Doxorubicin (Dox) was coupled by four published methods to a murine monoclonal antibody (MAb) developed against human pulmonary adenocarcinoma. Dox immunoconjugates made with this murine IgG1 MAb, 44-3A6, were evaluated for anti-tumor activity against the human lung cancer cell line, A549. Dox was attached to the MAb (1) by an oxidized dextran T40 intermediate, (2) using dilute glutaraldehyde cross-linking, (3) with an acid-sensitive linker using cis-aconitic anhydride and EDCI, a water-soluble carbodiimide, and (4) using EDCI alone. The biological activity of the different conjugates was compared in vitro by antigen binding (whole-cell RIA) versus serial dilutions of unconjugated MAb cytotoxicity (dye exclusion/viability) versus long dilutions of the various Dox conjugates, Dox and mixtures of Dox and MAb. Preincubation of antigen-expressing tumor cells (A549) for 2 h with excess (250 micrograms/ml) unconjugated MAb prior to conjugate exposure was attempted in order to block the cytotoxic effect. The number of Dox molecules conjugated to 44-3A6 (molar incorporation ratio or MIR) ranged from 3 to 10/1 for carbodiimide linkage and up to 63/1 using the dextran intermediate. Cis-aconityl-derivatized Dox conjugates contained an average of 22 mol Dox/mol immunoglobulin, but drug incorporation was quite variable from experiment to experiment. Dilute glutaraldehyde cross-linking produced an average MIR of 10/1. After repeated attempts to minimize drug and/or MAb precipitation, the percentage decrease (versus MAb) in immunoreactivity of the drug conjugates ranged from 2 to 42% and was dependent on the coupling method and extent of aggregate formation in the preparation. Loss of biological activity (antigen binding and cytotoxicity) was significant when aggregation and precipitation occurred. There were additional losses (17-25%) after sterile filtration through low protein-binding (0.22 microns) filters. Immunoconjugates produced by glutaraldehyde cross-linking were reproducibly 5-10 times more potent against antigen-bearing tumor cells than Dox, and showed selectivity for inhibiting the viability of antigen-positive A549 cell line. Noncovalent mixtures of 44-3A6 and Dox were slightly more potent than Dox. Immunoconjugates produced by the aconityl method and the dextran intermediates were less effective than Dox, the glutaraldehyde-mediated conjugates or Dox and 44-3A6 mixtures. The unconjugated MAb was not cytotoxic when tested at concentrations up to 500 micrograms/ml. Blocking studies using 'cold', unlabeled MAb were of limited success.(ABSTRACT TRUNCATED AT 400 WORDS)