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.

Evaluation of low dose continuous infusion 5-fluorouracil in patients with advanced and recurrent renal cell carcinoma. A Southwest Oncology Group Study.

BACKGROUND: The response rate of metastatic renal cell cancer to cytotoxic therapy over the last 10 years has been 5.6%. Low dose continuous 5-fluorouracil (5-FU) has demonstrated efficacy in other cytotoxic refractory tumors, such as pancreas, colorectal, and recurrent breast. The Southwest Oncology Group undertook a Phase II trial of low dose, continuous 5-FU in metastatic renal cell cancer. METHODS: Sixty-one patients were entered in the study to receive 300 mg 5-FU/m2/day for 7 days via a central venous catheter and external programmable pump. The pump was refilled every 7 days. Pyridoxine (50 mg, orally) was administered prophylactically three times a day. RESULTS: A response of 5.2% (one complete response [CR] and two partial responses [PRs]) was achieved. The overall survival was 12 months. The duration of the CR is more than 30 months. Both PRs lasted 6 months. No survival advantage was noted with either prior nephrectomy or biologic therapy. The majority of toxicities were Grade 2: anemia, anorexia, diarrhea, nausea/vomiting, and stomatitis. No toxic deaths occurred. CONCLUSION: Low dose, continuous 5-FU demonstrated minimal activity in metastatic renal cancer.

Phase II evaluation of amonafide in advanced sarcoma: a Southwest Oncology Group study.

Soft tissue sarcomas are generally resistant to most chemotherapeutic agents, and individuals with advanced disease have a poor prognosis. We evaluated amonafide, a new drug that has significant activity against several tumor cell lines, to determine its activity against sarcomas. Amonafide was administered to 18 patients with advanced soft tissue sarcoma (16 of whom had received prior chemotherapy) at a dose of 300 mg/m2 over 60 min daily for 5 days. Courses were repeated every 21 days. Toxicity was mild, but no responses were observed. We conclude that amonafide is not an active agent in previously treated, advanced soft tissue sarcomas in the dose and schedule utilized.

5-fluorouracil and interferon-alpha-2a in advanced colorectal cancer. Results of two treatment schedules.

BACKGROUND: Potential synergy between 5-fluorouracil (5-FU) and interferon alpha-2a (IFN-alpha-2a) has been demonstrated in the treatment of colorectal carcinoma. Continuous low-dose infusion of 5-FU may have superior response rates to bolus 5-FU in these malignancies. This report presents results of two Phase II trials using these principles in colorectal cancer. METHODS: Forty-eight patients were entered onto two protocols; 18 were treated with 5-FU by a bolus infusion schedule with concurrent IFN-alpha-2a (Group 1). Thirty patients were treated with continuous low-dose 5-FU and IFN-alpha-2a thrice weekly (Group 2). RESULTS: The overall response rates were 33% (95% confidence interval [CI], 16-68%) and 33% (95% CI, 17-53%), respectively, for Groups 1 and 2. In Group 2, in 16 previously untreated patients, there was a response rate of 56% (95% CI, 30-80%). The median survival was 11 months and 6 months for Groups 1 and 2, respectively. Toxicity in Group 1 was as expected, except the incidence of central nervous system toxicity was low, with only one patient requiring dose reduction because of cerebellar ataxia. The toxicity in Group 2 was substantial, with four patients being removed from study because of toxicity and all patients treated for more than 2 months requiring dose reductions. The most common (67%) toxicity was mucositis, with 33% of those patients classified as Grade III or IV (Southwest Oncology Group criteria). Other major toxicities were fatigue and hand/foot syndrome. CONCLUSIONS: The first trial confirms previous response rate data for bolus injection 5-FU and IFN-alpha-2a. The second trial of low-dose continuous infusion 5-FU with IFN-alpha-2a demonstrates similar efficacy with substantially greater toxicity.

Prolonged, alternating chemotherapy for extensive small cell lung cancer. A Southwest oncology Group study.

BACKGROUND: Etoposide may be schedule dependent in small cell lung cancer (SCLC), and some data suggest a benefit for increased dose intensity in this disease. This study attempted to optimize dose intensity using an outpatient program that included prolonged, oral etoposide administration. METHODS: Cisplatin-etoposide (PE) and cyclophosphamide, doxorubicin, and vincristine (CAV) were alternated at monthly intervals in patients with extensive SCLC. PE was given as cisplatin 50 mg/m2 on days 1 and 8 intravenously (i.v.) and etoposide 50 mg/m2/day for 14 days by mouth. CAV was administered as cyclophosphamide 60 mg/m2/day for 21 days orally, doxorubicin 20 mg/m2/week for three doses, and vincristine 2 mg i.v. on day 1 only. At the end of 4 months, responding patients received an additional course of PE alternating with CAV, and whole-brain irradiation (3000 cGy in 15 fractions) was delivered to clinical complete responders (CR). RESULTS: Among 61 eligible patients, 4 achieved CR, and 11 had a partial remission, by strict Southwest Oncology Group criteria. An additional 20 patients demonstrated greater than 50% tumor shrinkage on one disease assessment but did not have confirming measurements at all sites 4 weeks later. The overall response rate was 57%, including the latter group. The toxicity was primarily hematologic, with three treatment-related deaths from neutropenic infection (5%). Grade 4 neutropenia (< 500/microliters) occurred in nine patients (15%) and Grade 4 thrombocytopenia (< 25,000/microliters), in three (5%). Analysis of the delivered dose intensity (in milligrams per square meter per week) as a function of that which was planned revealed a mean of 93% for all courses. CONCLUSIONS: Although substitution of prolonged oral etoposide in PE and oral cyclophosphamide in CAV proved to be feasible, these results suggest no advantage over those from other reported series using these alternating regimens in which the agents are pulsed. Experience with alternating PE-CAV for extensive SCLC is reviewed.

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.

A randomized phase II trial of trimetrexate or didemnin B for the treatment of metastatic or recurrent squamous carcinoma of the uterine cervix: a Southwest Oncology Group trial.

Patients with measurable metastatic or recurrent squamous carcinoma of the uterine cervix who had failed prior surgery or radiation therapy were enrolled on this randomized phase II study. Twenty-seven eligible patients were assigned to receive didemnin B at either 2.6 mg/m2 iv every 28 days (sixteen patients) or at 5.6 mg/m2 (eleven patients). Sixteen patients were assigned to receive 12 mg/m2/day iv trimetrexate for 5 days, repeated every 21 days. Toxicity for didemnin B was characterized by nausea and vomiting (78% of patients), anemia (59%), mild diarrhea (11%), and episodic hypersensitivity (three patients). Toxicity for trimetrexate included nausea and vomiting (69%), leukopenia (51%), mild thrombocytopenia (38%), anemia (63%), and diarrhea (31%). No antitumor responses were observed for either agent. Neither trimetrexate nor didemnin B at these doses and schedules is recommended for the treatment of advanced squamous carcinoma of the uterine cervix.

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.

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.

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.

Lack of common haplotype among four family members with late-onset Kaposi's sarcoma.

Kaposi's sarcoma is associated with an increased frequency of HLA-DR5. The hypothesized model of a susceptibility gene in linkage disequilibrium with DR5 may be tested by haplotype analysis in familial Kaposi's sarcoma. Our finding of no common haplotype among afflicted members of a family provides evidence against the hypothesized linkage.

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.

Immunotoxicity of multiple dosing regimens of free doxorubicin and doxorubicin entrapped in cardiolipin liposomes.

We have shown that doxorubicin entrapped in cardiolipin liposomes retain antitumour efficacy in mice but had diminished cardiac uptake and cardiotoxicity. Such liposomes are preferentially taken up by spleen. In a previous study we showed that a single dose of liposomal doxorubicin is not more toxic than free doxorubicin with regard to immunologic parameters including generation of cytotoxicity for histocompatibility alloantigens and mitogenic responsiveness. In the present study, we have explored clinically relevant multiple dosing at weekly intervals, 2, 3, or 4 times. Again, despite splenic localization of liposomal doxorubicin, the depressive effect on these immunological parameters is not greater than the effect of free drug, and, in addition, the damage is repaired earlier.

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.

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.

Augmentation of natural immunity and correlation with tumor response in melanoma patients treated with human lymphoblastoid interferon.

Thirty-two melanoma patients treated with lymphoblastoid alpha interferon (Wellferon) were studied for augmentation of five putative parameters of natural immunity including natural killing (NK), antibody-dependent cellular cytotoxicity (ADCC), cell-mediated inhibition of growth in culture of a murine tumor (GIA), and the size of the OKTIO+ and Leu 7+ subpopulations of peripheral blood mononuclear cells (OKTIO and Leu7). This study confirms and extends our previous conclusions that interferon increases GIA and OKTIO. The increases occurred at 24 hr after interferon, both early and late in the course of treatment, and were dose dependent. NK, ADCC, and Leu7 were activated in many patients individually and mean values for NK and Leu7 were increased in the population as a whole. Two patients with complete remission showed dramatically increased natural immunity by the parameters studied, but the pattern of increase was very different for each patient. The current study of lymphoblastoid alpha interferon demonstrates the immunomodulatory potential of interferon given to melanoma patients, but it fails to support the hypothesis that augmentation of these parameters of natural immunity by interferon may result in tumor responses.

Phase II trial of lymphoblastoid interferon in metastatic malignant melanoma.

Thirty-three patients with metastatic malignant melanoma, some of whom had received previous chemotherapy, immunotherapy, or radiotherapy, were entered in a trial of three different doses and schedules of administration of lymphoblastoid alpha interferon. The overall response rate was 9%; some patients had long-lasting response and two achieved complete response. There was no clear advantage to any one regimen. Dose-related flu-like syndrome, granulocytopenia, hepatocellular enzyme elevation, and anemia were observed as the most common toxic effects. Interferon deserves further study in the treatment of malignant melanoma.

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.

A Phase II trial of high-dose intravenous interferon alpha-2 in advanced colorectal cancer.

Twenty-one patients with metastatic colorectal cancer were treated with high-dose intravenous interferon alpha-2 (30-50 X 10(6) units/m2) administered daily for 5 consecutive days. Courses of therapy were repeated every 2 to 3 weeks. No tumor responses were seen among 15 evaluable patients. In two subjects, disease remained stable for 3 and 7 months, respectively. Toxicity was substantial and a de-escalation of dose was frequently required. Fevers, gastrointestinal symptoms, fatigue, leukopenia, and elevated serum transaminases were common. High-dose interferon was found to be ineffective in the treatment of metastatic colorectal cancer. A daily dose of 50 X 10(6) units/m2 was greater than the maximum tolerated dose in this group of patients.