Immunology 1992 76 433-438
Biological activity of mutants of human tumour necrosis factor-alpha R. KIRCHEIS, J. MILLECK, V. G. KOROBKO,* L. N. SHINGAROVA,* D. BEHNKEt & H. E. SCHMIDTJ Max Delbrick Center for Molecular Medicine, Berlin-Buch, Germany, *Institute for Bioorganic Chemistry, Moscow, Russia, tCentral Institute for Microbiology and Experimental Therapy, Jena, Germany and tBiochemical Technological Center,
Berlin-Buch, Germany Acceptedfor publication 8 March 1992
SUMMARY Point mutations in different regions of the tumour necrosis factor-alpha (TNF-a) molecule influence anti-tumour cytotoxic/cytostatic activities as well as haemorrhagic tumour necrosis, tumour regression and lethal toxicity in mice. Mutations in the C-terminal region in positions 150 and 155 markedly decrease cytotoxicity for murine L929 fibroblasts and human MCF7 mammary carcinoma cells. Competitive binding experiments with '25l-labelled TNF-ax revealed that the loss of cytotoxicity is caused by a loss of target cell binding. In contrast to the reduced activity against L929 and MCF7 cells, neither binding to nor cytostatic activity against the human myeloid leukaemia cell lines HL60 and U937 are affected. This target cell type-dependent behaviour is probably due to the fact that L929 and MCF7 cells express different types of TNF receptor compared with myeloid leukaemia cells. While a mutation in position 127 decreases the overall activity of TNF-a, a deletion of four Nterminal amino acids does not reduce biological activity. In vivo the TNF mutants differed in their anti-tumour effects and lethal toxicity, but a segregation of anti-tumour activity and toxicity was not observed.
INTRODUCTION Tumour necrosis factor-alpha (TNF-a), a 17,000 MW protein mainly produced by activated monocytes/macrophages, was originally detected by its ability to cause haemorrhagic necrosis of certain mouse tumours.' TNF-a also exerts a direct cytotoxic or cytostatic effect on some tumour cell lines.2 As a pleiotropic molecule TNF-oc mediates inflammatory reactions as well as tissue remodelling processes. It activates macrophages, granulocytes, osteoclasts, fibroblasts and endothelial cells.3-6 TNF-a also inhibits the growth of normal and leukaemic haematopoietic progenitor cells.7 At high concentrations TNF-a provokes a lethal shock syndrome and chronic exposure to TNF-oc leads to cachexia.8'9 As has been shown in studies with cancer patients systemic administration of TNF-cx has only a marginal therapeutical effect.'0 For the clinical application of TNF-a it is necessary to reduce its toxic side-effects while maintaining or increasing antitumour activity. One way to modulate TNF-associated effects relies upon genetic modification of the TNF molecule."-'3 Experiments with TNF-deletion mutants have shown the critical role of the C-terminal region for the biological activity of TNF.'4 '5 Gase et al. have demonstrated that single amino acid changes in the C-terminal region also result in a reduction of tumour cytotoxicity.'5 We have now extended this investigation Correspondence: Dr R. Kircheis, Biotechnology Research Center, Teikyo University, 907 Nogawa, Miyamae-ku, Kawasaki 216, Japan.
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to additional TNF-dependent effects, including anti-tumour activity and lethal toxicity in vivo and have tested whether or not changes of biological activity are due to an altered efficiency of binding to the target cells.
MATERIALS AND METHODS TNF mutants The N- and C-terminal amino acid sequences of the TNF-a mutants, used as well as the sequence of the native human TNFa16 are shown in Fig. 1. TNF 331, which lacks two N-terminal amino acids, is identical with the native human TNF-a regarding the biological activity (tested for cytotoxicity against L929 fibroblasts).'7"8 TNF 311 has a deletion of four N-terminal amino acids. In TNF 332, a Leu is exchanged for the Ile in position 155. TNF 338 has an additional exchange of Val by Ile in position 150. In TNF 336, the Glu in position 127 is replaced by a Gln residue. The TNF preparations were prepared as described previously.'5 All TNF preparations had a purity of >95% as shown by SDS-PAGE and immunoblotting with polyclonal or monoclonal anti-hTNF-a antibodies. The endotoxin level was
Biological activity of mutants of human tumour necrosis factor-alpha R. KIRCHEIS, J. MILLECK, V. G. KOROBKO,* L. N. SHINGAROVA,* D. BEHNKEt & H. E. SCHMIDTJ Max Delbrick Center for Molecular Medicine, Berlin-Buch, Germany, *Institute for Bioorganic Chemistry, Moscow, Russia, tCentral Institute for Microbiology and Experimental Therapy, Jena, Germany and tBiochemical Technological Center,
Berlin-Buch, Germany Acceptedfor publication 8 March 1992
SUMMARY Point mutations in different regions of the tumour necrosis factor-alpha (TNF-a) molecule influence anti-tumour cytotoxic/cytostatic activities as well as haemorrhagic tumour necrosis, tumour regression and lethal toxicity in mice. Mutations in the C-terminal region in positions 150 and 155 markedly decrease cytotoxicity for murine L929 fibroblasts and human MCF7 mammary carcinoma cells. Competitive binding experiments with '25l-labelled TNF-ax revealed that the loss of cytotoxicity is caused by a loss of target cell binding. In contrast to the reduced activity against L929 and MCF7 cells, neither binding to nor cytostatic activity against the human myeloid leukaemia cell lines HL60 and U937 are affected. This target cell type-dependent behaviour is probably due to the fact that L929 and MCF7 cells express different types of TNF receptor compared with myeloid leukaemia cells. While a mutation in position 127 decreases the overall activity of TNF-a, a deletion of four Nterminal amino acids does not reduce biological activity. In vivo the TNF mutants differed in their anti-tumour effects and lethal toxicity, but a segregation of anti-tumour activity and toxicity was not observed.
INTRODUCTION Tumour necrosis factor-alpha (TNF-a), a 17,000 MW protein mainly produced by activated monocytes/macrophages, was originally detected by its ability to cause haemorrhagic necrosis of certain mouse tumours.' TNF-a also exerts a direct cytotoxic or cytostatic effect on some tumour cell lines.2 As a pleiotropic molecule TNF-oc mediates inflammatory reactions as well as tissue remodelling processes. It activates macrophages, granulocytes, osteoclasts, fibroblasts and endothelial cells.3-6 TNF-a also inhibits the growth of normal and leukaemic haematopoietic progenitor cells.7 At high concentrations TNF-a provokes a lethal shock syndrome and chronic exposure to TNF-oc leads to cachexia.8'9 As has been shown in studies with cancer patients systemic administration of TNF-cx has only a marginal therapeutical effect.'0 For the clinical application of TNF-a it is necessary to reduce its toxic side-effects while maintaining or increasing antitumour activity. One way to modulate TNF-associated effects relies upon genetic modification of the TNF molecule."-'3 Experiments with TNF-deletion mutants have shown the critical role of the C-terminal region for the biological activity of TNF.'4 '5 Gase et al. have demonstrated that single amino acid changes in the C-terminal region also result in a reduction of tumour cytotoxicity.'5 We have now extended this investigation Correspondence: Dr R. Kircheis, Biotechnology Research Center, Teikyo University, 907 Nogawa, Miyamae-ku, Kawasaki 216, Japan.
433
to additional TNF-dependent effects, including anti-tumour activity and lethal toxicity in vivo and have tested whether or not changes of biological activity are due to an altered efficiency of binding to the target cells.
MATERIALS AND METHODS TNF mutants The N- and C-terminal amino acid sequences of the TNF-a mutants, used as well as the sequence of the native human TNFa16 are shown in Fig. 1. TNF 331, which lacks two N-terminal amino acids, is identical with the native human TNF-a regarding the biological activity (tested for cytotoxicity against L929 fibroblasts).'7"8 TNF 311 has a deletion of four N-terminal amino acids. In TNF 332, a Leu is exchanged for the Ile in position 155. TNF 338 has an additional exchange of Val by Ile in position 150. In TNF 336, the Glu in position 127 is replaced by a Gln residue. The TNF preparations were prepared as described previously.'5 All TNF preparations had a purity of >95% as shown by SDS-PAGE and immunoblotting with polyclonal or monoclonal anti-hTNF-a antibodies. The endotoxin level was
