Specific elimination of cytotoxic effector cells. I. Adsorptive behavior of effectors and their precursors and spleen cell monolayers.

Monolayers formed of normal mouse spleen cells attached to polystyrene coated with poly-L-lysine were tested for their ability to bind specifically antigen-reactive cells in normal or primed mouse spleen. 88 to greater than 98% of the activity of cytotoxic populations was removed by a single adsorption. However, normal spleen cells or spleen cells previously primed in vitro could not be depleted of their capacity to be sensitized, even when adsorption effectively removed all residual cytotoxic activity from the same previously primed population. In fact, exposure to an immunoadsorbent augmented the ultimate cytotoxicity generated in a nonspecific fashion. This augmentation was especially dramatic in the case of a previously primed population and may have reflected the removal of a nonspecific suppressor. If antigen-reactive precursors cannot be removed efficiently by adsorption, other approaches to the generation of tolerant lymphoid populations, such as specific suppression of precursor differentiation must be sought.

Abrogation of interferon-induced resistance to interferon-activated major histocompatibility complex-unrestricted killers by treatment of a melanoma cell line with 5-fluorouracil.

Advanced cancer responds clinically to combined therapy with recombinant interferon-alpha and 5-fluorouracil. Although the two agents may interact in the biosynthetic pathway for thymidine, we investigated, as an alternative mechanism, the regulation of susceptibility of the A375 human melanoma to natural killers activated by interferon. A375 were preincubated with 5-fluorouracil, interferon, or both sequentially prior to assay as targets for cell-mediated killing. Pretreatment of A375 with interferon decreased apparent lytic efficiency. 5-Fluorouracil alone increased the susceptibility of A375 to killing. Pretreatment of targets with 5-fluorouracil abrogated the resistance normally induced by interferon pretreatment. Thus, 5-fluorouracil modulates certain immunoregulatory effects of interferon-alpha. Thymidine does not block the effect of 5-fluorouracil. While fluorodeoxyuridine is relatively ineffective in this system, fluorouridine is more effective than 5-fluorouracil in abrogating the effect of interferon. These data suggest important interactions of 5-fluorouracil and interferon in pathways for protein synthesis. It is known that interferon both increases the activity of natural killers and increases resistance of tumors to natural killers. We have shown that 5-fluorouracil, by blocking the resistance, may allow the augmented natural killing to be effective. This observation provides an alternate hypothesis for the clinical activity of 5-fluorouracil and interferon in combination.

Cloning of human lymphocytes reactive with autologous leukemia cells.

The primed lymphocyte typing test has been used to detect leukemia-associated antigens, but interpretation has been difficult because of significant levels of reactivity with normal cells. Elimination of unwanted reactivities could be accomplished by (a) use of the patient's own lymphocytes as responders to the leukemia cells and (b) cloning of the responding cells. Cloning of antigen-activated human lymphocytes can be accomplished through the use of T-lymphocyte growth factor, which permits the long-term growth of antigen-activated lymphocytes. In the study reported here, the remission lymphocytes of a patient with acute myelogenous leukemia were sensitized in culture to the patient's own leukemic myeloblasts and then grown from wells containing one or a few replicating units. Sufficient cells of three clones were growth for further testing of specificity: one responded only to the sensitizing myeloblast but not to normal cells tested; one responded to the sensitizing myeloblasts and one allogeneic myeloblast but not to normal cells; and one responded to none of the cells tested, although it proliferated vigorously with growth factor alone. These results demonstrate the feasibility of cloning human lymphocytes putatively responsive to leukemia-associated antigens in order to improve their discriminatory capacity in the primed lymphocyte typing test. The response pattern observed was that expected of a clone responding to a leukemia-associated antigen.

Augmented immunity in cancer patients treated with alpha-interferon.

Thirty patients with metastatic colon or breast cancer were treated with recombinant alpha-interferon, clone A, 9 to 50 X 10(6) units/sq m, i.m., 3 times weekly for up to 4 months. Immunological parameters including natural killer activity, antibody-dependent cellular cytotoxicity, an assay of inhibition of tumor cell growth in culture, and quantification of leukocyte subsets were monitored serially. Statistically significant increases in the inhibition of tumor growth and in the proportion of peripheral blood mononuclear cells bearing the T10 marker were observed both early and late in the treatment course in the population as a whole (p less than 0.03 and p less than 0.0001, respectively). The true maximum effect in the assay of inhibition of tumor growth was probably higher, since the monitoring was not performed at peak activity for this assay. Other immune parameters, including natural killing, could not be shown to change consistently in the population as a whole, although interferon effects could be discerned easily in the activity profiles of some individual patients. The two patients with tumor response showed increased putative tumor immunity by these measures. These data confirm results previously published supporting the responsiveness of these parameters to interferon as administered clinically and may provide the basis for optimization of interferon dose and scheduling.

Treatment of cancer-associated hemolytic uremic syndrome with staphylococcal protein A immunoperfusion.

Plasma perfusion over filters containing staphylococcal protein A (SPA) was used to treat 11 patients with adenocarcinoma who developed a hemolytic uremic syndrome. Immunoperfusion resulted in complete clearance of pretreatment elevated levels of circulating immune complexes in eight of the 11 patients with normalization of complement values depressed at the start of the therapy in seven. A significant rise in platelets and erythrocyte counts was achieved in nine patients, and stabilization of progressive renal impairment was achieved in six. The response was incomplete and short lived in three patients with clinically evident tumor recurrence, whereas long-term control of the syndrome was demonstrated in seven patients in complete tumor remission (no recurrence with median follow-up of 9 months). SPA immunoperfusion appears to be an effective form of therapy for this otherwise fatal syndrome.

Evaluation of cisplatin intensity in metastatic non-small-cell lung cancer: a phase III study of the Southwest Oncology Group.

PURPOSE: To test the concept that cisplatin dose-intensity is important in the treatment of non-small-cell lung cancer (NSCLC), the Southwest Oncology Group (SWOG) performed a randomized trial comparing standard-dose cisplatin (SDCP) 50 mg/m2 days 1 and 8 on a 28-day cycle for eight cycles, high-dose cisplatin (HDCP) 100 mg/m2 days 1 and 8 for four cycles, and high-dose cisplatin plus mitomycin (HDCP-M) 8 mg/m2 day 1. To isolate the effects of dose-intensity versus total dose, the planned cumulative cisplatin dose was 800 mg/m2 in each arm. PATIENTS AND METHODS: Between July 1988 and April 1990, 356 patients were enrolled and 323 were eligible and assessable. All patients had metastatic, measurable disease, were chemotherapy-naive, and had a performance status (PS) of 0 to 2. RESULTS: Confirmed complete plus partial response rates were SDCP, 12%; HDCP, 14%; and HDCP-M, 27% (P < .05). Complete responses were uncommon (HDCP, 3%; HDCP-M, 4%) and were observed only in the high-dose arms. Progressive disease occurred more frequently in the SDCP arm (57%) compared with HDCP (38%) or HDCP-M (34%) (P < .05). However, there were no significant differences in median survival times (SDCP, 6.9 months; HDCP, 5.3 months; HDCP-M, 7.2 months; P = .53). The mean delivered dose-intensity for cisplatin was significantly greater in the high-dose arms: HDCP 41 mg/m2/wk and HDCP-M 39 mg/m2/wk, versus SDCP 23 mg/m2/wk (P = .05). The high-dose arms resulted in an increased incidence of ototoxicity, emesis, and myelosuppression, but similar degrees of renal toxicity and neuropathy compared with SDCP. CONCLUSION: This study does not confirm evidence of a steep clinical dose-response curve for cisplatin in NSCLC at the cisplatin dose-intensities achieved. The addition of mitomycin increases the response rate, but does not improve survival. Continued evaluation of new agents in this disease is warranted.

Phase II trial of recombinant DNA gamma-interferon in advanced colorectal cancer: a Southwest Oncology Group study.

Fifty evaluable patients with advanced colorectal cancer, but without prior chemotherapy or immunotherapy, were randomized to one of two schedules of recombinant gamma-interferon (rGIFN). Twenty-four evaluable patients received rGIFN as a 2-h intravenous infusion daily x 5 every other week at a starting dose of 4.0 x 10(6) IU/m2/day (arm I). Twenty-six evaluable patients received rGIFN as a 24-h continuous intravenous infusion daily x 5 every month at a starting dose of 2.6 x 10(6) IU/m2/day (arm II). Toxicities on both schedules included flu-like symptoms, fevers/rigors, nausea/vomiting, hypotension, leukopenia, hepatotoxicity, nephrotoxicity, diarrhea, anemia, confusion, and ileus. Toxicity appeared to be more severe on arm I. No antitumor responses were observed, with 95% confidence intervals of 0 to 14% for arm I and 0 to 13% for arm II.

Mechanisms of interaction of interferon and 5-fluorouracil in solid tumors.

Recent clinical trials demonstrate the combined activity of 5-fluorouracil (5-FU) and interferon (IFN) in advanced colon cancer. Several possibilities exist for explaining the interaction. Interferon may alter the pharmacokinetics of 5-FU infusion by increasing the steady state concentration. Interferon enhances the inhibitory effects of 5-FU for tumor cells in culture. This enhancement is blocked by thymidine. Interferon reduces the concentration of thymidylate synthetase, and this may account for the thymidine-reversible interaction. An alternative mechanism invokes the immunomodulatory effects of IFN. Interferon augments the activity of killer cells with possible anti-tumor activity, both in vitro and in vivo. Also, by increasing the expression of human leukocyte class I antigens, IFN reduces the sensitivity of tumor cell lines to cell-mediated killing, an effect termed resistance. 5-Fluorouracil reverses the resistance in a time- and dose-dependent manner. The effect is mediated through inhibition of protein synthesis, since thymidine cannot reverse it. Fluorouridine is more active in reversing resistance than fluorodeoxyuridine. 5-Fluorouracil also reverses the induction of human leukocyte antigens by IFN. Studies in the resistance model suggest that high doses of 5-FU by infusion for several days might be the optimal method for modulation of IFN-induced effects.

Polyarteritis nodosa, hairy cell leukemia and splenosis.

Appendiceal vasculitis and splenosis in a young women provided pathologic evidence that a systemic necrotizing vasculitis preceded the onset of hairy cell leukemia by at least six years. As in other patients who have had both diseases, florid polyarteritis nodosa developed during the course of the malignancy. However, the antecedent vasculitis, as well as the coincidence of these rare diseases, indicate that systemic vasculitis does not necessarily result from hairy cell leukemia as previously reported and suggest that the two diseases might share a common pathogenesis or predisposing factor.

Genetic analysis of 23 RhL-A histocompatibility antigens of the rhesus monkey.

The lymphocytotoxicity of 116 rhesus monkey alloantisera was evaluated in 92 unrelated rhesus monkeys and in 33 pedigreed rhesus families. The study was conducted using a standard complement-dependent microcytotoxocity assay. A computer-assisted chi2 analysis of the reactivity of these sera in unrelated monkeys generated 23 groups of highly correlated antisera. The two-locus model of the mammalian major histocompatibility complex was assumed for the monkey, and genetic criteria for RhL-A antigens were determined before study. Seventeen groups of antisera which had met these predetermined criteria in a previous study using a different random population of monkeys were confirmed in the present analysis. The remaining six groups also met these predetermined criteria, although the genetic data for two were incomplete. Of the 23 antigens defined, 12 appeared to be products of the A locus and 11 of the B locus. Nineteen were similar or identical to antigens previously described by us or by Balner and coworkers in The Netherlands. Two groups have not been previously described. A frequency analysis indicated that these 23 antigens represented approximately 75% of the total expression of the RhL-A-A and RhL-A-B loci.

Differential effect of human interferon on normal and neoplastic T- and B-cell lymphocytes.

The effect of human interferon (HIF) on the growth and function of normal and neoplastic proliferating human T and B cells was studied. Cell cycle analysis by flow-cytometry showed that up to 2000 units of HIF had little effect on the proliferating fraction (that is, the present S + G2 + M phases of the cell cycle) of normal T cells grown continuously wit growth-promoting factor from PHA-stimulated lymphocytes, while 500 units of HIF suppressed the percent S + G2 + M of the acute lymphocytic leukemia (ALL) T-cell line, MOLT, by 36%. Up to 2000 units of HIF had a moderate enhancing effect on the percent S + G2 + M of PWM-stimulated lymphocytes. In striking contrast, a single unit of HIF caused nearly 40% suppression of the percent S + G2 + M of Daudi, a Burkitt's lymphoma cell. One-thousand units of HIF did not decrease the number of antibody-forming normal cells, and up to 500 units of HIF did not inhibit the total Ig secreted by these cells. These results suggest that amounts of HIF affecting the proliferation of some neoplastic lymphoid cells has little effect on the proliferation of normal T and B cells. In addition, HIF does not appear to affect polyclonal induction of B-cell differentiation.

Comparative immunotoxicity of free doxorubicin and doxorubicin encapsulated in cardiolipin liposomes.

The immunologic and pharmacologic effects of free doxorubicin and of doxorubicin entrapped in liposomes were compared in mice at a dose of 20 mg/kg. Liposomes for encapsulation of doxorubicin were prepared by using 39.35 mumol drug, 19.65 mumol cardiolipin, 100 mumol phosphatidylcholine, 68.4 mumol cholesterol, and 38.9 mumol stearylamine. Pharmacologic disposition studies after a dose of 20 mg/kg demonstrated 7- to 10-fold higher drug concentrations in the spleen at all time points following administration of doxorubicin entrapped in cardiolipin liposomes than after the free drug. The levels in liver were 4- to 5-fold higher with liposomal drug, whereas the cardiac uptake with liposomal doxorubicin was significantly lower than with free drug. Mice were sacrificed on days 1, 8, 15, and 22 after drug administration and spleen cells were isolated for studies of sensitization to alloantigens for cell-mediated cytolysis and of proliferation in response to mitogens. Mice treated with free doxorubicin demonstrated a decrease of more than 50 fold (compared with saline control) in allospecific cytotoxic activity on day 15; normal levels were recovered by day 22. The animals treated with doxorubicin encapsulated in liposomes showed a similar but not more pronounced fall to low levels. The total lytic activity per spleen after free drug or drug encapsulated in liposomes was markedly reduced at day 8, but this activity was fully recovered by day 15 in animals receiving liposomal doxorubicin; in those receiving free drug it had not recovered fully even at day 22. The proliferative response to concanavalin A was affected by the two forms of doxorubicin in a pattern very similar to the cytotoxic response. The proliferative response to lipopolysaccharide was markedly depressed by doxorubicin delivered in either form, and the kinetics were not altered by the mode of administration. The concentration of doxorubicin in spleen was markedly increased with liposomal delivery, but did not result in greater toxicity than that of free drug according to the immunologic parameters evaluated.

Rejection of skin grafts after orthotopic tooth transplantation between RhLA-paired monkeys.

Statistical analysis of paired observations indicated that RhLA-A,B disparate teeth caused the accelerated rejection of tooth-donor skin grafts placed 21 to 24 mo after tooth allotransplantation, but not at 30 to 34 mo. Teeth transplanted between matched monkeys did not cause the accelerated rejection of skin grafts.

Therapy of advanced gastric carcinoma. The Georgetown-Lombardi Cancer Center experience.

Gastric carcinoma, despite a decreasing incidence in the United States over the past 40 years, is the seventh most common cause of cancer death in this country and remains a significant worldwide problem. The 5-fluorouracil, Adriamycin (doxorubicin), and mitomycin (FAM) chemotherapy regimen, which was initially reported by Georgetown in 1979, has become a standard for advanced gastric carcinoma with response rates in the 40% range. The FAM regimen as well as subsequent trials conducted at Georgetown and our current approach to management of this tumor are discussed. Despite a decade of intensive clinical research, we have not identified a modification or innovation that is superior to the original FAM.

Phase II study of recombinant alpha-interferon in malignant melanoma.

Ninety-seven evaluable patients with measurable, advanced, malignant melanoma were treated with recombinant alpha interferon in a cooperative phase II efficacy trial, whose primary objective was to estimate the response rate. Interferon (rIFN alpha-2a, Roferon-A) was injected subcutaneously daily for 70 days. Dose was escalated in four steps from three million units to 36 million units over ten days. Eight patients responded objectively and six patients (6%) had a complete response. The median duration of complete response was 11 months. Patients achieving complete response had only cutaneous, nodal, or pulmonary disease; some had extensive prior therapy; some could tolerate no more than three million units per day. Few patients could tolerate the target dose of 36 million units daily for 70 days. Limiting toxicity was primarily fatigue. Interferon in tolerable doses is effective in a small subset of patients with melanoma. Comparison of published trials of dacarbazine and recombinant alpha interferon indicates the two drugs have similar activity.

CoVal fusions: a therapeutic vaccine platform using heat shock proteins to treat chronic viral infection and cancer.

We have developed an immunization platform which combines heat shock proteins (Hsp) with protein antigens, such as viral or cancer targets, into a single recombinant fusion protein. Pre-clinical data demonstrate the ability of Hsp fusion proteins to induce antigen-specific cytotoxic T lymphocytes, Type 1 cytokines and anti-tumour immunity. One Hsp fusion protein, HspE7, is now in clinical development for therapy of diseases caused by human papillomavirus (HPV). HPV infection is associated with development of proliferative lesions (papillomas or warts) as well as malignant lesions (anogenital dysplasia and cancer). HspE7 has been shown in efficacy trials to be active against genital warts and anal dysplasia, and a trial is underway in another HPV indication, recurrent respiratory papillomatosis. Having observed therapeutic activity for our lead product HspE7 in humans, we are currently developing Hsp fusion proteins as therapeutic vaccines for other chronic viral infections. Potential targets include hepatitis B, herpes simplex, hepatitis C, and human immunodeficiency virus.

Definition of two LD antigens in rhesus monkeys.

Two rhesus (Macaca mulatta) monkey lymphocyte-defined (LD) antigens have been identified using two typing cells as stiumlators in a one-way mixed leukocyte culture (MLC) assay. An analysis of the genetic behavior of these LD antigens in six rhesus monkey families revealed that both antigens were linked with RhLA. One probable recombinant indicated that the LD locus lies outside the two known RhLA-SD loci and the locus which controls the serum protein, properdin B(Bf). These two antigens, LD1 and LD2, had observed gene frequencies of 0.07 and 0.25, respectively. Neither of these two new LD antigens was significantly associated with any serologically defined (SD) antigen.