Gary B. Thurman

Constant-Infusion Recombinant Interleukin-2 in Adoptive Immunotherapy of Advanced Cancer

Adoptive immunotherapy involving bolus-dose recombinant interleukin-2 (rIL-2) has been reported to induce tumor regression in some patients with cancer, but has been associated with severe fluid retention and cardiopulmonary stress. In an effort to preserve the efficacy but reduce the toxicity of this treatment, we used escalating doses of rIL-2 as a constant infusion rather than as a bolus dose. Forty-eight patients with advanced cancer received rIL-2 as a 24-hour infusion in five-day cycles separated by five-day periods of rest and leukapheresis. Eight patients were removed from the study before receiving cells activated in vitro. In the 40 who could be evaluated for their response, there were 13 partial responses (32.5 percent) and 2 minor responses. Partial responses were observed in Hodgkins disease (one of one), non-Hodgkins lymphoma (one of one), lung cancer (one of five), ovarian cancer (one of one), parotid cancer (one of two), renal cancer (three of six), and melanoma (five of ten). Responses were associated with a good performance status, a base-line lymphocyte count above 1400 per cubic millimeter, and an rIL-2-induced lymphocyte count of at least 6000. Optimal lymphocytosis required a priming dose of rIL-2 of 3 X 10(6) U per square meter of body-surface area per day, and 15 of 28 patients receiving this priming dose responded to treatment. A weight gain of more than 10 percent of total body weight (five patients) and dyspnea at rest (six patients) were unusual events restricted to patients with poorer pretreatment performance. We conclude that the administration of rIL-2 as a constant infusion may preserve the antineoplastic activity of adoptive immunotherapy while increasing the safety and comfort of patients.

In vitro stimulation of murine spleen cells using a microculture system and a multiple automated sample harvester

Abstract A microculture system is described to study the in vitro properties of murine spleen cells utilizing as few a 6 × 10 5 cells per culture. The use of a new multiple automated sample harvester enabled rapid multiple tests using relatively few cells. This new procedure was highly reproducible and diminished the source of many errors. Data are presented to show the many variables that are involved in studying the PHA and PWM responses of murine spleen cells in this microculture system. The procedure provides a relatively simple technique for studying the various in vitro effects of specific and non-specific stimulation of leukocytes especially when very few cells are obtainable or in small animals where pooling of the cells lead to masking individual variations.

Current Status of Thymosin and Other Hormones of the Thymus Gland

Publisher Summary This chapter discusses the current status of the chemistry, biology, and clinical applications of the well-defined thymic hormones. The first biologically active polypeptide to be isolated from among the highly acidic components of bovine thymosin fraction 5 has been termed thymosin α1. This peptide is highly active in amplifying T-cell immunity and is active in modulating the expression of terminal deoxynucleotidyl transferase. The ultimate application of the thymosins and other thymic hormones and factors in cancer treatment should be in providing a means of safely augmenting specific T lymphocyte functions in patients with diminished thymic-dependent immunity. In anergic cancer patients, thymic hormones may be of importance as an adjunct to conventional treatments by increasing T-cell function in response not only to tumor cells but also to pathogens, thus reducing the high incidence of infection that often accompanies cancer treatment.

Current status of thymosin research: evidence for the existence of a family of thymic factors that control T-cell maturation*..

Thymosin fraction 5 contains several distinct hormonal-like factors which are effective in partially or fully inducing and maintaining immune function. Several of the peptide components of fraction 5 have been purified, sequenced and studied in assay systems designed to measure T-cell differentiation and function. These studied indicate that a number of the purified peptides act on different subpopulations of T-cells (see Figure 1). Thymosin beta 3 and beta 4 peptides act on terminal deoxynucleotidyl transferase (TdT) negative precursor T-cells to induce TdT positive cells. Thymosin alpha 1 induces the formation of functional helper cells and conversion of Lyt- cells to Lyt 1+, 2+, 3+ cells. Thymosin alpha 7 induces the formation of functional suppressor T-cells and also converts Lyt- cells to Lyt 1+, 2+, 3+ cells. These studies have provided further evidence that the thymus secretes a family of distinct peptides which act at various sites of the maturation sequence of T-cells to induce and maintain immune function. Phase I and Phase II clinical studied with thymosin in the treatment of primary immunodeficiency diseases, autoimmune diseases, and cancer point to a major role of the endocrine thymus in the maintenance of immune balance and in the treatment of diseases characterized by thymic malfunction. It is becoming increasingly clear that immunological maturation is a process involving a complex number of steps and that a single factor initiating a single cellular event might not be reflected in any meaningful immune reconstitution unless it is the only peptide lacking. Given the complexity of the maturation sequence of T-cells and the increasing numbers of T-cell subpopulations that are being identified, it would be surprising if a single thymic factor could control all of the steps and populations involved. Rather, it would appear that the control of T-cell maturation and function involves a complex number of thymic-specific factors and other molecules that rigidly control the intermediary steps in the differentiation process.

Lymphokines, monoclonal antibodies, and other biological response modifiers in the treatment of cancer

Biologicals and biological response modifiers (BRMs) represent a new class of agents for cancer therapy. Historically, there have been many attempts to stimulate the immune response with nonspecific immunomodulators in the form of bacterial extracts, viruses, and chemicals. Although these approaches have occasionally proven useful under defined conditions in experimental models, their extension to the clinic has been largely unsuccessful. Recent advances in molecular biology and hybridoma technology have made available genetically engineered lymphokines and cytokines, as well as monoclonal antibodies, as highly purified biologicals for cancer treatment. These agents may act directly on tumor cells and/or may act on the patients own biological responses to induce an antitumor response. Selective defects in T‐cell function have recently been identified in cancer patients and in patients with acquired immunodeficiency syndrome (AIDS). Simultaneously, the availability of gamma interferon (γ‐IF) and interleukin‐2 (IL‐2) may allow for the selective correction of these T‐cell deficits, leading to restoration of the patients immune responses and perhaps correction of the clinical syndromes. Preliminary data suggest that γ‐IF and IL‐2 have in vitro activity on these T‐cell defects, and the preliminary evidence that these agents have activity in vivo will be reviewed. Extensive trials are being conducted at the National Cancer Institute with monoclonal antibodies as anticancer agents. Animal model experiments have demonstrated considerable antitumor activity of immunoconjugates using monoclonal antibodies tied to toxins. Preliminary clinical results suggest that T‐101 in leukemia and lymphoma and 9.2.27 in malignant melanoma may prove useful as specific reagents in the treatment of these disorders. While the antitumor effects with these antibodies have not been dramatic, our preliminary data in approximately 30 patients with leukemia, lymphoma, and melanoma clearly demonstrate the ability of intravenous monoclonal antibody to locate and specifically lable tumor cells bearing the target antigens. It has been possible to localize antibody on the tumor cells in melanoma deposits that are barely visible in the skin. These data and radioimaging data suggest a future role for immunoconjugates as anticancer agents.

An improved method for the generation of human lymphokine activated killer cells

In an effort to make interleukin-2/lymphokine-activated killer cell (IL-2/LAK) therapy safer for cancer patients, we examined the efficacy of using Fenwal PL732 bags as tissue culture flasks. These bags can be sterilly connected using tubing kits thus reducing the risk of contamination to the cells. Peripheral blood mononuclear cells were obtained from normal donors or cancer patients undergoing IL-2/LAK cell therapy. Following Ficoll-Hypaque purification, these cells were incubated in the presence of IL-2 in either PL732 plastic bags or standard tissue culture flasks. Our results showed that LAK cells could be generated from either normal donors or cancer patients in the bags as well as in the flasks. Comparisons were made of the LAK cell populations obtained from the two sources and showed that each was similar in terms of morphology as determined by Wright stain differentials. The populations of cells were also similar in regard to cell surface phenotype as determined by flow cytometric analysis. In addition, recoveries from either tissue culture vessel as well as cell viability of the LAK cells were comparable. Finally, the LAK cells obtained from both sources were assessed for cytolytic activity against the tumor cell lines K562 and Daudi. These results showed that the cytolytic activity of the LAK cells against these target cells was the same whether the cells were obtained from the flasks or the bags.

Utilization of purified human monocytes in the agarose droplet assay for measuring migration inhibitory factors

Human monocytes, highly purified by counter-current elutriation, are excellent indicator cells for evaluation of human migration inhibitory factors (MIFs). We have adapted the agarose droplet MIF assay initially developed for guinea pig peritoneal exudate cells to utilize human monocytes. The experimental variables have been evaluated and standardized to make this assay a quantitative and sensitive method for measuring MIF activity. The assay can be performed serum-free in RPMI 1640 medium without protein or hormone additives, thereby increasing the sensitivity and eliminating potential masking of MIF effects by serum components. Cryopreserved monocytes also performed well in this assay, migrating approximately the same distance per unit time and showing migration inhibition in response to inhibitory factors. This assay provides a powerful tool in evaluating MIF-like activities of various lymphokines and factors, and could be used to monitor the activity of fractions produced during the physicochemical separation of MIFs from lymphokine-containing supernatants.

Effect of thymosin on T-lymphocyte rosette formation in children with kwashiorkor

Abstract Thymosin fraction 5 causes a significant increase in vitro in the percentage of E-rosettes (T-cells) of children with kwashiorkor. It is speculated that thymosin fraction 5 may play an important role in reconstituting host immunity in children with malnutrition.

The generation of human lymphokine-activated killer cells in various serum-free media

Recent data generated in our laboratory indicates that human lymphokine-activated killer (LAK) cells can be induced in several commercially available serum-free medium formulations. Interestingly, LAK cells were also generated in RPMI 1640 medium plus 1000 U/ml IL-2 without addition of human serum. However, higher levels of cytolytic activity were generally obtained in the commercial serum-free formulations. Cytotoxic activity was observed both at 3 and 7 days against a LAK-sensitive cell line (Daudi). Maximal cytolytic activity was usually produced by 7 days of in vitro culture in 1000 U/ml of interleukin-2 (IL-2).

Tumor acquisition, propagation, and preservation. The culture of human colorectal cancer

Fourteen new colorectal cancer cell lines were developed as part of a tumor acquisition, propagation, and preservation program for biotherapy. Fifty‐six specimens were received. Nine cell lines were generated from biopsies; seven of these cell lines were from metastatic lesions. Five additional cell lines were developed from xenografts grown in nude mice. Biopsies that produced three of these xenografts gave rise to parallel culture cell lines. Biopsy‐derived and xenograft‐derived cell lines from the same tumor behaved similarly in culture and exhibited similar markers when assessed immunohistochemically. Collagen substrate was beneficial in the primary culture of 50% of the specimens tested. Collagen was required for the successful propagation of two cell lines.