Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-12-13
2002-12-24
Gambel, Phillip (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S387300, C530S388100, C530S388200, C530S389100, C530S389200, C424S130100, C424S133100, C424S135100, C424S137100, C424S141100, C424S145100, C424S158100
Reexamination Certificate
active
06498237
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to ligands which bind to human tumour necrosis factor alpha (TNF) in a manner such that upon binding the biological activity of TNF is modified. The type of modification shown here is distinct from previous descriptions of antibodies which bind to TNF alpha and inhibit all TNF alpha activity. The new discovery shows how the different activities of TNF alpha can be selectively inhibited or enhanced. In addition, the present invention-relates to a composition comprising a molecule bound to TNF and to methods of therapy utilising TNF and molecules active against TNF.
BACKGROUND OF THE INVENTION
Tumor necrosis factor alpha (TNF) is a product of activated macrophages first observed in the serum of experimental animals presensitized with
Bacillus Calmette
-
Guerin
or
Corynebacterium parvum
and challenged with endotoxin (LPS). Following the systematic administration of TNF haemorrhagic necrosis was observed in some transplantable tumours of mice while in vitro TNF caused cytolytic or cytostatic effects on tumour cell lines.
In addition to its host-protective effect, TNF has been implicated as the causative agent of pathological changes in septicemia, cachexia and cerebral malaria. Passive immunization of mice with a polyclonal rabbit serum against TNF has been shown to protect mice against the lethal effects of LPS endotoxin, the initiating agent of toxic shock, when administered prior to infection.
The gene encoding TNF has been cloned allowing the usefulness of this monokine as a potential cancer therapy agent to be assessed. While TNF infusion into cancer patients in stage 1 clinical trials has resulted in tumour regression, side-effects such as thrombocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension have also been reported. These quite significant side-effects associated with the clinical use of TNF are predictable in view of the many known effects of TNF, some of which are listed in Table 1.
TABLE 1
BIOLOGICAL ACTIVITIES OF TNF
-ANTI-TUMOUR
-ANTI-VIRAL
-ANTI-PARASITE
FUNCTION
cytotoxic action on tumour cells
pyrogenic activity
angiogenic activity
inhibition of lipoprotein lipase
activation of neutrophils
osteoclast activation
induction of endothelial, monocyte and tumour cell
procoagulant activity
induction of surface antigens on endothelial cells
induction of IL-6
induction of c-myc and c-fos
induction of EGF receptor
induction of IL-1
induction of TNF synthesis
induction of GM-CSF synthesis
increased prostaglandin and collagenase synthesis
induction of acute phase protein C3
Of particular importance is the activation of coagulation which occurs as a consequence of TNF activation of endothelium and also peripheral blood monocytes. Disseminated intravascular coagulation is associated with toxic shock and many cancers including gastro-intestinal cancer, cancer of the pancreas, prostate, lung, breast and ovary, melanoma, acute leukaemia, myeloma, myeloproliferative syndrome and myeloblastic leukaemia. Clearly modifications of TNF activity such that tumour regression activity remains intact but other undesirable effects such as activation of coagulation are removed or masked would lead to a more advantageous cancer therapy, while complete abrogation of TNF activity is sought for successful treatment of toxic shock.
Segregation of hormonal activity through the use-of site-specific antibodies (both polyclonal and monoclonal) can result in enhanced hormonal activity (Aston et al, 1989, Mol. Immunol. 26, 435). To date few attempts have been made to assign antigenicity or function to particular regions of the TNF molecule for which the three-dimensional structure is now known. Assignment of function to such regions would permit the development of MAbs and other ligands of therapeutic use. Polyclonal antibodies to amino acids 1 to 15 have been reported to block Hela R19 cell receptor binding by TNF (Socher et al, 1987, PNAS 84, 8829) whilst monoclonal antibodies recognising undefined conformational epitopes on TNF have been shown to inhibit TNF cytotoxicity in vitro (Bringman and Aggarwal, 1987, Hybridoma 6, 489). However, the effects of these antibodies on other TNF activities is unknown.
SUMMARY OF THE PRESENT INVENTION
The present inventors have produced panels of monoclonal antibodies active against human TNF and have characterised them with respect to their effects on the anti-tumour effect of TNF (both in vitro and in viva), TNF receptor binding, activation of coagulation (both in vitro and in viva) and defined their topographic specificities. This approach has led the inventors to show that different topographic regions of TNF alpha are associated with different activities. Therefore the inventors enable the identification of antibodies or ligands which selectively enhance or inhibit TNF alpha activity, thereby providing for improved therapeutic agents and regimes including TNF alpha.
In a first aspect the present invention consists in a ligand capable of binding to human TNF, the ligand being characterised in that when it binds to TNF the following biological activities of the TNF are inhibited:-
1. Tumour regression;
2. Induction of endothelial procoagulant;
3. Induction of tumour fibrin deposition;
4. Cytotoxicity; and
5. Receptor binding.
In a preferred embodiment of all aspects the present invention the ligand is selected from the group consisting of antibodies, F(ab) fragments, restructured antibodies (CDR grafted humanised antibodies) single domain antibodies (dAbs), single chain antibodies, serum binding proteins, receptors and natural inhibitors. The ligand may also be a protein or peptide which has been synthesised and which is analogous to one of the foregoing fragments. However, it is presently preferred that the ligand is a monoclonal antibody or F(ab) fragment thereof.
In a second aspect the present invention consists in ligand capable of binding to human TNF, the ligand being characterized in that when it binds to TNF the induction of endothelial procoagulant, tumour regression, induction of tumour fibrin deposition, cytotoxicity and receptor binding activities of the TNF are inhibited, the ligand binding to the TNF such that the epitope of the TNF defined by the topographic regions of residues 1-18(Val
1
-Arg
2
-Ser
3
-Ser
4
-Ser
5
-Arg
6
- Thr
7
-Pro
8
-Ser
9
-Asp
10
-Lys
11
-Pro
12
-Val
13
-Ala
14
-His
15
-Val
16
-Val
17
-Ala
18
), 58-65 (Ile
58
-Tyr
59
-Ser
60
- Gln
61
-Val
62
-Leu
63
-Phe
64
-Lys
65
), 115-125 (Tyr
115
-Glu
116
-Pro
117
-Ile
118
-Tyr
119
-Leu
120
-Gly
121
-Gly
122
- Val
123
-Phe
124
-Gln
125
) and 138-149 (Arg
138
-Pro
139
-Asp
140
-Tyr
141
-Leu
142
-Asp
143
-Phe
144
-Ala
145
- Glu
146
-Ser
147
-Gly
148
-Gln
149
), or the topographic region of residues 1-18, 108-128, or the topographic region of residues 56-79 (Tyr
56
-Leu
57
-Ile
58
-Tyr
59
-Ser
60
-Gln
61
-Val
62
-Leu
63
-Phe
64
- Lys
65
-Gly
66
-Gln
67
-Gly
68
-Cys
69
-Pro
70
-Ser
71
-Thr
72
-His
73
-Val
74
-Leu
75
-Thr
77
-His
78
-Thr
79
), 110-127 (Glu
110
-Ala
111
-Lys
112
-Pro
113
-Trp
114
-Tyr
115
-Glu
116
-Pro
117
-Ile
118
-Tyr
119
-Leu
120
-Gly
121
- Gly
122
-Val
123
-Phe
124
-Gln
125
-Leu
126
-Glu
127
) and 136-155 (Ile
136
-Asn
137
-Arg
138
-Pro
139
-Asp
140
- Tyr
141
-Leu
142
-Asp
143
-Phe
144
-Ala
145
-Glu
146
-Ser
147
-Gly
148
-Gln
149
-Val
150
-Tyr
151
-Phe
152
-Gly
153
-Ile
154
- Ile
155
) is substantially prevented from binding to naturally occuring biologically active ligands.
In a third aspect the present invention consists in a ligand which binds to human TNF in at least two regions selected from the group consisting predominantly of the topographic region of residues 1-20, the topographic region of residues 56-77, the topographic region of residues 108-127 and the topographic region of residues 138-149.
In a preferred embodiment of the third aspect of the present invention the ligand binds to human TNF in the topographic regions of residues 1-18, 58-65, 115-125 and 138-149. Such sequence regions are topographically represented in FIG.
23
.
In a further preferred embodiment of the thir
Aston Roger
Rathjen Deborah Ann
Gambel Phillip
Peptech Limited
Roark Jessica H.
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