Anti-CD3 monoclonal antibody treatment of patients with CD3-negative tumors: a phase IA/B study.

Anti-CD3 monoclonal antibodies induce the proliferation of human T-cells in vitro and activate specific and nonspecific cytolysis by human T-cell clones and human peripheral blood lymphocytes. In vivo administration of anti-CD3 prevents tumor growth of a UV-induced mouse fibrosarcoma. We conducted a phase I trial to determine the toxicity and immunomodulatory properties of low doses of anti-CD3 in 36 patients with cancer. In 23 patients, anti-CD3 was given i.v. over 3 h at 1, 10, 30, and 100 mcg/patient. Five other patients received anti-CD3 at 30 mcg by i.v. bolus. Patients were treated every 3 days for a total of four doses. An additional eight patients received anti-CD3 daily for 14 days at 3 mcg by i.v. bolus, 3-h infusion, or 24-h infusion. Dose-limiting toxicity was headache. Headache was often accompanied by signs and symptoms of meningeal irritation leading to performance of a lumbar puncture in nine patients. The opening pressure was usually elevated, and six patients had a cerebrospinal fluid lymphocytosis with an elevated protein. Increased levels of interleukin 6 were identified in the cerebrospinal fluid. The maximum tolerated dose by 3-h infusion was 30 mcg. There were no objective tumor responses. There was a dose-related increase in the number of peripheral blood lymphocytes expressing the T-cell activation antigen CD69 (Leu 23), but no changes were seen in CD25 (interleukin 2 receptor) expression, and no changes were observed in the serum levels of the soluble interleukin 2 receptor. Even at these low doses of anti-CD3, 8 of 16 patients tested developed human anti-mouse antibodies.

Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin 2 in patients with cancer: clinical and immunological effects.

We have performed a phase IB study of polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly-ICLC) in combination with interleukin 2 (IL-2) in 25 patients with a variety of cancers. Patients received weekly or biweekly poly-ICLC by i.m. injection, at doses ranging from 0.01 to 1.0 mg/m2, for 1 month. This was followed by 2 months of outpatient therapy with biweekly i.m. poly-ICLC in combination with IL-2 (3 x 10(6) units/m2) given i.v. by 24-h continuous infusion twice weekly, using a portable infusion pump. No objective tumor responses were observed. Toxicity was moderate at all poly-ICLC doses tested and increased only slightly following the addition of IL-2. No increases in peripheral blood natural killer (NK) activity were observed after treatment with poly-ICLC alone. However, high dose poly-ICLC (greater than or equal to 0.3 mg/m2) in combination with IL-2 resulted in NK activity greater than that seen using the same dose of IL-2 in combination with lower poly-ICLC doses. Increases in the number and percentage of CD56+ cells were evident only after initiation of IL-2 therapy and were unaffected by the poly-ICLC dose. In the majority of patients, these increases were preferentially associated with the subset of CD56+ cells coexpressing CD8, while the CD56+/CD16+ population was elevated to a lesser extent. Moderate increases in serum neopterin levels and 2',5'-oligoadenylate synthetase activity in peripheral blood mononuclear cells were noted at 72 h following initial treatment with 1.0 mg/m2 poly-ICLC. No induction of alpha or gamma interferon was detected. This study shows that the addition of poly-ICLC to a well tolerated IL-2 regimen can significantly enhance NK activity. Poly-ICLC can be used to enhance IL-2-induced NK lytic activity without increases in the dose and, therefore, the toxicity of IL-2 treatment.

Poly I:C accelerates development of diabetes mellitus in diabetes-prone BB rat.

We developed a new experimental model of accelerated diabetes mellitus in the genetically susceptible diabetes-prone BB rat with the administration of the IFN-alpha inducer poly I:C. With this model, there was both an increased incidence and accelerated onset of insulin-dependent-diabetes in poly I:C-treated animals compared with saline-treated controls. All twelve rats administered poly I:C (5 micrograms/gm body weight 3 times/wk) developed diabetes by 57 days of age (100%) compared with 1 of 27 (3.7%) saline-treated controls. Furthermore, the development of diabetes was accelerated in the poly I:C-treated group (mean age +/- SE at onset 52.8 +/- 0.58 days) compared with saline-treated controls (89.3 +/- 2.4 days, P less than 0.01). Additionally, poly I:C-treated rats had higher mean serum IFN-alpha levels than saline-treated rats at weeks 2 and 3 of treatment (210 vs. 27 and 183 vs. 25 U/ml, respectively, P less than 0.001). Poly I:C treatment of 5 Wistar rats, the parental strain, which is not susceptible to diabetes, did not result in insulitis, diabetes, or hyperglycemia. The histopathologic findings of insulitis and decreased immunoreactive islet insulin in poly I:C-accelerated diabetic BB rats and in BB rats with spontaneous diabetes suggest a similar pathophysiology.

Poly I:C induces development of diabetes mellitus in BB rat.

Polyinosinic polycytidilic acid (poly I:C), an inducer of alpha-interferon, accelerates the development of diabetes in diabetes-prone (DP) BioBreeding (BB) rats. This study investigates the effect of administering poly I:C to a diabetes-resistant (DR) strain of BB rats. We compared the incidence of diabetes, the degree of insulitis, the number of NK cells, helper-inducer cells, cytotoxic-suppressor cells, Ia+ T cells, RT6.1+ T cells, and NK cell bioactivity in DR rats treated with saline and with a 5 micrograms/g body wt (poly-5) dose and a 10 micrograms/g body wt (poly-10) dose of poly I:C. The incidence of diabetes was also compared with that of DP rats receiving poly-5. We found that both doses of poly I:C significantly induce the development of diabetes in the DR BB rat. However, treatment of DR rats with the higher dose induces a greater rate of development of diabetes and earlier onset of diabetes than the lower poly-5 dose. The rate of diabetes development and the mean age of onset were similar in poly-10-treated DR and poly-5-treated DP rats. A significant degree of insulitis occurred in all the poly I:C-treated DR rats, even those not developing diabetes. Peripheral blood NK cell number was greater in poly I:C than in saline-treated rats, after 2 wk of treatment and when killed. The percentage of OX19+ peripheral blood mononuclear cells expressing RT6.1 allotype or Ia antigen were similar in poly I:C- and saline-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly I:C induction of alpha-interferon in the diabetes-prone BB and normal Wistar rats. Dose-response relationships.

Although the administration of a fixed dose of the alpha-interferon (alpha-IFN) inducer, polyinosinic polycytidilic acid (poly I:C), accelerates the development of diabetes in DP-BB rats, no reports have characterized the dose-response relationship of poly I:C with serum alpha-IFN levels and the development of diabetes. This study examines the dose-response relationships of poly I:C with the induction of serum alpha-IFN and the development of diabetes in DP-BB and normal Wistar rats. Also tested in this study is the hypothesis that the lack of development of diabetes in poly I:C-treated normal Wistar rats is attributable to a deficient alpha-IFN response. Using poly I:C doses of 0.5, 1.5, 5, and 10 micrograms/g body weight, a direct dose-response relationship was observed in DP-BB rats with the serum alpha-IFN response. Moreover, all doses of poly I:C accelerated the onset of diabetes in BB rats. Serum alpha-IFN levels inversely correlated with time of onset of diabetes (P < 0.01). Also, BB rats with higher levels of serum alpha-IFN were associated with earlier onset of diabetes (P < 0.001). Poly I:C-induced serum alpha-IFN levels were significantly lower in diabetic than in nondiabetic BB rats. In normal Wistar rats, although all doses of poly I:C significantly increased serum alpha-IFN levels, diabetes was not induced. The results of this study indicate that poly I:C administration elevates serum alpha-IFN and accelerates the development of diabetes in BB rats at even very low doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of acetyl-n-ser-asp-lys-pro (AcSDKP) on hematopoietic progenitor cells in short-term and long-term murine bone marrow cultures.

The tetrapeptide AcSDKP is a potent inhibitor of hematopoietic stem cell proliferation. Its activity was systematically examined in murine long-term bone marrow cultures (LTBMC) and short-term liquid cultures in the presence or absence of exogenous cytokines. The effects of AcSDKP on the production of granulocyte-macrophage colony-forming cells (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in LTBMCs were examined. AcSDKP was added daily to LTBMCs at various concentrations (10-3--10-16M) for up to 5 weeks. AcSDKP inhibited the entry of progenitor cells into S phase as measured by 3H-thymidine suicide assay and the absolute number of progenitor cells with peak activity at 10-12 M with less activity seen at higher or lower concentrations. The number of nonadherent CFU-GM per LTBMC was unchanged from control values at 1 week of treatment with AcSDKP but was significantly depressed at weeks 3 and 5. In contrast, HPP-CFC progenitor cells were decreased throughout the treatment period, and the numbers of CFU-GM and HPP-CFC in S phase were significantly decreased throughout the treatment period. Maximum S-phase inhibitory activity was observed at 10-12 M AcSDKP. AcSDKP had no effect on the number of adherent CFU-GM or HPP-CFC, cellularity per culture or percent of adherent progenitor cells in S phase. Murine short-term bone marrow cultures were also treated with AcSDKP in the presence or absence of cytokines (interleukin-3 [LI-3], stem cell factor [SCF], or granulocyte colony-stimulating factor [G-CSF]) for various time periods. Dose-response studies showed maximum effects at 10-12 M AcSDKP when no cytokines were added and 10-14 M AcSDKP when exogenous cytokines were added. These studies indicate that the concentration of the tetrapeptide critical in obtaining an effect on hematopoietic progenitor cells, and furthermore, we report that the presence of cytokines or stromal cells also affects the response of progenitor cells to AcSDKP.

Inhibition of hematopoiesis in long-term marrow cultures established on adherent layers from AcSDKP-treated dogs.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) interferes with G1/S phase progression in hematopoietic precursors. We investigated the effect of AcSDKP on in vitro and in vivo hematopoiesis in a canine model. AcSDKP, added daily for 2 weeks to long-term marrow culture (LTMC) at concentrations > 10(-8)M, reversibly inhibited colony-forming unit granulocyte/macrophage (CFU-GM) formation (p < 0.001 and p < 0.05 for 10(-6) and 10(-7)M, respectively). Inhibition was more profound when AcSDKP addition was begun at the initiation rather than at the time of recharging the cultures. Next, seven dogs were given AcSDKP in vivo at 50 (n = 2), 250 (n = 2), or 500 micrograms/kg/day (n = 3) via continuous infusion for 7 days. No adverse effects were observed. LTMCs were established on days -9, -2, +7, and +28 of AcSDKP. One week later (days -2, +5, +14, and +35), adherent layers were recharged with fresh autologous marrow, and CFU-GM in nonadherent cells was assayed weekly beginning 1 week after recharging. The cumulative number of CFU-GM harvested from LTMCs was dependent upon the time of initiation of LTMC. The difference between day -2 (adherent layer pre-AcSDKP; recharge on AcSDKP) and day +7 culture (adherent layer on AcSDKP; recharge after discontinuation of AcSDKP, p < 0.001) suggested an effect of AcSDKP on the adherent stromal layer. Ex vivo hematopoiesis partially recovered following discontinuation of AcSDKP, although CFU-GMs were still reduced in LTMCs established on day +28. Normal nonadherent cells recharged onto allogeneic adherent/layers obtained during AcSDKP treatment grew significantly fewer CFU-GM than cultures on adherent cells obtained before AcSDKP treatment (p < 0.05). Therefore, these data suggest that AcSDKP affects not only hematopoietic cells but also cells of the adherent layer.

In vivo protective effects of tetrapeptide AcSDKP, with or without granulocyte colony-stimulation factor, on murine progenitor cells after sublethal irradiation.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) demonstrated hemato-protective activity in mice after sublethal irradiation (7 GY). Bone marrow interleukin-3 (IL-3)-responsive colony-forming cells (CFC and high proliferative potential colony-forming cells (HPP-CFC) were significantly (p < 0.05) increased by day 10 after irradiation in mice receiving a continuous infusion of 1000 ng/day of AcSDKP compared to irradiated control mice. The maximum protective effect for bone marrow progenitors was achieved when AcSDKP was administered for 3 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days after AcSDKP infusion in irradiated mice, significantly increased numbers of IL-3 responsive CSF-only control mice. In addition, platelets were significantly (p < 0.05) increased in mice receiving AcSDKP and G-CSF on days 18 and 21 after irradiation compared with mice receiving G-CSF alone. We conclude that ACSDKP has a radioprotective effect in vivo for progenitor cells, and that time of initiation and duration of AcSDKP administration relative to irradiation are crucial for these effects. Further, AcSDKP has a significant additive protective effect not only for progenitor cells but also for platelets when given in combination with G-CSF. We suggest that these in vivo observations provide a basis on which to design optimal clinical hypothesis and protocols.

Phase I/IB study of polyadenylic-polyuridylic acid in patients with advanced malignancies: clinical and biologic effects.

The synthetic polynucleotide polyadenylic-polyuridylic acid (polyA:polyU) has shown antitumor activity in murine studies and human breast cancer. PolyA:polyU was evaluated in 25 cancer patients receiving weekly intravenous doses between 3 and 600 mg/m2. PolyA:polyU was well tolerated up to 600 mg/m2, with no doselimiting toxicity (all < grade 3). Side effects included mild elevation in temperature, fatigue, and mild hyperglycemia. No changes outside of the normal range in hematocrit, WBC count, platelet count, total bilirubin, or alkaline phosphatase were observed. Of 25 patients, 18 completed at least one cycle of 6 weeks, and 5 completed two cycles (median 6 weeks). Four patients had stable disease over 11-13 weeks of treatment, and no clinical responses were observed. At 24 h after the first treatment, there were no significant increases in biologic response (beta 2-microglobulin and neopterin in serum, or 2',5'-oligoadenylate synthetase in peripheral blood mononuclear cells). A small increase in beta 2-microglobulin was observed 24 h after the week 3 treatment (1.1-fold, p < 0.01). By the third week of treatment, 2-5A synthetase levels decreased slightly (to 80% of baseline, p < 0.01). No changes in cytokines IL-6, IL-12, tumor necrosis factor (TNF), or IL-2 receptor in serum were detected after 24 h of treatment. Thus, at these doses, polyA:polyU had no marked modulation on biologic responses in vivo, although this preparation significantly induced 2-5A synthetase in peripheral blood mononuclear cells in vitro. PolyA:polyU was well tolerated. An MTD was not reached but was greater than 600 mg/m2 on this weekly schedule.

Activity of acetyl-Ser-Asp-Lys-Pro (AcSDKP) on human hematopoietic progenitor cells in short-term and long-term bone marrow cultures.

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent inhibitor of hematopoietic stem cell proliferation. We examined the effects of AcSDKP on the production of granulocyte-macrophage colony-forming cells (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in human long-term bone marrow (LTBM) cultures and CFU-GM and erythroid burst-forming cells (BFU-e) in short-term liquid cultures. The addition of AcSDKP in short-term bone marrow cultures resulted in a maximum depression of the total number of progenitor cells as well as the number of progenitor cells entering cell cycle following culture with 10(-12) to 10(-14) M AcSDKP and 10(-14) M AcSDKP when exogenous cytokines (GM-CSF, IL-3, or SCF) were added. AcSDKP was added daily to LTBM cultures at various concentrations (10(-8) M to 10(-16) M) for up to 5 weeks. In these LTBM culture studies, AcSDKP inhibited the entry of nonadherent progenitor cells into S phase and decreased the number of nonadherent progenitor cells with peak activity at 10(-12) M. In contrast, AcSDKP had no effect on the number of adherent CFU-GM, HPP-CFC, or cellularity per culture or percent of adherent progenitor cells in S phase. These studies indicate that the concentration of the tetrapeptide is critical to the activity of AcSDKP on human hematopoietic progenitor cells. Furthermore, we report that the presence of cytokines or stromal cells also affects the response of progenitor cells to AcSDKP. These results will aid in determining kinetic properties of AcSDKP for the development of clinical protocols to protect normal human hematopoietic stem and progenitor cells following cycle-specific chemotherapy agents.

Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents.

The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively.

In vivo haemoprotective activity of tetrapeptide AcSDKP combined with granulocyte-colony stimulating factor following sublethal irradiation.

We report that acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), which removes progenitor cells from cell cycle, in combination with granulocyte-colony stimulating factor (G-CSF) can significantly improve myelorestoration following irradiation (7 Gy). Peripheral blood, spleen and bone marrow (BM) cell recovery and progenitor cell reconstitution [IL-3-responsive colony-forming cells (CFC) and high proliferative potential colony-forming cells (HPP-CFC)] were studied. Studies on the optimal schedule of AcSDKP administration revealed maximal effects on progenitor cells when AcSDKP was administered as a continuous infusion for 3 d starting 24 h prior to irradiation and used in combination with G-CSF. The numbers of CFC and HPP-CFC in the BM were significantly increased following irradiation in mice receiving AcSDKP and G-CSF as compared to either drug alone. The numbers of CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on days 10 and 14 as compared to AcSDKP alone, but not G-CSF. Similarly, CFC and HPP-CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on day 18 as compared to mice receiving PBS and G-CSF. These studies suggest that AcSDKP in combination with G-CSF may have potential for the protection of progenitor cells in patients undergoing intensive chemo- and/or radiotherapy.

Evidence for the involvement of monocyte-derived toxic oxygen metabolites in the lymphocyte dysfunction of Hodgkin's disease.

This study was performed to see if adherent cell-derived toxic oxygen metabolites contribute to the suppression of mononuclear cell blastogenic responses in Hodgkin's disease. Peripheral blood mononuclear cells from 10 patients with Hodgkin's disease were stimulated in culture with the mitogen PHA in the presence of the prostaglandin inhibitor indomethacin and the antioxidants catalase or vitamin E. Patient lymphocytes showed significant increases in PHA-induced proliferation at all PHA doses when cultured with indomethacin. Further augmentation of lymphocyte proliferation was achieved with the addition of catalase or vitamin E to indomethacin in the culture system. The increases in proliferation seen on culture with these agents were greatest in patients with more depressed initial PHA responses. When adherent cells were removed before culture, the agents no longer facilitated increases in proliferation. These data suggest that abnormal lymphocyte proliferative responses seen in Hodgkin's disease may result in part from the excessive production of toxic oxygen metabolites as well as prostaglandins by adherent cell populations.

In vivo radioprotective effect of AcSDKP on canine myelopoiesis.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) interferes with G1/S-phase progression, and the resulting cell cycle arrest is thought to protect hematopoietic stem cells against injury by cycle-active cytotoxic agents. We investigated the radioprotective effect of AcSDKP in a canine radiation model. Dogs were given total-body irradiation (TBI) at an exposure rate of 10 cGy/min, either without further therapy (control) or with administration of AcSDKP at 0.05-500 micrograms/ kg/24 h beginning before and continuing until after completion of TBI. At 400 cGy of TBI, one of 28 control dogs and one of eight AcSDKP-treated dogs recovered hematopoiesis (p = 0.40). At 300 cGy, seven of 21 control dogs recovered hematopoiesis compared with five of five AcSDKP-treated dogs (p = 0.01). In dogs given 300 cGy and AcSDKP, the granulocyte nadirs were less profound (p = 0.04) and occurred later (p = 0.04) than among controls; platelet kinetics did not differ. These data suggest, therefore, that AcSDKP provides a radioprotective effect in dogs exposed to 300 cGy TBI. Such an effect might be beneficial in recipients of intensive radiation therapy. Conceivably, the effect on hematopoietic recovery could be amplified by growth factor administration after irradiation.

Immunomodulatory effects of interferon-gamma in patients with metastatic malignant melanoma.

Interferon-gamma (IFN-gamma) is a potent monocyte/macrophage activating agent that in animal models exhibits a bell-shaped dose-response curve of immunomodulatory activity and antitumor efficacy. Previous clinical trials of IFN-gamma conducted at the maximal tolerated dose (MTD) have been associated with low response rates that may have been due to failure to treat at an optimal immunomodulatory dose (OID). The objective of this study was to test the hypothesis that optimal immunomodulatory activity of IFN-gamma in patients with metastatic melanoma would be obtained at a dose below the MTD. Groups of five patients each were given daily subcutaneous injections of IFN-gamma at doses of 0.01, 0.1, or 0.25 mg/m2. In vivo immunomodulation was assessed by serial measurement of serum neopterin and by flow cytometry. IFN-gamma doses of 0.1 or 0.25 mg/m2 induced significantly greater immunomodulation of monocyte-associated immune parameters than 0.01 mg/m2. Changes in immunologic parameters included marked elevation of serum neopterin levels, significant increases in monocyte expression of CD64, beta 2-microglobulin, and HLA-ABC, and decreased monocyte expression of CD14. The most dramatic decreases in CD14 expression were observed on monocytes obtained from patients treated at 0.25 mg/m2. The 0.25-mg/m2 dose group had significantly lower white blood cell counts on day 14. No bell-shaped curve of immunologic response was observed over the dosage range tested. Based on the similarity of the immunologic effects at 0.1 and 0.25 mg/m2, treatment at the MTD of IFN-gamma (0.25 mg/m2) represents treatment at the OID for patients with metastatic malignant melanoma.

Loss of interferon antibodies during prolonged continuous interferon-alpha 2a therapy in hairy cell leukemia.

Although highly active in hairy cell leukemia (HCL), interferons (IFN) are not curative in this disease; current data indicate that prolonged IFN therapy will be necessary to control disease in the majority of patients. We previously observed acquired IFN resistance in association with neutralizing IFN-alpha 2a antibodies in small numbers of patients with HCL. This finding suggests that the requisite long-term therapy may be compromised if there is an increasing incidence over time of neutralizing antibodies. We performed a follow-up study of IFN antibodies in our patients receiving continuous IFN therapy. All 16 patients who were previously antibody negative remained so. Surprisingly, all nine patients who previously had non-neutralizing IFN antibodies became antibody negative after a median of 14.5 months. Moreover, 3 of 10 patients who had neutralizing antibodies became antibody negative and five had only non-neutralizing antibodies a median of 10 months from the time neutralizing antibody had first been detected. Only two patients had persisting neutralizing antibodies. Inhibition of neopterin synthesis, inhibition of generation of 2', 5' oligoadenylate synthetase activity, and inability to detect IFN in serum after subcutaneous injection of IFN-alpha 2a was observed only in the one patient tested with neutralizing IFN antibodies confirming that these antibodies have functional significance in vivo. We conclude that, although neutralizing IFN antibodies inhibit the effectiveness of IFN in vivo, these antibodies are produced only transiently during long-term therapy. The long-term effectiveness of this drug will not likely be affected in most patients by neutralizing antibody.