Effects of the intermolecular toxin-monoclonal antibody linkage on the in vivo stability, immunogenicity and anti-leukemic activity of B43 (anti-CD19) pokeweed antiviral protein immunotoxin.

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 vivo chemical, immunological, and biological stability, (b) in vivo immunogenicity, and (c) in vivo anti-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 the in vivo pharmacokinetic features, immunogenicity, and anti-leukemic activity of anti-CD19 PAP immunotoxins that were prepared with different heterobifunctional crosslinking agents.

Accessory receptors regulate coupling of the T-cell receptor complex to tyrosine kinase activation and mobilization of cytoplasmic calcium in T-lineage acute lymphoblastic leukemia.

T-lineage acute lymphoblastic leukemia (T-ALL) cells have abundant cytoplasmic CD3/Ti but express low amounts on the cell surface and are deficient in CD3/Ti-mediated signal transduction. Nevertheless, plating T-ALL cells on dishes containing immobilized anti-CD3 monoclonal antibodies with a source of growth factors induced the expression of CD25 (interleukin-2 receptor alpha chain) and stimulated the formation of blast colonies in 12 of 14 cases studied. The proliferative response to CD3 ligation was modulated by the presence of antibodies to the CD2, CD4, or CD8 accessory T-cell receptors. The effect of these accessory receptors on signal transduction mediated by CD3/Ti was next investigated by monitoring cytoplasmic calcium concentration [( Ca2+]i) and by measuring tyrosine phosphorylation after stimulation. Crosslinking CD3, CD2, CD4, or CD8 alone did not induce cytoplasmic calcium mobilization in T-ALLs, but crosslinking the accessory receptors with CD3/Ti induced calcium responses in three of the T-ALLs and enhanced calcium responses in three of the T-ALL cell lines, including HPB-ALL, MOLT-4, and CEM. Crosslinking CD4 but not CD2 with CD3/Ti greatly enhanced tyrosine phosphorylation of multiple substrates in comparison with crosslinking either CD4 or CD3/Ti separately on both normal mature T cells and the CEM T-ALL cell line. Thus, CD4 regulates CD3/Ti signal transduction in T-ALL cells through the tyrosine phosphorylation of substrates whereas CD2 may regulate [Ca2+]i signal transduction through a separate mechanism.

Production of a pokeweed antiviral protein (PAP)-containing immunotoxin, B43-PAP, directed against the CD19 human B lineage lymphoid differentiation antigen in highly purified form for human clinical trials.

We describe a standardized method for the preparation and purification of a potent immunotoxin against B-lineage leukemia/lymphoma cells, constructed with the ribosome inhibitory single chain plant toxin pokeweed antiviral protein (PAP) and a murine IgG1 monoclonal antibody (MoAb) specific for the human B lineage differentiation antigen CD19 for human clinical trials. PAP was prepared from spring leaves of Phytolacca americana plants by ammonium sulfate precipitation and purified to homogeneity by successive steps of ion exchange chromatography. B43 MoAb was produced in vitro by hollow fiber technology and purified to homogeneity by affinity chromatography. PAP toxin and B43 MoAb were modified via their free amino groups prior to their intermolecular conjugation. 2-iminothiolane was used to introduce reactive sulfhydryl groups into PAP and N-succinimidyl 3-(2-pyridyldithio) propionate was used to introduce 2-pyridyl disulfide bonds into B43 MoAb. Modified PAP was reacted with modified B43 MoAb resulting in a sulfhydryl-disulfide exchange reaction and yielding disulfide linked PAP-B43 MoAb conjugates, which we refer to as B43-PAP immunotoxin. B43-PAP immunotoxin was subjected to preparative gel filtration chromatography and cation exchange chromatography to obtain a highly purified, sterile, and pyrogen-free immunotoxin preparation with less than 5% free antibody contamination and less than 0.5% free PAP contamination. The final product displayed a high affinity for and a very potent anti-leukemic activity against B lineage leukemia cells. With slight modifications, the procedures detailed in this report should be generally applicable to preparation of other PAP-MoAb conjugates for treatment of cancer or AIDS.