NeuroMab™ Anti-CD14 BBB Shuttle Antibody,Clone NR3355P

Figure 1 shows the expression of recombinant IgG2/4 hybrid antibody.

(AC) Ab concentration (mg/ml; s) and Ab productivity (pg/cell/day; d) in cell culture supernatants were determined by ELISA during 12 days of fed-batch expression. Data are presented as mean and SEM for rMil2 (A; n=12), r18D11 (B; n=5) and raNIP (C; n=2). (D) 500 ng of each purified antibody was subjected to reducing or non-reducing SDS-PAGE and stained with Coomassie blue. (E) Non-reducing SDS-PAGE and immunoblotting of the same sample (10 ng) using an Ab specific for the human IgG2 hinge region.

Figure 2 shows the in vitro binding of recombinant IgG2/4 hybrid antibodies to complement and Fc receptors.

Increasing concentrations of rMil2 (s), r18D11 (0) or raNIP (N) were mixed with (A) immobilized human C1q, (B) human Fcg receptor FcgRI, (C) FcgRIIa (allotype His131), (D) FcgRIIb, (E) FcgRIIIa (allotype Val158), (F) FcgRIIIb and (G, H) human (hFcRn) or (I, J) porcine FcRn (pFcRn), acidic (pH 6.0) and neutral pH (pH 7.4).

Figure 3 shows the functional characterization of anti-pig CD14 Ab rMil2.

(A) Whole pig blood together with 150 mg/ml FITC-conjugated Mil2 (FITC-Mil2) and increasing concentrations of unlabeled rMil2 (s), control IgG2/4 (eculizumab) (N), original clone Mil2 (d) Incubation or mIgG2b isotype control (n). (B and C) In the presence of increasing concentrations of rMil2 (s), Mil2 (d), control IgG2/4 (N) or mIgG2b (n). (D) Pig whole blood was incubated with increasing concentrations of rMil2 or Mil2, or ctrl IgG2/4 or mIgG2b isotype controls (up to 71.4 mg/ml). (E) Blood slides of samples containing 71.4 mg/ml rMil2 or Mil2 were stained with a nuclear stain and studied by light microscopy. (F) Pig whole blood incubated with 50 mg/ml rMil2 (s), Mil2 (d), control IgG2/4 (N) or mIgG2b (n).

Figure 4 shows the effect of the combination of rMil2 and the C5 inhibitor OmCI on the inflammatory response in porcine blood in vitro.

Analysis of the cytokine (A) TNF in plasma. (B) Interleukin-1b. (C) IL-8. (D) TF expression on granulocytes was measured by flow cytometry and expressed as median fluorescence intensity (MFI).

Figure 5 shows the in vivo application of anti-pig CD14 antibodies Mil2 and rMil2.

Healthy neonatal piglets (AD) were infused intravenously with increasing amounts of the original clone Mil2 (n=1) or rMil2 (n=1) and observed for 50 min. (A) After an initial small dose given within the first 10 minutes, Mil2 or rMil2 was administered every 5 minutes for 35 minutes. (B) Saturation of endogenous CD14 binding sites as a function of rMil2(s) or Mil2(d) concentration measured by flow cytometry. (C) Heart rate (HR) was recorded in real time throughout the experiment. (D) Platelet counts are given as a function of rMil2(s) or Mil2(d) concentrations. (EI). One piglet received a bolus dose of rMil2 prior to intravenous infusion of the bacteria, and the other piglet received saline as a control.