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Conjugates of tumor-reactive antibody and toxins (immunotoxins) have been used to eradicate tumor cells in vitro and in vivo. Such immunotoxins are highly effective in killing murine leukemic cells in infiltrated bone marrow and should be useful in the bone marrow rescue approach for the treatment of cancer. Similar immunotoxins injected parentally can help to induce prolonged remissions in leukemic mice, and antigen-containing immunotoxins can induce immunologic unresponsiveness in vitro in normal murine splenocytes. Thus, long-range goals for the parental use of immunotoxins include the killing of cancer cells in vivo and the modulation of the immune response for therapeutic purposes.

Highly specific antibodies (affinity-purified or hybridoma) directed against cell surface immunoglobulins on normal or neoplastic murine B lymphocytes were covalently coupled to the A chain of the plant toxin ricin. Such conjugates containing antibodies specific for IgM, for either of the two alloes of IgD, or for the idiotype of the B cell tumor BCLI rapidy bound m vitro to cells expressing the corresponding surface antigen and inhibited protein synthesis in such cells. The results demonstrate that A chain-coupled anti-idiotype antibody may be useful as a tumor-specific cytotoxic agent.The possibility of utilizing the exquisite specificity of antibodies to direct cytotoxic agents to tumor cells has been considered since the studies of Ehrlich (1). Studies usipg drug-antibody conjugates for this purpose have been hampered by the difficulty of raising antiserum specific for tumor cells, the inability in preparing purified antibody for drug conjugation, and the problems of preserving both pharmacologic and antibody activity after production of hybrid molecules (2). Several recent developments suggest that these obstacles can be surmounted and that the time is ripe for a reinvestigation of the above approach. First, the development of techniques for the generation of somatic cell hybrids secreting monoclonal antibody (3) and successful application of affinity chromatography of conventional antisera (4) make it possible to prepare highly purified antibody. Second, it has been demonstrated that the idiotype of the cell surface Ig of a B cell tumor represents a clonally expressed tumor specific marker (5). Finally, many potent protein toxins such as ricin, abrin, and diphtheria toxin have been shown to consist of a toxic portion (A chain) covalently bound to a portion (B chain) that can bind to surface moieties on cells and thereby facilitate entry of the toxic peptide into the cell (6, 7). The internalized toxic peptide kills the cells by catalytic inhibition of protein synthesis. By substituting specific antibody for the B chain, it should be possible to direct the peptide to cells for which the antibody is specific.In the present study, affinity-purified or hybridoma antibodies directed against immunoglobulin isotypes, allotypes, or idiotypes expressed on the surface of normal or malignant murine B lymphocytes were conjugated to the A chain of ricin. Such antibody conjugates were incubated with cells cultured in vitro and their cytotoxic effects were evaluated. The results indicate that the hybrid molecules maintain both their antigen-binding capacity and their toxic properties and that minute amounts of the conjugates are effective in specifically killing target cells in vitro. (8), Ricin A chain was concentrated by vicuum dialysis against 10 mM phosphate-buffered saline (Pi/NaCl), pH 7.0, containing 0.05% 2-mercaptoethanol. Thiol groups were introduced into affinity-purified or monoclonal antibodies (9) by using a 30-fold excess of N-succinimidyl-3-(3-pyridyl dithio)propionate (Pharmacia); After introdu...

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