NeuroMab™ Anti-TLR2 BBB Shuttle Antibody(NRZP-1022-ZP2654)

Fig.1 Inhibitory effect of mAb T2.5 on host activation by microbial challenge in vivo.

Mice were pretreated i. p. with 1 mg mAb T2.5 (solid bars) or left untreated (empty bars). Mice were challenged after 1 h with P3CSK4 and D-galactosamine (i. p.), as well as sacrificed 2 h or 4 h later (n=4 for each group at each time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c), and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005, ***p<0.001, student's t-test for unconnected samples).

Fig.2 Effects of mAb T2.5 administration on viability upon TLR2-specific systemic challenging.

Mice were pretreated i. p. with 1 mg mAb T2.5 (solid bars) or left untreated (empty bars). Mice were challenged after 1 h with P3CSK4 and D-galactosamine (i. p.), as well as sacrificed 2 h or 4 h later (n=4 for each group at each time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c), and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005, ***p<0.001, student's t-test for unconnected samples).

Fig.3 Dose dependent binding of T2.5 to mTLR2ECD versus failed binding of TL2.1 to the same antigen in ELISA.

Mice were pretreated i. p. with 1 mg mAb T2.5 (solid bars) or left untreated (empty bars). Mice were challenged after 1 h with P3CSK4 and D-galactosamine (i. p.), as well as sacrificed 2 h or 4 h later (n=4 for each group at each time point). Serum concentrations of TNFα (a), GROα/KC (human IL-8 homologue) (b), IL-6 (c), and IL-12p40 (d) were analyzed by ELISA (*p<0.05, **p<0.005, ***p<0.001, student's t-test for unconnected samples).

Fig.4 Lack of interaction between TL2.1 and murine TLR2 in Immunoprecipitation and subsequent immunoblot analysis.

Lysates of 1×106 murine RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody as indicated, and 20 μl of protein G beads (Santa Cruz, Calif., USA) were mixed for o. n. precipitation. Immune complexes were analyzed by immunoblot analysis with either Flag- or murine TLR2-specific antiserum.

Fig.5 Inhibitory effect of recombinant single chain and partially humanized hT2.5 antibody on lipopeptide induced cell activation. HT2.5 was overexpressed in HEK293 cells and supernatant collected.

Lysates of 1×106 murine RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody as indicated, and 20 μl of protein G beads (Santa Cruz, Calif., USA) were mixed for o. n. precipitation. Immune complexes were analyzed by immunoblot analysis with either Flag- or murine TLR2-specific antiserum.

Fig.6 Application of mAb T2.5 for specific detection of TLR2.

Lysates of 1×106 murine RAW264.7 macrophages or HEK293 cells, 1 μg of each antibody as indicated, and 20 μl of protein G beads (Santa Cruz, Calif., USA) were mixed for o. n. precipitation. Immune complexes were analyzed by immunoblot analysis with either Flag- or murine TLR2-specific antiserum.