Influence of antidrug antibodies on plectasin efficacy and pharmacokinetics

Abstract

Plectasin is a 4.4-kDa antimicrobial peptide with the potential to be a treatment of infections caused by gram-positive bacteria. Since plectasin is a large molecule compared to conventional antibiotics, the development of antidrug antibodies (ADAs) could be anticipated. The immunogenic properties of plectasin were assessed through immunization studies. In mice treated for 5 days with one to two daily subcutaneous doses of plectasin, no antibody response was observed. If the animals were immunized again, after a rest period, low levels of antibodies developed in approximately half the animals. Additionally, mice were immunized with plectasin in Freund's incomplete adjuvant (FIA). Ninety-two percent of these mice developed ADAs after repeated immunizations, with two-thirds having high levels of antibodies. An agar diffusion bioassay showed that sera from animals immunized with plectasin did not inhibit the efficacy of the drug, while hyperimmune sera from animals in which an immune response was provoked by immunization with plectasin in FIA reduced the efficacy of plectasin at the lowest concentration tested. Studies in the murine peritonitis model showed an excellent efficacy of plectasin for the treatment of Streptococcus pneumoniae infections both in naïve animals and in animals with ADAs. No difference in bacterial counts was seen when the animals were treated with plectasin at 2.5 mg/kg of body weight, a dose below the expected therapeutic level. When animals were treated with plectasin at 0.625 mg/kg, the effect was reduced but not neutralized in animals with high levels of ADAs. No animals showed signs of hypersensitivity or injection site reactions toward plectasin, and the half-life of the compound did not vary between animals with and without antibodies.

FIG. 1.

IgG response (measured by ELISA) in mice after a single dose (A) or repeated daily dosing (B) s.c. with plectasin. Imm., immunization period. The cutoff value is defined as 1.625 times the OD value for naïve animals.

FIG. 2.

IgG response (measured by ELISA) after repeated s.c. immunizations in animals immunized with plectasin in FIA (n = 12) (A) or plectasin alone (n = 12) (B). Imm, immunization occasion.

FIG. 3.

Mouse peritonitis model. Log CFU counts of S. pneumoniae in peritoneal wash 4 h after treatment with plectasin. Half of the animals were treated with 0.625 mg/kg (A), and the other half were treated with 2.5 mg/kg (B). Group 1, preimmunized with vehicle; group 2, preimmunized with adjuvant (FIA); group 3, preimmunized with plectasin at 10 mg/kg; group 4, preimmunized with 2.5 mg/kg plectasin in FIA. Untreated animals served as controls for bacterial growth in the peritoneal fluid. The median numbers of CFU/ml were compared between groups with and without antibodies against plectasin. The bacterial counts in none of the groups differed significantly. The counts for all groups were significantly different (P < 0.05) from the count for the untreated control group. The dashed lines represent the numbers of CFU at the start of treatment.

FIG. 4.

Mouse peritonitis model. Log CFU counts of S. pneumoniae in peritoneal wash 4 h after treatment with 0.625 mg/kg or 2.5 mg/kg plectasin. Colony counts are shown for animals that were preimmunized with plectasin (with or without adjuvant) and that developed either low or high (greater than five times the cutoff value) levels of antibodies compared to those in the control animals immunized with either vehicle or adjuvant alone. Animals with high antibody levels treated with low doses of plectasin had CFU counts significantly higher than those in animals with low levels of antibodies and controls. **, P < 0.01; *, P < 0.05.

FIG. 5.

Elimination of plectasin in serum after s.c. dosing of mice with 10 mg/kg. Prior to the kinetics study, mice were immunized with either vehicle, adjuvant (FIA), or plectasin in FIA on four occasions. Mice immunized with plectasin developed various levels of ADAs, noted as plectasin high (antibody level greater than five times the cutoff level) and plectasin low (antibody levels close to the cutoff value).