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A cocktail of humanized anti–pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

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Science Translational Medicine  02 Dec 2015:
Vol. 7, Issue 316, pp. 316ra195
DOI: 10.1126/scitranslmed.aad0966
  • Fig. 1. Humanized, chimeric, and murine antibodies have similar binding affinities to PTx.

    (A) SDS-PAGE comparing purified murine, chimeric (ch), and humanized (hu) variants of 1B7 and 11E6 in reduced and nonreduced forms as indicated. Molecular weight standards (kD) are shown in lane 1. (B) Indirect ELISA comparing the binding of all three versions of each antibody to PTx: murine (triangle), chimeric (circle), humanized (square), and isotype control (×) antibodies are shown. The absorbance data were normalized such that the maximum signal is 1.0; each sample was run in duplicate, and each assay was performed at least three times with different protein preparations.

  • Fig. 2. hu1B7 and hu11E6 antibodies have higher anti-PTx titers than high-titer P-IVIG.

    (A) hu1B7 and hu11E6 antibodies and high-titer P-IVIG were assessed for binding to PTx by ELISA. An ELISA plate was coated with PTx, blocked, and incubated with the indicated concentrations of P-IVIG, hu1B7, or hu11E6. (B) Sandwich ELISA was used to assess simultaneous binding of hu1B7 and hu11E6 antibodies according to the scheme indicated with goat anti-mouse–Fc–horseradish peroxidase conjugate (Gαm-Fc-HRP) used for detection. Each sample was run in duplicate, and each assay was repeated at least three times.

  • Fig. 3. The binary combination of hu1B7 and hu11E6 antibodies is synergistic and more potent than P-IVIG in vitro.

    The CHO cell clustering assay was used to determine the molar ratio of antibodies to PTx required for complete neutralization of CHO morphology changes. (A) Different ratios of hu1B7 and hu11E6 were compared to determine the most potent ratio. (B) An equimolar ratio of hu1B7 and hu11E6 was compared to the same ratio of murine antibodies and P-IVIG. Results depict average of three replicate experiments; statistical significance (*P < 0.05) was determined by single-factor analysis of variance (ANOVA) and Tukey’s test with α = 0.05. Ab, antibody.

  • Fig. 4. Prophylactic treatment with humanized antibodies protects mice against pertussis.

    Mice (n = 6) were each administered 20 μg of antibody intraperitoneally 2 hours before infection with 5 × 106 CFU B. pertussis D420 bacteria. (A to C) The infection severity was assessed on day 10 by (A) CD45+ leukocyte counts (WBC), (B) weight gain, and (C) bacterial colonization of the lungs. No bacteria were recovered from uninfected animals. Means ± SE are shown; significance (*P < 0.05, **P < 0.01, and ***P < 0.001) versus PBS treatment is indicated, using Tukey’s simultaneous test. Additionally, only P-IVIG–treated mice had WBC counts distinguishable from uninfected naïve mice (P < 0.01); mice treated with hu1B7, ch1B7, or the antibody cocktail had lower WBC counts than those treated with P-IVIG at equivalent doses (P < 0.05). Only mice treated with P-IVIG and ch11E6 exhibited reduced weight gain relative to uninfected mice (P < 0.05).

  • Fig. 5. Therapeutic treatment with antibody cocktail reduces leukocytosis and accelerates bacterial clearance in baboons.

    Weanling baboons were inoculated with 109 to 1010 CFU B. pertussis D420 bacteria on day 0. On day 3 after infection (indicated by arrow), animals in the treatment group (n = 4) were administered hu1B7 and hu11E6 antibodies intravenously, whereas control animals (n = 4) were given nothing. (A to C) All animals were subsequently monitored for (A) WBC count, (B) bacteria recovered by nasopharyngeal wash, and (C) coughing on the days indicated. Groups shown include controls (solid black icons, dashed line, C1 to C4), treated animals that were mildly colonized (gray icons, dotted black line, M1 and M2), and treated animals that were heavily colonized (hollow icons, solid gray line, H1 and H2). (D) Serum anti-Fha titers for individual animals were used to assess endogenous immune responses. Titers were normalized to the maximum response observed for serum from a historical control baboon, collected 3 weeks after experimental infection. Serum was not available for animal C3.

  • Table 1. Biochemical characterization of 1B7 and 11E6 antibodies.

    nd, not determined.

    Melting temperature (°C),
    second transition
    Kd, competition ELISA (nM)
    (no. of exp.)
    Kd, SPR (nM)
    χ2
    On-rate × 105,
    SPR (s−1 M−1)
    Off-rate × 10−4,
    SPR (s−1)
    m1B774.8 ± 0.70.4 ± 0.2 (5)0.7 ± 0.2 (0.32)1.7 ± 0.31.2 ± 0.3
    ch1B778.1 ± 0.50.5 ± 0.3 (3)0.5 ± 0.4 (0.74)1.5 ± 0.10.8 ± 0.5
    hu1B779.0 ± 0.31.2 ± 0.7 (6)0.7 ± 0.5 (0.75)0.9 ± 0.20.7 ± 0.5
    m11E667.3 ± 0.45 ± 1 (3)2.4 ± 0.9 (2.20)1.3 ± 0.73 ± 3
    ch11E669.4 ± 0.45 ± 2 (4)ndndnd
    hu11E674.4 ± 0.47 ± 3 (5)2.3 ± 0.7 (1.25)0.8 ± 0.41.7 ± 0.7

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/316/316ra195/DC1

    Fig. S1. Specific activity of humanized antibody variants.

    Fig. S2. Antibody thermal stability.

    Fig. S3. Competition ELISA to assess solution binding affinities of purified antibodies.

    Fig. S4. Binding kinetics of antibody-PTx interactions.

    Fig. S5. Pilot murine protection data with recent human clinical isolate D420 and m1B7.

    Fig. S6. Concentrations of anti-PTx antibodies in baboons.

    Fig. S7. Detection of the hu1B7-hu11E6 combination in the nasopharyngeal wash of baboons.

    Fig. S8. Histopathological analysis of lung tissue.

    Table S1. Humanized antibodies are more similar to the human repertoire than the original murine antibodies.

    Table S2. Baboon model challenge details.

    Table S3. Tabulated mouse challenge data.

    Table S4. Tabulated baboon challenge data.

  • Supplementary Material for:

    A cocktail of humanized anti–pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

    Annalee W. Nguyen, Ellen K. Wagner, Joshua R. Laber, Laura L. Goodfield, William E. Smallridge, Eric T. Harvill, James F. Papin, Roman F. Wolf, Eduardo A. Padlan, Andy Bristol, Michael Kaleko,* Jennifer A. Maynard*

    *Corresponding author. E-mail: maynard{at}che.utexas.edu (J.A.M.); mkaleko{at}syntheticbiologics.com (M.K.)

    Published 2 December 2015, Sci. Transl. Med. 7, 316ra195 (2015)
    DOI: 10.1126/scitranslmed.aad0966

    This PDF file includes:

    • Fig. S1. Specific activity of humanized antibody variants.
    • Fig. S2. Antibody thermal stability.
    • Fig. S3. Competition ELISA to assess solution binding affinities of purified antibodies.
    • Fig. S4. Binding kinetics of antibody-PTx interactions.
    • Fig. S5. Pilot murine protection data with recent human clinical isolate D420 and m1B7.
    • Fig. S6. Concentrations of anti-PTx antibodies in baboons.
    • Fig. S7. Detection of the hu1B7-hu11E6 combination in the nasopharyngeal wash of baboons.
    • Fig. S8. Histopathological analysis of lung tissue.
    • Table S1. Humanized antibodies are more similar to the human repertoire than the original murine antibodies.
    • Table S2. Baboon model challenge details.
    • Table S3. Tabulated mouse challenge data.
    • Table S4. Tabulated baboon challenge data.

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