Lynn E. Spitler

Therapy of metastatic malignant melanoma using xomazyme® Mel, a murine monoclonal anti-melanoma ricin A chain immunotoxin☆

An approach to therapy of malignant disease is the use of monoclonal antibodies conjugated to a cytotoxic substance. Such cytotoxic substances may include chemotherapeutic agents, radionuclides or ribosomal inhibiting proteins. One example of the latter is ricin A chain, an enzyme which binds to ribosomes and inhibits protein synthesis, thereby causing cell death. The A chain is an extremely potent cell toxin, and the monoclonal antibody targets to the tumor cells thus making the product specific for the tumor, while sparing normal tissue. This report summarizes results of over 3 years of work sponsored by XOMA Corporation using its murine monoclonal anti-melanoma ricin A chain immunotoxin. Characteristics of the antibody used in the construct of this immunotoxin are shown in Table 1. The antibody is an IgG2a murine monoclonal antibody. It reacts with high molecular weight melanoma antigens which are present in all melanomas tested by XOMA to date. The antibody has no significant cross reactivity with surface antigens on normal tissues. This immunotoxin has been administered as a single course of therapy to over 100 patients with metastatic melanoma. Side effects have been well defined and are illustrated in Table 2. They consist of the following:

Experimental allergic encephalitis: Study of cellular immunity to the encephalitogenic determinant☆

Abstract Guinea pigs were tested for cellular immunity to the encephalitogenic tryptophan peptide, the major encephalitogenic determinant of central nervous system basic protein, representing residues 114 to 122 of the molecule. Guinea pigs sensitized with human basic protein regularly developed experimental allergic encephalitis, but did not show cellular immunity to the encephalitogenic tryptophan peptide as measured by skin test reactivity, lymphocyte stimulation, or macrophage migration inhibition, although they did show cellular immunity to the immunizing antigen, human basic protein. Animals sensitized with the synthetic tryptophan peptide also regularly develop clinical and histologic features of experimental allergic encephalitis, and show cellular immunity to the peptide but not to human basic protein. The work of others indicates that, in guinea pigs sensitized with the whole basic protein, there are determinants for cellular immunity located near the encephalitogenic tryptophan peptide. The test peptides used in these studies all included amino acid residues of the basic protein not included in the encephalitogenic tryptophan peptide used in our study. Our work indicates that the encephalitogenic peptide is not one of the determinants for cellular immunity in the basic protein molecule. Since cellular immunity to the disease-producing determinant of the molecule could not be demonstrated, this work further suggests that cellular immunity, as measured by the three tests described herein, may not necessarily be correlated with production of experimental allergic encephalitis.

Penetration of anti-melanoma immunotoxin into multicellular tumor spheroids and cell kill effects

SummaryIn order to gain a better understanding of the interaction between immunotoxins and tumor cells at the level of three-dimensional tumor mass, we evaluated the cell kill effects of monoclonal antimelanoma-antibody/ricin-A-chain immunotoxin (ITN) on melanoma cells in multicellular tumor spheroids (MTS) as well as the penetration of ITN into MTS. For Minor melanoma cells in monolayer the ITN exerted cytotoxic effects after as little as 1 h of exposure. Increasing exposure time resulted in progressive increases in cytotoxic activity. In contrast, the cell kill effects of ITN were markedly delayed and reduced when Minor cells were in MTS. The ITN cytotoxic effects on the melanoma MTS were more than 100 fold less than those in monolayer. Patterns of ITN-induced cytotoxicities for Minor and for another melanoma cell line, DND-1A, were comparable. The native ricin A was more active against PC-10 squamous lung cancer cells than Minor cells, whereas the ITN was more cytotoxic against Minor cells than PC-10 cells, thus exhibiting selectivity. An autoradiographic study revealed time-dependent penetration of radiolabeled ITN from the surface of Minor MTS into the core. Incubation for 1 h resulted in the penetration of ITN into only the two or three outer layers of the Minor MTS, and low grain counts. Prolonged exposure resulted in inhomogeneous penetration of ITN into almost the entire melanoma MTS. Penetration of ITN into PC-10 MTS was extremely poor. The reduced cytotoxicity of ITN on melanoma cells in MTS as compared to cells grown in monolayer appears to correlate with its inhomogeneous distribution in the MTS. The delayed cytotoxicity of ITN is also consistent with its slow penetration into the core of the MTS.

Immunotherapy of Melanoma

Are there tumor-associated transplantation antigens (TATAs) on human tumor cells? If there are, are human subjects capable of mounting an immune response to those TATAs? Does clinical malignancy develop because of a failure of the normal immunological response to the tumor? The answers to these very basic questions of tumor immunology are unknown at present, and it seems unlikely that they will be answered in the near future.