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Abstract
To design hybrid tumour necrosis factor-alpha (TNF-alpha) applicable to systemic anti-tumour therapeutic use, we assessed the relationships among the molecular size of hybrid TNF-alpha, in vitro bioactivity and in vivo anti-tumour potency. Natural human TNF-alpha was covalently modified with polyethylene glycol (PEG) of various number-average molecular weights (Mn = 2000, 5000, 12,000). The in vitro bioactivity of PEG-modified TNF-alpha s decreased with an increase in the degree of PEG modification, irrespective of the molecular weight of PEG. This decrease in the specific bioactivity markedly increased with an increase in the molecular weight of the attached PEG. The in vivo anti-tumour effects of the hybrid TNF-alpha s with a molecular size from 100 to 110 kDa, which had more than 50% of specific bioactivity of native TNF-alpha, were significantly superior to other PEG-TNF-alpha s. These hybrid TNF-alpha s showed over ten times greater anti-tumour effects than native TNF-alpha. Thus, the molecular size, which was determined by the degree of PEG modification and PEG molecular weight, influences the specific activity and anti-tumour effects of hybrid TNF-alpha.
Highlights
We indicated that the molecular size of hybrid TNF-cx, which is determined by the steric hindrance resulting from the molecular weight of attached polyethylene glycol (PEG) and the degree of PEG modification, may influence its specific activity and in vivo anti-tumour activity
Natural human tumour necrosis factor-a (TNF-a) was chemically modified by end point attachment with PEG of various molecular weights via the formation of an amino bond between lysine amino groups of TNF-a and the terminal succinimidyl succinate group of PEG
These results strongly indicated that the molecular size of PEG-TNF-ac, that is, the steric hindrance determined by the degree of PEG modification as well as the molecular weight of PEG, is a very important factor to consider in designing hybrid TNF-x
Summary
MaterialsNatural human TNF-a was kindly supplied by Hayashibara Biological Laboratories Inc. (Okayama, Japan). Natural human TNF-a was kindly supplied by Hayashibara Biological Laboratories Inc. PEG; number -average molecular weights = 2000, 5000 and 12 000) were supplied by Nippon Oil and Fats (Tokyo, Japan). Other reagents and solvents were of analytical grade. Male ddY mice (5 weeks old) were purchased from SLC (Hamamatsu, Japan). L-M cells were generously provided by Mochida Pharmaceutical Co., Ltd. L-M cells were serially subcultured in Eagle's minimum essential medium containing 10% fetal calf serum (FCS; Filtron, Brooklyn, USA). Sarcoma-180 cells were maintained intraperitoneally by serial passages in male ddY mice
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