody sample against a certain protein was divided by the individual binding signals of the reference antibody MOR03207 to obtain the binding rate (BR). Next, the cumulative binding rate (CBR) of all proteins excluding the control (25 in total) was calculated: a CBR of 150 or less indicates IgG with no detectable nonspecific binding. A value above 150 indicates IgG with increased nonspecific binding compared to the reference antibody MOR03207.

[0416] result The results for MAB#1 and MAB#2 are summarized in Figure 10. In short, no significant nonspecific binding was detected for any of the proteins tested. Very low (MAB#2) and low (MAB#1) binding to bovine transferrin were observed. Binding to bovine transferrin was confirmed in the Octet-based binding assay, but binding to rat and cynomolgus monkey transferrin was not detected (data not shown). Therefore, the observed binding to bovine transferrin was considered insignificant.

[0417] Final antibody format and safety C5aR is expressed on various immune cells, including leukocytes, neutrophils, and lymphocytes, and human IgG1 Fc-mediated depletion of these cells needs to be prevented to avoid unwanted side effects. Therefore, the final IgG format of anti-C5aR antibodies needs to be suppressive in terms of their ability to induce any effector function during therapeutic intervention.

[0418] Both MAB#1 and MAB#2 contain five amino acid substitutions in the Fc region of human IgG1f, namely L234A, L235E, G237A, A330S, and P331S (hIgG1f_AEASS, numbered according to the EU index) to eliminate antibody-inducible effector function. The clinical safety of this format for C5aR antibody therapy has been described in the art (Wagner F et al., Annals of the Rheumatic Diseases. 2014;73:499.doi:10.1136 / annrheumdis-2014-eular.2156).

[0419] As outlined below, various assays, including binding studies to the Fcγ receptor or C1q, and in vitro ADCC and ADCP assays, confirmed that effector function induction ability was lacking in MAB#1 and MAB#2.

[0420] Example 10: Binding of MAB#1 and MAB#2 to FcRn receptors using Octet method Using the Octet HTX instrument (ForteBIO, Pall Life Sciences), apparent K2 receptors for immobilized neonatal Fc receptors (FcRn) from different species were analyzed. D The determination was performed at 27°C at pH 6.0 and 7.2. 0.5 nm biotinylated human, cynomolgus monkey, mouse, and rat FcRn were captured on a streptavidin (SA) sensor (ForteBIO, Pall Life Sciences). Kinetic measurements were performed using eight different concentrations of IgG (3n serial dilutions, 1000–0.46 nM) in Octet buffer (PBS, 0.05% / v / v) Tween-20 and 0.1% (w / v) BSA, with an association time of 240 s and a dissociation time of 180 s. After each cycle, the sensor was regenerated to remove the bound ligand / antibody complex (HBS-EP+, 2 × 30 s at pH 8.0). To determine apparent affinity, all sensorgrams were fitted using Data Analysis Software 10.0 (ForteBIO, Pall Life Sciences), and the data were fitted to a steady-state model.

[0421] result The results are summarized in Table 16. MAB#1 and MAB#2 revealed the apparent binding affinity to FcRn from species with different expected affinity ranges. (This was equivalent to the isotype control antibody MOR03207 IgG1f, and physiological binding behavior for human FcRn could be confirmed for both IgG molecules; that is, binding at neutral pH (7.2) was undetectable. Therefore, the five mutations introduced into the Fc region of the antibodies did not adversely affect human FcRn binding.)

[0422] [Table 29]

[0423] Example 11: Binding of MAB#1 and MAB#2 to human Fcγ receptor using Octet method Using Octet (ForteBIO, Pall Life Sciences), K via IgG capture setting at 27°C D The determination was made. 2.0 nm IgG, diluted in Octet assay buffer (PBS, 0.05% (v / v) Tween-20, 0.1% (w / v) BSA), was captured on a Protein A sensor (ForteBIO, Pall Life Sciences). Kinetic measurements were performed using seven concentrations of Fc gamma receptor (2n serial dilutions) in assay buffer. After each cycle, the sensor was regenerated to remove the bound ligand / antibody complex (2 × 30 s in 10 mM Gly / HCl, pH 1.5). All sensorgrams were fitted using Data Analysis Software 10.0 (ForteBIO, Pall Life Sciences). on and k off Determine the rate constant, K D Used to calculate the parameter R. max I set it up locally and fitted the 1:1 joined model with the raw data.

[0424] result The results are summarized in Table 17. For all of the Fcγ receptors tested, binding of MAB#1 and MAB#2 was either not detected at all or only very weak, confirming that the mutation introduced into the human IgG1 Fc region is effective in eliminating Fcγ receptor binding.

[0425] [Table 30]

[0426] Example 12: Binding of MAB#1 and MAB#2 to C1q using Octet method Using Octet (Fortebio Pall Life Sciences), apparent (divalent) K via IgG capture setting at 27°C D The determination was made. 2.0 nM IgG diluted in Octet buffer was captured on anti-hu Fab CH1 kappa / lambda (BAC) immobilized on a streptavidin (SA) sensor (ForteBIO, Pall Life Sciences). Kinetic measurements were performed using eight concentrations of C1q (3n serial dilutions, 500–0.69 nM) in Octet assay buffer (see above) with 240 s association and 240 s dissociation times. After each cycle, the sensor was regenerated to remove the bound ligand / antibody complex (2 × 50 mM NaOH, 1 × 10 mM Gly / HCl, pH 1.5, 30 s each). All sensorgrams were fitted using Data Analysis Software 9 (ForteBIO, Pall Life Sciences) to determine apparent affinity. The data were fitted to a steady-state model.

[0427] result The results are summarized in Table 18. As expected, binding of MAB#1 and MAB#2 to C1q (isolated from pooled human plasma) was not observable, confirming that the mutation introduced into the human IgG1 Fc region is effective in eliminating C1q binding.

[0428] [Table 31]

[0429] Example 13: In vitro activity of ADCC and ADCP of MAB#1 method Following the manufacturer's instructions, ADCC and ADCP activity against MAB#1 was tested using Promega ADCC and ADCP reporter bioassays (Cat#G7017 and Cat#G988A, respectively). This kit uses modified Jurkat cells as effector cells. These cells stably express either the FcyRIIIa receptor for ADCC, the FcγRIIa_H receptor for the V158 (high affinity) mutant and ADCP, or an NFAT response element that promotes firefly luciferase expression. Human C5aR-expressing CHO Flp-In cells were used as target cells. Binding of effector cells to the target via an antibody bridge (e.g., via MAB#1 or MAB#2) initiates a cascade of events in the NFAT pathway, resulting in the expression of firefly luciferase protein. This enzymatic reaction produces luminescence, which is proportional to the luciferase concentration, and this concentration directly correlates with ADCC or ADCP activity. ADCC or ADCP activity for MAB#1 was analyzed by plotting the mean signal against the background value.

[0430] In the case of MAB#1, since the wild-type human IgG1f version was not available, we included wild-type IgG1f and the in-house human anti-C5aR control antibody Fc-silent human IgG1f_AEASS version as positive controls. Furthermore, we included the isotype control antibody MOR03207 in both Fc-silent (hIgG1_AEASS) and wild-type (hIgG1f) versions as negative controls.

[0431] result The results are summarized in Table 19 and shown in Figures 8A (ADCP) and B (ADCC) for an IgG concentration of 10 μg / ml. MAB#1, like the Fc-silent version of the anti-C5aR control antibody and the Fc-silent version of MOR03207, did not induce FcγRIIIa or FcγRIIa_H activation of the NFAT pathway in modified effector cells in the presence of C5aR-overexpressing CHO cells. The wild-type (non-silent) version of the C5aR-specific control antibody clearly induced luciferase production in modified Jurkat cells in the presence of C5aR-expressing CHO cells.

[0432] In summary, this experiment clearly confirmed that mutations introduced into the wild-type human IgG1 Fc region are efficient in inhibiting ADCC and ADCP activity.

[0433] [Table 32]

[0434] Functional Features of MAB#1 and MAB#2 The neutralizing activity of MAB#1 and MAB#2 was analyzed in different in vitro assays monitoring C5a-inducible activation of C5aR.

[0435] Since high levels of C5a have been described under pathophysiological conditions, the ability of C5aR antagonist antibodies to neutralize the high concentrations of C5a that may be locally present at the disease site is expected to provide beneficial therapeutic effects in vivo. Therefore, in vitro experiments were also set up to reflect such in vivo pathological conditions.

[0436] Example 14: PathHunter® β-Arrestin Assay (DiscoveRx) method: Following the manufacturer's instructions, the PathHunter® β-arrestin assay was performed using DiscoverRx. Briefly, human C5a-inducible human C5aR activity was measured using β-galactosidase enzyme fragment complementation by detecting the interaction between activated C5aR and β-arrestin.

[0437] β-arrestin recruitment was induced using recombinant human C5a, and enzyme activity was measured using a chemiluminescent detection reagent from DiscoverRx. Chinese hamster ovary cells (CHO) expressing a modified version of human C5aR were seeded overnight, and serial dilutions of human C5a (R&D Systems) were added and incubated at 37°C and 5% CO2 for 1.5 hours (titration curves were constructed in the absence of an antagonist). In parallel, the titration curve for human C5a in the presence of a C5aR-specific antibody was determined (fixed IgG concentration 50 nM). To do this, IgG was added to the cells, followed by stimulation with human C5a, and incubated at 37°C and 5% CO2 for 1 hour. Results were expressed as relative luminescence units. Titration curves for human C5a in the presence and absence of antagonistic IgG were constructed via GraphPad Prism.

[0438] For the β-arrestin assay, the shift to higher doses (i.e., horizontally to the right on the dose axis) in the dose-response curve for human C5a was compared for MAB#1, MAB#2, and RefMAB#1 (Figure 4A). Furthermore, the % inhibition at three escalating C5a concentrations (1.2 nM, 11 nM, and 100 nM) was calculated and compared (Figure 4B).

[0439] result The results from the β-arrestin assay are shown in Figures 4A and 4B, and summarized in Tables 20 and 21. In the absence of antibody, the mean EC2 was measured. 50 A dose-response curve was obtained for human C5a at a concentration of 2.9 nM (Figure 4A).

[0440] When human C5a was treated with MAB#1 or MAB#2 at a final IgG concentration of 50 nM, a significant dose-response curve shift of more than 12-fold occurred to higher doses of human C5a. More precisely, in the presence of 50 nM MAB#1 or MAB#2, the mean EC2 50 A dose-response curve was obtained for human C5a at a concentration of 37 nM (Figure 4A, Table 20). In other words, the presence of MAB#1 or MAB#2 significantly reduces the C5aR activation-inducing ability of human C5, or, in the presence of MAB#1 or MAB#2, human C5a requires at least a 12-fold higher concentration to induce the same C5aR activity as its activity in the absence of the antibody.

[0441] This observation was also reflected when the percentage inhibition at three increasing C5a concentrations was calculated (Figure 4B). With 1.2 nM human C5a, all three test antibodies, MAB#1, MAB#2, and RefMAB#1, demonstrated equivalent inhibition of over 80% of C5a-inducible C5aR activity at a 50 nM IgG concentration. However, when a concentration approximately 10 times higher than that of human C5a (11 nM) was used to activate C5aR, RefMAB#1 was only able to neutralize about 50% of the C5a-inducible C5aR activity. This was quite different from MAB#1 and MAB#2, which were still able to neutralize up to 80% of the C5a-inducible C5aR activity.

[0442] This effect was even more pronounced when a concentration 100 times higher than that of human C5a (100 nM) was used to activate C5aR. In this case, RefMAB#1 showed almost no neutralizing activity, while MAB#1 and MAB#2 were still able to neutralize human C5a-inducible C5aR activity by approximately 40% and 30%, respectively.

[0443] Therefore, both MAB#1 and MAB#2 are effective in neutralizing pathophysiological C5a concentrations in vitro, and are significantly more potent than RefMAB#1.

[0444] [Table 33]

[0445] [Table 34]

[0446] Example 15: Inhibition of neutrophil and monocyte activation by MAB#1 and MAB#2 - CD11b assay The neutralizing potency of MAB#1 and MAB#2 was further determined in a functional CD11b whole blood assay corresponding to a more physiological setting. CD11b combines with CD18 to form an integrin Mac-1 complex, which acts as a multiligand receptor. CD11b is constitutively expressed on the surface of >50% of peripheral blood leukocytes; upon leukocyte activation, its expression is upregulated through the fusion of CD11b-containing secretory granules to the cell membrane. Therefore, CD11b expression is widely used as a marker of leukocyte activation both in vivo and in vitro.

[0447] C5a acts as a potent activator of human neutrophils and monocytes, inducing upregulation of the surface antigen CD11b. Therefore, we investigated the ability of MAB#1 and MAB#2 to prevent C5a-induced activation of granulocytes and monocytes by evaluating CD11b levels in whole blood-derived granulocytes and monocytes. This experiment was carried out essentially as described in U.S. Patent Application Publication No. 2013 / 0295116 (NOVO NORDISK).

[0448] method In the first assay setup, heparinized whole blood was mixed with IgG (serial diluent) and incubated at 37°C and 5% CO2 for 20 minutes. Human C5a at a standard concentration of 15 nM was added and incubated at 37°C and 5% CO2 for 20 minutes. Anti-CD11b-PE or isotype control antibody MOR03207 was added, and the plate was incubated at 37°C and 5% CO2 for 20 minutes. Finally, erythrocyte lysis buffer was added, incubated at room temperature in the dark for 15 minutes, washed the cells, and resuspended them in the lysis buffer.

[0449] In the second experimental setup, as described above, human C5a was added at a more clinically relevant pathophysiological concentration of 150 nM without modifying the remaining assay settings.

[0450] To investigate the receptor residence time of MAB#1, the assay was further adapted by extending the incubation time of heparinized whole blood with IgG from 20 minutes to 300 minutes.

[0451] Fluorescence was measured using a FACS array or Novocyte instrument. Samples were gated to remove dead cells and debris. Monocytes and granulocytes were identified and gated according to their FSC and SSC profiles. The median fluorescence intensity (MFI) of gated granulocytes and / or monocytes in the CD11b-PE channel (Yellow-A) was calculated. Results were expressed as a percentage of inhibition (% inhibition). Maximum CD11b expression (MFI) Max ) was the mean MFI of cells incubated with C5a, but without IgG. Minimum (background) CD11b expression (MFI) Min The mean MFI of cells incubated without C5a and without IgG was shown. The formula used to calculate the % inhibition for each sample was as follows: % inhibition = 100 - (((MFI 試料 -MFI Min )) / ((MFI Max -MFI Min ))×100)

[0452] We evaluated the data using FlowJo, input it into GraphPad Prism (v4.0), and fitted it to a sigmoid dose-response curve using nonlinear regression, and then performed IC. 50 I calculated it.

[0453] Results for MAB#1 using 15nM C5a ligand Table 22 summarizes the results for MAB#1 from the CD11b whole blood assay. For both gating cell populations (monocytes and granulocytes), IC in the single-order nM range was observed. 50The values ​​were determined, and there was a maximum inhibition of approximately 88% against granulocytes and 83% against monocytes.

[0454] [Table 35]

[0455] Results for MAB#1, MAB#2, and RefMAB#1 using 15nM vs. 150nM C5a ligands. In addition to the addition of 15 nM human C5a for granulocyte stimulation (as described above), a 10-fold higher concentration (150 nM) of ligand was used to mimic higher pathophysiological ligand concentrations.

[0456] Dose titrations of MAB#1, MAB#2, and RefMAB#1 were performed again, and inhibition curves were created using GraphPad Prism via a nonlinear regression function. The results are quantitatively expressed as "antagonist concentration required to induce 50% inhibition."

[0457] The results from this CD11b whole blood assay are shown in Figures 5A and 5B.

[0458] Figure 5A shows the neutralizing activity of MAB#1, MAB#2, and RefMAB#1 at 15 nM C5a, and Figure 5B shows the individual neutralizing activity at 150 nM C5a. As a quantitative reading, the IgG concentration required to achieve 50% inhibition of CD11b upcontrol was calculated and the results are shown below the x-axis in both figures.

[0459] Overall, similar observations were made regarding the β-arrestin assay (Example 14) when using incremental C5a concentrations in a gated CD11b whole blood assay on granulocytes as the initial target cells. To block up to 50% of the effect of 15 nM human C5a, a 12 nM IgG concentration was required for MAB#1 and RefMAB#1, while 17 nM was sufficient for MAB#2. However, significantly different amounts of antagonist were required to block 50% of the CD11b upregulation induced by a 10-fold higher C5a concentration (150 nM). For MAB#1, a 42 nM IgG concentration was sufficient to inhibit 50% of C5a-inducible activation of C5aR, while 7-fold higher RefMAB#1 (291 nM) was required to achieve the same blocking effect. For MAB#2, a 114 nM IgG concentration was sufficient.

[0460] Therefore, the same ranking of the three IgGs for potency (MAB#1 > MAB#2 > RefMAB#1) as already observed in the β-arrestin assay can be performed, and both MAB#1 and MAB#2 appeared to be very effective in neutralizing pathophysiological C5a concentrations in vitro, being significantly more potent than RefMAB#1.

[0461] Results for MAB#1 - Effects on receptor residence time Seow and his collaborators (Seow V et al., Sci.Rep.2016;6:24575) reported that C5a production may be localized to the cell membrane and significantly high over short but repeated periods. Therefore, it was suggested that C5aR antagonists with long residence times may be advantageous in systems involving rapid transient signaling. It was also concluded that prolonged receptor residence time, as measured in vitro, can be interpreted as an indication of duration of action and degree of in vivo efficacy.

[0462] To compare the receptor residence times of the two antibodies under similar settings as published by Seow and his collaborators, log-dose inhibition curves over 20-minute and 300-minute IgG incubations with granulocytes and monocytes present in whole blood were evaluated as described above.

[0463] The results from this experimental setup of the CD11b whole blood assay are shown in Figures 6A–D. Surprisingly, a significant increase in neutralizing activity over long incubation periods was observed in MAB#1. As shown in Figures 6A and C, the IC calculated against MAB#1 for both granulocyte and monocyte populations is shown. 50 The concentration decreased to approximately one-sixth over time (IC). 50 The values ​​are summarized in Table 23. In the case of RefMAB#1, no shift or decrease in the inhibition curve was observed after 300 minutes (Figures 6B and 6D).

[0464] [Table 36]

[0465] In short, the combination of effective neutralization of pathophysiological C5a levels and long-term elevation of efficacy is expected to be beneficial in vivo.

[0466] Example 16: C5a-induced migration of neutrophils The C5a-inducible chemotaxis of purified neutrophils was analyzed. This experiment was basically performed as described in U.S. Patent Application Publication No. 2013 / 0295116.

[0467] method Neutrophils were isolated and purified from the whole blood of three different human donors using the MACSxpress Whole Blood Neutrophil Isolation Kit, human (Miltenyi Biotec, Cat#130-104-434) and the MACSxpress Erythrocyte Depletion Kit, human (Miltenyi Biotec, CAT#130-098-196).

[0468] The efficacy of IgG in inhibiting hC5a-dependent neutrophil migration was analyzed using Boyden chamber technology with FluoroBlok® 3.0 μM pore size 96-well plates. The Boyden chamber pore membranes were coated with 1 mg / mL human fibrinogen at 37°C for 2 hours. After washing, the membranes were blocked with a solution containing 2% bovine serum albumin (BSA) at 37°C for 1 hour. Purified neutrophils were then stained with calcein and divided into groups with and without antagonist IgG (100 and 600 nM). 5 Individual stained cells were added to the upper compartment of a Boyden chamber. hC5a (R&D Systems, 10 nM) was applied to the lower compartment of the Boyden chamber. The plate was measured at 485 / 538 nm every 5 minutes at 37°C for 60 minutes using a plate reader (Tecan M1000Pro). The migration ability of calcein-stained neutrophils to the lower chamber was determined by measuring the neutrophils that passed through the fluoroblock membrane. The results are expressed as kinetic migration curves (5-60 minutes) and percentage inhibition calculated at selected time points (15, 25, and 35 minutes).

[0469] The formula used to calculate the % inhibition was as follows: % inhibition = 100 - (((RFU 試料 -RFU Min )) / ((RFU Max -RFU Min ))×100)

[0470] result Using neutrophils from three different human donors, the mean percentage inhibition of neutrophil migration at selected time points with two test IgG concentrations was calculated from three independent trials.

[0471] As shown in Figure 7, almost complete inhibition of C5a-induced neutrophil migration was achieved with MAB#1 15 minutes after migration. At 35 minutes after migration, and at the maximum IgG concentration tested (600 nM), MAB#1 still exhibited >50% inhibition. The negative isotype control antibody MOR03207 did not show any inhibitory effect on neutrophil migration.

[0472] Example 17: C5a-induced release of cytokines by macrophages Macrophages release pro-inflammatory or anti-inflammatory cytokines upon activation, depending on their polarization. Therefore, we evaluated whether blocking the C5a / C5aR interaction with MAB#1 affects C5a-induced cytokine release by M1 and M2 macrophages. Cytokine ELISA was performed to detect the production of anti-inflammatory IL-10 by M2 macrophages and pro-inflammatory IL-12 by M1 macrophages.

[0473] method In short, peripheral blood mononuclear cells (PBMCs) were prepared from whole blood of healthy human donors by density gradient centrifugation using Biocoll isolation solution and SepMate tubes (Stemcell). Monocytes were isolated from PBMCs using a CD14+ selection kit (Miltenyi Biotech) and cultured in RPMI supplemented with 10% FCS and 1×GlutaMax in 96-well plates. Monocytes plated overnight with LPS (20 ng / ml) and IFNγ (50 ng / ml) were polarized to M1 macrophages at 37°C for 24 hours, pre-warmed with MAB#1 (30 nM) for 30 minutes, and then treated with C5a (15 nM) for another 24 hours. This included M1 macrophages stimulated with C5a in the absence of MAB#1 and untreated controls. After 24 hours, the cell supernatant was analyzed by IL-12 / IL23 DuoSet ELISA (R&D Systems) according to the manufacturer's instructions. To generate M2 macrophages, monocytes were pre-differentiated into macrophages by culturing in RPMI / 10% FCS supplemented with M-CSF for 5 days, and M-CSF, IL-4, IL-13, and IL-6 (40 ng / ml each) were added on the 5th day. C5a (15 nM) + / - MAB#1 (30 nM) was added daily from the 5th to the 9th day. On the 9th day, the cell supernatant was analyzed by IL-10 DuoSet ELISA (R&D Systems) according to the manufacturer's instructions.

[0474] result While exposure to C5a led to a decrease in IL-12 production in M1 macrophages, no decrease in IL-12 levels was observed after pretreatment with MAB#1 compared to the untreated control. On the other hand, treatment with MAB#1 inhibited C5a-induced IL-10 production in M2 macrophages (see Figure 11).

[0475] In conclusion, MAB#1 efficiently inhibits C5a-induced production of anti-inflammatory IL-10 by M2 macrophages and restores pro-inflammatory IL-12 production by M1 macrophages, thereby clarifying the in vitro mechanism of action of MAB#1.

[0476] Pharmacokinetics Example 18: Pharmacokinetics The pharmacokinetic profile of MAB#1 was evaluated in male Han-Wistar rats (n=3) after a single intravenous (iv) administration of 10 mg / kg IgG.

[0477] method Plasma samples were collected from each animal via orbital venous plexus or mandibular vein puncture at the following time points: before administration, and 0.083, 1, 3, 8, 24, 48, 72, 96, 168, 240, 336, and 504 hours after administration.

[0478] The free bioactive MAB#1 concentration in rat plasma was determined using a ligand binding assay based on MSD. Briefly, a biotinylated N-terminal human C5aR peptide was coated onto the surface of a 96-well streptavidin-MSD plate. Binding samples were detected using a drug-specific anti-idiotic ECL-labeled antibody. Based on the free drug concentration in plasma, the pharmacokinetic properties of MAB#1 were evaluated using non-compartmental data analysis (NCA).

[0479] result The mean plasma concentrations over time are shown in Figure 9. In a total of three animals, the mean maximum plasma concentration of MAB#1 after a single IV dose was observed 5 minutes (i.e., 0.083 hours) after administration (T max (i.e., the time point of the first sample collection after administration). The mean volume (Vz) of the 106 mL / kg distribution was between the plasma volume and the extracellular volume (Davies et al., 1993). The mean final elimination half-life after IV administration was determined to be 9.0 days, and the mean total clearance was determined to be 0.341 mL / h / kg.

[0480] Overall, MAB#1 revealed a typical pharmacokinetic profile of human IgG1 antibody in rat plasma without cross-reactivity to rodent C5aR. No signs of anti-drug antibody (ADA)-mediated clearance were detected.

Claims

1. A pharmaceutical composition for the treatment of a subject suffering from a disease or disorder associated with an undesirable presence of C5aR, comprising an isolated antibody or antibody fragment that specifically binds to human C5aR and a pharmaceutically acceptable carrier or excipient, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR is a) Heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO: 27, HCDR 2 of SEQ ID NO: 28, HCDR 3 of SEQ ID NO: 29, Light chain complementarity determination region (LCDR) 1 of SEQ ID NO: 32, LCDR 2 of SEQ ID NO: 33, and LCDR 3 of SEQ ID NO: 34, or b) HCDR1 of SEQ ID NO: 27, HCDR2 of SEQ ID NO: 39, HCDR3 of SEQ ID NO: 40, LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 33, and LCDR3 of SEQ ID NO: 34 A pharmaceutical composition comprising, wherein the disease or disorder is at least one of an autoimmune disease, an inflammatory disease, or a proliferative disease.

2. The pharmaceutical composition according to claim 1, wherein the disease or disorder is an autoimmune disease.

3. The pharmaceutical composition according to claim 1, wherein the disease or disorder is an inflammatory disease.

4. The pharmaceutical composition according to claim 1, wherein the disease or disorder is a proliferative disorder.

5. The disease or disorder is rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, lupus nephritis, type 1 diabetes, Graves' disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease, interstitial lung disease, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjögren's syndrome, autoimmune nephritis, Good's disease. The pharmaceutical composition according to claim 1, wherein the condition is at least one of Pasture 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, antiphospholipid antibody syndrome, atherosclerosis, C3 glomerulopathy and IgA nephropathy, ischemia / reperfusion injury, peritonitis or sepsis.

6. The isolated antibody or antibody fragment that specifically binds to human C5aR is a) The variable heavy chain region (VH) of SEQ ID NO: 35 and the variable light chain region (VL) of SEQ ID NO: 36, or b) VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43 A pharmaceutical composition according to any one of claims 1 to 5, comprising:

7. The isolated antibody or antibody fragment that specifically binds to human C5aR is a) The heavy chain (HC) of SEQ ID NO: 37 and the light chain (LC) of SEQ ID NO: 38, or b) HC of SEQ ID NO: 44 and LC of SEQ ID NO: 45 A pharmaceutical composition according to any one of claims 1 to 5, comprising:

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR is of the human IgG1 class.

9. The pharmaceutical composition according to claim 8, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR comprises amino acid substitutions L234A, L235E, G237A, A330S and P331S numbered according to the EU index.

10. The pharmaceutical composition according to any one of claims 1 to 5, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR does not substantially induce effector function in vitro.

11. The pharmaceutical composition according to any one of claims 1 to 5, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR is specific to human C5aR and cross-reacts with cynomolgus monkey C5aR.

12. The pharmaceutical composition according to any one of claims 1 to 5, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR is a monoclonal antibody.

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the isolated antibody or antibody fragment that specifically binds to human C5aR is a human or chimeric antibody.

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