Siri Juell

Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin.

Pseudomonas exotoxin (PE)–containing immunotoxins (ITs) act by arresting protein synthesis and promoting apoptosis, but the mechanisms of the induced apoptosis and the relationship to protein synthesis inhibition is not well elucidated. We studied these effects in MA‐11 human breast cancer cells treated with 425.3PE, an unmodified PE covalently linked to the 425.3 antibody, which targets the EGF receptor. This IT induced efficient inhibition of protein synthesis with simultaneous induction of apoptosis. Thus, treatment of cells with 10 ng/ml of IT for 5 hr caused 85% inhibition of protein synthesis in parallel with caspase‐3, ‐8 and ‐9 activation and PARP inactivation. Even after 72 hr of IT treatment, preincubation with the broad‐spectrum caspase inhibitor z‐VAD‐FMK caused a significant increase in cell survival without affecting IT‐induced protein synthesis inhibition. Interestingly, a combination of z‐VAD‐FMK and the cathepsin B/L inhibitor z‐FA‐FMK prevented completely IT‐induced cell death in MA‐11 cells after 24 hr, indicating that cathepsin activation may be important for optimal induction of IT‐induced cell death. IT treatment caused after 2.5 hr a significant decrease in the level of the antiapoptotic protein Mcl‐1 but not of Bcl‐2 and Bcl‐XL. Furthermore, Mcl‐1 expression was not sensitive to caspase inhibitors but was totally prevented by the lactacystin proteasome inhibitor, suggesting that IT‐induced apoptosis may be triggered by a reduction in the Mcl‐1 level. Mitochondrial membrane potential (ΔΨ mito) decreased concurrently with caspase activation, showing the involvement of ΔΨ mito as a regulator of IT‐induced apoptosis. Our results demonstrate that 425.3PE‐mediated cell death involves simultaneous induction of apoptosis and protein synthesis inhibition in MA‐11 cells, thus contributing to an understanding of the mechanisms involved in IT‐induced apoptosis.

Intratumoral immunotoxin treatment of human malignant brain tumors in immunodeficient animals

Treatment of malignant brain tumors remains a clinical challenge. New treatment modalities are under investigation and among these are intratumoral infusion of immunotoxins that bind to specific cell surface molecules on the malignant cells. We have compared the efficacy of the 425.3‐PE immunotoxin (which targets the epidermal growth factor [EGF] receptor) with the well‐known immunotoxin Tfn‐CRM107 (which targets the transferrin receptor), for the treatment of subcutaneous and intracranial human gliomas in nude animals. Bolus intratumoral administration of 1 μg Tfn‐CRM107 or 425.3‐PE into sc U87Mg tumors in nude mice reduced the tumor volume to 29 and 79%, respectively, of that in the control group 18 days after start of treatment. Higher doses of Tfn‐CRM107 were toxic to the animals, whereas 425.3‐PE was tolerated, with a dose‐response relationship of up to 8 μg, a dose that reduced the tumor volume to 2% of control. In nude rats, treatment of intracerebral U87Mg tumors with Tfn‐CRM107 proved ineffective and doses above 10 ng/animal were toxic to tumor‐bearing rats. In contrast, intratumoral administration of 4 μg 425.3‐PE increased symptom‐free survival from 23 days to 40 days, with 2/9 surviving more than 90 days. We have recently shown that immunodeficient rats inoculated intracerebrally with precultured glioblastoma biopsy specimens develop highly infiltrative brain tumors. Direct interstitial infusion of immunotoxins into such tumors reduced the number of animals with detectable tumors at autopsy after 3 months, from 8/9 in the control animals to 4/6 and 2/6 in animals treated with Tfn‐CRM107 and 425.3‐PE, respectively. In conclusion, the anti‐EGF receptor immunotoxin 425.3‐PE exhibited promising efficacy, comparable to or better than that of Tfn‐CRM107, an immunotoxin that in early clinical trials has been found to give responses in patients with brain tumors.

Systemic immunotoxin treatment inhibits formation of human breast cancer metastasis and tumor growth in nude rats

Adjuvant chemotherapy in breast cancer patients has had limited success, which is possibly because of lack of effect on non‐proliferating cells accompanied by the emergence of drug‐resistant cell clones. Since immunotoxins (ITs) are known to exert proliferation‐independent cytotoxicity, we investigated the efficacy of systemically administered anti‐carcinoma ITs in nude rat models, simulating micrometastatic disease.

AMP-activated protein kinase protects against anti–epidermal growth factor receptor-Pseudomonas exotoxin A immunotoxin-induced MA11 breast cancer cell death

We have shown previously that our 425.3PE immunotoxin inhibits protein synthesis and induces apoptosis in human breast cancer cells. In attempts to further elucidate the intracellular pathways implicated in its cellular effects, we found that the immunotoxin induced an initial stress response, which rapidly caused an imbalance in the cellular energy status with an increase in reactive oxygen species. The AMP-activated protein kinase (AMPK), a sensor of increased cellular AMP/ATP ratio, was activated by 425.3PE. An immunotoxin-induced activation of c-Jun NH2-terminal kinase (JNK) preceded and overlapped caspase-mediated cleavage of the α-subunit of AMPK in a time- and dose-dependent manner. The JNK activation occurred already at a dose level too low to induce any detectable changes in the apoptotic machinery or protein synthesis. In contrast, cycloheximide, even at a concentration causing a 90% inhibition of protein synthesis, did neither affect the ATP level nor activate JNK and AMPK. Pretreatment of the cells with the specific AMPK inhibitor compound C and JNK inhibitor SP600125 blocked activation of AMPK and JNK, respectively, and subsequently sensitized the cells to 425.3PE-induced cell death. Whereas the antioxidant N-acetyl-l-cysteine blocked the generation of reactive oxygen species and activation of JNK and AMPK, it did not block immunotoxin-induced apoptosis. Together, the results show that 425.3PE induces several parallel signaling events, observed initially as an early activation of survival pathways, protecting the cells against the toxic effects of the immunotoxin, followed by subsequent apoptosis induction and protein synthesis inhibition. Conceivably, therapeutic manipulation of the signaling intermediates AMPK and JNK might provide a means to maximize the anticancer effects of the 425.3 immunotoxin. [Mol Cancer Ther 2006;5(4):1050–9]

Phase I trial of EpCAM-targeting immunotoxin MOC31PE, alone and in combination with cyclosporin

Background:A phase I trial was performed to determine the maximum tolerated dose (MTD), safety, pharmacokinetics and immunogenicity of the anti-EpCAM immunotoxin (IT) MOC31PE in cancer patients. An important part of the study was to investigate whether the addition of Sandimmune (cyclosporin, CsA) suppressed the development of anti-IT antibodies.Methods:Patients with EpCAM-positive metastatic disease were eligible for treatment with intravenous MOC31PE using a modified Fibonacci dose escalation sequence. Maximum tolerated dose was first established without, then with intravenously administered CsA.Results:Sixty-three patients were treated with MOC31PE in doses ranging from 0.5 to 8 μg kg−1. Maximum tolerated dose was 8 μg kg−1 for MOC31PE alone, and 6.5 μg kg−1 when combined with CsA. The dose-limiting adverse event was reversible liver toxicity. No radiological complete or partial responses were observed, whereas stable disease was seen in 36% of the patients receiving MOC31PE only. The pharmacokinetic profile of MOC31PE was characterised by linear kinetics and with a half-life of ∼3 h. The addition of CsA delayed the generation of anti-IT antibodies.Conclusions:Intravenous infusion of MOC31PE can safely be administered to cancer patients. Immune suppression with CsA delays the development of anti-MOC31PE antibodies. The antitumour effect of MOC31PE warrants further evaluation in EpCAM-positive metastatic disease.