Afuco™ Anti-Human NTRK1 ADCC Recombinant Antibody (MNAC13), ADCC Enhanced (CAT#: AFC-668CL)

Figure 1 MNAC13 specifically binds TrkA and leads to inhibition of NGF-dependent signaling.

(A) The MNAC13 antibody (here represented as monovalent antigen binding fragment) binds to TrkA extracellular domain (ECD), preventing TrkA from being activated by its natural ligand NGF. (B) MNAC13 does not bind to the p75 receptor: Surface Plasmon Resonance experiments were performed by using a BIACore instrument (BIACore, Uppsala, Sweden). (C) Western blot results showing that MNAC13 leads to reduction of phospho-TrkA levels after NGF stimulation in mouse 3T3 cells overexpressing TrkA. (D) Western blot results showing that MNAC13 leads to inhibition of NGF-dependent phosphorylation of the PLC 1 adaptor protein in mouse 3T3 cells overexpressing TrkA. In C and D, cells were incubated for 60 min at 37°C in the presence of MNAC13 monoclonal antibody or irrelevant monoclonal antibody SV5 (mock), or no monoclonal antibody (No Mab). Antibodies were used at two different concentrations: 300 g/ml (h) and 200 g/ml (l). NGF at 100 ng per milliliter was added for 10 min at 37°C after preincubation with monoclonal antibodies. In the negative control, NGF treatment was omitted.

Ugolini, G., Marinelli, S., Covaceuszach, S., Cattaneo, A., & Pavone, F. (2007). The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.Proceedings of the National Academy of Sciences, 104(8), 2985-2990.

Figure 2 Analgesic effects of MNAC13 on inflammatory pain in CD1 mice.

(A) Comparison between licking behavior responses of saline (SAL: n 7) and irrelevant IgG (60 g per mouse, n 10) s.c.-injected mice, during the early and late phases of formalin test. (B) Dose/response effect of MNAC13, s.c.-injected into the right hindpaw 18 h before testing, on the early (0-15 min) and late (15-40 min) phase of formalin test (IgG: 60 g, n 9; MNAC13 mAb: 0.9 g, n 8; 1.875 g, n

Ugolini, G., Marinelli, S., Covaceuszach, S., Cattaneo, A., & Pavone, F. (2007). The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.Proceedings of the National Academy of Sciences, 104(8), 2985-2990.

Figure 3 Antiallodynic effects measured with a dynamic plantar aesthesiometer after four i.p. injections of 50 g per mouse MNAC13 (from day 3 to day 6 after ligation of the sciatic nerve) in CCI mice tested for 14 days (IgG, n 7; MNAC13, 50 g per mouse, n 5)

(A) and after eight i.p. injections of 30 or 70 g per mouse MNAC13 (from day 3 to day 10 after ligation of the sciatic nerve) in CCI mice tested from day 3 up to day 31 (B). (IgG, n 11; MNAC13, 30 g per mouse, n 8; MNAC13, 70 g per mouse, n 12.) Black arrows represent the antibody i.p. administrations. BL (baseline) refers to mechanical threshold before operation. Black lines represent mechanical thresholds of sham-operated controls (n 6). Data are presented as means SEM. *, P 0.05; **, P 0.01 vs. IgG-injected mice (Tukey/Kramer).

Ugolini, G., Marinelli, S., Covaceuszach, S., Cattaneo, A., & Pavone, F. (2007). The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.Proceedings of the National Academy of Sciences, 104(8), 2985-2990.

Figure 4 The combined effect of ineffective doses of MNAC13 (1 g per mouse) and morphine (Mo 1 mg/kg)

(A) or fentanyl (FEN 5 g/kg) (B) results in analgesia on inflammatory pain similar to analgesia induced by effective doses of the opiates (morphine 2.5 mg/kg and fentanyl 10 g/kg). The anti-TrkA antibody shows synergistic action with the opiates both in the early (0-15 min) and late (15-40 min) phases of formalin test. Irrelevant IgG (1 g per mouse) and saline-injected (Sal) mice are used as control groups for MNAC13 and opioids, respectively. Naloxone methiodide (Nx-met 10 mg/kg) does not modify the response to MNAC13 plus morphine combination. (A) IgG Sal, n 8; MNAC13 Sal, n 9; IgG Mo 1 mg/kg, n 9; MNAC13 Mo 1 mg/kg, n 11; MNAC13 Nx-met Mo 1 mg/kg, n 9; IgG Mo 2.5 mg/kg, n 9; (B) IgG Sal, n 10; MNAC13 Sal, n 11; IgG FEN 5 g/kg, n 6; MNAC13 FEN 5 g/kg, n 6; IgG FEN 10 g/kg, n

Ugolini, G., Marinelli, S., Covaceuszach, S., Cattaneo, A., & Pavone, F. (2007). The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.Proceedings of the National Academy of Sciences, 104(8), 2985-2990.