Humanized complement 5A receptor 1 antibody and method of use thereof

Abstract

The present disclosure provides, inter alia, two different formats of humanized antibodies against human complement component 5a receptor 1. The present disclosure also provides a method of treating a subject having a dysfunction of the C5a / C5aR1 axis pathway, including but not limited to ANCA-associated vasculitis, comprising administering to a subject in need thereof an effective amount of an antibody, or a nucleic acid encoding an antibody, that binds to C5aR1 as described herein, wherein administration results in a reduction in symptoms associated with the C5a / C5aR1-associated dysfunction in the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 137,089, filed on 13 January 2021, and U.S. Provisional Patent Application No. 63 / 274,748, filed on 2 November 2021, the disclosures of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Compositions and methods for reducing autoimmune diseases and disorders associated with complement 5a / complement 5a receptor 1C5a / C5aR1-mediated immune inflammation are disclosed. The C5a-C5aR1 axis is of particular interest for therapeutic interventions to block neutrophil attraction to local sites, inhibit neutrophil activation and vasodeogenesis. Compositions and methods disclosed herein may include the step of administering a C5aR1 antagonist, and methods for treating subjects requiring such treatment. [Overview of the project] [Means for solving the problem]

[0003] This disclosure provides, in particular, anti-C5aR1 antibodies with increased specificity to C5aR1, and the therapeutic use of such antibodies in effectively treating diseases or disorders related to C5 and its receptors, such as ANCA vasculitis, typical hemolytic uremic syndrome, age-related macular degeneration, rheumatoid arthritis, sepsis, severe burns, antiphospholipid syndrome, asthma, lupus nephritis, Goodpasture syndrome, and chronic obstructive pulmonary disease. As described herein, this disclosure is in part based on the identification of humanized anti-C5aR1-specific antibodies that bind to specific regions of site I and / or site II of anti-C5aR1 and significantly reduce cross-reactivity to C5aR2 or any other G protein-coupled receptor. In particular, the anti-C5aR1 antibodies of this disclosure have high binding affinity to C5aR1 (e.g., less than 50 nM K) D) and C5aR2 are characterized by minimal cross-reactivity. This is important because the C5aR1 antibody of this disclosure enables potent inhibition of C5aR1 signaling in the presence of high C5a concentrations. As a result, the C5aR1 antibody of this disclosure can be used at lower doses to achieve therapeutic effects compared to other anti-C5aR1 or C5a antibodies. This is demonstrated by the remarkably high potency observed in functional assays compared to prior art antibodies described herein. Furthermore, the highly potent site I C5aR1 antibody of this disclosure competes with each other with respect to site I, and the highly potent site II C5aR1 antibody of this disclosure competes with each other on site II. Furthermore, this 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 a bispecific antibody (e.g., a biparatopic antibody) (but not two site II antibodies or two site II antibodies) significantly enhances activity. The anti-C5aR1 antibodies of the invention disclosed herein promise more potent treatment of complement-mediated diseases and disorders, particularly ANCA vasculitis.

[0004] Furthermore, this disclosure provides anti-C5aR1 antibodies, in particular, including Fc variants with significantly reduced ADCC, ADCP, and CDC function. As described herein, the anti-C5aR1 antibodies of this disclosure include novel mutational combinations that inactivate binding to all FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and C1q while maintaining their ability to bind to FcRn.

[0005] In some embodiments, the C5aR1 antibody provided herein has a wild-type IgG4 Fc domain. In some embodiments, the C5aR1 antibody provided herein has a modified IgG4 Fc domain. In some embodiments, the modified C5aR1 antibody includes a Fab arm exchange mutation. In some embodiments, the modified C5aR1 antibody further includes an Fc silencing mutation.

[0006] The C5a-C5aR1 axis is of particular interest for therapeutic interventions that block neutrophil attraction to local sites and inhibit neutrophil activation and vascular destruction. Compositions and methods disclosed herein may include the step of administering a C5aR1 antagonist, and methods for treating subjects requiring such treatment.

[0007] In one embodiment, the disclosure presents an antibody or antibody-conjugated fragment that binds to at least one of the sequences of C5aR1, including the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0008] In some embodiments, the antibody or its antigen-binding fragment binds to SEQ ID NO: 1 of C5aR1. In some embodiments, the antibody or its antigen-binding fragment binds to SEQ ID NO: 2 of C5aR1. In some embodiments, the antibody or its antigen-binding fragment binds to SEQ ID NO: 3 of C5aR1.

[0009] In one embodiment, the present disclosure presents an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14.

[0010] In some embodiments, VH includes an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 14. In some embodiments, VH includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 14.

[0011] In one embodiment, the disclosure presents an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), including a light chain variable region (VL), wherein the VL includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 25.

[0012] In some embodiments, VH includes an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, VH includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 25.

[0013] In one embodiment, the disclosure presents an antibody or antigen-binding fragment thereof comprising a VH region, wherein the VH comprises three heavy chain complementarity-determining regions (HCDRs), and the HCDR1, HCDR2, and HCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 6(NYWMH), 7(YLNPSSGYTKYAQKFQG), and 8(SGGDNYGNPYYFDR), respectively.

[0014] In one embodiment, the present disclosure presents an antibody or antigen-binding fragment thereof comprising a VL region, the VL comprising three light chain complementarity-determining regions (LCDRs), wherein the LCDR1, LCDR2, and LCDR3 sequences each comprise the amino acid sequences of SEQ ID NO: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT).

[0015] In one embodiment, the present disclosure presents an antibody or antigen-binding fragment comprising: a VH region, wherein VH comprises three heavy chain complementarity-determining regions (HCDRs), and the HCDR1, HCDR2, and HCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG), and 8 (SGGDNYGNPYYFDR); and a VL region, wherein VL comprises three light chain complementarity-determining regions (LCDRs), and the LCDR1, LCDR2, and LCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT).

[0016] In one embodiment, the antibody or antigen-binding fragment thereof according to the present disclosure further comprises an Fc region.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof according to the present disclosure further comprises an Fc region, the Fc domain being independently selected from IgG1, IgG2, IgG3, and IgG4.

[0018] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits the interaction between complement component 5a (C5a) and C5aR1.

[0019] In one embodiment, the antibody or its antigen-binding fragment does not bind to C5aR2 or any other GPCR.

[0020] In one embodiment, the antibody or its antigen-binding fragment is humanized.

[0021] In one embodiment, the VH or VL of an antibody or an antigen-binding fragment thereof is modified to increase the stability of the molecule.

[0022] In one embodiment, the antibody or an antigen-binding fragment thereof comprises a serine or tyrosine mutation at position 96 of SEQ ID NO: 5 or SEQ ID NO: 25.

[0023] In one embodiment, the antibody or an antigen-binding fragment thereof does not cross-react with mouse C5aR1.

[0024] In one embodiment, the antibody or an antigen-binding fragment thereof binds to C5aR1 with an affinity of 10 pM to 50 nM.

[0025] In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 100 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 90 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 80 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 75 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 70 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 65 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 60 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 60 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (K D ) that binds to C5aR1 of less than about 55 nM. In some embodiments, the antibody or an antigen-binding fragment thereof has a dissociation constant (KD ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 45 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 40 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 35 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 30 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 25 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 20 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 15 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 10 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 8 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 5 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 3 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 1 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 0.5 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of less than about 0.1 nM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) less than about 100 pM. D) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) less than about 80 pM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) less than about 50 pM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) less than about 25 pM. D ) binds to C5aR1. In some embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) less than about 10 pM. D ) binds to C5aR1. In one embodiment, the antibody or its antigen-binding fragment binds to C5aR1 with an affinity of 0.16 nM or less.

[0026] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis.

[0027] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 0.1 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 0.5 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 1 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 3 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 5 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 7 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 10 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 15 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 20 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 25 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 30 nM C5a concentration. In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of at least 40 nM C5a concentration. In one embodiment, an 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 another embodiment, an 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 yet another embodiment, an 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, an 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 another embodiment, an 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 yet another embodiment, an 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.

[0028] In one embodiment, the antibody or its antigen-binding fragment inhibits C5a-mediated C5aR1 Gα signaling.

[0029] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits calcium signaling.

[0030] In one embodiment, an antibody that binds to C5aR1 or its antigen-binding fragment inhibits CD11b expression.

[0031] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits neutropenia.

[0032] In one embodiment, an antibody or its antigen-binding fragment that binds to C5aR1 inhibits β-arrestin signaling.

[0033] In one embodiment, an antibody that binds to C5aR1 or an antigen-binding fragment thereof inhibits ROS production in neutrophils.

[0034] In one embodiment, the antibody or its antigen-binding fragment is stable for up to one week at approximately 4°C after one or more freeze-thaw cycles. In some embodiments, the antibody or its antigen-binding fragment is stable for up to one week at approximately 1 to 15°C after one or more freeze-thaw cycles. In some embodiments, the antibody or its antigen-binding fragment is stable for up to one week at approximately 2 to 10°C after one or more freeze-thaw cycles. In some embodiments, the antibody or its antigen-binding fragment is stable for up to two weeks at approximately 3 to 8°C after one or more freeze-thaw cycles.

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

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

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

[0038] In one embodiment, the disclosure includes nucleic acids encoding any antibody or antigen-binding fragment thereof as described herein.

[0039] In one embodiment, the disclosure includes cells comprising nucleic acids encoding any antibody or antigen-binding fragment thereof as described herein.

[0040] In one embodiment, the present disclosure encompasses a method for producing an antibody or an antigen-binding fragment thereof as described herein, the method comprising culturing a host cell containing a nucleic acid encoding the antibody or the antigen-binding fragment thereof, wherein the cell is cultured under conditions that enable the production of the antibody or the antigen-binding fragment thereof.

[0041] In one embodiment, the disclosure comprises a method for treating an autoimmune disease using an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1) and includes a heavy chain variable region (VH), wherein VH includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14 and / or an antibody or antigen-binding fragment that binds to human complement component 5a receptor 1 (C5aR1) and includes a light chain variable region (VL), wherein VL includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 25.

[0042] In one embodiment, a method for treating an autoimmune disease includes using an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is an antibody or antigen-binding fragment that binds to human complement component 5a receptor 1 (C5aR1) and includes a heavy chain variable region (VH), wherein VH includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14, and the antibody or antigen-binding fragment is an antibody or antigen-binding fragment that binds to human complement component 5a receptor 1 (C5aR1) and includes a light chain variable region (VL), wherein VL includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15.

[0043] In one embodiment, the present disclosure comprises a method for treating an autoimmune disease using an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is bound to complement component 5a receptor 1 (C5aR1), and the antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (HCDRs), where the HCDR1, HCDR2, and HCDR3 sequences each contain the amino acid sequences of SEQ ID NOs. 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG), and 8 (SGGDNYGNPYYFDR), respectively, and three light chain complementarity-determining regions (LCDRs), where the LCDR1, LCDR2, and LCDR3 sequences each contain the amino acid sequences of SEQ ID NOs. 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT), respectively.

[0044] In one embodiment, the present disclosure includes a method for treating an autoimmune disease using an antibody or an antigen-binding fragment thereof, which comprises SEQ ID NO: 4 or an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 4.

[0045] In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 87% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 4.

[0046] In one embodiment, the present disclosure includes a method for treating an autoimmune disease using an antibody or an antigen-binding fragment thereof, which comprises SEQ ID NO: 5 or an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 5.

[0047] In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 87% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or its antigen-binding fragment includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 4.

[0048] In one embodiment, the present disclosure encompasses diseases caused by neutropenia induced using antibodies or antigen-binding fragments thereof as described herein.

[0049] In one embodiment, neutropenia is caused by high levels of C5a.

[0050] In one embodiment, the disease is ANCA vasculitis or lupus.

[0051] In one embodiment, the disorder is rheumatoid arthritis.

[0052] In one embodiment, the disorder is kidney damage.

[0053] In one embodiment, the present disclosure includes a method for inhibiting C5a signaling using a monoclonal antibody that binds to C5aR1, comprising a heavy chain variable region (VH) (VH comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14) and a light chain variable region (VL) (VL comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 25).

[0054] In one embodiment, the disclosure comprises a biparatopic antibody or an antigen-binding fragment thereof comprising two pairs of antigen-binding domains, wherein the first antigen-binding domain comprises VH1 and VL1, which bind to C5aR1 by SEQ ID NO: 3, and the second antigen-binding domain comprises VH2 and VL2, which bind to C5aR1 by SEQ ID NO: 1 or SEQ ID NO: 2.

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

[0056] In one embodiment, the biparatopic antibody or its antigen-binding fragment comprises SEQ ID NO: 14 or VH1 which is at least 90% identical to SEQ ID NO: 14.

[0057] In one embodiment, the biparatopic antibody or its antigen-binding fragment comprises VL, where 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 having at least 75% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 84% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 comprises an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, VL1 includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 15.

[0058] In one embodiment, the biparatopic antibody or its antigen-binding fragment comprises VH2, which 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, VH2 comprises an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 16.

[0059] In one embodiment, the biparatopic antibody or its antigen-binding fragment comprises VL2, which 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 having at least 75% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 84% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 comprises an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VL2 includes an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 17. In some embodiments, VH2 includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 17.In some embodiments, VL2 includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 17.

[0060] In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment, comprising a heavy chain of SEQ ID NO: 12 or a heavy chain identical to at least 85% of the amino acid sequence of SEQ ID NO: 12, which binds to SEQ ID NO: 3. In some embodiments, the heavy chain comprises an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 84% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain comprises an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the heavy chain includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 12.

[0061] In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising a light chain of SEQ ID NO: 13 or a light chain identical to at least 85% of the amino acid sequence of SEQ ID NO: 13, which binds to SEQ ID NO: 3. In some embodiments, the light chain comprises an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 78% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 84% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain comprises an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the light chain includes an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 13.

[0062] In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain at least 85% identical to the amino acid sequence of SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or a light chain at least 85% identical to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain at least 90% identical to the amino acid sequence of SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or a light chain at least 90% identical to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain at least 92% identical to the amino acid sequence of SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or a light chain at least 92% identical to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain at least 95% identical to the amino acid sequence of SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or a light chain at least 95% identical to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain at least 99% identical to the amino acid sequence of SEQ ID NO: 12, and a light chain comprising SEQ ID NO: 13, or a light chain at least 99% identical to the amino acid sequence of SEQ ID NO: 13.

[0063] In one embodiment, the present invention provides an anti-C5aR1 biparatopic antibody comprising, in particular, the heavy chain of SEQ ID NO: 71 and the 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 contains a light chain that is at least 85% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 90% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 92% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 95% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 97% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 98% identical to SEQ ID NO: 72. In some embodiments, the anti-C5aR1 biparatopic antibody contains a light chain that is at least 99% identical to SEQ ID NO: 72.

[0064] In one embodiment, the present invention provides an anti-C5aR1 antibody comprising, in particular, the heavy chain of SEQ ID NO: 69 and the 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 that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 90% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 92% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 85% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 95% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 97% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 98% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody comprises a heavy chain that is at least 99% identical to that of SEQ ID NO: 69. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 85% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 90% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 92% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 85% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 95% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 97% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 98% identical to SEQ ID NO: 70. In some embodiments, the anti-C5aR1 antibody contains a light chain that is at least 99% identical to SEQ ID NO: 70.

[0065] In one embodiment, the disclosure includes a biparatopic antibody or its antigen-binding fragment, wherein the heavy chain comprises VH1 linked to an Fc domain.

[0066] In one embodiment, the Fc domain of a biparatopic antibody or its antigen-binding fragment is further ligated to scFv, which includes VH2, containing the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16, and / or VL2, containing the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17.

[0067] In one embodiment, the Fc domain is independently selected from IgG1, IgG2, IgG3, and IgG4.

[0068] In one embodiment, the scFv is linked to the Fc domain via a linker.

[0069] In one embodiment, the linker includes at least 5 amino acids, each containing an amino acid sequence having one of the sequence numbers 26-37. In one embodiment, the linker includes at least 3 amino acids, each containing an amino acid sequence having one of the sequence numbers 26-37. In one embodiment, the linker includes at least 4 amino acids, each containing an amino acid sequence having one of the sequence numbers 26-37. In one embodiment, the linker includes at least 6 amino acids, each containing an amino acid sequence having one of the sequence numbers 26-37. In one embodiment, the linker includes at least 7 amino acids, each containing an amino acid sequence having one of the sequence numbers 26-37.

[0070] In one embodiment, VH2 and VL2 of the biparatopic antibody, including SEQ ID NOs. 16 and SEQ ID NOs. 17, are linked to each other via a linker.

[0071] In one embodiment, the linker includes 1 to 10 repeats of SEQ ID NO: 31.

[0072] In one embodiment, the VH2 and VL2 of the biparatopic antibodies, which include an amino acid sequence that is 90% identical to SEQ ID NO: 16 and SEQ ID NO: 17, or SEQ ID NO: 16 and SEQ ID NO: 17, further include one or more mutations to improve the thermal stability of the biparatopic antibody.

[0073] In one embodiment, mutations to improve the thermal stability of the biparatopic antibody include the incorporation of cysteine ​​at position 559 and position 630 of SEQ ID NO: 12.

[0074] In one embodiment, a biparatopic antibody or its antigen-binding fragment that binds to C5aR1 inhibits the interaction between complement component 5a (C5a) and human C5aR1.

[0075] In one embodiment, the biparatopic antibody or its antigen-binding fragment does not bind to C5aR2.

[0076] In one embodiment, the biparatopic antibody is humanized.

[0077] In one embodiment, the biparatopic antibody does not cross-react with mouse C5aR1.

[0078] In one embodiment, the biparatopic antibody is stable for up to two weeks at 4°C after one freeze-thaw cycle. In some embodiments, the biparatopic antibody or its antigen-binding fragment is stable for up to two weeks at approximately 1–15°C after one or more freeze-thaw cycles. In some embodiments, the biparatopic antibody or its antigen-binding fragment is stable for up to two weeks at approximately 2–10°C after one or more freeze-thaw cycles. In some embodiments, the biparatopic antibody or its antigen-binding fragment is stable for up to two weeks at approximately 3–8°C after one or more freeze-thaw cycles.

[0079] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis.

[0080] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of high C5a concentrations.

[0081] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 10 nM.

[0082] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits C5a-mediated C5aR1 Gα signaling.

[0083] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits β-arrestin signaling.

[0084] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits ROS production in neutrophils.

[0085] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits calcium signaling.

[0086] In one embodiment, a biparatopic antibody that binds to C5aR1 inhibits CD11b expression.

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

[0088] In one embodiment, the present disclosure includes nucleic acids encoding biparatopic antibodies described herein.

[0089] In one embodiment, the disclosure includes cells containing nucleic acids encoding the biparatopic antibodies described herein.

[0090] In one embodiment, the present disclosure comprises a method for producing a biparatopic antibody or its antigen-binding fragment as described herein, the method comprising culturing a host cell containing a nucleic acid encoding the antibody or its antigen-binding fragment, wherein the cell is cultured under conditions that enable the production of the antibody or its antigen-binding fragment.

[0091] In one embodiment, the present disclosure comprises a method for treating an autoimmune disease using a biparatopic antibody, the antibody being bound to human complement component 5a receptor 1 (C5aR1) and comprising: VH1 and VL1, which are a first VH and a first VL, with VH1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 14 or SEQ ID NO: 14, and VL1 comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 15 or SEQ ID NO: 15; VH2 and VL2, which are a second VH and a second VL, with VH2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 16 or SEQ ID NO: 16, and VL2 comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 17 or SEQ ID NO: 17.

[0092] In one embodiment, the present disclosure comprises a method for treating an autoimmune disease, which is caused by neutropenia, using an antibody or antigen-binding fragment thereof as described herein.

[0093] In one embodiment, neutropenia is caused by high levels of C5a.

[0094] In one embodiment, the disease is ANCA vasculitis or lupus.

[0095] In one embodiment, the disorder is rheumatoid arthritis.

[0096] In one embodiment, the disorder is kidney damage.

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

[0098] In one embodiment, the antibody is a monospecific or biparatopic C5aR1 antibody from any of the preceding embodiments, comprising a constant domain of modified IgG1, IgG4, or IgG2.

[0099] In one embodiment, antibody C5aR1 contains a modified IgG4 Fc domain.

[0100] In one embodiment, the modified IgG4 Fc domain includes substitutions at positions F234, L235 and / or D265.

[0101] In one embodiment, the IgG4 Fc substitution at position F234 is a hydrophobic amino acid selected from alanine, valine, leucine, isoleucine, phenylalanine, or tryptophan.

[0102] In one embodiment, the IgG4 Fc substitution at position F234 is valine.

[0103] In one embodiment, the IgG4 Fc substitution at position L235 is an acidic amino acid.

[0104] In one embodiment, the IgG4 Fc substitution at position L235 is an acidic amino acid selected from glutamic acid or aspartic acid.

[0105] In one embodiment, the IgG4 Fc substitution at the L235 position is aspartic acid.

[0106] In one embodiment, the IgG4 Fc substitution at position D265 is a nonpolar amino acid.

[0107] In one embodiment, the IgG4 Fc substitution at position D265 is a nonpolar amino acid selected from alanine, cysteine, glycine, isoleucine, leucine, methionine, and valine.

[0108] In one embodiment, the IgG4 Fc substitution at position D265 is glycine.

[0109] In one embodiment, any of the antibodies in the embodiments described above further includes substitution at S228.

[0110] In one embodiment, the substitution in S228 is proline.

[0111] In one embodiment, the antibody is a monospecific or biparatopic C5aR1 antibody from any of the prior embodiments, comprising a modified IgG4 constant domain including a combination of F234V, L235E, and D265G substitutions.

[0112] In one embodiment, the present disclosure provides a method for reducing or preventing antibody-dependent cell injury, antibody-dependent phagocytosis, and / or complement-dependent cell injury using a single-specific or biparatopic C5aR1 antibody from any of the prior embodiments.

[0113] In one embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising the heavy chain variable region (VH) of SEQ ID NO: 14, the light chain variable region (VL) of SEQ ID NO: 25, and substitutions F234V, L235E, and D265G.

[0114] In one embodiment, the present disclosure includes an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising a heavy chain variable region (VH) including HCDR1 of SEQ ID NO: 6 (NYWMH), HCDR2 of SEQ ID NO: 7 (YLNPSSGYTKYAQKFQG), and HCDR3 of SEQ ID NO: 8 (SGGDNYGNPYYFDR); a light chain variable region (VL) including LCDR1 of SEQ ID NO: 9 (RASQSIVHSNGNTYLH), LCDR2 of SEQ ID NO: 10 (KVSNRFS), and LCDR3 of SEQ ID NO: 11 (AQYTLVPLT); and a modified Fc domain including substitutions F234V, L235E, and D265G.

[0115] In one embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising the heavy chain of SEQ ID NO: 69 and the light chain of SEQ ID NO: 70.

[0116] In one embodiment, the present disclosure relates to 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), The compound comprises a heavy chain containing HCDR1 of SEQ ID NO: 6 (NYWMH), HCDR2 of SEQ ID NO: 7 (YLNPSSGYTKYAQKFQG), and HCDR3 of SEQ ID NO: 8 (SGGDNYGNPYYFDR), a modified Fc domain including substitutions of F234V, L235E, and D265G, and an scFv containing LCDR4 of SEQ ID NO: 22 (RSSQSLVHSNGNTYLN), LCDR5 of SEQ ID NO: 23 (KVSNRLS), LCDR6 of SEQ ID NO: 24 (SQSTHVPYT), HCDR4 of SEQ ID NO: 18 (AYAMS), HCDR4 of SEQ ID NO: 19 (SISTGGNTYYADSVKG), and HCDR5 of SEQ ID NO: 20 (GYQRFSGFAY), wherein the scFv contains a biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), linked to the modified Fc domain.

[0117] In one embodiment, the present disclosure includes a first light chain variable region (VL) of SEQ ID NO: 15, a first heavy chain variable region (VH) of SEQ ID NO: 14, a modified Fc domain including substitutions F234V, L235E, and D265G, and an scFv including 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. scFv includes a biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), which is linked to a modified Fc domain.

[0118] In one embodiment, the disclosure includes a biparatopic or antigen-binding fragment thereof that binds to the complement component 5a receptor (C5aR1), comprising the light chain of SEQ ID NO: 72 and the heavy chain of SEQ ID NO: 71.

[0119] In one embodiment, the disclosure includes an scFv 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, 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 comprises a biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), linked to a modified Fc domain. In one embodiment, the biparatopic antibody or its antigen-binding fragment comprises a modified Fc domain including substitutions F234V, L235E, and D265G, thereby linking the scFv to the modified Fc domain.

[0120] In one embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that binds to the complement component 5a receptor (C5aR1), comprising the heavy chain variable region (VH) of SEQ ID NO: 43 and the light chain variable region (VL) of SEQ ID NO: 48. In one embodiment, the antibody further comprises a modified Fc domain including substitutions F234V, L235E, and D265G.

[0121] In one embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising the heavy chain variable region (VH) of SEQ ID NO: 14 and the light chain variable region (VL) of SEQ ID NO: 15. In one embodiment, the antibody further comprises a modified Fc domain including substitutions F234V, L235E, and D265G. In embodiments of the present invention, for example, the following items are provided. (Item 1) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14. (Item 2) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising a light chain variable region (VL), wherein the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 25. (Item 3) An antibody or antigen-binding fragment thereof comprising a VH region, wherein the VH region comprises three heavy chain complementarity-determining regions (HCDRs), and the HCDR1, HCDR2, and HCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 6(NYWMH), 7(YLNPSSGYTKYAQKFQG), and 8(SGGDNYGNPYYFDR). (Item 4) An antibody or antigen-binding fragment thereof comprising a VL region, wherein the VL comprises three light chain complementarity-determining regions (LCDRs), and the LCDR1, LCDR2, and LCDR3 sequences each comprise the amino acid sequences of SEQ ID NO: 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT). (Item 5) An antibody or antigen-binding fragment thereof, comprising: a VH region, wherein the VH comprises three heavy chain complementarity-determining regions (HCDRs), and the HCDR1, HCDR2, and HCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 6 (NYWMH), 7 (YLNPSSGYTKYAQKFQG), and 8 (SGGDNYGNPYYFDR); and a VL region, wherein the VL comprises three light chain complementarity-determining regions (LCDRs), and the LCDR1, LCDR2, and LCDR3 sequences each comprise the amino acid sequences of SEQ ID NOs. 9 (RASQSIVHSNGNTYLH), 10 (KVSNRFS), and 11 (AQYTLVPLT). (Item 6) The antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the antibody or antigen-binding fragment thereof further comprises an Fc region. (Item 7) The antibody or antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the Fc domain is independently selected from IgG1, IgG2, IgG3, and IgG4. (Item 8) The antibody or antigen-binding fragment thereof described in any one of items 1 to 7, wherein the antibody that binds to C5aR1 inhibits the interaction between complement component 5a (C5a) and C5aR1. (Item 9) The antibody described above does not bind to C5aR2, and is an antibody or antigen-binding fragment thereof as described in any one of items 1 to 8. (Item 10) The antibody or antigen-binding fragment described in any one of items 1 to 9 is humanized. (Item 11) The antibody or antigen-binding fragment thereof according to any one of the preceding items, wherein the VH or VL is modified to enhance molecular stability. (Item 12) The antibody or antigen-binding fragment thereof as described in item 11, wherein the VL contains serine or tyrosine at position 96 of SEQ ID NO: 5 or SEQ ID NO: 25. (Item 13) The antibody or antigen-binding fragment described in any one of items 1 to 12, wherein the antibody or antigen-binding fragment does not cross-react with mouse C5aR1. (Item 14) The antibody described above binds to C5aR1 with an affinity of 10 pM to 50 nM, and is an antibody or antigen-binding fragment thereof as described in any one of items 1 to 13. (Item 15) The antibody described above binds to C5aR1 with an affinity of 0.16 nM or less, and is an antibody or antigen-binding fragment described in any one of items 1 to 14. (Item 16) The antibody that binds to C5aR1 inhibits neutrophil chemotaxis, as described in any one of items 1 to 15, or the antigen-binding fragment thereof. (Item 17) The antibody or antigen-binding fragment thereof according to any one of items 1 to 15, wherein the antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 10 nM. (Item 18) The antibody or antigen-binding fragment thereof described in any one of items 1 to 15, wherein the antibody that binds to C5aR1 inhibits C5a-mediated C5aR1 Gα signaling. (Item 19) The antibody or antigen-binding fragment thereof described in any one of items 1 to 15, wherein the antibody that binds to C5aR1 inhibits calcium signaling. (Item 20) The antibody that binds to C5aR1 inhibits CD11b expression, and is an antibody or antigen-binding fragment thereof as described in any one of items 1 to 15. (Item 21) The antibody that binds to C5aR1 inhibits neutropenia, and is an antibody or antigen-binding fragment thereof as described in any one of items 1 to 15. (Item 22) The antibody or antigen-binding fragment thereof described in any of items 1 to 15, wherein the antibody that binds to C5aR1 inhibits β-arrestin signaling. (Item 23) The antibody or antigen-binding fragment thereof, as described in any of items 1 to 15, wherein the antibody that binds to C5aR1 inhibits ROS production in neutrophils. (Item 24) The antibody described in any one of the preceding items, or its antigen-binding fragment, is stable at 4°C for up to two weeks in a single freeze-thaw cycle. (Item 25) A nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in any one of the preceding items. (Item 26) Cells containing the nucleic acids listed in item 23. (Item 27) A method for producing an antibody or an antigen-binding fragment thereof as described in any one of the preceding items, comprising: culturing a host cell containing a nucleic acid encoding the antibody or the antigen-binding fragment thereof; and culturing the cell under conditions that enable the production of the antibody or the antigen-binding fragment thereof. (Item 28) A method for treating an autoimmune disease, comprising administering an antibody or an antigen-binding fragment thereof to a subject requiring the treatment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) that binds to complement component 5a receptor 1 (C5aR1) and has an amino acid sequence having at least 90% identity with SEQ ID NO: 14, and a light chain variable region (VL) that has an amino acid sequence having at least 90% identity with SEQ ID NO: 25. (Item 29) A method for treating an autoimmune disease, comprising administering an antibody or an antigen-binding fragment thereof to a subject requiring the treatment, wherein the antibody or antigen-binding fragment is bound to human complement component 5a receptor 1 (C5aR1), the antibody or antigen-binding fragment comprises a heavy chain variable region (VH), the VH is at least 90% identical to SEQ ID NO: 14, and the antibody or antigen-binding fragment bound to human complement component 5a receptor 1 (C5aR1) comprises a light chain variable region (VL), the VL is at least 90% identical to SEQ ID NO: 15. (Item 30) A method for treating an autoimmune disease, comprising administering an antibody or an antigen-binding fragment thereof to a subject requiring such treatment, wherein the antibody or antigen-binding fragment thereof binds to complement component 5a receptor 1 (C5aR1), and Heavy chains including HCDR1 containing SEQ ID NO: 6 (NYWMH), HCDR2 containing SEQ ID NO: 7 (YLNPSSGYTKYAQKFQG), and HCDR3 containing SEQ ID NO: 8 (SGGDNYGNPYYFDR), and A method comprising a light chain, comprising LCDR1 containing sequence number 9 (RASQSIVHSNGNTYLH), LCDR2 containing sequence number 10 (KVSNRFS), and LCDR3 containing sequence number 11 (AQYTLVPLT). (Item 31) A method for treating an autoimmune disease, comprising administering an antibody or an antigen-binding fragment thereof to a subject requiring the treatment, wherein the antibody or antigen-binding fragment comprises the heavy chain of SEQ ID NO: 4 or a heavy chain of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 4. (Item 32) A method for treating an autoimmune disease, comprising administering an antibody or an antigen-binding fragment thereof to a subject requiring the treatment, wherein the antibody or antigen-binding fragment comprises the light chain of SEQ ID NO: 5 or a light chain of an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 5. (Item 33) A method for treating a disease caused by neutropenia, comprising administering to a subject in need of the treatment an antibody or an antibody-conjugated fragment thereof as described in any one of items 1 to 24. (Item 34) The method according to any one of item 33, wherein the neutropenia is caused by high levels of C5a. (Item 35) The method according to any one of items 28 to 33, wherein the disease is ANCA vasculitis or lupus. (Item 36) The method according to any one of items 28 to 33, wherein the aforementioned disorder is rheumatoid arthritis. (Item 37) The method according to any one of items 28 to 33, wherein the aforementioned disorder is a kidney disorder. (Item 38) A method for inhibiting C5a signaling using a monoclonal antibody that binds to C5aR1, wherein the monoclonal antibody is A heavy chain variable region (VH) containing an amino acid sequence having at least 90% identity with SEQ ID NO: 14, A method comprising a light chain variable region (VL) having an amino acid sequence having at least 90% identity with SEQ ID NO: 25. (Item 39) The first antigen-binding domain, which includes VH1 and VL1 that bind to C5aR1 in Sequence ID No. 3, A biparatopic antibody or its antigen-binding fragment comprising a second antigen-binding domain containing VH2 and VL2 that bind to C5aR1 in SEQ ID NO: 1 or SEQ ID NO: 2. (Item 40) A biparatopic antibody or its antigen-binding fragment comprising a first antigen-binding domain containing VH1 and VL1 that bind to C5aR1 in SEQ ID NO: 1 or SEQ ID NO: 2, and a second antigen-binding domain containing VH2 and VL2 that bind to C5aR1 in SEQ ID NO: 3. (Item 41) The biparatopic antibody or antigen-binding fragment thereof according to item 39 or item 40, wherein VH1 contains the amino acid sequence of SEQ ID NO: 14 or is at least 90% identical to the amino acid sequence of SEQ ID NO: 14. (Item 42) The biparatopic antibody or antigen-binding fragment thereof according to item 39 or item 40, wherein VL1 contains 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. (Item 43) The biparatopic antibody or antigen-binding fragment thereof according to item 39 or item 40, wherein the VH2 contains 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. (Item 44) The biparatopic antibody or antigen-binding fragment thereof according to item 39 or item 40, wherein VL2 contains 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. (Item 45) A biparatopic antibody or its antigen-binding fragment containing the heavy chain of SEQ ID NO: 12, or a heavy chain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12. (Item 46) A biparatopic antibody or its antigen-binding fragment containing the light chain of SEQ ID NO: 13, or a light chain identical to at least 85% of the amino acid sequence of SEQ ID NO: 13. (Item 47) A biparatopic antibody or its antigen-binding fragment comprising the heavy chain of SEQ ID NO: 12, or a heavy chain identical to at least 85% of the amino acid sequence of SEQ ID NO: 12, and further comprising the light chain of SEQ ID NO: 13, or a light chain identical to at least 85% of the amino acid sequence of SEQ ID NO: 13. (Item 48) A biparatopic antibody or antigen-binding fragment thereof according to any one of items 39 to 47, wherein the heavy chain comprises VH1 linked to the Fc domain. (Item 49) A biparatopic antibody or antigen-binding fragment thereof as described in item 48, further ligated to scFv, comprising VH2, in which the Fc domain contains the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16, and / or VL2, in which the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17. (Item 50) The biparatopic antibody or antibody or antigen-binding fragment thereof as described in item 48 or item 49, wherein the Fc domain is independently selected from IgG1, IgG2, IgG3, and IgG4. (Item 51) The biparatopic antibody or antibody or antigen-binding fragment thereof as described in item 49, wherein the scFv is linked to the Fc domain via a linker. (Item 52) The linker contains an amino acid sequence having any of SEQ ID NOs: 26 to 37. A biparatopic antibody as described in item 51, containing at least 5 amino acids. (Item 53) The biparatopic antibody according to item 49, wherein VH2 containing SEQ ID NO: 16 and VL2 containing SEQ ID NO: 17 are linked to each other via a linker. (Item 54) The biparatopic antibody according to item 53, wherein the linker contains 1 to 10 repeats of SEQ ID NO: 31. (Item 55) The biparatopic antibody according to item 49, wherein the VH2 and VL2 further comprise one or more mutations for improving the thermal stability of the biparatopic antibody. (Item 56) The biparatopic antibody or antigen-binding fragment thereof as described in item 55, wherein the mutation includes the incorporation of cysteine ​​at position 559 and position 630 of SEQ ID NO: 12. (Item 57) The biparatopic antibody or antigen-binding fragment thereof, as described in any one of items 39 to 56, wherein the antibody that binds to C5aR1 inhibits the interaction between complement component 5a (C5a) and human C5aR1. (Item 58) The aforementioned antibody is a biparatopic antibody or antigen-binding fragment thereof, as described in any one of items 39 to 57, wherein the antibody does not bind to C5aR2. (Item 59) The aforementioned antibody or antigen-binding fragment is a humanized biparatopic antibody or antigen-binding fragment as described in any one of items 39 to 58. (Item 60) The aforementioned antibody or antigen-binding fragment does not cross-react with mouse C5aR1, and is a biparatopic antibody or antigen-binding fragment as described in any one of items 39 to 59. (Item 61) The antibody is a biparatopic antibody or antigen-binding fragment thereof as described in any one of items 39 to 60, which is stable at 4°C for up to two weeks in a single freeze-thaw cycle. (Item 62) The antibody that binds to C5aR1 inhibits neutrophil chemotaxis, and is a biparatopic antibody or antigen-binding fragment thereof as described in any one of items 39 to 61. (Item 63) The biparatopic antibody or antigen-binding fragment thereof according to any one of items 39 to 62, wherein the antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a high C5a concentration. (Item 64) The biparatopic antibody or antigen-binding fragment thereof according to any one of items 39 to 62, wherein the antibody that binds to C5aR1 inhibits neutrophil chemotaxis in the presence of a C5a concentration of at least 10 nM. (Item 65) The biparatopic antibody or antigen-binding fragment thereof according to any one of items 39 to 64, wherein the antibody that binds to C5aR1 inhibits C5a-mediated C5aR1 Gα signaling. (Item 66) The antibody that binds to C5aR1 inhibits calcium signaling, and is a biparatopic antibody or antigen-binding fragment thereof as described in any one of items 39 to 65. (Item 67) The antibody that binds to C5aR1 inhibits CD11b expression, and is a biparatopic antibody or antigen-binding fragment thereof as described in any one of items 39 to 66. (Item 68) The antibody that binds to C5aR1 inhibits neutropenia, and is a biparatopic antibody or antigen-binding fragment thereof as described in any one of items 39 to 67. (Item 69) The antibody that binds to C5aR1 inhibits β-arrestin signaling, and is a biparatopic antibody or antigen-binding fragment thereof as described in any of items 39 to 61. (Item 70) The antibody that binds to C5aR1 inhibits ROS production in neutrophils, and is a biparatopic antibody or antigen-binding fragment thereof as described in any of items 39 to 61. (Item 71) A nucleic acid encoding a biparatopic antibody as described in any one of items 39-68. (Item 72) Cells containing nucleic acids as described in item 71. (Item 73) A method for producing a biparatopic antibody or an antigen-binding fragment according to any one of items 39 to 70, comprising culturing a host cell containing a nucleic acid encoding the antibody or an antigen-binding fragment thereof, and culturing the cell under conditions that enable the production of the antibody or the antigen-binding fragment thereof. (Item 74) A method for treating an autoimmune disease using a biparatopic antibody, wherein the antibody binds to the human complement component 5a receptor 1 (C5aR1), a. VH1 and VL1, which are the first VH and first VL, wherein VH1 contains an amino acid sequence having at least 90% identity with SEQ ID NO: 14, and VL1 contains an amino acid sequence having at least 90% identity with SEQ ID NO: 15, b. A method comprising VH2 and VL2, which are a second VH and a second VL, wherein VH2 contains an amino acid sequence having at least 90% identity with SEQ ID NO: 16 and VL2 contains an amino acid sequence having at least 90% identity with SEQ ID NO: 17. (Item 75) A method for treating a disease caused by neutropenia, comprising using an antibody or antigen-binding fragment thereof from a biparatopic antibody described in any of the preceding items. (Item 76) The method according to any one of items 73-75, wherein the neutropenia is caused by high levels of C5a. (Item 77) The method according to any one of items 73 to 76, wherein the disease is ANCA vasculitis or lupus. (Item 78) The method according to any one of items 73 to 77, wherein the aforementioned disorder is rheumatoid arthritis. (Item 79) The method described in any of items 73 to 78, wherein the aforementioned disorder is a kidney disorder. (Item 80) The method according to either item 73-78 or 27-33, wherein the aforementioned disability is a stroke. (Item 81) The antibody is a monospecific or biparatopic C5aR1 antibody as described in any of the preceding items, comprising a constant domain of modified IgG1, IgG4, or IgG2. (Item 82) The antibody described in item 81, wherein the aforementioned C5aR1 contains a modified IgG4 Fc domain. (Item 83) The antibody according to item 82, wherein the modified IgG4 Fc domain includes substitutions at positions F234, L235 and / or D265. (Item 84) The antibody described in item 83, wherein the IgG4 Fc substitution at position F234 is a hydrophobic amino acid selected from alanine, valine, leucine, isoleucine, phenylalanine, or tryptophan. (Item 85) The antibody described in item 83, wherein the IgG4 Fc substitution at position F234 is valine. (Item 86) The antibody described in item 83, wherein the IgG4 Fc substitution at position L235 is an acidic amino acid. (Item 87) The antibody described in item 86, wherein the IgG4 Fc substitution at position L235 is an acidic amino acid selected from glutamic acid or aspartic acid. (Item 88) The antibody described in item 86, wherein the IgG4 Fc substitution at position L235 is aspartic acid. (Item 89) The antibody described in item 83, wherein the IgG4 Fc substitution at position D265 is a nonpolar amino acid. (Item 90) The antibody described in item 89, wherein the IgG4 Fc substitution at position D265 is a nonpolar amino acid selected from alanine, cysteine, glycine, isoleucine, leucine, methionine, and valine. (Item 91) The antibody described in item 89, wherein the IgG4 Fc substitution at position D265 is glycine. (Item 92) The antibody described above is the antibody described in any of the preceding items, further comprising substitution at S228. (Item 93) The antibody described in item 92, wherein the substitution in S228 is proline. (Item 94) The antibody is a monospecific or biparatopic C5aR1 antibody as described in any of the preceding items, comprising a modified IgG4 constant domain including a combination of F234V, L235E, and D265G substitutions. (Item 95) A method for reducing or preventing antibody-dependent cell injury, antibody-dependent phagocytosis, and / or complement-dependent cell injury, using a monospecific or biparatopic C5aR1 antibody as described in any of items 81-94. (Item 96) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), The heavy chain variable region (VH) of sequence number 14, The light chain variable region (VL) of sequence number 25, An antibody or antigen-binding fragment thereof comprising a modified Fc domain containing substituted F234V, L235E, and D265G. (Item 97) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), HCDR1 with sequence number 6 (NYWMH), HCDR2 with sequence number 7 (YLNPSSGYTKYAQKFQG), and HCDR3 with sequence number 8 (SGGDNYGNPYYFDR) The heavy chain variable region (VH) includes, The light chain variable region (VL) includes LCDR1 of sequence number 9 (RASQSIVHSNGNTYLH), LCDR2 of sequence number 10 (KVSNRFS), and LCDR3 of sequence number 11 (AQYTLVPLT), An antibody or antigen-binding fragment thereof comprising a modified Fc domain containing substituted F234V, L235E, and D265G. (Item 98) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), comprising the heavy chain of SEQ ID NO: 69 and the light chain of SEQ ID NO: 70. (Item 99) A biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), A light chain containing LCDR1 of sequence number 9 (RASQSIVHSNGNTYLH), LCDR2 of sequence number 10 (KVSNRFS), and LCDR3 of sequence number 21 (AQSTLVPLT), A heavy chain containing HCDR1 of sequence number 6 (NYWMH), HCDR2 of sequence number 7 (YLNPSSGYTKYAQKFQG), and HCDR3 of sequence number 8 (SGGDNYGNPYYFDR), Modified Fc domains including substitutions F234V, L235E, and D265G, Includes scFv containing LCDR4 of SEQ ID NO: 22 (RSSQSLVHSNGNTYLN), LCDR5 of SEQ ID NO: 23 (KVSNRLS), LCDR6 of SEQ ID NO: 24 (SQSTHVPYT), HCDR4 of SEQ ID NO: 18 (AYAMS), HCDR5 of SEQ ID NO: 19 (SISTGGNTYYADSVKG), and HCDR6 of SEQ ID NO: 20 (GYQRFSGFAY). A biparatopic antibody or its antigen-binding fragment, wherein the scFv is linked to the modified Fc domain. (Item 100) A biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), The first light chain variable region (VL) of sequence number 15, The first heavy chain variable region (VH) of sequence number 14, Modified Fc domains including substitutions F234V, L235E, and D265G, Includes scFv which includes the second light chain variable region (VL) of SEQ ID NO: 17 and the second heavy chain variable region (VH) of SEQ ID NO: 16, A biparatopic antibody or its antigen-binding fragment, wherein the scFv is linked to the modified Fc domain. (Item 101) A biparatopic antibody or antigen-binding fragment thereof that binds to complement component 5a receptor (C5aR1), comprising the light chain of SEQ ID NO: 72 and the heavy chain of SEQ ID NO: 71. (Item 102) A biparatopic antibody or its antigen-binding fragment that binds to the complement component 5a receptor (C5aR1), The first light chain variable region (VL) of sequence number 25, The first heavy chain variable region (VH) of sequence number 14, Includes scFv which includes the second light chain variable region (VL) of SEQ ID NO: 17 and the second heavy chain variable region (VH) of SEQ ID NO: 16, A biparatopic antibody or its antigen-binding fragment, wherein the scFv is linked to the Fc domain. (Item 103) A biparatopic antibody or antigen-binding fragment thereof according to item 102, further comprising a modified Fc domain containing substituted F234V, L235E, and D265G, wherein the scFv is linked to the modified Fc domain. (Item 104) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), The heavy chain variable region (VH) of sequence number 43, An antibody or its antigen-binding fragment, comprising the light chain variable region (VL) of SEQ ID NO: 48. (Item 105) The antibody described in item 104, further comprising a modified Fc domain including substituted F234V, L235E, and D265G. (Item 106) An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), The heavy chain variable region (VH) of sequence number 14, An antibody or its antigen-binding fragment, comprising the light chain variable region (VL) of SEQ ID NO: 15. (Item 107) The antibody described in item 106, further comprising a modified Fc domain including substituted F234V, L235E, and D265G. [Brief explanation of the drawing]

[0122] [Figure 1] This is a schematic diagram illustrating the pathogenesis of ANCA vasculitis. [Figure 2] This is a schematic diagram illustrating two types of antibodies described in this disclosure. [Figure 3A] This is an illustrative schematic diagram of an exemplary biparatopic antibody containing scFv ligated to the heavy chain Fc domain of Fab as described herein. [Figure 3B] This is a schematic diagram of an exemplary biparatopic antibody containing scFv linked to the light chain of Fab as described herein. [Figure 4A] This is an exemplary graph showing the binding of exemplary humanized site II antibodies (c2139) and exemplary biparatopic antibodies (c2137-e1711) to C5aR1 using ELISA, as described in this disclosure. [Figure 4B] This is an exemplary graph showing the binding of different batches of exemplary site II antibody (c2139) and exemplary biparatopic antibody (c2137-e1711) in U937-C5aR1 cells. [Figure 4C] This graph shows the binding of different batches of exemplary site II antibodies (c2139) and exemplary biparatopic antibodies (c2137-e1711) to human neutrophil cells. [Figure 5] This is an exemplary graph showing the binding of exemplary humanized site II antibodies (c2139) and biparatopic antibodies (c2137-e1711) to C5aR2 using the ELISA described herein. [Figure 6A]This is an exemplary graph showing inhibition of G-alpha signaling using the GeneBLAzer assay, employing the exemplary humanized site II antibody, c2139, and the exemplary biparatopic antibody, c2137-e1711, against C5aR1, as described herein, in the presence of 10 nM C5a. Avacopan is shown as a positive control. [Figure 6B] This is an exemplary graph showing inhibition of G-alpha signaling using the GeneBLAzer assay, which utilizes the exemplary humanization site II, c2139 antibody, and the exemplary biparatopic antibody, c2137-e1711, against C5aR1, as described in this disclosure, in the presence of 100 nM C5a. Avacopan is shown as a positive control. [Figure 6C] This is an exemplary graph showing inhibition of G-alpha signaling using the GeneBLAzer assay with the exemplary humanized site II antibody, c2139, and the exemplary biparatopic antibody, c2137-e1711 C5aR1, described herein, in the presence of 10 nM C5a. Anti-C5aR1 control Ab is shown as a positive control. [Figure 6D] This is an exemplary graph showing inhibition of G-alpha signaling using the GeneBLAzer assay, which utilizes the exemplary humanization site II, c2139 antibody, and the exemplary biparatopic antibody, c2137-e1711, against C5aR1 as described in this disclosure, in the presence of 100 nM C5a. The anti-C5aR1 control Ab is shown as a positive control. [Figure 7A] This is an exemplary graph showing inhibition of calcium signaling using the exemplary humanized site II antibody described herein in the presence of increasing concentrations of C5a and increasing concentrations of the antibody. [Figure 7B] This is an exemplary graph showing the inhibition of calcium signaling using the exemplary humanized biparatopic antibody described herein in the presence of increasing concentrations of C5a and increasing concentrations of the antibody. [Figure 7C]This is an exemplary graph showing inhibition of calcium signaling using avacopan, a known C5aR1 inhibitor described herein, in the presence of increasing concentrations of C5a and increasing concentrations of the antibody. [Figure 8A] This is an exemplary graph showing neutrophil chemotaxis inhibition using the exemplary humanized site II antibody c2139 described herein in the presence of increasing concentrations of C5a and increasing concentrations of the antibody. [Figure 8B] This is an exemplary graph showing neutrophil chemotaxis inhibition using the humanized biparatopic antibody c2137-e1711 described herein in the presence of increasing concentrations of C5a and increasing concentrations of the antibody. [Figure 8C] This is an exemplary graph showing neutrophil chemotaxis inhibition using avacopan, a known C5aR1 inhibitor described herein, in the presence of increasing concentrations of C5a and increasing concentrations of antibody. [Figure 9A] This is an exemplary graph showing the inhibition of CD11b expression in neutrophils by exemplary humanized site II antibody, c2139, and exemplary biparatopic antibody, c2137-e1711, compared to avacopan, in the presence of 100 nM C5a and increasing antibody concentrations. [Figure 9B] This is an exemplary graph showing the inhibition of CD11b expression in neutrophils by the exemplary humanized site II antibody, c2139, and the exemplary biparatopic antibody, c2137-e1711 described herein, compared to avacopan, in the presence of 10 nM exemplary humanized site II antibody and exemplary biparatopic antibody, as well as increasing concentrations of C5a. [Figure 9C] This is an exemplary graph showing the inhibition of CD11b expression in neutrophils by the exemplary humanized site II antibody, c2139, and the exemplary biparatopic antibody, c2137-e1711 described herein, compared with 10 nM and 100 nM anti-C5aR1 control abs in the presence of 10 nM exemplary humanized site II antibody and exemplary biparatopic antibody, as well as increasing concentrations of C5a. [Figure 10A]This is an exemplary graph showing the inhibition of β-arrestin recruitment in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at doses of 1 nM, compared to 10 nM avacopan, in the presence of 1 nM C5a. [Figure 10B] This is an exemplary graph showing the inhibition of β-arrestin recruitment in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at doses of 1 nM, compared to 10 nM avacopan, in the presence of 10 nM C5a. [Figure 10C] This is an exemplary graph showing the inhibition of β-arrestin recruitment in the presence of exemplary humanized C5aR1 antibodies c2139 and c2137-e1711 at doses of 1 nM, compared to 10 nM avacopan, in the presence of 100 nM C5a. [Figure 11] This is an exemplary graph showing inhibition of ROS signaling in ANCA(-) and ANCA(+) cells compared to c2139, c2137-e1711, motabizumab, and avacopan. [Figure 12A] This is an exemplary graph showing the internal migration of an exemplary humanized anti-10nM C5aR1 antibody in C5aR1-U937 cells at 0, 6, and 12 hours, visualized by an amine-conjugated antibody containing a pH-sensitive fluorescent dye. [Figure 12B] This is an exemplary graph showing the internal migration of an exemplary humanized anti-10nM C5aR1 antibody in U937 cells at 0, 6, and 12 hours, visualized by an amine-conjugated antibody containing a pH-sensitive fluorescent dye. [Figure 12C] This is an exemplary graph showing the internal migration of an exemplary humanized anti-100nM C5aR1 antibody in C5aR1-U937 cells at 0, 6, and 12 hours, visualized by an amine-conjugated antibody containing a pH-sensitive fluorescent dye. [Figure 12D] This is an exemplary graph showing the internal migration of an exemplary humanized anti-100nM C5aR1 antibody in U937 cells at 0, 6, and 12 hours, visualized by an amine-conjugated antibody containing a pH-sensitive fluorescent dye. [Figure 13A] This is an illustrative graph showing the binding (cross-reaction) of an exemplary humanized C5aR1 antibody to squirrel monkeys. [Figure 13B] This is an illustrative graph showing the binding (cross-reactivity) of an exemplary humanized C5aR1 antibody to dogs. [Figure 14A] This is a schematic diagram illustrating an exemplary study design in squirrel monkeys, showing the timing of blood collection and administration. [Figure 14B] This is an exemplary scatter plot of the percentage change in neutrophil count from baseline in the vehicle, exemplary humanized site II antibody, and avacopan. [Figure 14C] This is a bar graph showing the percentage change in neutrophil count from baseline in the vehicle, an exemplary humanized site II antibody, and avacopan. [Figure 15A] This is a schematic diagram illustrating an exemplary study design in hC5aR1 mice, showing the timing of blood collection and administration. [Figure 15B] This is an illustrative scatter plot of the percentage change in neutrophil count from baseline in the vehicle, humanized site II antibody, and avacopan. [Figure 15C] This is an illustrative bar graph showing the percentage change in neutrophil count from baseline in the vehicle, humanized site II antibody, and avacopan. [Figure 16A] This is an exemplary graph showing the 21-day PK profiles of exemplary tetravalent (biparatopic) and monospecific antibodies. [Figure 16BCD] [Figure 16B] An exemplary graph showing the 500-hour PK profile of motavizumab in mouse serum. [Figure 16C] An exemplary graph showing the 500-hour PK profile of c2139 in mouse serum. [Figure 16D] An exemplary graph showing the 500-hour PK profile of c2137-e1711 in mouse serum. [Figure 17A] This is an illustrative graph showing the affinity curves of a single-specific C5aR1 antibody (c2139-Fcmod) and a C5aR1 biparatopic antibody (c2137-e1711-Fcmod). [Figure 17B] This is an example graph showing the kinetic parameters of the C5aR1 biparatopic antibody (c2137-e1711-Fcmod). [Figure 17C] This is an illustrative graph showing the binding of a single-specific C5aR1 antibody (c2139-Fcmod) and a C5aR1 biparatopic antibody (c2137-e1711-Fcmod) to C5aR2. [Figure 18A] This demonstrates increased internal migration of C5aR1 antibodies. Figure 18A is an exemplary graph showing internal migration of the monospecific antibody c2139 several hours after dissociation. Figure 18B is an exemplary graph showing increased internal migration of the biparatopic antibody c2137-e1711 during association. [Figure 18B] This demonstrates increased internal migration of C5aR1 antibodies. Figure 18A is an exemplary graph showing internal migration of the monospecific antibody c2139 several hours after dissociation. Figure 18B is an exemplary graph showing increased internal migration of the biparatopic antibody c2137-e1711 during association. [Figure 19A] This shows inhibition of Gα signaling in the presence of the C5aR1 Fc modified antibody. Figure 19A shows Gα signaling in the presence of 10 nM C5a and the C5aR1 Fc modified antibody. Figure 19B shows Gα signaling in the presence of 100 nM C5a and the C5aR1 Fc modified antibody. [Figure 19B] This shows inhibition of Gα signaling in the presence of the C5aR1 Fc modified antibody. Figure 19A shows Gα signaling in the presence of 10 nM C5a and the C5aR1 Fc modified antibody. Figure 19B shows Gα signaling in the presence of 100 nM C5a and the C5aR1 Fc modified antibody. [Figure 20A]Figure 20A is an exemplary series of graphs showing the inhibition of calcium signaling in U937-C5aR1 cells in the presence of the C5aR1 Fc-modified antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 20A is a dose-response curve showing an exemplary graph of the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibodies described herein in the presence of increasing concentrations of antibody and 100 nM C5a. Figure 20B is an inhibition percentage graph showing the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibodies described herein in the presence of antibody and 100 nM C5a. [Figure 20B] Figure 20A is an exemplary series of graphs showing the inhibition of calcium signaling in U937-C5aR1 cells in the presence of the C5aR1 Fc-modified antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 20A is a dose-response curve showing an exemplary graph of the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibodies described herein in the presence of increasing concentrations of antibody and 100 nM C5a. Figure 20B is an inhibition percentage graph showing the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibodies described herein in the presence of antibody and 100 nM C5a. [Figure 21AB] The following shows the inhibition of calcium signaling in U937-C5aR1 cells compared to human neutrophils. Figure 21A shows the inhibition of calcium signaling in U937-C5aR1 cells in the presence of 10 nM C5a. Figure 21B shows the inhibition of calcium signaling in U937-C5aR1 cells in the presence of 100 nM C5a. Figure 21C shows the inhibition of calcium signaling in human neutrophils in the presence of 10 nM C5a. Figure 21D shows the inhibition of calcium signaling in human neutrophils in the presence of 100 nM C5a. [Figure 21CD]The following shows the inhibition of calcium signaling in U937-C5aR1 cells compared to human neutrophils. Figure 21A shows the inhibition of calcium signaling in U937-C5aR1 cells in the presence of 10 nM C5a. Figure 21B shows the inhibition of calcium signaling in U937-C5aR1 cells in the presence of 100 nM C5a. Figure 21C shows the inhibition of calcium signaling in human neutrophils in the presence of 10 nM C5a. Figure 21D shows the inhibition of calcium signaling in human neutrophils in the presence of 100 nM C5a. [Figure 22A] This summarizes the saturation percentage and F norm after 1 hour of incubation of U937-C5aR1 cells incubated with increasing concentrations of antibodies (c2139-Fcmod and c2137-e1711-Fcmod) and 100 nM C5a. [Figure 22B] This summarizes the saturation percentage and F standard after 3 hours of incubation of U937-C5aR1 cells incubated with increasing concentrations of antibodies (c2139-Fcmod and c2137-e1711-Fcmod) and 100 nM C5a. [Figure 23A] The inhibition of C5a-mediated β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod is shown. Figure 23A shows the dose response of inhibition of β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod. Figure 23B shows the percentage of inhibition of β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod. [Figure 23B] The inhibition of C5a-mediated β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod is shown. Figure 23A shows the dose response of inhibition of β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod. Figure 23B shows the percentage of inhibition of β-arrestin signaling by c2137-e1711-Fcmod and c2139-Fcmod. [Figure 24A]Figure 24A shows inhibition of chemotaxis in stable C5aR1-U937 cells after treatment with C5aR1 antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 24B shows inhibition of chemotaxis in stable C5aR1-U937 cells in the presence of 1 nM, 3.16 nM, and 10 nM c2139-Fcmod and increasing concentrations of C5a. Figures 24C-24D show inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of c2137-e1711-Fcmod, 1 nM, 3.16 nM, and 10 nM anti-C5aR1 control Ab and avacopan. [Figure 24B] Figure 24A shows inhibition of chemotaxis in stable C5aR1-U937 cells after treatment with C5aR1 antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 24B shows inhibition of chemotaxis in stable C5aR1-U937 cells in the presence of 1 nM, 3.16 nM, and 10 nM c2139-Fcmod and increasing concentrations of C5a. Figures 24C-24D show inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of c2137-e1711-Fcmod, 1 nM, 3.16 nM, and 10 nM anti-C5aR1 control Ab and avacopan. [Figure 24C]Figure 24A shows inhibition of chemotaxis in stable C5aR1-U937 cells after treatment with C5aR1 antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 24B shows inhibition of chemotaxis in stable C5aR1-U937 cells in the presence of 1 nM, 3.16 nM, and 10 nM c2139-Fcmod and increasing concentrations of C5a. Figures 24C-24D show inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of c2137-e1711-Fcmod, 1 nM, 3.16 nM, and 10 nM anti-C5aR1 control Ab and avacopan. [Figure 24D] Figure 24A shows inhibition of chemotaxis in stable C5aR1-U937 cells after treatment with C5aR1 antibodies, c2137-e1711-Fcmod and c2139-Fcmod, compared to avacopan and anti-C5aR1 control Ab. Figure 24B shows inhibition of chemotaxis in stable C5aR1-U937 cells in the presence of 1 nM, 3.16 nM, and 10 nM c2139-Fcmod and increasing concentrations of C5a. Figures 24C-24D show inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of c2137-e1711-Fcmod, 1 nM, 3.16 nM, and 10 nM anti-C5aR1 control Ab and avacopan. [Figure 25A] Figure 25A shows inhibition of CD11b signaling in response to treatment with c2137-e1711-Fcmod and c2139-Fcmod. Figure 25B shows inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonistic antibody and 10 nM C5a. [Figure 25B]Figure 25A shows inhibition of CD11b signaling in response to treatment with c2137-e1711-Fcmod and c2139-Fcmod. Figure 25B shows inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonistic antibody and 10 nM C5a. [Figure 26A] This shows the inhibition of ROS signaling in ANCA(-) and ANCA(+) cells compared to c2139-Fcmod, c2137-e1711-Fcmod, motabizumab, and avacopan. Figure 26A shows the inhibition of ROS production with increasing concentrations of monospecific C5aR1 antibody. Figure 26B shows the inhibition of ROS production with increasing concentrations of biparatopic C5aR1 antibody. [Figure 26B] This shows the inhibition of ROS signaling in ANCA(-) and ANCA(+) cells compared to c2139-Fcmod, c2137-e1711-Fcmod, motabizumab, and avacopan. Figure 26A shows the inhibition of ROS production with increasing concentrations of monospecific C5aR1 antibody. Figure 26B shows the inhibition of ROS production with increasing concentrations of biparatopic C5aR1 antibody. [Figure 27A] This is a schematic diagram illustrating an exemplary study design in hC5aR1 mice, showing the timing of blood collection and administration. [Figure 27B] This is an exemplary scatter plot of the percentage change in neutrophil count from baseline in vehicle, c2139, c2139-Fcmod, c2137-e1711, and c2137-e1711-Fcmod. [Figure 27C] These are illustrative bar graphs showing the percentage change in neutrophil count from baseline in vehicle, c2139, c2139-Fcmod, c2137-e1711, and c2137-e1711-Fcmod. [Figure 28A] This is a graphical description of infarct size in mouse brains after treatment with the prescribed dose of Fc-modified antibody, compared to 1 mg / kg of PMX53. [Figure 28B]This graph shows the infarct size in mouse brains after treatment with the prescribed dose of Fc-modified antibody, compared to 1 mg / kg of PMX53. [Figure 29A] These are graphs showing the pharmacokinetics of the Fc-modified antibody. Figure 29A shows the percentage of Fc-modified C5aR1 antibody in serum over 500 hours. Figure 29B shows the average concentration of antibody in μg per 1 ml of serum over 500 hours. [Figure 29B] These are graphs showing the pharmacokinetics of the Fc-modified antibody. Figure 29A shows the percentage of Fc-modified C5aR1 antibody in serum over 500 hours. Figure 29B shows the average concentration of antibody in μg per 1 ml of serum over 500 hours. [Figure 30A] Figure 30 shows the PK and PD studies of Fc-modified C5aR1 antibodies compared to MVZ-IgG4. Figure 30A shows the dose-response curve of c2139Fcmod in serum over 200 hours. Figure 30B shows the dose-response curve of c2137-e1711-Fcmod in serum over 200 hours. Figure 30C compares c2139Fcmod, c2137-e1711-Fcmod, and MVZ-IgG4. Figure 30D shows the in silico graph of c2139 at three different concentrations over 500 hours. Figure 30E shows the in silico graph of c2137-e1711 at three different concentrations over 500 hours. Figure 30F shows the in silico graph of isotype control antibodies at a concentration of 20 mg / kg over 500 hours. [Figure 30B]Figure 30 shows the PK and PD studies of Fc-modified C5aR1 antibodies compared to MVZ-IgG4. Figure 30A shows the dose-response curve of c2139Fcmod in serum over 200 hours. Figure 30B shows the dose-response curve of c2137-e1711-Fcmod in serum over 200 hours. Figure 30C compares c2139Fcmod, c2137-e1711-Fcmod, and MVZ-IgG4. Figure 30D shows the in silico graph of c2139 at three different concentrations over 500 hours. Figure 30E shows the in silico graph of c2137-e1711 at three different concentrations over 500 hours. Figure 30F shows the in silico graph of isotype control antibodies at a concentration of 20 mg / kg over 500 hours. [Figure 30C] Figure 30 shows the PK and PD studies of Fc-modified C5aR1 antibodies compared to MVZ-IgG4. Figure 30A shows the dose-response curve of c2139Fcmod in serum over 200 hours. Figure 30B shows the dose-response curve of c2137-e1711-Fcmod in serum over 200 hours. Figure 30C compares c2139Fcmod, c2137-e1711-Fcmod, and MVZ-IgG4. Figure 30D shows the in silico graph of c2139 at three different concentrations over 500 hours. Figure 30E shows the in silico graph of c2137-e1711 at three different concentrations over 500 hours. Figure 30F shows the in silico graph of isotype control antibodies at a concentration of 20 mg / kg over 500 hours. [Figure 30DEF]Figure 30 shows the PK and PD studies of Fc-modified C5aR1 antibodies compared to MVZ-IgG4. Figure 30A shows the dose-response curve of c2139Fcmod in serum over 200 hours. Figure 30B shows the dose-response curve of c2137-e1711-Fcmod in serum over 200 hours. Figure 30C compares c2139Fcmod, c2137-e1711-Fcmod, and MVZ-IgG4. Figure 30D shows the in silico graph of c2139 at three different concentrations over 500 hours. Figure 30E shows the in silico graph of c2137-e1711 at three different concentrations over 500 hours. Figure 30F shows the in silico graph of isotype control antibodies at a concentration of 20 mg / kg over 500 hours. [Figure 31] This graph shows the percentage change in neutrophil count in human C5aR1 mice induced by exemplary C5aR1 antibodies c2139-Fcmod and c2137-e1711-Fcmod at different doses. [Figure 32A] This is a graphical representation of the internal migration of exemplary C5aR1 antibodies, c2139-Fcmod and c2137-e1711-Fcmod. Figure 32A shows the fluorescence intensities of c2139-Fcmod and c2137-e1711-Fcmod in U937 cells at 0, 6, and 24 hours. Figure 32B shows the internal migration of both c2137-e1711-Fcmod and c2139-Fcmod in live cells observed by Nikon confocal experiment over 300 minutes. [Figure 32B] This is a graphical representation of the internal migration of exemplary C5aR1 antibodies, c2139-Fcmod and c2137-e1711-Fcmod. Figure 32A shows the fluorescence intensities of c2139-Fcmod and c2137-e1711-Fcmod in U937 cells at 0, 6, and 24 hours. Figure 32B shows the internal migration of both c2137-e1711-Fcmod and c2139-Fcmod in live cells observed by Nikon confocal experiment over 300 minutes. [Modes for carrying out the invention]

[0123] definition Antibody: As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunoglobulin (Ig) molecule containing an immunoactive portion, i.e., an antigen-binding site that binds to (immunely reacts with) an antigen. To “bind” or “immunely react 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 dAb (domain antibodies), single-chain, Fab, Fab', F(ab')2 fragments, scFv, and Fab expression libraries. Antibodies may be whole antibodies, immunoglobulins, or antibody fragments.

[0124] Antibody-dependent cell injury: As used herein, the term “antibody-dependent cell injury” or “ADCC” refers to the lysis of human target cells by the antibody according to the present invention in the presence of effector cells.

[0125] Fab Arm Exchange: The term "Fab Arm Exchange" refers to the phenomenon in which IgG4 antibodies can exchange "half-halves," and is referred to herein as Fab Arm Exchange. In particular, with bispecific molecules or biparatopic molecules, this can lead to the generation of functionally monovalent antibodies with unknown specificity, and therefore, a decrease in therapeutic efficacy. To inhibit Fab Arm Exchange, mutations can be introduced into the Fc domain. The S228P mutation is known to prevent IgG4 FAE to undetectable levels both in vitro and in vivo.

[0126] Fc domain: As used herein, the term “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the natural sequence and the Fc region of variants. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0127] Humanized Antibodies: The term "humanized antibody" includes non-human (e.g., mouse) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof containing minimal non-human (e.g., mouse) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determinant region (CDR) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capabilities (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0128] Increase in ADCC: The term "increase in ADCC" is defined as either an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and / or a decrease in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase in ADCC is compared to the ADCC measured in an acceptable assay recognized in the art.

[0129] Monoclonal Antibodies: The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for any naturally occurring variations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigen site. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0130] Multispecific antibody: As used herein, the term “multispecific antibody” refers to a binding molecule, antibody, or antigen-binding fragment thereof that has the ability to specifically bind to two or more different epitopes on the same or different targets.

[0131] Biparatopic antibody: As used herein, the term “biparatopic antibody” refers to a multispecific antibody that has the ability to bind to two different non-overlapping epitopes on the same target antigen molecule.

[0132] K i or K d :When used in this specification, the term “K d When used herein, “$$ refers to a dissociation constant of a particular antibody-antigen interaction known in the art, and will be applied to a composition in question as a parameter of the binding affinity of the targeting moiety to its homologous ligand.

[0133] IC50: As used herein, the term "IC50" refers to the concentration required to inhibit half of the maximum biological response of a ligand agonist, and is generally determined by a competitive binding assay.

[0134] EC50: As used herein, the term "EC50" refers to the half-maximal effective concentration. The term EC50 refers to the concentration of a drug, antibody, or toxic substance that induces an intermediate response between baseline and maximum after a specified exposure time. More simply, EC50 can be defined as the concentration required to obtain 50% of the desired effect.

[0135] C5a: As used herein, the term "C5a" refers to complement component 5a.

[0136] C5aR1: As used herein, the term “C5aR1” refers to complement component 5a receptor 1. In some embodiments, human C5aR1 includes SEQ ID NO: 38. In some embodiments, certain amino acids of human C5aR1 including SEQ ID NO: 38 have native variants such as (N2D and N279K) shown in lowercase in Table 1.

[0137] Linker: As used herein, the term “linker” refers to a molecule or group of molecules (such as monomers or polymers) that connects two molecules and often helps to place the two molecules into a preferred configuration. Many strategies can be used to covalently bond molecules to one another. These include, but are not limited to, polypeptide bonds between the N-terminus and C-terminus of a protein or protein domain, disulfide bond-mediated bonding, and chemical crosslinking reagent-mediated bonding. 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, a linker peptide may mainly consist of the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length suitable for linking two molecules so that the two molecules take the correct stereochemistry relative to each other, so that they retain the desired activity. A length suitable for this purpose includes at least one and no more than 30 amino acid residues. In one embodiment, the linker is about 1 to 30 amino acid lengths. In another embodiment, the linker is about 1 to 15 amino acid lengths. Furthermore, the amino acid residues selected for inclusion in the linker peptide should exhibit properties that do not significantly hinder the polypeptide's activity.

[0138] Neutrophils: As used herein, the term “neutrophil” refers to the major class of white blood cells in the peripheral blood. Neutrophils play a vital role in engulfing and killing extracellular pathogens.

[0139] scFv: As used herein, the term "scFv" refers to a fusion protein of the variable region of the heavy (VH) and light (VL) chains of an immunoglobulin, conjugated to a short linker peptide of 10 to approximately 25 amino acids.

[0140] Fab: As used herein, the term "Fab" refers to an antibody fragment comprising a portion of an intact antibody, including its antigen-binding region or its variable region.

[0141] Neutropenia: As used herein, the term “neutropenia” refers to a low neutrophil count. For example, in human subjects, neutropenia may range from an ANC (absolute neutrophil count) of less than 500 to less than 1500. (ANC is measured in cells per microliter of blood). In mice, neutropenia is measured in blood 1 mm³ 3 It is defined as <10 neutrophils per unit.

[0142] In vitro: As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as a test tube or reaction vessel or cell culture, rather than within a multicellular organism.

[0143] In vivo: As used herein, the term “in vivo” refers to events occurring within multicellular organisms such as humans and non-human animals. In terms of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0144] Subject: As used herein, the term “subject” means human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is human. The subject may be a patient, meaning a person who visits a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject is afflicted with or susceptible to a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0145] Dysfunction: As used herein, the term “dysfunction” refers to abnormal function. Dysfunction of a molecule (e.g., a protein) may be caused by an increase or decrease in the activity associated with such molecule. Dysfunction of a molecule may be caused by defects in the molecule itself, or by defects in other molecules that directly or indirectly interact with or modulate the molecule.

[0146] Derivative: As used herein, the term “derivative” means an antibody, or, as used in relation to a C5aR1 antibody, a portion having a portion of the sequence of the original molecule that retains at least some of the function and / or properties of the original molecule.

[0147] Identity: As used herein, the term “identity” refers to the relationship between sequences of two or more polypeptide molecules or nucleic acid molecules known in the art, comparing the sequences of these molecules. The relationship is determined by doing so. In the art, “identity” also means the degree of sequence relevance between nucleic acid molecules or polypeptides, and in some cases means multiple nucleotide sequences or multiple sequences. It may be determined by the matching of amino acid sequence strings. “Identity” means between the smaller sequences of two or more sequences and a gap alignment (if any) addressed by a particular mathematical model or computer program (i.e., “algorithm”). The percentage of identity matching is measured.

[0148] Similarity or Resemblance: As used herein, the term “similarity” is used in the art with respect to the relevant concepts, but in contrast to “identity,” “similarity” refers to both identity and matching of conserved substitutions. If two polypeptide sequences have, for example, 10 identical amino acids out of 20, with the rest being all non-conservative substitutions, then both the percentage of identity and the percentage of similarity are 50%. In the same example, if there are five more conservative substitutions, the percentage of identity remains 50%, but the percentage of similarity is 75%. Thus, when there are conservative substitutions, the percentage of similarity between two polypeptides is higher than the percentage of identity between those two polypeptides.

[0149] Treatment: As used herein, the terms “to treat,” “treatment,” or “to treat” mean any method used to partially or completely reduce, improve, alleviate, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who show no signs of the disease and / or only early signs of the disease, with the aim of reducing the risk of developing a disease-related condition.

[0150] Vector: The term "vector" refers to a polynucleotide (usually DNA) used to artificially deliver exogenous genetic material to another cell capable of replicating or expressing it. Non-exclusive exemplary vectors include plasmids, viral vectors, cosmids, and artificial chromosomes. Such vectors can originate from a variety of sources, including bacteria and viral sources. A non-exclusive exemplary viral source for plasmids is adeno-associated virus.

[0151] Various aspects of this disclosure are described in detail in the following sections. The use of these sections is not intended to limit this disclosure. Each section may apply to any aspect of this disclosure. In this application, unless otherwise stated, the use of “or” means “and / or.” As used herein, the singular “a,” “an,” and “the” refer to both singular and plural objects unless the context otherwise explicitly indicates. Detailed description of the invention

[0152] This disclosure describes antibodies, nucleic acids, and systems for producing them, as well as their use and methods for treating diseases associated with dysfunction of 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.

[0153] Lupus nephritis, IgA nephropathy, myasthenia gravis, macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, neuropathic pain, COVID-19 infection, allergic asthma, chronic obstructive pulmonary disease, bullous pemphigoid, pyoderma gangrenosum, and psoriasis.

[0154] ANCA-associated vasculitis is a group of diseases characterized by the destruction and inflammation of small blood vessels (granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, and microscopic polyangiitis). Antineutrophil cytoplasmic autoantibodies (ANCAs) are the cause of ANCA-associated vasculitis. Experimental data from animal models and in vitro experiments demonstrate that primed neutrophils are activated by ANCA and produce C5a that engages with the C5a receptor on the neutrophils. This attracts more neutrophils, which are then primed one after another for activation by ANCA. The binding of C5a to C5aR1 may play a central role in the pathogenesis of ANCA-associated vasculitis. A general schematic diagram of ANCA-associated vasculitis is shown in Figure 1. Standard treatment is immunosuppressive therapy with glucocorticoids, and these therapies are substantially associated with short-term and long-term toxicity. Avacopan, a small molecule C5aR1 binding antagonist, has recently been approved by the FDA for use in ANCA-associated vasculitis. However, in vitro data suggest that the avacopan antagonistic effect of C5a can be overcome by high concentrations of C5a. It is known that C5a concentrations at inflammatory sites in active AAV can reach 100 nM. Under such conditions, it is known that avacopan-mediated inhibition of C5aR1 can be overcome. To counteract disorders associated with dysfunction of this pathway, there is a need for the development of strong inhibitors of the C5a / C5aR1 axis. This disclosure provides compositions and methods for the treatment of C5a / C5aR1 axis dysfunction-related disorders using biological C5aR1 antagonists.

[0155] In some embodiments, this specification provides two groups of exemplary antibodies that bind to and antagonize C5aR1. Schematic diagrams of the two antibody groups in this disclosure are shown in Figure 2.

[0156] The energy of an agonist that binds to the extracellular domain transmits allosteric conformational changes to the transmembrane and intracellular domains, enabling G protein binding and signal transduction. C5aR1 has two known agonists: C5a and C5a desArg The C-terminal arginine is rapidly cleaved by carboxypeptidase N to form C5a desArg Because it forms a short half-life in serum, C5a binds to C5aR1 with reduced affinity and exhibits biased signaling. desArg Unlike C5a, it does not signal via the β-arrestin pathway and does not stimulate granulocyte release. However, C5a desArg Since it stimulates neutrophil chemotaxis, C5a is used to prevent neutrophil migration to the site of inflammation. desArg Blocking the bond is also interesting. C5a desArg Signal transduction supports chemotaxis and makes it resistant to desensitization (for example, neutrophils use C5a desArg (Instead, it continues to migrate until it reaches a high concentration of C5a). In one embodiment, an orthosteric antagonist that blocks C5a binding (e.g., an antibody molecule described herein) also blocks C5a desArg It also inhibits (e.g., blocks) the binding. In some embodiments, inhibition of C5a binding is required at the site of inflammation, while C5a desArg Inhibition of this pathway is required in the periphery and can prevent neutrophils from migrating to the site of inflammation. Targeted C5aR1 typically leaves the membrane invasion complex pathway (C5b) intact. Design of monospecific C5aR1 antagonists

[0157] In some embodiments, the antibodies presented herein bind to a site defined by SEQ ID NO: 1 or SEQ ID NO: 2, also referred to as "site I." Site I typically comprises the N-terminal residue of C5aR1 (e.g., the N-terminal 37 residues) and forms a flexible random coil structure as defined by SEQ ID NO: 1 or SEQ ID NO: 2. An antibody molecule that binds to site I can typically bind to all residues of site I or a subset thereof. For example, an antibody molecule that binds to site I comes into contact with one or more residues of site I. In one embodiment, site I generally comprises a number of sulfated residues (e.g., sulfated tyrosine is present) and a number of Asp residues.

[0158] In some embodiments, the antibodies presented herein bind to a site defined by SEQ ID NO: 3, also called "site II." Site II typically includes the extracellular loop 2 (ECL2) and transmembrane residues that form the vestibule of C5aR1. In one embodiment, the antibody molecule that binds to site II binds to ECL2 but not to ECL1 and / or ECL3, or does not bind substantially to them. In another embodiment, the antibody molecule that binds to site II binds to both ECL2 and ECL1 but not to ECL3, or does not bind substantially to it.

[0159] In some embodiments, the antibody molecules described herein are designed to target site II, as defined by the amino acids in SEQ ID NO: 3. In some embodiments, the amino acids comprising R175-G189 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E180-P183 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E180-P184 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E178-P183 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, one or more residues of C5aR1 (SEQ ID NO: 38) R35, H101, V176, V177, R178, E179, E180, Y181, F182, P183, P184, K185, L187, D191, S193, H194, E266, P267, S268, F272, L273, and / or K276 are important for the binding of the site II antibody described herein. In some embodiments, one or more residues of SEQ ID NO: 38 E180, Y181, F182, and / or P183 are extremely important epitopes for the binding of the site II antibody described herein. In one embodiment, the amino acid residue W102 of SEQ ID NO: 38 is extremely important for the binding of the site II antibody described herein.

[0160] Sequence IDs 1, 2, and 3 are shown in Table 1. Table 1. Amino acid sequences of site I, site II, and human C5aR1. Variants (N2D and N279K) are shown in lowercase. [Table 1]

[0161] In some embodiments, the exemplary humanization site I (SEQ ID NO: 1 or SEQ ID NO: 2)-binding antibodies presented herein include a heavy chain variable region (HCVR or VH) containing the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39-43 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and / or a light chain variable region (LCVR or VL) containing the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44-49 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 the exemplary site I-binding antibodies. In some embodiments, three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, are located within the HCVR or VH (SEQ ID NO: 16 or SEQ ID NO: 39-43). In some embodiments, the three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, are located within LCVR or VL (SEQ ID NO: 17 or SEQ ID NO: 44-49). Note that exemplary humanized full-length site I-binding antibodies may include any combination of VH selected from the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39-43 and VL selected from the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44-49.

[0162] Please note that the humanized full-length site I-binding antibody may contain any combination of VH, selected from the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39-43, or from an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 39-43, and VL, selected from the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44-49, or from an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17 or SEQ ID NO: 44-49.

[0163] In some embodiments, the exemplary site-I binding antibody, e1711, contains the heavy chain of SEQ ID NO: 17, or an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, and 99% identical to that of SEQ ID NO: 17. In some embodiments, the exemplary site-I binding antibody, e1711, contains the light chain of SEQ ID NO: 16, or an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, and 99% identical to that of SEQ ID NO: 16.

[0164] In some embodiments, the exemplary site-I binding antibody, e1711, contains the heavy chain of SEQ ID NO: 43, or an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, and 99% identical to that of SEQ ID NO: 43. In some embodiments, the exemplary site-I binding antibody, e1711, contains the light chain of SEQ ID NO: 48, or an amino acid sequence that is 80%, 85%, 90%, 95%, 98%, and 99% identical to that of SEQ ID NO: 48.

[0165] In some embodiments, the exemplary site-I binding antibody e1711 presented herein comprises an e11L variable light chain and an e11H variable heavy chain, each comprising a heavy chain containing 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 thereto, and / or a light chain containing 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 thereto. Table 2a. Exemplary variable regions of humanized site-I binding antibodies. [Table 2] Table 2b. Exemplary heavy and light chains of humanized site I-binding antibodies [Table 3-1] [Table 3-2]

[0166] In some embodiments, the exemplary site II-binding antibodies presented herein include HCVR or VH containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 50-59 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and / or LCVR or VL containing the amino acid sequence of SEQ ID NO: 60-68 or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. Table 3 summarizes the amino acid sequences of the exemplary site II-binding antibodies VH and VL. In some embodiments, three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, are located within HCVR or VH (SEQ ID NO: 50-59). In some embodiments, three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, are located within LCVR or VL (SEQ ID NO: 60-68).

[0167] It should be noted that humanized full-length site II-binding antibodies may include any combination of VH, selected from amino acid sequences that have 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 50-59, or any of the amino acid sequences among SEQ ID NO: 14 or SEQ ID NO: 50-59, and VL, selected from amino acid sequences that have 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 25, or SEQ ID NO: 60-68, or any of the amino acid sequences among SEQ ID NO: 15, SEQ ID NO: 25, or SEQ ID NO: 60-68. Table 3. Variable regions of exemplary humanized site-II bound antibodies. [Table 4-1] [Table 4-2] [Table 4-3]

[0168] In some embodiments, the exemplary site II-binding antibody includes a combination of c39L (SEQ ID NO: 14) and c21H (SEQ ID NO: 25) for producing antibody c2139.

[0169] In some embodiments, the exemplary site-II binding antibody c2139 presented herein comprises a c39L variable light chain and a c21H variable heavy chain, each comprising a heavy chain containing 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 thereto, and / or a light chain containing 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 thereto.

[0170] In some embodiments, the exemplary site-II binding antibody c2137 presented herein comprises a c37L variable light chain and a c21H variable heavy chain, each comprising a heavy chain containing 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 thereto, and / or a light chain containing 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 thereto.

[0171] In some embodiments, the exemplary site II (SEQ ID NO: 3)-conjugated antibody, c2139, presented herein comprises a heavy chain containing HCVR or VH, where HCVR or VH comprises three heavy chain complementarity-determining regions, HCDR1, HCDR2, and HCDR3, each containing SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In some embodiments, the exemplary site II-conjugated antibody presented herein further comprises LCVR or VL, where LCVR or VL comprises three light chain complementarity-determining regions, LCDR1, LCDR2, and LCDR3, each containing SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, or comprises a sequence that differs from the amino acids of SEQ ID NOs: 6, 7, 8, 9, 10, and / or 11 by no more than 5, 4, 3, 2, or 1 amino acid. The sequences of the heavy chain, light chain, and HCDR and LCDR are shown in Table 4a.

[0172] In some embodiments, the exemplary site-II binding antibody c2139 presented herein includes HCVR or VH containing the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and / or LCVR or VL containing the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0173] In one embodiment, an exemplary antibody molecule, c2139, capable of binding to complement component 5a receptor 1 (C5aR1), is described herein, which competes with C5aR1 antibody molecules capable of binding to one or more amino acid residues at site II (SEQ ID NO: 3).

[0174] In some embodiments, the exemplary site II-binding antibody contains glycine or alanine at position 89 of VH. In some embodiments, the exemplary site II-binding antibody contains tyrosine at position 91. In some embodiments, the exemplary site II-binding antibody contains tyrosine at position 91 and glycine or alanine at position 89 of VH. Table 4a. Amino acid sequences of the heavy chain, light chain, HCDR, and LCDR of exemplary humanized C5aR1 site II antibodies. [Table 5-1] [Table 5-2]

[0175] In some embodiments, the HCVR and LCVR of exemplary site II-binding antibodies, such as c2139, are further linked to an Fc domain. The Fc domain further comprises CH2 and CH3 domains linked to each other via a linker. However, immediate disclosure also includes site II-binding biomolecules lacking an Fc domain.

[0176] 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 and C5a, and thus antagonizing the C5a / C5aR1 axial pathway.

[0177] In some embodiments, effective inhibition of C5a / C5aR1 antibodies is measured by inhibition of Gα signaling, inhibition of neutrophil chemotaxis, inhibition of CD11b expression, and inhibition of calcium signaling.

[0178] In some embodiments, exemplary site I or site II binding antibodies against C5aR1 do not bind to C5aR2 or other GPCRs, or bind substantially not.

[0179] In some embodiments, exemplary site I or site II-binding antibodies against C5aR1 can bind to human neutrophils.

[0180] In some embodiments, exemplary site I or site II-binding antibodies against C5aR1 inhibit β-arrestin signaling.

[0181] In some embodiments, exemplary site I or site II binding antibodies against C5aR1 inhibit ROS production in neutrophils.

[0182] In some embodiments, exemplary site I or site II-binding antibodies against C5aR1 undergo internal migration. In some embodiments, internal migration takes at least 6 hours. In some embodiments, internal migration takes at least 12 hours. In some embodiments, internal migration takes less than 6 hours. Design of multispecific C5aR1 antagonists

[0183] In some embodiments, the antibodies presented herein are multispecific antibodies. In one embodiment, the multispecific antibody molecule is a multiparatopic antibody molecule, for example, it comprises multiple immunoglobulin variable region sequences, where a first immunoglobulin variable region sequence of the plurality has binding specificity to a first epitope, and a second immunoglobulin variable region sequence of the plurality has binding specificity to a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a polymer protein). A bispecific antibody or biparatopic antibody has specificity to two or fewer antigens or epitopes. A bispecific or biparatopic antibody molecule is typically characterized by a first immunoglobulin variable region sequence having binding specificity to a first epitope, and a second immunoglobulin variable region sequence having binding specificity to a second epitope. In one embodiment, a bispecific antibody molecule or biparatopic antibody molecule comprises a semi-antibody or fragment thereof having binding specificity to a first epitope and a semi-antibody or fragment thereof having binding specificity to a second epitope. In one embodiment, the first and second epitopes are located on the same antigen, e.g., 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., the N-terminal region as described herein), and the second epitope is located on C5aR1 (e.g., site II, e.g., 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.

[0184] In some embodiments, the biparatopic antibody includes a VH and VL pair from Table 2a. In some embodiments, the biparatopic antibody includes a VH and VL pair from Table 3. In some embodiments, the biparatopic antibody includes a VH and VL pair from Table 2a, as well as a VH and VL pair from Table 3. In some embodiments, the biparatopic antibody includes a VH selected from amino acid sequences having 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 50-59, or any of the amino acid sequences among SEQ ID NO: 14 or SEQ ID NO: 50-59, and a VL selected from amino acid sequences having 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 25, or SEQ ID NO: 60-68, or any of the amino acid sequences among SEQ ID NO: 15, SEQ ID NO: 25, or SEQ ID NO: 60-68. In some embodiments, the biparatopic antibody comprises VH selected from the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 39-43, or from amino acid sequences that are 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16 or SEQ ID NO: 39-43, and VL selected from the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 44-49, or from amino acid sequences that are 80%, 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17 or SEQ ID NO: 44-49.

[0185] In some embodiments, one epitope of the biparatopic antibody described herein is designed to target the sulfated N-terminal peptide or site I of C5aR1, as defined by SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the site I residue targeted by the antibody molecule described herein is sulfated. In some embodiments, one or more amino acid residues T8 (threonine 8), D10 (aspartic acid 10), Y11 (tyrosine 11), Y14 (tyrosine 14), and / or D15 (aspartic acid 15) are critical site I epitope contact sites. In some embodiments, sulfation at Y11 and / or Y14 is critical for binding of the site I antibody molecule described herein. In some embodiments, the core epitope spans 12 amino acids from T7 to D18 of SEQ ID NO: 38 for binding of the site I antibody molecule described herein. In some embodiments, the core epitope spans amino acids T8-D18 of SEQ ID NO: 38 for binding to the site I antibody molecule described herein.

[0186] In some embodiments, the antibody molecules described herein are designed to target site II, as defined by the amino acids in SEQ ID NO: 3. In some embodiments, the amino acids comprising R175-G189 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E180-P183 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E180-P184 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, the amino acids comprising E178-P183 in SEQ ID NO: 38 are the core epitopes for binding to the site II antibody molecules described herein. In some embodiments, one or more residues of C5aR1 (SEQ ID NO: 38) R35, H101, V176, V177, R178, E179, E180, Y181, F182, P183, P184, K185, L187, D191, S193, H194, E266, P267, S268, F272, L273, and / or K276 are important for the binding of the site II antibody described herein. In some embodiments, one or more of E180, Y181, F182, and / or P183 of SEQ ID NO: 38 are extremely important epitopes for the binding of the site II antibody described herein. In one embodiment, the amino acid residue W102 of SEQ ID NO: 38 is extremely important for the binding of the site II antibody described herein.

[0187] In some embodiments, the biparatopic antibodies presented herein comprise a Fab-Fc and a single-chain variable region fragment (scFv), where the Fc is linked to the scFv via a linker. In some embodiments, the Fab domain binds to site I, and the scFv binds to site II.

[0188] In some embodiments, the biparatopic antibodies presented herein comprise a Fab-Fc and a single-chain variable region fragment (scFv). In some embodiments, the Fab domain is bound to site II, the scFv is bound to site I, and the Fc is linked to the scFv via a linker.

[0189] In some embodiments, the biparatopic antibodies presented herein are tetravalent antibodies containing a heavy chain, the heavy chain containing VH-Fc linked to an scFV domain, as shown in Figure 3A.

[0190] In some embodiments, the site II binding arm of the biparatopic antibody contains glycine or alanine at position 89 of VH. In some embodiments, the site II binding arm of the biparatopic antibody contains tyrosine at position 91. In some embodiments, the site II binding arm of the biparatopic antibody contains tyrosine at position 91 and glycine or alanine at position 89 of VH.

[0191] In some embodiments, the biparatopic antibodies presented herein are tetravalent antibodies containing a light chain, the light chain containing a VL linked to an scFv domain, as shown in Figure 3B.

[0192] In some embodiments, the biparatopic antibody is a bispecific antibody with a two-arm single-chain Fab-Fc design and includes a "knobs-in-holes" (KiH) mutation in the CH3 domain to assemble two half-antibodies (a common Fc heterodimer and unique VH-CH and VL-CL domains). In some embodiments, the KiH mutation includes the ability to create a knob using the T366Y mutation in one CH3 domain and a hole using the Y407T mutation in the other CH3 domain. In some embodiments, the knob can be created using the F405A mutation in one CH3 domain and a hole using the T394W mutation in the other CH3 domain. In some embodiments, the knob can be created using the T366W mutation in one CH3 domain and a hole using the Y407A mutation in the other CH3 domain. In some embodiments, biparatopic scFv-Fc molecules can be prepared using knob-hole techniques (e.g., including hole mutations: Y349C, T366S, L368A, Y407V and knob mutations: S354C, T366W).

[0193] In some embodiments, the biparatopic antibody comprises the antibody format shown in Table 4b. Table 4b. Format of Biparatopic Antibodies [Table 6]

[0194] 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).

[0195] In some embodiments, the Fc domain used in this application includes or is derived from IgG, IgM, IgE, or Fc moieties. In addition to the KiH mutation described above, the Fc domain includes the 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 the 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 the S228P mutation.

[0196] In some embodiments, the biparatopic antibody comprises two scFv regions linked to each other via a linker, where the first scFv binds to site I and the second scFv binds to site II.

[0197] In some embodiments, the Fab-Fc-linker-scFv biparatopic antibody is a tetravalent antibody comprising the heavy chain sequence of SEQ ID NO: 12 and the 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.

[0198] In some embodiments, the Fab-Fc-linker-scFv biparatopic antibody is a tetravalent antibody comprising the heavy chain sequence of SEQ ID NO: 12 and the 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.

[0199] Table 4c shows the heavy chain and light chain sequences of the biparatopic antibody. Table 4c. Amino acid sequences of the heavy and light chains of the humanized C5aR1 biparatopic antibody. [Table 7-1] [Table 7-2]

[0200] In some embodiments, the biparatopic has two variable regions, variable region 1 and variable region 2. Each variable region includes a variable heavy chain and a variable light chain.

[0201] In exemplary embodiments, the variable region 1 includes a variable heavy chain 1 (VH1) containing SEQ ID NO: 14 and a variable light chain 1 (VL) containing SEQ ID NO: 15. In some embodiments, the variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1 containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1 containing the amino acid sequence of SEQ ID NO: 15.

[0202] In some exemplary embodiments, variable region 2 includes a variable heavy chain 2 (VH2) and a variable light chain 2 (VL2), each containing the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, variable region 2 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, variable region 2 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2 containing the amino acid sequence of SEQ ID NO: 17.

[0203] In some exemplary embodiments, a 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.

[0204] In some exemplary embodiments, VH1 comprises three HCDRs, HCDR1, HCDR2, and HCDR3, which include the amino acid sequences of SEQ ID NOs. 6, 7, and 8, or comprises sequences that differ from the amino acids of SEQ ID NOs. 6, 7, and / or 8 by no more than 5, 4, 3, 2, or 1 amino acid.

[0205] In some exemplary embodiments, VH2 includes three HCDRs, HCDR4, HCDR5, and HCDR6, which include the amino acid sequences of SEQ ID NOs. 18, 19, and 20, or includes sequences that differ from the amino acids of SEQ ID NOs. 18, 19, and / or 20 by no more than 5, 4, 3, 2, or 1 amino acid.

[0206] In some exemplary embodiments, VL1 includes three LCDRs, LCDR1, LCDR2, and LCDR3, which include the amino acid sequences of SEQ ID NOs. 9, SEQ ID NOs. 10, and SEQ ID NOs. 21, or includes sequences that differ from the amino acids of SEQ ID NOs. 9, 10, and / or 21 by no more than 5, 4, 3, 2, or 1 amino acid.

[0207] In some exemplary embodiments, VL2 includes three LCDRs, LCDR4, LCDR5, and LCDR6, which include the amino acid sequences of SEQ ID NOs. 22, 23, and 24, or includes sequences that differ from the amino acids of SEQ ID NOs. 22, 23, and / or 24 by no more than 5, 4, 3, 2, or 1 amino acid. Table 5 presents the amino acid sequences of VH1, VH2, VL1, VL2, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6.

[0208] In some exemplary embodiments, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6 contain amino acid sequences that differ from the amino acid sequences of SEQ ID NOs. 16-21 by no more than 5 amino acid residues. Table 5. Amino acid sequences of the variable domain and CDR of the humanized C5aR1 biparatopic antibody (c2137-e1711) [Table 8]

[0209] In some exemplary embodiments, the variable region of the biparatopic antibody includes variable region 1, which includes VH1 and VL1, each containing the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1 containing the amino acid sequence of SEQ ID NO: 17.

[0210] In some exemplary embodiments, the variable region of the biparatopic antibody includes a variable region 2, which includes VH2 containing SEQ ID NO: 14 and VL2 containing SEQ ID NO: 15. In some embodiments, the variable region 2 includes a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2 containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the variable region 2 includes a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2 containing the amino acid sequence of SEQ ID NO: 15.

[0211] In some exemplary embodiments, VH1 includes three HCDRs, HCDR1, HCDR2, and HCDR3, which include the amino acid sequences of SEQ ID NOs. 18, 19, and 20, or includes sequences that differ from the amino acids of SEQ ID NOs. 18, 19, and / or 20 by no more than 5, 4, 3, 2, or 1 amino acid.

[0212] In some exemplary embodiments, VH2 includes three HCDRs, HCDR4, HCDR5, and HCDR6, which include the amino acid sequences of SEQ ID NOs. 6, 7, and 8, or includes sequences that differ from the amino acids of SEQ ID NOs. 6, 7, and / or 8 by no more than 5, 4, 3, 2, or 1 amino acid.

[0213] In some exemplary embodiments, VL1 includes three LCDRs, LCDR1, LCDR2, and LCDR3, which include the amino acid sequences of SEQ ID NOs. 22, 23, and 24, or includes sequences that differ from the amino acids of SEQ ID NOs. 22, 23, and / or 24 by no more than 5, 4, 3, 2, or 1 amino acid.

[0214] In some exemplary embodiments, VL2 includes three LCDRs, LCDR4, LCDR5, and LCDR6, which include the amino acid sequences of SEQ ID NOs. 9, SEQ ID NOs. 10, and SEQ ID NOs. 21, or includes sequences that differ from the amino acids of SEQ ID NOs. 9, 10, and / or 21 by no more than 5, 4, 3, 2, or 1 amino acid. Table 5 presents the amino acid sequences of VH1, VH2, VL1, VL2, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6.

[0215] In some exemplary embodiments, HCDR1, HCDR2, HCDR3, HCDR4, HCDR5, HCDR6, LCDR1, LCDR2, LCDR3, LCDR4, LCDR5, and LCDR6 contain amino acid sequences that differ from the amino acid sequences of SEQ ID NOs. 16-21 by no more than 5 amino acid residues.

[0216] In some exemplary embodiments, the variable region of the biparatopic antibody includes variable region 1, which includes VH1 and VL1, each containing the amino acid sequences of SEQ ID NO: 14 and SEQ ID NO: 25, respectively. In some embodiments, variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH1 containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, variable region 1 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL1 containing the amino acid sequence of SEQ ID NO: 25.

[0217] In some exemplary embodiments, the variable region of the biparatopic antibody includes a variable region 2, which includes VH2 and VL2, each containing the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, the variable region 2 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VH2 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the variable region 2 contains a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of VL2 containing the amino acid sequence of SEQ ID NO: 17.

[0218] In some exemplary embodiments, a biparatopic antibody comprising VH1 of SEQ ID NO: 14, VL1 of SEQ ID NO: 25, VH2 of SEQ ID NO: 16, and VL2 of SEQ ID NO: 17 is referred to as c2139-e1711.

[0219] In some exemplary embodiments, the exemplary biparatopic antibody that binds to C5aR1 binds to one or more of the 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.

[0220] In some exemplary embodiments, the exemplary biparatopic antibody that binds to C5aR1 binds to amino acid residues R175-G189 of SEQ ID NO: 38.

[0221] In some exemplary embodiments, the exemplary biparatopic antibody that binds to C5aR1 binds to amino acid residues E180-P183 of SEQ ID NO: 38.

[0222] In some exemplary embodiments, the exemplary biparatopic antibody that binds to C5aR1 binds to amino acid residues E180-P184 of SEQ ID NO: 38.

[0223] In some exemplary embodiments, the exemplary biparatopic antibody that binds to C5aR1 binds to amino acid residues E178-P183 of SEQ ID NO: 38.

[0224] In some embodiments, exemplary biparatopic antibodies against C5aR1 inhibit β-arrestin signaling.

[0225] In some embodiments, exemplary biparatopic antibodies against C5aR1 inhibit ROS production in neutrophils.

[0226] In some embodiments, the exemplary biparatopic antibody against C5aR1 undergoes internal migration. In some embodiments, internal migration takes at least 6 hours. In some embodiments, internal migration takes at least 12 hours. In some embodiments, internal migration takes less than 6 hours.

[0227] 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 the stability of an antibody is to increase the thermal transition midpoint (Tm), which is measured by differential scanning calorimetry (DSC). Generally, protein Tm correlates with its stability and inversely correlates with its susceptibility to unfolding and denaturation in solution, as well as with degradation processes that depend on the protein's unfolding tendency. Numerous studies have found correlations between the ranking of the 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 PharmSci. 5E8:2003, Bedu-Addo et al. (2004) Pharm. Res. 21:1353-61, Zhang et al. (2004) J. Pharm. Sci. 93:3076-89). Formulation studies suggest that Fab Tm influences the long-term physical stability of the corresponding mAb.

[0228] 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.

[0229] In some exemplary embodiments, the strategic introduction of π-stacking interactions with aromatic amino acids (AAs) such as tryptophan (TRP), tyrosine (TYR), phenylalanine (PHE), and histidine (HIS) plays a role in enhancing the thermal stability of antibodies.

[0230] In some embodiments, the strategic introduction of salt bridges between amino acid side chains with opposite positive or negative total electronic charges, i.e., Glu or Asp versus Arg or Lys (at neutral pH), enhances the stability of proteins, particularly antibodies.

[0231] In some exemplary embodiments, a monospecific antibody or a biparatopic antibody includes 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 includes cysteine ​​at position 559 and position 630 of SEQ ID NO: 12 to enhance thermal stability.

[0232] In some embodiments, the Tm of the exemplary biparatopic antibody exceeds 65°C. In some embodiments, the Tm of the exemplary biparatopic antibody exceeds 60°C. The Tm of the exemplary biparatopic antibody exceeds 55°C. In some embodiments, the Tm of the exemplary biparatopic antibody exceeds 50°C.

[0233] Several types of biparatopic antibody molecules are produced by crosslinking the binding domains or antigen-binding fragments at site I and site II to create bispecific antibodies. Suitable crosslinking agents include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide) or homobifunctional (e.g., disuccinimidyl sulfite) linkers having two distinct reactive groups separated by a suitable spacer. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0234] In some embodiments, peptide linkers are used to link scFv antibodies or single-chain antibodies to the Fc domain of Fab. Some examples of suitable linkers include a single glycine (G) residue, a diglycine 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), and a peptide with eight glycine residues (GGGGGGGG; SEQ ID NO: 30). Other combinations of amino acid residues may also be used, such as 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 residues, Val residues, and dipeptides RTQP, SS, TK, SL, TKGPS, TVAAP, and QPKAA. The examples given above are not intended to limit the scope of this disclosure in any way, and linkers containing randomly selected amino acids 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 proteins. For further descriptions of linker sequences, see, for example, WO2012 / 135345.

[0235] The identity and sequence of amino acid residues in a linker can vary depending on the type of secondary structural elements required to be achieved within the linker. For example, glycine, serine, and alanine are best suited for linkers with maximum flexibility. Several combinations of glycine, proline, threonine, and serine are useful when a more rigid and elongated linker is needed. Depending on the desired properties, any amino acid residue can be considered as a linker in combination with other amino acid residues to construct a larger peptide linker as needed. Design of the Fc variant

[0236] In some embodiments, the monospecific and multispecific (including bispecific or biparatopic) C5aR1 antibodies presented herein contain variations or mutations in the Fc region. In some embodiments, the Fc variant or mutant reduces the ability to undergo 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 certain embodiments, the Fc domain is an IgG1 Fc domain. In other embodiments, the Fc domain is an IgG4 Fc domain. In more specific embodiments, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P.

[0237] In some embodiments, the Fc region includes a human IgG4 Fc region containing one or more mutations selected from the group consisting of S228P, L234V, L235A, G237A, D265G, A330S, P331S, L328R, H268A, and N297Q mutations (as specified according to Kabat, et al. (1991)). In some cases, the human IgG4 Fc variant has a total of up to 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 includes the F234V, L235E, and D265G mutations. In some embodiments, the Fc region includes the F234V, L235E, D265G, and S228P mutations.

[0238] In some embodiments, the Fc variant exhibits reduced binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits ablate binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits elimination of phagocytosis compared to the wild-type human IgG Fc region.

[0239] Antibody-dependent cell-mediated cytotoxicity (CMC), also referred to herein as ADCC, is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells containing antigens and subsequently kill them. Antibody-dependent cell-mediated phagocytosis (ADCP), also referred to herein as ADCP, is a form of cytotoxicity in which secreted Ig is secreted onto Fc receptors (FcRs) present on specific phagocytic cells (e.g., macrophages), enabling these phagocytic cells to specifically bind to target cells containing antigens and subsequently engulf and digest them. Ligand-specific high-affinity IgG antibodies directed at the surface of target cells can stimulate cytotoxic cells or phagocytic cells and can be used for such killing. In some embodiments, polypeptide constructs containing the Fc variants described herein exhibit reduced ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, polypeptide constructs containing the Fc variant described herein exhibit a reduction of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, antibodies containing the Fc variant described herein exhibit removal of ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region.

[0240] In some embodiments, the Fc variant exhibits reduced binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits ablate binding to the target Fc receptor compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits reduced phagocytosis compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits elimination of phagocytosis compared to the wild-type human IgG Fc region.

[0241] Antibody-dependent cell-mediated cytotoxicity (DCD), also referred to herein as ADCC, is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells and neutrophils), enabling these cytotoxic effector cells to specifically bind to target cells containing antigens and subsequently kill them. Antibody-dependent cell-mediated phagocytosis (DCP), also referred to herein as ADCP, is a form of cytotoxicity in which secreted Ig is secreted to Fc receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and / or FcγRIIIb) present on specific phagocytic cells (e.g., macrophages), enabling these phagocytic cells to specifically bind to target cells containing antigens and subsequently engulf and digest them. Ligand-specific high-affinity IgG antibodies directed at the surface of target cells can stimulate cytotoxic cells or phagocytic cells and can be used for such killing. In some embodiments, polypeptide constructs containing the Fc variant described herein exhibit a reduction in ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, polypeptide constructs containing the Fc variant described herein exhibit a reduction in ADCC or ADCP of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to polypeptide constructs containing the wild-type Fc region. In some embodiments, antibodies containing the Fc variant described herein exhibit removal of ADCC or ADCP compared to polypeptide constructs containing the wild-type Fc region.

[0242] Exemplary single-specificity site II (SEQ ID NO: 3) conjugated antibodies containing the Fc variant are also described in this disclosure as c2139-F c Also known as mod. Exemplary biparatopic antibodies containing the Fc variant are also referred to in this disclosure as c2137-e1711-Fcmod. The sequences of monospecific antibodies and biparatopic antibodies containing the Fc variant are listed in Table 6. Table 6-Fc-modified exemplary monospecific and biparatopic antibody sequences. [Table 9-1] [Table 9-2] [Table 9-3]

[0243] In some embodiments, the monospecific anti-C5aR1 antibody contains 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 with SEQ ID NO: 69.

[0244] In some embodiments, the monospecific anti-C5aR1 antibody contains 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 with SEQ ID NO: 70.

[0245] In some embodiments, the biparatopic anti-C5aR1 antibody contains 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 with SEQ ID NO: 71.

[0246] In some embodiments, the biparatopic anti-C5aR1 antibody contains 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 with SEQ ID NO: 72.

[0247] In some embodiments, the monospecific anti-C5aR1 antibody contains 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 with SEQ ID NO: 79.

[0248] In some embodiments, the monospecific anti-C5aR1 antibody contains 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 with SEQ ID NO: 81.

[0249] In some embodiments, the monospecific anti-C5aR1 antibody contains 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 with SEQ ID NO: 82. Use of monospecific and biparatopic C5aR1 antagonists for the treatment of disease

[0250] This specification describes methods for treating diseases associated with C5a / C5aR1 axis dysfunction. Accordingly, in some embodiments, monospecific antibodies and / or biparatopic antibodies against C5aR1 described herein are suitable for treating subjects with C5a / C5aR1 axis-related dysfunction, such as ANCA-associated vasculitis.

[0251] 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 related to neutrophil recruitment, activation, and / or NETosis. In one embodiment, the disorder is related to complement system activation and / or coagulation system activation. In one embodiment, the disorder is related to a C5aR-mediated inflammatory response. In one embodiment, the disorder is related to monocyte chemotactic protein-1 (MCP-1) and / or renal inflammation. In one embodiment, the disorder is related to chemotaxis (e.g., chemotactic priming). In one embodiment, the disorder is related to endothelial damage.

[0252] A treatment method involves administering an antibody described herein to a subject in need of it. In one embodiment, an exemplary disorder, such as a C5aR1-related disorder, can be treated using an antibody capable of binding to one or more amino acid residues of C5aR1 at site II (SEQ ID NO: 3).

[0253] In one embodiment, exemplary disorders, such as C5aR1-related disorders, can be treated using an antibody that can compete with an antibody that binds to C5aR1 at site I (SEQ ID NO: 1 or SEQ ID NO: 2) or site II (SEQ ID NO: 3).

[0254] In one embodiment, exemplary disorders, such as C5aR1-related disorders, can be treated using antibodies that can compete with antibodies binding to C5aR1 at site I (SEQ ID NO: 1 or SEQ ID NO: 2) and site II (SEQ ID NO: 3).

[0255] The monospecific antibodies and / or biphase antibodies described herein can be used to treat any disease associated with dysfunction related to the C5a / C5aR1 axis.

[0256] In some embodiments, exemplary disorders, such as C5aR1-related disorders, can be treated using monospecific antibodies or biparatopic antibodies capable of binding to C5aR1, the antibodies binding to C5aR1 with affinity from 10 pM to 50 nM.

[0257] In some embodiments, the antibody can be administered intravenously, subcutaneously, intradermally, or intramuscularly to a subject that requires it.

[0258] In some embodiments, the nucleic acids encoding the monospecific or biparatopic antibodies described herein can be administered to subjects requiring them using appropriate delivery methods. Several methods for delivering nucleic acids are known in the literature. For example, the rAAV vectors encoding the monospecific or biparatopic antibodies described herein can be administered to subjects by intravenous, intraperitoneal, subcutaneous, or intradermal administration. In some embodiments, delivery of the antibody-encoding nucleic acids can be achieved using a "gene gun," a bioristic particle delivery system, or nonviral lipid nanoparticles.

[0259] In some embodiments, the monospecific antibodies or biparatopic antibodies described herein can be administered to subjects in need in combination with additional therapeutic agents. In some embodiments, the additional therapeutic agent is a small molecule such as avacopan, a corticosteroid, or an immunosuppressant. Exemplary corticosteroids include, but are not limited to, prednisolone, hydrocortisone, prednisone, dexamethasone, or cortisone. Exemplary immunosuppressants include, but are not limited to, methotrexate, azathioprine, mycophenolate mofetil, or cyclophosphamide.

[0260] In some embodiments, the monospecific or biparatopic antibodies described herein are superior to avacopan monotherapy in reducing neutropenia in vivo.

[0261] In some embodiments, the monospecific or biparatopic antibodies described herein are superior to avacopan in that they antagonize C5aR1 in the presence of a higher concentration of C5a of 100 nM.

[0262] In some embodiments, the monospecific antibodies or biparatopic antibodies described herein are superior to avacopan in inhibiting Gα signaling in vitro.

[0263] In some embodiments, the monospecific or biparatopic antibodies described herein are superior to avacopan in inhibiting calcium signaling in vitro and in vivo.

[0264] In some embodiments, the monospecific or biparatopic antibodies described herein are superior to avacopan in inhibiting neutrophil chemotaxis in vitro and in vivo.

[0265] In some embodiments, the monospecific antibodies or biparatopic antibodies described herein are stable in serum for up to 500 hours after injection.

[0266] In some embodiments, the monospecific or biparatopic antibodies described herein are superior to avacopan in inhibiting CD11b expression in vitro and in vivo. Nucleic acids, vectors, and methods for producing them

[0267] This disclosure also features nucleic acids comprising nucleotide sequences encoding antibody molecules (e.g., the heavy chain variable region and light chain variable region and CDR of an antibody molecule), as described herein.

[0268] For example, this disclosure features first and second nucleic acids encoding the heavy chain variable region and light chain variable region, respectively, of an antibody molecule selected from one or more antibody molecules disclosed herein, such as an antibody molecule or a portion of an antibody molecule, such as a variable region of any of the antibodies disclosed herein. The nucleic acids may include a nucleotide sequence encoding any one of the amino acid sequences in the table herein, or a sequence substantially identical thereto (for example, a sequence that is at least about 85%, 90%, 95%, 99%, or more identical thereto, or a sequence that differs from the sequences shown in the table herein by no more than 3, 6, 15, 30, or 45 nucleotides).

[0269] In certain embodiments, the nucleic acid comprises nucleotide sequences encoding at least one, two, or three CDRs derived from a heavy chain variable region having the amino acid sequences listed in the table herein, or sequences substantially homologous thereto (e.g., sequences identical to them by at least about 85%, 90%, 95%, 99%, or more, and / or sequences having one or more substitutions, e.g., conserved substitutions). In one embodiment, the nucleic acid comprises nucleotide sequences encoding at least one, two, or three CDRs derived from a light chain variable region having the amino acid sequences listed in the table herein, or sequences substantially homologous thereto (e.g., sequences identical to them by at least about 85%, 90%, 95%, 99%, or more, and / or sequences having one or more substitutions, e.g., conserved substitutions). In one embodiment, the nucleic acid comprises nucleotide sequences encoding at least one, two, three, four, five, or six CDRs derived from heavy and light chain variable regions having the amino acid sequences listed in the Tables herein, or sequences substantially homologous thereto (e.g., sequences that are at least about 85%, 90%, 95%, 99%, or more identical thereto, and / or sequences having one or more substitutions, e.g., conserved substitutions).

[0270] The nucleic acids disclosed herein include deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may be single-stranded or double-stranded, and if single-stranded, they may be coding or non-coding (antisense) strands. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogs. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Nucleic acids may be recombinant polynucleotides, or polynucleotides of genomic, cDNA, semi-synthetic, or synthetic origin that do not occur naturally or are linked to other polynucleotides in an unnatural configuration.

[0271] In some embodiments, this application features host cells and vectors containing nucleic acids as described herein. The nucleic acids may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail below. vector

[0272] Vectors comprising nucleotide sequences encoding antibody molecules described herein (e.g., variable regions and CDRs of the heavy and light chains of antibody molecules) are further provided herein.

[0273] In one embodiment, the vector comprises nucleic acids described herein. For example, the vector may comprise first and second nucleic acids encoding the heavy chain variable region and light chain variable region, respectively, of an antibody molecule selected from one or more antibody molecules disclosed herein, such as an antibody molecule or a part of an antibody molecule, such as a variable region listed in any of Tables 1 to 4.

[0274] In certain embodiments, the vector comprises nucleotide sequences encoding at least one, two, or three CDRs derived from a heavy chain variable region having the amino acid sequences listed in the table herein, or sequences substantially homologous thereto (e.g., sequences identical to them by at least about 85%, 90%, 95%, 99%, or more, and / or sequences having one or more substitutions, e.g., conserved substitutions). In one embodiment, the vector comprises nucleotide sequences encoding at least one, two, or three CDRs derived from a light chain variable region having the amino acid sequences listed in the table herein, or sequences substantially homologous thereto (e.g., sequences identical to them by at least about 85%, 90%, 95%, 99%, or more, and / or sequences having one or more substitutions, e.g., conserved substitutions). In one embodiment, the vector comprises nucleotide sequences encoding at least one, two, three, four, five, or six CDRs derived from heavy and light chain variable regions having amino acid sequences listed in the Tables herein, or sequences substantially homologous thereto (e.g., sequences that are at least about 85%, 90%, 95%, 99%, or more identical thereto, and / or sequences having one or more substitutions, e.g., conserved substitutions).

[0275] Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semlik Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0276] Furthermore, cells in which DNA has been stably incorporated into their chromosomes may be selected by introducing one or more markers that enable the selection of transfected host cells. These markers may provide, for example, prototropy to a trophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker genes may be directly ligated to the expressed DNA sequence or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0277] Once an expression vector or DNA sequence containing a construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques may be used, such as plasma fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. In the case of plasma fusion, cells are grown in culture medium and screened for appropriate activity.

[0278] 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 this description depending on the specific expression vector and mammalian host cell used. cell

[0279] This disclosure also provides cells (e.g., host cells) containing nucleic acids encoding antibody molecules described herein. For example, a host cell may contain a nucleotide sequence encoding an amino acid sequence listed in any of Tables 1 to 5, a nucleic acid molecule having a substantially homologous sequence (e.g., a sequence that is at least about 85%, 90%, 95%, 99% or more identical thereto, and / or a sequence that can hybridize under the stringency conditions described herein), or a portion of one of such nucleic acids.

[0280] In one embodiment, a host cell is genetically engineered to contain a nucleic acid encoding an antibody molecule as described herein.

[0281] In certain embodiments, host cells are genetically engineered using expression cassettes. The term “expression cassette” refers to a nucleotide sequence that can influence gene expression in a host that matches such a sequence. Such cassettes may include promoters, open reading frames with or without introns, and termination signals. Additional factors necessary or useful for producing expression, such as inducible promoters, may also be used.

[0282] This disclosure also provides host cells containing the vectors described herein.

[0283] The cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Preferred eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Preferred insect cells include, but are not limited to, Sf9 cells. In one embodiment, the cells (e.g., host cells) are isolated cells. Method of administration

[0284] The compositions of the present invention can be formulated in any suitable form, such as liquid, semi-solid, and solid dosage forms, including liquid solutions (e.g., injectable and insoluble solutions), dispersants, or suspensions. The optimal form for any composition depends on the intended mode of administration, the properties of the composition or combination, and the therapeutic or other intended use. A typical mode of delivery of the compositions of the present invention is by parenteral administration (e.g., intravenous administration). In one embodiment, the compositions of the present invention are administered to a human patient by intravenous infusion or injection.

[0285] In some embodiments, the Disclosure provides compositions comprising antibody molecules described herein, for example, formulated with a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable composition. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, isotonic agent, and absorption retarder. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or dermal administration (e.g., by injection or infusion).

[0286] As used herein, the terms “parenteral administration” and “administered parenterally” typically mean, but are not limited to, modes of administration other than enteral and topical administration by injection, including, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Therapeutic compositions should typically be sterile and stable under manufacturing and storage conditions. Compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high concentrations of antibodies. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody moiety) in the required amount in a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and lyophilization, which yield a powder to which any additional desired components from a pre-sterilized filtered solution are added to the active ingredient. Appropriate fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin to maintain the particle size required in the case of dispersion, and by the use of a surfactant. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, for example, monostearate and gelatin. The antibody molecules described herein can be administered by various methods. In many therapeutic, prophylactic, or diagnostic applications, the appropriate route / mode of administration is intravenous injection or infusion, which is several known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. [Examples]

[0287] Other features, purposes, and advantages of this disclosure will become apparent in the following embodiments. However, it should be understood that these embodiments illustrate embodiments of this disclosure, but are given only as examples and not as limitations. Various changes and modifications within the scope of this disclosure will become apparent to those skilled in the art from the embodiments. Example 1. Dynamics analysis of a humanized antibody that antagonistizes C5aR1.

[0288] This example describes the binding affinity and specificity of the site II monospecific antibody and biparatopic antibody described herein to C5aR1 and C5aR2. (a) Binding affinity

[0289] The binding affinity of antibodies to the target receptor C5aR1 was determined using an ELISA assay. C5aR1 VLPs were immobilized at a concentration of 30 μg / mL on a MaxiSorp ELISA plate and incubated overnight at 4°C. The following morning, the plate was washed three times with 1×PBS and blocked with 100 μL of PBSA (1×PBS containing 3% BSA) for 30 minutes. Serial titration of anti-C5aR1 antibody was performed in the presence of PBSA and incubated at room temperature for 1 hour. The plate was washed six times with PBSA. Anti-human-HRP was diluted in PBSA and added to all wells, and incubated at room temperature for 45 minutes. The plate was washed six times with PBS. TMB substrate was added to all wells and incubated for 10 minutes, after which stop solution (0.1 M sulfuric acid) was added. Absorbance at 450 nm was measured using a standard plate reader. EC50 values ​​for antibody titrations in nM were generated using a 4-parameter curve fitting. The affinity curves for the monospecific c2139 antibody and the biparatopic c2137-e1711 antibody are shown in Figure 4A.

[0290] Under the conditions described above, it was observed that the monospecific C5aR1 antibody bound to C5aR1 with an affinity of approximately 0.16 nM, while the biparatopic antibody bound to C5aR1 with an affinity of approximately 0.22 nM.

[0291] In a different configuration, the binding of site II antibodies, c2139, and the biparatopic antibody c2137-e1711, from two different antibody batches was assayed for binding to U937-C5aR1 cells (Figure 4B) and human neutrophils (Figure 4C). Table 6 shows the EC50 of U937-C5aR1 bound to different protein batches of different anti-C5aR1 antibodies (c2139 and c2137-e1711). Table 6. EC50 of U937-C5aR1 bound to exemplary C5aR1 antibody [Table 10]

[0292] Table 7 shows the EC50 of human neutrophils bound to different protein batches of different anti-C5aR1 antibodies (c2139 and c2137-e1711). Table 7. EC50 of human neutrophils bound to exemplary C5aR1 antibodies. [Table 11]

[0293] Motabizumab, a non-C5aR1 antibody, was used as a control. Both the site II antibody, c2139, and the biparatopic antibody c2137-e1711 were observed to bind to U937 and human neutrophils across multiple antibody preparation lots. (b) Binding specificity

[0294] The specificity of the C5aR1 antagonist antibody was determined by measuring the affinity of the anti-C5aR1 antibody for C5aR2. C5aR2 VLP was immobilized at a concentration of 30 μg / mL on a MaxiSorp ELISA plate and incubated overnight at 4°C. The following morning, the plate was washed three times with 1×PBS and blocked with 100 μL of PBSA (1×PBS containing 3% BSA) for 30 minutes. Serial titration of the anti-C5aR1 antibody was performed in the presence of PBSA and incubated at room temperature for 1 hour. The plate was washed six times with PBSA. Anti-human-HRP was diluted with PBSA and added to all wells, and incubated at room temperature for 45 minutes. The plate was washed six times with PBS. TMB substrate was added to all wells and incubated for 10 minutes, after which stop solution (0.1 M sulfuric acid) was added. Absorbance at 450 nm was measured with a standard plate reader. EC50 values ​​for antibody titrations in nM were generated using a 4-parameter curve fitting.

[0295] The binding data for monospecific antibodies and biparatopic antibodies are shown in Figure 5. It was observed that the exemplary C5aR1 antagonist antibody did not bind to C5aR2 under the conditions described above. Overall, the data from this example demonstrate that the anti-C5aR1 antibody of the present invention binds to C5aR1 with high affinity and to C5aR2 with immeasurable affinity, indicating that the C5aR1 antibody is indeed specific to C5aR1. Example 2. Inhibition of Gα signaling

[0296] This example illustrates the functional aspects of the C5aR1 monospecific antibody and biparatopic antibody described herein in relation to the inhibition of Gα signaling using the GeneBLazer assay.

[0297] The GeneBLAzer assay kit and C5aR1 cell line are commercially available from Thermo Fisher (catalog number K1544). The assay was performed as recommended by the manufacturer. Briefly, the antibody or antagonist was incubated at 37°C for 30 minutes at increasing concentrations. Then, C5a was added to the cells and incubated for a further 4-5 hours at 37°C. Next, the β-lactamase substrate was added and incubated at room temperature for 2 hours. Fluorescence measurements were taken in each well with excitation / emission of 409 / 460 (blue) and 409 / 530 (green). The increase in the ratio of blue to green was proportional to C5aR1 activation and was used to calculate the percentage of cell activation in each well. The C5aR1 activation profile was monitored under increasing antagonist concentrations. IC50 was determined by plotting the percentage of signal against the concentration of the antagonist.

[0298] The Gα signaling assays of monospecific and biparatopic antibodies are shown in Figure 6A as a function of antibody concentration at 10 nM C5a. In an alternative setup, a known C5aR antibody, anti-C5aR1 control Ab, is used as a positive control for inhibition of Gα signaling (Figure 6C).

[0299] The Gα signaling assays of monospecific and biparatopic antibodies are shown in Figure 6B as a function of antibody concentration at 100 nM C5a. In a different setting, a known C5aR antibody, an anti-C5aR1 control Ab, is 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. Exemplary antibodies were observed to retain superior inhibition compared to avacopan and anti-C5aR1 control Ab at all C5a concentrations tested. Example 3. Inhibition of calcium signaling

[0300] This example illustrates the functional aspects of the C5aR1 monospecific antibody and biparatopic antibody described herein for inhibiting C5a-mediated calcium signaling using a calcium flux assay.

[0301] The efficacy of antibodies was evaluated to inhibit intracellular calcium release in stable C5aR1-U937 cells expressing C5aR1 or stable neutrophils isolated from whole blood in the presence of C5a. Changes in cytoplasmic calcium concentration were detected using a calcium flux assay with a calcium-sensitive dye. Cells were incubated with an esterified (inactive) calcium dye. The dye permeated the cell membrane and became activated upon entering the cell. The active form of this dye fluoresced after binding to intracellular calcium, and this fluorescence was used to determine C5aR1 signaling in response to C5a addition. Specifically, stable C5aR1-U937 or neutrophils isolated from whole blood were stained with the Thermo Fisher Fluo-4 Direct calcium assay kit at 37°C for 1 hour. The antibody or antagonist was then incubated with the cells for 30 minutes. Baseline fluorescence, with excitation at 494 nm and emission at 516 nm, was measured for 15 seconds. C5a was added to cells, and fluorescence was measured over 4 minutes. The response ratio was calculated using the baseline reading before C5a stimulation and the maximum signal after C5a stimulation. Calcium flux assays were performed using engineered cells stably expressing C5aR1, as well as human neutrophils.

[0302] The inhibition of C5a-mediated calcium signaling assays by monospecific (Figure 7A) and biparatopic antagonist antibodies (Figure 7B) is plotted as calcium flux as a function of C5a concentration with increasing antibody concentration. Furthermore, the inhibition of C5a-mediated calcium signaling by avacopan, a known C5aR1 antagonist, using the protocol described above, is illustrated in Figure 7C. The tested antibodies were observed to be superior to avacopan in inhibiting calcium signaling. Example 4. Inhibition of neutrophil chemotaxis

[0303] This example describes the functional aspects of the C5aR1 monospecific antibody and biparatopic antibody described herein for the inhibition of neutrophil chemotaxis, which is known to be induced by C5aR1 activity, using a Boyden chamber.

[0304] First, stable C5aR1-U937 cells were seeded in the upper chamber of a 96 Transwell plate, either without antibody (antagonist) or in the presence of 1, 10, or 100 nM antibodies (antagonists). The bottom chamber was 10 -6 ~10 -9.5 The system contained semi-logarithmic titrations of C5a (chemotactic factor) in the M range. The upper and lower chambers were separated by a membrane. C5a was expected to induce migration of U937 cells. However, the use of a C5aR1 antagonist was expected to inhibit this migration. Dose-dependent inhibition of chemotaxis was determined using exemplary antibodies at increasing concentrations. Furthermore, it was determined whether inhibition of chemotaxis could be overcome by using high concentrations of C5a, using increasing concentrations of chemotactic factor (C5a). In parallel, avacopan, a known C5aR1 inhibitor, was also used to evaluate the inhibition of neutrophil chemotaxis and the possibility of overcoming chemotaxis in the presence of excess C5a.

[0305] Next, human neutrophils were seeded in the upper chamber of a 96 Transwell plate, either without antibody (antagonist) or in the presence of 1, 10, or 100 nM antibodies (antagonists). The bottom chamber was 10 -6 ~10 -9.5The sample contained semi-logarithmic titrations of C5a (chemotactic factor) in the M range. The upper and lower chambers were separated by a membrane. C5a was expected to induce migration of U937 cells. However, the use of a C5aR1 antagonist was expected to inhibit this migration. Dose-dependent inhibition of chemotaxis was determined using increasing concentrations of the antibody. Furthermore, it was determined whether the inhibition of chemotaxis could be overcome by using high concentrations of C5a, using increasing concentrations of the chemotactic factor (C5a). In parallel, avacopan, a known C5aR1 inhibitor, was also used to evaluate the inhibition of neutrophil chemotaxis and the possibility of overcoming chemotaxis in the presence of excess C5a.

[0306] Figures 8A-8C show the amount of fluorescence intensity relative to C5a concentration in the presence of different concentrations of anti-C5aR1 antibody, demonstrating the inhibition of chemotaxis by anti-C5aR1 antibody. Generally, anti-C5aR1 antibody was observed to inhibit cell chemotaxis in a dose-dependent manner. Furthermore, unlike avacopan, it was observed that the inhibition of neutrophil chemotaxis by anti-C5aR1 antibody could not be overcome in the presence of excess C5a. Example 5. Inhibition of CD11b expression

[0307] This example describes the functional aspects of the C5aR1 monospecific antibody and biparatopic antibody described herein for inhibiting CD11b expression.

[0308] The integrin receptor CD11b is recruited to the neutrophil surface in response to C5a activation and mediates intravascular crawling prior to neutrophil migration. CD11 is involved in numerous adhesion-related associations between cells, including monocytes, macrophages, natural killer (NK) cells, and granulocytes.

[0309] Figures 9A-9B show the percentage of CD11b expression relative to antibody (C5aR1 antagonist) concentration in the presence of 100 nM C5a. The antibody was observed to potently inhibit CD11b across 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. Table 8. IC50 of CD11b expression inhibition using exemplary C5aR1 antibodies [Table 12]

[0310] In a different setting, the inhibition of the anti-C5aR1 antibodies disclosed herein in human neutrophils was compared using 10 nM and 100 nM anti-C5aR1 control antibodies as controls (Figure 9C). Furthermore, avacopan was observed to be partially potent in inhibiting CD11b. Moreover, this antibody was observed to be a superior inhibitor to the anti-C5aR1 control antibody. Example 6. Inhibition of β-arrestin recruitment

[0311] This example describes the functional aspects of the C5aR1 monospecific antibody and the biparatopic antibody described herein for inhibiting β-arrestin recruitment.

[0312] To characterize the lead mAb as a complete antagonist of C5aR1, the inventors aimed to evaluate the potential of the lead mAb to inhibit β-arrestin 2 recruitment. The β-arrestin assay utilized a luciferase reporter system with two unique subunits. One subunit fused to C5aR1, and the other fused to β-arrestin 2. In close proximity, the two subunits formed an enzyme that produced a luminescence signal, which was used as a substitute for C5aR1-mediated β-arrestin 2 recruitment. Exemplary antibodies significantly reduced C5aR1-mediated β-arrestin 2 recruitment at two different C5a concentrations, 1 nM and 10 nM. Figures 10A–10C are graphs showing the inhibition of the fluorescence signal after the addition of 1 nM (Figure 10A), 10 nM (Figure 10B), and 100 nM (Figure 10C) C5a. Table 9 shows the IC50 for β-arrestin inhibition mediated by c2139, c2137-e1711, and avacopan. Table 9. IC50 of monospecific antibodies or biparatopic antibodies for β-arrestin signaling [Table 13]

[0313] Compared to the small molecule C5aR1 inhibitor avacopan, exemplary antibodies, c2139 and c2137-e1711, showed superior signal inhibition even when avacopan was used at 10 times higher concentrations than the antibodies. In particular, when the C5a concentration increased to 10 nM, avacopan lost its potency, while c2139 and c2137-e1711 maintained >75% inhibition of β-arrestin II recruitment. The data suggested that exemplary C5aR1 antibodies, c2139 and c2137-e1711, inhibited β-arrestin at 10 times lower concentrations than avacopan. Example 7. Inhibition of ROS production by humanized anti-C5aR1 antibody.

[0314] This example demonstrates that exemplary humanized monospecific antibodies (c2139) and exemplary biparatopic antibodies (c2137-e1711) reduce reactive oxygen species (ROS).

[0315] ROS production by neutrophils was detected using the Cellular ROS Detection Assay Kit (Abcam) according to the manufacturer's manual. RBC-lysed WB cells were mixed in 100 mL of buffer at a rate of 3 × 10⁶ 5 The solution was diluted to the concentration of individual cells. Pretreatment with a ROS inhibitor (N-acetyl-L-cysteine) was performed on the negative control group at 37°C and 5% CO2 for 30 minutes. Subsequently, a ROS detection antibody was added to the antibody cocktail for flow cytometry-based detection of neutrophil ROS production. A ROS inducer (pyocyanin) was added to all groups, incubated for 30 minutes, and then captured on a cytometer. Figure 11 shows ROS production in neutrophils in the ANCA- and ANCA+ groups in response to treatment with anti-C5aR1 antibody.

[0316] c2139 and c2137-e1711 were observed to inhibit ROS production by ANCA. Example 8. Internal transfer of humanized anti-C5aR1 antibody in U937 cells.

[0317] This example demonstrates the internal transfer of humanized monospecific antibodies and biparatopic C5aR1 antibodies in hC5ar1-U937 cells. Humanized monospecific anti-C5aR1 antibody, e1711, and biparatopic antibody, c2139-e1711 were conjugated to pH-sensitive dyes that fluoresce brightly at low pH but not at neutral pH. The conjugated antibodies were incubated with U937 cells and hC5aR1 knock-in U937 cells. Figures 12A-12D show the fluorescence intensity after 24 hours of incubation with each conjugated antibody. Figures 12A-12B show the fluorescence intensity of U037 cells or U937-C5aR1 cells using 10 nM antibody, and Figures 12C-12D show the fluorescence intensity of U037 cells or U937-C5aR1 cells using 100 nM antibody. Example 9. Cross-reactivity of humanized anti-C5aR1 antibody

[0318] This example demonstrates the binding of the humanized anti-C5aR1 antibody, e1711, and the biparatopic antibody, c2139-e1711, to C5aR1 in lisza and dogs.

[0319] Cell surface binding and flow cytometry, 10 -7.5 ~10 -12 The procedure was performed using semi-logarithmic titration of anti-C5aR1 antibodies in the M range. As shown in Figures 13A-13B, the humanized anti-C5aR1 antibodies bound to squirrel monkey and canine C5aR1. Example 10. Inhibition of neutropenia in squirrel monkeys

[0320] This example demonstrates the inhibition of neutropenia in squirrel monkeys by a C5aR1 antibody. Neutropenia is caused by the rapid expression of cell surface CD11b, which leads to transient adhesion of neutrophils to vascular endothelium, thereby reducing the peripheral neutrophil count. Squirrel monkeys are a physiologically appropriate model for evaluating neutropenia compared to mice, as their neutrophil count is similar to that of humans (50-70%), while mice have only 10-20%. Furthermore, blood can be collected from squirrel monkeys at multiple possible time points, allowing for a longer study period. In addition, cross-reactivity with squirrel monkey C5aR1 was observed, as shown in Figure 13A.

[0321] Figure 14A shows the experimental design in squirrel monkeys to evaluate the reduction of neutropenia. Briefly, three groups of squirrel monkeys were evaluated for inhibition of neutropenia. In Group 1, eight squirrel monkeys were intravenously injected with PBS (vehicle). In Group 2, eight squirrel monkeys were intravenously injected with an exemplary antibody at a dose of 10 mg / kg. In Group 3, eight squirrel monkeys were intravenously injected with avacopan at a dose of 30 mg / kg. One hour after injection of PBS, exemplary antibody, or avacopan, the squirrel monkeys were injected with 0.1 mg / kg of human C5a. Blood samples were collected 1 minute, 5 minutes, 15 minutes, 2 hours, and 6 hours after human C5a injection. The percentage change in neutrophils and the mean change in neutrophils were calculated for all three groups and are shown as scatter plots in Figure 14B. The mean change in neutrophils is shown as a bar graph in Figure 14C.

[0322] Administration of human C5a was observed to result in a rapid, transient neutropenia caused by neutrophil adhesion to endothelial cells. The vehicle (PBS) group showed severe neutropenia, with a mean 89% decrease in neutrophil count from baseline 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment using an exemplary anti-C5aR1 antibody (23% vs. 89% in the vehicle). Furthermore, neutrophil counts began to disappear between 2 and 6 hours. Neutrification was observed with avacopan. Neutrification was not observed with c2139. The avacopan group also responded with a mean 59% decrease in neutrophils. The exemplary anti-C5aR1 antibody was observed to have a superior response rate compared to avacopan. Example 11. Inhibition of neutropenia in human C5aR1 transgenic mice.

[0323] This example demonstrates the inhibition of neutropenia in transgenic human C5aR1 mice by a C5aR1 antibody. The exemplary C5aR1 antibody does not cross-react with mouse C5aR1. Transgenic human C5aR1 (hC5aR1) knock-in mice were generated in Jackson Laboratory using CRISPR technology.

[0324] Figure 15A shows the experimental design in mice to evaluate the reduction of neutropenia. Briefly, five groups of hC5aR1 mice were evaluated for inhibition of neutropenia. In Group 1, five hC5aR1 mice were intravenously injected with PBS (vehicle). In Group 2, five hC5aR1 mice were intravenously injected with an exemplary monospecific anti-C5aR1 antibody, c2139, at a dose of 20 mg / kg. In Group 3, five hC5aR1 mice were intravenously injected with an exemplary biparatopic anti-C5aR1 antibody, c2137-e1711, at a dose of 20 mg / kg. In Group 4, five hC5aR1 mice were intravenously injected with a non-C5aR1 antibody (motavizumab) at a dose of 20 mg / kg. In Group 5, five hC5aR1 mice were intravenously injected with avacopan at a dose of 30 mg / kg. One hour after injection of PBS, exemplary antibody, motavizumab, or avacopan, the hC5aR1 mice were injected with 0.1 mg / kg of human C5a. Blood samples were collected 1 minute, 5 minutes, and 2 hours after human C5a injection. The probability percentage of neutrophils and the mean change in neutrophils were calculated for all three groups and are shown in Figures 15B-15C. The mean change in neutrophils is shown in Figure 15C. Neutrophil cell counts were evaluated by gating CD45+ / CD11b+ / Ly6G+ cells.

[0325] Administration of human C5a was observed to result in a rapid, transient neutropenia caused by neutrophil adhesion to endothelial cells. The vehicle (PBS) group showed severe neutropenia, with a mean 89% decrease in neutrophil count from baseline at 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment using exemplary anti-C5aR1 antibody (23% vs. 89% in the vehicle). The avacopan group also responded with a mean 59% decrease in neutrophils.

[0326] Exemplary anti-C5aR1 antibodies were observed to exhibit superior response rates compared to avacopan. Administration of human C5a was observed to result in rapid, transient neutropenia in human C5aR1 mice. The vehicle (PBS) group showed severe neutropenia, with a mean 76% decrease in neutrophil count from baseline at 1 minute after C5a injection. Significant inhibition of neutropenia was observed with pretreatment using an exemplary monospecific C5aR1 antibody (11% vs. 89% in the vehicle). Complete inhibition was observed against the exemplary biparatopic C5aR1 antibody. The avacopan group also responded with a mean 10% decrease in neutrophils. Example 12. Pharmacokinetic study of exemplary C5aR1 antibodies in hC5aR1 mice

[0327] This example demonstrates a pharmacokinetic (PK) study of an exemplary C5aR1 antibody in mice.

[0328] Exemplary monospecific and biparatopic antibodies were intravenously injected into Tg32 mice at a dose of 5 mg / kg. Serum antibody levels were evaluated over 500 hours. Figure 16A shows the pharmacokinetic properties of the exemplary antibodies. Figures 16B–16D show the 500-hour stability of anti-C5aR1 antibodies in the serum of five different mice. Monospecific (c2139–Figure 16C) and biparatopic (c2137–e1711–Figure 16D) antibodies were observed to be stable in serum and to persist similarly in Tg32 mice. TMDD was observed in hC5aR1 transgenic mice with both c2139 and c2137–e1711. Tetravalent antibodies showed slightly lower serum persistence compared to monospecific antibodies. Motavizumab was used as a control. Example 13. Prediction of pharmacokinetic studies of exemplary C5aR1 antibodies in humans.

[0329] Nonlinear penetrative cross-pollination (PK) has been observed for known anti-C5aR1 antibodies (Lee et al, 2006, PMID 16980984). A >90% receptor occupancy rate was observed over 4.3 weeks with an IV dose of 10 mg / kg, and a >90% receptor occupancy rate was observed over 1.8 weeks with a subcutaneous dose of 4 mg / kg. Based on affinity data for this humanized anti-C5aR1 antibody, model simulations showed nonlinear clearance of antibody ids from 0.1 to 10 mg / kg, which are predicted to result in a >90% receptor occupancy rate 24 days after IV administration.

[0330] Regardless of bioavailability, a subcutaneous dose of 4 mg / kg is predicted to maintain >90% receptor occupancy for approximately 7–13 days.

[0331] Tetravalent antibodies (biparatopic) were observed to have slightly lower persistence in serum compared to monospecific leads. Example 14: Stability of exemplary C5aR1 antibody at 4.4°C

[0332] This example demonstrates the stability of exemplary C5aR1 antibodies (both monospecific and biparatopic) at 4°C with one thawing cycle in between.

[0333] Both monospecific and biparatopic antibodies were incubated at 4°C for up to 14 days. After one thawing cycle at 37°C, the amount of intact antibody was assessed by gel filtration, undenatured PAGE, and dynamic light scattering (DLS). Table 10 shows the percentage of antibody aggregates (HMW) compared to the percentage of intact antibody. Table 10. Stability of C5aR1 bispecific antibody after one freeze-thaw cycle at room temperature / 37°C. [Table 14-1] [Table 14-2]

[0334] Both monospecific antibodies and biparatopic antibodies were observed to be stable at 4°C and RT for up to two weeks and one freeze / thaw cycle. Example 15 - Dynamic analysis of a humanized antibody that antagonists C5aR1 and has a modified Fc domain.

[0335] This embodiment describes the binding affinity and specificity of the site II monospecific and biparatopic antibodies described herein against C5aR1 and C5aR2 having modified Fc domains. The Fc domain modification was introduced to counteract effector functions such as ADCC or ADCP. Furthermore, the modified Fc domain includes mutations to prevent Fab arm exchange. Table 11 summarizes the Fc modifications in the C5aR1 antibody. Table 11 - Overview of monospecific and biparatopic antibodies C5aR1 with modified Fc domains. [Table 15]

[0336] The binding affinity of monospecific antibodies and biparatopic antibodies having modified Fc domains was calculated as described in Example 1. The monospecific antibody having a modified Fc domain is referred to below as "c2139-F c The term "mod" refers to a biparatopic antibody having a modified Fc domain, and hereafter it is referred to as "c2137-e1711-F c It's called "mod." (a) Binding affinity

[0337] Monospecific C5aR1 antibody (c2139-Fcmod) and C5aR1 biparatopic antibody (c2137-e1711-F c The affinity curve modulo (mod) is shown in Figure 17A.

[0338] Under the conditions described above, the monospecific C5aR1 antibody, c2139-Fcmod, binds to C5aR1 with an affinity of approximately 0.31 nM, and the biparatopic antibody of C5aR1, c2137-e1711-F cThe modulo molecule was observed to bind to C5aR1 with an affinity of approximately 0.34 nM.

[0339] An exemplary kinetic assay for binding to C5aR1-expressing cells is shown (Figure 17B). Kinetic measurements were fitted after the association phase and subsequent dissociation phase following 2-3 doses. The EC50 of the Fc-modified antibody was observed to be similar to that of the Fc-unmodified antibody. For example, as can be seen from Table 12, the biparatopic antibodies c2137-e1711 and c2137-e1711-F c The modal EC50 was approximately 1 nM in both U937-C5aR1 cells, approximately 1 nM and 2 nM in neutrophils, and 0.5 nM and 0.6 mM in macrophages. Tables 12-c2137-e1711 and c2137-e1711-F c Comparison of mod EC50 [Table 16] (b) Binding specificity

[0340] The specificity of the Fc-modified C5aR1 antagonist antibody was determined by measuring the affinity of the anti-C5aR1 antibody for C5aR2. This was determined as described in Example 1.

[0341] Figure 17C is c2137-e1711-F c Affinity curves for determining the specificity of mod and c2139-Fcmod are shown. It was observed that the Fc-modified antibody did not show affinity for C5aR2. Example 16 - Internal transfer of C5aR1 antibody.

[0342] This example aims to verify that the internal migration of C5aR1 antibodies is specific and not due to nonspecific clustering of membrane immunoglobulins. Sodium azide is a metabolic inhibitor that inhibits internal migration or endocytosis, which are energy-dependent processes. Any metabolic dependence of the internal migration of exemplary C5aR1 antibodies was probed using 10 nM C5aR1 antibodies in the presence or absence of 20 mM sodium azide. Exemplary C5aR1 antibodies were observed to undergo metabolically based internal migration. For example, c2139 underwent internal migration several hours after dissociation, and c2139-e1711 underwent internal migration during association. The dissociation constant of the monospecific C5aR1 antibody c2139 in the presence of 20 mM sodium azide was 1.43 × 10⁻¹⁴. -5 The dissociation constant is 3.4 × 10⁻⁶ / second, while the dissociation constant without sodium azide is 3.4 × 10⁻⁶ / second. -5 The value was / second. The association constant of the biparatopic C5aR1, c2137-e1711 in the presence of 20 mM sodium azide is 2.15 × 10⁻¹⁵. 4 The dissociation constant is / mol / second, while the dissociation constant without sodium azide is 4.38 × 10⁻⁶. 3 The value was / mol / second. Figures 18A-18B demonstrate the increased internal migration of the C5aR1 antibody. Figure 18A is an exemplary graph showing the internal migration of the monospecific antibody c2139 several hours after dissociation. Figure 18B is an exemplary graph showing the increased internal migration of the biparatopic antibody c2137-e1711 during association.

[0343] Nonspecific clustering of membrane immunoglobulins was observed not to be solely involved in the internal translocation of exemplary c5aR1 antibodies. Example 17: Inhibition of Gα signaling by Fc-modified C5aR1 antibody

[0344] This example illustrates the functional aspects of the Fc-modified C5aR1 monospecific antibody and biparatopic antibody described herein for the inhibition of Gα signaling using the GeneBLazer assay.

[0345] This experiment was carried out as detailed in Example 2. Figures 19A-19B show the C5aR1 Fc modified antibody-c2137-e1711-F c mod and c2139-F c The results of inhibiting Gα signaling in the presence of mod (mod) compared to abacopan and anti-C5aR1 control Ab are shown. c2137-e1711-F c Both mod and c2139-Fcmod were observed to potently inhibit Gα signaling in a dose-dependent manner.

[0346] Table 13 summarizes the inhibition of C5a at 10 nM and 100 nM concentrations compared to avacopan and anti-C5aR1 control Ab. c2137-e1711-F c Mod and c2139-Fcmod were observed to retain superior inhibition even at higher concentrations of C5a compared to abacopan and anti-C5aR1 control Ab. Table 13 - c2137-e1711-F in the presence of C5a compared to abacopan and anti-C5aR1 control Ab c mod and c2139-F c Modification inhibition [Table 17] * Extrapolated data - Previous data using Avacopan suggest that it is not very potent at high C5a concentrations. Example 18 - Inhibition of calcium signaling by Fc-modified C5aR1 antibody

[0347] This example illustrates the functional aspects of the C5aR1 Fc-modified monospecific antibody and biparatopic antibody described herein for the inhibition of C5a-mediated calcium signaling using a calcium flux assay.

[0348] This experiment was carried out as described in Example 3. Figures 20A-20B show the C5aR1 Fc modified antibody-c2137-e1711-F compared with avacopan and anti-C5aR1 control Ab in U937-C5aR1 anti-C5aR1 cells.c mod and c2139-F c This shows the results of inhibiting calcium signaling in the presence of mod. c2137-e1711-F c mod and c2139-F c Mod was observed to inhibit calcium flux more potently than abacopan and anti-C5aR1 control Ab. c2137-e1711-F c mod and c2139-F c It was observed that at least 10–100× was required to reach the inhibition level indicated by mod. The Fc-modified C5aR1 antibody was observed to inhibit calcium signaling in human neutrophils and U937-C5aR1 cells. Figure 20A is a dose-response curve showing an exemplary graph illustrating the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibody described herein in the presence of increasing concentrations of antibody and 100 nM C5a. Figure 20B is an inhibition percentage graph showing the inhibition of calcium signaling using the exemplary Fc-modified humanized site II antibody described herein in the presence of antibody and 100 nM C5a.

[0349] Figures 21A-21D show inhibition in U937-C5aR1 cells compared to human neutrophils. Figure 21A shows inhibition of calcium signaling in U937-C5aR1 cells in the presence of 10 nM C5a. Figure 21B shows inhibition of calcium signaling in U937-C5aR1 cells in the presence of 100 nM C5a. Figure 21C shows inhibition of calcium signaling in human neutrophils in the presence of 10 nM C5a. Figure 21D shows inhibition of calcium signaling in human neutrophils in the presence of 100 nM C5a.

[0350] Furthermore, after incubation with the indicated concentrations of antagonistic antibodies at various incubation times (e.g., 1 hour and 3 hours), Fc-modified antibodies were probed for inhibition of C5a-mediated calcium signaling in U937-C5aR1 cells. Figures 22A-22B summarize the saturation levels of U937-C5aR1 cells with each inhibitor. Figure 22A shows the increasing concentrations of antibodies (c2139-F) after 1 hour of incubation. c mod and c2137-e1711-F c Figure 22B summarizes the saturation percentage and F standard of U937-C5aR1 cells incubated with mod) and 100 nM C5a. Figure 22B shows the increasing concentrations of antibody (c2139-F) after 3 hours of incubation. c mod and c2137-e1711-F c This summarizes the saturation percentage and F standard of U937-C5aR1 cells incubated with modulo and 100 nM C5a.

[0351] Figure 22A is c2137-e1711-F c mod and c2139-F c Figure 22B shows the inhibitory dose-response of calcium signaling by mod. c2137-e1711-F c mod and c2139-F c This shows the percentage of inhibition of calcium signaling by modalities.

[0352] c2137-e1711-F c The mod was observed to be more potent than abacopan and the anti-C5aR1 control Ab. Furthermore, c2139-F c The modulus reached a saturation point with a short antagonist incubation time. This saturation is somewhat alleviated by a longer antagonist incubation time. This phenomenon is related to c2139-F. c Observed only in mod, and observed in the precursor c2139. Example 19 - Inhibition of β-arrestin signaling by Fc-modified C5aR1 antibody This example describes functional aspects of Fc-modified C5aR1 monospecific antibodies and the biparatopic antibodies described herein for inhibiting β-arrestin recruitment.

[0353] Details of the experiment for determining this β-arrestin recruitment are described in Example 6. Figures 23A to 23B show c2137-e1711-F c mod and c2139-F c mod summarizes the inhibition of C5a-mediated β-arrestin signaling. c2137-e1711-F c mod and c2139-F c mod was observed to more potently block β-arrestin recruitment to C5aR1 than avacopan and the anti-C5aR1 control Ab. Table 14 summarizes K D for inhibition of β-arrestin recruitment. Table 14-K D

Table 18

[0354] This example describes functional aspects of the Fc-modified C5aR1 monospecific antibodies and biparatopic antibodies described herein for inhibiting neutrophil chemotaxis known to be induced by C5aR1 activity using a Boyden chamber.

[0355] Details of the experiment for determining this neutrophil chemotaxis are described in Example 4. Figures 24A to 24D show C5aR1 antibody, c2137-e1711-F c mod, and c2139-F c mod shows inhibition of chemotaxis in C5aR1-U937 stable cells after treatment with mod. c2137-e1711-Fcmod and c2139-F c mod was observed to inhibit chemotaxis more potently than avacopan and the anti-C5aR1 control Ab. Figure 24A shows 1 nM, 3.16 nM, and 10 nM c2139-Fc shows inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of mod and increasing concentrations of C5a. Figure 24B shows 1 nM, 3.16 nM, and 10 nM C5a in the presence of c2137-e1711-F c mod and increasing concentrations of C5a, inhibition of chemotaxis in C5aR1-U937 stable cells. Figures 24C-24D show that c2137-e1711-F c in the presence of mod, shows inhibition of chemotaxis in C5aR1-U937 stable cells in the presence of 1 nM, 3.16 nM and 10 nM anti-C5aR1 control Ab and avacopan, respectively. Example 21 - Inhibition of CD11b expression by Fc-modified C5aR1 antibodies

[0356] This example describes functional aspects of the Fc-modified C5aR1 monospecific antibodies and biparatopic antibodies described herein with regard to inhibition of CD11b expression.

[0357] Details of this experiment determining CD11 expression are described in Example 5. Figures 25A-25B show that c2137-e1711-F c mod and c2139-F c mod inhibits CD11b signaling in response to treatment. c2137-e1711-F c mod and c2139-F c mod was observed to potently inhibit CD11b expression over a wide range of C5a concentrations compared to avacopan and anti-C5aR1 control Ab. Figure 25A shows inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibody and 10 nM C5a. Figure 25B shows inhibition of CD11b signaling in the presence of increasing concentrations of C5aR1 antagonist antibody and 100 nM C5a. Example 22 - Inhibition of ROS production by Fc-modified C5aR1 antibodies

[0358] This example illustrates that the exemplary humanized Fc-modified monospecific antibody (c2139-F c mod) and the exemplary biparatopic antibody (c2137-e1711-F cThis shows that modulo (modulation) reduces reactive oxygen species (ROS).

[0359] Details of the experiment determining this ROS production are described in Example 7. Figures 26A-26B show c2139-F compared to c2139, c2137-e1711, anti-C5aR1 control Ab, and abacopan. c mod, c2137-e1711-F c This shows inhibition of ROS production in RBC-lysed WB cells treated with mod. c2139-F c mod, and c2137-e1711-F c Mod was observed to maintain low respiratory burst activity in neutrophils, similar to abacopan and anti-C5aR1 control Ab. Furthermore, c2139-F c mod, and c2137-e1711-F c The mod exhibited reduced respiratory burst activity compared to the previous forms c2139 and c2137-e1711. Figure 26A shows the inhibition of ROS production by increasing concentrations of monospecific C5aR1 antibody (Fc modified). Figure 26B shows the inhibition of ROS production by increasing concentrations of biparatopic C5aR1 antibody (Fc modified). Example 23. Inhibition of neutropenia in human C5aR1 transgenic mice by Fc-modified C5aR1 antibody.

[0360] This example demonstrates the inhibition of neutropenia in transgenic human C5aR1 mice by an Fc-modified C5aR1 antibody. As described above (Example 11), the exemplary C5aR1 antibody does not cross-react with mouse C5aR1. Transgenic human C5aR1 (hC5aR1) knock-in mice were generated in Jackson Laboratory using CRISPR technology.

[0361] Figure 27A shows the experimental design in mice to evaluate the reduction of neutropenia. Details of this mouse in vivo assay are described in Example 11. The probability percentage and mean change in neutrophils were calculated for all groups and are shown in Figures 27B-27C. The mean change in neutrophils is shown in Figure 27B. The change in neutrophil count is shown in Figure 27C. The new Fc-silenced leads were observed to be as potent as the unsilenced c2139 and c2137-e1711. The vehicle (PBS) group showed severe neutropenia, with a mean 65% reduction in neutrophil count from baseline at 1 minute after C5a injection. In the vehicle control group, one animal did not respond to C5a. Example 24. Stroke protection in a mouse model

[0362] This experiment showed that c2139-F was found in a mouse model of stroke. c mod, and c2137-e1711-F c Mod demonstrated the ability to protect infarct volume. Neutrophil activity and cerebral infiltration have been well documented in the pathology of stroke and traumatic brain injury, including acute models.

[0363] Each of 20 mg / kg contains c2139-F c mod, and c2137-e1711-F c The mod was administered to tMCAO mice. 1 mg / kg of PMX53 was used as a control. The brain was resected, sectioned, and stained with TTC. The stained sections were analyzed by imaging.

[0364] c2139-F c mod, and c2137-e1711-F c Mice treated with mod were observed to offer protection against stroke by significantly reducing infarct volume compared to the vehicle group. c2137-e1711-F c Mod treatment showed a significant reduction in infarct volume compared to the vehicle group. c2139-F cThe mod treatment showed a reduction in infarct volume compared to the vehicle group. The PMX53 treatment (serving as a positive control and comparator) showed only a slight reduction in infarct volume compared to the vehicle group. Figure 28A shows a graphical description of the reduction in infarct volume. Figure 28B shows c2139-F c mod, c2137-e1711-F c The graph shows the infarct volume after treatment using mod and PMX53. Example 25. Pharmacokinetic study of exemplary C5aR1 antibody in hC5aR1 mice with a modified Fc domain.

[0365] This example demonstrates a pharmacokinetic (PK) study of exemplary C5aR1 using a modified Fc-domain antibody in mice. Tg32 mice are a transgenic model in which human FcRn replaces native mouse FcRn. FcRn is required for bidirectional transport of abs across the cell barrier and influences PK. Tg32 mice with human FcRn have been extensively studied and are thought to correlate with PK in humans. The goal of this experiment was to perform FcRn-mediated recycling in the IgG-scFv vs. IgG format and evaluate the effect of introducing Fc-domain modification on FcRn-mediated recycling.

[0366] Exemplary monospecific antibodies and biparatopic antibodies were intravenously injected into Tg32 mice at a dose of 5 mg / kg. The percentage of antibodies in serum was determined using assays recognized in the art. MVZ-IgG4-VFc17, a motabizumab antibody with the same Fc modification as the IgG4 Fc and C5aR1 antibodies, was used as a control. Further PK / PD analysis was performed using CERTARA.

[0367] C-max (the highest concentration of antibody in the blood was similar for both monospecific and biparatopic antibodies) was observed. Furthermore, the antibody half-life was c2139-F c mod, and c2137-e1711-F c Similarities were observed in the mod.

[0368] The amount of antibodies in the serum was evaluated over 500 hours. Table 15 shows the dosage and sample collection intervals for the pharmacokinetic study. Table 15 - Exemplary doses and sample collection schedules for C5aR1 Fc modified antibodies to determine the pharmacokinetics of individual antibodies. [Table 19]

[0369] Figures 29A and 29B show the pharmacokinetic characteristics of the C5aR1-Fc modified antibody. Figure 29A is a graph showing the percentage of Fc-modified C5aR1 antibody in serum over 500 hours. Figure 29B is a graph showing the average concentration of antibody in μg per 1 ml of serum over 500 hours. These observations are from c2137-e1711-F c mod efficiently engages with hFcRn and c2139-F c This demonstrated that the antibodies were recycled in a similar manner to mods. Furthermore, the PK data was similar to that of C5aR1 antibodies with unmodified Fc domains. Table 16 shows the PK parameters of exemplary Fc-modified C5aR1 antibodies. Table 16 - Pharmacokinetic parameters of the Fc-modified C5aR1 antibody. [Table 20] Example 26 - Pharmacokinetics of Fc-modified C5aR1 antibody administered multiple times in human C5aR1 knock-in mice

[0370] This example demonstrates a repeated-dose pharmacokinetic (PK) study of an exemplary C5aR1 antibody using modified Fc domain antibody hC5aR1 mice.

[0371] Table 17 summarizes the drug regimens for the antibodies tested. Target-mediated pharmacokinetics (TMDD) are evident for both exemplary C5aR1 antibodies with modified Fc domains. TMDD is the phenomenon in which a drug binds to its pharmacological target site (such as a receptor) with high affinity, influencing its pharmacokinetic (PK) properties. Target binding and subsequent removal of the drug-target complex can affect both the distribution and removal of the drug, potentially leading to dose-dependent nonlinearity in PK.

[0372] TMDD is most commonly observed as a linear PK at high dose levels and as a non-linear PK at low dose levels.

[0373] As expected for antibodies with TMDD, dose-dependent clearance was observed. At 5 mg / kg and 0.5 mg / kg, the maximum concentrations of each antibody were similar for both antibodies. max Furthermore, the half-lives (T) of each antibody were determined at 5 mg / kg and 0.5 mg / kg. 0.5 ) were found to be similar. At 20 mg / kg, c2139 had a better half-life (T) than c2137-e1711. 0.5 ) had. Table 17 - Exemplary doses and sample collection schedules for C5aR1 Fc modified antibodies to determine the pharmacokinetics of individual antibodies. [Table 21]

[0374] Target binding and subsequent removal of the drug-target complex can affect both drug distribution and removal, potentially leading to nonlinearity of PK in a dose-dependent manner. Figures 30A–30F demonstrate that antibody persistence is dose-dependent. Table 18 shows the PK parameters of exemplary Fc-modified C5aR1 antibodies. Figure 30A is a graphical representation of the dose-response curve of c2139-Fcmod in serum over 200 hours. Figure 30B is a graphical representation of the dose-response curve of c2137-e1711-Fcmod in serum over 200 hours. Figure 30C is a comparison of c2139Fcmod, c2137-e1711-Fcmod, and MVZ-IgG4. Figure 30D is a graphical representation of c2139 in silico at three different concentrations over 500 hours. Figure 30E is a graphical representation of c2137-e1711 in silico at three different concentrations over 500 hours. Figure 30F shows an in silico graph of isotype control antibodies at a concentration of 20 mg / kg over 500 hours. Non-naive mice were administered mAbs multiple times, once weekly, for a total of 4 weeks. Both c2139Fcmod and c2137-e1711-Fcmod were observed to maintain exposure with weekly IV administration of 5 mg / kg. No severe outcomes were observed with weekly mAb administration for several weeks.

[0375] Table 18 - PK parameters of exemplary Fc-modified C5aR1 antibodies. [Table 22]

[0376] Example 27 - Inhibition of neutropenia in human C5aR1 KI mice by low-dose Fc-modified C5aR1 antibody. This example demonstrates that c2139-Fcmod and c2137-e1711-Fcmod can inhibit neutropenia in human C5aR1 knock-in mice at low doses.

[0377] Table 19 summarizes the drug regimens for the antibodies tested and the bleeding time to determine neutropenia. Figure 31 shows the neutrophil changes from baseline (-5 minute bleeding) 1 minute after C5a administration.

[0378] c2139-Fcmod and c2137-e1711-Fcmod were observed to potently inhibit C5a-induced neutropenia compared to vehicle controls, even at a dose of 0.1 mg / kg. Table 19. Dosage regimens [Table 23-1] [Table 23-2] Example 28 - Internal transfer of Fc-modified C5aR1 antibody

[0379] This example demonstrates the internal transfer of Fc-modified humanized monospecific antibodies and biparatopic C5aR1 antibodies in hC5ar1-U937 cells.

[0380] 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 is nonfluorescent at neutral pH. The conjugated antibodies were incubated with U937 cells and hC5aR1 knock-in U937 cells. Figure 32A shows the fluorescence intensity after 6 hours and 24 hours of incubation with each conjugated antibody.

[0381] Exemplary Fc-modified C5aR1 antibodies were observed to undergo metabolic-based internal translocation. Figure 32B shows the internal translocation of both c2137-e1711-Fcmod and c2139-Fcmod in live cells observed by Nikon confocal experiment over 300 minutes. Equivalents and range

[0382] Those skilled in the art will recognize many equivalents to the specific embodiments of the present disclosure described herein, or can confirm this by routine experimentation alone. The scope of the present disclosure is not intended to be limited to the above description, but rather to be as described in the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to complement component 5a receptor 1 (C5aR1), wherein the antibody or antigen-binding fragment comprises a VH region and a VL region, the VH region comprising three heavy chain complementarity-determining regions (HCDRs), the HCDR1, HCDR2, and HCDR3 sequences each comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, and the VL region comprising three light chain complementarity-determining regions (LCDRs), the LCDR1, LCDR2, and LCDR3 sequences each comprising the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively.

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14, and the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

25.

3. The antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment further comprises an Fc region.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the Fc region is independently selected from IgG1, IgG2, IgG3, and IgG4.

5. The antibody that binds to C5aR1, i. Inhibit the interaction between complement component 5a (C5a) and C5aR1, ii. Do not bond to C5aR2. iii. Inhibits neutrophil chemotaxis, iv. Inhibit C5a-mediated C5aR1 Gα signaling. v. Inhibits calcium signaling, vi. Inhibit CD11b expression, vii. Inhibit neutropenia, viiii. Inhibits β-arrestin signaling, and / or ix. Inhibit ROS production in neutrophils. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is humanized.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody binds to C5aR1 with a dissociation constant of 10 pM to 50 nM.

8. The antibody that binds to C5aR1, i. Inhibit the interaction between complement component 5a (C5a) and human C5aR1. ii. Do not bond to C5aR2. iii. Inhibits neutrophil chemotaxis, iv. Inhibit C5a-mediated C5aR1 Gα signaling. v. Inhibits calcium signaling, vi. Inhibit CD11b expression, vii. Inhibit neutropenia, viiii. Inhibits β-arrestin signaling, and / or ix. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which inhibits ROS production in neutrophils.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is humanized.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is stable at 4°C for up to two weeks in a single freeze-thaw cycle.

11. A nucleic acid encoding an antibody according to any one of claims 1 to 10.

12. A cell comprising the nucleic acid described in claim 11.

13. A method for producing an antibody or an antigen-binding fragment according to any one of claims 1 to 10, comprising: culturing a host cell containing a nucleic acid encoding the antibody or an antigen-binding fragment thereof; and culturing the cell under conditions that enable the production of the antibody or an antigen-binding fragment thereof.

14. A composition comprising an antibody according to any one of claims 1 to 10, for use in a method for treating an autoimmune disease requiring treatment.

15. The composition according to claim 14, wherein the autoimmune disease is ANCA vasculitis, lupus, rheumatoid arthritis, kidney disorder, or stroke.

16. The antibody according to any one of claims 3 to 10, wherein the antibody comprises a modified IgG4 Fc region.

17. The modified IgG4 Fc region is replaced (i) One or more mutations selected from the group consisting of S228P, L234V, L235A, G237A, D265G, A330S, P331S, L328R, H268A and N297Q mutations; or (ii) F234V, L235E, and D265G mutations; or (iii) F234V, L235E, D265G, and S228P mutations The antibody according to claim 16, comprising:

18. The antibody according to claim 17, wherein the antibody comprises a modified IgG4 Fc region including a combination of F234V, L235E, and D265G substitutions.

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