Karl Erik Hellstrom

22 – COLONY INHIBITION AND CYTOTOXICITY ASSAYS

Publisher Summary nThis chapter describes the colony inhibition technique and the cytotoxic test. The colony inhibition technique has a high sensitivity and reproducibility, while the cytotoxic test has the additional advantage that it needs small amounts of material and modest skill for the final counting. Method for colony inhibition test was first worked out to study cytotoxic humoral antibodies. Test for humoral cytotoxic antibody involves trypsinizing the target cells adhering to the bottle mildly and briefly. Next step involves adding a few ml of calf serum or medium with calf serum to inactivate the trypsin. One should add 0.1 ml of the serum being tested for cytotoxic antibody to all the wells except those in one column, which will serve as controls. This serum may be diluted as desired in complete Waymouths medium.

Quantitative imaging of mouse L-6 monoclonal antibody in breast cancer patients to develop a therapeutic strategy

L-6, a mouse IgG2a anti-adenocarcinoma monoclonal antibody (MoAb) with favorable immunopathology and mouse biokinetics, was evaluated for cancer radioimmunotherapy by pharmacokinetic studies in 10 patients with breast cancer. The effect of escalating the preinfused protein dose was studied in two patients at each level, using 50, 100, 150, 200 and 400 mg of unlabeled L-6 prior to a 10 mCi imaging dose of 131I L-6. Quantitative imaging, and blood and urine clearances were obtained. After the 50 mg preinfusion, rapid blood clearance and lung extraction of the radiopharmaceutical occurred immediately post injection. Greater preload amounts of L-6 were associated with an increase in the intercept of the slow phase of the blood clearance from 17 to 22% injected dose (ID) with 50 mg to 70 to 80% ID with 400 mg (P less than 0.01). Lung uptake of the radiopharmaceutical immediately post injection decreased from 15 to 19% ID (50 mg) to 6 to 8% ID (400 mg). Tumors were visualized only after larger L-6 preloads, but in these patients small chest tumors contained 0.6-1.2% ID (0.1% ID/g maximum). This study suggests that L-6 reactive sites that are readily available in the lung can be saturated, so that a subsequent dose of I-131 L-6 is delivered to the tumor. This approach provides a new strategy for developing an effective method for radioimmunotherapy using a MoAb that has some cross-reactivity. Quantitative imaging contributed to detection of the cross-reactivity and the strategy for overcoming it.

Recent Advances in Antitumor Vaccines

Immunization with anti-idiotypic antibodies can induce cell-mediated and humoral antitumor immunity in animal models. This immunity can sometimes cause tumor destruction. However, more needs to be learned about how best to induce the type of immune response that is responsible for tumor destruction, since the presence of anti-idiotypic antibodies has been shown occasionally to enhance, rather than to inhibit, tumor growth. There is evidence suggesting that immunization of human cancer patients with Ab2 can have therapeutic benefit, and also that patients who mount a vigorous Ab2 response following treatment with an Ab1 may do clinically better than those who do not make any Ab2. Although the generation of Ab2 related to infused antitumor Ab1 does not cause tumor rejection in the majority of patients, and although the clinical data from patients given Ab2 are scarce, the suggestion that Ab2 may cause destruction of human cancers indicates that further work in this area may become rewarding.

Mouse Antibodies to Carcinomas

Many monoclonal antibodies (Mabs) have been created by immunizing mice with human carcinomas. In the vast majority of cases, these Mabs define tumor-associated differentiation antigens (TADAs), which have relative but not absolute specificity for neoplastic cells (Reisfeld, 1986; Roth, 1986; Sikorska, Shuster, and Gold, 1988; Bast, 1989; K. E. Hellstrom and Hellstrom, 1989a). Although more tumor-specific Mabs would be preferable, many potential therapeutic uses of the Mabs are already available, and several have been tested clinically and given promising results.

CYTOTOXIC LYMPHOCYTES, BLOCKING SERUM FACTORS AND “UNBLOCKING” ANTIBODIES IN CANCER PATIENTS

Publisher Summary This chapter explains cytotoxic lymphocytes, blocking serum factors, and unblocking antibodies in cancer patients. The majority of tumors in experimental animals have tumor-specific antigens, which can be detected with a variety of techniques. Both humoral and cell-mediated immune reactions against tumors have been identified. A major part of the immunological defense against cancer is cell-mediated as is apparent also from the fact that animals (or human patients) whose cell-mediated immunity is depressed are more prone to develop cancers than are immunologically capable individuals. Some studies have indicated that murine bladder tumors may provide good animal models: bladder papillomas and carcinomas of the rat cross-react antigenically, also when they have been induced by different carcinogenic agents, and they behave similarly, therefore, to bladder carcinomas of man. Chemically induced bladder carcinomas of mice also share a common antigen. According to preliminary studies in rats, it is possible to delay the appearance of primary chemically induced bladder papillomas by immunizing the animals with bladder tumor tissue before the chemical carcinogen is applied. These studies indicate that an immunological reaction against common bladder tumor antigens may be beneficial to the host.

Immunity to Human Tumor Antigens

It has been well established during the last 10–15 years that most (all?) experimentally induced animal tumors possess specific antigens against which an immunity can be demonstrated by in vivo and in vitro techniques (Old and Boyse1964; Sjogren 1965; Klein 1966; Hellstrom and Hellstrom, 1969 b, 1970 b).Evidence that human neoplasms are antigenic as well has been forthcoming more recently. Some of this evidence will be summarized here, primarily by providing references. No attempts will be made to go into details, since these are covered in two recent review articles (Hellstrom and Hellstrom 1969 b, 1970 b, c; Klein 1970; Gold 1970).