WO1993018148A2 - Tumor necrosis factor with modified channel activity - Google Patents

Tumor necrosis factor with modified channel activity Download PDF

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Publication number
WO1993018148A2
WO1993018148A2 PCT/US1993/002475 US9302475W WO9318148A2 WO 1993018148 A2 WO1993018148 A2 WO 1993018148A2 US 9302475 W US9302475 W US 9302475W WO 9318148 A2 WO9318148 A2 WO 9318148A2
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tnf
residue
group
amino acid
replacement amino
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PCT/US1993/002475
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WO1993018148A3 (en
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Bernadine J. Wisnieski
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Wisnieski Bernadine J
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to modified forms of tumor necrosis factors- ⁇ and - ⁇ (TNF- ⁇ and - ⁇ ) and related TNF- li e molecules. More particularly, the invention relates to modified forms of TNF having enhanced or diminished channel activity as compared to unmodified TNF.
  • Tumor necrosis factor- ⁇ and - ⁇ are polypeptide secretion products produced primarily by activated macrophages and lymphocytes. TNF plays a pivotal role in inflammatory responses, as well as in infectious and neoplastic disease states. TNF- ⁇ has been shown to be identical to cachectin. TNF-/3 is sometimes called lymphotoxin (LT) . Human TNF- ⁇ is expressed as a 233 residue prohormone and is secreted as a mature protein of 157 residues (17,356 kDa) after cleavage of a 76-amino acid long pro-peptide. The amino acid sequences of several species forms of mature TNF- ⁇ are known, viz.
  • TNF- ⁇ human, murine, rat, rabbit, feline, ovine, goat, bovine, and porcine.
  • the amino acid sequence of TNF- ⁇ for each of these species is provided in Appendix. A. A comparison of the various sequences of TNF- ⁇ from the above species indicates that the amino acid sequence of TNF- ⁇ is highly conserved, i.e., the sequences for TNF- ⁇ from the above non-human species are very similar to the sequence of human TNF- ⁇ .
  • human TNF- ⁇ is encoded as a 203 residue prohormone and is secreted after cleavage in two forms, i.e. a mature protein of 171 residues (24 kDa) and a mature protein of 148 residues (20 kDa) .
  • Mature human TNF- ⁇ is glycosylated, but glycosylation is not required for activity.
  • the amino acid sequences of several species forms of mature TNF-/3 are known, viz. human, bovine, murine, and rabbit. The amino acid sequence of the larger mature form of TNF- ⁇ for each of these species is provided in Appendix A.
  • TNF- ⁇ amino acid sequence of TNF- is highly conserved, i.e., the sequences for TNF-3 from the above non-human species are very similar to the sequence of human TNF- / 3. It has further been shown by x-ray crystallography that the secreted form of TNF- ⁇ consists of three monomers of mature TNF- ⁇ associated in the form of a 52,500 kDa trimer (Sprang, S. T. and Eck, M. J. , Current Research in Protein Chemistry, Chapter 35: "Subunit Interactions and Function of Tumor Necrosis Factor," pp. 383-394 (1990)) .
  • TNF- ⁇ is known to bind to at least two forms of TNF receptor and to insert into target membranes. Acidic pH is known to enhance TNF insertion into target membranes. TNF- ⁇ has been similarly shown to form trimers and to bind and insert into target membranes. The 3-D structure of the TNF- ⁇ trimer is also known (Eck et al. , The Journal of Biological Chemistry, 267, 2119-2122 (1992)) . Both forms of TNF have been shown to play pivotal roles in inflammatory responses, as well as in infectious and neoplastic disease states.
  • Trimers of unmodified TNF are shown to bind and insert into target membranes and to form ion channels therein (Kagan, B. L. , Baldwin, R. L. , Munoz, D. and isnieski, B. J. , Science vol xx, pp-pp (1992), "Formation of Ion- Permeable Channels by Tumor Necrosis Factor- ⁇ ,” incorporated herein by reference) .
  • the invention is a modified form of tumor necrosis factor (TNF) .
  • TNF tumor necrosis factor
  • the modified form of TNF may have amino acid substitutions, additions, and/or deletions, and/or chemically modified amino acids for enhancing, diminishing, and/or modulating the channel activity of such modified TNF within target membranes.
  • enhancement, diminution, and/or modulation of the channel activity occurs while such modified TNF retains a capacity to bind to one or more TNF receptors.
  • enhancement, diminution, and/or modulation of the channel activity occurs while such modified TNF retains a capacity to trimerize with itself, with other forms of modified TNF, and/or with corresponding forms of unmodified TNF.
  • the invention also includes methods for using such modified forms of TNF.
  • Preferred modes of achieving the enhancement, diminution, and/or modulation of channel activity include: the enlargement or reduction of the cross-sectional size of the channel for controlling or modulating the cross- sectional size of channel transportants; lining the channel with amino acid residues having a preponderance of positive or negative charges for enhancing or diminishing the channel permeability with respect to molecules having net positive or negative charge and for modulating the transport of neutral molecules as compared to charged molecules; controlling the process of channel formation, including channel dilation and closure.
  • a trimer is covalently held intact by one or more cysteine-cysteine linkages connecting the individual subunits within the trimer.
  • the modified form of TNF has a channel activity that is substantially enhanced, diminished, and/or modulated with respect to the channel activity of a corresponding form of unmodified TNF.
  • the channel activity of the modified TNF is measured within a black lipid membrane system, described infra.
  • X-ray crystallographic studies of trimerized TNF show that the TNF trimer exhibits an approximate three-fold axial symmetry. The symmetry axis of rotation of the trimer defines the approximate center of a channel region.
  • X-ray crystallography also identifies the amino acid residues that line the channel region.
  • amino acid residues that lie in or adjacent to the channel region are denominated as channel residues.
  • amino acid substitutions, additions, and/or deletions and the chemical modifications of amino acids of the modified TNF are directed to channel residues, i.e., amino acid residues that are shown by x-ray crystallography to lie in or adjacent to the symmetry axis of the trimer.
  • the modified form of TNF retains an ability to bind to one or more TNF receptors.
  • the binding assay may be performed in an in vitro assay as described infra.
  • the modified form of TNF undergoes a trimerization reaction similar to that of unmodified TNF.
  • the modified form of TNF may trimerize with itself, with other forms of modified TNF, and/or with unmodified forms of TNF.
  • Preferred means for the ascertainment of such trimerization of TNF include standard in vitro assays such as separation by high performance liquid chromatography (HPLC) on. gel exclusion (sizing) columns or by ascertaining the electrophoretic mobility of such TNF on a native gel or by. ascertaining its electrophoretic mobility on a denaturing gel after treatment with crossing linking agents.
  • Fig. 1 (a) is an exploded view in perspective of three monomers or subunits of unmodified TNF, illustrating two background subunits and one foreground subunit, the three TNF subunits being in a dissociated form.
  • the topology of the subunits is not intended to be precisely representative.
  • Fig. 1 (b) is a plan view of the dissociated subunits • of TNF shown in Fig. 1 (a) , viewed from above.
  • Fig. 2 (a) is a perspective of the two background TNF subunits illustrated in Fig. 1 (a) , showing the association of those two subunits of TNF as a dimer and the partial formation of a channel. The foreground subunit is omitted. The topology of this TNF dimer and of the partial formation of the channel is not intended to be precisely representative.
  • Fig. 2 (b) is a plan view of the background dimer of TNF illustrated in Fig. 2 (a) and of the dissociated foreground subunit illustrated in Fig. 1 (a) , viewed from above
  • Fig. 3 (a) is a perspective of the foreground subunit and of the two background subunits of TNF illustrated in Fig. 1 (a) , showing the association of all three subunits of TNF as a trimer. The view of the channel is blocked by the foreground subunit. The topology of this trimer is not intended to be precisely representative.
  • Fig. 3 (b) is a plan view of the trimer of TNF illustrated in Fig. 3 (a) , viewed from above, showing the formation of a channel.
  • Fig. 4 (a) illustrates a sectional view of the trimer of unmodified TNF illustrated in Fig. 3 (a) prior to its insertion into a target membrane. The cross-section passes through the channel of the trimer. The topology of this trimer is not intended to be precisely representative.
  • Fig. 4 (b) is a sectional view of the trimer illustrated in Fig. 4 (a) after such trimer has inserted into a target membrane. Note that the channel dilates after the insertion of the trimer into the target membrane.
  • Fig. 5 (a) is a sectional view of a trimer of modified TNF having a channel that is wider than the channel of the unmodified TNF illustrated in Fig. 4 (a) .
  • Fig. 5 (b) is a sectional view of the trimer of modified TNF illustrated in Fig. 5 (a) after such trimer has inserted into a target membrane. Note that the wide channel dilates even further after the trimer has inserted into the target membrane.
  • Fig. 6 (a) is a sectional view of a trimer of modified TNF having a channel that is narrower than the channel of the unmodified TNF illustrated in Fig. 4 (a) .
  • Fig. 6 (b) is a sectional view of the trimer of modified TNF illustrated in Fig. 6 (a) after such trimer has inserted into a target membrane. Note that the narrow channel of the trimer dilates when the trimer has inserted into the target membrane.
  • unmodified TNF monomers (1) form a compact trimer.
  • these trimers have an approximate three-fold axis of symmetry, i.e., the structures of the three TNF subunits (1) forming the trimer are not precisely identical to one another.
  • X-ray crystallographic studies also tell us that the trimers have a channel-like structure (2) extending approximately along the axis of symmetry.
  • the channel activity of a TNF trimer may be determined by several means.
  • the pore size and channel activity with respect to uncharged solutes may be determined by the use of multilamellar vesicles systems.
  • Channel activity of the TNF can be correlated with the passive transport of uncharged solutes through the TNF channel.
  • Evidence for the passive transport of solutes into a multilamellar vesicle can be derived from observations of the swelling of such multilamellar vesicles. Transport of solutes into a vesicle induces a change in the osmolarity of the fluid in the vesicle lumen.
  • Detection of swelling of the multilamellar vesicles may be obtained by means of light scattering measurements.
  • the rate of swelling will depend upon the activity of the TNF channel with respect to the size of the external solute. Solutes too large to penetrate the channel will not cause the vesicles to swell.
  • the channel activity of unmodified forms of TNF with respect to multilamellar vesicles may be compared with the corresponding channel activity of modified forms of TNF.
  • An observation of differential swelling of multilamellar vesicles with respect to modified and unmodified forms of TNF is a preferred example .of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
  • the channel activity with respect to ions or- other charged molecules may be ascertained by studying the conductivity of planar membranes (3) .
  • Solvent free membranes (3) may be prepared as described by M. Montal, Methods in Enzymology, 32 , 545 (1974) .
  • Squalene (Sigma) or squalane (Fluka) may be employed to coat a hole (100-200 ⁇ m diameter) in a Teflon partition.
  • Monolayers may be spread from mixtures of soybean phosphatidylethanolamin (40%) , soybean- phosphatidylcholine (40%) (Pelham, AL) , and bovine phosphatidylserine (20%) (Avanti) .
  • This lipid mixture can also be mixed 1:1 with asolectin (Y. Dagawa and E. Racker, Journal of Biological Chemistry, 246, 5477 (1971)) .
  • Membranes (3) should be stable within the voltage range of ⁇ 100 mV for at least 10 minutes before the addition of TNF.
  • the aqueous phase should include 100 mM NaCl, 2 mM di ethylglutaric acid-(pH 4.5) or 5 mM tris (pH 7.2) as buffer, 2 mM MgCl 2 , and 1 mM EDTA.
  • Voltage-clamp conditions should be employed.
  • a battery-driven stimulator may be employed to apply voltages and a eithley 427 current amplifier may be employed to measure current. Output of the current measurement may be displayed on an oscilloscope and recorded on a chart recorder.
  • the cis compartment, to which TNF should be added, is defined as ground.
  • Voltages refer to the trans compartment, opposite the TNF-containing side and analogous to the cytosol of a target cell.
  • Silver/silver chloride electrodes may be employed to connect the solutions to the electronics.
  • 3 M KC1/ agar salt bridges may be employed in connection with measurements involving salt gradients.
  • V ⁇ 40 mV V ⁇ 40 mV
  • V > 40 mV the current rises within seconds to a new, higher steady state, which is steeply dependent on the membrane voltage.
  • the conductance decreases rapidly to approximately zero.
  • the current remains approximately zero at most negative voltages, whereas current values increase sharply at positive voltages.
  • the conductance induced by TNF is due to the formation of ion-permeable channels. Observed single- channel conductances are heterogeneous, but may be grouped into two main classes, viz. one class centered at approximately 5-10 pS, and a second larger class ranging from approximately 100 - 1000 pS. The most frequently observed event is the 5 pS class.
  • channels can occasionally form at pH 7.2, channel formation is enhanced by lowering the pH of the aqueous phase containing TNF.
  • the TNF channels exhibit preferential permeability for cations over anions, but not absolutely so.
  • a TNF-treated membrane (3) showed a reversal potential of approximately 25-30 mV.
  • the channel activity of unmodified forms of TNF with respect to planar membranes (3) may be compared with the corresponding channel activity of modified forms of TNF.
  • An observation of differential conductance of planar membranes (3) with respect to modified and unmodified forms of TNF is a preferred example of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
  • the channel activity of TNF may be ascertained with respect to cancer cells.
  • Binding is allowed to proceed for 2 hours at 4°C. Then 10 ⁇ l of 20 M ouabain in water or 10 ⁇ l of water alone may be added, and the samples incubated for 13 minutes at 37°C. Then, 10 ⁇ l of 22 NaCl [10 ⁇ M stock, 200 ⁇ Ci/ l (Amersham) ] may be added to each sample, and incubation at 37°C may be continued for 10 minutes. Ice- cold PBS (0.8 ml; 10 mM sodium phosphate and 150 mM NaCl) may be added to stop the flux of 22 Na + . The cells may then be pelleted in a microcentrifuge (Bechman) and washed twice with 1 ml of PBS.
  • Bechman microcentrifuge
  • the channel activity of unmodified forms of TNF with respect to cancer cells may be compared with the corresponding channel activity of modified forms of TNF.
  • An observation of differential sodium ion permeability of human U937 histiocytic lymphoma cells with respect to modified and unmodified forms of TNF is a preferred example of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
  • Binding Assay with respect to TNF Target A binding assay may be performed as described by
  • the binding assay may be performed with any of a large number of naturally occurring human cell types which express one or more of the TNF receptors.
  • the binding assay may be performed with model cells such as human U937 histiocytic lymphoma cells -or with COS-1 cells that have been transfected with the 1.3 kb gene for the 55 kDa TNF receptor. After 2-3 days in culture, the transfected COS-1 cells may be detached with EDTA and • tested for binding by 125 I-TNF- ⁇ or 125 I-TNF-3.
  • the cells are ' washed, resuspended at 2.8 x 10 6 cells/milliliter , and incubated with various concentrations of 125 I-TNF- ⁇ or 125 I- TNF-3 in the absence and presence of a 500-fold excess of cold TNF- ⁇ or TNF- ⁇ , respectively, for 2 hours at 4°C.
  • the bound radioactivity is then counted in a gamma counter. Nonspecific binding is subtracted to obtain the net specific binding of the 125 I-TNF- ⁇ or 125 I-TNF-3 to the transfected COS-1 cells.
  • An alternative method for performing the binding assay is described by Coffman et al. (Lymphokine Research, 1_, 371-383 (1988) .
  • modified and unmodified forms of TNF may undergo a trimerization reaction.
  • the modified form of TNF may trimerize with itself, with other forms of modified TNF, and/or with unmodified forms of TNF.
  • Preferred means for the ascertainment of such trimerization of TNF include standard in vitro assays such as separation of the monomer and trimer forms by high performance liquid chromatography (HPLC) on gel exclusion (sizing) columns.
  • HPLC high performance liquid chromatography
  • the sizes of modified and unmodified forms of TNF may be ascertained from their electrophoretic mobilities on a native gel or they may be ascertained by contrasting their electrophoretic mobilities on a denaturing gel after treatment with cross-linking agents.
  • TNF- ⁇ from 8 other species, including porcine, bovine, goat, ovine, feline, rabbit, rat and murine.
  • Some forms of non-human TNF- ⁇ are known to be glycosylated, but glycosylation is not required for activity. It can be anticipated that TNF- ⁇ will be found in further species as well and will be similarly sequenced.
  • TNF- ⁇ can be shown to have channel activity when it is inserted into a planar lipid membrane (3) .
  • the amino acid residues that line or face this channel i.e. the primary "channel residues” include the following, viz.: Lys 11 , Leu 57 , Tyr 59 , Lys 9 *, Lys 112 , Glu 116 , Tyr 119 , Gly 121 , lie 155 , and Leu 157 .
  • these primary "channel residues" are encompassed within a larger group of amino acids designated as channel liners, some of which merely contact the channel via the carbonyl oxygens and amide NH groups of the peptide chain.
  • these channel liners include residues number 11, 55-59, 98-125, and 151-157. Because of the close sequence homology between human TNF- ⁇ and non- human forms of TNF- ⁇ , the important channel residues with respect to non-human forms of TNF- ⁇ have the same sequence numbers as given above for human TNF- ⁇ . It can be anticipated that, of those mutein forms of human and non- human TNF- ⁇ that exhibit modified channel activity, the important channel residues for such mutein forms of TNF- ⁇ will also have the same sequence numbers as given above for human TNF- ⁇ . However, the particular amino acids that occupy such sequence sites may vary from one non-human species to the next or from one mutein form to another.
  • the indicated parent amino acid is the amino acid found in human TNF- ⁇ with respect to that particular sequence number.
  • the parent amino acid with respect to non-human forms of TNF- ⁇ and with respect to various mutein forms of TNF- ⁇ may differ from the indicated patent amino acid. In such instances, the parent amino acid may be determined by referring to the sequence number for the particular channel residue being substituted.
  • Preferred amino acid substitutions of channel residues with respect to TNF- ⁇ include the following:
  • Trp Trp, Phe, Ser, Thr, Ala, Met
  • Trp Trp, Ser, Thr, Ala, Met, Cys, Phe, & Tyr
  • TNF-/3 from 3 other species, including bovine, rabbit, and murine. It can be anticipated that TNF-9 will be found in further species as well and will be similarly sequenced.
  • TNF- ⁇ can be shown to have channel activity when it is inserted into a planar lipid membrane (3) .
  • the amino acid residues that line or face this channel include the following, viz.: Lys 28 , Phe 74 , Tyr 76 , Lys 119 , Glu 127 , His 131 , T y r i34 ⁇ Gly 136 , Phe 169 , and Leu 171 .
  • these primary "channel residues" are encompassed within a larger group of amino acids designated as channel liners, some of which merely contact the channel via the carbonyl oxygens and amide NH groups of the peptide chain.
  • these channel liners include residues number 28, 72-76, 119-140, and 165-171. Because of the close sequence homology between human TNF- ⁇ and non-human forms of TNF-/3, the important channel residues with respect to non-human forms of TNF-3 have the same sequence numbers as given above for human TNF- ⁇ . It can be anticipated that, of those mutein forms of human and non-human TNF- / 3 that exhibit altered channel activity, the important channel residues for such mutein forms of TNF-/3 will also have the same sequence numbers as given above for human TNF-/3. However, the particular amino acids that occupy such sequence sites may vary from one non-human species to the next or from one mutein form to another.
  • the indicated parent amino acid is the amino acid found in human TNF- ⁇ with respect to that particular sequence number.
  • the parent amino acid with respect to non-human forms of TNF- / 3 and with respect to various mutein forms of TNF- / 3 may differ from the indicated patent amino acid. In such instances, the parent amino acid may be determined by referring to the sequence number for the particular channel residue being substituted.
  • Preferred amino acid substitutions of channel residues with respect to TNF- / 3 include the following:
  • Trp Trp, Gly, Pro, Tyr, Phe, & Met
  • Trp Trp, Ser, Thr, Ala, Met, Cys,
  • Trp Trp, Phe, Ser, Thr, Ala, Met
  • Trp Trp, Ser, Thr, Ala, Met, Cys,
  • Trp Trp, Ser, Thr, Ala, Cys, Phe,
  • TNF- ⁇ TNF- Lys 11 corresponds to Lys 28
  • Leu 57 corresponds to Phe 74
  • Tyr 59 corresponds to Tyr 76
  • Lys 98 corresponds to Lys 119
  • Lys 112 corresponds to Glu 127
  • Glu 116 corresponds to His 131
  • Tyr 119 corresponds to Tyr 134
  • Gly 121 corresponds to Gly 136 lie 155 corresponds to Phe 169
  • Leu 157 corresponds to Leu 171
  • the above list of channel residues for TNF- ⁇ and - ⁇ are correlated because these channel residues are similarly positioned within their respective channels and because an amino acid substitution of correlated channel residues tends to cause correlated changes of channel activity with respect to both TNF- ⁇ and - ⁇ .
  • a substitution of a long chain aliphatic for Gly 121 (TNF- ⁇ ) and Gly 136 (TNF- ⁇ ) will tend to occlude the channel and will tend to diminish the channel activity of- both TNF- ⁇ and - ⁇ .
  • the removal of bulky amino acids and the substitution of short chain amino acids tends to broaden the cross-sectional diameter of the channel and frequently, causes an increase in channel activity.
  • Cross-link Subunits within a TNF Trimer X-ray crystallographic studies on the trimers of both TNF- ⁇ and - ⁇ indicate the usage of several salt bridges to bond together the respective TNF subunits.
  • the amino acid residues for these salt linkages can be substituted with cysteines in order to form covalent cysteine-cysteine linkages between respective TNF subunits.
  • TNF- ⁇ examples include the following:
  • the interchain salt linkage between Lys 98 of one subunit and Glu 116 of an adjacent subunit can be modified by substituting Cys for both residues #98 and #116.
  • Cys 98 of each subunit will form a covalent disulfide bond with the Cys 116 on the adjacent subunit.
  • the resultant disulfide bond can be disrupted under reducing conditions.
  • TNF subunit may be modified by Cys substitutions to form a modified form of TNF having Cys 103 and Cys 104 to form three interchain disulfide bonds for stabilizing the TNF trimer.
  • the interchain salt linkage between Lys 11 of one TNF subunit and the carboxy terminal Leu 157 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified form of TNF having Cys" and Cys 157 to form three interchain disulfide bonds for stabilizing the TNF trimer.
  • TNF1-SS-TNF2 to TNF2-SS-TNF3 to TNF3-SS-TNF1.
  • TNF- ⁇ include the following: A. A pH dependent interchain salt linkage between Lys 119 of one subunit and His 131 of an adjacent subunit can be modified by substituting Cys for both residues #119 and #131. When the resulting modified form of TNF trimerizes under non- reducing conditions, three interchain covalent linkages will form, viz. the Cys 119 of each subunit will form a covalent disulfide bond with the Cys 131 on the adjacent subunit. The resultant disulfide bond can be disrupted under reducing conditions, but, unlike the salt bridge, will be relatively independent of pH.
  • interchain linkage between Ser 117 of one TNF subunit and His 135 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified TNF having Cys 117 and Cys 135 to form three interchain disulfide bonds for stabilizing the TNF trimer.
  • interchain salt linkage between Lys 28 of one TNF subunit and the carboxy terminal Leu 171 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified form of
  • TNF having Cys 28 and Cys 171 to form three interchain disulfide bonds for stabilizing the TNF trimer.
  • Tyr 134 residues can be substituted with Cys 134 .
  • no salt bridge is replaced and only one interchain disulfide bond results under non-reducing conditions.
  • this single disulfide bond may switch from one subunit pair to another, e.g., from TNF1-SS-TNF2 to TNF2-SS-TNF3 to TNF3-SS- TNF1.
  • TNF Channel for Diminishing or Modulating Channel Activity A number of reagents may be employed to interact selectively with channel residues of modified and unmodi- fied forms of TNF so as to form an occlusion within the channel for diminishing its activity.
  • one or more candidate forms of modified TNF- ⁇ and/or - ⁇ are formed by substituting one or more channel residues with replacement amino acids.
  • the preferred channel residues and the respective preferred amino acid substitutions for such channel residues are indicated above.
  • a preferred mode for effecting such amino acid substi ⁇ tutions by means of site-specific mutagenesis is described by Kunkel, T. A.- (Proceedings of the National Academy of Sciences, U.S.A., J32 . , 488-492).
  • Kunkel teaches that plas- mid pHTP320 may be digested with Spel and Hindlll to iso- late the DNA fragment containing the TNF gene, and may be subcloned into the Hindlll and Xbal sites of phage M13mpl9. From this recombinant phage, single-stranded DNA may be prepared as a template containing uracils for mutagenesis by using E. coli CJ236. Appropriate mutagenic oligonucleotides (approximately 20-mers) corresponding to the desired amino acid substitution may be chemically synthesized with a DNA synthesizer. For each mutagenesis, .
  • approximately 200 ng (0.1 pmol) of a template containing uracil may be mixed with 4 pmol of 5'-phosphorylated mutagenic oligonucleotide in 10 ⁇ l of an annealing buffer (20 M Tris-HCl, pH 7.4; 2 mM MgCl 2 ; 50 mM NaCl) .
  • the reaction mixture may be heated at 70°C for 10 minutes, and then cooled at a rate of approximately l°C/min. until 30°C.
  • reaction mixture After addition of DNA polymerase, Klenow fragment (2.5 units) , T4 DNA ligase (5 units) and 1 ⁇ l of lOx synthesis buffer (5mM each dNTP; lOmM ATP; 100 mM Tris-HCL, pH 7.4; 50 mM MgCl 2 ; 20 mM dithiothreitol) , the reaction mixture may ⁇ be sequentially incubated at 0°C for 5 minutes, 25°C for 5 minutes and 37°C for 90 minutes. A sample of the ligation • reaction may be employed to transform competent E. coli JM105 cells.
  • single-stranded DNA may be extracted from the plaques and identified by nucleotide sequencing.
  • the replicative form of the mutant may be digested with restriction endonucleases Clal and Hindlll.
  • the fragment containing the mutagenized TNF coding sequence may be subcloned into an expression plasmid pHTP320 in place of the TNF gene.
  • the transformants having an expression plasmid for the modified form of TNF may be incubated in LB medium supplemented with ampicillin (50 ⁇ g/ l) at 37°C overnight and then the cultures inoculated in M9 medium supplemented with 0.5% casamino acids and ampicillin (50 ⁇ g/ml) . After 3 hours, 3-indoleacrylic acid (20 ⁇ g/ml) may be added to induce the E. coli trp promoter and cultivation may be further continued at 37°C for 20 hours. Cells may then be disrupted by lysozyme-digestion and freezing-thawing as described by Nagata et al. Nature, 284, 316-320 (1980) .
  • the disrupted cells may then be centrifuged to obtain a clarified supernatant as E. coli extracts.
  • the candidate TNF may then be purified from the E. coli extract according to the method of Yamada et al., Journal of Biotechnology, 2, 141-153 (1985) .
  • the purified form of candidate TNF may then tested to determine if it retains an ability to form TNF trimers.
  • the protocol for testing trimerization is given above.
  • the purified form of candidate TNF may also be tested to determine if the TNF has an ability to achieve intimate contact with a target that includes both a membrane (3) and one or more TNF receptors.
  • the protocols for determining TNF receptor binding and channel formation are given above.
  • the purified form of candidate TNF may also be tested to determine if the TNF, when in intimate contact with the target, achieves a modified effect. Examples of such modified effects are given above, however, they must be of • a type caused by formation of a modified channel activity.
  • the modified channel activity must materially differ from corresponding unmodified channel activities caused by the corresponding unmodified TNF.
  • a modified form of TNF may be selected from one or more of the candidate forms of TNF.
  • the modified form of TNF must have been determined to be able to form TNF trimers; it must be able to achieve intimate contact with the target; and it must be able to achieve a modified effect upon a target by virtue of a modified channel activity.
  • TNF modified form of TNF
  • it may be made in purified form and in commercial quantities.
  • the permeability exhibited by a TNF target membrane (3) may be regulated by contacting the target membrane with a modified form of TNF.
  • the modified form of TNF should have a reconstructed channel (5 or 7) , as compared to unmodified TNF, as described above, for regulating channel activity.
  • modified form of TNF (6) is selected that forms a reconstructed channel (7) so as to result in a reduction of the channel activity as compared to the unmodified form of TNF. Accordingly, the insertion of such modified form of TNF in the target membrane (3) serves to reduce the channel activity.
  • a modified form of TNF (4) is selected that forms a reconstructed channel (5) so as to result in an enhancement of the channel activity as compared to the unmodified form of TNF, as indicated above. Accordingly, the insertion of this form of modified TNF in the target membane (3) serves to enhance the channel activity.
  • a modified form of TNF having a reconstructed channel (7) for reducing channel activity within a target membrane (3) may be employed for inhibiting the binding of unmodified TNF to one or more TNF receptors attached to a target membrane (3) .
  • a modified form of TNF having a channel reconstructed for reducing channel activity, as compared to unmodified TNF is contacted with one or more of TNF receptors under conditions for permitting binding between the modified form of TNF and the TNF receptor.
  • the modified form of TNF acts as an antagonist or competitive inhibitor of the unmodified TNF with respect to binding to TNF receptor.
  • Channel Blockers and Activators Molecules may be designed to interact specifically with the channel region of modified and unmodified forms of TNF. Some interactions will occlude the channel and block its channel activity. Some interactions will dilate the channel and enhance its channel activity.
  • NAME/KEY Tumor Necrosis Factor (porcine)
  • Xaa is inserted after residue No. 70 of bovine TNF and the sequence numbering is augmented by 1 starting with residue No. 71 in order to maximize the sequence homology with human TNF.
  • NAME/KEY Tumor Necrosis Factor (goat)
  • NAME/KEY Tumor Necrosis Factor (ovine)
  • X PUBLICATION INFORMATION
  • NAME/KEY Tumor Necrosis Factor (rabbit)
  • NAME/KEY Tumor Necrosis Factor (murine)
  • Xaa are inserted after residue No. 34 and No. 61 of murine lymphotoxin and the sequence numbering is augmented by 1 starting with residue No. 35 and again augmented by 1 starting with residue No. 62 in order to maximize the sequence homology with human lymphotoxin.
  • Xaa are inserted after residue No. 4 of murine lymphotoxin and the sequence numbering is augmented by 2 starting with residue No. 5 in order to maximize the sequence homology with human lymphotoxin.

Abstract

An improved form of tumor necrosis factor (TNF) can be employed to regulate TNF channel activity. The identity of the amino acids which line the channel of the TNF molecule and which exert significant control over the activity of the channel are disclosed. The improved form of TNF includes amino acid substitutions, additions, and/or deletions, and/or chemically modified amino acid residues within the channel region for enhancing, diminishing, and/or modulating its channel activity within target membranes. The modified form of TNF is capable of trimerization and of achieving intimate contact with a target membrane containing one or more types of TNF receptor. Contacting target membranes with forms of TNF having modified channel activities can be employed to regulate the permeability and/or response of the target membrane. Greater control over the regulation of the permeability and/or response of target membranes can be achieved with modified forms of TNF as compared to unmodified TNF.

Description

Description
TUMOR NECROSIS FACTOR WITH MODIFIED CHANNEL ACTIVITY
Technical Field
The invention relates to modified forms of tumor necrosis factors-α and -β (TNF-α and -β) and related TNF- li e molecules. More particularly, the invention relates to modified forms of TNF having enhanced or diminished channel activity as compared to unmodified TNF.
Background Art
Tumor necrosis factor-α and -β (TNF) are polypeptide secretion products produced primarily by activated macrophages and lymphocytes. TNF plays a pivotal role in inflammatory responses, as well as in infectious and neoplastic disease states. TNF-α has been shown to be identical to cachectin. TNF-/3 is sometimes called lymphotoxin (LT) . Human TNF-α is expressed as a 233 residue prohormone and is secreted as a mature protein of 157 residues (17,356 kDa) after cleavage of a 76-amino acid long pro-peptide. The amino acid sequences of several species forms of mature TNF-α are known, viz. human, murine, rat, rabbit, feline, ovine, goat, bovine, and porcine. The amino acid sequence of TNF-α for each of these species is provided in Appendix. A. A comparison of the various sequences of TNF-α from the above species indicates that the amino acid sequence of TNF-α is highly conserved, i.e., the sequences for TNF-α from the above non-human species are very similar to the sequence of human TNF-α.
Similarly, human TNF-β is encoded as a 203 residue prohormone and is secreted after cleavage in two forms, i.e. a mature protein of 171 residues (24 kDa) and a mature protein of 148 residues (20 kDa) . Mature human TNF-β is glycosylated, but glycosylation is not required for activity. The amino acid sequences of several species forms of mature TNF-/3 are known, viz. human, bovine, murine, and rabbit. The amino acid sequence of the larger mature form of TNF-β for each of these species is provided in Appendix A. A comparison of the various sequences of TNF-^ from the above species indicates that the amino acid sequence of TNF- is highly conserved, i.e., the sequences for TNF-3 from the above non-human species are very similar to the sequence of human TNF-/3. It has further been shown by x-ray crystallography that the secreted form of TNF-α consists of three monomers of mature TNF-α associated in the form of a 52,500 kDa trimer (Sprang, S. T. and Eck, M. J. , Current Research in Protein Chemistry, Chapter 35: "Subunit Interactions and Function of Tumor Necrosis Factor," pp. 383-394 (1990)) . TNF-α is known to bind to at least two forms of TNF receptor and to insert into target membranes. Acidic pH is known to enhance TNF insertion into target membranes. TNF- β has been similarly shown to form trimers and to bind and insert into target membranes. The 3-D structure of the TNF-β trimer is also known (Eck et al. , The Journal of Biological Chemistry, 267, 2119-2122 (1992)) . Both forms of TNF have been shown to play pivotal roles in inflammatory responses, as well as in infectious and neoplastic disease states.
Disclosure of Invention
Trimers of unmodified TNF are shown to bind and insert into target membranes and to form ion channels therein (Kagan, B. L. , Baldwin, R. L. , Munoz, D. and isnieski, B. J. , Science vol xx, pp-pp (1992), "Formation of Ion- Permeable Channels by Tumor Necrosis Factor-α," incorporated herein by reference) .
The invention is a modified form of tumor necrosis factor (TNF) . The modified form of TNF may have amino acid substitutions, additions, and/or deletions, and/or chemically modified amino acids for enhancing, diminishing, and/or modulating the channel activity of such modified TNF within target membranes. However such enhancement, diminution, and/or modulation of the channel activity occurs while such modified TNF retains a capacity to bind to one or more TNF receptors. Furthermore, such enhancement, diminution, and/or modulation of the channel activity occurs while such modified TNF retains a capacity to trimerize with itself, with other forms of modified TNF, and/or with corresponding forms of unmodified TNF. The invention also includes methods for using such modified forms of TNF.
Preferred modes of achieving the enhancement, diminution, and/or modulation of channel activity include: the enlargement or reduction of the cross-sectional size of the channel for controlling or modulating the cross- sectional size of channel transportants; lining the channel with amino acid residues having a preponderance of positive or negative charges for enhancing or diminishing the channel permeability with respect to molecules having net positive or negative charge and for modulating the transport of neutral molecules as compared to charged molecules; controlling the process of channel formation, including channel dilation and closure. In a preferred embodiment, a trimer is covalently held intact by one or more cysteine-cysteine linkages connecting the individual subunits within the trimer.
The modified form of TNF has a channel activity that is substantially enhanced, diminished, and/or modulated with respect to the channel activity of a corresponding form of unmodified TNF. In a preferred mode for determining the enhancement, diminution, and/or modulation of channel activity of a modified form of TNF, the channel activity of the modified TNF is measured within a black lipid membrane system, described infra. X-ray crystallographic studies of trimerized TNF show that the TNF trimer exhibits an approximate three-fold axial symmetry. The symmetry axis of rotation of the trimer defines the approximate center of a channel region. X-ray crystallography also identifies the amino acid residues that line the channel region. Amino acid residues that lie in or adjacent to the channel region are denominated as channel residues. In the preferred mode, the amino acid substitutions, additions, and/or deletions and the chemical modifications of amino acids of the modified TNF are directed to channel residues, i.e., amino acid residues that are shown by x-ray crystallography to lie in or adjacent to the symmetry axis of the trimer.
The modified form of TNF retains an ability to bind to one or more TNF receptors. In a preferred mode for determining the binding activity of the modified TNF with respect to one or more TNF receptors, the binding assay may be performed in an in vitro assay as described infra.
The modified form of TNF undergoes a trimerization reaction similar to that of unmodified TNF. The modified form of TNF may trimerize with itself, with other forms of modified TNF, and/or with unmodified forms of TNF. Preferred means for the ascertainment of such trimerization of TNF include standard in vitro assays such as separation by high performance liquid chromatography (HPLC) on. gel exclusion (sizing) columns or by ascertaining the electrophoretic mobility of such TNF on a native gel or by. ascertaining its electrophoretic mobility on a denaturing gel after treatment with crossing linking agents.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 (a) is an exploded view in perspective of three monomers or subunits of unmodified TNF, illustrating two background subunits and one foreground subunit, the three TNF subunits being in a dissociated form. The topology of the subunits is not intended to be precisely representative.
Fig. 1 (b) is a plan view of the dissociated subunits • of TNF shown in Fig. 1 (a) , viewed from above. Fig. 2 (a) is a perspective of the two background TNF subunits illustrated in Fig. 1 (a) , showing the association of those two subunits of TNF as a dimer and the partial formation of a channel. The foreground subunit is omitted. The topology of this TNF dimer and of the partial formation of the channel is not intended to be precisely representative.
Fig. 2 (b) is a plan view of the background dimer of TNF illustrated in Fig. 2 (a) and of the dissociated foreground subunit illustrated in Fig. 1 (a) , viewed from above
Fig. 3 (a) is a perspective of the foreground subunit and of the two background subunits of TNF illustrated in Fig. 1 (a) , showing the association of all three subunits of TNF as a trimer. The view of the channel is blocked by the foreground subunit. The topology of this trimer is not intended to be precisely representative.
Fig. 3 (b) is a plan view of the trimer of TNF illustrated in Fig. 3 (a) , viewed from above, showing the formation of a channel. Fig. 4 (a) illustrates a sectional view of the trimer of unmodified TNF illustrated in Fig. 3 (a) prior to its insertion into a target membrane. The cross-section passes through the channel of the trimer. The topology of this trimer is not intended to be precisely representative. Fig. 4 (b) is a sectional view of the trimer illustrated in Fig. 4 (a) after such trimer has inserted into a target membrane. Note that the channel dilates after the insertion of the trimer into the target membrane. Fig. 5 (a) is a sectional view of a trimer of modified TNF having a channel that is wider than the channel of the unmodified TNF illustrated in Fig. 4 (a) .
Fig. 5 (b) is a sectional view of the trimer of modified TNF illustrated in Fig. 5 (a) after such trimer has inserted into a target membrane. Note that the wide channel dilates even further after the trimer has inserted into the target membrane.
Fig. 6 (a) is a sectional view of a trimer of modified TNF having a channel that is narrower than the channel of the unmodified TNF illustrated in Fig. 4 (a) .
Fig. 6 (b) is a sectional view of the trimer of modified TNF illustrated in Fig. 6 (a) after such trimer has inserted into a target membrane. Note that the narrow channel of the trimer dilates when the trimer has inserted into the target membrane.
DETAILED DESCRIPTION
As illustrated in Fig.'s 3 (a) & (b) and Fig.'s 4 (a) & (b) , unmodified TNF monomers (1) form a compact trimer. According to x-ray crystallographic studies, these trimers have an approximate three-fold axis of symmetry, i.e., the structures of the three TNF subunits (1) forming the trimer are not precisely identical to one another. X-ray crystallographic studies also tell us that the trimers have a channel-like structure (2) extending approximately along the axis of symmetry.
Determination of TNF Channel Activity with respect to Neutral Solutes
The channel activity of a TNF trimer may be determined by several means. In a preferred method, the pore size and channel activity with respect to uncharged solutes may be determined by the use of multilamellar vesicles systems. Channel activity of the TNF can be correlated with the passive transport of uncharged solutes through the TNF channel. Evidence for the passive transport of solutes into a multilamellar vesicle can be derived from observations of the swelling of such multilamellar vesicles. Transport of solutes into a vesicle induces a change in the osmolarity of the fluid in the vesicle lumen. Detection of swelling of the multilamellar vesicles may be obtained by means of light scattering measurements. The rate of swelling will depend upon the activity of the TNF channel with respect to the size of the external solute. Solutes too large to penetrate the channel will not cause the vesicles to swell. The channel activity of unmodified forms of TNF with respect to multilamellar vesicles may be compared with the corresponding channel activity of modified forms of TNF. An observation of differential swelling of multilamellar vesicles with respect to modified and unmodified forms of TNF is a preferred example .of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
Determination of TNF Channel Activity with respect to Ions In an alternative preferred method, the channel activity with respect to ions or- other charged molecules, may be ascertained by studying the conductivity of planar membranes (3) . Solvent free membranes (3) may be prepared as described by M. Montal, Methods in Enzymology, 32 , 545 (1974) . Squalene (Sigma) or squalane (Fluka) may be employed to coat a hole (100-200 μm diameter) in a Teflon partition. Monolayers may be spread from mixtures of soybean phosphatidylethanolamin (40%) , soybean- phosphatidylcholine (40%) (Pelham, AL) , and bovine phosphatidylserine (20%) (Avanti) . This lipid mixture can also be mixed 1:1 with asolectin (Y. Dagawa and E. Racker, Journal of Biological Chemistry, 246, 5477 (1971)) . Capacitance measurements may be employed to monitor bilayer formation from the apposition of the two monolayers. After membrane formation, the conductance (g=I/V) of the unmodified membrane should be ohmic and should lie within the approximate range of 5 to 10 pS. Membranes (3) should be stable within the voltage range of ±100 mV for at least 10 minutes before the addition of TNF. The aqueous phase should include 100 mM NaCl, 2 mM di ethylglutaric acid-(pH 4.5) or 5 mM tris (pH 7.2) as buffer, 2 mM MgCl2, and 1 mM EDTA. Voltage-clamp conditions should be employed. A battery-driven stimulator may be employed to apply voltages and a eithley 427 current amplifier may be employed to measure current. Output of the current measurement may be displayed on an oscilloscope and recorded on a chart recorder. The cis compartment, to which TNF should be added, is defined as ground. Voltages refer to the trans compartment, opposite the TNF-containing side and analogous to the cytosol of a target cell. In ion selectivity measurements, salt gradients may be imposed across the membrane and the zero-current reversal potential E, where I = g(V-E) , may be measured. Silver/silver chloride electrodes may be employed to connect the solutions to the electronics. 3 M KC1/ agar salt bridges may be employed in connection with measurements involving salt gradients.
Upon addition of unmodified TNF to a final concentration of 100 ng/ l, the membrane current I (and therefore conductance, g=I/V) remains nearly zero at small voltages (absolute values of
V < 40 mV) . At larger positive voltages (V > 40 mV) , the current rises within seconds to a new, higher steady state, which is steeply dependent on the membrane voltage. When the polarity of the voltage is reversed, the conductance decreases rapidly to approximately zero. The current remains approximately zero at most negative voltages, whereas current values increase sharply at positive voltages. The conductance induced by TNF is due to the formation of ion-permeable channels. Observed single- channel conductances are heterogeneous, but may be grouped into two main classes, viz. one class centered at approximately 5-10 pS, and a second larger class ranging from approximately 100 - 1000 pS. The most frequently observed event is the 5 pS class. Although channels can occasionally form at pH 7.2, channel formation is enhanced by lowering the pH of the aqueous phase containing TNF. The TNF channels exhibit preferential permeability for cations over anions, but not absolutely so. In a tenfold concentration gradient of NaCl, a TNF-treated membrane (3) showed a reversal potential of approximately 25-30 mV. The channel activity of unmodified forms of TNF with respect to planar membranes (3) may be compared with the corresponding channel activity of modified forms of TNF. An observation of differential conductance of planar membranes (3) with respect to modified and unmodified forms of TNF is a preferred example of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
Determination of TNF Channel Activity with Cells In an alternative preferred mode, the channel activity of TNF may be ascertained with respect to cancer cells. The addition of unmodified recombinant human TNF to human U937 histiocytic lymphoma cells increases 22Na+ uptake by
100 to 300%, in the presence or absence of ouabain. Human U937 cells (American Type Culture Collection, ATCC) are washed four times in buffer A (100 mM choline chloride, 25 mM MgCl2, 5 mM KC1, and 20 mM Hepes, with the addition of 7.17 mM NaOH to adjust the pH to 7.2) Each sample, containing 2xl06 cells, may be resuspended in 200 μl of buffer A after the last wash and equilibrated at 4°C for 15 minutes before the addition of 1 μg TNF (2 μl of a 0.5 mg/ml stock in 10 mM sodium phosphate and 0.2 M NaCl; pH 7) or 2 μl of buffer alone. Binding is allowed to proceed for 2 hours at 4°C. Then 10 μl of 20 M ouabain in water or 10 μl of water alone may be added, and the samples incubated for 13 minutes at 37°C. Then, 10 μl of 22NaCl [10 μM stock, 200 μCi/ l (Amersham) ] may be added to each sample, and incubation at 37°C may be continued for 10 minutes. Ice- cold PBS (0.8 ml; 10 mM sodium phosphate and 150 mM NaCl) may be added to stop the flux of 22Na+. The cells may then be pelleted in a microcentrifuge (Bechman) and washed twice with 1 ml of PBS. Aliquots (10 μl) of the first and last supernatants are then removed for counting. Pelleted cells may be solubilized by incubation for 15 minutes with 100 μl of 0.5% Triton X-100 in buffer A at 37°C. Solubilized cells and supernatant aliquots may then be mixed with 10 ml of liquid scintillant and counted at the 1C setting of a Beckman scintillation counter. Na+ uptake values may be based on the presence of 9.365 mM Na+ (radioactive plus cold) . This assay is a modification of that of J.B. Smith and E. Rozengurt, Proceedings of the National Academy of Sciences, U.S.A., 15. r 5560 (1978) . The channel activity of unmodified forms of TNF with respect to cancer cells may be compared with the corresponding channel activity of modified forms of TNF. An observation of differential sodium ion permeability of human U937 histiocytic lymphoma cells with respect to modified and unmodified forms of TNF is a preferred example of a modified "effect" which a modified TNF may have upon a TNF target by virtue of its modified channel activity.
Binding Assay with respect to TNF Target A binding assay may be performed as described by
Loetscher et al. (Cell, .61, 351-359 (1990) , as modified herein. The binding assay may be performed with any of a large number of naturally occurring human cell types which express one or more of the TNF receptors. Alternatively, the binding assay may be performed with model cells such as human U937 histiocytic lymphoma cells -or with COS-1 cells that have been transfected with the 1.3 kb gene for the 55 kDa TNF receptor. After 2-3 days in culture, the transfected COS-1 cells may be detached with EDTA and • tested for binding by 125I-TNF-α or 125I-TNF-3. The cells are' washed, resuspended at 2.8 x 106 cells/milliliter , and incubated with various concentrations of 125I-TNF-α or 125I- TNF-3 in the absence and presence of a 500-fold excess of cold TNF-α or TNF-β, respectively, for 2 hours at 4°C. The bound radioactivity is then counted in a gamma counter. Nonspecific binding is subtracted to obtain the net specific binding of the 125I-TNF-α or 125I-TNF-3 to the transfected COS-1 cells. An alternative method for performing the binding assay is described by Coffman et al. (Lymphokine Research, 1_, 371-383 (1988) .
Detection of Trimerization Both modified and unmodified forms of TNF may undergo a trimerization reaction. The modified form of TNF may trimerize with itself, with other forms of modified TNF, and/or with unmodified forms of TNF. Preferred means for the ascertainment of such trimerization of TNF include standard in vitro assays such as separation of the monomer and trimer forms by high performance liquid chromatography (HPLC) on gel exclusion (sizing) columns. Alternatively, the sizes of modified and unmodified forms of TNF may be ascertained from their electrophoretic mobilities on a native gel or they may be ascertained by contrasting their electrophoretic mobilities on a denaturing gel after treatment with cross-linking agents.
SUBSTITUTIONS FOR TNF-α An amino acid sequence 157 residues long for an active form of human TNF-α is listed in Appendix A. Other forms of human TNF-α may have a greater or lesser number of amino acid residues, i.e. there may be deletions or additions at either end of the peptide sequence. Furthermore, there are a number of muteins of human TNF-α having one or more substitutions, deletions, and/or additions. However," the numbering of the amino acid sequence of all such alternate forms of human TNF-α may be adapted so as to correspond to the number of the sequence for human TNF-α given in Appendix A.
Also listed in Appendix A are amino acid sequences for TNF-α from 8 other species, including porcine, bovine, goat, ovine, feline, rabbit, rat and murine. Some forms of non-human TNF-α are known to be glycosylated, but glycosylation is not required for activity. It can be anticipated that TNF-α will be found in further species as well and will be similarly sequenced.
There is a close sequence homology between human TNF-α and TNF-α from non-human sources. Where the total number of amino acids within a sequence of a non-human TNF-α is less than the total number of amino acids within human TNF- α, one or more blank insertions have been introduced into the sequence numbering of the non-human TNF-α. The insertions of such blanks into the sequence numbering of non-human forms of TNF-α is done in such a way so as to maximize the sequence homology between human TNF-α and non- human TNF-α. The sequence numbering found in Appendix A and employed herein with respect to non-human forms of TNF- α is that sequence numbering that yields the greatest sequence homology between each non-human form of TNF-α and human TNF-α.
As indicated in the assay described above, TNF-α can be shown to have channel activity when it is inserted into a planar lipid membrane (3) . With respect to human TNF-α, the amino acid residues that line or face this channel, i.e. the primary "channel residues," include the following, viz.: Lys11, Leu57, Tyr59, Lys9*, Lys112, Glu116, Tyr119, Gly121, lie155, and Leu157. However, these primary "channel residues" are encompassed within a larger group of amino acids designated as channel liners, some of which merely contact the channel via the carbonyl oxygens and amide NH groups of the peptide chain. For TNF-α these channel liners include residues number 11, 55-59, 98-125, and 151-157. Because of the close sequence homology between human TNF-α and non- human forms of TNF-α, the important channel residues with respect to non-human forms of TNF-α have the same sequence numbers as given above for human TNF-α. It can be anticipated that, of those mutein forms of human and non- human TNF-α that exhibit modified channel activity, the important channel residues for such mutein forms of TNF-α will also have the same sequence numbers as given above for human TNF-α. However, the particular amino acids that occupy such sequence sites may vary from one non-human species to the next or from one mutein form to another. Listed below are preferred amino acid substitutions with respect to channel residues of TNF-α for enhancing, diminishing, or modulating its channel activity. In each instance, the indicated parent amino acid is the amino acid found in human TNF-α with respect to that particular sequence number. The parent amino acid with respect to non-human forms of TNF-α and with respect to various mutein forms of TNF-α may differ from the indicated patent amino acid. In such instances, the parent amino acid may be determined by referring to the sequence number for the particular channel residue being substituted.
Preferred amino acid substitutions of channel residues with respect to TNF-α include the following:
1. Residue No.: 11 Lys
First preference: Glu, Arg, Cys, Asp, Gin, Asn, Ser,Thr, ' His
Second Preference: Val, Leu, lie, & Ala Third Preference: Trp, Gly, Pro, Tyr, Phe, & Met
2. Residue No.: 57 Leu First preference: Trp, Ser, Thr, Ala, Met, Cys,
Phe, & Tyr Second Preference: Arg, Glu, Lys, Asp, Gin, &' Asn
Third Preference: Gly, Val, lie, His, & Pro
3. Residue No.: 59 Tyr First preference: Trp, Ser, Thr, Ala, Met, &
Cys
Second Preference: Arg, Glu, Lys, Asp, Gin, & Asn
Third Preference: Gly, Val, lie, Leu, His, & Pro
4. Residue No.: 98 Lys
First preference: Arg, Cys, Glu, Asp, Gin, Asn,
Ser, Thr, & His Second Preference: Val, Leu, lie, & Ala Third Preference: Trp, Met, Gly, Pro, Tyr, & Phe
5. Residue No.: 112 Lys
First preference: Arg, Cys, Asp, Gin, Asn, Ser,
Thr, Glu, & His
Second Preference: Val, Leu, He, & Ala Third Preference: Trp, Gly, Pro, Tyr, & Phe
6. Residue No.: 116 Glu
First preference: Lys, Arg, Cys, Asp, Gin, Asn,
Ser, His, & Thr
Second Preference: Leu, He, & Ala Third Preference: Trp, Met, Gly, Pro, Tyr, &
Phe
7. Residue No.: 119 Tyr
First preference: Trp, Phe, Ser, Thr, Ala, Met,
& Cys Second Preference: Arg, Glu, Lys, Asp, Gin, &
Asn
Third Preference: Gly, Val, He, Leu, & Pro
8. Residue No.: 121 Gly
First preference: Ala, Val, Ser, & Thr Second Preference: Pro, He, Leu, & His Third Preference: Trp, Tyr, Phe, Cys, Met, Lys,
Glu, Arg, Gin, Asp, & Asn
9. Residue No.: 155 He
First preference:. Trp, Ser, Thr, Ala, Met, Cys, Phe, & Tyr
Second Preference: Arg, Glu, Lys, Asp, Gin, & Asn
Third Preference: Gly, Val, His, & Pro
10. Residue No.: 157 Leu First preference: Trp, Ser, Thr, Ala, Cys, &
Tyr
Second Preference: Arg, Glu, Lys, Asp, & Asn Third Preference: Gly, He, & His
SUBSTITUTIONS FOR TNF-θ
An amino acid sequence 171 residues long for an active form of human TNF-/3 (lymphotoxin) is listed in Appendix A. Other forms of human TNF-3 may have a greater or lesser number of amino acid residues, i.e. there may be deletions or additions at either end of the peptide sequence.
Furthermore, there are a number of muteins of human TNF-/3 having one or more substitutions, deletions, and/or additions. However, the numbering of the amino acid sequence of all such alternate forms of human TNF-/3 may be adapted so as to correspond to the numbering of the sequence for human TNF-/3 given in Appendix A.
Also listed in Appendix A are the amino acid sequences for TNF-/3 from 3 other species, including bovine, rabbit, and murine. It can be anticipated that TNF-9 will be found in further species as well and will be similarly sequenced.
There is a close sequence homology between human TNF-/3 and non-human TNF-3. Where the total number of amino acids within a sequence of non-human TNF-/3 is less than the total number of amino acids within human TNF-/3, one or more blank insertions have been introduced into the sequence numbering of the non-human form of TNF-/3. Such insertions are positioned so as to maximize the sequence homology between human TNF-jø and non-human TNF- . The sequence numbering found in Appendix A and employed herein with respect to non-human forms of TNF-/3 is that sequence numbering that yields the greatest sequence homology between such non- human forms of TNF-jS and human TNF-jS.
TNF-β can be shown to have channel activity when it is inserted into a planar lipid membrane (3) . With respect to human TNF-β, the amino acid residues that line or face this channel, i.e. the "channel residues," include the following, viz.: Lys28, Phe74, Tyr76, Lys119, Glu127, His131, Tyr i34 ^ Gly136, Phe169, and Leu171. However, these primary "channel residues" are encompassed within a larger group of amino acids designated as channel liners, some of which merely contact the channel via the carbonyl oxygens and amide NH groups of the peptide chain. For TNF- these channel liners include residues number 28, 72-76, 119-140, and 165-171. Because of the close sequence homology between human TNF-β and non-human forms of TNF-/3, the important channel residues with respect to non-human forms of TNF-3 have the same sequence numbers as given above for human TNF-β. It can be anticipated that, of those mutein forms of human and non-human TNF-/3 that exhibit altered channel activity, the important channel residues for such mutein forms of TNF-/3 will also have the same sequence numbers as given above for human TNF-/3. However, the particular amino acids that occupy such sequence sites may vary from one non-human species to the next or from one mutein form to another.
Listed below are preferred amino acid substitutions with respect to channel residues of TNF-/3 for enhancing, diminishing, or modulating its channel activity. In each instance, the indicated parent amino acid is the amino acid found in human TNF-β with respect to that particular sequence number. The parent amino acid with respect to non-human forms of TNF-/3 and with respect to various mutein forms of TNF-/3 may differ from the indicated patent amino acid. In such instances, the parent amino acid may be determined by referring to the sequence number for the particular channel residue being substituted.
Preferred amino acid substitutions of channel residues with respect to TNF-/3 include the following:
1. Residue No.: 28 Lys
First preference: Glu, Arg, Cys, Asp, Gin, Asn, Ser, Thr, & His
Second Preference: Val, Leu, He, & Ala
Third Preference: Trp, Gly, Pro, Tyr, Phe, & Met
2. Residue No.: 74 Phe First preference: Trp, Ser, Thr, Ala, Met, Cys,
Leu, & Tyr Second Preference: Arg, Glu, Lys, Asp, Gin, & Asn
Third Preference: Gly, Val, He, His, & Pro 3. Residue No.: 76 Tyr
First preference: Trp, Ser, Thr, Ala, Met, Cys,
& Phe Second Preference: Arg, Glu, Lys, Asp, Gin, & Asn Third Preference: Gly, Val, He, Leu, His, &
Pro
4. Residue No.: 119 Lys
First preference: Arg, Cys, Glu, Asp, Gin, Asn,
Ser, Thr, & His Second Preference: ' Val, Leu, He, & Ala
Third Preference: Trp, Met, Gly, Pro, Tyr, & Phe
5. Residue No.: 127 Glu
First preference: Arg, Cys, Asp, Gin, Asn, Ser, Thr, Lys, & His
Second Preference: Val, Leu, lie, Met, & Ala Third Preference: Trp, Gly, Pro, Tyr, & Phe Residue No.: 131 His
First preference: Lys, Arg, Cys, Asp, Gin, Asn,
Ser, Glu, & Thr
Second Preference: Leu, He, Val, & Ala Third Preference: Trp, Met, Gly, Pro, Tyr, &
Phe 7. Residue No.: 134 Tyr
First preference: Trp, Phe, Ser, Thr, Ala, Met,
& Cys
Second Preference: Arg, Glu, Lys, Asp, Gin, His,
& Asn
Third Preference: Gly, Val, He, Leu, & Pro
8. Residue No.: 136 Gly
First preference: Ala, Val, Ser, & Thr Second Preference: Pro, He, Leu, & His Third Preference: Trp, Tyr, Phe, Cys, Met, Lys,
Glu, Arg, Gin, Asp, & Asn
9. Residue No.: 169 Phe
First preference: Trp, Ser, Thr, Ala, Met, Cys,
He, & Tyr
Second Preference: Arg, Glu, Lys, Asp, Gin, Leu,
& Asn
Third Preference: Gly, Val, His, & Pro 10 Residue No.: 171 Leu
First preference: Trp, Ser, Thr, Ala, Cys, Phe,
& Tyr
Second Preference: Arg, Glu, Lys, Asp, Gin, &
Asn Third Preference: Gly, Pro, Val, He, Met, &
His
MECHANISMS WHEREBY CHANNEL ACTIVITY MAY BE CONTROLLED The channel activity of unsubstituted forms.of TNF-α and —β share many broad similarities. Similarly, there is a correspondence between the specific channel residues' of TNF-α and -β . The correspondence between the channel residues of human TNF-α and -β is as follows:
TNF-α TNF- Lys11 corresponds to Lys28
Leu57 corresponds to Phe74
Tyr59 corresponds to Tyr76
Lys98 corresponds to Lys119
Lys112 corresponds to Glu127 Glu116 corresponds to His131
Tyr119 corresponds to Tyr134
Gly121 corresponds to Gly136 lie155 corresponds to Phe169
Leu157 corresponds to Leu171 The above list of channel residues for TNF-α and -β are correlated because these channel residues are similarly positioned within their respective channels and because an amino acid substitution of correlated channel residues tends to cause correlated changes of channel activity with respect to both TNF-α and -β . For example a substitution of a long chain aliphatic for Gly121 (TNF-α) and Gly136 (TNF- β) will tend to occlude the channel and will tend to diminish the channel activity of- both TNF-α and -β . Similarly, the removal of bulky amino acids and the substitution of short chain amino acids tends to broaden the cross-sectional diameter of the channel and frequently, causes an increase in channel activity.
Amino Acid Substitutions which Employ Cysteine-Cysteine Bonding to
Cross-link Subunits within a TNF Trimer X-ray crystallographic studies on the trimers of both TNF-α and -β indicate the usage of several salt bridges to bond together the respective TNF subunits. The amino acid residues for these salt linkages can be substituted with cysteines in order to form covalent cysteine-cysteine linkages between respective TNF subunits.
Examples for TNF-α include the following:
A. The interchain salt linkage between Lys98 of one subunit and Glu116 of an adjacent subunit can be modified by substituting Cys for both residues #98 and #116. When the resulting modified form of TNF trimerizes under non-reducing conditions, three interchain covalent linkages will form, viz. the Cys98 of each subunit will form a covalent disulfide bond with the Cys116 on the adjacent subunit. The resultant disulfide bond can be disrupted under reducing conditions.
B. Similarly, the interchain salt linkage between Arg103 of one TNF subunit and Glu104 of an adjacent
TNF subunit may be modified by Cys substitutions to form a modified form of TNF having Cys103 and Cys104 to form three interchain disulfide bonds for stabilizing the TNF trimer. C. Similarly, the interchain salt linkage between Lys11 of one TNF subunit and the carboxy terminal Leu157 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified form of TNF having Cys" and Cys157 to form three interchain disulfide bonds for stabilizing the TNF trimer.
D. The Tyr119 residues that protrude into the middle of the TNF channel can be substituted with Cys119. However, in this case no salt bridge is replaced and only one interchain disulfide bond results under non-reducing conditions. However, this single disulfide bond may switch from one subunit pair to another, e.g., from
TNF1-SS-TNF2 to TNF2-SS-TNF3 to TNF3-SS-TNF1. Examples for TNF-β include the following: A. A pH dependent interchain salt linkage between Lys119 of one subunit and His131 of an adjacent subunit can be modified by substituting Cys for both residues #119 and #131. When the resulting modified form of TNF trimerizes under non- reducing conditions, three interchain covalent linkages will form, viz. the Cys119 of each subunit will form a covalent disulfide bond with the Cys131 on the adjacent subunit. The resultant disulfide bond can be disrupted under reducing conditions, but, unlike the salt bridge, will be relatively independent of pH.
B. Similarly, the interchain linkage between Ser117 of one TNF subunit and His135 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified TNF having Cys117 and Cys135 to form three interchain disulfide bonds for stabilizing the TNF trimer.
C. Similarly, the interchain salt linkage between Lys28 of one TNF subunit and the carboxy terminal Leu171 of an adjacent TNF subunit may be modified by Cys substitutions to form a modified form of
TNF having Cys28 and Cys171 to form three interchain disulfide bonds for stabilizing the TNF trimer.
D. The Tyr134 residues can be substituted with Cys134. However, in this case no salt bridge is replaced and only one interchain disulfide bond results under non-reducing conditions. However, it is possible that this single disulfide bond may switch from one subunit pair to another, e.g., from TNF1-SS-TNF2 to TNF2-SS-TNF3 to TNF3-SS- TNF1.
Chemical Modification of TNF Channel for Diminishing or Modulating Channel Activity A number of reagents may be employed to interact selectively with channel residues of modified and unmodi- fied forms of TNF so as to form an occlusion within the channel for diminishing its activity.
A Method for Making Modified Forms of TNF-α and/or -β Having a Reconstructed Channel with One or More Amino Acid Substitutions, As Compared to Unmodified TNF-α and/or -β For Regulating Channel Activity Firstly, one or more candidate forms of modified TNF-α and/or -β are formed by substituting one or more channel residues with replacement amino acids. The preferred channel residues and the respective preferred amino acid substitutions for such channel residues are indicated above. A preferred mode for effecting such amino acid substi¬ tutions by means of site-specific mutagenesis is described by Kunkel, T. A.- (Proceedings of the National Academy of Sciences, U.S.A., J32., 488-492). Kunkel teaches that plas- mid pHTP320 may be digested with Spel and Hindlll to iso- late the DNA fragment containing the TNF gene, and may be subcloned into the Hindlll and Xbal sites of phage M13mpl9. From this recombinant phage, single-stranded DNA may be prepared as a template containing uracils for mutagenesis by using E. coli CJ236. Appropriate mutagenic oligonucleotides (approximately 20-mers) corresponding to the desired amino acid substitution may be chemically synthesized with a DNA synthesizer. For each mutagenesis,. approximately 200 ng (0.1 pmol) of a template containing uracil may be mixed with 4 pmol of 5'-phosphorylated mutagenic oligonucleotide in 10 μl of an annealing buffer (20 M Tris-HCl, pH 7.4; 2 mM MgCl2; 50 mM NaCl) . The reaction mixture may be heated at 70°C for 10 minutes, and then cooled at a rate of approximately l°C/min. until 30°C. After addition of DNA polymerase, Klenow fragment (2.5 units) , T4 DNA ligase (5 units) and 1 μl of lOx synthesis buffer (5mM each dNTP; lOmM ATP; 100 mM Tris-HCL, pH 7.4; 50 mM MgCl2; 20 mM dithiothreitol) , the reaction mixture may¬ be sequentially incubated at 0°C for 5 minutes, 25°C for 5 minutes and 37°C for 90 minutes. A sample of the ligation • reaction may be employed to transform competent E. coli JM105 cells. Without previous selection by hybridization assay with the mutated oligonucleotide, single-stranded DNA may be extracted from the plaques and identified by nucleotide sequencing. The replicative form of the mutant may be digested with restriction endonucleases Clal and Hindlll. The fragment containing the mutagenized TNF coding sequence may be subcloned into an expression plasmid pHTP320 in place of the TNF gene. The transformants having an expression plasmid for the modified form of TNF may be incubated in LB medium supplemented with ampicillin (50 μg/ l) at 37°C overnight and then the cultures inoculated in M9 medium supplemented with 0.5% casamino acids and ampicillin (50 μg/ml) . After 3 hours, 3-indoleacrylic acid (20 μg/ml) may be added to induce the E. coli trp promoter and cultivation may be further continued at 37°C for 20 hours. Cells may then be disrupted by lysozyme-digestion and freezing-thawing as described by Nagata et al. Nature, 284, 316-320 (1980) . The disrupted cells may then be centrifuged to obtain a clarified supernatant as E. coli extracts. The candidate TNF may then be purified from the E. coli extract according to the method of Yamada et al., Journal of Biotechnology, 2, 141-153 (1985) .
The purified form of candidate TNF may then tested to determine if it retains an ability to form TNF trimers. The protocol for testing trimerization is given above. The purified form of candidate TNF may also be tested to determine if the TNF has an ability to achieve intimate contact with a target that includes both a membrane (3) and one or more TNF receptors. The protocols for determining TNF receptor binding and channel formation are given above. The purified form of candidate TNF may also be tested to determine if the TNF, when in intimate contact with the target, achieves a modified effect. Examples of such modified effects are given above, however, they must be of • a type caused by formation of a modified channel activity. The modified channel activity must materially differ from corresponding unmodified channel activities caused by the corresponding unmodified TNF.
A modified form of TNF may be selected from one or more of the candidate forms of TNF. The modified form of TNF must have been determined to be able to form TNF trimers; it must be able to achieve intimate contact with the target; and it must be able to achieve a modified effect upon a target by virtue of a modified channel activity.
After the modified form of TNF is selected, it may be made in purified form and in commercial quantities.
Method for Regulating the Channel Activity with respect to a Target Membrane
The permeability exhibited by a TNF target membrane (3) may be regulated by contacting the target membrane with a modified form of TNF. The modified form of TNF should have a reconstructed channel (5 or 7) , as compared to unmodified TNF, as described above, for regulating channel activity. In a preferred mode, modified form of TNF (6) is selected that forms a reconstructed channel (7) so as to result in a reduction of the channel activity as compared to the unmodified form of TNF. Accordingly, the insertion of such modified form of TNF in the target membrane (3) serves to reduce the channel activity. In an alternative preferred mode, a modified form of TNF (4) is selected that forms a reconstructed channel (5) so as to result in an enhancement of the channel activity as compared to the unmodified form of TNF, as indicated above. Accordingly, the insertion of this form of modified TNF in the target membane (3) serves to enhance the channel activity.
Method for Inhibiting "the Binding of Unmodified TNF to one or more TNF Receptors
Attached to a Target Membrane A modified form of TNF having a reconstructed channel (7) for reducing channel activity within a target membrane (3) may be employed for inhibiting the binding of unmodified TNF to one or more TNF receptors attached to a target membrane (3) . In a preferred mode, a modified form of TNF having a channel reconstructed for reducing channel activity, as compared to unmodified TNF, is contacted with one or more of TNF receptors under conditions for permitting binding between the modified form of TNF and the TNF receptor. The modified form of TNF acts as an antagonist or competitive inhibitor of the unmodified TNF with respect to binding to TNF receptor.
Regulation of Channel Activity by
Channel Blockers and Activators Molecules may be designed to interact specifically with the channel region of modified and unmodified forms of TNF. Some interactions will occlude the channel and block its channel activity. Some interactions will dilate the channel and enhance its channel activity.
APPENDIX A
SEQUENCE LISTING FOR TUMOR NECROSIS FACTOR (1) GENERAL INFORMATION:
(i) APPLICANT: Wisnieski, Bernadine J.
(ii) TITLE OF INVENTION: Tumor Necrosis Factor with Modified Ion Channel
(iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Donald G. Lewis
(B) STREET: 8328 Regents Road #1E (C) CITY: San Diego
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 92122
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 M storage
(B) COMPUTER: VE System 386
(C) OPERATING SYSTEM: MS-DOS 5
(D) SOFTWARE: Word Perfect
(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: unknown
(B) FILING DATE: 12 March 1993
(C) CLASSIFICATION: unknown
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/852,625 (B) FILING DATE: 12 March 1992
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Donald G. Lewis
(B) REGISTRATION NUMBER: 28636
(C) REFERENCE/DOCKET NUMBER: BJW-2
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (619) 554-2421
(B) TELEFAX: (619) 554-6312
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids (B) TYPE: amino acids
(C) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (human) (x) PUBLICATION INFORMATION:
(A) AUTHORS: Pennica D., Nedwin G. E. , Hayflick, J.S., Seeburg P.H. , Derynck, R. , Palladino, M.A., Kohr, W.J., et al.
(B) TITLE: Human Tumor Necrosis Factor: Precursor Structure, Expression and Homology to Lymphotoxin (C) JOURNAL: Nature
(D) VOLUME: 312
(E) PAGES: 724-729
(F) DATE: 1984
(G) RELEVANT RESIDUES IN SEQ ID NO:l: 1-157 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His 10 15
Val Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu Asn
20 25 30
Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp
35 40 45
Asn Gin Leu Val Val Pro Ser Glu Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Ser Thr His Val Leu
65 75 80 Leu Thr His Thr He Ser Arg He Ala Val Ser Tyr Gin Thr Lys
85 90 95
Val Asn Leu Leu Ser Ala He Lys Ser Pro Cys Gin Arg Glu Thr
100 105 110
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro He Tyr Leu
115 120 125
Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu
130 135 140
He Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val
145 150 155 Tyr Phe Gly He He Ala Leu
155
(3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (porcine)
(B) OTHER INFORMATION: A blank residue designated by "Xaa" is inserted after residue No. 7 of porcine TNF and the sequence numbering is augmented by 1 starting with residue No. 8 in order to maximize the sequence homology with human TNF.
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Pauli, U. Beutler, B. , and Peterhans, S.
(B) TITLE: Porcine Tumor Necrosis Factor-α:Cloning with the Polymerase Chain Reaction and Determination of the Nucleotide Sequence
(C) JOURNAL: Gene
(D) VOLUME: 81
(E) PAGES: 185-191
(F) DATE: 1989 (G) RELEVANT RESIDUES IN SEQ ID NO:2: 1-157 (includes one blank)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Leu Arg Ser Ser Ser Gin Thr Xaa Ser Asp Lys Pro Val Ala His
5 10 15
Val Val Ala Asn Val Lys Ala Glu Gly Gin Leu Gin Trp Gin Ser
20 25 30
Gly Tyr Ala Asn Ala Leu Leu Ala Asn Gly Val Lys Leu Lys Asp
35 40 45
Asn Gin Leu Val Val Pro Thr Asp Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Arg Gly Gin Gly Cys Pro Ser Thr Asn Val Phe
65 70 75 Leu Thr His Thr He Ser Arg He Ala Val Ser Tyr Gin Thr Lys
80 85 90
Val Asn Leu Leu Ser Ala He Lys Ser Pro Cys Gin Arg Glu Thr
95 100 105
Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro He Tyr Leu
110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Asp Asp Arg Leu Ser Ala Glu 125 130 135
He Asn Leu Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val 140 145 150
Tyr Phe Gly He He Ala Leu
155
(4) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids (C) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein . (ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (bovine)
(B) OTHER INFORMATION: A blank residue designated by
"Xaa" is inserted after residue No. 70 of bovine TNF and the sequence numbering is augmented by 1 starting with residue No. 71 in order to maximize the sequence homology with human TNF.
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Niitsu, Y. and Watanabe, N.
(B) TITLE: Cytokines and Receptors - Their Functions, Structures and Cloning of Code Genes. Tumor Necrosis
Factor.
(C) JOURNAL: Nippon Rinsho (D) VOLUME: 46
(E) PAGES: 1041-1049
(F) DATE: 1988
(G) RELEVANT RESIDUES IN SEQ ID NO:3: 1-157 (includes one blank)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Leu Arg Ser Ser Ser Gin Ala Ser Ser Asn Lys Pro Val Ala His
5 10 15 Val Val Ala Asp He Asn Ser Pro Gly Gin Leu Arg Trp Trp Asp
20 25 30
Ser Tyr Ala Asn Ala Leu Met Ala Asn Gly Val Gin Leu Glu Asp
35 40 45
Asn Gin Leu Val Val Pro Ala Glu Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Arg Gly Gin Gly Cys Pro Xaa Pro Pro Pro Val
65 70 75
Leu Thr His Thr He Ser Arg He Ala Val Ser Tyr Gin Thr Lys
80 85 90
Val Asn He Leu Ser Ala He Lys Ser Pro Cys His Arg Glu Thr
95 100 105 Pro Glu Trp Ala Glu Ala Lys Pro Trp Tyr Glu Pro He- Tyr Gin
110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Asp Asp Arg Leu Ser Ala Glu
125 130 135
He Asn Leu Pro Asp Tyr Leu Asp Tyr Ala Glu Ser Gly Gin Val 140 145 150
Tyr Phe Gly He He Ala Leu 155
(5) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (goat)
(B) OTHER INFORMATION: A blank residue designated by "Xaa" is inserted after residue No. 107 of goat TNF and the sequence numbering is augmented by 1 starting with residue No. 108 in order to maximize the sequence homology with human TNF. (x) PUBLICATION INFORMATION: (A) JOURNAL: Submitted to EMBL Data Bank X14828
(B) DATE: March 1989
(G) RELEVANT RESIDUES IN SEQ ID NO:4: 1-157 (includes one blank)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : Leu Arg Ser Ser Ser Gin Ala Ser Ser Asn Lys Pro Val Ala His
5 10 15
Val Val Ala Asn He Ser Ala Pro Gly Gin Leu Arg Trp Gly Asp
20 25 30
Ser Tyr Ala Asn Ala Leu Lys Ala Asn Gly Val Glu Leu Lys Asp
35 40 45
Asn Gin Leu Val Val Pro Thr Asp Gly. Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Arg Gly His Gly Cys Pro Ser Thr Pro Leu Phe
65 70 75 Leu Thr His Thr He Ser Arg He Ala Val Ser Tyr Gin Thr Lys
80 85 90
Val Asn He Leu Ser Ala He Lys Ser Pro Cys His Arg Glu Thr
95 100 105
Pro Glu Xaa Ala Glu Ala Lys Pro Trp Tyr Glu Pro He Tyr Gin
110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu 125 130 135
He Asn Gin Pro Glu Tyr Leu Asp Tyr Ala Glu Ser Gly Gin Val
140 145 150 Tyr Phe Gly He He Ala Leu
155
(6) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids (B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (ovine) (X) PUBLICATION INFORMATION:
(A) AUTHORS: Young, A.J., Hay, J.B. , and Chan, J.Y.C.
(B) TITLE: Primary Structure of Ovine Tumor Necrosis Factor Alpha cDNA.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 18
(E) PAGE: 6723 (F) DATE: 1990
(G) RELEVANT RESIDUES IN SEQ ID NO:5: 1-157 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Leu Arg Ser Ser Ser Gin Ala Ser Asn Asn Lys Pro Val Ala His
5 10 15
Val Val Ala Asn Leu Ser Ala Pro Gly Gin Leu Arg Trp Gly Asp
20 25 30
Ser Tyr Ala Asn Ala Leu Met Ala Asn Gly Val Glu Leu Lys Asp
35 40 45 Asn Gin Leu Val Val Pro Thr Asp Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Arg Gly His Gly Cys Pro Ser Thr Pro Leu Phe
65 70 75
Leu Thr His Thr He Ser Arg He Ala Val Ser Tyr Gin Thr Lys
80 85 90
Val Asn He Leu Ser Ala He Lys Ser Pro Cys His Arg Glu Thr 95 100 105
Leu Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro He Tyr Gin 110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu
125 130 135 lie Asn Leu Pro Glu Tyr Leu Asp Tyr Ala Glu Ser Gly Gin Val
140 145 ' 150
Tyr Phe Gly He He Ala Leu 155
(7) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (feline) (X) PUBLICATION INFORMATION:
(A) AUTHORS: McGraw, R. A., Coffee, B.W. , Otto, C ., Drews, R.T. and Rawling, CA.
(B) TITLE: Gene Sequence of Feline Tumor Necrosis Factor α.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 18
(E) PAGE: 5564
(F) DATE: 1990 (G) RELEVANT RESIDUES IN SEQ ID NO:6: 1-157
( i) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His 5 10 15
Val Val Ala Asn Pro Glu Ala Glu Gly Gin Leu Gin Arg Leu Ser 20 25 30
Figure imgf000037_0001
He Asn Leu Pro Ala Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val 140 145 150
Tyr Phe Gly He He Ala Leu
155 (8) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (rabbit)
(B) OTHER INFORMATION: A blank residue designated by "Xaa" is inserted after residue No. 70 of rabbit TNF and the sequence numbering is augmented by 1 starting with residue No. 71 in order to maximize the sequence homology with human TNF.
(X) PUBLICATION INFORMATION: (A) AUTHORS: Ito, H. , Shirai, T. , Yamamoto, S., Akira, M. , Kawahara, S., Todd, C.W. and Wallace, R.B.
(B) TITLE: Molecular Cloning of the Gene Encoding Rabbit Tumor Necrosis Factor.
(C) JOURNAL: DNA . (D) VOLUME: 5
(E) PAGES: 157-165
(F) DATE: 1986
(G) RELEVANT RESIDUES IN SEQ ID NO:7: 1-157 (includes one blank)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu Arg Ser Ala Ser Arg Ala Leu Ser Asp Lys Pro Leu Ala His
5 10 15
Val Val Ala Asn Pro Gin Val Glu Gly Gin Leu Gin Trp Leu Ser
20 25 30
Gin Arg Ala Asn Ala Leu Leu Ala Asn Gly Met Lys Leu Thr Asp
35 40 45 Asn Gin Leu Val Val Pro Ala Asp Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Ser Gly Gin Gly Cys Arg Xaa Ser Tyr Val Leu
65 70 75
Leu Thr His Thr Val Ser Arg Phe Ala Val Ser Tyr Pro Asn Lys
80 85 90
Val Asn Leu Leu Ser Ala He Lys Ser Pro Cys His Arg Glu Thr 95 - 100 105
Pro Glu Glu Ala Glu Pro Met Ala Trp Tyr Glu Pro He Tyr Leu
110 115 120 Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Thr Glu
125 130 135
Val Asn Gin Pro Glu Tyr Leu Asp Leu Ala Glu Ser Gly Gin Val
140 145 150
Tyr Phe Gly He He Ala Leu
155 (9) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE: (A) NAME/KEY: Tumor Necrosis Factor (rat)
(B) OTHER INFORMATION: A blank residue designated by "Xaa" is inserted after residue No. 70 of rat TNF and the sequence numbering is augmented by 1 starting with residue No. 71 in order to maximize the sequence homology with human TNF.
(X) PUBLICATION INFORMATION: (A) AUTHORS: Shirai, T. , Shimizu, N. , Horiguchi,
S. , and Ito, H.
(B) TITLE: Cloning and Expression in Escherichia coli of the gene for Rat
(C) JOURNAL: Agric. Biol. Chem.
(D) VOLUME: 53
(E) PAGES: 1733-1736
(F) DATE: 1989 (G) RELEVANT RESIDUES IN SEQ ID NO:8: 1-157 (includes one blank)
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Arg Ser Ser Ser Gin Asn Ser Ser Asp Lys Pro Val Ala His
5 10 15
Val Val Ala Asn His Gin Ala Glu Glu Gin Leu Glu Trp Leu Ser
20 25. 30
Gin Arg Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp
35 40 45 Asn Gin Leu Val Val Pro Ala Asp Gly Leu Tyr Leu He Tyr Ser
50 55 60
Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Xaa Asp Tyr Val Leu 65 70 75
Leu Thr His Thr Val Ser Arg Phe Ala He Ser Tyr Gin Glu Lys
80 85 90 Val Ser Leu Leu Ser Ala He Lys Ser Pro Cys Pro Lys Asp Thr
95 100 105
Pro Glu Gly Ala Glu Leu Lys Pro Trp Tyr Glu Pro Met Tyr'Leu
110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Leu Leu Ser Ala Glu
125 130 135
Val Asn Leu Pro Lys Tyr Leu Asp He Thr Glu Ser Gly Gin Val 140 145 150
Tyr Phe Gly Val He Ala Leu
155 (10) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Tumor Necrosis Factor (murine)
(B) OTHER INFORMATION: A blank residue designated by "Xaa" is inserted after residue No. 70 of murine TNF and the sequence numbering is augmented by 1 starting with residue No. 71 in order to maximize the sequence homology with human TNF.
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Caput, D J, Beutler, B. Hartog, K. Thayer, R. , Brown-Shi er, S_. and
Cerami, A. (B) TITLE: Identification of a Common Nucleotide
Sequence in the 3 '-Untranslated Region of mRNA Molecules Specifying Inflammatory Mediators. (C) JOURNAL: Proc. National Academy of Science,
U.S.A.
(D) VOLUME: 83 (E) PAGES: 1670-1674
(F) DATE: 1986
(G) RELEVANT RESIDUES IN SEQ ID NO:9: 1-157 (includes one blank)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: murine TNF Leu Arg Ser Ser Ser Gin Asn Ser Ser Asp Lys Pro Val Ala His
5 10 15
Val Val Ala Asn His Gin Val Glu Glu Gin Leu Glu Trp Leu Ser
20 25 30
Gin Arg Ala Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp
35 40 45
Asn Gin Leu Val Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser
50 55 60
Gin Val Leu Phe Lys Gly Gin Gly Cys Pro Xaa Asp Val Val Leu
65 70 75 Leu Thr His Thr Val Ser Arg Phe Ala He Ser Tyr Gin Glu Lys
80 85 90
Val Asn Leu Leu Ser Ala Val Lys Ser Pro Cys Pro Lys Asp Thr
95 100 105
Pro Glu Gly Ala Glu Leu Lys Pro Trp Tyr Glu Pro He Tyr Leu
110 115 120
Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Gin Leu Ser Ala Glu 125 130 135
Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val
140 145 150 Tyr Phe Gly Val He Ala Leu
155 (11) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE: (A) NAME/KEY: Lymphotoxin (human)
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Hedwin, G.E., Naylor, S.L.,
Sakaguchi, A.Y., Smith, D. , Nedwin, J."J. , Pennica, D. , Goeddel, D.V. , et al. (B) TITLE: Human Lymphotoxin and Tumor Necrosis
Factor: Structure, Homology and Chromosomal Localization.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(E) PAGES: 6261-6373 . (F) DATE: 1985
(G) RELEVANT RESIDUES IN SEQ ID NO: 10: 1-171 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10 human It
Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gin Thr Ala Arg
5 10 15
Gin His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala
20 25 30
Ala His Leu He Gly Asp Pro Ser Lys Gin Asn Ser Leu Leu Trp
35 40 45 Arg Ala Asn Thr Asp Arg Ala Phe Leu Gin Asp Gly Phe Ser Leu
50 55 60 Ser Asn Asn Ser Leu Gly He Tyr Phe Val
65 75
Tyr Ser Gin Val Val Tyr Ser Pro Lys Ala
80 90
Thr Ser Ser Pro Leu Val Gin Leu Phe Ser
95 105
Ser Gin Tyr Pro Phe Ser Ser Gin Lys Met
110 120
Val Tyr Pro Gly Leu His Ser Met Tyr His
125 135
Gly Ala Ala Phe Gin Gin Leu Ser Thr His
140 150
Thr Asp Gly He Pro Pro Ser Thr Val Phe
155
Figure imgf000043_0001
165
Phe Gly Ala Phe Ala Leu
170
(12) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Lymphotoxin (bovine) (X) PUBLICATION INFORMATION:
(A) AUTHORS: Niitsu, Y. and Watanabe, N.
(B) TITLE: Cytokines and Receptors - Their Functions, Structures and Cloning of Code Genes. Tumor Necrosis
Factor.
(C) JOURNAL: Nippon Rinsho (D) VOLUME : 46
(E) PAGES: 1041-1049
(F) DATE: 1988
(G) RELEVANT RESIDUES IN SEQ ID NO:11: 1-171 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: bovine It
Leu Arg Gly He Gly Leu Thr Pro Ser Ala Ala Gin Pro Ala His
10 15
Gin Gin Leu Pro Thr Pro Phe Thr Arg Gly Thr Leu Lys Pro Ala
20 25 30
Ala His Leu Val Gly Asp Pro Ser.Thr Gin Asp Ser Leu Arg Trp
35 40 45
Arg Ala Asn Thr Asp Arg Ala Phe Leu Arg His Gly Phe Ser Leu
50 55 60
Ser Asn Asn Ser Leu Leu Val Pro Thr Ser Gly Leu Tyr Phe Val
65 70 75
Tyr Ser Gin Val Val Phe Ser Gly Arg Gly Cys Phe Pro Arg Ala
80 85 90
Thr Pro Thr Pro Leu Tyr Leu Ala His Glu Val Gin Leu Phe Ser
95 100 105
Pro Gin Tyr Pro Phe His Val Pro Leu Leu Ser Ala Gin Lys Ser
110 115 120
Val Cys Pro Gly Pro Gin Gly Pro Trp Val Arg Ser Val Tyr Gin
125 130 135
Gly Ala Val Phe Leu Leu Thr Arg Gly Asp Gin Leu Ser Thr His
140 145 150
Thr Asp Gly He Ser His Leu Leu Leu Ser Pro Ser Ser Val Phe
155 160 165
Phe Gly Ala Phe Ala Leu
170
(13) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids (B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Lymphotoxin (rabbit)
(B) OTHER INFORMATION: Two blank residues designated by
"Xaa" are inserted after residue No. 34 and No. 61 of murine lymphotoxin and the sequence numbering is augmented by 1 starting with residue No. 35 and again augmented by 1 starting with residue No. 62 in order to maximize the sequence homology with human lymphotoxin.
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Ito, H. , Shirai, T. , Yamamoto, S., Akira, M. , Kawahara, S., Todd, C.W. and Wallace, R.B. (B) TITLE: Molecular Cloning of the Gene Encoding
Rabbit Tumor Necrosis Factor.
(C) JOURNAL: DNA (D) VOLUME: 5
(E) PAGES: 157-165
(F) DATE: 1986
(G) RELEVANT RESIDUES IN SEQ ID NO:12: 1-171 (includes
2 blanks)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Leu Pro Gly Ala Gin Phe Pro Pro Ser Ala Ala Arg Asn Ala Gin
5 10 . 15
Gin Arg Leu Gin Lys His Phe Gly His Ser Thr Leu Lys Pro Ala 20 25 30
Ala His Leu Val Xaa Asp Pro Ser Ala Gin Asp Ser Leu Arg Trp
35 40 45 Arg Ala Asn Thr Asp Arg Ala Phe Leu Ala His Gly Phe Ser Leu
50 55 60 Ser Asn Xaa Phe Pro Cys Gly Pro Ser Ser Gly Leu Tyr Phe Val
65 70 75
Tyr Ser Gin Val Val Phe Ser Gly Glu Gly Cys Ser Pro Lys Ala
80 85 90
Val Pro Thr Pro Leu Tyr Leu Ala His Glu Val His Leu Phe Ser
95 100 105 Ser Gin Tyr Ser Phe His Val Pro Leu Leu Ser Ala Gin Lys Ser
110 115 120
Val Cys Pro Gly Pro Gin Gly Pro Trp Val Arg Ser Val Tyr Gin
125 130 135
Gly Ala Val Phe Leu Leu Thr Gin Gly Glu Gin Leu Ser Thr His
140 145 150
Thr Asp Gly He Ala His Leu Leu Leu Ser Pro Ser Ser Val Phe 155 160 165
Phe Gly Ala Phe Ala Leu
170 (14) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Lymphotoxin (murine)
(B) OTHER INFORMATION: Two blank residues designated by
"Xaa" are inserted after residue No. 4 of murine lymphotoxin and the sequence numbering is augmented by 2 starting with residue No. 5 in order to maximize the sequence homology with human lymphotoxin.
(X) PUBLICATION INFORMATION: (A) AUTHORS: Li, C-B., Gray, R.W. , Lin, P-F. , McGrath,
K.M. and Ruddle, F.H. , Ruddle, N.H. (B) TITLE: Cloning and Expression of Murine Lymphotoxin cDNA.
(C) . JOURNAL: J. Immunology
(D) VOLUME: 138
(E) PAGES: 4496-4501 (F) DATE: 1987
(G) RELEVANT RESIDUES IN SEQ ID NO: 13: 1-171 (includes two blanks) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Leu Ser Gly Val Xaa Xaa Arg Phe Ser Ala Ala Arg Thr Ala His
5 10 15 Pro Leu Pro Gin Lys His Leu Thr His Gly He Leu Lys Pro Ala
20 25 -30
Ala His Leu Val Gly Tyr Pro Ser Lys Gin Asn Ser Leu Leu Trp
35 40 45
Arg Ala Ser Thr Asp Arg Ala Phe Leu Arg His Gly Phe Ser Leu
50 55 60
Ser Asn Asn Ser Leu Leu He Pro Thr Ser Gly Leu Tyr Phe Val 65 70 75
Tyr Ser Gin Val Val Phe Ser Gly Glu Ser Cys Ser Pro Arg Ala
80 85 90 He Pro Thr Pro He Tyr Leu Ala His Glu Val Gin Leu Phe Ser
95 100 105
Ser Gin Tyr Pro Phe His Val Pro Leu Leu . Ser Ala Gin Lys Ser 110 115 120
Val Tyr Pro Gly Leu Gin Gly Pro Trp Val Arg Ser Met Tyr Gin
125 130 135
Gly Ala Val Phe Leu Leu Ser Lys Gly Asp Gin Leu Ser Thr His 140 145 * 150
Thr Asp Gly He Ser His Leu His Phe Ser Pro Ser Ser Val Phe
155 160 165 Phe Gly Ala Phe Ala Leu
170

Claims

What is claimed is:
1. A method for making a modified form of TNF-α having a reconstructed channel, as compared to unmodified TNF-α, for regulating channel activity, the method comprising the following steps:
Step A: forming one or more candidate forms of modified TNF-α by substituting one or more channel residues with replacement amino acids, the channel residues being selected from the group consisting of the following sequence numbers: residue #11; residue #57; residue # 59; residue #98; residue #112; residue # 116; residue #119; residue #121; residue #155; and residue #157; the sequence numbers being defined with respect to unmodified forms of human TNF-α, the replacement amino acid for residue #11 ' being selected from the group consisting of: Glu, Arg, Cys, Asp, Gin, Asn, Ser, Thr, and His; the replacement amino acid for residue #57 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Phe, and Tyr; the replacement amino acid for residue #59 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met,
Cys, and Phe; the replacement amino acid for residue #98 being selected from the group consisting of: Arg, Cys, Glu, Asp, Gin, Asn, Ser, Thr, and His; the replacement amino acid for residue #112 being selected from the group consisting of: Arg, Cys, Asp Gin, Asn, Ser, Thr, Glu and His; the replacement amino acid for residue #116 being selected from the group consisting of: Lys, Arg, Cys, Asp, Gin, Asn, Ser, His, and Thr; the replacement amino acid for residue #119 being selected from the group consisting of: Trp, Phe, Ser, Thr, Ala, Met, and Cys; the replacement amino acid for residue #121 being selected from the group consisting of: Ala, Val, Ser, and Thr; the replacement amino acid for residue #155 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Phe, and Tyr; and the replacement amino acid for residue #157 being selected from the group consisting of: Trp, Ser, Thr, Ala, Cys, and Tyr; Step B: determining whether the candidate form of TNF-α.has an ability to form TNF trimers;
Step C: determining whether the candidate form of TNF-α has an ability to achieve intimate contact with a target that includes both a membrane and one or more TNF receptors; Step D: determining whether the candidate form of TNF-α, when in intimate contact with the target, achieves a modified effect, the modified effect being of a type caused by a modified channel activity of the candidate form of TNF-α, the modified channel activity materially differing from corresponding unmodified channel activities of unmodified TNF-α; then
Step E: selecting the modified form of TNF-α from one or more of the candidate forms of TNF-α, the modified form of TNF-α having been determined in said Step B to be able to form TNF trimers, in said Step C to be able to achieve intimate contact with the target, and in said Step D to be able to achieve the modified effect by virtue of the modified channel activity; and then
Step F: making the modified form of TNF-α selected in said Step E in purified form and in commercial quantities.
2. A method for making a modified form of TNF-α as described in claim 1 wherein: in said Step A: the replacement amino acid for residue #11 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #57 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #59 . being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #98 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #112 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #116 being further selected from the group consisting of: Leu, He, and Ala; the replacement amino acid for residue #119 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #121 being further selected from the group consisting of: Pro, He, Leu, and His; the replacement amirio acid for residue #155 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; and . the replacement amino acid for residue #157 being further selected from the group consisting of: Arg, Glu, Lys, Asp, and Asn.
3. A method for making a modified form of TNF-α as described in claim 2 wherein: in said Step A: the replacement amino acid for residue #11 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, Phe, and Met; the replacement amino acid for residue #57 being further selected from the group consisting of: Gly, Val, He, His, and Pro; the replacement amino acid for residue #59 being further selected from the group consisting of: Gly, Val, Leu, He, His, and Pro; the replacement amino acid for residue #98 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #112 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, and
Phe; the replacement amino acid for residue #116 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #119 being further selected from the group consisting of: Gly, Val, He, Leu, and Pro; the replacement amino acid for residue #121 being further selected from the group consisting of: Trp, Tyr, Phe, Cys, Met, Lys, Glu, Arg, Gin, Asp, and Asn; the replacement amino acid for residue #155 being further selected from the group consisting of: Gly, Val, His, and Pro; and the replacement amino acid for residue #157 being further selected from the group consisting of: Gly, He, and His.
4. A method for making a modified form of TNF-α as described in claim 3 wherein: in said Step D: the modified effect achieved by the modified TNF-α with the target being of a type caused by a modified channel activity that is materially reduced as compared to the corresponding unmodified channel activity of unmodified TNF-α; and in said Step E: selecting as the modified form of TNF-α a candidate form of TNF-α that is further determined to achieve the modified effect by virtue of the modified channel activity that is materially reduced.
5. A method for making a modified form of TNF-α as described in claim 3 wherein:
in said Step D: the modified effect achieved by the modified TNF-α with the target being of a type caused by a modified channel activity that is materially enhanced as compared to the corresponding unmodified channel activity of unmodified TNF-α; and in said Step E: selecting as the modified form of TNF-α a candidate form of TNF-α that is further determined to achieve the modified effect by virtue of the modified channel activity that is materially enhanced. 6. A modified form of TNF-α having a reconstructed channel, as compared to unmodified TNF-α, for regulating channel activity, the modified form of TNF-α being constructed by the following steps:
Step A: forming one or more candidate forms of modified TNF-α by substituting one or more channel residues with replacement amino acids, the channel residues being selected from the group consisting of the following sequence numbers: residue #11; residue #57; residue # 59; residue #98; residue #112; residue #
116; residue #119; residue #121; residue #155; and residue #157; the sequence numbers being defined with respect to unmodified forms of human TNF-α, the replacement amino acid for residue #11 being selected from the group consisting of: Glu, Arg, Cys, Asp, Gin, Asn, Ser, Thr, and His; the replacement amino acid for residue #57 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Phe, and Tyr; the replacement amino acid for residue #59 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, and Phe; the replacement amino acid for residue #98 being selected from the group consisting of: Arg, Cys, Glu, Asp, Gin,
Asn, Ser, Thr, and His; the replacement amino acid for residue #112 being selected from the group consisting of: Arg, Cys, Asp, Gin, Asn, Ser, Thr, Glu, and His; the replacement amino acid for residue #116 being selected from the group consisting of: Lys, Arg, Cys, Asp, Gin, Asn, Ser, His, and Thr; the replacement amino acid for residue #119 being selected from the group consisting of: Trp, Phe, Ser, Thr, Ala, Met, and Cys; the replacement amino acid for residue #121 being selected from the group consisting of: Ala, Val, Ser, and Thr; the replacement amino acid for residue #155 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Phe, and Tyr; and the replacement amino acid for residue #157 being selected from the group consisting of: Trp, Ser, Thr, Ala, Cys, and Tyr; Step B: determining whether the candidate form of TNF-α has an ability to form TNF trimers; Step C: determining whether the candidate form of TNF-α has an ability to achieve intimate contact with a target that includes both a membrane and one or more TNF receptors;
Step D: determining whether the candidate form of TNF-α, when in intimate contact with the target, achieves a modified effect, the modified effect being of a type caused by a modified channel activity of the candidate form of TNF-α, the modified channel activity materially differing from corresponding unmodified channel activities of unmodified TNF-α; then Step E: selecting the modified form of TNF-α from one or more of the candidate forms of TNF-α, the modified form of TNF-α having been determined in said Step B to be able to form TNF trimers, in said Step C to be able to achieve intimate contact with the target, and in said Step D to be able to achieve the modified effect by virtue of the modified channel activity; and then Step F: making the modified form of TNF-α selected in said Step E in purified form and in commercial quantities.
7. A modified form of TNF-α as described in claim 6 wherein: in said Step A: the replacement amino acid for residue #11 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino- acid for residue #57 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #59 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #98 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #112 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #116 being further selected from the group consisting of: Leu, He, and Ala; the replacement amino acid for residue #119 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #121 being further selected from the group consisting of: Pro, He, Leu, and His; the replacement amino acid for residue #155 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; and the replacement amino acid for residue #157 being further selected from the group consisting of: Arg, Glu, Lys, Asp, and Asn.
8. A modified form of TNF-α as described in claim 7 wherein: in said Step A: the replacement amino acid for residue #11 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, Phe, and Met; the replacement amino acid for residue #57 being further selected from the group consisting of: Gly, Val, He, His, and Pro; the replacement amino acid for residue #59 being further selected from the group consisting of: Gly, Val, Leu, He, His, and Pro; the replacement amino acid for residue #98 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #112 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #116 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid- for residue #119 being further selected from the group consisting of: Gly, Val, He, Leu, and Pro; the replacement amino acid for residue #121 being further selected from the group consisting of: Trp, Tyr, Phe, Cys, Met, Lys, Glu, Arg, Gin, Asp, and Asn; the replacement amino acid for residue #155 being further selected from the group consisting of: Gly, Val, His, and Pro; and the replacement amino acid for residue #157 being further selected from the group consisting of: Gly, He, and His.
9. A modified form of TNF-α as described in claim 8 wherein: in said Step E: determining whether the candidate form of TNF-α has a channel activity that is materially reduced as compared to the corresponding channel activity of unmodified TNF- α and that is employable for reducing the channel activity of the target membrane; and in said Step F: designating as the modified form of TNF-α a candidate form of TNF-α that is determined to have a channel activity that is materially reduced with respect to the corresponding channel activity of unmodified TNF- α.
10. A modified form of TNF-α as described in claim 8 wherein: in said Step E: determining whether the candidate form of TNF-α has a channel activity that is materially enhanced as compared to the corresponding channel activity of unmodified TNF- α and that is employable for enhancing the channel activity of the target membrane; and in said Step F: designating as the modified form of TNF-α a candidate form of TNF-α that is determined to have a channel activity that is materially enhanced with respect to the corresponding channel activity of unmodified TNF- α.
11. A modified form of TNF-α comprising: a trimer of three monomers of modified TNF-α, two or more of the three monomers of modified TNF-α within said trimer being covalently bonded to one another by means of disulfide bonds.
12. A modified form of TNF-α as described in claim 11 wherein: the disulfide bond being formed under non-reducing conditions by cysteine molecules introduced by substitution into the modified TNF-α molecule, the cysteine substitutions being introduced into residues selected from the group consisting of the following sequence numbers: the pair of residues #98 and #116; the pair of residues #103 and #104; the pair of residues #11 and #157; and the single residue #119; the sequence numbers being defined with respect to unmodified forms of human TNF-α.
13. A method for making a modified form of TNF-3 having a reconstructed channel, as compared to unmodified TNF-3, for regulating channel activity, the method comprising the following steps: Step A: forming one or more candidate forms of modified TNF-/3 by substituting one or more channel residues with replacement amino acids, the channel residues being selected from the group consisting of the following sequence numbers: residue #28; residue #74; residue # 76; residue #119; residue #127; residue # 131; residue #134; residue #136; residue #169; and residue #171; the sequence numbers being defined with respect to unmodified forms of human
TNF-/3, the replacement amino acid for residue #28 being selected from the group consisting of: Glu, Arg, Cys, Asp, Gin,
Asn, Ser, Thr, and His; the replacement amino acid-for residue #74 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Leu, and Tyr; the replacement amino acid for residue #76 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met,
Cys, and Phe; the replacement amino acid for residue #119 being selected from the group consisting of: Arg, Cys, Glu, Asp, Gin,
Asn, Ser, Thr, and His; the replacement amino acid for residue #127 being selected from the group consisting of: Arg, Cys, Asp, Gin, Asn,
Ser, Thr, Lys, and His; the replacement amino acid for residue #131 being selected from the group consisting of: Lys, Arg, Cys, Asp,
Gin, Asn, Ser, Glu, and Thr; the replacement amino acid for residue #134 being selected from the group consisting of: Trp, Phe, Ser, Thr, Ala, Met, and Cys; the replacement amino acid for residue #136 being selected from the group consisting of: Ala, Val, Ser, and Thr; the replacement amino acid for residue #169 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, He, and Tyr; and the replacement amino acid for residue #171 being selected from the group consisting of: Trp, Ser, Thr, Ala, Cys,
Phe, and Tyr; Step B: determining whether the candidate form of
TNF-Θ has an ability to form TNF trimers; Step C: determining whether the candidate form of TNF-3 has an ability to achieve intimate contact with a target that includes both a membrane and one or more TNF receptors; Step D: determining whether the candidate form of TNF-/3, when in intimate contact with the target, achieves a modified effect, the modified effect being of a type caused by a modified channel activity of the candidate form of TNF-3, the modified channel activity materially differing from corresponding unmodified channel activities of unmodified TNF-/3; then
Step E: selecting the modified form of TNF-/3 from one or more of the candidate forms of TNF-/3, the modified form of TNF-3 having been determined in said Step B to be able to form TNF trimers, in said Step C to be able to achieve intimate contact with the target, and in said Step D to be able to achieve the modified effect by virtue of the modified channel activity; and then Step F: making the modified form of TNF-3 selected in said Step E in purified form and in commercial quantities.
14. A method for making a modified form of TNF-3 as described in claim 13 wherein: in said Step A: the replacement amino. acid for residue #28 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #74 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #76 being further selected from the group consisting"of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #119 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #127 being further selected from the group consisting of: Val, Leu, He, Met, and Ala; the replacement amino acid for residue #131 being further selected from the group consisting of: Leu, He, Val, and Ala; the replacement amino acid for residue #134 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, His, and Asn; the replacement amino acid for residue #136 being further selected from the group consisting of: Pro, He, Leu, and His; the replacement amino acid for residue #169 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, Leu, and Asn; and the replacement amino acid for residue #171 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin,. and Asn.
15. A method for making a modified form of TNF-3 as described in claim 14 wherein: in said Step A: the replacement amino acid for residue #28 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, Phe, and Met; the replacement amino acid for residue #74 being further selected from the group consisting of: Gly, Val, He, His, and
Pro; the replacement amino acid for residue #76 being further selected from the group consisting of: Gly, Val, He, Leu, His, and Pro; the replacement amino acid for residue #119 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #127 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #131 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #134 being further selected from the group consisting of: Gly, Val, He, Leu, and
Pro; the replacement amino acid for residue #136 being further selected from the group consisting of: Trp, Tyr, Phe, Cys, Met, Lys, Glu, Arg, Gin, Asp, and Asn; the replacement amino acid for residue #169 being further selected from the group consisting of: Gly, Val, His, and Pro; and the replacement amino acid for residue #171 being further selected from the group consisting of: Gly, Pro, Val, Met, He, and His.
16. A method for making a modified form of TNF- as described in claim 15 wherein: in said Step D: the modified effect achieved by the modified TNF-β with the target being of a type caused by a modified channel activity that is materially reduced as compared to the corresponding unmodified channel activity of unmodified TNF-/3; and in said Step E: selecting as the modified form of TNF-/3 a candidate form of TNF-/3 that is further determined to achieve the modified effect by virtue of the modified channel activity that is materially reduced.
17. A method for making a modified form of TNF-/3 as described in claim 15 wherein: in said Step D: the modified effect achieved by the modified TNF-/3 with the target being of a type caused by a modified channel activity that is materially enhanced as compared to the corresponding unmodified channel activity of unmodified TNF-/3; and in said Step E: selecting as the modified form of TNF- a candidate form of TNF-β that is further determined to achieve the modified effect by virtue of the modified channel activity that is materially enhanced.
18. A modified form of TNF-/3 having a reconstructed channel, as compared to unmodified TNF- , for regulating channel activity, the modified form of TNF-yS being constructed by the following steps: Step A: forming one or more candidate forms of modified TNF- by substituting one or more channel residues with replacement amino acids, the channel residues being selected from the group consisting of the following sequence numbers: residue #28; residue #74; residue # 76; residue #119; residue #127; residue # 131; residue #134; residue #136; residue #169; and residue #171; the sequence numbers being defined with respect to unmodified forms of human TNF-3, the replacement amino acid for residue #28 being selected from the group consisting of: Glu, Arg, Cys, Asp, Gin,
Asn, Ser, Thr, and His; the replacement amino acid for residue #74 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, Leu, and Tyr; the replacement amino acid for residue #76 being selected from the group consisting of: Trp, Ser, Thr, Ala, Met, Cys, and Phe; the replacement amino acid for residue #119 being selected from the group consisting of: Arg, Cys, Glu, Asp, Gin, Asn, Ser, Thr, and His; the replacement amino acid for residue #127 being selected from the group consisting of: Arg, Cys, Asp, Gin, Asn, Ser, Thr, Lys, and His; the replacement amino acid for residue #131 being selected from the group consisting of: Lys, Arg, Cys, Asp,
Gin, Asn, Ser, Glu, and Thr; the replacement amino acid 'for residue #134 being selected from the group consisting of: Trp, Phe, Ser, Thr, Ala, Met, and Cys; the replacement amino acid for residue #136 being selected from the group consisting of: Ala, Val, Ser, and Thr; the replacement amino acid for residue #169 being selected from the group. consisting of: Trp, Ser, Thr, Ala, Met, Cys, He, and Tyr; and the replacement amino acid for residue #171 being selected from the group consisting of: Trp, Ser, Thr, Ala, Cys,
Phe, and Tyr; Step B: determining whether the candidate form of
TNF- has an ability to form TNF trimers; Step C: determining whether the candidate form of TNF-β has an ability to achieve intimate contapt with a target that includes both a membrane and one or more TNF receptors; Step D: determining whether the candidate form of TNF-9, when in intimate contact with the target, achieves a modified effect, the modified effect being of a type caused by a modified channel activity of the candidate form of TNF-β, the modified channel activity materially differing from corresponding unmodified channel activities of unmodified TNF-β; then Step E: selecting the modified form of' TNF-3 from one or more of the candidate forms of TNF-/3, the modified form of TNF-3 having been determined in said Step B to be able to form TNF trimers, in said Step C to be able to achieve intimate contact with the target, and in said Step D to be able to achieve the modified effect by virtue of the modified channel activity; and then
Step F: making the modified form of TNF-/3 selected in said Step E in purified form and in commercial quantities.
19. A modified form of TNF-3 as described in claim 18 wherein: in said Step A: the replacement amino acid for residue #28 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #74 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #76 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn; the replacement amino acid for residue #119 being further selected from the group consisting of: Val, Leu, He, and Ala; the replacement amino acid for residue #127 being further selected from the group consisting of: Val, Leu, He, Met, and Ala; the replacement amino acid for residue #131 being further selected from the group consisting of: Leu, He, Val, and Ala; the replacement amino acid for residue #134 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, His, and Asn; the replacement amino acid for residue #136 being further selected from the group consisting of: Pro, He, Leu, and His; the replacement amino acid for residue #169 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin,
Leu, and Asn; and the replacement amino acid for residue #171 being further selected from the group consisting of: Arg, Glu, Lys, Asp, Gin, and Asn.
20. A modified form of TNF-0 as described in claim 19 wherein: in said Step A: the replacement amino acid for residue #28 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, Phe, and Met; the replacement amino acid for residue #74 being further selected from the group consisting of: Gly, Val, He, His, and Pro; the replacement amino acid for residue #76 being further selected from the group consisting of: Gly, Val, He, Leu, His, and Pro; the replacement amino acid for residue #119 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #127 being further selected from the group consisting of: Trp, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #131 being further selected from the group consisting of: Trp, Met, Gly, Pro, Tyr, and Phe; the replacement amino acid for residue #134 being further selected from the group consisting of: Gly, Val, He, Leu, and Pro; the replacement amino acid for residue #136 being further selected from the group consisting of: Trp, Tyr, Phe, Cys, Met,
Lys, Glu, Arg, Gin, Asp, and Asn; the replacement amino acid for residue #169 being further selected from the group consisting of: Gly, Val, His, and Pro; and the replacement amino acid for residue #171 being further selected from the group consisting of: Gly, Val, Met, He, and His.
21. A modified form of TNF-/3 as described in claim 20 wherein: in said Step E: determining whether the candidate form of TNF-/3 has a channel activity that is materially reduced as compared to the corresponding channel activity of unmodified TNF- β and that is employable for reducing the channel activity of the target membrane; and in said Step F: designating as the modified form of TNF-/3 a candidate form of TNF-/3 that is determined to have a channel activity that is materially reduced with respect to the corresponding channel activity of unmodified TNF- β -
22. A modified form of TNF-β as described in claim 20 - wherein: in said Step E: determining whether the candidate form of TNF-/3 has a channel activity that is materially enhanced as compared to the corresponding channel activity of unmodified TNF- β and that is employable for enhancing the channel activity of the target membrane; and in said Step F: designating as the modified form of TNF- a candidate form of TNF-/3 that is determined to have a channel activity that is materially enhanced with respect to the corresponding channel activity of unmodified TNF- β -
23. A modified form of TNF-/3 comprising: a trimer of three molecules of modified TNF- , each molecule of modified TNF-3 within said trimer being covalently bonded to the other two molecules therein by means of disulfide bonds, for holding said trimer together.
24. A modified form of TNF-/3 as described in claim 23 wherein: the disulfide bond is formed under non-reducing conditions by cysteine molecules introduced by substitution into the modified TNF-/3 molecule, the cysteine substitutions being introduced into residues selected from the group consisting of the following sequence numbers: the pair of residues #119 and #131; the pair of residues #117 and #135; the pair of residues #28 and #171; and the single residue #134; the sequence numbers being defined with respect to unmodified forms of human TNF-/3.
25. A method for regulating the permeability of a TNF target membrane, the method comprising: contacting the target membrane with a modified form . of TNF, the modified form of TNF having a reconstructed channel, as compared to unmodified TNF, for regulating channel activity.
26. A method for regulating the permeability of a TNF target membrane as described in claim 25 wherein: the modified form of TNF having a reduced channel activity as compared to the unmodified form of TNF and the insertion of the modified form of TNF serving to reduce the channel activity exhibited with the target membrane.
27. A method for regulating the permeability of a TNF target membrane as described in claim 25 wherein: the modified form of TNF having an enhanced channel activity as compared to the unmodified form of TNF and the insertion of the modified form of TNF serving to enhance the channel activity exhibited with the target membrane.
28. A method for inhibiting the binding of unmodified TNF to one or more TNF receptors attached to a target membrane, the method employing a modified form of TNF having a reconstructed channel for reducing channel activity within a target membrane, the method comprising: contacting the modified form of TNF with one or more of the TNF receptors under conditions for permitting binding between the modified form of TNF and the TNF receptor, the modified form of TNF having a reconstructed channel, as compared to unmodified TNF, for reducing channel activity.
PCT/US1993/002475 1992-03-12 1993-03-12 Tumor necrosis factor with modified channel activity WO1993018148A2 (en)

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US8742079B2 (en) 2007-08-20 2014-06-03 Protalix Ltd. Saccharide-containing protein conjugates and uses thereof
US9194011B2 (en) 2009-11-17 2015-11-24 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
US9708595B2 (en) 2009-11-17 2017-07-18 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
US10280414B2 (en) 2009-11-17 2019-05-07 Protalix Ltd. Stabilized α-galactosidase and uses thereof
US10870842B2 (en) 2009-11-17 2020-12-22 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
WO2011107992A3 (en) * 2010-03-02 2011-11-10 Protalix Ltd. Multimeric forms of therapeutic proteins and uses thereof
US9732333B2 (en) 2011-01-20 2017-08-15 Protalix Ltd. Nucleic acid construct for expression of alpha-galactosidase in plants and plant cells

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