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In vitro measurements of the immune response in patients with cancer can be divided into those that estimate nonspecific and those that estimate tumor-specific immune responses. Contained herein is a review of these measurements, especially as they relate to studies that have been reported in patients with transitional cell carcinoma (TCC). In vitro tumor-specific immunity has been extensively examined in TCC using the lymphocyte-mediated microcytotoxicity assay, but subsequent observations on this assay have seriously jeopardized the validity of those early findings. Recent modifications of this assay have permitted longitudinal studies of lymphocyte cytotoxicity in TCC patients, and clinical correlations suggest that this modified assay may detect important immunological events. To date, however, a clinically useful classification of the TCC patient based on in vitro measurement of immune responses has not been achieved, although many promising areas still require investigation.

Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.

Human lymphocyte immunity to tumor-derived target cells was estimated by titrating lymphocyte concentration to achieve a 50% reduction of target cell survival. This lymphocyte titration assay gave estimates of cytotoxicity that were different from those obtained with the conventional cell-mediated cytotoxicity assays but were more proportional to lymphocyte activity. Estimates of cytotoxicity obtained using the lymphocyte titration assay were reproducible upon repeated testin over the course of several months and were relatively unaffected by two- to fourfold variations in target cell concentration. Target cell-specific cytotoxicity was reproducible but often did not appear to be tumor specific.