Introduction: The complement system anaphylatoxin C5a is a critical player in inflammation. By binding to complement C5a receptor 1 (C5aR1/CD88), C5a regulates many cellular functions, mainly as a potent pro-inflammatory inducer. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). Methods: Initial C5aR1 hybridoma clone selection was performed with a cell-binding study in human whole blood. In-house C5aR1 mAb assessment for C5aR1 inhibition was done via the iLite® C5a assay. C5aR1 mAb specificity was investigated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. Physiological C5aR1 inhibition was assessed via a C5a-driven calcium flux assay and stimulation assay based on isolated polymorphonuclear leukocytes (PMNs) and a whole blood model stimulated with Escherichia coli. Results: The supernatant of hybridoma clones targeting the N-terminal section of C5aR1 displayed efficient binding to C5aR1 in whole blood, which was confirmed for purified mAbs. The C5aR1 mAb 18-41-6 was selected following the assay of in-house C5aR1 mAbs via the iLite® C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab’)2 preparations of mAb 18-41-6 were found to efficiently abrogate C5a-induced calcium flux in neutrophils and to significantly reduce the upregulation of the activation markers CD11b (neutrophils, monocytes) and CD66b (neutrophils). Conclusion: Our results demonstrate that mAb 18-41-6 is a valuable tool for investigating the C5a-C5aR1 axis and a potential therapeutic candidate for inflammatory disease treatment.

The complement system is part of the innate immune system, consisting of many soluble and membrane-bound proteins [1]. Components of the complement system are conserved in mammals [2], and certain complement factors are also present in invertebrate species [3]. It is considered an initial defense barrier against invading microorganisms [4]. However, emerging evidence indicates that the complement system’s functions are far more versatile and complex, ranging from canonical roles in immune defense and inflammation to important functions in neuronal development and tissue regeneration [5, 6]. The recently described intracellular complement system, the complosome, seems to play instrumental roles in immune cell regulation and metabolism [7].

The complement system is a crucial player in initiating and amplifying inflammatory processes due to the cleavage of C3 and C5 into the respective anaphylatoxins, namely, C3a and C5a [8]. C5a is a potent anaphylatoxin with strong pro-inflammatory properties mediated through its binding to complement C5a receptor 1 (C5aR1/CD88). To a less-understood degree, C5a interacts with complement C5a receptor 2 (C5aR2 or C5L2), influencing C5aR1 signaling [9]. C5aR1 is classified as a G protein-coupled receptor (GPCR) consisting of 350 amino acids organized into seven transmembrane domains, with an extracellular N-terminus and cytosolic C-terminus [10]. It is expressed by a plethora of immune and nonimmune cells in different tissues and organs, particularly in polymorphonuclear neutrophils, monocytes, and macrophages [11]. Upon binding of C5a to C5aR1, GPCR-mediated signaling pathways are activated, regulating immune cell activation, migration, and production and release of antimicrobial compounds and cytokines [12, 13]. Neutrophils play a central role in the initial cellular response to an infection [14], which makes the C5a-C5aR1 axis a critical component of the swift initial cellular response against invading bacterial and fungal pathogens [15, 16].

Due to the strong inflammatory potential of C5, both C5a and C5aR1 are attractive pharmaceutical candidates to dampen C5a-C5aR1 axis-driven inflammatory diseases [17]. Inhibition of C5 has been approved for the treatment of diseases like atypical hemolytic uremic syndrome [18, 19], myasthenia gravis [19, 20], and paroxysmal nocturnal hemoglobinuria (PNH) [19, 21]. Studies targeting C5aR1 in ANCA-associated vasculitis have shown promising results [22] and led to a recent FDA approval of avacopan, a small-molecule C5aR1 inhibitor [23]. Of great interest are initial findings regarding a potential treatment strategy focusing on the C5a-C5aR1 axis by targeting C5a in COVID-19 [24] and also in hyperinflammatory conditions like sepsis [25]. The present study aimed to produce novel and improved monoclonal antibodies (mAbs) blocking C5aR1, intended for research and clinical application.

Generation of Hybridoma Clones Producing C5aR1 mAbs

Immunization of BALB/c × NMRI mice was carried out essentially as previously described [26]. Mice were subcutaneously immunized three times with 25 μg of respective C5aR1-specific peptide: MNSFNYTTPDYGHYDDFDTLDLNTPVDKTSNTLRVP (C5aR1 1-36, N-terminal region), PILFTSIVQHHHWPFGGAA (C5aR1 90-109, first extracellular loop), RVVREEYFPPKVL (C5aR1 175-188, second extracellular loop), SFLEPSSPTFLLLNKLD (C5aR1 266-282, third extracellular loop). Individual peptides were coupled to diphtheria toxoid, which was adsorbed to Al(OH)3. Before immunization, peptide-toxoid complexes were combined 1:1 with Freund’s incomplete adjuvant. Antibody fusion was conducted as previously documented [27]. Antibody clones were preselected for recognition of the respective immunization peptide. Selected mAbs by screening were first purified from bovine serum-supplemented culture supernatants (for binding studies on whole blood), and afterward, serum-free culture supernatants by protein G affinity chromatography using the Äkta start liquid protein chromatography system (Cytiva, Uppsala, Sweden). The isotype of selected mAbs was determined utilizing the IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit (Roche, Basel, Switzerland, cat. no.: 11493027001), according to the supplier’s instructions. F(ab’)2 fragments of C5aR1 mAb 18-41-6 were prepared via a kit (Thermo Fisher Scientific, Waltham, MA, USA, cat. no.: 44980).

Screening and Selection of C5aR1 Hybridoma Clones on Whole Blood

Healthy donor blood was drawn into K2EDTA-coated blood vials (Greiner Bio-One, Kremsmünster, Austria, cat. no.: 454246). 40 µL of blood was transferred to 5 mL FACS tubes (Corning, Corning, NY, USA, cat. no.: 352052). Red blood cell (RBC) lysis was performed using 1 mL of RBC lysing reagent (Thermo Fisher Scientific, cat. no.: HYL250) and following incubation for at least 10 min at room temperature (RT) in the dark. The supernatant was removed by centrifugation (500 g, 5 min, at 4°C), and all samples were washed once with 1 mL of DPBS (Merck, Darmstadt, Germany, cat. no.: D8537), with 0.5% BSA (Merck, cat. no.: A8327) added (DPBS/BSA). To each respective sample, 50 μL of either DPBS/BSA, 5 μg/mL IgG1k isotype mAb (Becton Dickinson, Franklin Lakes, NJ, USA, cat. no.: 557273), 5 μg/mL IgG2ak isotype mAb (BioLegend, San Diego, CA, USA, cat. no.: 401501), 5 μg/mL C5aR1 mAb S5/1 (BioLegend, cat. no.: 344302), or undiluted C5aR1 hybridoma supernatant was added. Samples were incubated for 15 min at 4°C (dark). Subsequently, all samples were washed once with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). Afterward, 50 μL of either DPBS/BSA or 5 μg/mL of goat anti-mouse IgG, human ads-PE antibody (SouthernBiotech, Birmingham, AL, USA, cat. no.: 1030-09S) was added to the respective samples for detection. Samples were incubated for 15 min at 4°C (dark). Afterward, samples were washed once with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). Following, samples were fixed with 300 μL of 4% formaldehyde (VWR, Radnor, PA, USA, cat. no.: 9713.1000) for 20 min at RT (dark). Samples were afterward washed twice with DPBS/BSA (500 g, 5 min, at 4°C). Pelleted cells were resuspended in DPBS/BSA and analyzed with a BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG) by recording 100,000 total events per sample. The assay was repeated three times with the blood of a different anonymous healthy donor for each repeat; data points were acquired in singlets.

Assessment of C5aR1 Recognition by Purified C5aR1 mAbs on Whole Blood

Healthy donor blood was drawn into K2EDTA-coated blood vials (Greiner Bio-One, cat. no.: 454246). 40 µL of blood was added to 5 mL FACS tubes (Corning, cat. no.: 352052). RBC lysis was conducted as described before. Likewise, samples were washed once with 1 mL of DPBS/BSA after RBC lysis (500 g, 5 min, at 4°C). To the corresponding samples, 50 μL of either DPBS/BSA, 5 μg/mL IgG1k isotype mAb (Becton Dickinson, cat. no.: 557273), 5 μg/mL IgG2ak isotype mAb (BioLegend, cat. no.: 401501), 5 μg/mL C5aR1 mAb S5/1 (BioLegend, cat. no.: 344302), or 5 μg/mL of respective serum-purified, in-house C5aR1 mAb was added. Samples were incubated for 15 min at 4°C (dark). Afterward, samples were washed once with 1 mL DPBS/BSA (500 g, 5 min, at 4°C). Following, 50 μL of either DPBS/BSA or 5 μg/mL of goat anti-mouse IgG, human ads-PE antibody (SouthernBiotech, cat. no.: 1030-09S) was added. Samples were incubated for 15 min at 4°C in the dark as done before. Samples were washed three consecutive times with 1 mL of DBPS/BSA (500 g, 5 min, at 4°C) before adding 50 μL of stain master mix per sample, containing 0.5 μL CD14 BB700 (Becton Dickinson, cat. no.: 566465), 0.5 μL CD15 BV605 (BioLegend, cat. no.: 323032), 0.25 μL CD16 BV711 (BioLegend, cat. no.: 302044), and 1 μL CD45 SBV790 (Bio-Rad Laboratories, Hercules, CA, USA, cat. no.: MCA87SBV790). Samples were incubated for 15 min at 4°C (dark). Samples were washed once with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C) before fixation with 300 μL 4% formaldehyde (VWR, cat. no.: 9713.1000) for 20 min at RT (dark). Following, samples were washed twice with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). Samples were resuspended in DPBS/BSA and analyzed via a BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG). Per sample, 100,000 total events were recorded. Compensation was conducted with beads (Thermo Fisher Scientific, cat. no.: 01-3333-42), using the aforementioned antibodies for staining. For compensation, the goat anti-mouse IgG, human ads-PE antibody (SouthernBiotech, cat. no.: 1030-09S) was replaced with CD11b PE (BioLegend, cat. no.: 301306). The assays were repeated three times with the blood of a different anonymous healthy donor per repeat; data points were acquired in singlets.

Assay of C5aR1 mAbs and Inhibitors on iLite® C5a Assay Ready Cells

The assay was conducted according to the supplier’s application note (Svar Life Science, Malmö, Sweden). C5aR1 mAbs (serum-free purified, in-house C5aR1 mAbs, mAb S5/1 [BioLegend, cat. no.: 344302], mAb 347214 [R&D systems, Minneapolis, MN, USA, cat. no.: MAB3648], avdoralimab [Nordic BioSite, Täby, Sweden, cat. no.: 155-10-969]), C5aR1 inhibitors (W-54011 [R&D systems, cat. no.: 5455], PMX-53 [kindly supplied by Trent Woodruff, School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, QLD, Australia], avacopan [MedChemExpress, Monmouth Junction, NJ, USA, cat. no.: HY-17627]), and controls (IgG1k isotype mAb [Becton Dickinson, cat. no.: 557273], IgG2ak isotype mAb [BioLegend, cat. no.: 401501]) were diluted in DMEM, containing 9% heat-inactivated (HI) FBS and 1% Pen/Strep (Svar Life Science, cat. no.: 5811) and added to a 96-well assay plate (Tecan, Männedorf, Switzerland, cat. no.: 30122300) in a volume of 20 μL. One vial per plate of iLite® C5a Assay Ready Cells (Svar Life Science, cat. no.: BM4075) was thawed inside a 37°C water bath for 1 min. Of the vial, 250 μL of the cell suspension was transferred to 37°C prewarmed 5.75 mL of DMEM (9% HI-FBS, 1% Pen/Strep) (Svar Life Science, cat. no.: 5811) per assay plate. 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 pm) in sample buffer. Following, the plate was incubated at 37°C and 5% CO2 for 5 h. Afterward, the plate was rested at RT for 10 min. 80 µL of firefly luciferase (FL) substrate (Svar Life Science, cat. no.: Dual Glo) was added to each well of the plate, followed by agitation at RT for 10 min (dark). The FL luminescence signal of the assay plate was acquired by a GloMax Explorer luminometer (Promega, Madison, WI). 80 µL of Renilla luciferase (RL) substrate (Promega, cat. no.: E313B), diluted 1:100 in FL stopping buffer (Promega, cat. no.: E314B), was added to each well of the plate. The assay plate was agitated for 10 min at RT (dark). The assay plate was read again to acquire the RL luminescence signal. Data were collected in duplicates across multiple assay plates; each assay plate was repeated three times.

Establishment of Human C5aR1his- and C5aR2his-Expressing Flp-In™-CHO Cells

The sequence of C5aR1 (NCBI Reference Sequence: NP_001727.2) and C5aR2 (NCBI Reference Sequence: NP_001258678.1), with an added polyhistidine-tag (his) at the C-terminus, was synthesized by GeneArt (Thermo Fisher Scientific) and cloned into the pcDNA™5/FRT expression vector (Thermo Fisher Scientific, cat. no.: V601020) by Gibson assembly. Flp-In™-CHO cells (Thermo Fisher Scientific, cat. no.: R75807) were treated with a 100 μL transfection master mix, containing either 0.05 μg pcDNA™5/FRT-C5aR1his plasmid or 0.05 μg pcDNA™5/FRT-C5aR2his plasmid, 0.45 μg of pOG44 plasmid (Thermo Fisher Scientific, cat. no.: V600520), and 50 μL of 1:25 diluted Lipofectamine™ 2000 (Thermo Fisher Scientific, cat. no.: 11668019) per well according to the manufacturer’s instructions, using RPMI-1640 medium (Merck, cat. no.: R5886) as diluent. Transfected cells were essentially cultured according to the supplier’s recommendations. Selection of transfected cells was performed with 1 mg/mL of hygromycin B (Thermo Fisher Scientific, cat. no.: 10687010).

Examination of C5aR1-Specficity of mAb 18-41-6 on C5aR1his- and C5aR2his-Transfected Flp-In™-CHO Cells

Non-transfected Flp-In™-CHO cells, C5aR1his-transfected Flp-In™-CHO cells, and C5aR2his-transfected Flp-In™-CHO cells were cultured according to the suppliers’ recommendation in Ham’s F-12 Nutrient Mix medium (Thermo Fisher Scientific, cat. no.: 11765054), with in-house added 10% FBS (Merck, cat. no.: F7524), 0.5% L glutamine (Thermo Fisher Scientific, cat. no.: 25030-024), and 1% of Pen/Strep (Thermo Fisher Scientific, cat. no.: 15140-122) (complete Ham’s F-12 Nutrient Mix medium). In addition, respective selection antibiotics Zeocin™ (Thermo Fisher Scientific, cat. no.: R25005) and Hygromycin B (Thermo Fisher Scientific, cat. no.: 10687010) were supplemented at the concentration of 100 μg/mL (Zeocin™, for culturing non-transfected Flp-In™-CHO cells) and 500 μg/mL (hygromycin B, for culturing C5aR1his-, and C5aR2his-transfected Flp-In™-CHO cells). On the day of the experiment, the medium of the cells was removed, cells were washed with PBS (Thermo Fisher Scientific, cat. no.: 10010-023), and subsequently detached with TrypLE™ Express Enzyme (Thermo Fisher Scientific, cat. no.: 12604-013). The process was stopped by the addition of complete Ham’s F-12 Nutrient Mix medium, followed by the removal of supernatant by centrifugation (500 g, 5 min, at RT). Cells were washed once with DPBS (Merck, cat. no.: D8537) (500 g, 5 min, at RT). The supernatant was removed, and pelleted cells were resuspended in DPBS (Merck, cat. no.: D8537). Per sample, 2.5 × 105 cells in 50 μL were administered to a 5 mL FACS tube (Corning, cat. no.: 352052). Following, 50 μL of viability staining master mix was added to each sample, containing 1 μL of eBioscience™ Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, cat. no.: 65-0866-14) in DPBS (Merck, cat. no.: D8537). Samples were incubated for 30 min at 4°C (dark). Following, samples were washed twice with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). To each respective sample, 50 μL of either DPBS/BSA, 10 μg/mL IgG1k isotype mAb (Becton Dickinson, cat. no.: 557273), 10 μg/mL IgG2ak isotype mAb (BioLegend, cat. no.: 401501), 10 μg/mL C5aR1 mAb S5/1 (BioLegend, cat. no.: 344302), 10 μg/mL mAb 18-41-6, or 10 μg/mL C5aR2 mAb 1D9-M12 (BioLegend, cat. no.: 342402) was added. Samples were incubated for 10 min at 4°C (dark). Samples were washed once with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). Thereafter, 50 μL of either DPBS/BSA or 10 μg/mL goat anti-mouse IgG, human ads-PE antibody (SouthernBiotech, cat. no.: 1030-09S) was added to the respective samples. Afterward, samples were incubated for 10 min at 4°C (dark), followed by washing with 1 mL DPBS/BSA (500 g, 5 min, at 4°C). Cells were resuspended in DPBS/BSA prior to FACS analysis with a BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG). Samples were analyzed by recording at least 20,000 total events per sample. Beads (Thermo Fisher Scientific, cat. no.: 01-3333-42) were used to create compensation controls, using CD11b PE (BioLegend, cat. no.: 301306), instead of goat anti-mouse IgG, human ads-PE (SouthernBiotech, cat. no.: 1030-09S). Amine-reactive beads (Thermo Fisher Scientific, cat. no.: A10346) were used to create compensation controls stained with eBioscience™ Fixable Viability Dye eFluor™ 506 (Thermo Fisher Scientific, cat. no.: 65-0866-14). The assay was repeated three times; data points were acquired in singlets.

Functional Assessment of mAb 18-41-6 in a C5a-Driven Polymorphonuclear leukocyte Calcium Flux Assay

Blood of healthy donors was drawn into K2EDTA-coated blood vials (Greiner Bio-One, cat. no.: 455045) and equilibrated to RT. An equal volume of equilibrated blood was layered on a Polymorphprep™ solution layer (Axis-Shield, Dundee, UK, cat. no.: AXS-1114683), with subsequent centrifugation (300 g, 50 min, at 20°C), and without centrifuge breaking. After centrifugation, the lower cell phase, containing polymorphonuclear leukocytes (PMNs), was isolated. The cells were washed with an equal volume of 1:2 diluted HEPES buffer (Region H Apoteket, Copenhagen, Denmark, cat. no.: 864492) with centrifugation at 400 g, 10 min, at 20°C. The supernatant was removed, and the cell pellet was resuspended in HBSS (Thermo Fisher Scientific, cat. no.: 14025-092). To determine individual WBC concentrations, the cell suspension was analyzed via the XP-300™ hematology analyzer (Sysmex, Kobe, Japan). The cell suspension was diluted in HBSS (Thermo Fisher Scientific, cat. no.: 14025-092), and 100 μL, containing 1.0 × 106 PMNs, were added to a 5 mL FACS tube (Corning, cat. no.: 352052). Each sample was stained in the dark for 30 min at 37°C with 100 μL of 10 μm Calbryte™ 520 AM (AAT Bioquest, Pleasanton, CA, USA, cat. no.: 20653), diluted in HBSS (Thermo Fisher Scientific, cat. no.: 14025-092), with added 0.5% BSA (Merck, cat. no.: A8327) (HBSS/BSA), with an additionally added 0.04% Pluronic® F-127 (AAT Bioquest, cat. no.: 20053) (HBSS/BSA [+0.04% Pluronic® F-127]). Following, samples were washed once with 1 mL of HBSS/BSA (+0.04% Pluronic® F-127) (500 g, 5 min, at 20°C). The supernatant was removed, and samples were stained in the dark for 15 min at RT with 50 μL of master mix containing 2 μL CD15 BV605 (BioLegend, cat. no.: 323032), 1 μL CD16 BV711 (BioLegend, cat. no.: 302044), and 1 μL CD45 SBV790 (Bio-Rad Laboratories, cat. no.: MCA87SBV790) in HBSS/BSA (+0.04% Pluronic® F-127). Following, samples were washed once with 1 mL of HBSS/BSA (+0.04% Pluronic® F-127) (500 g, 5 min, at 20°C) before adding 50 μL of either HBSS/BSA (+0.04% Pluronic® F-127), IgG1k isotype mAb (BioLegend, cat. no.: 401408), C5aR1 mAb 18-41-6, C5aR1 F(ab’)2 18-41-6, or avacopan (TargetMol Chemicals, Boston, MA, USA, cat. no.: T8223), at a final concentration of approximately 80 nm and/or 20 μm (after addition of 300 μL buffer and 100 μL of C5a/buffer). Incubation with these reagents was carried out in the dark for 10 min at RT. Following, 300 μL of HBSS/BSA (+0.04% Pluronic® F-127) was added to all samples. Samples analysis was conducted with a BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG) by recording ungated events for 5 min and 30 s per sample. After initial baseline acquisition for 1 min, 100 μL of either HBSS/BSA (+0.04% Pluronic® F-127) or 500 ng/mL C5a (Complement Technology, Tyler, TX, USA, cat. no.: A144) dilution was added to the respective samples at the final concentration of approximately 100 ng/mL (corresponding molarity: 9.62 nm). Sample recording continued for 2 min before adding 100 μL of either HBSS/BSA (+0.04% Pluronic® F-127) or 6 μm ionomycin (Merck, cat. no.: I3909) dilution at the final concentration of approximately 1 μm. The samples were continuously recorded for another 2 min (5 min and 30 s total, with 2 × 15-s time windows for the addition of stimulant or buffer). Compensation was conducted with beads (Thermo Fisher Scientific, cat. no.: 01-3333-42), using the aforementioned antibodies for staining. Compensation for Calbryte™ 520 AM staining was carried out with stained cells. The assay was repeated three times with the blood of a different anonymous healthy donor for each repeat. Data were acquired in singlets.

Investigation of C5aR1-Inhibitory Activity of C5aR1 mAb 18-41-6 on C5a-Stimulated PMNs

PMNs were isolated from healthy donor blood, as described previously. 50 µL of PMN dilution in HBSS (Thermo Fisher Scientific, cat. no.: 14025-092), containing 5.0 × 105 PMNs, were added to each respective 5 mL FACS tube (Corning, cat. no.: 352052). To each sample, 25 μL of either HBSS/BSA, IgG1k isotype mAb (BioLegend, cat. no.: 401408), C5aR1 mAb 18-41-6, C5aR1 mAb 18-41-6-based F(ab’)2 fragments, or avacopan (TargetMol Chemicals, Boston, MA, cat. no.: T8223) was added, at a final concentration of 300 nm and/or 75 μm (after addition of C5a). This varied for the titration experiments regarding C5aR1 F(ab’)2 18-41-6 and avacopan (300 nm–1.23 nm final concentration, based on a three-fold dilution series). Samples were incubated for 10 min at RT. Following, 25 μL of either HBSS/BSA or C5a (Complement Technology, cat. no.: A144) dilution at the final concentration of 10 ng/mL (corresponding molarity: 961.54 pm) was added to the respective samples. Samples were consequently incubated for 10 min at 37°C (except baseline control [Tb] samples). Afterward, samples were transferred to an ice-water bath (4°C equivalent) and stained with 50 μL of stain master mix, containing: 5 μL CD11 b PE (BioLegend, cat. no.: 301306), 1 μL CD15 BV605 (BioLegend, cat. no.: 323032), 0.5 μL CD16 BV711 (BioLegend, cat. no.: 302044), and 5 μL CD66b BV421 (BioLegend, cat. no.: 392916) in DPBS/BSA. Samples were incubated for 10 min inside the ice-water bath. Consequently, samples were washed with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). The supernatant was removed, and all samples were fixated for 20 min at RT via the addition of 300 μL of 4% formaldehyde (VWR, cat. no.: 9713.1000) (dark). Afterward, samples were washed twice with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). Samples were resuspended in DPBS/BSA prior to FACS analysis with BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG). Per sample, 100,000 ungated events were recorded. Beads (Thermo Fisher Scientific, cat. no.: 01-3333-42) were used to create compensation controls, using previously mentioned antibodies for staining. Both assays (controls, titration) were repeated three times with the blood of different anonymous healthy donors, with two donors (control assay) and one donor (titration assay) per repeat, respectively. Data points were acquired in singlets.

Functional Assessment of mAb 18-41-6 in Bacteria-Stimulated Human Whole Blood

The ex vivo whole blood model was implemented according to a previously established model [15]. Blood was drawn into sterile blood vials (Greiner Bio-One, cat. no.: 454241) containing lepirudin (Refludan®) (Celgene, Windsor, UK) as an anticoagulant at the final concentration of 44 μg/mL. Immediately after the blood sampling, the tubes were inverted and put inside an ice-water bath (4°C equivalent). A volume of 390 μL blood was combined with 45 μL of either DPBS (Merck, cat. no.: D8662), IgG1k isotype mAb (BioLegend, cat. no.: 401408), C5aR1 mAb 18-41-6, C5aR1 mAb 18-41-6-based F(ab’)2 fragments, or avacopan (TargetMol Chemicals, cat. no.: T8223) in sterile 1.8 mL cryogenic tubes (Thermo Fisher Scientific, cat. no.: 363401) at a final concentration of 400 nm and/or 100 μm (after addition of bacterial stimulant). Different concentrations were used for titration experiments regarding C5aR1 F(ab’)2 18-41-6 and avacopan (400 nm–12.5 nm final concentration, based on a two-fold dilution series). Samples were incubated for 5 min inside an ice-water bath before 15 μL of either DPBS (Merck, cat. no.: D8662) or HI E. coli (American Type Culture Collection, Manassas, VA, USA, cat. no.: 33572, strain LE392) (final concentration of 1.0 × 107 cells/mL) was added to the samples.

All tubes were then incubated at 37°C under agitation (Boule Medical, Spånga, Sweden, Mixer 820) for 15 min, except baseline control (Tb) samples. 40 µL of blood was added to 5 mL FACS tubes (Corning, cat. no.: 352052), containing 6.4 μL of stop solution, resulting in a final concentration of 20 mm EDTA (Merck, cat. no.: 03690, pH adjusted to 7.4) and 10.3 mm sodium citrate (Becton Dickinson, cat. no.: 367714). The samples were treated with 1 mL of RBC lysis reagent (Thermo Fisher Scientific, cat. no.: HYL250) and incubated for at least 10 min at RT in the dark. FACS tubes were centrifuged (500 g, 5 min, at 4°C), and the supernatant was removed, followed by a wash step with 1 mL of DPBS/BSA. Samples were resuspended by adding 50 μL stain master mix containing 5 μL CD11b PE (BioLegend, cat. no.: 301306), 0.5 μL CD14 BB700 (Becton Dickinson, cat. no.: 566465), 0.5 μL CD15 BV605 (BioLegend, cat. no.: 323032), 0.25 μL CD16 BV711 (BioLegend, cat. no.: 302044), 1 μL CD45 SBV790 (Bio-Rad Laboratories, cat. no.: MCA87SBV790), and 5 μL CD66b BV421 (BioLegend, cat. no.: 392916) diluted in DPBS/BSA. Samples were incubated for 15 min at 4°C (dark). Afterward, samples were washed once with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C). The supernatant was removed, and cells were fixed in 300 μL of 4% formaldehyde (VWR, cat. no.: 9713.1000) for 20 min at RT (dark). Samples were washed twice with 1 mL of DPBS/BSA (500 g, 5 min, at 4°C) and afterward resuspended in DPBS/BSA for analysis. FACS samples were analyzed via a BD FACSCelesta™ flow cytometer (Becton Dickinson, standard configuration BVYG). Per sample, at least 70,000 ungated events were recorded. Beads (Thermo Fisher Scientific, cat. no.: 01-3333-42) were used to create compensation controls, using previously mentioned antibodies for staining. Both assays (controls, titration) were repeated three times with the blood of different anonymous healthy donors, using two blood donors (control assay) or one blood donor (titration assay) per repeat. Data points were collected in singlets.

Data Analysis

Flow cytometric data were analyzed using the software “FlowJo” (version 10.9.0). Samples were analyzed with the sets of gates displayed in the online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000535084). To analyze calcium flux data, the built-in “kinetics” platform was used. Statistical analyses were performed with the software “GraphPad Prism” (version 9.4.1). The built-in analysis “Nonlinear regression (curve fit)” and “Sigmoidal, 4PL, X is log (concentration)” formula were used for curve analysis, with no additional constraints. To determine whether obtained curve fitting parameters were significant between C5aR1 inhibitors, datasets were analyzed by nonlinear curve fitting with extra sum-of-squares F test comparison (p > 0.05). Statistical analysis of C5aR1 inhibition was conducted via one-way ANOVA, using matched data, assumed Gaussian distribution, and non-assumed sphericity (“RM one-way ANOVA with Geisser-Greenhouse correction”), including Dunnett’s test (“Dunnett’s multiple comparisons test, with individual variances computed for each comparison”) to compare multiple C5aR1 inhibitors to the same control.

Screening and Selection of C5aR1 Hybridoma Clones on Whole Blood

Hybridoma supernatants of all C5aR1-targeting clones were tested for binding to white blood cells. Granulocytes and monocytes have been described to strongly express C5aR1, making them ideal targets for binding assessments [28, 29]. In total, ten hybridoma supernatants were defined as positive for C5aR1 detection (MFI hybridoma supernatant ≥2 × MFI secondary antibody control for both granulocyte and monocyte population) (Fig. 1; online suppl. Fig. 1). Strong positive signals were only recorded for hybridoma clones obtained from the immunization against the N-terminal peptide region of C5aR1 (hybridoma clones 18-41-1 to 18-41-15). No positive binding clones were observed for any other selected topical region of C5aR1 (first extracellular loop [hybridoma clones 18-39-1 to 18-39-8], second extracellular loop [hybridoma clones 18-40-1 to 18-40-10], and third extracellular loop [hybridoma clones 18-36-1 to 18-36-4]). The selected ten hybridoma clones were expanded in serum-containing media and purified by protein G affinity chromatography for confirmation of C5aR1 detection. Later, all clones were cultured in serum-free media for assessment of inhibitory properties against C5aR1.

Fig. 1.

Screening of individual hybridoma supernatants on whole blood. Corresponding immunization peptide regions (1–36, 90–109, 175–188, and 266–282) of individual clones are presented below. Negative controls (IgG1k isotype mAb, IgG2ak isotype mAb) and a positive control (C5aR1 detection: mAb S5/1) were included. Data are presented as fold increase based on the secondary antibody control (secondary control). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were acquired in singlets. Data are presented as mean ± SD.

Fig. 1.

Screening of individual hybridoma supernatants on whole blood. Corresponding immunization peptide regions (1–36, 90–109, 175–188, and 266–282) of individual clones are presented below. Negative controls (IgG1k isotype mAb, IgG2ak isotype mAb) and a positive control (C5aR1 detection: mAb S5/1) were included. Data are presented as fold increase based on the secondary antibody control (secondary control). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were acquired in singlets. Data are presented as mean ± SD.

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Assessment of C5aR1 Recognition by Purified C5aR1 mAbs on Whole Blood

Selected, bovine serum-grown C5aR1-targeting mAbs were tested for binding to neutrophils (CD45+, CD14−, CD15+, CD16+) and monocytes (CD45+, CD14+, CD15−) in whole blood (Fig. 2; online suppl. Fig. 2). All clones displayed high MFI signals on both cell populations, indicating successful C5aR1 recognition. All tested C5aR1-targeting mAbs were chosen for serum-free purification and analysis via the iLite® cell-based assay platform, investigating C5aR1 inhibition. It must be noted that C5aR1 mAb addition caused the upregulation of certain gating markers on neutrophils (CD14) and monocytes (CD15), while CD16 was reduced on neutrophils.

Fig. 2.

Screening of selected, purified C5aR1 mAbs on whole blood. Negative controls (IgG1ak isotype mAb and IgG2ak isotype mAb) and a positive control (C5aR1 detection: mAb S5/1) were included. Data are presented as a fold increase based on the secondary antibody control (secondary control). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were acquired in singlets. Data are presented as mean ± SD.

Fig. 2.

Screening of selected, purified C5aR1 mAbs on whole blood. Negative controls (IgG1ak isotype mAb and IgG2ak isotype mAb) and a positive control (C5aR1 detection: mAb S5/1) were included. Data are presented as a fold increase based on the secondary antibody control (secondary control). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were acquired in singlets. Data are presented as mean ± SD.

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Assay of C5aR1 mAbs and Inhibitors on iLite® C5a Assay Ready Cells

All C5aR1-targeting mAbs were tested via the iLite® cell-based assay platform to assess C5aR1 inhibition. The iLite® C5a Assay Ready Cells display modified C5aR1 on their surface, which, upon activation via C5a, induces the expression of FL. These cells also express RL constitutively. iLite® C5a Assay Ready Cells were first incubated with the respective C5aR1-targeting mAb before stimulation with C5a. To compare in-house C5aR1-targeting mAbs to commercially available C5aR1 inhibitors, the mAb 347214, mAb S5/1, avdoralimab, and peptidic (PMX-53) and non-peptidic (W-54011, avacopan) C5aR1 inhibitors were included in the assays (Fig. 3). C5aR1 mAb 18-41-6 displayed the best combination of lowest IC50 value (2.19 nm) and best-fit bottom parameter (16.51% control FL signal) of all tested in-house C5aR1 mAbs, which was lower than the commercial C5aR1 mAb 347214 (IC50: 3.32 nm, best-fit bottom: 23.98% control FL signal) and mAb S5/1 (IC50: 6.01 nm, best-fit bottom: 23.53% control FL signal) (online suppl. Table 1). Only the use of avdoralimab (IC50: 1.88 nm, best-fit bottom: 10.38% control FL signal) resulted in a lower IC50 value and best-fit bottom parameter than C5aR1 mAb 18-41-6, although this difference was not statistically significant for both IC50 (p = 0.3434) and best-fit bottom parameter (p = 0.0635). Compared to other commercial C5aR1 mAbs, mAb 18-41-6 application resulted in a significant change of IC50 compared to mAb S5/1 (p = <0.0001), but not for mAb 347214 (p = 0.0862). The use of mAb 18-41-6 did not alter the obtained best-fit bottom parameter significantly compared to both mAb 347214 (p = 0.2351) and mAb S5/1 (p = 0.2977). For small-molecule C5aR1 inhibitors, mAb 18-41-6 displayed a significantly improved IC50 compared to W-54011 (p = <0.0001), PMX-53 (p = <0.0001), and avacopan (p = <0.0001). However, application of small-molecule C5aR1 inhibitors resulted in a significantly improved best-fit bottom parameter for W-54011 (p = 0.0309), PMX-53 (p = 0.0005), and avacopan (p = 0.0034) compared to mAb 18-41-6. Obtained RL signals did not indicate sample toxicity caused by any of the tested C5aR1 inhibitors. However, lower RL values were observed for W-54011, PMX-53, and avacopan at high doses (online suppl. Fig. 3). Incubation of C5aR1-targeting mAbs without C5a showed no C5aR1-mediated cell activation (FL signal increase) by direct antibody binding to the receptor in a small trial.

Fig. 3.

Assay of C5aR1 inhibitors on iLite® C5a Assay Ready Cells, displaying commercial mAb and small-molecule C5aR1 inhibitors (a) and in-house C5aR1 mAbs (b). Data were normalized to a stimulation control (added C5a) and analyzed by nonlinear curve fitting. Data points were collected in duplicates across multiple assay plates; each assay plate was repeated three times. Data are presented as mean ± SD.

Fig. 3.

Assay of C5aR1 inhibitors on iLite® C5a Assay Ready Cells, displaying commercial mAb and small-molecule C5aR1 inhibitors (a) and in-house C5aR1 mAbs (b). Data were normalized to a stimulation control (added C5a) and analyzed by nonlinear curve fitting. Data points were collected in duplicates across multiple assay plates; each assay plate was repeated three times. Data are presented as mean ± SD.

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Examination of C5aR1-Specficity of mAb 18-41-6 on C5aR1his- and C5aR2his-Transfected Flp-In™-CHO Cells

C5aR1 mAb 18-41-6 was confirmed to bind to Flp-In™-CHO cells transfected with C5aR1his but not to Flp-In™-CHO cells transfected with C5aR2his. No ectopic expression of C5aR1 or C5aR2 was observed in non-transfected Flp-In™-CHO cells (Fig. 4; online suppl. Fig. 4). Transfection of Flp-In™-CHO cells with C5aR1his resulted in a substantial increase in secondary antibody PE MFI for C5aR1 mAbs S5/1 and 18-41-6, confirming that both mAbs detect the receptor adequately. At the same time, C5aR2his-transfected Flp-In™-CHO cells were shown to bind C5aR2 mAb 1D9-M12 but not C5aR1 mAbs S5/1 and 18-41-6, confirming the specificity of mAb 18-41-6 for C5aR1.

Fig. 4.

Assessment of mAb 18-41-6 on non-transfected Flp-In™-CHO cells, C5aR1his-transfected Flp-In™-CHO cells, and C5aR2his-transfected Flp-In™-CHO cells. Negative controls (IgG1k isotype mAb and IgG2ak isotype mAb) and positive controls (C5aR1 detection: mAb S5/1; C5aR2 detection: mAb 1D9-M12) were included. Data are presented as fold change based on secondary antibody control (secondary control). The experiment was repeated three times; data points were obtained in singlets. Data are presented as mean ± SD.

Fig. 4.

Assessment of mAb 18-41-6 on non-transfected Flp-In™-CHO cells, C5aR1his-transfected Flp-In™-CHO cells, and C5aR2his-transfected Flp-In™-CHO cells. Negative controls (IgG1k isotype mAb and IgG2ak isotype mAb) and positive controls (C5aR1 detection: mAb S5/1; C5aR2 detection: mAb 1D9-M12) were included. Data are presented as fold change based on secondary antibody control (secondary control). The experiment was repeated three times; data points were obtained in singlets. Data are presented as mean ± SD.

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Functional Assessment of mAb 18-41-6 in a C5a-Driven PMN Calcium Flux Assay

Individual C5aR1 inhibitors were assessed for inhibiting C5a-mediated calcium flux in isolated PMNs. Neutrophils were identified by their respective markers (CD45+, CD15+, and CD16+) (online suppl. Fig. 5). It could be shown that both full-size mAb and F(ab’)2 fragments of C5aR1 mAb 18-41-6 efficiently abrogated C5aR1 activation by C5a alongside avacopan (Fig. 5). Sample stimulation with ionomycin confirmed the selectivity of tested C5aR1 inhibitors, as strong ionomycin-mediated calcium flux could be observed independently from C5aR1 inhibition.

Fig. 5.

Assay of C5aR1 inhibitors within a C5a-driven PMN calcium flux assay. A negative inhibition control (IgG1k isotype mAb) and a positive inhibition control (20 μm avacopan) were included. After first recording the baseline, either C5a or buffer was added, followed by the addition of either ionomycin or buffer. Data are presented as fold increase based on the initially recorded baseline (mean FITC detector MFI). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were obtained in singlets. Data are presented as mean + SD (one-sided) for better visualization.

Fig. 5.

Assay of C5aR1 inhibitors within a C5a-driven PMN calcium flux assay. A negative inhibition control (IgG1k isotype mAb) and a positive inhibition control (20 μm avacopan) were included. After first recording the baseline, either C5a or buffer was added, followed by the addition of either ionomycin or buffer. Data are presented as fold increase based on the initially recorded baseline (mean FITC detector MFI). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were obtained in singlets. Data are presented as mean + SD (one-sided) for better visualization.

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Investigation of C5aR1-Inhibitory Activity of C5aR1 mAb 18-41-6 on C5a-Stimulated PMNs

Assays were conducted to examine the impact of C5aR1 mAb 18-41-6, mAb 18-41-6-based F(ab’)2 preparations, and avacopan on C5a-stimulated, isolated PMNs. Neutrophils were identified by their physical parameters (FSC and SSC) and gating markers (CD15+ and CD16+), while also cell activation was investigated (CD11b and CD66b) (online suppl. Fig. 6). Both C5aR1 mAb 18-41-6 mAb (CD11b: p = 0.0003, CD66b: p = <0.0001) and F(ab’)2 preparations (CD11b: p = 0.0003, CD66b: p = <0.0001), as well as avacopan (CD11b: p = 0.0004, CD66b: p = <0.0001), were shown to significantly inhibit the C5a-driven upregulation of CD11b and CD66b on neutrophils (Fig. 6a–c) at a concentration of 300 nm compared to a stimulation control. Titration of F(ab’)2 preparations of C5aR1 mAb 18-41-6 versus equimolar concentrations of avacopan resulted in comparable CD11b and CD66b reduction profiles (Fig. 6d, e). F(ab’)2 preparations of mAb 18-41-6 displayed efficient inhibition of CD11b (IC50: 31.52 nm, best-fit bottom: 42.31% control MFI) and CD66b (IC50: 76.96 nm, best-fit bottom: 49.12% control MFI) (online suppl. Table 2). Statistical comparison of F(ab’)2 preparations of mAb 18-41-6 to equimolar concentrations of avacopan was not performed due to obtained unstable nonlinear curve fitting parameters for neutrophilic CD11b and CD66b for avacopan.

Fig. 6.

Assay of C5aR1 inhibitors on C5a-stimulated PMNs (a–c), followed by titration of mAb 18-41-6-based F(ab’)2 fragments versus equimolar concentrations of avacopan (d, e). a–c A negative control for C5aR1 inhibition (IgG1k isotype mAb) and a positive control (75 μm avacopan) were included. Data points were normalized to a stimulation control sample (T10stim) with added C5a. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T10) are depicted. Titration curves of mAb 18-41-6-based F(ab’)2 fragments and avacopan were analyzed by nonlinear curve fitting. The experiments were repeated three times with the blood of two different anonymous healthy donors (a–c) or one different anonymous healthy donor per repeat (d, e); data points were acquired in singlets. Data are presented as mean ± SD.

Fig. 6.

Assay of C5aR1 inhibitors on C5a-stimulated PMNs (a–c), followed by titration of mAb 18-41-6-based F(ab’)2 fragments versus equimolar concentrations of avacopan (d, e). a–c A negative control for C5aR1 inhibition (IgG1k isotype mAb) and a positive control (75 μm avacopan) were included. Data points were normalized to a stimulation control sample (T10stim) with added C5a. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T10) are depicted. Titration curves of mAb 18-41-6-based F(ab’)2 fragments and avacopan were analyzed by nonlinear curve fitting. The experiments were repeated three times with the blood of two different anonymous healthy donors (a–c) or one different anonymous healthy donor per repeat (d, e); data points were acquired in singlets. Data are presented as mean ± SD.

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Functional Assessment of mAb 18-41-6 in Bacteria-Stimulated Human Whole Blood

A previously described ex vivo lepirudin whole blood model [15] was utilized to determine the C5aR1-blocking efficacy of C5aR1-targeting mAb 18-41-6 in blood stimulated with bacteria. Lepirudin-anticoagulated blood was incubated with IgG1k isotype mAb, mAb 18-41-6, F(ab’)2 preparations of mAb 18-41-6, or avacopan before incubation with E. coli. Blood was analyzed for CD11b and CD66b expression to assess the activation state while differentiating in neutrophil (CD45+, CD14–, CD15+, CD16+) and monocyte (CD45+, CD14+, CD15–) populations (online suppl. Fig. 7). F(ab’)2 preparations of C5aR1 mAb 18-41-6 displayed efficient and significant CD11b (p = 0.0092) and CD66b (p = 0.0060) reduction on neutrophils at 400 nm, which was more pronounced compared to a nonsignificant reduction of CD11b (p = 0.2101) and CD66b (p = 0.3162) by an equimolar concentration of avacopan compared to a stimulation control (Fig. 7a–d). In addition, monocytic CD11b was significantly reduced by both F(ab’)2 preparations of C5aR1 mAb 18-41-6 (p = 0.0020) and avacopan (p = 0.0095) compared to a stimulation control. However, blood treatment with full-size mAb 18-41-6 did result in a nonsignificant increase for neutrophilic CD11b (p = 0.4005) and monocytic CD11b (p = 0.9196), as well as in a significant increase in CD66b (p = 0.0400) compared to a stimulation control. Titration of F(ab’)2 preparations of C5aR1 mAb 18-41-6 versus an equimolar concentration of avacopan revealed a strikingly improved C5aR1 inhibition in the nanomolar concentration range compared to avacopan (Fig. 7e–g), resulting in effective C5aR1 inhibition via F(ab’)2 preparations of C5aR1 mAb 18-41-6 in the intermediate nanomolar range for neutrophilic CD11b (IC50: 40.60 nm, best-fit bottom: 67.20% control MFI) and CD66b (IC50: 36.95 nm, best-fit bottom: 72.12% control MFI), as well for monocytic CD11b (IC50: 19.24 nm, best-fit bottom: 81.73% control MFI) (online suppl. Table 3). Statistical comparison of F(ab’)2 preparations of C5aR1 mAb 18-41-6 versus equimolar concentrations of avacopan for either neutrophilic CD11b and CD66b, as well as monocytic CD11b inhibition was omitted due to either unstable fitting parameters (neutrophilic CD66b) or low coefficient of determination values (neutrophilic and monocytic CD11b) obtained for avacopan.

Fig. 7.

Assay of C5aR1 inhibitors in whole blood stimulated by HI E. coli. a–d A negative control (IgG1k isotype mAb) and a positive control (100 μm avacopan) for C5aR1 inhibition were included. Data are normalized to a stimulation control (T15stim) with added HI E. coli. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T15) are depicted. e–g Nonlinear curve fitting was employed to analyze mAb 18-41-6-based F(ab’)2 fragments and avacopan titration curves. Experiments were repeated three times, with the blood of two different anonymous healthy donors (a–d) or one different anonymous healthy donor per repeat (e–g); data points were obtained in singlets. Data are presented as mean ± SD.

Fig. 7.

Assay of C5aR1 inhibitors in whole blood stimulated by HI E. coli. a–d A negative control (IgG1k isotype mAb) and a positive control (100 μm avacopan) for C5aR1 inhibition were included. Data are normalized to a stimulation control (T15stim) with added HI E. coli. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T15) are depicted. e–g Nonlinear curve fitting was employed to analyze mAb 18-41-6-based F(ab’)2 fragments and avacopan titration curves. Experiments were repeated three times, with the blood of two different anonymous healthy donors (a–d) or one different anonymous healthy donor per repeat (e–g); data points were obtained in singlets. Data are presented as mean ± SD.

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This study presents the functional characterization of potent in-house C5aR1-targeting mAbs that display binding on neutrophils and monocytes and strong inhibitory properties against C5aR1. After initial screening of hybridoma clones on whole blood, only mAbs targeting the N-terminal section of C5aR1 resulted in above-threshold MFIs. Subsequent purification of selected mAbs confirmed efficient C5aR1 detection. All purified C5aR1 mAbs were then tested in a commercially available iLite® C5a assay. Remarkably, one of our in-house produced C5aR1-targeting mAbs (mAb 18-41-6) matched or outperformed commercial C5aR1-blocking mAbs (mAb S5/1, mAb 347214) in both IC50 and efficacy (best-fit bottom). Only the therapeutic C5aR1 mAb avdoralimab (IPH5401, Innate Pharma) [30] displayed improved IC50 and maximal inhibition (best-fit bottom) compared to in-house C5aR1 mAb 18-41-6, though both were not statistically significant. As avdoralimab has been extensively optimized for its therapeutic purpose, a comparison to in-house murine C5aR1 mAbs might not be entirely relevant, as we, at this stage, have yet to make any structural improvements toward a therapeutic-grade reagent. When comparing potent in-house C5aR1-targeting mAbs with small-molecule C5aR1 inhibitors, a significantly improved IC50 for mAb 18-41-6 was observed. Comparison of maximal inhibition (best-fit bottom) favored small-molecule C5aR1 inhibitors, which were able to reduce the observed FL signal significantly better than mAb 18-41-6. However, it must be noted that much higher concentrations of drug were necessary to achieve this level of FL inhibition than was achievable for individual mAbs. The bivalent nature of antibodies must also be considered, as C5aR1 antibodies may block two C5aR1. This has not been taken into consideration for the calculation of IC50 parameters. Based on the iLite assay results, the most promising C5aR1-targeting mAb was C5aR1 mAb 18-41-6 in terms of IC50 and efficacy (best-fit bottom).

Subsequently, we confirmed that C5aR1 mAb 18-41-6 exclusively detected C5aR1, but not C5aR2, on C5aR1his- and C5aR2his-overexpressing Flp-In™-CHO cells. A calcium flux assay, based on isolated PMNs, was used to demonstrate the efficient inhibition of C5a-mediated C5aR1 activation by C5aR1 mAb 18-41-6 and C5aR1 mAb 18-41-6-based F(ab’)2 preparations. The functionality of C5aR1 mAb 18-41-6 was investigated, utilizing both isolated PMNs and an ex vivo lepirudin human whole blood model. In both models, the application of full-size C5aR1 mAb 18-41-6 and corresponding F(ab’)2 preparations influenced activation marker expression. Within the PMN C5a assay, the application of full-size C5aR1 mAb 18-41-6 and corresponding mAb 18-41-6-based F(ab’)2 preparations resulted in a significant activation marker reduction (neutrophilic CD11b and CD66b) compared to the stimulation control, alongside avacopan.

In bacteria-stimulated whole blood, application of mAb 18-41-6-based F(ab’)2 preparations resulted in a significant activation marker reduction for neutrophilic CD11b and CD66b, while an equimolar concentration of avacopan did not result in a significant reduction compared to the stimulation control. In comparison with the stimulation control, both avacopan and 18-41-6-based F(ab’)2 preparations reduced monocytic CD11b significantly. Application of full-size mAb 18-41-6 did not reduce cell activation markers but led to a nonsignificant increase of neutrophilic and monocytic CD11b and a significant increase of neutrophilic CD66b after application compared to the stimulation control.

Similarly, the sole application of in-house C5aR1 mAbs to white blood cells influenced marker expression of neutrophils (CD14 and CD16) and monocytes (CD15) visibly compared to the buffer control within the cell binding assay. This is believed to be due to signaling caused by the Fc region of bound antibodies. Employment of C5aR1 mAb 18-41-6-based F(ab’)2 preparations significantly reduced observed activation marker expression for both CD11b and CD66b on neutrophils and CD11b on monocytes in bacteria-stimulated whole blood, compared to the stimulation control, supporting the need for Fc-targeted modifications, as this was not seen for the full-size mAb.

Titration of C5aR1 mAb 18-41-6-based F(ab’)2 preparations versus equimolar concentrations of avacopan revealed a remarkable efficiency of F(ab’)2 preparations for C5aR1 inhibition in both C5a-stimulated, isolated PMNs and an bacteria-stimulated, ex vivo whole blood model, resulting in low nanomolar IC50 parameters for both models. Similar activation marker reduction profiles were obtained for both C5aR1 inhibitors for C5a-stimulated PMNs. Differences were noticed for C5aR1 inhibition of bacteria-stimulated whole blood, where improved activation marker reduction via C5aR1 mAb 18-41-6-based F(ab’)2 preparations at low to intermediate nanomolar concentrations was observed compared to equimolar concentrations of avacopan.

Thus, the C5aR1 mAb 18-41-6 could be an excellent candidate to investigate C5aR1 inhibition under physiological conditions in vitro and ex vivo and potentially a therapeutical candidate after optimization and humanization. It must be noted that the cross-reactivity to other species has yet to be determined.

Interestingly, despite conducting peptide immunizations with synthetic peptides of multiple topologically accessible regions of C5aR1, only immunizations targeting the N-terminus of C5aR1 resulted in recognizing and inhibiting mAbs, which indicates the difficulty of developing mAbs, targeting multiple regions of native C5aR1. However, structural constraints of extracellular loops may result in challenges when linear peptides are used for immunization. All tested in-house mAbs interfere with C5a binding to C5aR1, as the N-terminal amino acids of C5aR1 are instrumental for C5a binding [31].

Currently, the C5a-C5aR1 axis is being investigated for treatment options in many inflammation-driven diseases, with a particular interest in COVID-19 [24]. Eculizumab has been successfully used to reduce morbidity and mortality in patients with complement-driven diseases, like PNH [21] and atypical hemolytic uremic syndrome, among others, by preventing C5 cleavage into C5a and C5b. Whether this effect primarily occurs via inhibition of C5a generation, the formation of the terminal complement complex, or both is still unresolved. In PNH, it is most likely that C5b-9 formation is the most important mediator of this hemolytic disease [32], but in several other diseases, C5a might be the most important mediator.

Despite the high efficacy of assayed small-molecule C5aR1 antagonists (W-54011, PMX-53, avacopan), mAbs could still be a valid option for clinical C5aR1 inhibition, as mAbs are generally highly specific for their target, might have a longer half-life, and less off-target effects [33]. Noteworthy is also the residence time of C5aR1 inhibitors on the target, which has been reported to be short for C5aR1 inhibitor W-54011 [34], raising questions on how effective small-molecule C5aR1 inhibitors are compared to mAbs in sustaining C5aR1 inhibition.

In addition, the use of multiple C5aR1 inhibitors might be necessary for complete C5aR1 signaling abrogation. In the context of the recently discovered complosome [35], signaling via extracellular and intracellular C5aR1 must be considered. An obstacle in targeting intracellular C5aR1 is the necessity of the respective C5aR1 inhibitor to be able to traverse the cellular membrane efficiently. Recently, a particular small-molecule C5aR1 inhibitor has been reported to be able to diffuse through the cellular membrane and abrogate intracellular C5aR1 signaling [35]. Antibodies are more difficult to transport across cell membranes, which mostly requires additional cell treatment, physical delivery methods, and/or attachment of antibodies to other compounds for enhanced uptake [36]. With future improved delivery methods, antibodies will certainly be more applicable for intracellular targets.

Nevertheless, interference with the C5a-C5aR1 axis must be considered carefully and tested thoroughly in each disease condition, as not only inflammatory but also anti-inflammatory responses might be mediated via C5aR1 [37], which is further complexed by its interaction with C5aR2 [9]. In addition, inhibition of C5aR1, C5, or C5a might increase the risk for certain microbial infections, which has been reported for the C5 inhibitor eculizumab [38].

In conclusion, we have described the generation of C5aR1 mAb 18-41-6, which was able to match or outperform commercial C5aR1 mAbs in a commercial iLite® C5a assay. Assay of C5aR1 mAb 18-41-6-based F(ab’)2 preparations revealed a significant reduction of activation marker expression within a C5a-stimulated PMN assay and E. coli-stimulated ex vivo whole blood model compared to the respective stimulation control. Titrations of C5aR1 mAb 18-41-6-based F(ab’)2 preparations in bacteria-stimulated whole blood revealed a substantial improvement of the expression of cell activation and inflammatory markers at low to intermediate nanomolar concentrations compared to equimolar concentrations of avacopan. This underlines a potential advantage of C5aR1 mAbs and mAb-based F(ab’)2 fragments compared to small-molecule C5aR1 inhibitors. With additional structural optimizations, we suggest C5aR1 mAb 18-41-6 is a promising candidate for not only experimental C5aR1 inhibition but also for therapeutic targeting of C5aR1.

The authors thank Bettina Eide Holm (Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 76311, Copenhagen University Hospital – Rigshospitalet, Copenhagen, Denmark), as well as Camilla Schjalm and Karin Ekholt McAdam (Department of Immunology, Oslo University Hospital and University of Oslo, Oslo, Norway) for their excellent technical support. Furthermore, the authors thank Trent Woodruff (School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, QLD, Australia) for kindly providing PMX-53. Finally, the authors greatly thank Anna Pramhed (Svar Life Science, Malmö, Sweden) for kindly supplying iLite® C5a Assay Ready Cells and the great technical support.

Human whole blood was obtained from anonymous donors via the Blood Bank at Copenhagen University Hospital – Rigshospitalet in Copenhagen. Informed written consent was not obtained and required. Ethical approval was not required for this study in accordance with local and national guidelines.

The Danish Animal Experiments Inspectorate has approved the experimental animal procedures. Procedures were carried out in accordance with the Danish Animal Welfare Act for the Care and Use of Animals for Scientific Purposes (approval number ID 2019-15-0201-00090). All procedures followed the recommendations of the animal facilities at the University of Copenhagen.

The authors have no conflicts of interest to declare.

L.C., T.E.M., and P.G. are supported by the EU MSCA-ITN CORVOS grant (860044). P.G. is further funded by the Austrian Science Fund (FWF) excellence program HOROS (W12530), the Carlsberg Foundation (CF20-476 0045), the Novo Nordisk Foundation (NFF205A0063505, NNF20SA0064201), and the Svend Andersen Research Foundation (SARF2021). A.R. is supported by the Novo Nordisk Foundation (NNF18SA0034956) by participating in the BRIDGE – Translational Excellence Programme.

L.C., M.-O.S., R.B.-O., T.E.M., P.G., and A.R. designed the following study. M.-O.S. established the hybridoma cell lines, producing C5aR1 monoclonal antibodies. C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells were established by L.C. and R.B.-O. L.C. designed the experiments. L.C. and I.M. performed the experiments. L.C. analyzed the data. L.C. made the first draft of the manuscript. All authors carefully revised the manuscript. All authors approved the final draft.

All data generated and analyzed during this study are obtainable by inquiry from the corresponding authors.

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