A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead.

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin-ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

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.

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.

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.

Histamine dihydrochloride protects against early alcohol-induced liver injury in a rat model.

Inflammation of the liver may be caused by a variety of factors that include infectious agents and toxins. Reactive oxygen species (ROS) generated by the NADPH oxidase in Kupffer cells and infiltrating leukocytes play an important role in the pathogenesis of early alcohol-induced hepatitis. Histamine dihydrochloride (histamine) suppresses the generation of ROS through the histamine type-2 receptor (H2 receptor). Histamine was studied as a potential protective treatment against early alcohol-induced liver injury in an experimental hepatitis model. Female Wistar rats were given ethanol (5 g/kg) intragastrically by gavage once daily for 4 weeks, while a control group not receiving ethanol was fed an isocaloric high-fat diet. Animals receiving ethanol had elevated serum levels of alanine and aspartate transaminase (ALT/AST) and developed steatosis, inflammation, and necrosis of the liver. Histamine treatment (0.5 or 5.0 mg/kg, twice daily) protected against this liver injury as evident by normal serum transaminase levels and significantly reduced liver pathology scores. Ranitidine (10 mg/kg), an H2 receptor antagonist, blocked the protective effect of histamine, indicating that the histamine effect is predominantly mediated through the H2 receptor. In conclusion, these results suggest that histamine protects against early alcohol-induced liver injury in rats.

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.

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.