Afuco™ Anti-Influenza Virus M2e ADCC Recombinant Antibody (TCN-032), ADCC Enhanced (CAT#: AFC-488CL)

Figure 1 Anti-M2e mAbs TCN-032 and TCN-031 bind virus particles and virus-infected cells but not M2e-derived synthetic peptide.

(A) Purified in fluenza virus (A/Puerto Rico/8/34) was coated at 10 μ g/mL on ELISA wells and binding of anti-M2e mAbs TCN-031, TCN-032, ch14C2, and the HCMV mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc. (B) The 23mer synthetic peptide of M2 derived from A/Fort Worth/1/50 was coated at 1 μ g/mL on ELISA wells and binding of mAbs TCN-031, TCN-032, ch14C2, and 2N9 were evaluated as in A. (C) MDCK cells were infected with A/Puerto Rico/8/34 (PR8) and subsequently stained with mAbs TCN-031, TCN-032, ch14C2 and the HCMV mAb 5J12. Binding of antibodies was detected using Alexa fl uor 647-conjugated goat anti- Human IgG H&L antibody and quanti fi ed by fl ow cytometry. (D) HEK 293 cells stably transfected with the M2 ectodomain of A/Fort Worth/1/50 (D20) were stained with transient transfection supernatant containing mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FMAT for binding to M2 in the presence or absence of 5 μ g/mL M2e peptide. Mock-transfected cells are 293 cells stably transfected with vector alone.

Grandea, A. G., Olsen, O. A., Cox, T. C., Renshaw, M., Hammond, P. W., Chan-Hui, P. Y., ... & Pekosz, A. (2010). Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.Proceedings of the National Academy of Sciences, 107(28), 12658-12663.

Figure 2 Therapeutic efficacy of anti-M2 mAbs TCN-031 and TCN-032 in mice.

Mice (n = 10) were infected by intranasal inoculation with 5 × LD 50 A/Vietnam/ 1203/04 (H5N1) (A and B) or (n = 5) with 5 × LD 50 A/Puerto Rico 8/34 (H1N1) (C and D), followed by 3 i.p. injections with mAbs at 24, 72, and 120 h postinfection (a total of three mAb injections per mouse) and weighed daily for 14 d.

Grandea, A. G., Olsen, O. A., Cox, T. C., Renshaw, M., Hammond, P. W., Chan-Hui, P. Y., ... & Pekosz, A. (2010). Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.Proceedings of the National Academy of Sciences, 107(28), 12658-12663.

Figure 3 Binding of anti-M2e mAbs TCN-031 and TCN-032 to M2 mutants indicates the epitope is located in the highly conserved N terminal of M2e.

Mutants with alanine substituted at each position of the M2 ectodomain of A/Fort Worth/1/50 (D20) (A) or 40 wild-type M2 mutants, including A/Vietnam/1203/04 (VN) and A/Hong Kong/483/97 (HK) (B), were transiently transfected into 293 cells. The identity of each wild-type M2 mutant is listed in Table S4. Transfected cells were stained with mAbs TCN-031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2 at 24 h posttransfection. The mAbs TCN-031 and TCN-032 do not bind variants with amino acid substitutions at positions 1, 4, or 5 of M2e. (C) The deduced epitope for TCN-031 and TCN-032 occurs in a highly conserved region of M2e and is distinct from that found for ch14C2.

Grandea, A. G., Olsen, O. A., Cox, T. C., Renshaw, M., Hammond, P. W., Chan-Hui, P. Y., ... & Pekosz, A. (2010). Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.Proceedings of the National Academy of Sciences, 107(28), 12658-12663.

Figure 4 Anti-M2e mAbs TCN-031 and TCN-032 bind cells that have been infected with H1N1 A/California/4/09.

MDCK cells were infected with Influenza A strain H1N1 A/Memphis/14/96, H1N1 A/California/4/09, or mock infected. Twenty-four hours postinfection cells were stained with mAbs TCN031, TCN-032, or the control ch14C2 and analyzed by FACS for binding to M2.

Grandea, A. G., Olsen, O. A., Cox, T. C., Renshaw, M., Hammond, P. W., Chan-Hui, P. Y., ... & Pekosz, A. (2010). Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.Proceedings of the National Academy of Sciences, 107(28), 12658-12663.

Figure 5 A composite graph of daily influenza symptoms and viral load as determined by qPCR.

Median total daily influenza symptom scores and viral shedding. Abbreviation: TCID50, median tissue culture infective dose.

Figure 6 No differences in cytokine levels were detected between the TCN-032 and placebo groups.

Serum cytokine levels at day 3. Abbreviations: IFN, interferon; IL-8, interleukin 8; IL-10, interleukin 10; TNF, tumor necrosis factor.

Ramos, E. L., Mitcham, J. L., Koller, T. D., Bonavia, A., Usner, D. W., Balaratnam, G., ... & Swiderek, K. M. (2014). Efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza. The Journal of infectious diseases, 211(7), 1038-1044.