Table 3

Cytotoxic potency (EC50) of rLF preparations in the presence of different concentrations of rPA in the macrophage lysis assay (MLA) using J774A.1 cells

rLF preparationaEC50 [ng/ml; means (SD) from 6 tests] at the indicated rPA concn (ng/ml)b:
1520305080100120140
rLF-A6 (1.1)3.8 (0.7)2.5 (0.4)1.7 (0.3)1.1 (0.2)1 (0.2)0.9 (0.2)ND
rLF-HMALNRNRNR6.5 (1.2)5.1 (0.8)4.3 (0.6)4 (0.5)3.7 (0.4)
rLF-HMAENRNRNRNRNR16.1 (2.9)13.8 (2.1)12.1 (2.1)
rLF-d8.1 (1.7)5.7 (0.9)4.6 (0.2)3.9 (1.7)ND1.5 (1)NDND
  • a Different concentrations of each rLF preparation were evaluated for cytotoxic potency in the presence of different concentrations of rPA (rLF-A, native LF sequence; rLF-HMAE and rLF-HMAL contain additional N-terminal Met and His residues and were produced by Emergent BioSolutions and List Biological Laboratories, respectively; rLF-d was derived from rLF-HMAE digested with N-terminal dipeptidase, which removed 2 or 4 N-terminal residues).

  • b rLF dose-response curves that demonstrated 100% cytotoxicity were used to calculate the EC50 using a sigmoidal dose-response variable slope fit analysis of log10(rLF) concentration curves using GraphPad Prism 5.04. Data were from a single rLF lot that was representative of 4 lots of rLF-HMAE, 2 lots ofrLF-d, and 4 lots ofrLF-A with similar results. ND, not determined; NR, not reported (EC50s of curves that did not reach 100% cytotoxicity were not reported, because accurate comparisons of curves require similar degrees of cytotoxicity).