of neoplasia was realized. A major breakthrough in such techniques was the discovery of the cytokine, IL-2, originally known as T-cell growth factor (Morgan et al., 1976). This discovery allowed the expansion or growth of T cells and NK cells from relatively small numbers taken from the blood of the tumor-bearing host.

Lymphokine-Activated Killer Cells

The realization of the methodology mentioned above in both animals and humans came about within a decade of the discovery of T-cell growth factor. Mononuclear cells were isolated from blood by a continuous-flow cell separator, allowing the reinfusion of mononuclear cell-depleted blood back into the same patient. Some 10 to 14 L of whole blood could be processed from a patient within 4 hours (Rosenberg et al., 1987). These cells were then subjected to one of several schemes, an example of which is noted in Figure 19.35. The adherent lymphokine-activated killer (LAK) cells, which were adherent to the culture dish, were those expanded by growth in cell culture. These cells are predominantly derived from NK cells (cf. Whiteside and Herber-man, 1990). Cumulative doses up to 1.5 x 1011 cells were administered intravenously over a period of 4 to 14 days together with IL-2 (Rosenberg et al., 1987). The principal neoplasms treated in this way were melanoma and renal cell carcinoma, with the average response from eight different studies of 16% and 22% respectively (Chang and Shu, 1992). Unfortunately, a variety of toxicities were associated with this type of therapy, and thus additional other modalities have been investigated.

Tumor-Infiltrating Lymphocytes

In an attempt to enhance the specificity of the LAK cell therapy, Rosenberg and colleagues studied a system in mice in which lymphocytes occurring directly within neoplasms, termed tumor-infiltrating lymphocytes (TILs), were cultured with IL-2 (Rosenberg et al., 1986). Their studies showed that in mice bearing micrometastases from various types of neoplasms, TILs are 50 to 100 times more effective in their therapeutic potency than are LAK cells. In humans, 60% to 70% of patients bearing a variety of different types of neoplasms allowed for the expansion of TIL successfully. Unfortunately, since it takes about 2 months to expand the population of TIL to therapeutic levels, the original neoplasm has usually grown significantly during this period in those cases in which this technique has been tried (cf. Schiltz et al., 1997). IL-2 is also adminis-

Figure 19.33 Basic steps in the production of a monoclonal antibody, beginning with the immunization of mice with the antigen of interest. Spleen cells that express the immunoglobulin (Ig+) of interest as well as express an enzyme used for selection, hypoxanthine pyrophosphorylribosyltransferase (HPRT+), are fused with malignant myeloma cells maintained in culture. The latter are HPRT- and Ig-. Fusion of these cells results in self fusion of both myeloma and spleen cells seen on the right and left respectively, as well as a hybrid between the two cell types, seen in the middle. Utilization of a selection medium (HAT) containing both hypoxanthine and thymine as well as amethopterin allows the selection of myeloma hybrids that can bypass the amethopterin block by the presence of HPRT and thymidine kinase from the spleen cell. a

The fused myeloma cell is unable to do this and dies, while the fused spleen cells tend not to grow. One d then clones the fused cell termed a hybridoma, each clone producing only a single antibody species for the reasons discussed earlier in this chapter. From this, one may obtain large amounts of monoclonal antibody by culture of the hybridoma cells or inoculation into mice as an ascites, which also produces large amounts of the monoclonal antibody. Hybridoma cells may be frozen for future use. (Adapted from Levy, 1985, with permission of the author and publisher.)

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