Henry C. Stevenson

Treatment of advanced non-Hodgkin's lymphoma with recombinant leukocyte A interferon

We report the results of a trial of recombinant leukocyte A interferon in previously treated patients with non-Hodgkins lymphoma who were no longer responsive to chemotherapy. Patients received recombinant leukocyte A interferon (50 X 10(6) U per square meter of body-surface area) by intramuscular injection three times weekly for three months or longer. Forty-five patients were enrolled in the study, and 37 were evaluated for a response. Thirteen of 24 (54 per cent) evaluable patients with low-histologic-grade non-Hodgkins lymphoma had objective responses (nine partial responses and four histologically confirmed complete responses). Two of six (33 per cent) with intermediate-grade lymphoma responded (one partially and one completely), and one of seven (14 per cent) with high-grade lymphoma had a partial response. The median duration of responses was eight months. Four of the five complete responders have continued to receive maintenance interferon and have been in complete remission for 3, 7, 9, and 12 months, respectively; one had a recurrence at a site of previous disease seven months after interferon had been stopped. Side effects were noted in most patients. All 16 responders had been heavily pretreated with combination chemotherapy, including doxorubicin in 8 of the 16. These results suggest that recombinant leukocyte A interferon may be an effective new therapy for some patients with low- and intermediate-grade non-Hodgkins lymphoma.

A phase I trial of recombinant gamma interferon in patients with cancer

SummaryA total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-γ), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m2 of IFN (1 mg≥10×106 units of IFN) escalating to 10 mg/m2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chils, fatigue, anorexia, and granulocytopenia. The influenzalike symptoms were very similar to those encountered with IFN-α but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48–72 h following administration of rIFN-γ. Absolute granulocyte counts only rarely dropped below 1000 mm3. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4–8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.

An improved technique for the negative selection of large numbers of human lymphocytes and monocytes by counterflow centrifugation-elutriation

Abstract Human unfractionated mononuclear leukocyte (UFMNL) preparations and T-cell-depleted (TCD) mononuclear leukocyte preparations, obtained from 400 ml of whole peripheral blood (PB) or from continuous-flow centrifugation leukapheresis (CFCL) concentrates, were cleanly separated by counterflow centrifugation-elutriation (CCE) into highly purified lymphocyte and monocyte populations, each with excellent recovery and function. Lymphocytes isolated from UFMNL preparations ranged in number from 1.88 × 10 8 to 2.22 × 10 9 with mean purities in excess of 93%. Lymphocytes isolated from TCD preparations yielded up to 1.90 × 10 9 lymphocytes devoid of all detectable sheep RBC receptor-bearing cells with mean purity and recovery in excess of 93 and 90%, respectively. Monocytes negatively selected from UFMNL preparations obtained from whole PB resulted in yields of 1.08 × 10 8 monocytes with 92% purity and 94% recovery; UFMNL obtained from CFCL concentrates resulted in yields of 4.22 × 10 8 monocytes with 92% purity. TCD preparations obtained from PB yielded 7.19 × 10 7 monocytes in 95% purity and 94% recovery, and from CFCL concentrates, 6.21 × 10 8 monocytes in 94% purity and 79% recovery. Recovered lymphocytes and monocytes have excellent overall viability, and the purified monocytes have normal phagocytic and chemotactic functional capabilities. Counterflow centrifugation-elutriation (CCE) is a valuable technique for obtaining monocyte and monocyte-depleted (i.e., lymphocyte) cell preparations that are negatively selected in high yield, with excellent purity and viability.

Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

Elutriator‐purified human monocytes were cultured in a serum‐free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate.

Human Blood Monocytes: Characterization of Negatively Selected Human Monocytes and Their Suspension Cell Culture Derivatives

Normal human monocytes were negatively selected from leucapheresis cell suspensions by countercurrent centrifugation–elutriation in high yield with a mean purity of 93.5%. The combination of the novel methods of negative cell selection and suspension cell culture has provided the opportunity to study serially over several days the morphologic and functional changes of monocytes from a single donor as they matured in culture to typical macrophages. Human monocytes nearly double in size during the first week of culture, experiencing near daily increases in cell volume. This was associated with changes in the ultrastructure of these cells, including the development of numerous small knob‐like projections on the cell membrane and the proliferation of microtubules and filamentous Structures within the cell cytoplasm during the first 6 days of culture. Peroxidase activity declined during the first 4 days of culture, whereas 5′‐nucleotidase activity was acquired during the first 48 h of culture. Lysozyme activity in the cultures increased from day 2 to day 6 of culture. The phagocytic capacity of monocytes for IgG‐coated erythrocytes increased dramatically during the first week of culture, but the cytotoxic capability of monocytes against similar targets in an antibody‐dependent cytotoxicity assay declined to nearly hall of base‐line levels by day 2 of culture and remained at this diminished level during subsequent days in culture.

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.

A system for obtaining large numbers of cryopreserved human monocytes purified by leukapheresis and counter-current centrifugation elutriation (CCE).

A system has been developed for the isolation of large numbers of unfractionated mononuclear cells from single, well characterized normal individuals and for the separation by elutriation of these cells into populations of greater than 90% pure monocytes and greater than 99% pure lymphocytes. The total number of monocytes obtained from a single donor averaged about 550 million. After cryopreservation and thawing of these cells, the viability remained greater than 90%, 80% of original cells were recovered, and the ability to ingest antibody-coated targets was comparable to that of fresh monocytes. The cells remained sterile without the use of antibiotics and were suitable for long-term culture. The monocytes that were isolated and cryopreserved by these procedures functioned reproducibly as inhibitors of tumor cell growth and in an assay of responsiveness to monocyte migration inhibitory factor (MIF).

Differential Ability of Human Blood Monocyte Subsets to Release Various Cytokines

We have shown that two human monocyte subsets can be isolated from the peripheral blood of healthy donors; these subsets possess different morphological, cytochemical, functional, and in vivo trafficking properties [1]. In this report, these two subsets were further characterized. One subset (intermediate monocytes, IM) has been shown to have significantly lower acid phosphatase activity and total cellular protein content as well as lower peroxidase activity when compared with another subset (regular monocytes, RM). The overall activation status of the two subsets (as determined by their alkaline phosphodiesterase activity) was identical. We also examined the capacity of these subsets to release various cytokines with or without polyriboinosinic and poly‐ ribocytidylic acid (Poly l:C) stimulation. There was no appreciable difference in their ability to release interferon (IFN), interleukin 1 (IL‐1), and prostaglandin E (PGE) without stimulation, while IM produced slightly, but significantly, higher amounts of colony‐stimulating factor (CSF) than RM. The amount of IFN released by IM in response to poly l:C was approximately three times higher than the amount of IFN released by RM. IL‐1 was also released in higher amounts by IM than by RM in response to poly l:C. IM were also found to release more CSF than RM in response to poly l:C. In contrast, it was noted that IM secrete significantly less PGE response to poly l:C than do RM. These findings indicate that two purified human monocyte subsets, distinguishable by maturation markers, differ significantly in their ability to release various cytokines after stimulation; this difference may be relevant to potential in vivo roles of these immunoregulatory cells.

Polymyxin B causes coordinate inhibition of phorbol ester-induced C-kinase activity and proliferation of B lymphocytes

Lymphocytes were found to be rich in phospholipid/Ca2+-dependent (C-kinase) activity. Addition of polymyxin B (PMB) to in vitro assays of endogenous and exogenous phosphorylation resulted in profound inhibition of C-kinase activity. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) directly activated C-kinase, leading to increased phosphorylation of the same substrates. TPA also stimulated proliferation of B cells as assessed by 3H-thymidine uptake, and PMB strongly inhibited this effect. This coordinate inhibition of TPA-induced phosphorylation and mitogenesis indicates that PMB is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.

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.