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Considerable heterogeneity in the amount of surface antigen can regularly be demonstrated by cytofluorometric analysis among genetically identical cells in a tumor clone. We have used monoclonal idiotype-specific antibodies to investigate the patterns of change in amounts of an idiotypic tumor antigen and how such changes affect the immune escape of malignant B cells expressing this antigen. By the use of fluorescence-activated cell sorting, we separated cells expressing either very large or very small amounts of the idiotypic target antigen and then analyzed these subpopulations of tumor cells at various times after isolation for expression of idiotype. We found that the differences in amount of antigen expression were not heritable, and that over a period of about 7 days of continuous growth in vitro, the fluorescence-activated cell sorted populations gradually came to express normal amounts of idiotype. The mechanisms regulating the quantity of surface idiotype were independent of those affecting the amounts of other membrane-associated molecules such as H-2 antigen. Furthermore, these nonheritable intraclonal differences in amounts of antigen expression were unrelated to stages of the cell cycle but clearly did affect the susceptibility of the cells from immune lysis. Thus, tumor cells expressing the lowest amount of surface idiotype were much more resistant to the lytic effects of antiidiotypic antibody and complement but had the same cell cycle distribution as did unseparated cells. These results demonstrate that nonheritable, non-cell cycle-related heterogeneity in amount of tumor antigen expression can significantly determine which cells in a cloned malignant cell population preferentially escape monoclonal tumor-specific antibody therapy.

We investigated the effect of three monoclonal anti-idiotype antibodies (anti-M104E) on various functions of MOPC 104E myeloma cells in vitro. The antibodies used were N-20-2 [immunoglobulin M (IgM), BALB/c], SJL18-1 [IgM, BALB/c X SJL F1], and CD3-2 [immunoglobulin G1 (IgG1), BALB/c X A/J F1]. The two IgM antibodies were very efficient in blocking surface M104E IgM as shown by rosette inhibition, whereas the IgG1 isotype was not very effective. The reexpression of surface M104E IgM was different from antibody to antibody. The secretion of M104E IgM by MOPC 104E cells was partially blocked by the two IgM antibodies, but the IgG1 antibody had no effect. All three anti-idiotype antibodies inhibited the stem cell renewal activity of MOPC 104E cells assayed by colony formation assay. On the other hand, in suspension culture, the two IgM antibodies inhibited the growth of MOPC 104E cells in the absence of complement or effector cells of antibody-dependent cellular cytotoxicity, but IgG1 antibody had no effect. The starting tumor inoculum size was critical in the observations of the effects seen on both the growth and the colony-forming activity of MOPC 104E cells. The results of this study show the functional differences between various monoclonal anti-idiotype antibodies and also indicate that some anti-idiotype antibodies can inhibit the growth of MOPC 104E myeloma cells directly without any help of complement or effector cells of antibody-dependent cellular cytotoxicity.