Humanized complement 5A receptor 1 antibodies and methods of use thereof

Abstract

The present disclosure provides, inter alia, two different formats of humanized antibodies against human complement component 5a receptor I. The disclosure also provides a method of treating a subject having a C5a / C5aR1 axis pathway dysfunction, including but not limited to ANCA-associated vasculitis, comprising administering to the subject in need thereof an effective amount of an antibody described herein or a nucleic acid encoding an antibody that binds to C5aR1, and wherein administration causes a reduction in the symptoms associated with C5a / C5aR1 associated dysfunction in the subject.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 137,089, filed on January 13, 2021, and U.S. Provisional Application No. 63 / 274,748, filed on November 2, 2021, the disclosures of each of which are hereby incorporated by reference in their entirety. Background Art

[0003] Disclosed are compositions and methods for reducing autoimmune diseases and conditions associated with complement 5a / complement 5a receptor 1, C5a / C5aR1-mediated immune inflammation. The C5a-C5aR1 axis has attracted considerable attention for therapeutic intervention to block neutrophil attraction to local sites, inhibit neutrophil activation, and inhibit vascular damage. The compositions and methods disclosed herein may comprise the step of administering a C5aR1 antagonist, as well as methods for treating subjects in need of such treatment. Summary of the Invention

[0004] The present disclosure provides, inter alia, anti-C5aR1 antibodies with increased specificity for C5aR1 and the therapeutic use of these antibodies in effectively treating diseases or conditions associated with C5 and its receptors, such as ANCA vasculitis, classic hemolytic uremic syndrome, age-related macular degeneration, rheumatoid arthritis, sepsis, severe burns, antiphospholipid syndrome, asthma, lupus nephritis, Goodpasture's syndrome, and chronic obstructive pulmonary disease. As described herein, the present disclosure is based, in part, on the identification of humanized anti-C5aR1-specific antibodies that bind to certain regions at Site I and / or Site II of C5aR1 and have significantly reduced cross-reactivity with C5aR2 or any other G protein-coupled receptor. Specifically, the anti-C5aR1 antibodies of the present disclosure are characterized by having high binding affinity for C5aR1 (e.g., having a K of less than 50 nM). D) and minimal cross-reactivity with C5aR2. This is significant because the C5aR1 antibodies of the present disclosure allow for effective inhibition of C5aR1 signaling in the presence of high C5a concentrations. Therefore, the C5aR1 antibodies of the present disclosure can be used at lower doses to achieve therapeutic effects relative to other anti-C5aR1 antibodies or C5a antibodies. As described herein, this is demonstrated by the unexpectedly high efficacy observed in functional assays relative to prior art antibodies. In addition, the highly effective Site I C5aR1 antibodies of the present disclosure compete with each other for Site I, and the highly effective Site II C5aR1 antibodies of the present disclosure compete with each other for Site II. In addition, the present disclosure provides methods and compositions for inhibiting C5aR1 and / or C5a signaling by targeting both Site I and Site II of C5aR1. Simultaneous targeting of Site I and Site II can significantly enhance inhibitory activity. For example, a combination of Site I and Site II antibodies or bispecific antibodies (e.g., biparatopic), rather than two Site II or two Site II antibodies, significantly enhances activity. The inventive anti-C5aR1 antibodies of the present disclosure promise more effective treatments for complement-mediated diseases and disorders, particularly ANCA vasculitis.

[0005] In addition, the present disclosure provides, inter alia, anti-C5aR1 antibodies comprising Fc variants having significantly reduced ADCC, ADCP, and CDC functions. As described herein, the anti-C5aR1 antibodies of the present disclosure comprise a novel combination of mutations that eliminate binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q while maintaining their ability to bind to FcRn.

[0006] In some embodiments, the C5aR1 antibodies provided herein have a wild-type IgG4 Fc domain. In some embodiments, the C5aR1 antibodies provided herein have a modified IgG4 Fc domain. In some embodiments, the modified C5aR1 antibodies include Fab arm swap mutations. In some embodiments, the modified C5aR1 antibodies further include Fc silent mutations.

[0007] The C5a-C5aR1 axis is of interest for therapeutic intervention to block neutrophil attraction to local sites, inhibit neutrophil activation and vascular destruction. The compositions and methods disclosed herein may comprise the step of administering a C5aR1 antagonist, as well as methods of treating a subject in need of such treatment.

[0008] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to at least one of the sequences of C5aR1, the antibody or antigen-binding fragment thereof comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO: 1 of C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO: 2 of C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO: 3 of C5aR1.

[0010] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein VH comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14.

[0011] In some embodiments, the VH comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0012] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising a light chain variable region (VL), wherein the VL comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:25.

[0013] In some embodiments, the VL comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 78% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 88% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence having at least 92% identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the VL comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 25.

[0014] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a VH region, wherein the VH comprises three heavy chain complementarity determining regions (HCDRs), wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the amino acid sequences of SEQ ID Nos: 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG) and 8 (SGGDNYGNPYYFDR), respectively.

[0015] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a VL region, wherein the VL comprises three light chain complementarity determining regions (LCDRs), wherein LCDR1, LCDR2 and LCDR3 sequences have SEQ ID Nos: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS) and 11 (AQYTLVPLT), respectively.

[0016] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a VH region, wherein the VH comprises three heavy chain complementarity determining regions (HCDRs), wherein the HCDR1, HCDR2 and HCDR3 sequences comprise the amino acid sequences of SEQ ID Nos: 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG) and 8 (SGGDNYGNPYYFDR), respectively; and a VL region, wherein the VL comprises three light chain complementarity determining regions (LCDRs), wherein the LCDR1, LCDR2 and LCDR3 sequences have SEQ ID Nos: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS) and 11 (AQYTLVPLT), respectively.

[0017] In one embodiment, the antibody or antibody or antigen-binding fragment thereof according to the present disclosure further comprises an Fc region.

[0018] In one embodiment, the antibody or antibody or antigen-binding fragment thereof according to the present disclosure further comprises an Fc region, wherein the Fc domains are independently selected from IgG1, IgG2, IgG3 and IgG4.

[0019] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits the interaction of complement component 5a (C5a) and C5aR1.

[0020] In one embodiment, the antibody or antigen-binding fragment thereof does not bind to C5aR2 or any other GPCR.

[0021] In one embodiment, the antibody or antigen-binding fragment thereof is humanized.

[0022] In one embodiment, the VH or VL of the antibody or antigen-binding fragment thereof has been modified to enhance the stability of the molecule.

[0023] In one embodiment, the antibody or antigen-binding fragment thereof comprises a serine or tyrosine mutation at position 96 of SEQ ID NO: 5 or SEQ ID NO: 25.

[0024] In one embodiment, the antibody or antigen-binding fragment thereof does not cross-react with mouse C5aR1.

[0025] In one embodiment, the antibody or antigen-binding fragment thereof binds C5aR1 with an affinity between 10 pM and 50 nM.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof has a dissociation constant (K) of less than about 100 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 90 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 80 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 75 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 70 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 65 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 60 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 60 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 55 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 50 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 45 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 40 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 35 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 30 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 25 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 20 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 15 nM.D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 10 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 8 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 5 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 3 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 1 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 0.5 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 0.1 nM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 100 pM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 80 pM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 50 pM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 25 pM. D ) binds to C5aR1. In some embodiments, the antibody or antigen-binding fragment thereof binds to C5aR1 with a dissociation constant (K) of less than about 10 pM. D ) binds to C5aR1. In one embodiment, the antibody or antigen-binding fragment thereof binds to C5aR1 with an affinity of 0.16 nM or less.

[0027] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 0.1 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 0.5 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 1 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 3 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 5 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 7 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 10 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 15 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 20 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 25 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 30 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 40 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 50 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 60 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 70 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 80 nM. In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 90 nM.In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 100 nM.

[0029] In one embodiment, the antibody or antigen-binding fragment thereof inhibits C5a-mediated C5aR1 Ga signaling.

[0030] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits calcium signaling.

[0031] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits CD11b expression.

[0032] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits neutropenia.

[0033] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits beta-arrestin signaling.

[0034] In one embodiment, the antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits ROS production in neutrophils.

[0035] In one embodiment, the antibody or antigen-binding fragment thereof is stable at about 4°C for up to 1 week under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 1 to 15°C for up to 1 week under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 2 to 10°C for up to 1 week under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 3 to 8°C for up to 2 weeks under one or more freeze-thaw cycles.

[0036] In one embodiment, the antibody or antigen-binding fragment thereof is stable at about 4°C for up to 2 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 1 to 15°C for up to 2 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 2 to 10°C for up to 2 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 3 to 8°C for up to 2 weeks under one or more freeze-thaw cycles.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof is stable at about 4°C for up to 4 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 1 to 15°C for up to 4 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 2 to 10°C for up to 4 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 3 to 8°C for up to 4 weeks under one or more freeze-thaw cycles.

[0038] In one embodiment, the antibody or antigen-binding fragment thereof is stable at about 4°C for up to 8 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 1 to 15°C for up to 8 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 2 to 10°C for up to 8 weeks under one or more freeze-thaw cycles. In some embodiments, the antibody or antigen-binding fragment thereof is stable at about 3 to 8°C for up to 8 weeks under one or more freeze-thaw cycles.

[0039] In one aspect, the disclosure encompasses a nucleic acid encoding any of the antibodies or antigen-binding fragments thereof described herein.

[0040] In one aspect, the disclosure encompasses a cell comprising a nucleic acid encoding any of the antibodies or antigen-binding fragments thereof described herein.

[0041] In one aspect, the present disclosure encompasses a method of making an antibody or antigen-binding fragment thereof described herein, the method comprising: culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof, and culturing the cell under conditions that allow production of the antibody or antigen-binding fragment thereof.

[0042] In one aspect, the present disclosure encompasses a method of treating an autoimmune disease using an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14, and / or the antibody or antigen-binding fragment thereof that binds human complement component 5a receptor 1 (C5aR1) comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25.

[0043] In one embodiment, a method of treating an autoimmune disease encompasses the use of an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to human complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 14, and the antibody or antigen-binding fragment thereof that binds to human complement component 5a receptor 1 (C5aR1) comprises a light chain variable region (VL), wherein the VL comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 15.

[0044] In one aspect, the present disclosure encompasses a method of treating an autoimmune disease using an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (HCDRs), wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the amino acid sequences of SEQ ID Nos: 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG), and 8 (SGGDNYGNPYYFDR), respectively, and three light chain complementarity determining regions (LCDRs), wherein the LCDR1, LCDR2, and LCDR3 sequences have SEQ ID Nos: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT), respectively.

[0045] In one aspect, the present disclosure encompasses a method of treating an autoimmune disease using an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 4 or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 4.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 87% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 93% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0047] In one aspect, the present disclosure encompasses a method of treating an autoimmune disease using an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 5 or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 5.

[0048] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 70% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 75% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 78% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 82% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 87% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence that has at least 93% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0049] In one embodiment, the present disclosure encompasses a disease caused by neutropenia caused by the use of an antibody or antigen-binding fragment thereof described herein.

[0050] In one embodiment, the neutropenia is caused by high levels of C5a.

[0051] In one embodiment, the disease is ANCA vasculitis or lupus.

[0052] In one embodiment, the disorder is rheumatoid arthritis.

[0053] In one embodiment, the disorder is a kidney disorder.

[0054] In one aspect, the present disclosure encompasses a method of inhibiting C5a signaling using a monoclonal antibody that binds to C5aR1, the monoclonal antibody comprising a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14; and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 25.

[0055] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof, comprising two pairs of antigen-binding domains, wherein the first antigen-binding domain comprises VH1 and VL1, wherein VH1 and VL1 bind C5aR1 at SEQ ID NO:3, and wherein the second antigen-binding domain comprises VH2 and VL2, wherein VH2 and VL2 bind C5aR1 at SEQ ID NO:1 or SEQ ID NO:2.

[0056] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof, comprising two pairs of antigen binding domains, wherein the antigen binding domain comprises VH1 and VL1, wherein VH1 and VL1 bind C5aR1 at SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the second antigen binding domain comprises VH2 and VL2, wherein VH2 and VL2 bind C5aR1 at SEQ ID NO: 3.

[0057] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a VH1 comprising or at least 90% identical to SEQ ID NO:14.

[0058] In one embodiment, a biparatopic antibody or antigen-binding fragment thereof comprises a VL, wherein VL1 comprises the amino acid sequence of SEQ ID NO: 15 or is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 84% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 15.

[0059] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a VH2, wherein the VH2 comprises the amino acid sequence of SEQ ID NO: 16 or is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH2 comprises an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 16.

[0060] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a VL2, wherein VL2 comprises the amino acid sequence of SEQ ID NO: 17 or is at least 90% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 84% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 88% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 92% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VH2 comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 17.

[0061] In one aspect, the present disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 3, the biparatopic antibody or antigen-binding fragment thereof comprising SEQ ID NO: 12 or a heavy chain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 84% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0062] In one aspect, the present disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds to SEQ ID NO: 3, the biparatopic antibody or antigen-binding fragment thereof comprising SEQ ID NO: 13 or a light chain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 78% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 82% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 84% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 86% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 88% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 13.

[0063] In one aspect, the present disclosure encompasses a biparatopic antibody, or antigen-binding fragment thereof, comprising a heavy chain comprising SEQ ID NO: 12, or an amino acid sequence at least 85% identical to SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or an amino acid sequence at least 85% identical to SEQ ID NO: 13. In one aspect, the present disclosure encompasses a biparatopic antibody, or antigen-binding fragment thereof, comprising a heavy chain comprising SEQ ID NO: 12, or an amino acid sequence at least 90% identical to SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or an amino acid sequence at least 90% identical to SEQ ID NO: 13. In one aspect, the present disclosure encompasses a biparatopic antibody, or antigen-binding fragment thereof, comprising a heavy chain comprising SEQ ID NO: 12, or an amino acid sequence at least 92% identical to SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or an amino acid sequence at least 92% identical to SEQ ID NO: 13. In one aspect, the present disclosure encompasses a biparatopic antibody, or antigen-binding fragment thereof, comprising a heavy chain comprising SEQ ID NO: 12, or an amino acid sequence at least 95% identical to SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or an amino acid sequence at least 95% identical to SEQ ID NO: 13. In one aspect, the present disclosure encompasses a biparatopic antibody, or antigen-binding fragment thereof, comprising a heavy chain comprising SEQ ID NO: 12, or an amino acid sequence at least 99% identical to SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or an amino acid sequence at least 99% identical to SEQ ID NO: 13.

[0064] In one aspect, the present invention provides, inter alia, an anti-C5aR1 biparatopic antibody comprising a heavy chain of SEQ ID NO: 71 and a light chain of SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 85% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 90% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 92% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 97% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 98% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a heavy chain that is at least 99% identical to SEQ ID NO: 71. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 85% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 90% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 92% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 95% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 97% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 98% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody comprises a light chain that is at least 99% identical to SEQ ID NO: 72.

[0065] In one aspect, the present invention provides, inter alia, an anti-C5aR1 antibody comprising a heavy chain of SEQ ID NO: 69 and a light chain of SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 85% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 90% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 92% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 85% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 97% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 98% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 99% identical to SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 85% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 90% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 92% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 85% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 95% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 97% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 98% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody comprises a light chain that is at least 99% identical to SEQ ID NO: 70.

[0066] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof, wherein the heavy chain comprises a VH1 linked to an Fc domain.

[0067] In one embodiment, the Fc domain of the biparatopic antibody or antigen-binding fragment thereof is further linked to a scFv comprising: a VH2 comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16, and / or a VL2 comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17.

[0068] In one embodiment, the Fc domains are independently selected from IgG1, IgG2, IgG3, and IgG4.

[0069] In one embodiment, the scFv is linked to the Fc domain via a linker.

[0070] In one embodiment, the linker comprises at least 5 amino acids comprising an amino acid sequence having any one of SEQ ID NOs: 26-37. In one embodiment, the linker comprises at least 3 amino acids comprising an amino acid sequence having any one of SEQ ID NOs: 26-37. In one embodiment, the linker comprises at least 4 amino acids comprising an amino acid sequence having any one of SEQ ID NOs: 26-37. In one embodiment, the linker comprises at least 6 amino acids comprising an amino acid sequence having any one of SEQ ID NOs: 26-37. In one embodiment, the linker comprises at least 7 amino acids comprising an amino acid sequence having any one of SEQ ID NOs: 26-37.

[0071] In one embodiment, the VH2 and VL2 of the biparatopic antibody comprising SEQ ID NO: 16 and SEQ ID NO: 17 are connected to each other via a linker.

[0072] In one embodiment, the linker comprises 1 to 10 repeats of SEQ ID NO:31.

[0073] In one embodiment, the VH2 and VL2 of the biparatopic antibody comprising amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17 or 90% identical thereto further comprises one or more mutations to improve the thermal stability of the biparatopic antibody.

[0074] In one embodiment, the biparatopic antibody mutations to improve thermal stability of the biparatopic antibody comprise incorporation of cysteines at position 559 of SEQ ID NO: 12 and at position 630 of SEQ ID NO: 12.

[0075] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof that binds to C5aR1 inhibits the interaction of complement component 5a (C5a) with human C5aR1.

[0076] In one embodiment, the biparatopic antibody or antigen-binding fragment thereof does not bind to C5aR2.

[0077] In one embodiment, the biparatopic antibody is humanized.

[0078] In one embodiment, the biparatopic antibody does not cross-react with mouse C5aR1.

[0079] In one embodiment, the biparatopic antibody is stable at 4°C for up to 2 weeks with one freeze-thaw cycle. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof is stable at about 1 to 15°C for up to 2 weeks with one freeze-thaw cycle. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof is stable at about 2 to 10°C for up to 2 weeks with one freeze-thaw cycle. In some embodiments, the biparatopic antibody or antigen-binding fragment thereof is stable at about 3 to 8°C for up to 2 weeks with one freeze-thaw cycle.

[0080] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis.

[0081] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of high C5a concentrations.

[0082] In one embodiment, the biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 10 nM.

[0083] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits C5a-mediated C5aR1 Ga signaling.

[0084] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits beta-arrestin signaling.

[0085] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits ROS production in neutrophils.

[0086] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits calcium signaling.

[0087] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits CD11b expression.

[0088] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutropenia.

[0089] In one aspect, the disclosure encompasses a nucleic acid encoding a biparatopic antibody described herein.

[0090] In one aspect, the disclosure encompasses a cell comprising a nucleic acid encoding a biparatopic antibody described herein.

[0091] In one aspect, the disclosure encompasses a method of making a biparatopic antibody described herein, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof, and culturing the cell under conditions that allow production of the antibody or antigen-binding fragment thereof.

[0092] In one aspect, the disclosure encompasses a method of treating an autoimmune disease using a biparatopic antibody, wherein the antibody binds human complement component 5a receptor 1 (C5aR1), comprising: a first VH and a first VL: VH1 and VL1, wherein VH1 comprises SEQ ID NO: 14, or an amino acid sequence with at least 90% identity to SEQ ID NO: 14, and wherein VL1 comprises SEQ ID NO: 15, or an amino acid sequence with at least 90% identity to SEQ ID NO: 15; and a second VH and a second VL: VH2 and VL2, wherein VH2 comprises SEQ ID NO: 16, or an amino acid sequence with at least 90% identity to SEQ ID NO: 16, and VL2 comprises SEQ ID NO: 17, or an amino acid sequence with at least 90% identity to SEQ ID NO: 17.

[0093] In one embodiment, the present disclosure encompasses a method of using the antibodies or antigen-binding fragments thereof described herein to treat an autoimmune disease caused by neutropenia.

[0094] In one embodiment, the neutropenia is caused by high levels of C5a.

[0095] In one embodiment, the disease is ANCA vasculitis or lupus.

[0096] In one embodiment, the disorder is rheumatoid arthritis.

[0097] In one embodiment, the disorder is a kidney disorder.

[0098] In one embodiment, the disorder is stroke.

[0099] In one embodiment, the monospecific or biparatopic C5aR1 antibody of any one of the preceding embodiments, wherein the antibody comprises a modified IgG1, IgG4, or IgG2 constant domain.

[0100] In one embodiment, antibody C5aR1 comprises a modified IgG4 Fc domain.

[0101] In one embodiment, the modified IgG4 Fc domain comprises substitutions at positions F234, L235 and / or D265.

[0102] In one embodiment, the IgG4 Fc substitution at position F234 is to a hydrophobic amino acid selected from alanine, valine, leucine, isoleucine, phenylalanine, or tryptophan.

[0103] In one embodiment, the IgG4 Fc at position F234 is substituted with valine.

[0104] In one embodiment, the IgG4 Fc at position L235 is substituted with an acidic amino acid.

[0105] In one embodiment, the IgG4 Fc at position L235 is substituted with an acidic amino acid selected from glutamic acid or aspartic acid.

[0106] In one embodiment, the IgG4 Fc at position L235 is substituted with aspartic acid.

[0107] In one embodiment, the IgG4 Fc at position D265 is substituted with a non-polar amino acid.

[0108] In one embodiment, the IgG4 Fc substitution at position D265 is to a non-polar amino acid selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, and valine.

[0109] In one embodiment, the IgG4 Fc at position D265 is substituted with glycine.

[0110] In one embodiment, the antibody of any one of the previous embodiments further comprises a substitution at S228.

[0111] In one embodiment, the substitution at S228 is proline.

[0112] In one embodiment, the monospecific or biparatopic C5aR1 antibody of any one of the preceding embodiments, wherein the antibody comprises a modified IgG4 constant domain comprising a combination of F234V, L235E, and D265G substitutions.

[0113] In one embodiment, the present disclosure provides a method of reducing or preventing antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity using the monospecific or biparatopic C5aR1 antibody of any of the previous embodiments.

[0114] In one aspect, the present disclosure encompasses an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), comprising: a heavy chain variable region (VH) of SEQ ID NO: 14; a light chain variable region (VL) of SEQ ID NO: 25; and a modified Fc domain comprising substitutions F234V, L235E, and D265G.

[0115] In one aspect, the present disclosure encompasses an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), comprising: a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID NO: 6 (NYWMH), a HCDR2 of SEQ ID NO: 7 (YLNPSSGYTKYAQKFQG), and a HCDR3 of SEQ ID NO: 8 (SGGDNYGNPYYFDR); a light chain variable region (VL) comprising a LCDR1 of SEQ ID NO: 9 (RASQSIVHSNGNTYLH), a LCDR2 of SEQ ID NO: 10 (KVSNRFS), and a LCDR3 of SEQ ID NO: 11 (AQYTLVPLT); and a modified Fc domain comprising substitutions F234V, L235E, and D265G.

[0116] In one aspect, the present disclosure encompasses an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising the heavy chain of SEQ ID NO:69 and the light chain of SEQ ID NO:70.

[0117] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds complement component 5a receptor (C5aR1), the biparatopic antibody or antigen-binding fragment thereof comprising: a light chain comprising LCDR1 of SEQ ID NO:9 (RASQSIVHSNGNTYLH), LCDR2 of SEQ ID NO: 10 (KVSNRFS), and LCDR3 of SEQ ID NO: 21 (AQSTLVPLT); and

[0118] a heavy chain comprising a HCDR1 of SEQ ID NO: 6 (NYWMH), a HCDR2 of SEQ ID NO: 7 (YLNPSSGYTKYAQKFQG), and a HCDR3 of SEQ ID NO: 8 (SGGDNYGNPYYFDR); a modified Fc domain comprising substitutions F234V, L235E, and D265G; and a scFv comprising a LCDR4 of SEQ ID NO: 22 (RSSQSLVHSNGNTYLN), a LCDR5 of SEQ ID NO: 23 (KVSNRLS), a LCDR6 of SEQ ID NO: 24 (SQSTHVPYT), a HCDR4 of SEQ ID NO: 18 (AYAMS), a HCDR5 of SEQ ID NO: 19 (SISTGGNTYYADSVKG), and a HCDR6 of SEQ ID NO: 20 (GYQRFSGFAY), wherein the scFv is linked to the modified Fc domain.

[0119] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds complement component 5a receptor (C5aR1), the biparatopic antibody or antigen-binding fragment thereof comprising: a first light chain variable region (VL) of SEQ ID NO: 15; a first heavy chain variable region of SEQ ID NO: 14; a modified Fc domain comprising substitutions F234V, L235E, and D265G; and a second light chain variable region (VL) comprising SEQ ID NO: 17 and a second heavy chain variable region (VH) of SEQ ID NO: 16,

[0120] wherein the scFv is linked to a modified Fc domain.

[0121] In one aspect, the disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds complement component 5a receptor (C5aR1) comprising the light chain of SEQ ID NO:72 and the heavy chain of SEQ ID NO:71.

[0122] In one aspect, the present disclosure encompasses a biparatopic antibody or antigen-binding fragment thereof that binds complement component 5a receptor (C5aR1), the biparatopic antibody or antigen-binding fragment thereof comprising: a first light chain variable region (VL) of SEQ ID NO: 25; a first heavy chain variable region (VH) of SEQ ID NO: 14; and an scFv comprising a second light chain variable region (VL) of SEQ ID NO: 17 and a second heavy chain variable region (VH) of SEQ ID NO: 16, wherein the scFv is linked to an Fc domain. In one embodiment, the biparatopic antibody or antigen-binding fragment thereof comprises a modified Fc domain comprising substitutions F234V, L235E, and D265G such that the scFv is linked to the modified Fc domain.

[0123] In one aspect, the present disclosure encompasses an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) of SEQ ID NO: 43; and a light chain variable region (VL) of SEQ ID NO: 48. In one embodiment, the antibody further comprises a modified Fc domain comprising substitutions F234V, L235E, and D265G.

[0124] In one aspect, the present disclosure encompasses an antibody or antigen-binding fragment thereof that binds complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) of SEQ ID NO: 14; and a light chain variable region (VL) of SEQ ID NO: 15. In one embodiment, the antibody further comprises a modified Fc domain comprising substitutions F234V, L235E, and D265G. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 Figure 1 is an exemplary schematic diagram showing the pathogenesis of ANCA vasculitis.

[0126] Figure 2 is an exemplary schematic diagram of two classes of antibodies described in this disclosure.

[0127] Figure 3A is an exemplary schematic diagram of an exemplary biparatopic antibody described herein comprising a scFv linked to the heavy chain Fc domain of a Fab. Figure 3B is a schematic diagram of an exemplary biparatopic antibody comprising a scFv linked to a light chain of a Fab as described herein.

[0128] Figure 4AExemplary graphs showing binding of an exemplary humanized site II antibody (c2139) and an exemplary biparatopic antibody (c2137-el711) to C5aR1 using ELISA as described in the present disclosure. Figure 4B Exemplary graphs showing binding of different batches of an exemplary site II antibody (c2139) and an exemplary biparatopic antibody (c2137-e1711) in U937-C5aR1 cells. Figure 4C Figure 2 is a graph showing binding of different batches of an exemplary site II antibody (c2139) and an exemplary biparatopic antibody (c2137-el711) in human neutrophils.

[0129] Figure 5 Exemplary graphs showing binding of an exemplary humanized site II antibody (c2139) and a biparatopic antibody (c2137-el711) to C5aR2 using ELISA as described in the present disclosure.

[0130] Figure 6A Exemplary graphs demonstrating inhibition of G-alpha signaling using exemplary humanized site II antibody c2139 and exemplary biparatopic antibody c2137-e1711 directed against C5aR1 as described in this disclosure using GeneBLAzer analysis in the presence of 10 nM C5a. Avacopan is shown as a positive control. Figure 6B Exemplary graphs demonstrating inhibition of G-alpha signaling using an exemplary humanized site II c2139 antibody and an exemplary biparatopic antibody directed against C5aR1, c2137-e1711 as described in this disclosure using GeneBLAzer analysis in the presence of 100 nM C5a. Avacopam is shown as a positive control. Figure 6C Exemplary graphs demonstrating inhibition of G-alpha signaling using exemplary humanized site II antibody c2139 and exemplary biparatopic antibody c2137-el711 directed against C5aR1 as described in this disclosure using GeneBLAzer analysis in the presence of 10 nM C5a. An anti-C5aR1 control Ab is shown as a positive control. Figure 6D Exemplary graphs demonstrating inhibition of G-alpha signaling using exemplary humanized site II c2139 antibodies and exemplary biparatopic antibodies to C5aR1, c2137-el711 as described in this disclosure using GeneBLAzer analysis in the presence of 100 nM C5a. An anti-C5aR1 control Ab is shown as a positive control.

[0131] Figure 7A

[00145] Exemplary graphs demonstrating inhibition of calcium signaling using exemplary humanized Site II antibodies as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of antibody. Figure 7B

[00145] Exemplary graphs demonstrating inhibition of calcium signaling using exemplary humanized biparatopic antibodies as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of antibody. Figure 7C

[00145] An exemplary graph showing inhibition of calcium signaling using avacopan (a known C5aR1 inhibitor) as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of an antibody.

[0132] Figure 8A

[00145] An exemplary graph showing inhibition of neutrophil chemotaxis using exemplary humanized site II antibody c2139 as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of antibody. Figure 8B

[00145] An exemplary graph demonstrating inhibition of neutrophil chemotaxis using an exemplary humanized biparatopic antibody c2137-el711 as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of antibody. Figure 8C

[00145] An exemplary graph showing the inhibition of neutrophil chemotaxis using avacopam (a known C5aR1 inhibitor) as described in the present disclosure in the presence of increasing concentrations of C5a and increasing concentrations of antibodies.

[0133] Figure 9A To illustrate exemplary graphs showing inhibition of CD11b expression in neutrophils by exemplary humanized site II antibody c2139 and exemplary biparatopic antibody c2137-e1711 as described herein, compared to avacopam, in the presence of 100 nM C5a and increasing antibody concentrations. Figure 9B To illustrate exemplary graphs showing inhibition of CD11b expression in neutrophils by exemplary humanized Site II antibody c2139 and exemplary biparatopic antibody c2137-e1711 as described herein, as compared to avacopam, in the presence of 10 nM of exemplary humanized Site II antibody and exemplary biparatopic antibody and increasing concentrations of C5a. Figure 9C To illustrate exemplary graphs showing inhibition of CD11b expression in neutrophils by exemplary humanized Site II antibody c2139 and exemplary biparatopic antibody c2137-e1711 as described herein, compared to 10 nM and 100 nM anti-C5aR1 control Ab and in the presence of 10 nM exemplary humanized Site II antibody and exemplary biparatopic antibody and increasing concentrations of C5a.

[0134] Figure 10AExemplary graphs showing inhibition of beta arrestin recruitment in the presence of 1 nM C5a compared to 10 nM avacopam in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at a dose of 1 nM each. Figure 10B Exemplary graphs showing inhibition of beta arrestin recruitment in the presence of 10 nM C5a compared to 10 nM avacopam in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at a dose of 1 nM each. Figure 10C Exemplary graph showing inhibition of beta arrestin recruitment in the presence of 100 nM C5a compared to 10 nM avacopam in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at a dose of 1 nM each.

[0135] Figure 11 To show exemplary graphs for inhibition of ROS signaling in ANCA(-) and ANCA(+) cells compared to c2139, c2137-e1711, motavizumab, and avacopan.

[0136] Figure 12A To illustrate exemplary graphs showing internalization of an exemplary humanized anti-10 nM C5aR1 antibody in C5aR1-U937 cells after 0, 6, and 12 hours as observed by an amine-bound antibody with a pH-sensitive fluorescent dye. Figure 12B To illustrate exemplary graphs showing internalization of an exemplary humanized anti-10 nM C5aR1 antibody in U937 cells after 0, 6, and 12 hours as observed by an amine-bound antibody with a pH-sensitive fluorescent dye. Figure 12C To illustrate exemplary graphs showing internalization of an exemplary humanized anti-100 nM C5aR1 antibody in C5aR1-U937 cells after 0, 6, and 12 hours as observed by an amine-bound antibody with a pH-sensitive fluorescent dye. Figure 12D To illustrate exemplary graphs showing internalization of an exemplary humanized anti-100 nM C5aR1 antibody in U937 cells after 0, 6, and 12 hours as observed by an amine-bound antibody with a pH-sensitive fluorescent dye.

[0137] Figure 13A is an exemplary graph showing binding (cross-reactivity) of an exemplary humanized C5aR1 antibody to squirrel monkey. Figure 13B is an exemplary graph showing the binding (cross-reactivity) of an exemplary humanized C5aR1 antibody to dog.

[0138] Figure 14A Exemplary schematic diagram of the study design in squirrel monkeys showing the timing of blood draws and drug administration. Figure 14B is an exemplary scatter plot of percent change from baseline in neutrophil counts for vehicle, an exemplary humanized site II antibody, and avacopam. Figure 14C Bar graph showing percent change from baseline in neutrophil counts for vehicle, exemplary humanized site II antibodies, and avacopam.

[0139] Figure 15A Exemplary schematic diagram of the study design in hC5aR1 mice showing the timing of blood draws and drug administration. Figure 15B Exemplary scatter plot of percent change from baseline in neutrophil counts for vehicle, humanized site II antibody, and avacopam. Figure 15C Exemplary bar graph of percent change from baseline in neutrophil counts for vehicle, humanized site II antibody, and avacopam.

[0140] Figure 16A is an exemplary graph showing the 21-day PK profiles of exemplary tetravalent antibodies (biparatopic antibodies) and monospecific antibodies. Figure 16B is an exemplary graph showing the 500-hour PK profile of motavizumab in mouse serum. Figure 16C is an exemplary graph showing the 500-hour PK profile of c2139 in mouse serum. Figure 16D is an exemplary graph showing the 500-hour PK profile of c2137-e1711 in mouse serum.

[0141] Figure 17A To display the monospecific C5aR1 antibody (c2139-Fcmod) and the C5aR1 biparatopic antibody (c2137-e1711-F c mod). Figure 17B To display the C5aR1 biparatopic antibody (c2137-e1711-F c Exemplary graphs of kinetic parameters for mod). Figure 17C To display the monospecific C5aR1 antibody (c2139-Fcmod) and the C5aR1 biparatopic antibody (c2137-e1711-F c Exemplary scheme of binding of mod) to C5aR2.

[0142] Figures 18A to 18B showed increased internalization of the C5aR1 antibody. Figure 18A is an exemplary graph showing internalization of monospecific antibody c2139 several hours after dissociation. Figure 18B is an exemplary graph showing increased internalization of the biparatopic antibody c2137-e1711 during association.

[0143] Figures 19A to 19B This indicates that Gα signaling is inhibited in the presence of a C5aR1 Fc-modified antibody. Figure 19A Ga signaling in the presence of C5aR1 Fc-modified antibodies in the presence of 10 nM C5a is shown. Figure 19B Ga signaling in the presence of 100 nM C5a and in the presence of C5aR1 Fc-modified antibody is shown.

[0144] Figures 20A to 20B To illustrate the effect of C5aR1 Fc-modified antibody c2137-e1711-F on the expression of C5aR1 Fc in U937-C5aR1 cells compared with avacopam and anti-C5aR1 control Ab. c Exemplary series of graphs showing inhibition of calcium signaling in the presence of c2139-Fcmod and c2139-Fcmod. Figure 20A To present an exemplary graph showing dose response curves demonstrating inhibition of calcium signaling using an exemplary Fc-modified humanized site II antibody as described in the present disclosure in the presence of increasing concentrations of antibody and 100 nM C5a. Figure 20B To show the percent inhibition of calcium signaling using an exemplary Fc-modified humanized Site II antibody as described in this disclosure in the presence of antibody and 100 nM C5a.

[0145] Figures 21A to 21D Demonstrates inhibition of calcium signaling in U937-C5aR1 cells compared to human neutrophils. Figure 21A Demonstrated inhibition of calcium signaling in U937-C5aR1 cells in the presence of 10 nM C5a. Figure 21B Demonstrated inhibition of calcium signaling in U937-C5aR1 cells in the presence of 100 nM C5a. Figure 21C Demonstrated inhibition of calcium signaling in human neutrophils in the presence of 10 nM C5a. Figure 21D Demonstrates inhibition of calcium signaling in human neutrophils in the presence of 100 nM C5a.

[0146] Figure 22A Summary of percent saturation and F norm for U937-C5aR1 cells incubated with increasing concentrations of antibodies (c2139-Fcmod and c2137-e1711-Fcmod) and 100 nM C5a after 1 hour incubation. Figure 22B Summary of percent saturation and F norm for U937-C5aR1 cells incubated with increasing concentrations of antibodies (c2139-Fcmod and c2137-e1711-Fcmod) and 100 nM C5a after 3 h incubation.

[0147] Figures 23A to 23B Display by c2137-e1711-F c Inhibition of C5a-mediated β-arrestin signaling by c2139-Fcmod and c2139-Fcmod. Figure 23A Display by c2137-e1711-F c Dose-response of inhibitory effects of mod and c2139-Fcmod on β-arrestin signaling. Figure 23B Display by c2137-e1711-F c Percent inhibition of β-arrestin signaling by mod and c2139-Fcmod.

[0148] Figures 24A to 24D Compared with avaxome and anti-C5aR1 control Ab, the C5aR1 antibody, c2137-e1711-F c Inhibition of chemotaxis in C5aR1-U937 stable cells after mod and c2139-Fcmod treatment. Figure 24A Inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of 1 nM, 3.16 nM, and 10 nM c2139-Fcmod and increasing concentrations of C5a is shown. Figure 24B Inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of 1 nM, 3.16 nM, and 10 nM C5a c2137-e1711-Fcmod and increasing concentrations of C5a are shown. Figures 24C to 24D Shown is the inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of c2137-e1711-Fcmod, in the presence of 1 nM, 3.16 nM and 10 nM anti-C5aR1 control Ab and avacopam, respectively.

[0149] Figures 25A to 25B Showcase response to c2137-e1711-F c mod and c2139-F c Mod treatment inhibits CD11b signaling. Figure 25A Inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibodies and 10 nM C5a is demonstrated. Figure 25B Inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibodies and 100 nM C5a is demonstrated.

[0150] Figures 26A to 26B Display and c2139-F c mod、c2137-e1711-F cInhibition of ROS signaling in ANCA(-) cells and ANCA(+) cells by mod, motavizumab, and avaclopan. Figure 26A Inhibition of ROS production by increasing concentrations of monospecific C5aR1 antibodies is demonstrated. Figure 26B Inhibition of ROS production by increasing concentrations of the biparatopic C5aR1 antibody is demonstrated.

[0151] Figure 27A Exemplary schematic diagram of the study design in hC5aR1 mice showing the timing of blood draws and drug administration. Figure 27B Exemplary scatter plot of percent change from baseline in neutrophil counts for vehicle, c2139, c2139-Fcmod, c2137-e1711, and c2137-e1711-Fcmod. Figure 27C Exemplary bar graph of percent change from baseline in neutrophil counts for vehicle, c2139, c2139-Fcmod, c2137-e1711, and c2137-e1711-Fcmod.

[0152] Figure 28A Figure 2 is a graphical representation of infarct size in mouse brains following treatment with the indicated doses of Fc-modified antibodies compared to 1 mg / kg PMX53. Figure 28B Figure 2 is a graphical representation of infarct size in mouse brains following treatment with the indicated doses of Fc-modified antibodies compared to 1 mg / kg PMX53.

[0153] Figures 29A to 29B Schematic representation of the pharmacokinetics of Fc-modified antibodies. Figure 29A Figure 2 is a graphical representation of the percentage of Fc-modified C5aR1 antibodies that persist in serum for 500 hours. Figure 29B Graph showing the mean concentrations in μg antibody / ml serum over 500 hours.

[0154] Figures 30A to 30F Figure 2 is a graphical representation of the PK and PD studies of the Fc-modified C5aR1 antibody compared with MVZ-IgG4. Figure 30A Graph showing the dose-response curve of c2139Fcmod in serum for 200 hours. Figure 30B Graph showing the dose-response curve of c2137-e1711-Fcmod in serum for 200 hours. Figure 30C Comparison of c2139Fcmod, c2137-e1711-Fcmod and MVZ-IgG4. Figure 30D Figure 2 is a computer simulation of three different concentrations of c2139 over a 500-hour period. Figure 30EFigure 3 is a computer simulation of three different concentrations of c2137-e1711 over a 500-hour period. Figure 30F Figure 2 is a computer simulation of an isotype control antibody at a concentration of 20 mg / Kg over a period of 500 hours.

[0155] Figure 31 Figure 2 is a graphical representation of the percent change in neutrophil counts in human C5aR1 mice using different doses of exemplary C5aR1 antibodies c2139-Fcmod and c2137-e1711-Fcmod.

[0156] Figures 32A to 32B Schematic representation of the internalization of exemplary C5aR1 antibodies, c2139-Fcmod, and c2137-e1711-Fcmod. Figure 32A The fluorescence intensities of c2139-Fcmod and c2137-e1711-Fcmod in U937 cells at 0, 6, and 24 hours are shown. Figure 32B Shown are the internalization of both c2137-e1711-Fcmod and c2139-Fcmod in living cells as observed by Nikon confocal experiment over a 300 min period.

[0157] definition

[0158] Antibody: As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that binds (immunoreacts with) an antigen. "Binding" or "immunoreacting with" means that the antibody reacts with one or more desired antigenic determinants. Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAbs (domain antibodies), single chains, Fab, Fab', F(ab')2 fragments, scFv, and Fab expression libraries. Antibodies can be whole antibodies, immunoglobulins, or antibody fragments.

[0159] Antibody-dependent cellular cytotoxicity: As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the lysis of human target cells by an antibody according to the present invention in the presence of effector cells.

[0160] Fab arm exchange: The term "Fab arm exchange" refers to the phenomenon in which IgG4 antibodies can exchange 'half molecules', an activity referred to herein as Fab arm exchange. Particularly in bispecific or biparatopic molecules, this results in functional monovalent antibodies with unknown specificity and, therefore, potentially reduced therapeutic efficacy. Mutations can be introduced into the Fc domain to inhibit Fab arm exchange. The S228P mutation is known to prevent IgG4 FAEs from reaching undetectable levels in vitro and in vivo.

[0161] Fc domain: As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is based on 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.

[0162] Humanized antibody: The term "humanized antibody" includes non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof, that contain minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0163] Increased ADCC: The term "increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above and / or a decrease in the concentration of antibody required to achieve half of the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase in ADCC is relative to ADCC measured using an acceptable art-recognized assay.

[0164] Monoclonal Antibody: The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0165] Multispecific antibody: As used herein, the term "multispecific antibody" refers to a binding molecule, antibody, or antigen-binding fragment thereof that is capable of specifically binding to two or more different epitopes on the same or different targets.

[0166] Biparatopic antibody: As used herein, the term "biparatopic antibody" refers to a multispecific antibody capable of binding to two different non-overlapping epitopes on the same target antigen molecule.

[0167] K i or K d : As used herein, the term "K d ” refers to the dissociation constant for a particular antibody-antigen interaction as known in the art, and for the subject compositions will apply as a parameter for the binding affinity of the targeting moiety to its cognate ligand.

[0168] IC50: As used herein, the term "IC50" refers to the concentration required to inhibit half of the maximal biological response of a ligand agonist and is generally determined by a competition binding assay.

[0169] EC50: As used herein, the term "EC50" refers to half-maximal effective concentration. The term EC50 refers to the concentration of a drug, antibody, or toxin that induces a response halfway between baseline and maximum after a specified exposure time. More simply, the EC50 can be defined as the concentration required to achieve 50% of the desired effect.

[0170] C5a: As used herein, the term "C5a" refers to complement component 5a.

[0171] C5aR1: As used herein, the term "C5aR1" refers to complement component 5a receptor 1. In some embodiments, human C5aR1 comprises SEQ ID NO: 38. In some embodiments, certain amino acids of human C5aR1 comprising SEQ ID NO: 38 have natural variants, for example (N2D and N279K), which are shown in lowercase in Table 1.

[0172] Linker: As used herein, the term "linker" refers to a molecule or group of molecules (e.g., a monomer or polymer) that connects two molecules and is generally used to place the two molecules in a preferred configuration. Many strategies can be used to covalently link molecules together. These strategies include, but are not limited to, polypeptide linkage between the N-terminus and C-terminus of a protein or protein domain, linkage via disulfide bonds, and linkage via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond produced by recombinant technology or peptide synthesis. In some embodiments, the linker may contain amino acid residues that provide flexibility. Thus, the linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length sufficient to connect the two molecules in a manner that allows the two molecules to present the correct conformation relative to each other, such that they retain the desired activity. Suitable lengths for this purpose include at least one and no more than 30 amino acid residues. In one embodiment, the linker is about 1 to 30 amino acids in length. In another embodiment, the linker is about 1 to 15 amino acids in length. In addition, the amino acid residues selected to be included in the linker peptide should exhibit properties that do not significantly interfere with the activity of the polypeptide.

[0173] Neutrophils: As used herein, the term "neutrophils" refers to the major class of white blood cells in peripheral blood. Neutrophils play an important role in phagocytosis and killing of extracellular pathogens.

[0174] scFv: As used herein, the term "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, connected to a short linker peptide of 10 to about 25 amino acids.

[0175] Fab: As used herein, the term "Fab" refers to an antibody fragment that comprises a portion of an intact antibody, including the antigen binding or variable region thereof.

[0176] Neutropenia: As used herein, the term "neutropenia" refers to a low neutrophil count. For example, in human subjects, neutropenia can range from less than 500 ANC to less than 1500 ANC (absolute neutrophil count). ANC is measured as the number of cells per microliter of blood. Neutropenia in mice is defined as <10 neutrophils / mm 3 blood.

[0177] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0178] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal. In the context of a cell-based system, the term can be used to refer to events that occur within a living cell (as opposed to, for example, an in vitro system).

[0179] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include both prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a human who goes to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or condition, but may or may not display symptoms of the disease or condition.

[0180] Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease in an activity associated with that molecule. Dysfunction of a molecule can be caused by defects associated with the molecule itself or with other molecules that directly or indirectly interact with or regulate the molecule.

[0181] Derivative: As used herein, the term "derivative" when used in connection with an antibody or C5aR1 antibody refers to a portion of the sequence of an original molecule that retains at least some of the functions and / or properties of the original molecule.

[0182] Consistency: As used herein, the term "consistency" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as known in the art, which compares the sequences of these molecules. The relationship is determined by performing. In the art, "consistency" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, and in some cases more than one nucleotide sequence or more than one. It can be measured by the match between amino acid sequence strings. "Consistency" means between a gap alignment (if any) solved by a specific mathematical model or computer program (i.e., "algorithm") and a smaller sequence of two or more sequences. The percentage of consistency match is measured.

[0183] Similarity or resemblance: As used herein, the term "similarity" is used in the art with respect to related concepts, but in contrast to "identity" and "similarity," to refer to both identity and conservative substitution matching. If two polypeptide sequences have, for example, 10 identical amino acids out of 20 amino acids and the rest are non-conservative substitutions, then both the percent identity and percent similarity are 50%. In the same example, if there are 5 or more conservative substitutions, then the percent identity is still 50%, but the percent similarity is 75%. Therefore, if there are conservative substitutions, then the percent similarity between the two polypeptides is higher than the percent identity between the two polypeptides.

[0184] Treatment: As used herein, the terms "treat," "treatment," and "treating" refer to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of a disease and / or only exhibit early signs of a disease in order to reduce the risk of developing pathologies associated with the disease.

[0185] Vector: The term "vector" refers to a polynucleotide (usually DNA) that is used to artificially carry foreign genetic material to another cell where the foreign genetic material can be replicated or expressed. Non-limiting exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. These vectors can be derived from a variety of sources, including bacterial and viral sources. A non-limiting exemplary viral source of plasmids is adeno-associated virus.

[0186] Various aspects of the present disclosure are described in detail in the following sections. The use of sections is not intended to limit the present disclosure. Each section may apply to any aspect of the present disclosure. In this application, unless otherwise indicated, the use of "or" means "and / or". As used herein, unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" include both singular and plural referents. DETAILED DESCRIPTION

[0187] The present disclosure describes antibodies, nucleic acids, and systems for their manufacture, as well as uses and methods for treating diseases associated with dysfunctional C5a / C5aR1 axis signaling, particularly autoimmune diseases such as, but not limited to, ANCA-associated vasculitis, lupus, rheumatoid arthritis, inflammatory bowel disease, C3 glomerulopathy (C3G), hidradenitis suppurativa (HS), and atypical hemolytic uremic syndrome.

[0188] Lupus nephritis, IgA nephropathy, myasthenia gravis, macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, neuralgia, COVID-19 infection, allergic asthma, chronic obstructive pulmonary disease, bullous pemphigoid, pyoderma gangrenosum, and psoriasis.

[0189] ANCA-associated vasculitis is a group of diseases characterized by destruction and inflammation of small blood vessels (granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, and microscopic polyangiitis). Anti-neutrophil cytoplasmic autoantibodies (ANCA) are the cause of ANCA-associated vasculitis. Experimental data in animal models and in vitro experiments show that primed neutrophils are activated by ANCA, which produces C5a that engages with the C5a receptor on neutrophils. This attracts and in turn triggers more neutrophils to be activated by ANCA. C5a bound to C5aR1 may play a major role in the pathogenesis of ANCA-associated vasculitis. A general schematic diagram of ANCA vasculitis is shown in Figure 1 Standard treatment is immunosuppressive therapy with glucocorticoids; these therapies are associated with a number of short-term and long-term toxicities. Avacopam, a small molecule C5aR1 binding antagonist, was recently accepted by the FDA for use in ANCA-associated vasculitis. However, in vitro data suggest that avacopam antagonism of C5a can be overcome by high concentrations of C5a. It is known that C5a concentrations at sites of inflammation in active AAV can reach up to 100 nM. It is known that under these conditions, avacopam inhibition of C5aR1 can be overcome. There is a need to develop robust inhibitors of the C5a / C5aR1 axis for use against conditions associated with dysfunction of this pathway. The present disclosure provides compositions and methods for treating conditions associated with C5a / C5aR1 axis dysfunction using biological C5aR1 antagonists.

[0190] In some aspects, two sets of exemplary antibodies that bind to and antagonize C5aR1 are provided herein. Schematic diagrams of the two sets of antibodies in this disclosure are shown in Figure 2 middle.

[0191] The energy from agonist binding to the extracellular domain transmits allosteric conformational changes to the transmembrane and intracellular domains, allowing G-protein binding and signaling. C5aR1 has two known agonists: C5a and C5a desArg C5a has a short half-life in serum because the C-terminal arginine is rapidly cleaved by carboxypeptidase N to form C5a desArg , which binds to C5aR1 with reduced affinity and exhibits biased signaling. desArg Does not signal the β-arrestin pathway and does not stimulate granulocyte release. However, C5adesArg does stimulate neutrophil chemotaxis, so there is also interest in blocking C5a desArg Binds to prevent neutrophils from migrating to sites of inflammation. C5a desArg Signaling favors chemotaxis and is less susceptible to desensitization (e.g., neutrophils continue to migrate until high concentrations of C5a are reached, rather than C5a desArg In one embodiment, an orthosteric antagonist (e.g., an antibody molecule as described herein) that blocks C5a binding also inhibits (e.g., blocks) C5a desArg In some embodiments, it is desirable to inhibit C5a binding at the site of inflammation while also inhibiting C5a in the periphery. desArg It also prevents neutrophils from migrating to sites of inflammation. Targeting C5aR1 generally leaves the membrane attack complex pathway (C5b) unaffected.

[0192] Design of monospecific C5aR1 antagonists

[0193] In some embodiments, the antibodies provided herein bind to the site defined by SEQ ID NO: 1 or SEQ ID NO: 2, also referred to as "Site I". Site I generally includes the N-terminal residues of C5aR1 (e.g., the N-terminal 37 residues) and forms a flexible random coil structure defined by SEQ ID NO: 1 or SEQ ID NO: 2. Antibody molecules that bind to Site I generally bind to all or a subset of residues in Site I. For example, antibody molecules that bind to Site I make contacts with one or more residues in Site I. In one embodiment, Site I generally includes a plurality of sulfated residues (e.g., sulfated tyrosine residues) and a plurality of Asp residues.

[0194] In some embodiments, the antibodies provided herein bind to the site defined by SEQ ID NO: 3, also referred to as "Site II". Site II generally includes additional cellular loop 2 (ECL2) and transmembrane residues that form the vestibule of C5aR1. In one embodiment, an antibody molecule that binds to Site II binds to ECL2 but does not bind or does not substantially bind to ECL1 and / or ECL3. In one embodiment, an antibody molecule that binds to Site II binds to ECL2 and ECL1 but does not bind or does not substantially bind to ECL3.

[0195] In some embodiments, the antibody molecules described herein are designed to target Site II defined by the amino acids of SEQ ID NO: 3. In some embodiments, amino acids encompassing R175 to G189 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E180 to P183 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E180 to P184 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E178 to P183 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, one or more of residues R35, H101, V176, V177, R178, E179, E180, Y181, F182, P183, P184, K185, L187, D191, I93, H194, E266, P267, S268, F272, L273, and / or K276 of C5aR1 (SEQ ID NO: 38) are critical for binding to the Site II antibodies described herein. In some embodiments, one or more of residues E180, Y181, F182, and / or P183 of SEQ ID NO: 38 are critical epitopes for binding to the Site II antibodies described herein. In one embodiment, amino acid residue W102 of SEQ ID NO: 38 is critical for binding to the Site II antibodies described herein.

[0196] The sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are provided in Table 1.

[0197] Table 1. Amino acid sequences of Site I, Site II and human C5aR1. Variants (N2D and N279K) are shown in lower case.

[0198]

[0199]

[0200] In some aspects, the exemplary humanized site 1 (SEQ ID NO: 1 or SEQ ID NO: 2) binding antibodies provided herein include a heavy chain variable region (HCVR or VH) comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39 to SEQ ID NO: 43, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and / or a light chain variable region (LCVR or VL) comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44 to SEQ ID NO: 49, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. Table 2a summarizes the amino acid sequences of the VH and VL of exemplary site 1 binding antibodies. In some embodiments, the three heavy chain complementarity determining regions, HCDR1, HCDR2, and HCDR3, are found within the HCVR or VH (SEQ ID NO: 16 or SEQ ID NO: 39 to SEQ ID NO: 43). In some embodiments, the three light chain complementarity determining regions, LCDR1, LCDR2, and LCDR3, are found within the LCVR or VL (SEQ ID NO: 17 or SEQ ID NO: 44 to SEQ ID NO: 49). It should be noted that exemplary humanized full-length site I binding antibodies may include any combination of a VH selected from the amino acid sequence of SEQ ID NO: 16 or SEQ ID NOs: 39-43, and a VL selected from the amino acid sequence of SEQ ID NO: 17 or SEQ ID NOs: 44-49.

[0201] It should be noted that the humanized full-length site I binding antibody may include any combination of the following: a VH selected from the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39-43 or any one of the amino acid sequences that are 80%, 85%, 90%, 95%, 98%, 99% identical to SEQ ID NO: 16 or SEQ ID NO: 39-43, and a VL selected from the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44-49 or any one of the amino acid sequences that are 80%, 85%, 90%, 95%, 98%, 99% identical to SEQ ID NO: 17 or SEQ ID NO: 44-49.

[0202] In some embodiments, exemplary site I binding antibody e1711 comprises a heavy chain of SEQ ID NO: 17, or an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17. In some embodiments, exemplary site I binding antibody e1711 comprises a light chain of SEQ ID NO: 16, or an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16.

[0203] In some embodiments, exemplary site I binding antibody e1711 comprises a heavy chain of SEQ ID NO: 43, or an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 43. In some embodiments, exemplary site I binding antibody e1711 comprises a light chain of SEQ ID NO: 48, or an amino acid sequence 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 48.

[0204] In some embodiments, exemplary site I-binding antibodies e1711 provided herein comprise an e11L variable light chain and an e11H variable heavy chain comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:77, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:77, and / or a light chain comprising the amino acid sequence of SEQ ID NO:78, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:78.

[0205] Table 2a. Variable regions of exemplary humanized site I binding antibodies

[0206]

[0207]

[0208] Table 2b. Heavy and light chains of exemplary humanized site I binding antibodies

[0209]

[0210] In some aspects, exemplary site II binding antibodies provided herein comprise: a HCVR or VH comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 50 to SEQ ID NO: 59, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and / or a LCVR or VL comprising the amino acid sequence of SEQ ID NO: 60 to SEQ ID NO: 68, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. Table 3 summarizes the amino acid sequences of the VH and VL of exemplary site II binding antibodies. In some embodiments, the three heavy chain complementary determining regions, HCDR1, HCDR2, and HCDR3, are found within the HCVR or VH (SEQ ID NO: 50 to SEQ ID NO: 59). In some embodiments, the three light chain complementary determining regions, LCDR1, LCDR2, and LCDR3, are found within the LCVR or VL (SEQ ID NO: 60 to SEQ ID NO: 68).

[0211] It should be noted that a humanized full-length site II binding antibody may include any combination of: a VH selected from the amino acid sequence of SEQ ID NO: 14 or SEQ ID NOs: 50-59 or having at least 85%, 90%, 95%, 99% or 100% identity to an amino acid sequence of SEQ ID NO: 14 or any one of SEQ ID NOs: 50-59, and a VL selected from the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 25 or any one of SEQ ID NOs: 60-68 or having at least 85, 90, 95, 99 or 100% identity to an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 25 or any one of SEQ ID NOs: 60-68.

[0212] Table 3. Variable regions of exemplary humanized site II binding antibodies

[0213]

[0214]

[0215] In some aspects, an exemplary site II binding antibody includes the combination of c39L (SEQ ID NO: 14) and c21H (SEQ ID NO: 25) to generate antibody c2139.

[0216] In some embodiments, the exemplary site II binding antibody c2139 provided herein comprises a c39L variable light chain and a c21H variable heavy chain, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:4, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:4, and / or a light chain comprising the amino acid sequence of SEQ ID NO:5, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:5.

[0217] In some embodiments, the exemplary site II binding antibodies c2137 provided herein comprise a c37L variable light chain and a c21H variable heavy chain, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:4, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:4, and / or a light chain comprising the amino acid sequence of SEQ ID NO:75, or a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:75.

[0218] In some aspects, the exemplary site II (SEQ ID NO: 3) binding antibody c2139 provided herein comprises a heavy chain comprising a HCVR or VH, wherein the HCVR or VH comprises three heavy chain complementary determining regions, HCDR1, HCDR2, and HCDR3, comprising SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In some embodiments, the exemplary site II binding antibodies provided herein further comprise a LCVR or VL, wherein the LCVR or VL comprises three light chain complementary determining regions, LCDR1, LCDR2, and LCDR3, comprising SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; or comprising a sequence that differs by no more than 5, 4, 3, 2, or 1 amino acid from the amino acids of SEQ ID NO: 6, 7, 8, 9, 10, and / or 11. The sequences of the heavy chains, light chains, and HCDRs and LCDRs are provided in Table 4a.

[0219] In some aspects, the exemplary site II binding antibody c2139 provided herein comprises: a HCVR or VH comprising the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 14, and / or a LCVR or VL comprising the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 25.

[0220] In one aspect, described herein is an exemplary antibody molecule c2139 that is capable of binding to complement component 5a receptor 1 (C5aR1), wherein the antibody molecule competes with C5aR1 antibody molecules that are capable of binding to one or more amino acid residues in position II (SEQ ID NO: 3).

[0221] In some embodiments, exemplary Site II binding antibodies comprise a glycine or alanine at position 89 of VH. In some embodiments, exemplary Site II binding antibodies comprise a tyrosine at position 91. In some embodiments, exemplary Site II binding antibodies comprise a tyrosine at position 91 and a glycine or alanine at position 89 of VH.

[0222] Table 4a. Amino acid sequences of heavy chains, light chains, HCDRs, and LCDRs of exemplary humanized C5aR1 site II antibodies

[0223]

[0224] In some embodiments, the HCVR and LCVR of an exemplary Site II binding antibody, such as c2139, are further linked to an Fc domain. The Fc domain further comprises a CH2 domain and a CH3 domain connected to each other by a linker. However, the present disclosure also encompasses Site II binding biomolecules that do not contain an Fc domain.

[0225] In some embodiments, exemplary site I or site II binding antibodies against C5aR1 bind to C5aR1 with a dissociation constant (Kd) of 10 pM to 50 nM, thereby inhibiting the association of C5aR1 with C5a, thereby antagonizing the C5a / C5aR1 axis pathway.

[0226] In some embodiments, effective inhibition of C5a / C5aR1 antibodies is measured by inhibition of Ga signaling, inhibition of neutrophil chemotaxis, inhibition of CD11b expression, and inhibition of calcium signaling.

[0227] In some embodiments, exemplary Site I or Site II binding antibodies against C5aR1 do not bind or do not substantially bind to C5aR2 or other GPCRs.

[0228] In some embodiments, exemplary Site I or Site II binding antibodies against C5aR1 are capable of binding to human neutrophils.

[0229] In some embodiments, an exemplary site I or site II binding antibody against C5aRl inhibits beta-arrestin signaling.

[0230] In some embodiments, exemplary site I or site II binding antibodies against C5aR1 inhibit ROS production in neutrophils.

[0231] In some embodiments, an exemplary site I or site II binding antibody against C5aR1 is internalized. In some embodiments, internalization takes at least 6 hours. In some embodiments, internalization takes at least 12 hours. In some embodiments, internalization takes less than 6 hours.

[0232] Design of multispecific C5aR1 antagonists

[0233] In some aspects, the antibodies provided herein are multispecific antibodies. In one embodiment, the multispecific antibody molecule is a multiparatope antibody molecule, for example, it includes multiple immunoglobulin variable region sequences, wherein the first immunoglobulin variable region sequence of the multiple immunoglobulin variable region sequences has binding specificity to the first epitope and the second immunoglobulin variable region sequence of the multiple immunoglobulin variable region sequences has binding specificity to the second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). Bispecific or biparatope antibodies have specificity for no more than two antigens or epitopes. Bispecific or biparatope antibody molecules are generally characterized by a first immunoglobulin variable region sequence having binding specificity to the first epitope and a second immunoglobulin variable region sequence having binding specificity to the second epitope. In one embodiment, bispecific or biparatope antibody molecules include a half antibody or fragment thereof having binding specificity to the first epitope and a half antibody or fragment thereof having binding specificity to the second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope is located on C5aR1 (e.g., Site I, e.g., including the N-terminal region as described herein), and the second epitope is located on C5aR1 (e.g., Site II, e.g., including ECL2 as described herein). In one embodiment, the antibody is a biparatopic antibody that binds to Site I (SEQ ID NO: 1 or SEQ ID NO: 2) and Site II (SEQ ID NO: 3) of C5aR1.

[0234] In some aspects, the biparatopic antibody comprises a pair of VH and VL from Table 2a. In some embodiments, the biparatopic antibody comprises a pair of VH and VL from Table 3. In some embodiments, the biparatopic antibody comprises a pair of VH and VL from Table 2a and a pair of VH and VL from Table 3. In some aspects, the biparatopic antibody comprises a VH selected from the group consisting of, or having at least 85%, 90%, 95%, 99%, or 100% identity to, an amino acid sequence of SEQ ID NO: 14 or SEQ ID NOs: 50-59, and a VL selected from the group consisting of, or having at least 85%, 90%, 95%, 99%, or 100% identity to, an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 25 or SEQ ID NOs: 60-68. In some aspects, the biparatopic antibody comprises a VH selected from the group consisting of an amino acid sequence of SEQ ID NO: 16 or SEQ ID NOs: 39-43, or any one of amino acid sequences that are 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NOs: 39-43, and a VL selected from the group consisting of an amino acid sequence of SEQ ID NO: 17 or SEQ ID NOs: 44-49, or any one of amino acid sequences that are 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17 or SEQ ID NOs: 44-49.

[0235] In some embodiments, one epitope of the biparatopic antibodies described herein is designed to target the sulfated N-terminal peptide or Site 1 of C5aR1 defined by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Site 1 residues targeted by the antibody molecules described herein are sulfated. In some embodiments, one or more of amino acid residues T8 (Threonine 8), D10 (Aspartic Acid 10), Y11 (Tyrosine 11), Y14 (Tyrosine 14), and / or D15 (Aspartic Acid 15) are key Site 1 epitope contact points. In some embodiments, sulfation at Y11 and / or Y14 is critical for binding to the Site 1 antibody molecules described herein. In some embodiments, the core epitope spans 12 amino acids from T7 to D18 of SEQ ID NO: 38 for binding to the Site 1 antibody molecules described herein. In some embodiments, the core epitope spans amino acids from T8 to D18 of SEQ ID NO: 38 for binding to the Site 1 antibody molecules described herein.

[0236] In some embodiments, the antibody molecules described herein are designed to target Site II defined by the amino acids of SEQ ID NO: 3. In some embodiments, amino acids encompassing R175 to G189 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E180 to P183 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E180 to P184 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, amino acids encompassing E178 to P183 of SEQ ID NO: 38 are core epitopes for binding to Site II antibody molecules described herein. In some embodiments, one or more of residues R35, H101, V176, V177, R178, E179, E180, Y181, F182, P183, P184, K185, L187, D191, I93, H194, E266, P267, S268, F272, L273, and / or K276 of C5aR1 (SEQ ID NO: 38) are critical for binding to the Site II antibodies described herein. In some embodiments, one or more of residues E180, Y181, F182, and / or P183 of SEQ ID NO: 38 are critical epitopes for binding to the Site II antibodies described herein. In one embodiment, amino acid residue W102 of SEQ ID NO: 38 is critical for binding to the Site II antibodies described herein.

[0237] In some embodiments, the biparatopic antibodies provided in the present disclosure comprise a Fab-Fc and a single-chain variable fragment (scFv), wherein the Fc is connected to the scFv via a linker. In some embodiments, the Fab domain binds to Site I and the scFv binds to Site II.

[0238] In some embodiments, the biparatopic antibodies provided herein comprise a Fab-Fc and a single-chain variable fragment (scFv). In some embodiments, the Fab domain binds to Site II and the scFv binds to Site I, wherein the Fc is connected to the scFv via a linker.

[0239] In some embodiments, the biparatopic antibodies provided herein are tetravalent antibodies comprising a heavy chain comprising a VH-Fc linked to a scFV domain, such as Figure 3A As shown in .

[0240] In some embodiments, the Site II binding arm of the biparatopic antibody comprises a glycine or alanine at position 89 of VH. In some embodiments, the Site II binding arm of the biparatopic antibody comprises a tyrosine at position 91. In some embodiments, the Site II binding arm of the biparatopic antibody comprises a tyrosine at position 91 and a glycine or alanine at position 89 of VH.

[0241] In some embodiments, the biparatopic antibodies provided herein are tetravalent antibodies comprising a light chain comprising a VL linked to a scFV domain, such as Figure 3B As shown in .

[0242] In some embodiments, biparatopic antibodies are bispecific antibodies with a two-arm single-chain Fab-Fc design that include "knobs-in-hole" (KiH) mutations in the CH3 domains to assemble two half antibodies (a common Fc heterodimer and distinct VH-CH and VL-CL domains). In some embodiments, the KiH mutations include a T366Y mutation in one CH3 domain that can be used to create a knob, while a Y407T mutation in the other CH3 domain can be used to create a hole. In some embodiments, a F405A mutation in one CH3 domain can be used to create a knob, while a T394W mutation in the other CH3 domain can be used to create a hole. In some embodiments, a T366W mutation in one CH3 domain can be used to create a knob, while a Y407A mutation in the other CH3 domain can be used to create a hole. In some embodiments, biparatopic scFv-Fc molecules can be generated using knob-hole technology (e.g., comprising hole mutations: Y349C, T366S, L368A, Y407V; knob mutations: S354C, T366W).

[0243] In some embodiments, the biparatopic antibodies comprise the antibody formats described in Table 4b.

[0244] Table 4b. Format of biparatopic antibodies

[0245] (Site II) Fab-IgG-scFv (Site I) (Site II) Fab-IgG-Linker-VL (Site I)-Linker-VH (Site I) (Site II) Fab-IgG-Linker-VH (Site I)-Linker-VL (Site I) (Site I) Fab-IgG-scFv (Site II)

[0246] In one embodiment, the biparatopic anti-C5aR1 antibody molecule comprises two heavy chain variable regions and two light chain variable regions. In one embodiment, the anti-C5aR1 antibody molecule comprises Fab, F(ab')2, Fv, Fd, or a single-chain Fv fragment (scFv).

[0247] In some embodiments, the Fc domain used in this application includes or is derived from an IgG, IgM, IgE, or Fc portion. In addition to the KiH mutation described above, the Fc domain includes an S228P mutation. In some embodiments, S228P enhances the homogeneity of the antibody. In some embodiments, the Fc domain includes or is derived from an IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain is derived from or includes an IgG4 Fc domain. In some embodiments, the Fc domain is derived from or includes an IgG4 Fc domain having an S228P mutation. In some embodiments, the Fc domain is derived from or includes an IgG1 Fc domain. In some embodiments, the Fc domain is derived from or includes an IgG1 Fc domain having an S228P mutation.

[0248] In some embodiments, a biparatopic antibody comprises two scFv regions connected to each other by a linker, wherein the first scFv binds to site I and the second scFv binds to site II.

[0249] In some embodiments, the Fab-Fc-linker-scFv biparatopic antibody is a tetravalent antibody comprising a heavy chain sequence of SEQ ID NO: 12 and a light chain sequence of SEQ ID NO: 13, or any sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 12 and SEQ ID NO: 13.

[0250] In some embodiments, the Fab-Fc-linker-scFv biparatopic antibody is a tetravalent antibody comprising a heavy chain sequence of SEQ ID NO: 12 and a light chain sequence of SEQ ID NO: 76, or any sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 12 and SEQ ID NO: 76.

[0251] Table 4c provides the sequences of the heavy and light chain sequences of the biparatopic antibodies

[0252] Table 4c. Amino acid sequences of heavy and light chains of humanized C5aR1 biparatopic antibodies

[0253]

[0254] In some embodiments, the biparatope has two variable regions: variable region 1 and variable region 2. Each variable region comprises a variable heavy chain and a variable light chain.

[0255] In an exemplary embodiment, variable region 1 comprises: a variable heavy chain 1 (VH1) comprising SEQ ID NO: 14 and a variable light chain 1 (VL1) comprising SEQ ID NO: 15. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1, which comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1, which comprises the amino acid sequence of SEQ ID NO: 15.

[0256] In some exemplary embodiments, variable region 2 includes a variable heavy chain 2 (VH2) and a variable light chain 2 (VL2), which respectively include the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17. In some embodiments, variable region 2 includes a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2, which includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, variable region 2 includes a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2, which includes the amino acid sequence of SEQ ID NO: 17.

[0257] In some exemplary embodiments, the biparatopic antibody comprising VH1 of SEQ ID NO: 14, VL1 of SEQ ID NO: 15, VH2 of SEQ ID NO: 16, and VL2 of SEQ ID NO: 17 is referred to as c2137-e1711.

[0258] In some exemplary embodiments, VH1 includes three HCDRs: HCDR1, HCDR2, and HCDR3, which include the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; or include a sequence that differs from the amino acids of SEQ ID NO:6, 7, and / or 8 by no more than 5, 4, 3, 2, or 1 amino acids.

[0259] In some exemplary embodiments, VH2 includes three HCDRs: HCDR4, HCDR5, and HCDR6, which include the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; or include a sequence that differs from the amino acids of SEQ ID NO: 18, 19, and / or 20 by no more than 5, 4, 3, 2, or 1 amino acids.

[0260] In some exemplary embodiments, VL1 includes three LCDRs: LCDR1, LCDR2, and LCDR3, which include the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 21; or include a sequence that differs from the amino acids of SEQ ID No: 9, 10, and / or 21 by no more than 5, 4, 3, 2, or 1 amino acids.

[0261] In some exemplary embodiments, VL2 includes three LCDRs: LCDR4, LCDR5, and LCDR6, which include the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or a sequence that differs from the amino acids of SEQ ID NO: 22, 23, and / or 24 by no more than 5, 4, 3, 2, or 1 amino acid. Table 5 provides the amino acid sequences of VH1, VH2, VL1, VL2, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6.

[0262] In some exemplary embodiments, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6 comprise an amino acid sequence that differs from the amino acid sequence of SEQ ID NOs: 16-21 by no more than 5 amino acid residues.

[0263] Table 5. Amino acid sequences of the variable domains and CDRs of the humanized C5aR1 biparatopic antibody (c2137-e1711)

[0264] HCDR1 NYWMH (SEQ ID NO: 6) HCDR2 YLNPSSGYTKYAQKFQG(SEQ ID NO:7) HCDR3 SGGDNYGNPYYFDR (SEQ ID NO: 8) HCDR4 AYAMS (SEQ ID NO: 18) HCDR5 SISTGGNTYYADSVKG(SEQ ID NO:19) HCDR6 GYQRFSGFAY (SEQ ID NO: 20) LCDR1 RASQSIVHSNGNTYLH (SEQ ID NO: 9) LCDR2 KVSNRFS (SEQ ID NO: 10) LCDR3 AQSTLVPLT (SEQ ID NO: 21) LCDR4 RSSQSLVHSNGNTYLN(SEQ ID NO:22) LCDR5 KVSNRLS (SEQ ID NO: 23) LCDR6 SQSTHVPYT (SEQ ID NO: 24)

[0265] In some exemplary embodiments, the variable region of the biparatopic antibody comprises variable region 1, wherein variable region 1 comprises VH1 and VL1 comprising the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1, which comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1, which comprises the amino acid sequence of SEQ ID NO: 17.

[0266] In some exemplary embodiments, the variable region of the biparatopic antibody comprises Variable Region 2, wherein Variable Region 2 comprises: VH2 comprising SEQ ID NO: 14 and VL2 comprising SEQ ID NO: 15. In some embodiments, Variable Region 2 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2, which comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, Variable Region 2 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2, which comprises the amino acid sequence of SEQ ID NO: 15.

[0267] In some exemplary embodiments, VH1 includes three HCDRs: HCDR1, HCDR2, and HCDR3, which include the amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; or include a sequence that differs from the amino acids of SEQ ID NO: 18, 19, and / or 20 by no more than 5, 4, 3, 2, or 1 amino acids.

[0268] In some exemplary embodiments, VH2 includes three HCDRs: HCDR4, HCDR5, and HCDR6, which include the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; or include a sequence that differs from the amino acids of SEQ ID NO:6, 7, and / or 8 by no more than 5, 4, 3, 2, or 1 amino acids.

[0269] In some exemplary embodiments, VL1 includes three LCDRs: LCDR1, LCDR2, and LCDR3, which include the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24; or include a sequence that differs from the amino acids of SEQ ID NO: 22, 23, and / or 24 by no more than 5, 4, 3, 2, or 1 amino acids.

[0270] In some exemplary embodiments, VL2 comprises three LCDRs: LCDR4, LCDR5, and LCDR6 comprising the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 21, or comprising a sequence that differs from the amino acids of SEQ ID NO: 9, 10, and / or 21 by no more than 5, 4, 3, 2, or 1 amino acid. Table 5 provides the amino acid sequences of VH1, VH2, VL1, VL2, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6.

[0271] In some exemplary embodiments, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6 comprise an amino acid sequence that differs from the amino acid sequence of SEQ ID NOs: 16-21 by no more than 5 amino acid residues.

[0272] In some exemplary embodiments, the variable region of the biparatopic antibody comprises variable region 1, wherein variable region 1 comprises VH1 and VL1 comprising the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 25, respectively. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, variable region 1 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1 comprising the amino acid sequence of SEQ ID NO: 25.

[0273] In some exemplary embodiments, the variable region of the biparatopic antibody comprises Variable Region 2, wherein Variable Region 2 comprises VH2 and VL2 comprising the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, Variable Region 2 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2, which comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, Variable Region 2 comprises a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2, which comprises the amino acid sequence of SEQ ID NO: 17.

[0274] In some exemplary embodiments, the biparatopic antibody comprising the VH1 of SEQ ID NO: 14, the VL1 of SEQ ID NO: 25, the VH2 of SEQ ID NO: 16, and the VL2 of SEQ ID NO: 17 is referred to as c2139-e1711.

[0275] In some exemplary embodiments, an exemplary biparatopic antibody that binds C5aR1 binds one or more of amino acid residues T8 (threonine 8), D10 (aspartic acid 10), Y11 (tyrosine 11), Y14 (tyrosine 14), and / or D15 (aspartic acid 15) of SEQ ID NO:38.

[0276] In some exemplary embodiments, exemplary biparatopic antibodies that bind C5aR1 bind to amino acid residues R175 to G189 of SEQ ID NO:38.

[0277] In some exemplary embodiments, an exemplary biparatopic antibody that binds C5aR1 binds to amino acid residues E180 to P183 of SEQ ID NO:38.

[0278] In some exemplary embodiments, exemplary biparatopic antibodies that bind C5aR1 bind to amino acid residues E180 to P184 of SEQ ID NO:38.

[0279] In some exemplary embodiments, exemplary biparatopic antibodies that bind C5aR1 bind to amino acid residues E178 to P183 of SEQ ID NO:38.

[0280] In some embodiments, exemplary biparatopic antibodies directed against C5aRl inhibit β-arrestin signaling.

[0281] In some embodiments, exemplary biparatopic antibodies against C5aRl inhibit ROS production in neutrophils.

[0282] In some embodiments, exemplary biparatopic antibodies against C5aR1 are internalized. In some embodiments, internalization takes at least 6 hours. In some embodiments, internalization takes at least 12 hours. In some embodiments, internalization takes less than 6 hours.

[0283] In some exemplary embodiments, monospecific antibodies and biparatopic antibodies may be modified or mutated to enhance the thermal stability of the antibody. The thermal stability of an antibody can be assessed by determining the aggregation onset temperature. One way to increase antibody stability is to increase the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (DSC). In general, the Tm of a protein is correlated with its stability and is inversely correlated with its susceptibility to unfolding and denaturation in solution, as well as degradation processes that depend on the tendency of the protein to unfold. Many studies have found a correlation between the degree of physical stability of formulations, measured as thermal stability by DSC, and physical stability measured by other methods (Maa et al. (1996) Int. J. Pharm. 140:155-68; Remmele et al. (1997) Pharm. Res. 15:200-8; Gupta et al. (2003) AAPS Pharm Sci. 5E8:2003; Bedu-Addo et al. (2004) Pharm. Res. 21:1353-61; Zhang et al. (2004) J. Pharm. Sci. 93:3076-89). Formulation studies have shown that Fab Tm is involved in the long-term physical stability of the corresponding mAb.

[0284] In some exemplary embodiments, the strategic introduction of disulfide bonds can stabilize monomeric and multi-subunit proteins and play a role in enhancing the thermal stability of antibodies.

[0285] In some exemplary embodiments, strategic introduction of π-stacking interactions with aromatic amino acids (AA), such as tryptophan (TRP), tyrosine (TYR), phenylalanine (PHE), and histidine (HIS), plays a role in enhancing the thermal stability of antibodies.

[0286] In some embodiments, strategic introduction of salt bridges between amino acid side chains with opposite positive or negative full electron charges, ie, (at neutral pH) Glu or Asp compared to Arg or Lys, enhances protein, particularly antibody, stability.

[0287] In some exemplary embodiments, the monospecific antibody or biparatopic antibody comprises one or more modifications that enhance thermal stability. In some embodiments, the modification that enhances thermal stability is the introduction of a cysteine ​​residue. In some embodiments, the biparatopic antibody comprises cysteines at position 559 of SEQ ID NO: 12 and at position 630 of SEQ ID NO: 12 to enhance thermal stability.

[0288] In some embodiments, exemplary biparatopic antibodies have a Tm greater than 65° C. In some embodiments, exemplary biparatopic antibodies have a Tm greater than 60° C., exemplary biparatopic antibodies have a Tm greater than 55° C. In some embodiments, exemplary biparatopic antibodies have a Tm greater than 50° C.

[0289] Some types of biparatopic antibody molecules are produced by cross-linking site I and site II binding domains or antigen binding fragments to produce bispecific antibodies. Suitable cross-linking agents include those that are heterobifunctional (having two differently reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). These linkers are available from Pierce Chemical Company, Rockford, 111.

[0290] In some embodiments, a peptide linker is used to connect an scFv or single-chain antibody to the Fc domain of a Fab. Several examples of suitable linkers include a single glycine (G) residue; a diethylene glycol peptide (GG); a tripeptide (GGG); a peptide with four glycine residues (GGGG; SEQ ID NO: 26); a peptide with five glycine residues (GGGGG; SEQ ID NO: 27); a peptide with six glycine residues (GGGGGG; SEQ ID NO: 28); a peptide with seven glycine residues (GGGGGGG; SEQ ID NO: 29); a peptide with eight glycine residues (GGGGGGGG; SEQ ID NO: 30). Other combinations of amino acid residues can be used, for example, peptide GGGGS (SEQ ID NO: 31), peptide GGGGSGGGGS (SEQ ID NO: 32), peptide GGGGSGGGGSGGGGS (SEQ ID NO: 33), peptide GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34), peptide GGSGSSGSGG (SEQ ID NO: 35), QRIEG (SEQ ID NO: 36), and peptide GQPKAAP (SEQ ID NO: 37). Other suitable linkers include single Ser and Val residues; dipeptides RTQP, SS, TK, SL, TKGPS, TVAAP, QPKAA. The examples listed above are not intended to limit the scope of the present disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable for binding to proteins. For further description of linker sequences, see, for example, WO2012135345.

[0291] The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structural element desired in the linker. For example, glycine, serine, and alanine are most preferably used for linkers with maximum flexibility. If a more rigid and extended linker is desired, some combination of glycine, proline, threonine, and serine is suitable. Depending on the desired properties, any amino acid residue can be considered as a linker in combination with other amino acid residues, as needed, to construct a larger peptide linker.

[0292] Design of Fc variants

[0293] In some aspects, the monospecific and multispecific (including bispecific or biparatopic) C5aR1 antibodies provided herein include variations or mutations in the Fc region. In some embodiments, the Fc variants or mutants reduce the ability to perform Fab arm exchange for the production of stable IgG1 or IgG4 bispecific antibodies. (See Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly an amino acid substitution S228P.

[0294] In some embodiments, the Fc region comprises a human IgG4 Fc region comprising one or more mutations selected from the group consisting of: S228P, L234V, L235A, G237A, D265G, A330S, P331S, L328R, H268A, and N297Q mutations (as indicated by Kabat et al., (1991)). In some cases, the human IgG4 Fc variant has a total of at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the wild-type human IgG4 sequence. In one embodiment, the Fc region comprises F234V, L235E, and D265G mutations. In some embodiments, the Fc region comprises F234V, L235E, D265G, and S228P mutations.

[0295] In some embodiments, the Fc variants exhibit reduced binding to a subject's Fc receptors compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit abrogated binding to a subject's Fc receptors compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit reduced phagocytosis compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit reduced phagocytosis compared to a wild-type human IgG Fc region.

[0296] Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells carrying the antigen and subsequently kill the target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain phagocytes (e.g., macrophages), enabling these phagocytic effector cells to specifically bind to target cells carrying the antigen and subsequently engulf and digest the target cells. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic cells or phagocytes and can be used for such killing. In some embodiments, polypeptide constructs comprising Fc variants as described herein exhibit reduced ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, polypeptide constructs comprising Fc variants as described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, antibodies comprising Fc variants as described herein exhibit abolished ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions.

[0297] In some embodiments, the Fc variants exhibit reduced binding to a subject's Fc receptors compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit abrogated binding to a subject's Fc receptors compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit reduced phagocytosis compared to a wild-type human IgG Fc region. In some embodiments, the Fc variants exhibit reduced phagocytosis compared to a wild-type human IgG Fc region.

[0298] Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb) present on certain phagocytes (e.g., macrophages), enabling these phagocytic effector cells to specifically bind to target cells carrying antigens and subsequently engulf and digest target cells. Ligand-specific high-affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic cells or phagocytes and can be used for such killing. In some embodiments, the polypeptide constructs comprising Fc variants as described herein exhibit reduced ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, the polypeptide constructs comprising Fc variants as described herein exhibit at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ADCC or ADCP reduction compared to polypeptide constructs comprising wild-type Fc regions. In some embodiments, antibodies comprising Fc variants as described herein exhibit eliminated ADCC or ADCP compared to polypeptide constructs comprising wild-type Fc regions.

[0299] An exemplary monospecific site II (SEQ ID NO: 3) binding antibody comprising an Fc variant is also referred to in this disclosure as c2139-F c Mod. Exemplary biparatopic antibodies comprising Fc variants are also referred to in this disclosure as c2137-el711-Fcmod. Sequences of monospecific and biparatopic antibodies comprising Fc variants are set forth in Table 6.

[0300] Table 6 - Sequences of exemplary monospecific and biparatopic antibodies with Fc modifications

[0301]

[0302]

[0303]

[0304] In some embodiments, the monospecific anti-C5aR1 antibody comprises a heavy chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:69.

[0305] In some embodiments, the monospecific anti-C5aR1 antibody comprises a light chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:70.

[0306] In some embodiments, the biparatopic anti-C5aR1 antibody comprises a heavy chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:71.

[0307] In some embodiments, the biparatopic anti-C5aR1 antibody comprises a heavy chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:72.

[0308] In some embodiments, the monospecific anti-C5aR1 antibody comprises a light chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:79.

[0309] In some embodiments, the monospecific anti-C5aR1 antibody comprises a light chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:81.

[0310] In some embodiments, the monospecific anti-C5aR1 antibody comprises a light chain amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:82.

[0311] Treating Disease with Monospecific and Biparatopic C5aR1 Antagonists

[0312] Described herein are methods of treating diseases associated with dysfunction of the C5a / C5aR1 axis. Thus, in some embodiments, the anti-C5aR1 monospecific and / or biparatopic antibodies described herein are useful for treating a subject suffering from a dysfunction associated with the C5a / C5aR1 axis, such as ANCA-associated vasculitis.

[0313] Exemplary disorders or conditions that can be treated or prevented by the antibody molecules described herein include, but are not limited to, C5aR1-related disorders or C5-related disorders. In one embodiment, the disorder is associated with neutrophil recruitment, activation, and / or NETosis. In one embodiment, the disorder is associated with complement system activation and / or coagulation system activation. In one embodiment, the disorder is associated with C5aR-mediated inflammatory response. In one embodiment, the disorder is associated with monocyte chemoattractant protein-1 (MCP-1) and / or renal inflammation. In one embodiment, the disorder is associated with chemotaxis (e.g., chemotaxis priming). In one embodiment, the disorder is associated with endothelial damage.

[0314] The treatment method comprises administering an antibody described herein to a subject in need thereof. In one embodiment, exemplary disorders (eg, C5aR1-associated disorders) can be treated using an antibody that binds to one or more amino acid residues of C5aR1 at site II (SEQ ID NO: 3).

[0315] In one embodiment, exemplary disorders (eg, C5aR1-associated disorders) can be treated using antibodies that compete with antibodies that bind to C5aR1 at site I (SEQ ID NO: 1 or SEQ ID NO: 2) or at site II (SEQ ID NO: 3).

[0316] In one embodiment, exemplary disorders (eg, C5aR1-associated disorders) can be treated using antibodies that compete with antibodies that bind to C5aR1 at Site I (SEQ ID NO: 1 or SEQ ID NO: 2) and at Site II (SEQ ID NO: 3).

[0317] The monospecific and / or biparatopic antibodies described herein can be used to treat any disease associated with dysfunction associated with the C5a / C5aR1 axis.

[0318] In some embodiments, exemplary disorders (eg, C5aR1-associated disorders) can be treated using monospecific or biparatopic antibodies that bind to C5aR1 with an affinity of 10 pM to 50 nM.

[0319] In some embodiments, the antibody may be administered to a subject in need thereof intravenously, subcutaneously, intradermally, or intramuscularly.

[0320] In some embodiments, nucleic acids encoding the monospecific or biparatopic antibodies described herein can be administered to a subject in need thereof using an appropriate delivery method. Several methods for delivering nucleic acids are known in the literature. For example, rAAV vectors encoding the monospecific or biparatopic antibodies described herein can be administered to the subject being administered by intravenous, intraperitoneal, subcutaneous, or intradermal administration. In some embodiments, delivery of nucleic acids encoding antibodies can be achieved using a "gene gun" (gene gun particle delivery system or non-viral lipid nanoparticles).

[0321] In some embodiments, the monospecific or biparatopic antibodies described herein and another therapeutic agent may be administered to a subject in need thereof. In some embodiments, the other therapeutic agent is a small molecule, such as avacopan, a corticosteroid, or an immunosuppressive drug. Exemplary corticosteroids include, but are not limited to, prednisolone, hydrocortisone, prednisone, dexamethasone, or cortisone. Exemplary immunosuppressive drugs include, but are not limited to, methotrexate, azathioprine, mycophenolate mofetil, or cyclophosphamide.

[0322] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopan monotherapy in reducing neutropenia in vivo.

[0323] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopam in antagonizing C5aR1 in the presence of C5a at a concentration of 100 nM or greater.

[0324] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopam in inhibiting Ga signaling in vitro.

[0325] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopam in inhibiting calcium signaling in vitro and in vivo.

[0326] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopam in inhibiting neutrophil chemotaxis in vitro and in vivo.

[0327] In some embodiments, the monospecific or biparatopic antibodies described herein are stable in serum for up to 500 hours after injection.

[0328] In some embodiments, the monospecific or biparatopic antibodies described herein are more effective than avacopam in inhibiting CD11b expression in vitro and in vivo.

[0329] Nucleic acids, vectors, and methods of manufacturing

[0330] The disclosure also features nucleic acids including nucleotide sequences that encode antibody molecules as described herein (eg, heavy and light chain variable regions and CDRs of the antibody molecules).

[0331] For example, the disclosure features a first and a second nucleic acid, which respectively encode the heavy and light chain variable regions of an antibody molecule or a portion of an antibody molecule selected from one or more of the antibody molecules disclosed herein (e.g., the antibody molecules of Table 1C), such as the variable region of any of the antibodies disclosed herein. The nucleic acid can include a nucleotide sequence encoding any of the amino acid sequences in the tables herein, or a sequence substantially identical thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto or that differs by no more than 3, 6, 15, 30, or 45 nucleotides from a sequence shown in the tables herein).

[0332] In certain embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one, two, or three CDRs from a heavy chain variable region having an amino acid sequence as set forth in a table herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions). In one embodiment, the nucleic acid comprises a nucleotide sequence encoding at least one, two, or three CDRs from a light chain variable region having an amino acid sequence as set forth in a table herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions). In one embodiment, the nucleic acid comprises a nucleotide sequence encoding at least one, two, three, four, five or six CDRs from heavy and light chain variable regions having an amino acid sequence as set forth in the Tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions)).

[0333] The nucleic acids disclosed herein comprise deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may be single-stranded or double-stranded, and if single-stranded, may be coding strands or non-coding (antisense) strands. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interspersed with non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by combining with a labeling component. Nucleic acids may be recombinant polynucleotides, or polynucleotides of genomic, cDNA, semisynthetic or synthetic origin that do not exist in nature or are linked to another polynucleotide in a non-natural arrangement.

[0334] In some aspects, the application features host cells and vectors containing the nucleic acids described herein. The nucleic acids can be present in a single vector or in separate vectors of the same host cell or separate host cells, as described in more detail below.

[0335] carrier

[0336] Further provided herein are vectors comprising a nucleotide sequence encoding an antibody molecule as described herein (eg, the heavy and light chain variable regions and CDRs of the antibody molecule).

[0337] In one embodiment, the vector comprises a nucleic acid described herein. For example, the vector can comprise a first and a second nucleic acid, each encoding a heavy chain and a light chain variable region of an antibody molecule or a portion of an antibody molecule selected from one or more of the antibody molecules disclosed herein (e.g., an antibody molecule described herein), such as a variable region of any one of Tables 1 to 4.

[0338] In certain embodiments, the vector comprises a nucleotide sequence encoding at least one, two, or three CDRs from a heavy chain variable region having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions). In one embodiment, the vector comprises a nucleotide sequence encoding at least one, two, or three CDRs from a light chain variable region having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions). In one embodiment, the vector comprises a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs from heavy and light chain variable regions having an amino acid sequence as set forth in the Tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto and / or having one or more substitutions (e.g., conservative substitutions)).

[0339] Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern Equine Encephalitis virus, and flaviviruses.

[0340] In addition, can select the cell that DNA is stably integrated into its chromosome by introducing one or more markers that allow to select transfection host cell.Marker can provide the original trophotype of for example auxotrophic host, biocide resistance (for example, antibiotic) or resistance to heavy metal (for example copper) etc. Selectable marker gene can be directly connected to dna sequence dna to be expressed or is introduced into same cell by cotransformation.The most preferably synthesis of mRNA also may need other elements.These elements can comprise splicing signal and transcription promoter, enhancer and termination signal.

[0341] Once an expression vector or DNA sequence containing a construct for expression is prepared, the expression vector can be transfected or introduced into an appropriate host cell. A variety of techniques can be used to achieve this, such as primary plasmid fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of primary plasmid fusion, cells are grown in culture and screened for appropriate activity.

[0342] The methods and conditions for culturing the resulting transfected cells and recovering the produced antibody molecules are known to those skilled in the art and can be modified or optimized based on the description herein, depending on the specific expression vector and mammalian host cell used.

[0343] cell

[0344] The present disclosure also provides cells (e.g., host cells) comprising nucleic acids encoding antibody molecules as described herein. For example, the host cell may comprise a nucleic acid molecule having a nucleotide sequence encoding an amino acid sequence described in any one of Tables 1 to 5, a sequence substantially homologous thereto (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto and / or capable of hybridizing under stringent conditions as described herein), or a portion of one of the nucleic acids.

[0345] In one embodiment, the host cell is genetically engineered to include a nucleic acid encoding an antibody molecule described herein.

[0346] In certain embodiments, host cells are genetically engineered using expression cassettes. The phrase "expression cassette" refers to a nucleotide sequence that is capable of affecting the expression of a gene in a host compatible with these sequences. These cassettes may contain a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful for achieving expression, such as inducible promoters, may also be used.

[0347] The present disclosure also provides host cells comprising the vectors described herein.

[0348] The cell may be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In one embodiment, the cell (e.g., host cell) is an isolated cell.

[0349] Application method

[0350] The compositions of the present invention can be formulated in any suitable form, such as liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions and the like. The most preferred form of any composition depends on the properties and therapeutic applications or other intended uses of the intended mode of administration, composition or combination. Typical delivery modes of the compositions of the present invention are by parenteral administration (e.g., intravenous administration). In one aspect, the compositions of the present invention are administered to human patients by intravenous infusion or injection.

[0351] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an antibody molecule described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents, and the like, that are physiologically compatible. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0352] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnical, intraspinal, epidural, and intrasternal injection and infusion. The therapeutic composition should generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound (i.e., antibody or antibody portion) into an appropriate solvent optionally with one or a combination of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. This can be achieved, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion and by using a surfactant to maintain the appropriate fluidity of the solution. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as monostearate and gelatin in the composition. The antibody molecules described herein can be administered in a variety of ways. Several methods are known in the art, and in many therapeutic, prophylactic or diagnostic applications, appropriate routes of administration / modes are intravenous injection or infusion. As will be appreciated by those skilled in the art, routes of administration and / or modes will vary depending on the desired results.

[0353] Examples

[0354] Other features, objectives, and advantages of the present disclosure will be apparent from the following examples. However, it should be understood that the examples, while indicating embodiments of the present disclosure, are provided by way of illustration and not limitation. Various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art from the examples.

[0355] Example 1. Kinetic Analysis of C5aR1 Antagonistic Humanized Antibodies

[0356] This example describes the binding affinity and specificity of the Site II monospecific and biparatopic antibodies described herein for C5aR1 and C5aR2.

[0357] (a) Binding affinity

[0358] The binding affinity of the antibodies to the target receptor C5aR1 was determined using ELISA analysis. C5aR1 VLPs were fixed to MaxiSorp ELISA plates at a concentration of 30 μg / mL and incubated overnight at 4°C. The next morning, the plates were washed three times with 1×PBS and blocked with 100 μL PBSA (1×PBS with 3% BSA) for 30 minutes. Serial titrations of anti-C5aR1 antibodies were performed in the presence of PBSA and incubated for 1 hour at room temperature. The plates were washed 6 times with PBSA. Anti-human HRP was diluted in PBSA, added to all wells and incubated for 45 minutes at room temperature. The plates were washed 6 times with PBS. TMB substrate was added to all wells and incubated for 10 minutes, followed by the addition of stop solution (0.1 M sulfuric acid). The absorbance at 450 nm was measured on a standard plate reader. EC50 values ​​(in nM) for antibody titrations were generated using a four-parameter curve fit. Affinity curves for the monospecific c2139 antibody and the biparatopic c2137-e1711 antibody are shown in Figure 4A middle.

[0359] It was observed that under the conditions described above, the monospecific C5aR1 antibody bound C5aR1 with an affinity of approximately 0.16 nM and the biparatopic antibody bound C5aR1 with an affinity of approximately 0.22 nM.

[0360] In another case, site II antibody c2139 and biparatopic antibody c2137-e1711 from two different antibody lots were analyzed with U937-C5aR1 cells ( Figure 4B ) and human neutrophils ( Figure 4C The EC50s of U937-C5aR1 binding to the anti-C5aR1 antibody were different for different protein batches (c2139 and c2137-e1711), which are shown in Table 6.

[0361] Table 6. EC50 of U937-C5aR1 binding to exemplary C5aR1 antibodies

[0362]

[0363] The EC50s of binding to human neutrophils of different protein lots (c2139 and c2137-e1711) of anti-C5aR1 antibodies were different and are shown in Table 7.

[0364] Table 7. EC50 binding to human neutrophils of exemplary C5aR1 antibodies

[0365]

[0366] The non-C5aR1 antibody motavizumab was used as a control. Binding of both site II antibody c2139 and biparatopic antibody c2137-e1711 to U937 and human neutrophils was observed across multiple antibody production batches.

[0367] (b) Binding specificity

[0368] The specificity of C5aR1 antagonist antibodies was determined by measuring the affinity of anti-C5aR1 antibodies for C5aR2. C5aR2 VLPs were immobilized to MaxiSorp ELISA plates at a concentration of 30 μg / mL and incubated overnight at 4°C. The next morning, the plates were washed three times with 1×PBS and blocked with 100 μL PBSA (1×PBS with 3% BSA) for 30 minutes. Serial titrations of anti-C5aR1 antibodies were performed in the presence of PBSA and incubated for 1 hour at room temperature. The plates were washed six times with PBSA. Anti-human HRP was diluted in PBSA, added to all wells, and incubated for 45 minutes at room temperature. The plates were washed six times with PBS. TMB substrate was added to all wells and incubated for 10 minutes, followed by the addition of stop solution (0.1 M sulfuric acid). The absorbance at 450 nm was measured on a standard plate reader. EC50 values ​​(in nM) for antibody titrations were generated using a four-parameter curve fit.

[0369] Binding data for monospecific and biparatopic antibodies are shown in Figure 5 It was observed that the exemplary C5aR1 antagonist antibodies did not bind to C5aR2 under the conditions described above. Overall, the data in this example show that the anti-C5aR1 antibodies of the invention bind to C5aR1 with high affinity and to C5aR2 with no measurable affinity, indicating that the C5aR1 antibodies are in fact specific for C5aR1.

[0370] Example 2. Inhibition of Gα signaling

[0371] This example describes functional aspects of the C5aR1 monospecific and biparatopic antibodies described herein for inhibition of Ga signaling analyzed using GeneBLazer.

[0372] GeneBLAzer assay kits and C5aR1 cell lines are available from Thermo Fisher (Cat. No. K1544). Assays were performed as recommended by the manufacturer. Briefly, the antibody or antagonist was incubated at increasing concentrations at 37°C for 30 minutes. C5a was then added to the cells and incubated at 37°C for an additional 4 to 5 hours. β-lactamase substrate was then added and incubated at room temperature for 2 hours. Fluorescence measurements were taken for each well with an excitation / emission of 409 / 460 (blue) and 409 / 530 (green). The increase in the blue to green ratio was proportional to C5aR1 activation and was used to calculate the percent activation of cells in each well. C5aR1 activation profiles were monitored at increasing antagonist concentrations. The IC50 was determined by plotting the percent signal versus antagonist concentration.

[0373] Figure 6A In the example shown, Gα signaling analysis of monospecific and biparatopic antibodies at 10 nM C5a as a function of antibody concentration is described. In another example, a known C5aR antibody (anti-C5aR1 control Ab) was used as a positive control for inhibition of Gα signaling ( Figure 6C ).

[0374] Figure 6B In the example shown, Gα signaling analysis of monospecific and biparatopic antibodies at 100 nM C5a as a function of antibody concentration is described. In another example, a known C5aR antibody (anti-C5aR1 control Ab) was used as a positive control for inhibition of Gα signaling ( Figure 6D ). The IC50 of the monospecific C5aR1 antagonist was observed to be 0.14 nM at 10 nM C5a and 0.19 nM at 100 nM C5a. The IC50 of the bispecific C5aR1 antagonist was 0.27 nM at 10 nM C5a and 0.28 nM at 100 nM C5a. It was observed that the exemplary antibodies maintained excellent inhibition compared to avacopam and the anti-C5aR1 control Ab at all concentrations of C5a tested.

[0375] Example 3. Inhibition of calcium signaling

[0376] This example describes functional aspects of the C5aR1 monospecific and biparatopic antibodies described herein for inhibition of C5a-mediated calcium signaling using a calcium flux assay.

[0377] The effectiveness of the ability of the antibody to inhibit the release of intracellular calcium in stable C5aR1-U937 cells expressing C5aR1 or neutrophils isolated from whole blood in the presence of C5a was assessed. Calcium flux analysis using calcium-sensitive dyes was used to detect cytosolic changes in calcium concentration. Cells were incubated with esterified (inactive) calcium dyes. The dye penetrates the cell membrane and becomes active once inside the cell. The active form of the dye becomes fluorescent after binding to intracellular calcium, and fluorescence is used to measure C5aR1 signaling in response to C5a addition. Specifically, stable C5aR1-U937 or neutrophils isolated from whole blood were stained with the Fluo-4 direct calcium assay kit from Thermo Fisher at 37°C for 1 hour. Subsequently, the antibody or antagonist was incubated with the cells for 30 minutes. The basal fluorescence excited at 494nm and emitted at 516nm was measured for 15 seconds. C5a was added to the cells and fluorescence was measured over a four-minute span. Response ratios were calculated using basal readings from before C5a stimulation and maximal signals after C5a stimulation.Calcium flux analysis was performed using engineered cells stably expressing C5aR1 and using human neutrophils.

[0378] Monospecific antibodies ( Figure 7A ) and biparatopic antagonistic antibodies ( Figure 7B ) inhibition of C5a-mediated calcium signaling assays is shown as a function of calcium flux as a function of increasing C5a concentration and antibody concentration. Figure 7C Inhibition of C5a-mediated calcium signaling with the known C5aR1 antagonist avacopam was demonstrated using the protocol described above. It was observed that the test antibodies were more effective than avacopam in inhibiting calcium signaling.

[0379] Example 4. Inhibition of Neutrophil Chemotaxis

[0380] This example describes the functional aspects of the C5aR1 monospecific and biparatopic antibodies described herein for use in inhibiting neutrophil chemotaxis known to be induced by C5aR1 activity using a Boyden Chamber.

[0381] First, C5aR1-U937 stable cells were seeded in the top chamber of a 96-well plate in the presence of no antibody (antagonist) or 1, 10, or 100 nM of each antibody (antagonist). -6 to 10 -9.5A semi-log titration of C5a (chemokine) was performed. The top chamber was separated from the bottom chamber by a membrane. C5a was expected to induce migration of U937 cells. However, it was expected that this migration would be inhibited using a C5aR1 antagonist. Increasing concentrations of the exemplary antibody were used to determine the dose-dependent inhibition of chemotaxis. In addition, increasing concentrations of the chemokine (C5a) were used to determine whether the inhibition of chemotaxis could be overcome by using high concentrations of C5a. In parallel, the known C5aR1 inhibitor avacopan was also used to assess the inhibition of neutrophil chemotaxis and the surmountability of chemotaxis in the presence of excess C5a.

[0382] Next, human neutrophils were seeded in the top chamber of a 96-well plate in the presence of no antibody (antagonist) or 1, 10, or 100 nM of each antibody (antagonist). -6 to 10 -9.5 A semi-log titration of C5a (chemokine) was performed. The top chamber was separated from the bottom chamber by a membrane. C5a was expected to induce migration of U937 cells. However, it was expected that this migration would be inhibited using a C5aR1 antagonist. Increasing concentrations of the antibody were used to determine the dose-dependent inhibition of chemotaxis. In addition, increasing concentrations of the chemokine (C5a) were used to determine whether the inhibition of chemotaxis could be overcome by using high concentrations of C5a. In parallel, the known C5aR1 inhibitor avacopan was also used to assess the inhibition of neutrophil chemotaxis and the surmountability of chemotaxis in the presence of excess C5a.

[0383] Figures 8A to 8C Fluorescence intensity in the presence of different concentrations of anti-C5aR1 antibodies is plotted against C5a concentration, indicating the inhibition of chemotaxis by anti-C5aR1 antibodies. It was observed that anti-C5aR1 antibodies generally inhibited cell chemotaxis in a dose-dependent manner. Furthermore, it was observed that, unlike avacopam, the inhibition of neutrophil chemotaxis by anti-C5aR1 antibodies was insurmountable in the presence of excess C5a.

[0384] Example 5. Inhibition of CD11b expression

[0385] This example describes the functional aspects of the C5aR1 monospecific and biparatopic antibodies described herein for inhibiting CD11b expression.

[0386] CD11b (integrin receptor) moves to the surface of neutrophils in response to C5a activation and mediates intravascular peristalsis prior to neutrophil translocation.CD11 is involved in numerous adhesion-related associations between cells such as monocytes, macrophages, natural killer (NK) cells, and granulocytes.

[0387] Figures 9A to 9BThe percentage of CD11b expression in the presence of 100 nM C5a is shown versus the antibody (C5aR1 antagonist) concentration. It was observed that the antibodies effectively inhibited CD11b over the entire range of expected physiological concentrations of C5a. Table 8 shows the IC50 of c2139 and c2137-e1711 in the presence of 100 nM C5a.

[0388] Table 8. IC50 for inhibition of CD11b expression using exemplary C5aR1 antibodies

[0389]

[0390]

[0391] In another instance, 10 nM and 100 nM anti-C5aR1 control Ab were used as controls to compare the inhibition of the anti-C5aR1 antibodies disclosed herein in human neutrophils ( Figure 9C ). It was observed that avacopan was only partially effective in inhibiting CD11b. It was further observed that the antibodies of the present invention were more effective inhibitors than the anti-C5aR1 control Ab.

[0392] Example 6. Inhibition of β-arrestin recruitment

[0393] This example describes the functional aspects of the C5aR1 monospecific and biparatopic antibodies described herein for inhibiting β-arrestin recruitment.

[0394] To characterize the lead mAb as a full antagonist of C5aR1, we aimed to assess its potential to inhibit β-arrestin2 recruitment. The β-arrestin assay utilizes a proprietary two-subunit luciferase reporter system: one subunit is fused to C5aR1 and one subunit is fused to β-arrestin2. When in close proximity, the two subunits form an enzyme that produces a fluorescent signal, which serves as a proxy for C5aR1-mediated β-arrestin2 recruitment. At two different C5a concentrations (1 nM and 10 nM), the exemplary antibody significantly reduced C5aR1-mediated β-arrestin2 recruitment. Figures 10A to 10C To demonstrate the addition of 1 nM ( Figure 10A )、10nM( Figure 10B ) and 100nM( Figure 10C ) Graph of the inhibition of fluorescent signal after C5a. Table 9 shows the IC50 for c2139, c2137-e1711, and avacopan-mediated inhibition of β-arrestin.

[0395] Table 9. IC50 of monospecific or biparatopic antibodies for β-arrestin signaling

[0396]

[0397] When compared to the small molecule C5aR1 inhibitor avacopam, exemplary antibodies c2139 and c2137-e1711 demonstrated superior signal inhibition, even when avacopam was used at a 10-fold higher concentration than the antibodies. Notably, avacopam lost efficacy as C5a concentration increased to 10 nM, while c2139 and c2137-e1711 maintained >75% inhibition of β-arrestin2 recruitment. The data demonstrate that exemplary C5aR1 antibodies (c2139 and c2137-e1711) inhibit β-arrestin at 10-fold lower concentrations than avacopam.

[0398] Example 7. Inhibition of ROS Generation by Humanized Anti-C5aR1 Antibody

[0399] This example demonstrates that an exemplary humanized monospecific antibody (c2139) and an exemplary biparatopic antibody (c2137-el711) reduce reactive oxygen species (ROS).

[0400] ROS production by neutrophils was detected using a cellular ROS detection assay kit (Abcam) following the manufacturer's instructions. RBC lysed WB cells were diluted to 3 × 10 in 100 mL of buffer. 5 The concentration of cells was 100 μg / mL. The negative control group was pretreated with a ROS inhibitor (N-acetyl-L-cysteine) at 37°C, 5% CO2 for 30 minutes. ROS detection antibodies were then added to the antibody mixture for detection of neutrophil ROS production based on flow cytometry. ROS inducer (pyocyanin) was added to all groups and incubated for 30 minutes before acquisition on a cell counter. Figure 11 Shown are ROS production in responding neutrophils in ANCA- and ANCA+ groups in response to treatment with anti-C5aR1 antibody.

[0401] It was observed that c2139 and c2137-e1711 inhibited ANCA-induced ROS production.

[0402] Example 8. Internalization of humanized anti-C5aR1 antibody in U937 cells

[0403] This example demonstrates internalization of humanized monospecific and biparatopic C5aR1 antibodies in hC5ar1-U937 cells.

[0404] Humanized monospecific anti-C5aR1 antibody e1711 and biparatopic antibody c2139-e1711 were conjugated to a pH-sensitive dye that fluoresces brightly at low pH but not at neutral pH. The bound antibodies were incubated with U937 cells and hC5aR1 knock-in U937 cells. Figures 12A to 12D Fluorescence intensity after 24 hours of incubation with each bound antibody is shown. Figures 12A to 12B shows the fluorescence intensity of U037 cells or U937-C5aR1 cells in the presence of 10 nM antibody; and Figures 12C to 12D Fluorescence intensity of U037 cells or U937-C5aR1 cells in the presence of 100 nM antibody is shown.

[0405] Example 9. Cross-reactivity of humanized anti-C5aR1 antibodies

[0406] This example demonstrates binding of the humanized anti-C5aRl antibody, e1711, and the biparatopic antibody c2139-e1711 to C5aRl in squirrel monkey and dog.

[0407] For the range between 10 -7.5 to 10 -12 Cell surface binding and flow cytometry were performed using half-log titrations of anti-C5aR1 antibodies. Figures 13A to 13B As shown in , anti-C5aR1 humanized antibodies bind to squirrel monkey and dog C5aR1.

[0408] Example 10. Inhibition of Neutropenia in Squirrel Monkeys

[0409] This example demonstrates the inhibition of neutropenia by C5aR1 antibodies in squirrel monkeys. Neutropenia is caused by the rapid expression of CD11b on the cell surface, which causes neutrophils to temporarily adhere to the vascular endothelium, thereby reducing peripheral neutrophil counts. Compared to mice, squirrel monkeys are a physiologically relevant model for evaluating neutropenia because neutrophil counts in squirrel monkeys are similar to those in humans (50 to 70%), while mice have only 10 to 20%. In addition, squirrel monkeys allow blood collection at multiple time points, thereby extending the duration of the study. Further cross-reactivity with squirrel monkey C5aR1 was observed, such as Figure 13A As shown in .

[0410] Figure 14AThe experimental design for evaluating the relief of neutropenia in squirrel monkeys is presented. Briefly, three groups of squirrel monkeys were evaluated for the suppression of neutropenia. In the first group, 8 squirrel monkeys were injected intravenously with PBS (vehicle). In the second group, 8 squirrel monkeys were injected intravenously with a demonstration antibody at a dose of 10 mg / kg. In the third group, 8 squirrel monkeys were injected intravenously with avacopam at a dose of 30 mg / kg. One hour after the injection of PBS, the demonstration antibody, or avacopam, 0.1 mg / kg of human C5a was injected into the squirrel monkeys. Blood was collected 1 min, 5 min, 15 min, 2 hours, and 6 hours after the injection of human C5a. The percentage change in neutrophils and the average change in neutrophils in all three groups were calculated and displayed in a scatter plot. Figure 14B The mean changes in neutrophils are shown in the bar graph. Figure 14C middle.

[0411] It was observed that administration of human C5a caused a rapid, transient neutropenia, which was caused by neutrophil adhesion to endothelial cells. The vehicle (PBS) group showed robust neutropenia, with an average 89% reduction in neutrophil counts relative to baseline 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment with the exemplary anti-C5aR1 antibody (23% compared to 89% for vehicle). It was further observed that neutrophil counts began to resolve between 2 and 6 hours. Neutrophilia was observed with avacopam. No neutrophilia was observed with c2139. The avacopam group also responded with a 59% average reduction in neutrophils. It was observed that the exemplary anti-C5aR1 antibody had a superior response rate when compared to avacopam.

[0412] Example 11. Suppression of Neutropenia in Human C5aR1 Transgenic Mice

[0413] This example demonstrates inhibition of neutropenia by C5aR1 antibodies in transgenic human C5aR1 mice. The exemplary C5aR1 antibodies do not cross-react with mouse C5aR1. At the Jackson Laboratory, CRISPR technology was used to generate transgenic human C5aR1 (hC5aR1) knock-in mice.

[0414] Figure 15AThe experimental design for evaluating the alleviation of neutropenia in mice is presented. Briefly, five groups of hC5aR1 mice were evaluated for inhibition of neutropenia. In the first group, five hC5aR1 mice were injected intravenously with PBS (vehicle). In the second group, five hC5aR1 mice were injected intravenously with the exemplary monospecific anti-C5aR1 antibody c2139 at a dose of 20 mg / kg. In the third group, five hC5aR1 mice were injected intravenously with the exemplary biparatopic anti-C5aR1 antibody c2137-e1711 at a dose of 20 mg / kg. In the fourth group, five hC5aR1 mice were injected intravenously with a non-C5aR1 antibody (motavizumab) at a dose of 20 mg / kg. In the fifth group, five hC5aR1 mice were injected intravenously with avacopam at a dose of 30 mg / kg. One hour after injection of PBS, exemplary antibodies, motavizumab, or avaxomeva, 0.1 mg / kg human C5a was injected into hC5aR1 mice. Blood was collected 1 minute, 5 minutes, and 2 hours after injection of human C5a. The percent change in neutrophils and the mean change in neutrophils were calculated for all three groups and displayed in scatter plots. Figures 15B to 15C The mean changes in neutrophils are shown in Figure 15C Neutrophil counts were assessed by gating CD45+ / CD11b+ / Ly6G+ cells.

[0415] It was observed that administration of human C5a caused a rapid, transient neutropenia, which was caused by neutrophil adhesion to endothelial cells. The vehicle (PBS) group showed robust neutropenia, with an average 89% reduction in neutrophil counts relative to baseline 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment with an exemplary anti-C5aR1 antibody (23% vs. 89% for vehicle). The avacopan group also responded with a 59% average reduction in neutrophils.

[0416] It was observed that the exemplary anti-C5aR1 antibodies had superior response rates when compared to avacopam. It was observed that administration of human C5a caused rapid, transient neutropenia in human C5aR1 mice. The vehicle (PBS) group showed robust neutropenia, with a mean 76% reduction in neutrophil counts relative to baseline 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment with the exemplary monospecific C5aR1 antibody (11% versus 89% for vehicle). Complete inhibition was seen with the exemplary biparatopic C5aR1 antibody. The avacopam group also responded with a mean 10% reduction in neutrophils.

[0417] Example 12. Pharmacokinetic Studies of Exemplary C5aR1 Antibodies in hC5aR1 Mice

[0418] This example demonstrates pharmacokinetic (PK) studies of an exemplary C5aR1 antibody in mice.

[0419] 5 mg / kg of exemplary monospecific and biparatopic antibodies were injected intravenously into Tg32 mice. The amount of antibody in the serum was assessed over a period of 500 hours. Figure 16A Pharmacokinetic properties of exemplary antibodies are presented. Figures 16B to 16D The stability of the anti-C5aR1 antibody in the serum of 5 different mice was demonstrated for 500 hours. Figure 16C ) antibodies and biparatopic (c2137-e1711- Figure 16D ) antibodies were stable in serum and equally persistent in Tg32 mice. In hC5aR1 transgenic mice, TMDD was observed with both c2139 and c2137-e1711. Compared to the monospecific antibodies, the tetravalent antibody had slightly reduced persistence in serum. Motavizumab was used as a control.

[0420] Example 13. Predictions for Pharmacokinetic Studies of Exemplary C5aR1 Antibodies in Humans

[0421] Nonlinear PK has been observed for known anti-C5aR1 antibodies (Lee et al., 2006, PMID 16980984). It has been reported that at a 10 mg / kg IV dose, >90% receptor occupancy was observed for 4.3 weeks, and at a 4 mg / kg subcutaneous dose, >90% receptor occupancy was observed for 1.8 weeks. Based on the affinity data for the humanized anti-C5aR1 antibodies of the present invention, model simulations showed that, following IV administration, the same predictions of nonlinear clearance between 0.1 and 10 mg / kg of the antibody resulted in >90% receptor occupancy for 24 days.

[0422] Regardless of bioavailability, a 4 mg / kg subcutaneous dose is predicted to have >90% receptor occupancy for approximately 7 to 13 days.

[0423] It was observed that the tetravalent antibody (biparatopic) had slightly reduced persistence in serum compared to the monospecific lead.

[0424] Example 14. Stability of exemplary C5aR1 antibodies at 4°C

[0425] This example demonstrates the stability of exemplary C5aRl antibodies (both monospecific and biparatopic) at 4°C with one freeze-thaw cycle.

[0426] Both monospecific and biparatopic antibodies were incubated at 4° C. for up to 14 days. After one thaw cycle at 37° C., the amount of intact antibody was assessed by gel filtration, native PAGE, and dynamic light scattering (DLS). Table 10 shows the percentage of antibody aggregates (HMW) compared to the percentage of intact antibody.

[0427] Table 10. Stability of C5aR1 bispecific antibodies after 1 thaw cycle at room temperature / 37°C.

[0428]

[0429] It was observed that both monospecific and biparatopic antibodies were stable at 4°C and RT for up to 2 weeks and one freeze / thaw cycle.

[0430] Example 15 - Kinetic Analysis of C5aR1 Antagonistic Humanized Antibodies with Modified Fc Domains.

[0431] This example describes the binding affinity and specificity of the site II monospecific and biparatopic antibodies described herein to C5aR1 and C5aR2 with modified Fc domains. Fc domain modifications were introduced to counteract effector functions, such as ADCC or ADCP. In addition, the modified Fc domains included mutations to prevent Fab arm exchange. Table 11 summarizes the Fc modifications in the C5aR1 antibody.

[0432] Table 11 - Summary of monospecific and biparatopic antibodies with modified Fc domains C5aR1 antibody.

[0433] Monospecific Biparatopic name <![CDATA[c2139-F c against]]> <![CDATA[c2137-e1711-F c against]]> <![CDATA[V H District]]> <![CDATA[V H Area c2137]]> <![CDATA[V H Area c2137-e1711]]> Fc silent mutation F234V, L235E, D265G F234V, L235E, D265G Fab arm exchange mutation S228P S228P

[0434] The binding affinities of the monospecific and biparatopic antibodies with modified Fc domains were calculated as described in Example 1. The monospecific antibodies with modified Fc domains are hereinafter referred to as "c2139-F c mod"; and the biparatopic antibody with a modified Fc domain is hereinafter referred to as "c2137-e1711-F c mod".

[0435] (a) Binding affinity

[0436] Figure 17A The monospecific C5aR1 antibody (c2139-Fcmod) and the C5aR1 biparatopic antibody (c2137-e1711-F c mod) affinity curve.

[0437] It was observed that under the conditions described above, the monospecific C5aR1 monospecific antibody c2139-Fcmod bound to C5aR1 with an affinity of approximately 0.31 nM and the C5aR1 biparatopic antibody c2137-e1711-F c mod binds C5aR1 with an affinity of approximately 0.34 nM.

[0438] An exemplary kinetic analysis of binding to cells expressing C5aR1 is shown in ( Figure 17B ). Kinetic measurements were fitted after 2 to 3 dose association phases followed by dissociation. It was observed that the EC50s of the Fc-modified antibodies were comparable to those of the Fc-unmodified antibodies. For example, as can be seen from Table 12, the biparatopic antibodies c2137-e1711 and c2137-e1711-F c The EC50s of mod were all approximately 1 nM in U937-C5aR1 cells, and approximately 1 nM and 2 nM in neutrophils and 0.5 nM and 0.6 mM in macrophages.

[0439] Table 12-c2137-e1711 and c2137-e1711-F c Comparison of EC50 of mods

[0440]

[0441]

[0442] (b) Binding specificity

[0443] The specificity of the Fc-modified C5aR1 antagonist antibodies was determined by measuring the affinity of the anti-C5aR1 antibodies for C5aR2. This was determined as described in Example 1.

[0444] Figure 17C Display assay c2137-e1711-F c Specific affinity curves of Fc mod and c2139-Fcmod. It was observed that the Fc-modified antibody did not show affinity for C5aR2.

[0445] Example 16 - Internalization of C5aR1 Antibody.

[0446] The examples attempt to demonstrate that internalization of the C5aR1 antibody is specific and not due to nonspecific aggregation of membrane immunoglobulins. Sodium azide is a metabolic inhibitor and inhibits internalization or endocytosis, as these are energy-dependent processes. 10 nM C5aR1 antibody was used in the presence or absence of 20 mM sodium azide to probe for any metabolic dependency on internalization of the exemplary C5aR1 antibody. The exemplary C5aR1 antibody was observed to undergo metabolism-based internalization. For example, c2139 undergoes internalization several hours after dissociation and c2139-e1711 undergoes internalization during association. The dissociation constant of the monospecific C5aR1 antibody c2139 in the presence of 20 mM sodium azide was 1.43×10 -5 / s, while the dissociation constant in the absence of sodium azide is 3.4 × 10 -5 The association constant of biparatopic C5aR1 (c2137-e1711) in the presence of 20 mM sodium azide was 2.15 × 10 4 / mol / s, while the dissociation constant in the absence of sodium azide is 4.38×10 3 / mol / second. Figures 18A to 18B showed increased internalization of the C5aR1 antibody. Figure 18A is an exemplary graph showing internalization of monospecific antibody c2139 several hours after dissociation. Figure 18B is an exemplary graph showing increased internalization of the biparatopic antibody c2137-e1711 during association.

[0447] It was observed that nonspecific aggregation of membrane immunoglobulins was not the only cause of internalization of the exemplary c5aR1 antibody.

[0448] Example 17 - Inhibition of Gα Signaling by Fc-Modified C5aR1 Antibody

[0449] This example describes functional aspects of the Fc-modified C5aR1 monospecific and biparatopic antibodies described herein for inhibition of Ga signaling analyzed using GeneBLazer.

[0450] This experiment was performed as described in detail in Example 2. Figures 19A to 19B The results showed that the Fc-modified antibody (c2137-e1711-F c mod and c2139-F c mod) inhibits Gα signaling. c Both mod and c2139-Fcmod potently inhibited Gα signaling in a dose-dependent manner.

[0451] Table 13 summarizes the inhibition at 10 nM and 100 nM C5a concentrations compared to avacopam and anti-C5aR1 control Ab. It was observed that c2137-e1711-F inhibited the C5a inhibition at 10 nM and 100 nM C5a concentrations compared to avacopam and anti-C5aR1 control Ab, even at higher C5a concentrations. c mod and c2139-Fcmod maintained excellent inhibition.

[0452] Table 13 - c2137-e1711-F in the presence of C5a compared to avacopam and anti-C5aR1 control Ab c mod and c2139-F c Inhibition *Extrapolated data - Previous data using avacopam suggest it is not extremely effective at high C5a concentrations

[0453] Example 18 - Inhibition of Calcium Signaling by Fc-Modified C5aR1 Antibody

[0454] This example describes the use of calcium flux analysis to analyze the functional aspects of the C5aR1 Fc-modified monospecific and biparatopic antibodies described herein for inhibition of C5a-mediated calcium signaling.

[0455] This experiment was performed as described in Example 3. Figures 20A to 20B The C5aR1 Fc-modified antibody (c2137-e1711-F c mod and c2139-F c mod) inhibits calcium signaling. c mod and c2139-F c mod inhibited calcium flux more effectively than avacopam and anti-C5aR1 control Ab. It was observed that at least 10 to 100 times was required to achieve the same level of inhibition by c2137-e1711-F c mod and c2139-F c It was observed that the Fc-modified C5aR1 antibody inhibited calcium signaling in human neutrophils and U937-C5aR1 cells. Figure 20A To present an exemplary graph showing dose response curves demonstrating inhibition of calcium signaling using an exemplary Fc-modified humanized site II antibody as described in the present disclosure in the presence of increasing concentrations of antibody and 100 nM C5a. Figure 20BTo show the percent inhibition of calcium signaling using an exemplary Fc-modified humanized Site II antibody as described in this disclosure in the presence of antibody and 100 nM C5a.

[0456] Figures 21A to 21D Demonstrates inhibition in U937-C5aR1 cells compared to human neutrophils. Figure 21A Demonstrated inhibition of calcium signaling in U937-C5aR1 cells in the presence of 10 nM C5a. Figure 21B Demonstrated inhibition of calcium signaling in U937-C5aR1 cells in the presence of 100 nM C5a. Figure 21C Demonstrates inhibition of calcium signaling in human neutrophils in the presence of 10 nM C5a. Figure 21D Demonstrates inhibition of calcium signaling in human neutrophils in the presence of 100 nM C5a.

[0457] Furthermore, inhibition of C5a-mediated calcium signaling by Fc-modified antibodies was probed in U937-C5aR1 cells after incubation with the indicated concentrations of antagonistic antibodies for various incubation times (eg, 1 hour and 3 hours). Figures 22A to 22B Saturation levels of U937-C5aR1 cells with each inhibitor are summarized. Figure 22A Summary After 1 hour incubation, the cells were incubated with increasing concentrations of antibody (c2139-F c mod and c2137-e1711-F c Saturation percentage and F norm of U937-C5aR1 cells incubated with 100 nM C5a (Fig. Figure 22B Summary After 3 hours of incubation, the cells were treated with increasing concentrations of antibody (c2139-F c mod and c2137-e1711-F c Saturation percentage and F norm of U937-C5aR1 cells incubated with 100 nM C5a (Fig.

[0458] Figure 22A Display by c2137-e1711-F c mod and c2139-F c Dose-response of MOD inhibition of calcium signaling. Figure 22B Display by c2137-e1711-F c mod and c2139-F c Percent inhibition of calcium signaling by MOD.

[0459] Observed, c2137-e1711-F cmod was more effective than avacopam and anti-C5aR1 control Ab. In addition, c2139-F c mod reached a saturation point at a shorter antagonist incubation time. This saturation was slightly reduced with the extension of antagonist incubation time. This phenomenon was only observed in c2139-F c mod and also observed in precursor c2139.

[0460] Example 19 - Inhibition of β-arrestin signaling by Fc-modified C5aR1 antibodies

[0461] This example describes the functional aspects of the Fc-modified C5aR1 monospecific and biparatopic antibodies described herein for inhibiting β-arrestin recruitment.

[0462] Experimental details for determining β-arrestin recruitment are described in Example 6. Figures 23A to 23B Overview by c2137-e1711-F c mod and c2139-F c Mod inhibits C5a-mediated β-arrestin signaling. c mod and c2139-F c Mod blocked β-arrestin recruitment to C5aR1 more effectively than avacopam and anti-C5aR1 control Ab. Table 14 summarizes the K for inhibition of β-arrestin recruitment D Table 14 - K of β-arrestin inhibition D

[0463] antagonists IC50 (nM) of 100nM C5a c2139-Fcmod 1.9 <![CDATA[c2137-e1711-F c against]]> 0.5 Avacropam 34 Anti-C5aR1 control Ab 12.7

[0464] Example 20 - Inhibition of Neutrophil Chemotaxis by Fc-Modified C5aR1 Antibody

[0465] This example describes the functional aspects of the Fc-modified C5aR1 monospecific and biparatopic antibodies described herein for use in inhibiting neutrophil chemotaxis known to be induced by C5aR1 activity using a Boyden Chamber.

[0466] Experimental details for determining neutrophil chemotaxis are described in Example 4. Figures 24A to 24D Displayed in C5aR1 antibody, c2137-e1711-F c mod and c2139-F c Inhibition of chemotaxis in C5aR1-U937 stable cells after mod treatment. c mod inhibited chemotaxis more effectively than avacopam and anti-C5aR1 control Ab. Figure 24A Displayed at 1nM, 3.16nM and 10nM c2139-F c mod and increasing concentrations of C5a inhibit chemotaxis in C5aR1-U937 stable cells. Figure 24B Displayed at 1nM, 3.16nM, and 10nM C5a c2137-e1711-F c mod and increasing concentrations of C5a inhibit chemotaxis in C5aR1-U937 stable cells. Figures 24C to 24D Displayed in c2137-e1711-F c Inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of mod, in the presence of 1 nM, 3.16 nM and 10 nM anti-C5aR1 control Ab and avacopam, respectively.

[0467] Example 21 - Inhibition of CD11b expression by Fc-modified C5aR1 antibody

[0468] This example describes the functional aspects of the Fc-modified C5aR1 monospecific and biparatopic antibodies described herein for inhibition of CD11b expression.

[0469] Experimental details for determining CD11 expression are described in Example 5. Figures 25A to 25B Showcase response to c2137-e1711-F c mod and c2139-F c Mod treatment inhibited CD11b signaling. It was observed that c2137-e1711-F c mod and c2139-F c mod effectively inhibited CD11b expression over a wide range of C5a concentrations. Figure 25A Inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibodies and 10 nM C5a is demonstrated. Figure 25B Inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibodies and 100 nM C5a is demonstrated.

[0470] Example 22 - Inhibition of ROS Generation by Fc-Modified C5aR1 Antibody

[0471] This example shows an exemplary humanized Fc-modified monospecific antibody (c2139-F c mod) and an exemplary biparatopic antibody (c2137-e1711-F c mod) reduces reactive oxygen species (ROS).

[0472] Experimental details for measuring ROS production are described in Example 7. Figures 26A to 26B Compared with c2139, c2137-e1711, anti-C5aR1 control Ab and avacopam, the effect of c2139-F c mod、c2137-e1711-F c Inhibition of ROS production in WB cells lysed by mod-treated RBCs. Similar to avacopam and anti-C5aR1 control Ab, c2139-F c mod and c2137-e1711-F c mod maintains low respiratory burst activity in neutrophils. In addition, compared with the previous forms c2139 and c2137-e1711, c2139-F c mod and c2137-e1711-F c The mod has reduced respiratory burst activity. Figure 26A Inhibition of ROS production by increasing concentrations of monospecific C5aR1 antibody (Fc modified) is shown. Figure 26B Inhibition of ROS production by increasing concentrations of the biparatopic C5aR1 antibody (Fc modified) is demonstrated.

[0473] Example 23. Suppression of Neutropenia in Human C5aR1 Transgenic Mice by Fc-Modified C5aR1 Antibodies

[0474] This example demonstrates the inhibition of neutropenia by an Fc-modified C5aR1 antibody in transgenic human C5aR1 mice. As described above (Example 11), the exemplary C5aR1 antibody does not cross-react with mouse C5aR1. At the Jackson Laboratory, CRISPR technology was used to generate transgenic human C5aR1 (hC5aR1) knock-in mice.

[0475] Figure 27A The experimental design for evaluating the reduction of neutropenia in mice is presented. The experimental details of this in vivo analysis of mice are described in Example 11. The percent change in neutrophils and the mean change in neutrophils in all groups were calculated and presented in Figures 27B to 27C The mean changes in neutrophils are shown in Figure 27B The changes in neutrophil counts are shown in Figure 27C The new Fc-silencing lead was observed to be just as effective as the non-silencing c2139 and c2137-e1711. The vehicle (PBS) group showed robust neutropenia, with a mean 65% reduction in neutrophil counts relative to baseline 1 minute after C5a injection. One animal in the vehicle control group did not respond to C5a.

[0476] Example 24. Stroke Protection in a Mouse Model

[0477] This experiment confirmed that 2139-F c mod and c2137-e1711-F c The ability of MOD to protect infarct volume in a mouse model of stroke Neutrophil activity and infiltration into the brain are well documented in acute models of stroke and traumatic brain injury pathology.

[0478] In tMCAO mice, 20 mg / kg of c2139-F was administered c mod and c2137-e1711-F c 1 mg / kg PMX53 was used as a control. The brain was excised, sliced, and stained with TTC. The stained cross sections were analyzed by imaging.

[0479] It was observed that compared with vehicle, c2139-F c mod and c2137-e1711-F c Mod-treated mice were protected from stroke by significantly reducing infarct volume. c Mod treatment showed a significant reduction in infarct volume. c mod treatment showed reduced infarct volume. PMX53 treatment (serving as a positive control and comparator) showed only slightly reduced infarct volume compared to the vehicle group. Figure 28A Graph showing reduction in infarct volume. Figure 28B Display c2139-F in use c mod、c2137-e1711-F c Graphical representation of infarct volume after mod and PMX53 treatment.

[0480] Example 25. Pharmacokinetic Study of Exemplary C5aR1 Antibodies in hC5aR1 Mice with Modified Fc Domains

[0481] This example shows a pharmacokinetic (PK) study of an exemplary C5aR1 antibody with a modified Fc domain in mice. Tg32 mice are a transgenic model in which human FcRn replaces the native mouse FcRn. FcRn is required for bidirectional transport of Abs across cell barriers, thereby affecting PK. Tg32 mice with human FcRn have been extensively studied and are believed to be relevant to PK in humans. The purpose of this experiment was to assess FcRn-mediated recycling of IgG-scFv and IgG formats and to assess any effects of introducing Fc domain modifications on FcRn-mediated recycling.

[0482] 5 mg / kg of exemplary monospecific and biparatopic antibodies were injected intravenously into Tg32 mice. The percentage of antibody in serum was determined using art-recognized assays. MVZ-IgG4-VFc17 (a motavizumab antibody with an IgG4 Fc and the same Fc modification as the C5aR1 antibody) was used as a control. Additional PK / PD analyses were performed using CERTARA.

[0483] C-max (maximum concentration of antibody in blood) was observed to be similar for both monospecific and biparatopic antibodies. c mod and c2137-e1711-F c mod, the half-lives of the antibodies are similar.

[0484] The amount of antibodies in the serum was assessed over a period of 500 hours.Table 15 shows the doses and sampling intervals used for the pharmacokinetic studies.

[0485] Table 15 - Dosing and sampling schedules for exemplary C5aR1 Fc-modified antibodies used to determine the pharmacokinetics of individual antibodies.

[0486]

[0487] Figures 29A to 29B Pharmacokinetic properties of C5aR1-Fc modified antibodies are shown. Figure 29A Figure 2 is a graphical representation of the percentage of Fc-modified C5aR1 antibodies that persist in serum for 500 hours. Figure 29B The mean concentrations in μg antibody / ml serum over 500 hours are shown in Figure 2. These observations show that c2137-e1711-F c mod binds efficiently to hFcRn and binds to c2139-F c Mod was recycled in a similar manner. In addition, the PK data were also similar to the PK of the C5aR1 antibody with an unmodified Fc domain. Table 16 shows the PK parameters of exemplary Fc-modified C5aR1 antibodies.

[0488] Table 16 - Pharmacokinetic parameters of Fc-modified C5aR1 antibodies.

[0489]

[0490]

[0491] Example 26 - Multi-dose Pharmacokinetics of Fc-Modified C5aR1 Antibodies in Human C5aR1 Knock-in Mice

[0492] This example demonstrates a multiple-dose pharmacokinetic (PK) study in mice of exemplary C5aR1 antibodies and the modified Fc domain antibody hC5aR1.

[0493] Table 17 summarizes the dosing regimens for the tested antibodies. Target-mediated drug disposition (TMDD) was evident for the two exemplary C5aR1 antibodies with modified Fc domains. TMDD is the extent to which a drug binds to its pharmacological target site (e.g., a receptor) with high affinity to the point of affecting its pharmacokinetic (PK) characteristics. Target binding and subsequent elimination of the drug-target complex can affect both drug distribution and elimination and produce nonlinearity in PK in a dose-dependent manner.

[0494] The main observed PK of TMDD was linear at high dose level, while the main observed PK was nonlinear at low dose level.

[0495] As expected for antibodies with TMDD, dose-dependent clearance was observed. The maximum concentration C for each antibody was found at 5 mg / Kg and 0.5 mg / Kg. max It was similar for both antibodies. In addition, at 5 mg / Kg and 0.5 mg / Kg, the half-life (T 0.5 At 20 mg / Kg, c2139 has a more preferred half-life (T 0.5 ).

[0496] Table 17 - Dosing and sampling schedules for exemplary C5aR1 Fc-modified antibodies used to determine the pharmacokinetics of individual antibodies.

[0497]

[0498]

[0499] Target binding and subsequent elimination of drug-target complexes may affect both drug distribution and elimination and produce nonlinearity in PK in a dose-dependent manner. Figures 30A to 30F Antibody persistence was shown to be dose dependent.Table 18 shows the PK parameters of exemplary Fc-modified C5aR1 antibodies. Figure 30A Graph showing the dose-response curve of c2139-Fcmod in serum for 200 hours. Figure 30B Graph showing the dose-response curve of c2137-e1711-Fcmod in serum for 200 hours. Figure 30C Comparison of c2139Fcmod, c2137-e1711-Fcmod and MVZ-IgG4. Figure 30DFigure 2 is a computer simulation of three different concentrations of c2139 over a 500-hour period. Figure 30E Figure 3 is a computer simulation of three different concentrations of c2137-e1711 over a 500-hour period. Figure 30F This is a computer simulation of an isotype control antibody at a concentration of 20 mg / kg over a 500-hour period. Multiple doses of mAb were administered weekly to non-native mice for a total of 4 weeks. It was observed that both c2139Fcmod and c2137-e1711-Fcmod maintained exposure at 5 mg / kg IV once weekly. No severe effects were observed with weekly mAb administration for several weeks.

[0500] PK parameters of exemplary Fc-modified C5aR1 antibodies

[0501]

[0502] Example 27 - Inhibition of Neutropenia in Human C5aR1 KI Mice by Low Dose of Fc-Modified C5aR1 Antibody

[0503] This example demonstrates that c2139-Fcmod and c2137-e1711-Fcmod can suppress neutropenia in human C5aR1 knock-in mice at low doses.

[0504] Table 19 summarizes the dosing regimens of the tested antibodies and the blood sampling times for neutropenia determination. The change in neutrophils relative to baseline at 1 min after C5a administration (-5 min bleed) is shown in Figure 31 middle.

[0505] It was observed that c2139-Fcmod and c2137-e1711-Fcmod effectively inhibited neutropenia induced by C5a administration compared to vehicle control even at a dose of 0.1 mg / kg.

[0506] Table 19. Dosage regimen

[0507]

[0508] Example 28 - Internalization of Fc-modified C5aR1 antibody

[0509] This example demonstrates internalization of Fc-modified humanized monospecific and biparatopic C5aR1 antibodies in hC5ar1-U937 cells.

[0510] The Fc-modified humanized monospecific anti-C5aR1 antibodies c2137-e1711-Fcmod and c2139-Fcmod were conjugated to a pH-sensitive dye (DyLight488) that fluoresces brightly at low pH but not at neutral pH. The bound antibodies were incubated with U937 cells and hC5aR1 knock-in U937 cells. Figure 32A Fluorescence intensity after 6 hours and 24 hours of incubation with each binding antibody is shown.

[0511] The exemplary Fc-modified C5aR1 antibody was observed to undergo metabolism-based internalization. Figure 32B Shown is the internalization of both c2137-e1711-Fcmod and c2139-Fcmod in living cells as observed by Nikon confocal experiments over a 300 min period.

[0512] Equivalents and scope

[0513] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the above description, but is set forth in the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said VH comprises three heavy chain complementarity-determining regions (HCDRs), wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the amino acid sequences of SEQ ID No: 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG), and 8 (SGGDNYGNPYYFDR), respectively; and a light chain variable region (VL), wherein said VL comprises three light chain complementarity-determining regions (LCDRs), wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the amino acid sequences of SEQ ID No: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT), respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VH comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

14.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein said VH comprises the amino acid sequence of SEQ ID NO:

14.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein said VL comprises an amino acid sequence having at least 95% identity with SEQ ID NO:

25.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein said VL comprises the amino acid sequence of SEQ ID NO:

25.

6. An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), said antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) of SEQ ID NO: 14; and a light chain variable region (VL) of SEQ ID NO:

25.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment thereof further comprises an Fc region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the Fc domain is independently selected from IgG1, IgG2, IgG3, and IgG4.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody that binds to C5aR1 inhibits the interaction between complement component 5a (C5a) and C5aR1.

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody does not bind to C5aR2.

11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or antigen-binding fragment is humanized.

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said VH or said VL has been modified to enhance the stability of the molecule.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein said VL comprises serine or tyrosine at position 96 of SEQ ID NO:

25.

14. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment does not cross-react with murine C5aR1.

15. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of the preceding claims.

16. A cell comprising the nucleic acid according to claim 15.

17. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-14.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1-14 for use in the manufacture of a medicament for the treatment of autoimmune diseases.

19. An antibody or antigen-binding fragment thereof according to any one of claims 1-14 for use in the manufacture of a medicament for the treatment of a disease caused by neutropenia.

20. The antibody or antigen-binding fragment thereof for use according to claim 19, wherein the neutropenia is caused by high levels of C5a.

21. The antibody or antigen-binding fragment thereof for use according to any one of claims 18-20, wherein the disease is ANCA vasculitis or lupus.

22. The antibody or antigen-binding fragment thereof for use according to any one of claims 18-20, wherein the disease is rheumatoid arthritis.

23. The antibody or antigen-binding fragment thereof for use according to any one of claims 18-20, wherein the disease is a kidney disorder.

24. The antibody or antigen-binding fragment thereof for use according to any one of claims 18-20, wherein the disease is a stroke.

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