High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting.

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

Improved leukemia-free survival after postconsolidation immunotherapy with histamine dihydrochloride and interleukin-2 in acute myeloid leukemia: results of a randomized phase 3 trial.

The primary objective of this phase 3 study was to determine whether postconsolidation immunotherapy with interleukin-2 (IL-2) and histamine dihydrochloride (HDC) improved the leukemia-free survival (LFS) of adult patients with acute myeloid leukemia (AML) in complete remission (CR). Three hundred twenty patients with AML (median age, 57 years; range, 18-84 years) were stratified by CR1 or subsequent CR (CR > 1) and randomly assigned to treatment with HDC/IL-2 or no treatment (control). Treatment comprised 10 21-day cycles with IL-2 (16 400 U/kg) plus HDC (0.5 mg); both compounds were administered by subcutaneous injection twice daily. Study arms were balanced for age, sex, previous treatment, leukemic karyotypes, time from CR to inclusion, and frequency of secondary leukemia. Three years after enrollment of the last patient, treatment with HDC/IL-2 was found to improve LFS over control in the study population (CR1 + CR > 1, n = 320; P < .01, log-rank test). For patients in CR1 (n = 261), treatment significantly improved LFS (P = .01) with 3-year LFS estimates of 40% (HDC/IL-2) compared with 26% (control). Side effects were typically mild to moderate. These results indicate that HDC/IL-2 treatment offers an efficacious and tolerable treatment for patients with AML in remission.

A role for transferrin receptor in triggering apoptosis when targeted with gambogic acid.

Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.

The discovery and mechanism of action of novel tumor-selective and apoptosis-inducing 3,5-diaryl-1,2,4-oxadiazole series using a chemical genetics approach.

A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics-based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-beta1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.

Histamine improves survival and protects against interleukin-2-induced pulmonary vascular leak syndrome in mice.

The therapeutic efficacy in the treatment of metastatic cancer with high doses of interleukin-2 (IL-2) has been limited by the onset of vascular leak syndrome (VLS) and related toxicities. VLS is characterized by an increase in vascular permeability and severe hypotension resulting in interstitial edema and organ failure. This study explores the protective effects of histamine dihydrochloride (HDC) against IL-2-induced toxicities in mice. Treatment with HDC administered before or after IL-2 (1.25 x 10(6) IU, BID) was shown to protect mice from VLS-related toxicities and mortality in a dose-dependent manner. Survival rates when HDC was added were 56, 75 and 81% at doses of 0.47, 4.7 and 47.0 mg/kg, respectively, compared to 42% survival with IL-2 alone. HDC protected against IL-2-induced macroscopic pulmonary lesions, reduced edema (up to 62% reduction in lung wet/dry weight ratio) and reduced capillary leakage into the lungs as measured by a reduction in Evans Blue dye content. In addition, the systemic effect on serum cytokine levels showed that HDC only moderately lowered IL-2 induced IFN-gamma, IL-6, IL-10, IL-18 and TNF-alpha. Serum levels of IL-1beta, IL-4 and IL-12 were not measurably induced by IL-2 treatment. HDC modulates many cellular functions including regulating cytokines and blocking immune-suppression caused by reactive oxygen species (ROS) generated by the NADPH oxidase. However, the protective effect of HDC on alleviating IL-2-induced pulmonary edema was not related to ROS inhibition. Our data indicate that HDC treatment improves survival and protects against IL-2 induced VLS independent of ROS regulation in mice.

Addition of histamine to interleukin 2 treatment augments type 1 T-cell responses in patients with melanoma in vivo: immunologic results from a randomized clinical trial of interleukin 2 with or without histamine (MP 104).

PURPOSE: Preclinical investigations suggest that histamine dihydrochloride (HDC) protects T cells and natural killer cells from inhibition by monocyte-derived reactive oxygen metabolites and synergizes with interleukin (IL) 2 in inducing T-cell activation. Here, we investigate whether this mechanism is operational in patients with melanoma treated with HDC as an adjunct to IL-2. EXPERIMENTAL DESIGN: Melanoma patients having liver metastases were treated with IL-2 with or without HDC within a randomized, multicenter, phase III trial. The effect of HDC on type 1 and type 2 T-cell cytokine production was investigated in peripheral blood samples from 19 patients with the use of intracellular cytokine flow cytometry. Melanoma-specific T-cell responses were analyzed in eight HLA-A2-positive patients. RESULTS: Frequencies of CD3+ T cells producing IFN-gamma (type 1 T cells) in response to phorbol myristate acetate/ionomycin increased (median, 1.8-fold) in patients receiving IL-2 plus HDC but not in those receiving IL-2 alone (P < 0.01 for comparison between arms). In contrast, the number of IL-13-producing type 2 T cells that increased in patients after treatment with IL-2 was not modulated by HDC. Melanoma- and tyrosinase-specific IFN-gamma and IL-13-producing T cells were detected in two of four HLA-A2-positive patients with melanoma following treatment with HDC + IL-2. CONCLUSIONS: Treatment of patients with stage IV melanoma with HDC in combination with IL-2 increases type 1 T-cell responses and may promote induction of melanoma-specific T cells. These effects are of relevance for tumor immunotherapy and provide a potential mechanism for the clinical efficacy of HDC added to IL-2.

Treatment with histamine dihydrochloride and interleukin-2 in patients with advanced metastatic malignant melanoma: a detailed safety analysis.

Intravenous high dose bolus therapy with interleukin-2 (IL2) is associated with low overall response rates (15%) and significant toxicity. Phase II and III trials of a lower dose subcutaneous regimen of IL2 administered alone (n = 152) or in combination with histamine dihydrochloride (n = 239) have recently been completed. This article describes a comprehensive safety and toxicity analysis of the results of these two trials. The phase III trial demonstrated a survival benefit in patients with liver metastases in the histamine/IL2 arm. Eligible patients had stage IV malignant melanoma with at least one measurable lesion. Toxicity was graded using the National Cancer Institute (NCI) common toxicity scale. All reported adverse events were included in the analysis. Almost all toxicities in each treatment group were NCI grade 1 or 2. The incidence of toxicities expected to occur with histamine treatment, such as hypotension/vasodilation, headache and injection site reaction, were higher among patients receiving histamine. With the exception of headache, the incidence of grade 3 or 4 toxicities was similar across the treatment groups. The addition of histamine to the subcutaneous IL2 regimen did not result in a difference in the incidence of drug interruption, dose modification or discontinuation. Study-related deaths were low and were not impacted by the addition of histamine to the IL2 regimen.

The health-related quality-of-life impact of histamine dihydrochloride plus interleukin-2 compared with interleukin-2 alone in patients with metastatic melanoma.

The aim of this study was to compare the quality-of-life (QOL) effects of interleukin-2 (IL-2) alone with those of IL-2 plus histamine dihydrochloride in the setting of a multicenter, randomized trial for patients with metastatic melanoma. QOL data were collected from July 1997 to March 2000 during a phase III trial comparing subcutaneous histamine plus IL-2 and IL-2 alone. Prior to each treatment cycle, patients completed the 76-item Quality-of-Well-Being Scale-Self-Administered (QWB-SA) questionnaire, the Overall State of Health (OSH) item, and the General Health Perception (GHP) item. A longitudinal data analysis using the generalized estimating equations approach was performed to compare changes in QWB-SA scores over time between treatment groups, and predicted QWB-SA scores from the regression analysis were used to calculate quality-adjusted survival duration over the 12-month study period. QOL analyses were conducted for all randomized patients (intent-to-treat overall population, ITT-OA) and all patients who had liver metastases at randomization (ITT-LM population). In the ITT-OA population, differences in QWB-SA scores over time between the histamine plus IL-2 group (150 patients) and the IL-2 alone group (151 patients) were not significant (P=0.511, type III F test). In the ITT-LM population (53 histamine plus IL-2 patients and 73 IL-2 alone patients), changes in QWB-SA scores over time favored the histamine plus IL-2 group (P=0.018, type III F-test). In both the ITT-OA and ITT-LM populations, QWB-SA scores deteriorated more quickly over time in the IL-2 alone group than in the histamine plus IL-2 group, resulting in a significant difference in median quality-adjusted survival duration in favor of the histamine plus IL-2 group by 31.3 days in the ITT-OA population (P=0.007, Mann-Whitney U-test), and 50.2 days in the ITT-LM population (P=0.011). OSH and GHP scores did not differ between groups. The addition of subcutaneous histamine dihydrochloride to IL-2 treatment improved median quality-adjusted survival duration and did not adversely affect QOL in patients with malignant melanoma.

Toxicology and toxicokinetics of acute and subchronic administration of histamine dihydrochloride in rats.

Histamine dihydrochloride is currently being evaluated as an adjuvant to immunotherapy regimens in neoplastic and infectious diseases. The no-observed-effect-level (NOEL), no-observable-adverse-effect-level (NOAEL), and pharmacokinetics of subcutaneously administered histamine dihydrochloride were determined via 5 and 28 day repeated dose studies in Sprague-Dawley rats. In the five day study, male rats received 0 (vehicle), 5, 30, 500, or 1000 mg/kg BID. Acute tissue damage was observed at one or more injection sites in the two highest dose groups after 24 h. At five days, animals in these groups displayed indications of pathological inflammation at the injection sites. In the 28 day study, male and female rats received 0 (vehicle), 0.5, 5, or 100 mg/kg BID. The most significant treatment-related pathological findings were signs of inflammation at the injection sites for animals in the 100 mg/kg BID group. Hematology and clinical chemistry changes in the highest dose groups in both studies were consistent with inflammation and anemia but were found to be reversible following a 14-day recovery. Plasma histamine levels were quantified from male and female animals receiving 0.5, 5, and 100 mg/kg injections on Day 1 and 28 of the twenty-eight day study. Cmax was achieved within 0.25 h and was dose-proportional. The elimination half-life and tmax were longer at the 100 mg/kg dose than the lower doses. No marked differences between genders or between Day 1 and 28 were found. Based on these findings, the NOEL and NOAEL were established at 0.5 mg/kg BID and 5 mg/kg BID, respectively. When converted to human equivalent dose, the NOAEL is 0.81 mg/kg which is 54 times the intended human dose. These studies support a wide safety margin for histamine dihydrochloride.

Pharmacokinetics of histamine dihydrochloride in healthy volunteers and cancer patients: implications for combined immunotherapy with interleukin-2.

Recent clinical trials in melanoma and leukemia have demonstrated potential for increased survival time and improved remission when histamine dihydrochloride is added to cytokine monotherapy. In the present study, the pharmacokinetics of subcutaneous histamine (1 mg) in 21 healthy subjects and 12 melanoma patients was determined via model-dependent methods. Drug-drug interactions with subcutaneous interleukin-2 (1.1 mg) were evaluated in a combined cohort of patients with melanoma (n = 8) or renal cell carcinoma (n = 4). Histamine dihydrochloride administered over 10 minutes in healthy subjects peaked at 18 minutes (Cmax 38 nmol/L), attained a distribution volume of 59 L, and was eliminated at 6%/min. The results were similar in a 20-minute infusion in melanoma patients. No gender effects were observed (p > 0.05). Interleukin-2 injected either 10 minutes prior to or 10 minutes following histamine dihydrochloride had no effect on histamine kinetics. Histamine dihydrochloride administered 10 minutes prior to injection of interleukin-2 also had no effect on interleukin-2 kinetics. Maximal concentration of interleukin-2 (2,442 pg/ml) occurred at 2.5 hours with an elimination half-life of 1.7 hours, area under the curve (AUC) of 15,746 pg x h/ml, and volume of distribution and plasma clearance of 194 L and 74 L/h, respectively. However, interleukin-2 Cmax (1,758 pg/ml) and AUC (12,448 pg x h/ml) were reduced when histamine dihydrochloride was infused 10 minutes following interleukin-2, likely due to the pharmacodynamic effects of histamine, including increased heart rate and reduced blood pressure. It is concluded that histamine dihydrochloride and interleukin-2 can be safely coadministered with minimal interaction.

Results from a randomized phase III study comparing combined treatment with histamine dihydrochloride plus interleukin-2 versus interleukin-2 alone in patients with metastatic melanoma.

PURPOSE: Reactive oxidative species (ROS) produced by phagocytic cells have been ascribed a role in the localized suppression of lymphocyte function within malignant tumors. Histamine has been shown to inhibit ROS formation and possibly synergize with cytokines to permit activation of natural killer cells and T cells. This study was designed to determine whether the addition of histamine to a subcutaneous (SC) regimen of interleukin-2 (IL-2) would improve the survival of metastatic melanoma patients. PATIENTS AND METHODS: A phase III, multicenter, randomized, parallel group study comparing IL-2 plus histamine with IL-2 alone was conducted in 305 patients with advanced metastatic melanoma. Patients were randomized to IL-2 (9 MIU/m(2) bid SC on days 1 to 2 of weeks 1 and 3, and 2 MIU/m(2) bid SC on days 1 to 5 of weeks 2 and 4) with or without histamine (1.0 mg bid SC days 1 to 5, weeks 1 to 4). The primary end point, survival, was prospectively applied to all randomized patients (intent-to-treat-overall population, ITT-OA) and all patients having liver metastases at randomization (ITT-LM population). Secondary end points included safety of the combined treatment, time to disease progression, and response rate. RESULTS: Combined treatment with histamine plus IL-2 significantly improved overall survival in the ITT-LM population (P =.004) and showed a trend for improved survival in the ITT population (P =.125). Grade 3 and 4 adverse events were comparable in the two arms. CONCLUSION: Use of histamine as an adjunct to IL-2 is safe, well tolerated, and associated with a statistically significant prolongation of survival compared with IL-2 alone in metastatic melanoma patients with liver involvement. Further trials to confirm and understand the role of histamine in this combination treatment are underway.