Continuous delivery of human type I interferons (α/β) has significant activity against acute myeloid leukemia cells in vitro and in a xenograft model

Abstract

IntroductionDespite significant advances in the treatment of acute myeloid leukemia (AML), long-term survival is limited to about 30% of patients; hence the urgent need for alternative treatment strategies. 1 Type I IFNs (IFN-␣ and IFN-␤) have shown considerable promise in the treatment of many solid tumors 2 and have proven to be highly effective in several hematologic malignancies, including chronic myeloid leukemia. 3 Although in vitro studies with type I IFNs have shown impressive cytotoxicity against AML cells, 4 their antitumor efficacy in vivo remains largely untested in animal models or in patients with AML, except for the United Kingdom Medical Research Council (UK MRC) AML 11 trial, in which maintenance treatment with IFN-␣ failed to prevent relapse or improve overall survival. 5 However, it remains unclear whether this lack of efficacy was related to poor antileukemic effect in vivo or a failure to maintain stable therapeutic IFN-␣ levels because of its short half-life. 6 In this study, we compare the antileukemic effects of IFN-␣ and ␤ against a variety of AML cell lines in a xenograft model. Two different schedules of administration were used: the traditional high-dose multiple bolus injection approach using recombinant IFN-␤, as well as continuous low-dose delivery, achieved here through gene transfer with adenoassociated virus (AAV) vectors. Materials and methods Cell culture and cell proliferation assaysThe AML cell lines HL-60, KG1␣, AML193, and OCI AML5 were cultured in RPMI medium with 10% fetal calf serum. GM-CSF at 100 pg/mL was added to the medium for AML193 and OCI AML5 cells. Frozen AML cells obtained from leukopheresis of patients with poor-risk AML were used for our primary AML experiments. Cell lines were seeded in 96-well plates at 20 000 cells per well to which varying concentrations of recombinant human IFN␤-1a or IFN␣-2b were added. Following stimulation for 3 or 5 days, cells were pulsed with 3 H-thymidine (0.5 Ci [0.0185 MBq] per well) for 4 hours, harvested, and 3 H-thymidine incorporation measured by scintillation counting. AAV2/8 hIFN-␤ and AAV2/8 hFIX vector productionAs previously described, recombinant AAV vectors pseudotyped with serotype 8 capsid were generated by transient transfection of 293T cells using either the pAV2 CAGG hIFN-␤ or pAV2 CAGG human factor IX (hFIX) control vector plasmids together with pAAV2/8 packaging and pHTGI adeno helper plasmids. 7 The AAV2/8 vectors were purified using an ion exchange chromatography method, and slot blot analysis was used to determine the titer of the vectors. Human IFN␣-2b and IFN␤-1a bioassayPlasma levels of human IFN-␣ and IFN-␤ were determined using a validated antiviral assay with a sensitivity of 0.5 IU/mL as described before. 8

Interferon-α in acute myeloid leukemia: an old drug revisited

“…14,15 Careful retrospective analysis of all clinical trials performed hitherto reveals that IFN-a indeed has the potential to induce clinically relevant antileukemic responses in AML patients. Such responses, however, have only been observed in a minority of patients, which has raised questions about the factors responsible for this inconsistency.…”

“…16 Clinical experience so far is mainly based on the use of these unmodified IFN formulations, hence providing a plausible explanation for the inconsistent and relatively modest clinical results achieved to date. 14 In recent years, IFN preparations with improved pharmacokinetic properties have been developed. Sustained serum IFN levels can nowadays be obtained by pharmacological modification of IFN, including genetic fusion of IFN-a with human serum albumin 17 or addition of a polyethylene glycol moiety (known as pegylation).…”

“…Sustained serum IFN levels can nowadays be obtained by pharmacological modification of IFN, including genetic fusion of IFN-a with human serum albumin 17 or addition of a polyethylene glycol moiety (known as pegylation). 16,18 On the basis of the results of the xenograft study described above, 14 it is highly expected that the use of these modified IFN preparations can unlock the therapeutic potential of IFN-a in acute leukemia. Pegylated IFN-a was recently put to the clinical test in a patient with AML superimposed on primary myelofibrosis.…”

Establishment of Leukemia Mouse Model Using Mouse-Derived A20 Leukemic Cells, and Detection of Tumor Cells in Bone Marrow

“…혈액 암의 새로운 치료법의 개발을 위해서 새로운 백혈병-림 프종 연구(leukemia-lymphoma research)를 위한 세포주와 동물 모델의 개발이 끊임없이 진행되어 왔으며, 1963년 림프종에서 유래한 세포주가 처음으로 개발된 이래 현재 까지 1,000여종의 세포가 보고되고 있다 (Hans et al, 2000). 백혈병 동물모델로는HL-60, K562 등 인간 골수성 백혈병 세포주를 면역 부전 마우스에 정맥 이식하거나 마 우스 유래의 B림프구인 A20 세포주를 동일계통 마우스 (syngenic mouse)에 정맥 이식방법이 많이 사용되고 있으 며, 이들은 간단한 동소위 종양이식이란 점에서 혈액암 동물모델로서의 가치가 높다 (Ratajczak et al, 1992;Benjamin et al, 2007). 백혈병 동물모델 형성을 위하여 HL-60, K562, KG-1, MO7E, TF-1 등 인간 백혈병 세포 주(human leukemia cell line)를 누드 마우스에 투여하여 급성골수성백혈병(acute myelogenous leukemia; AML) 동 물모델이 완성되었다 (Nara and Miyamoto, 1982;Machado et al, 1984;Janssen et al,1987;Krishnaraju et al, 1998;Kiser et al, 2001).…”

“…Each group consisted of 6-13 mice. ) 정도 투여했을 때보다도 종양모델이 쉽게 형성되 여 생존기간이 10여일 정도 단축시킬 수 있다는 점에서 장점을 가지고 있다 (Glass, 1996;Zeis, 1997;Benjamin, 2007;Liu, et al, 2007). 참고문헌…”