Supplementary Figure 1: Cell depletion activity of immunotoxins

(a) In vivo HSC depletion screen of candidate immunotoxins administered at 3 mg/kg in C57BL/6 mice. Bone marrow was harvested 8 days post-administration and HSCs quantified by flow cytometry. Non-treated C57BL/6 mice were used as controls. Data represent mean ± range (n = 2 mice/group, assayed individually). (b) Investigation of various ratios of biotinylated anti-CD45 antibody (clone 104) to streptavidin-saporin on in vivo HSC depletion activity assessed 8 days post-administration in C57BL/6 mice. Data represent mean ± SD (n = 4 mice/group, assayed individually). (c) Peripheral blood chimerism 4 months after competitive transplantation of bone marrow harvested from control or CD45-SAP conditioned mice demonstrates depletion of functional HSCs by CD45-SAP. Data represent mean ± SD (n = 5 mice/group, assayed individually). (d) CD45-SAP clone 104 does not deplete HSCs in congenic CD45.1 C57BL/6 mice. Data represent mean ± SD (n = 5 mice/group, assayed individually). (e) In vitro IC50 values against EL4 and EML cell lines after 72 hour incubation with CD45-SAP clones 104 and 30-F11. Data represent mean ± SD of 3 independent experiments. (f) In vivo HSC depletion by 3 mg/kg CD45-SAP created from clones 104 and 30-F11 assessed 8 days post-administration. Data represent mean ± SEM (n = 4 mice/group, assayed individually). (g) In vivo persistence 24 hours post-administration of AF488-labelled CD45 antibody clones 104 and 30-F11 in bone marrow progenitor (LKS) cells, peripheral blood white blood cells, and splenocytes. Data represent mean ± SD (n = 3 mice/group) of 1 of 2 independent experiments. * indicates p value <0.05; ** indicates p value <0.01; *** indicates p value <0.001; n.s. indicates not significant (p value >0.05). Statistics calculated using two-sided unpaired t test.