Fig 2: C5aR1 inhibition prevents anaphylactoid reactions induced by paclitaxel in mice.Effect of vehicle, DF3966A or Avacopan on paclitaxel-increased vascular permeability (A) (one-way ANOVA, effect of treatment F (4, 44) = 5.121; p = 0.0018 followed by Bonferroni’s multiple comparisons test) and plasma extravasation (B) (one-way ANOVA, effect of treatment F (4,44) = 7.258; p = 0.0001 followed by Bonferroni’s multiple comparisons test) in mice. Serum histamine (one-way ANOVA, effect of treatment F (3,36) = 7.590; p = 0.0005 followed by Bonferroni’s multiple comparisons test) (C) and SC5B-9 (one-way ANOVA, effect of treatment F (3,36) = 24.74; p < 0.0001 followed by Bonferroni’s multiple comparisons test) (D) concentration in mice receiving paclitaxel pre-treated with vehicle, DF3966A or Avacopan. Paclitaxel was intravenously administered at 20 mg/kg. Vehicle DF3966A (saline) and DF3966A (30 mg/kg, os) were administered 3 h before paclitaxel, while vehicle Avacopan and Avacopan (30 mg/kg, os) were administered 1 h before paclitaxel. ***P < 0.0005, **P < 0.005 and *P < 0.05 vs Cremophor group; +++P < 0.0005; ++P < 0.005 and +P < 0.05 vs paclitaxel. Data are shown as mean ± SEM of 10 animals per group.

Fig 3: Structure of the human C5a/MEDI7814 scFv complex. (A) Left: Overall complex structure showing C5a (orange) interacts primarily through the VHCDR3 and VLCDR2 of MEDI7814 scFv: variable heavy chain domain (salmon pink), VHCDRs (magenta), variable light chain domain (grey) and VLCDRs (blue), C5a glycosylation site N64 is shown as a stick. (B) Paratope residues (Kabat) of MEDI7814 scFv: VHCDR1 (S31, Y32), VHCDR3 (D96, D97, E100, W100c, Y100d, Y100e, G100f, M100g, D101), VLCDR1 (K31, Y32), VLCDR2 (D50, V51, N52, R53), VH framework 3 (R94), and VL framework 2 (L46, I48, F49). The framework amino acids are Vernier residues, important for the structural integrity of antibodies.50 (C) Discontinuous epitope on C5a recognized by MEDI7814 scFv comprises amino acid residues Y13-C21, D24 and G25, C27, R37, R40-C47 and F51, shown as orange sticks.

Fig 4: Overview of C5 complement activation. Complement C5 activation can occur either by the classical, lectin and alternate complement pathways to generate C5 convertases, or via proteases extrinsic to the complement pathway. C5 convertases or extrinsic proteases can cleave C5 to form proteolytic fragments C5a and C5b. C5a is a potent anaphylatoxin that is rapidly deactivated by removal of the C terminal arginine to form C5adesArg. C5a binds with high affinity (thick black arrows) to both C5aR1 and C5aR2 receptors, while C5adesArg binds with high affinity to the C5aR2 receptor and with low affinity (dashed arrow) to the C5aR1 receptor. The C5aR1 receptor primarily drives pro–inflammatory effects, while the C5aR2 receptor can mediate pro– or anti–inflammatory effects depending on its cellular context. In the absence of cell associated complement inhibitors, C5b interacts with a single copy of complement proteins C6, C7, C8 and a further 18 copies of C9 to form a pore within the cell membrane called the membrane attack complex (MAC).8 The MAC can lyse certain Gram–negative bacteria, promoting bacterial clearance, while host cells can avoid lysis due to the presence of membrane associated complement regulators.

Fig 5: MEDI7814 neutralizes C5a-C5aR1 receptor mediated CD11b upregulation in human whole blood. Representative results showing that (A) MEDI7814 (?) neutralizes endogenous C5a mediated neutrophil CD11b up-regulation in human whole blood with a geomean IC50 of 28 nM. Endogenous C5a was generated by activation of complement using E coli as described by Fung et al.45 Data points represent the mean ± standard error of the mean for 5 separate experiments. No inhibition of response was observed with the isotype irrelevant control antibody (?). (B) CD11b upregulation is mediated predominately via the C5aR1 receptor. 100 nM of an anti-C5aR1 receptor neutralizing antibody (anti-C5aR1 F(ab')2) inhibits C5a mediated CD11b up-regulation in whole human blood upon complement activation using E.coli. 100 nM of an anti-C5aR2 receptor neutralizing antibody (anti-C5aR2 F(ab')2) and an isotype negative control F(ab')2 showed no significant inhibition, indicating that CD11b up-regulation is mediated predominately through the C5aR1 receptor. 100 nM MEDI7814 inhibits CD11b up-regulation, whereas the isotype control antibody showed no inhibition. T0 and T10 represent CD11b level at time 0 and 10 minutes after blood was incubated at 37°C without E.coli. Individual data points from = 6 different donors are shown alongside the mean ± standard deviation for each test condition. ****p < 0.0001, by one-way ANOVA followed by Sidak's multiple comparisons test, (ns, not significant).