We have successfully constructed highly potent and selective anti-CD19 PAP immunotoxins using each of the three crosslinking agents, SPDP, LC-SPDP, or SMPT, to generate an intermolecular bridge between the B43 MoAb and PAP toxin moieties. These immunotoxins were selectively immunoreactive with and cytotoxic against CD19′B-lineage ALL cells. In this report, we compared (a)in vivochemical, immunological, and biological stability, (b)in uiuoimmunogenicity, and (c)in vivoanti-leukemic activity of various B43-PAP immunotoxin constructs. Our data recommend the use of SPDP and SMPT rather than LC-SPDP for generation of B43(anti-CD19)-PAP immunotoxins as clinical anti-leukemic agents. To our knowledge, this is the first comparative analysis of thein vivopharmacokinetic features, immunogenicity, and anti-leukemic activity of anti-CD19 PAP immunotoxins that were prepared with different heterobifunctional crosslinking agents.