NO304854B1 - Method of screening prophylactic agents or therapeutic agents for diseases caused by the production of mature tumor necrosis factor (TNF) hormone - Google Patents

Description

The present invention relates to a method for screening prophylactic agents or therapeutic agents for diseases caused by, aggravated by or associated with the production of mature tumor necrosis factor (TNF) hormone, or one or more lower molecular weight species of said mature protein hormone, which is produced from a TNF prohormone by convertase cleavage of said prohormone.

The present invention therefore relates to the field of immunology/biochemistry, and in particular the development of compositions and methods for the identification of inhibitors of protein-hormone formation and prophylactic and therapeutic uses of inhibitors for the treatment of diseases associated with elevated levels of hormones. The invention specifically facilitates the identification of compounds that can be used to treat various diseases, particularly sepsis, AIDS and autoimmune diseases, thereby providing the physician with alternative treatment regimens.

In the U.S. alone, nosocomial bacteremia develops in approximately 194,000 patients, and of these approximately 75,000 die. Maki, D.G., 1981, Nosocomial Infect., (Dikson, R.E., Ed.), page 183, Occupational Medical Books, U.S.A. Most of these deaths are due to six main gram-negative bacilli, and these are Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella, Enterobacter and Serratia. The current treatment for bacteremia is the administration of antibiotics, which unfortunately have limited effectiveness.

The exact pathology of bacteremia is not completely known, but it is known that bacterial endotoxins, lipopolysaccharides (LPS), are the primary causative agents. LPS consists of at least three significant antigenic regions, lipid A, the core polysaccharide and O-specific polysaccharide. The latter is also conditioned O-specific chain or only O-antigen. The O-specific chain region is a long-chain polysaccharide formed by repeating polysaccharide units. The number of polysaccharide units varies among different bacterial species, and can vary from one to as many as six or seven monosaccharide units. The O-specific chain varies among different gram-negative bacteria, while lipid A and the core polysaccharides are similar, if not identical.

Because LPS plays a major role in sepsis, various approaches have been attempted to neutralize its activity. At present, considerable work exists suggesting that antibody to LPS will soon be a valuable clinical adjunct to standard antibiotic therapy.

LPS initiates a cascade of biochemical events that ultimately cause the patient's death. It is believed that the second event, after the introduction of LPS, is the production of tumor necrosis factor (TNF) as a result of LPS stimulation of macrophage cells. Much work has been done to produce neutralizing antibodies against TNF or other molecules that can inhibit its septic effects. Antibodies to TNF are likely to have valuable clinical applications. Tracey, et al., 1987, Nature, 330-662.

TNF exists in both membrane-bound and soluble secreted forms. Decker, et al., 1987, J. of Immunol., 138-957; Kriegler, et al., 1988, Cell, 53-45. Human TNF has been cloned and shown to consist of a 17 kd polypeptide, plus an unusually long 76 amino acid putative signal leader sequence. The 17 kd molecule is a key agent involved in the initiation of the biochemical cascade responsible for sepsis. It has been suggested by Kriegler, et al., 1988, Cell, 53-45, that TNF may exist as both a membrane-bound 26 kd form and a soluble form corresponding to the 17 kd species. The 26 kd form is the precursor, or prohormone, to the mature 17 kd molecule. It has further been suggested by Kriegler, et al. above, that the two forms of TNF can have different biological effects.

Because TNF plays a key role in the development of sepsis, it is necessary to identify and develop anti-TNF prophylactic/therapeutic agents. As mentioned above, anti-TNF antibody appears promising and has been shown to be effective in baboons. However, these studies have included the use of non-human TNF and non-human TNF antibody. From a practical standpoint, non-human anti-TNF antibody will have limited therapeutic application, due to immunological rejection of the antibody by the patient's immune system. Human antibody, or genetically engineered antibody consisting of the human constant region and the murine variable region, is therefore preferred.

TNF, in addition to playing a critical role in sepsis, has recently been shown to be involved in the initiation of the expression of the human immunodeficiency virus in human cells that carry the virus latently. Folks et al., Proe. Nati. Acad. Pollock. United States, vol. 86, pp. 2365 (1989). Therefore, preventing or inhibiting the formation of 17 kd or lower molecular weight forms of TNF will be valuable prophylactically for the treatment of AIDS patients by preventing the virus expression that is latent in the patient.

TNF also plays a role in various -autoimmune diseases, especially arthritis. Duff, et al., 1987, International Conference on Tumor Necrosis Factor and Related Cytotoxins, vol. 175:10. Compounds or methods for inhibiting the TNF effect will therefore be used for the treatment of various diseases of immunological origin.

In addition to antibodies, other molecules with TNF inhibitory activity are being sought. Non-antibody TNF inhibitors are described by Seckinger, et al., 1988, J. Exp. Med., 167-151, and Seckinger, et al., 1989, J. Biol. Chem. 264-11966 and in European Patent Application No. 88830365.8, inventors Wallach, et al. The inhibitors are present in the urine of febrile patients and have been purified and shown to have molecular weights of approximately 27,000-33,000. Until now, no inhibitors have been shown to be effective for the treatment of sepsis.

From the above, it appears that it is necessary to identify and develop additional anti-TNF inhibitors, both antibody-based and other, which can be effectively used for the treatment of sepsis.

The present invention therefore relates to a method for screening prophylactic agents or therapeutic agents for diseases caused by, aggravated by or associated with the production of mature tumor necrosis factor (TNF) hormone, or one or more lower molecular weight species of said mature protein hormone, which is produced from a TNF prohormone by convertase cleavage of said prohormone,

characterized in that it includes the following steps:

a) contacting said TNF prohormone with an effective amount of said convertase; b) measuring the conversion of said TNF prohormone to said TNF mature hormone or one or more lower molecular weight species of said mature hormone; c) repeating steps (a) and (b) above, further comprising a molecule believed to be identified as a prophylactic or therapeutic disease caused by, aggravated by, or associated with production of said mature TNF hormone; d) measuring conversion of said TNF prohormone to said mature TNF hormone or lower molecular weight species in step (c); and e) comparing the amount of conversion of said prohormone from steps (b) and (d), wherein a smaller

cleavage in step (d) than in step (b) suggests that the molecule according to step (c) is potentially a

prophylactic agent or therapeutic agent for a disease caused by, aggravated by or associated with the production of mature TNF hormone or one or more lower molecular weight species of mature TNF hormone.

The purposes according to the invention will become apparent after a consideration of the detailed description of the invention shown below. Figure 1. panel A, shows the restriction map of the DNA sequence encoding 26 kd TNF. Panel B shows a hydrophobicity plot of 26 kd TNF, and panel C shows the DNA and amino acid sequences of the molecule. Figure 2 shows the transformation of 26 kd TNF by TNF convertase. Columns (A), (B) and (C) show different controls: TNF 6.8 cell lysate (A), 26 kd transcription/translation (B) and incubation (C) controls. Columns D, E and F show the conversion of transcribed/translationally generated 26 kd TNF to predominantly 17 kd TNF by the convertase either present in the HL 60 S-1 cytosol uninduced (D) and induced (E) fractions, or a P-1 pellet fraction prepared from induced cells . G is a blank column. Figure 3 shows the effect of the convertase inhibitors on the conversion of 26 kd TNF to the lower molecular weight forms, determined by gel electrophoresis. Columns A, B, C and D, according to panel 1, respectively show immunoprecipitation of a cell lysate of pFVXM-TNF6 transfected cell line TNF 6.8 (Kriegler, et al., 1988, in Cell, 53-45), immunoprecipitation of in vitro transcribed/translated 26 kd TNF, the action of (l-((3-((acetyloxyl )-7-methoxy-8-oxy-8-oxo-5-thio-l-azabicyclo [4,2,0]oct-2 -en-2-yl)carbonyl)morpholine, S,S-dioxide, (6R-cis) on the conversion of 26 kd TNF, and the conversion of 26 kd TNF in the absence of (l-((3-((acetyloxy)- 7-Methoxy-8-oxy-8-oxo-5-thio-1-azabicyclo[4,2,0]oct-2-en-2-yl)carbonyl)morpholine, S,S- dioxide, (6R-cis ).Columns A and B according to panel 2 show, respectively, immunoprecipitation of a cell lysate of the pFVXM-TNF6-transfected cell line TNF 6.8 (Kriegler, et al., 1988, in Cell, 53-45), and immunoprecipitation of in vitro transcribed/translated 26 kd TNF Columns C and D show conversion of 26 kd TNF in the presence and absence of 3,4-dichloroisocoumarin Columns E and F show conversion of 26 kd TNF in the presence and absence of elas tinal.

To facilitate understanding of the nature and scope of the present invention, several definitions regarding various aspects of the invention are presented below. It appears that these definitions are general in nature, and include meanings well known to those skilled in the art.

The terms "prohormone" and "mature hormone" have the following meanings. Prohormone should cover proteins, preferably of immunological origin, where a peptide segment of the protein has been removed during its in vivo production. Removal of the peptide provides the "mature" form of the hormone. The preferred embodiment of the invention is the 26 kd TNF prohormone, as discussed below, which is cleaved predominantly to a 17 kd mature form. Other cleavage products, on the other hand, are also formed from the prohormone, and these fall under the meaning of "mature" hormone. Finally, it is important to note that prohormones in addition to TNF should come within the scope of these definitions, and are considered part of the invention. Examples of prohormones are CSF and IL-1.

Sepsis is defined herein to mean a disease resulting from a gram-positive or gram-negative bacterial infection, the latter being mainly caused by bacterial endotoxin, lipopolysaccharide (LPS). It can be induced by at least six main gram-negative bacilli and these are Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella, Enterobacter and Serratia. TNF is a factor that is detectable in the early phase of the disease and that contributes to this process. The 17 kd molecule, or a shorter mutein known in this area, are the primary, active forms.

As used herein, TNF, which has a molecular weight of approximately 26,000, refers to the prohormone form of TNF. The amino-terminal peptide of the prohorm is known to vary in length depending on the species from which it is derived, while the propeptide segment of the molecule is highly conserved. In mice, approximately Sb% of the 79 amino acids that form the putative leader sequence of the prohormone are identical to the 79 known amino acids that comprise the prosequence of human TNF. It will be apparent to those skilled in the art that when reference is made below to TNF, which has a molecular weight of approximately 26,000, it will be known that what is being indicated is a molecule which is not derived from a particular species, and which may have a somewhat altered leader sequence compared to that the human sequence known in this area.

The term "convertase" or "TNF convertase" shall include an enzyme which is normally present in the body and which is responsible for the cleavage of 26 kd TNF into one or more species with a lower molecular weight. Convertase is essentially membrane-associated, despite the fact that significant activity is located in the cytosol. Another characteristic of convertase is that it is mainly associated with cells that produce TNF.

The term "membrane associated" as applied to TNF convertase denotes a form of the convertase that is substantially insoluble as indicated by the presence of most of the convertase activity in a 30,000 xg pellet fraction.

"Recombinant antibody" refers to antibody in which a portion of each of the heavy and light chain amino acid sequences is homologous to the corresponding sequence in the antibody

derived from a particular species, or belonging to a particular class, while the remaining segment of the chains are homologous to corresponding sequences in another. In a recombinant antibody, the variable region of both light and heavy chains is a mirror image of the variable regions of the antibody derived from one mammalian species, while the constant regions are homologous to the sequences in antibody derived from another.

In its most general form, the present invention relates to methods for identifying inhibitors of diseases associated with the production of mature hormones from their prohormone forms. The preferred embodiment of a prohormone is 26 kd TNF, which is cleaved into molecules of lower molecular weight, especially those having a molecular weight of 17,000, which are significantly involved in the formation of sepsis. Inhibitors that can interfere with the conversion of the 26 kd form to TNF are useful for the prevention or treatment of sepsis.

The assays described herein detect the conversion of a prohormone to its mature hormone form, the preferred embodiment being the enzymatic conversion of the 26 kd molecular weight form of TNF to preferably a 17,000 molecular weight form. The enzyme responsible for the conversion is called convertase. The invention is thus most easily presented in four parts. Part one shows the materials and methods for realizing the 26 kd form of TNF. Part two identifies the sources of TNF convertase, and methods for partial purification of the enzyme. Part three describes analyzes for the detection and identification of different convertase inhibitors. Finally, part four provides a description of ways of using the inhibitors to treat patients suffering from sepsis. Each of these parts will now be described separately.

Several patents/patent applications and scientific references are referred to below. The present invention is based on some of the materials and methods mentioned in these references, and these references are therefore incorporated by reference in their entirety.

It is important to note that any procedure that allows measuring the conversion of the 26 kd TNF species can be used to identify inhibitors of TNF. Although the recombinant systems described below make the 26 kd molecule obtainable in considerable quantities, and therefore facilitate assays for TNF inhibitors, it is noted that non-recombinant systems can also be used. It has further been shown that the 26 kd molecule can be identified in stimulated monocytes. This is described by Kriegler, et al., 1988, Cell, 53:45. A suitable assay procedure is to stimulate monocytes to produce the 26 kd molecule, and then, in the presence of convertase, measure the decay of the molecule to a lower molecular weight species, preferably the 17,000 molecular weight species.

With regard to the transformation of the 26,000 molecule, the convertase, which appears below, the molecule is cleaved at an internal site, and perhaps several. The main seat is at the boundary separating the secreted form of TNF, i.e. the 17,000 molecular weight species from the leader sequence. The sequence at this boundary is -Gln-Ala-Val-Arg-Ser-Ser-. The major cleavage occurs between alanine and valine, since valine is known to be the amino-terminal amino acid of the molecule with a molecular weight of 17,000. Several other species of TNF are produced by convertase, and these are thereby the products of the secondary cleavage sites. Regardless of whether 26,000 molecules are cleaved at one or more sites, this is, in terms of the identification of inhibitors for sepsis, of great importance because the present assay can record either the inhibition of the conversion of the 26,000 TNF form or the appearance of a form with lower molecular weight.

TNF, either the 26 kd or 17 kd forms, has been cloned and expressed in a number of systems. For example, the cloning of rabbit TNF is described in EP 146,026, published June 26, 1985 (Dainippon Pharmaceutical Co., Ltd.) and EP 148,311, published July 17, 1985 (Asahi Kasei Kogyo Kabushiki). The cloning of human TNF with 151 and 155 amino acids (2 and 6 less than the native form) is described in EP 155,549, published September 25, 1985 (Dainippon Pharmaceutical Co., Ltd.), and human TNF having 155 amino acids is described in EP 158,286, published October 16, 1985 (Asahi Kasei Kogyo Kabushiki Kaisha) and corresponding GB 1,158,829A, published November 20, 1985. The cloning of mature TNF (157 amino acids) and various modified forms (mutines) thereof is described in EP 168,214, published Jan. 15, 1986 (Genentech) and PCT US 85/01921, filed Oct. 3, 1985, (Cetus Corporation). The latter, PCT 85/01921, corresponds to WO 86/02381.

In addition, the U.S. shows patent no. 4,677,063 and 4,677,064 cDNA sequences encoding 26,000 and 17,000 forms of TNF, as well as muteins of these molecules.

The cDNA sequence encoding the 26 kd TNF species is preferably obtained from plasmid pBll, described in WO 86/02381; and the U.S. patents Nos. 4,677,063 and 4,677,064. The plasmid pBll contains the SV40 promoter operably linked to the TNF coding sequence, and is therefore useful for expression of the 26 kd TNF species in eukaryotic host cells. Another plasmid containing the entire sequence encoding 26 kd TNF, the species is additionally described in the above-mentioned U.S. Pat. patent application and patents. It is designated pE4. The plasmid pE4 has been deposited with the American Type Culture Collection, accession no. 39894.

The cDNA sequence encoding the 26 kd TNF species is present in plasmid pBll as a Pstl fragment. It is therefore easily removed and injected into any of a number of suitable expression systems. The preferred expression system is

plasmid pFVXM, which has been deposited with the American Type Culture Collection and has accession no. 67.103.

pFVXM is a retroviral vector that was derived from the plasmid pEVX, described by Kriegler, et al., 1984, in Cell, 38:483. pEVX has a Moloney murine leukemia virus-derived splice donor site 3' to 5' long terminal repeat. It was previously shown that this splice donor sequence reduces the yield of correctly spliced translation templates from retroviral constructs. pEVX was therefore constructed to remove the splice donor site, and replace it with an analogous Sma I fragment from the Harvey murine sarcoma virus genome, which lacks the Moloney murine leukemia virus splice donor sequence. The resulting vector, pFVXM, lacks the Moloney murine leukemia virus spliced donor sequence, and carries a viral packaging sequence. pFVXM has an appropriate Pst I site into which the DNA sequences encoding the 26 kd TNF species can be inserted.

Various biological materials are available as sources of convertase activity. These include tissues, cells or extracts, or fluids associated therewith, which are preferably, but not necessarily, of immunological origin. Furthermore, established cell lines can also be used. Suitable sources include human peripheral blood nuclear cells such as leukocytes or cell lines of leukocyte origin, preferably the cell line HL60. Due to ease of manipulation of established cell lines, preferred convertase source is HL60. Conversion of the 26 kd TNF species to the 17 kd species can be achieved by combining the 26 kd species with either intact HL60 cells, extracts derived therefrom or media in which the HL60 cells were grown, and which therefore contain convertase activity. In some cell types, convertase activity is present in the culture medium after appropriate stimulation as discussed below. Because the convertase activity is partly membrane-associated, it is possible to obtain a membrane fraction that can be used. The procedures for isolating monocytes are well known in the art, and there are also other methods for cultivating cell lines such as HL60. Monocytes can be prepared from peripheral blood by centrifugation first through Ficoll-paque and percoll ($49.2) using standard procedures. This provides an enriched population of monocytes and lymphocytes, and the monocytes can be further enriched by seeding the mixture of cells onto tissue culture dishes and incubating the cells for a time sufficient to allow the monocytes to adhere to the surface of the dishes. The lymphocytes are then washed off the dishes, leaving only adherent monocytes. These cells can then be used as is, or can be stimulated to produce higher levels of convertase using known monocyte activators, preferably lipopolysaccharide and phorbol myristate acetate. The cells can be fractionated, and either an extract or a membrane fraction can be prepared from them and used in the analyzes described below.

Inhibitors of convertase activity can also be prophylactic agents or therapeutic agents that can be used for the treatment of sepsis. They can be identified using the above assay, and further included in the assay reaction mixture compounds to be tested for inhibitory activity. A suitable assay consists of combining 26 kd TNF, convertase and a putative inhibitor. Those skilled in the art will appreciate that the inhibitory material may be added to the convertase before the convertase is added to TNF, or it may be added to TNF before, or immediately after, addition of the convertase. The order of addition may facilitate the identification of the inhibitors, but is not essential. If a compound has inhibitory activity, this can be seen by electrophoretic analysis of the solution, which will show a reduction in the amount of the 26 kd species, and the presence of TNF molecules with a lower molecular weight.

In addition to the identification of compounds with unsuspected anti-convertase activity, several compounds will have strong inhibitory activity, such as anti-convertase antibody, either polyclonal or monoclonal, or recombinant antibody, preferably humanized. Monoclonal antibody against convertase can be prepared using the general methods described by Kohler, G. and Milstein, C, 1975, Nature, 256-495, which have been modified in recent years as known in the art. These original studies involved the condensation of murine lymphocytes and drug-selectable plasmacytomas to produce hybridomas. The technique has therefore been used to produce hybrid cell lines that secrete human monoclonal antibodies. The latter procedures are generally described in Abrams, P., 1986, Methods in Enzymology, 121:107, but other modifications are known in the art. Regardless of whether murine or human antibody is produced, the cells that secrete the antibody are combined with the fusion partner and the cells are condensed with a suitable condensing agent, preferably polyethylene glycol, and more preferably polyethylene glycol 1000. The latter is added to a cell pellet containing antibody-secreting cells and the fusion partner in small amounts above a short period of time followed by gentle agitation. After addition of the fusion agent, the cell mixture is washed to remove the fusion agent and any cellular debris, and the cell mixture consisting of condensed and uncondensed cells is seeded into appropriate cell culture chambers containing selective growth medium. After a period of several weeks, hybrid cells are visible and can be identified for antibody production, and subcloned, to ensure the availability of a stable hybrid cell line.

The preferred antibody is human monoclonal antibody which can be produced from lymphocytes sensitized with convertase, either in vivo or in vitro, by immortalizing antibody-producing hybrid cell lines, thereby making a permanent source of the desired antibody available. In vivo immunization techniques are well known in the art, while in vitro techniques are generally described by Luben, R. and Mohler, M., 1980, Molecular Immunology, 17:635, Reading, D. Methods in Enzymology, 121 (part one): 18, or Voss, B., 1986, Methods in Enzymology, 121:27. A number of in vitro immunization systems have been shown to be effective for sensitization of human B cells. Reading, C, 1982, J. of Immun, Methods, 53:261.

It will be apparent to those skilled in the art that in view of immunizing individuals directly with convertase, lymphocytes may be isolated from individuals undergoing, or having undergone, a bacteremic attack. A fraction of these lymphocytes will be sensitized to the convertase, and can be used to produce permanent antibody-secreting hybrid cell lines. Immunocompromised human patients are e.g. generally susceptible to bacterial infections, especially those suffering from various malignancies, extensive burns, etc., and lymphocytes isolated therefrom may be a source of antibody-secreting cells.

Sensitized lymphocytes can be immortalized by viral transformation. The preferred viral transformation technique for human lymphocytes involves the use of Epstein-Barr virus. The virus can transform human B cells, and has been used to generate human monoclonal antibodies. Crawford, D. et al., 1983, J. of General Virology, 64:697; Kozbor, V. and Roder, J., 1983, J. Immun. Today, 4:72.

Another method by which sensitized lymphocytes can be immortalized consists of a combination of the above two techniques, which are viral transformation and cell fusion. The preferred combination consists of transforming antibody-secreting cells with Epstein-Barr virus, and then condensing the transformed cells into a suitable fusion partner. The fusion partner can be a mouse myeloma cell line, a heteromyeloma line or a human myeloma line or another immortalized cell line. PCT Patent Application No. 81/00957; Schlom et al., 1980, PNÅS USA, 77:6841; Croce et al., 1980, Nature, 288:488. The preferred fusion partner is a mouse-human heterohybrid, and more preferably the cell line is designated F3B6. This cell line has been deposited in the American Type Culture Collection, Accession No. HB8785. It was deposited on April 18, 1985. The methods for the formation of F3B6 are described in European Patent Application Publication No. 174.204.

Techniques that can be used for the application of Epstein-Barr virus transformation and the production of immortal antibody-secreting cell lines are presented by Roder, J. et al., 1986, Methods in Enzymology, 121:140. The procedure essentially consists of isolating Epstein-Barr virus from a suitable source, usually an infected cell line, and exposing target antibody-secreting cells to supernatants containing the virus. The cells are washed and cultured in an appropriate cell culture medium. Thereafter, virally transformed cells present in the cell culture can be identified by the presence of Epstein-Barr viral core antigen, and transformed antibody-secreting cells can be identified using standard methods known in the art.

It is to be noted that since the preferred embodiment of the present invention is the neutralization of anti-convertase monoclonal antibody, individually or in combination, the antibody(s) can be changed and still maintain biological activity. Included within the scope of the invention are therefore antibodies modified by reduction to fragments of different sizes, such as F(ab')2, Fab, Fv, etc. The hybrid cell lines that produce the antibody can also be considered as a source of DNA encoding the desired antibody that can be isolated and transferred into cells by known genetic techniques, to produce genetically engineered antibody. An example of the latter would be the production of single-chain antibody that has the antibody-combining site of the hybridomas described herein. Single chain antibody is described in U.S. Pat. Patent No. 4,704,692. Another example of genetically engineered antibody is recombinant or chimeric antibody. Recombinant antibody methods are shown in U.S. Pat. Patent No. 4,816,567, inventor Cabilly, et al.; Japanese Patent Application Serial No. 84169370, filed August 15, 1984; British Patent Application No. 8422238, filed on September 3, 1984 and Japanese Patent Application No. 85239543, filed on October 28, 1985. British Patent Application No. 867679, filed on March 27, 1986, also describes methods for the production of a modified antibody, wherein at least a portion of the complementary determining regions (CDRs) in the variable domains of light or heavy chains have been replaced by analogous portions of the CDRs from an antibody with a different specificity. Using the procedures described therein, it is possible to construct recombinant antibody having the CDR region of one species grafted onto the antibody of another species that has replaced the CDR region. The preferred embodiment in this case is a murine anti-convertase antibody CDR region that replaces the human antibody CDR region.

In addition to antibody, compounds that compete with 26 kd TNF for binding to convertase will inhibit or reduce the conversion of 26 kd TNF to the 17 kd form, and therefore be useful for drugs to treat sepsis. One such class of reagents consists of peptides or proteins, or other synthetic or naturally occurring compounds, which have similar convertase binding activity to the 26 kd form of TNF. Preferred within this class are peptides or proteins having amino acid sequences similar to those found at the boundary between the 26 kd TNF and the 17 kd species. This sequence is Gln-Ala-Val-Arg-Ser-Ser-Ser, where Ala is the residue present on the part of TNF that remains in the membrane after cleavage, and Val is the N-terminal amino acid of 17 kd TNF. It is important to note that the sequence shows that it consists of 7 amino acids, the smallest of which is a dipeptide sequence that is recognized by the convertase and is Ala-Val. Because the dipeptide sequence is required as part of an inhibitory peptide or protein, the latter may also have additional amino acids that enhance, or are necessary for, the dipeptide sequence to bind to the convertase and inhibit its enzymatic activity.

An alternative embodiment of a peptide/protein convertasein inhibitor is one having the amino acid sequence, a sequence functionally similar to: Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys- Pro-Val-Ala. This peptide covers both convertase cleavage sites, and will therefore prevent the formation of the 17 kd form of TNF and lower molecular weight forms. The first and dominant cleavage site is between alanine and valine at positions -1 and +1; and the second site is between proline and valine at positions +12 and +13. These positions correspond to the amino acid sequence shown in Figure 1.

Another class of competitive inhibitors consists of the compounds having the sequence shown above, i.e. Gln-Ala-Val-Arg-Ser-Ser-Ser or Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro -Ser-Asp-Lys-Pro-Val-Ala, but where the amino acids have been mutated or deleted to provide a non-cleavable substrate. A preferred embodiment of this peptide is a 26 kd non-cleavable TNF mutein, produced by standard site-specific mutagenesis techniques. Most preferred is a mutein in which alanine and/or valine is substituted or deleted.

The peptides described above can be prepared by techniques well known in the art, such as e.g. The Merrifield solid phase method described in Science, 232:341-347

(1985). The method can use commercially available synthesis equipment, such as a Biosearch 9500 automated peptide machine, where the cleavage of the blocked amino acids is achieved with hydrogen fluoride, and the peptides are purified by preparative HPLC using a Waters Delta Prep 3000 instrument, on a 15-20 pm Vydac C4 PrepPAK column.

Furthermore, it is important to be aware of the fact that the specificity of the convertase is similar to enzymes such as elastase, i.e. which preferentially cleave between neutrally charged amino acids such as between valine, proline and alanine residues. In addition to the peptide inhibitors mentioned above, various other inhibitors known to inhibit elastase will therefore also generally inhibit the enzyme that cleaves TNF. By using the assay described below, those compounds which inhibit the convertase can be identified. Various elastase inhibitors are commercially available, see for example Boehringer Mannheim Biochemicals catalogues, or are known in the field. Doherty, et al., 1986, Nature, 322:192; U.S. Patent Nos. 4,711,886, 4,797,396, 4,717,722 and 4,699,904. Preferred elastase inhibitors are modified cephalosporin antibiotics, as shown by Doherty et al., supra. More preferred is (1-((3-((acetyloxy)-7-methoxy-8-oxy-8-oxo-5-thio-1-azabicyclo[4.2.0]oct-2-en-2-yl)- carbonyl )morpholine, S,S-dioxide, (6R-cis) Also, Stetler et al., 1986, Nucleic Acids Research, 14:7883, describes a cDNA clone encoding an inhibitor of neutrophil elastase.

Most of the recombinant techniques described herein can be used to transform cells, prepare vectors, extract messenger RNA, etc., and are used in biotechnology, and most practitioners of these techniques are familiar with the standard materials and methods used. . The following paragraphs are set out as guidelines. Construction of suitable vectors containing the desired TNF coding sequence utilizes standard ligation and restriction techniques which are well known. Isolated vectors, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated in the desired form.

Site-specific DNA cleavage is performed by treatment with the appropriate restriction enzyme, under conditions generally known in the art, and details specified by the manufacturer of these commercially available restriction enzymes. See e.g. New England Biolabs, Product Catalog. Generally, approximately 1 pg of plasmid or DNA sequence is digested with one enzyme unit in approximately 20 µl of buffer solution. In the examples herein, an excess of restriction enzyme is usually used to ensure complete cleavage of the DNA substrate. Incubation times of about one hour to two hours at about 37°C may be used, although variations may be tolerated. After each incubation, protein is removed by phenol/chloroform extraction, and may be followed by ether extraction, and the nucleic acid removed from the aqueous the fractions by precipitation with ethanol followed by chromatography using a Sephadex G-50 spin column. If desired, size separation of the cleaved fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations can be found in Methods Enzymology, 1980, 65:499-560.

Restriction cleaved fragments can be blunt-ended by treatment with the large fragment of E.coli DNA polymerase I, i.e., the Klenow fragment, in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of approximately 15 to 25 minutes at 20 to 25°C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 10 mM dNTP. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with Sl nuclease results in hydrolysis of single-stranded parts.

Ligations are performed in 15-30 µl volumes under the following standard conditions and temperatures: 20mM Tris-Cl pH 7.5, 10

mM MgCl2, 10 mM DTT, 33 pg/ml BSA, 10 mM-50 mM NaCl and 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 4°C for

"sticky end" ligation, or for "butt end" ligation. Intermolecular "sticky end" ligations are typically performed at 33-100 pg/ml total DNA concentration. In butt-end ligations, the total DNA concentration of the ends is approximately 1 pM.

When constructing vectors using "vector fragments", the vector fragment is usually treated with bacterial alkaline phosphatase (BAP) to remove the 5'-phosphate and prevent religation of the vector. BAP platings are carried out at pH 8 in about 150 mM Tris, in the presence of Na+ and jflg+2 using about 1 unit of BAP per pg vector at 60°C for approximately 1 hour. The nucleic acid fragments are isolated by extracting the preparation with phenol/chloroform, followed by ethanol precipitation. Alternatively, religation can be prevented in vectors that have been double-cleaved by further restriction enzyme cleavage of the unwanted fragments.

In the constructs indicated below, correct ligations are confirmed by first transforming the appropriate E. coli strain with the ligation mixture. Successful transformants are selected by resistance to ampicillin, tetracycline or other antibiotics, or by the use of other markers depending on the method of plasmid concentration, which appears in this area. Miniprep DNA can be prepared from transformants by the method of D. Ish-Howowicz et al., 1981, Nucleic Acids Res., 9:2989, and analyzed by restriction and/or sequenced by the dideoxy method of F. Sanger et al., 1977, Pro. Nati. Acad. Pollock.

(USA), 74:5463, as further described by Messing et al., 1981, Nucleic Acids Res., 9:309, or by the method of Maxam et al., 1980 Methods in Enzymology, 65:499.

Host strains used for cloning in M13 consist of E.coli strains susceptible to phage infection, such as E.coli K12 strain DG98 is used. The DG98 strain has been deposited with ATCC, July 13, 1984, and has accession number 1965.

Depending on the host cell used, the transformation is performed using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described by S.N. Cohen, 1972 Proe. Nati. Acad. Sei (USA) 69:2110, or the RbCl 2 method described by Maniatis et al., 1984, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor press, p. 254, can be used for prokaryotes. Transfection can also be achieved using a modification of the calcium phosphate precipitation technique of Graham, F.L. et al., 1973 Virology 52:456 or Wigler et al., 1978, Cell, 14:725.

Synthetic oligonucleotides were prepared by the triester method of Matteucci et al., 1981, J. Am Chem. Soc. 103:3185, or using commercially available oligonucleotide synthesis equipment. Kinase treatment of single strands before fusion or for labelling, is achieved by applying an excess, preferably about 10 units of polynucleotide kinase to 0.1 nmol of substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 1- 2 mM ATP, 1.7 pmol gamma<32>P-ATP (2.9 mCi/mmol), 0.1 mM spermidine, 0.1 mM EDTA.

Mutagenesis can be accomplished using any number of procedures known in the art. These techniques are described by Smith, 1985, Annual Review and Genetics, 19:423, and modifications of some of the techniques are described in Methods in Enzymology, 154, Part E, (eds.) Wu and Grossman (1978), cat. 17, 18, 19 and 20. The preferred procedure is a modification of the Duplex site-directed mutagenesis method. The general procedure is described by Kramer et al., in Chapter 17 of Methods in Enzymology, supra.

Conventional M13 mutagenesis methods involve fusing a short, synthetic oligonucleotide to single-stranded M13 DNA, which has a cloned target coding sequence believed to be mutagenized. The oligonucleotide is nearly, but not completely, complementary to the target sequence, and has at least one mismatched nucleotide. After the fusion reaction, the remaining portion of single-stranded DNA must be filled in to provide heteroduplex DNA that can be transfected into a suitable host cell, enabling expression of the mutation. In the gap-holding duplex method, a partial DNA duplex is constructed that has only the target region exposed, unlike the conventional methods that have the target region and the rest of the single-stranded M13 DNA exposed. Like the conventional methods, a short oligonucleotide is fused to the target region and extended and ligated to produce a heteroduplex. Because only a small portion of the single-stranded DNA is available for hybridization in the gapped duplex method, the oligonucleotide is not fused to unwanted sites within the M13 genome. This method also has the additional advantage of introducing few errors during the formation of the heteroduplex, due to the fact that only a small region of DNA on one side of the target region needs to be filled.

The gapped duplex method specifically involves cloning the target DNA sequence into an appropriate M13 phage containing selectable markers, such as e.g. the stop codon amber mutation. The latter enables negative selection in a host cell that cannot suppress the effects of the mutation. The phage is preferably M13mp9 which contains two amber codons in the critical phage genes. The sequence encoding 26 kd TNF is therefore cloned into M13mp9 amber+, and single-stranded DNA is prepared therefrom using standard techniques. Next, double-stranded replicative form DNA from M13 GAP, a genetically engineered M13 derivative lacking the amber codons, is cleaved with the Hine II restriction enzyme. The base sequence of M13 GAP is similar to M13ml8, which lacks both the amber codons and the sequence between base pairs 6172 and 6323. This deletion flanks the multiple cloning sites of the M13mp series and forms a unique Hine II site. Gap-containing duplex DNA is generated using standard DNA/DNA hybridization techniques, consisting of single-stranded DNA that has the amber codons and a second strand of DNA from the Hine II cleaved M13 GAP that lacks both the amber codons and TNF-coding sequences. Thus, the only part of the duplex with GAP that is exposed is the 26 kd TNF target sequence. The desired oligonucleotide is fused to GAP-containing duplex DNA, and any remaining gaps are filled in with DNA polymerase and the nicks sealed with DNA ligase to form a heteroduplex. The latter is transfected, preferably in a mismatched repair-deficient host, and mixed phage are formed. From the mixing phage population, phage containing unmutated 26 kd TNF DNA, which also have the amber mutations, can be selected against, by infecting the mixed phage population into a host cell that cannot suppress the amber mutation. Clones can then be isolated for subjects containing the desired TNF mutation.

Compounds identified to have convertase inhibitory activity would also have prophylactic or therapeutic applications for the treatment of sepsis. Because the course of sepsis is associated with an increase in circulating TNF, these inhibitors can be used prophylactically in those cases where there is a risk of bacterial infection, especially in a preoperative setting. Likewise, in those cases where there is an early diagnosis of sepsis, the inhibitors will have beneficial, therapeutic effects for significantly reducing the amount of TNF that is produced.

Another medical application for inhibitors of convertase is for the treatment of AIDS. TNF has been shown to cause activation of latent human immunodeficiency virus. Folks et al., Proe. Nati. Acad. Pollock. United States, vol. 86, pp. 2365 (1989). Prevention or inhibition of the formation of 17 kd, or lower molecular weight forms of TNF by inhibition of convertase, may be valuable prophylaxis for the treatment of AIDS, and will preferably be used to treat patients infected with the virus which is in a latent phase.

Examples that illustrate the scope of the invention follow below.

EXAMPLE 1

Conversion of 26 kd TNF

Vector pFVXM, deposited to the American Type Culture Collection, accession no. 67,103, was used to produce a vector pFVXM-TNF6, which contains the DNA sequence encoding the 26 kd TNF species. To produce the latter vector, plasmid Bli, containing the cDNA sequence encoding the 26 kd TNF species, was treated with Pst I which cleaves the coding sequence. The fragment was purified using standard electrophoretic techniques. Next, the vector pFVXM was treated with Pst I, and the Pst I fragment from pBll containing the 26 kd coding sequence was inserted into the 1 polylinker region of the vector using standard techniques as described above, to produce pFVX-TNF6. pFVX-TNF6 was used to produce the cell line TNF 6.8, as described by Kriegler et al., (1988), above, or as described in U.S. Pat. patent application entitled "Cleavage Site Blocking Antibody to Prohormone Proteins and Uses Thereof", filed on the same day as the present patent application. This application is Cetus Case No. 2534, with inventors Kriegler and Perez.

TNF 6.8 expresses both 26 kd and 17 kd TNF. Figure 2 shows conversion of 26 kd TNF by the convertase activity present in the HL 60 cells. The production of labeled 26 kd TNF by in vitro transcription/translation and analysis by gel electrophoresis is described below in Example 2. It is noted that S-1 cytosol or the pellet fractions cause almost complete conversion of 26 kd TNF to a 17 kd form. Figure 2 also shows, for comparison, 26 kd and 17 kd TNF in a lysate of TNF 6.8 cells.

pFVXM and plasmid pBll were both amplified in E.coli strain HB101. Ligation of the fragments was performed using standard conditions. Plasmid DNA was isolated after the ligation procedure, and the correct orientation of TNF coding sequences was determined by restriction analysis.

Plasmid DNA was prepared according to the method of Birnboim and Doly, as described in Nucleic Acid Research, 7:1513

(1979). Plasmid DNA was banded twice in cesium chloride, and extensively dialyzed against TE buffer consisting of 10 mM Tris, pH 8.0 and 1 mM EDTA.

EXAMPLE 2

Convertase analysis

A. In vitro transcript. 1on/ translas. 1onsanalvse

The preferred assay procedure consists of in vitro transcription/translation of the 26 kd molecule, followed by treatment with convertase in the presence or absence of compounds being tested for convertase inhibitory activity. The procedure comprises in vitro transcription/translation of the 26 kd molecule present in plasmid Bli. The sequence is removed from pBll by Pst I digestion and inserted into the Pst I site of pGEM-3 (obtained from Promega Biotec). The resulting plasmid, designated pGEM-TNF14, was amplified in E.coli using established techniques, and plasmid DNA was prepared according to the method of Birnboim and Doly, described above. Plasmid DNA was in vitro transcribed by linearizing it with Hind III and linearized plasmid templates were used to generate capped transcripts with T7 RNA polymerase and an in vitro transcription kit supplied by Promega Biotec. The transcription was carried out using standard techniques as suggested by the dealer's instructions.

mRNA was in vitro translated in the presence of 35g_CyS-tein fov ^ producing <3>^S-cysteine-labeled 26 kd TNF. The procedure consisted of

using a rabbit reticulocyte lysate translation kit, also supplied by Promega Biotec, and according to the conditions suggested by the vendor.

<35>S-cysteine labeled 26 kd TNF was used to assay for convertase inhibitors as follows. 25 µl of in vitro translated material was combined with 250 µl of uninduced HL60 convertase activity, plus compounds to be assayed for inhibitory activity. The convertase was produced by harvesting 2 x 10<9>HL60 cells, and isolating the S-1 and P-30 fractions, a total of 18 and 6 ml. The 250 X P-30 fraction was used, although the S-1 fraction can also be used. The assay was performed at 30°C for 1 hour, essentially as described above. The reaction mixture was then immunoprecipitated with anti-TNF polyclonal antisera and protein A sepharose, pelleted and washed. The bound protein was eluted and electrophoresed. The gel was dried and exposed to X-ray film and then developed. Gel electrophoretic profiles of 26 kd TNF treated with varying dilutions of HL60 convertase showed compounds with inhibitory activity.

By applying the above analysis, it was determined that 3,4-dichloroisocoumarin and elastinal at concentrations of respectively 100 pg/ml and 5 mg/ml, inhibited the convertase. It was also shown that (1-((3-((acetyloxyl)-7-methoxy-8-oxy-8-oxo-5-thio-1-azabicyclo[4.2.0.]oct-2-en-2- yl)carbonyl)morpholine, S,S-dioxide, (6R-cis) at a concentration of 1 mM inhibits the convertase activity, these results are shown in Figure 3.

B. Monocvtt- analvse

In addition to the 26 kd TNF produced by the in vitro transcription/translation assay described above, stimulated monocytes that produce 26 kd TNF, as described by Kriegler, et al., 1988, Cell, 53:45, can thus be used as a source for the molecule. A suitable assay procedure is to stimulate monocytes, and then in the presence of convertase, measure the loss of 26 kd species to a lower molecular weight species, preferably 17 kd TNF.

Human monocytes are purified from human blood by centrifugation, and then enriched based on adherence of monocytes to the cell culture dishes. The centrifugation consisted of purification of the monocytes through Ficoll plaque and percoll ($49.2), available from Pharmacia. The dealer's recommended procedures are followed. Next, the mixture of cells resulting from the centrifugation step, consisting of monocytes and lymphocytes, is plated on tissue culture dishes containing RPMI medium supplemented with 20% fetal calf serum. The dishes are incubated for 30 minutes at 37 °C and then rinsed with the same medium. This treatment removed non-adherent lymphocytes, leaving only adherent monocytes.

Monocyte 26 kd TNF is radiolabeled as follows. The monocytes are incubated for 3 hours at 37°C in RPMI medium supplemented with 20% fetal calf serum, 100 ng/ml lipopolysaccharide and 10 µg/ml phorbol myristate acetate for 30 minutes at 37°C. The latter two compounds induce the expression of TNF. RPMI medium is cysteine-free, and fetal calf serum present is at a final concentration of 5%. The serum is dialysed before use to remove any cysteine present. After the 30 minute incubation period, 100 µl of Cl<35>S-cysteine is added and the cells radiolabeled for 3 hours at 37°C, after which they are lysed and used to assay for convertase activity. The steps for performing the assay, as well as identifying the inhibitors of convertase, are similar to those described above.

EXAMPLE 3

TNF mutein/ antibody/ peptide inhibitors with convertase activity The following compounds will have convertase inhibitory activity

tet, and can be prepared as follows and tested for inhibitory activity as described above.

A. Anti-convertase antibody

Antibody, either monoclonal or polyclonal, is produced which either neutralizes the enzymatic activity of the convertase, or which binds to the convertase and thereby sterically prevents the convertase from binding to 26 kd TNF. The procedure consists of immunization of an appropriate host animal with a membrane-containing fraction of convertase-producing HL60 cells. A sufficient amount of material should be used to trigger an immune response. This will usually consist of between 10 >jg to 10 mg per kg. body weight. The immunization can be carried out with adjuvant in a biologically acceptable buffer, known in the field. The best route of immunization can be determined experimentally, and the primary immunization can be followed by a more secondary immunization depending on the strength of the immune response to the original immunization. The presence of neutralizing anti-convertase antibodies in sera can be detected using the convertase assay described above, in which antisera are present in the assay mixture. Inhibition of the conversion of the 26 kd TNF species to a lower molecular weight species indicates the presence of neutralizing antibody. It is of course assumed that proper controls are performed to ensure that antisera from non-immunized animals are not inhibitory. Polyclonal antibody can be purified as described below.

Monoclonal antibody to the convertase can be produced using either in vivo or in vitro immunization techniques, and sensitized lymphocytes resulting therefrom can be used to produce hybrid cell lines secreting the appropriate monoclonal antibody. Rodents, preferably of murine origin or human antibody, are most preferred. The in vivo immunization procedure comprises sensitization of lymphocytes to convertase by immunization of either mice or humans, and isolation therefrom of the antibody-secreting cell fraction and immortalization of the cells therein by one or more procedures. An alternative embodiment is to isolate lymphocytes that have already been sensitized to the convertase from septic patients, as described above.

(i ) Murine antibody

For in vivo immunization of mice, the procedure of Kohler and Milstein, described in Nature, 256:495 (1975) can be followed, or modified procedures such as those shown by Fendly, et al., 1987, Hybridoma, 6:359 , Buck, et al., 1988, in Vitro, 18:377. In vitro techniques are generally described by Luben, R. and Mohler, M., 1980, Molecular Immunology, 17:635, Reading, C. Methods in Enzymology, 121 (part one): 18, or Voss. B., 1986, Methods in Enzymology, 121:27.

Mice are immunized with 1 mg/ml of a membrane-containing fraction of HL-60 cells, previously shown to be positive for convertase activity. The immunization is carried out in complete Freund's adjuvant. Two additional immunizations, or boosters, are performed monthly without adjuvant, and one month after the last boost, the mice are given an I.V. boost of 10 jjg membrane-containing material. Three days after I.V. boost, the mice are sacrificed, the spleens removed and spinocytes isolated and condensed into an immortalized drug-selectable myeloma partner cell line. Many such myeloma lines are known in this field, and most cannot grow in HAT supplemented cell culture medium. A typical myeloma cell line is SP-2/0Ag 14. The hybridomas are thereby formed by combining splenocytes and myeloma cells in a 5:1 ratio, which generally consists of 2 x 10^ myeloma cells to 1 x 10<7>splenocytes. The cell mixture is pelleted, media removed and fusion achieved by addition of 1.0 ml 40% (v/v) solution of polyethylene glycol 1500 by dropwise addition over 60 seconds at room temperature, followed by a 60 second incubation at 37°C. To the cell suspension with gentle agitation, 9 ml of Dulbecco's modified Eagle's medium is added over 5 minutes. Cell clumps in the mixture are gently resuspended, cells washed to remove any residual PEG, and seeded into microtitre dishes at approximately 2 x 10 cells/well in DMEM supplemented with 2056 fetal calf serum. After 24 hours, the cells are fed a 2x solution of hypoxanthine and azaserine selection medium.

Media from wells showing positive cell growth can be screened for neutralization by monoclonal antibody against convertase. The preferred assay is the convertase assay described in example 2 above, in which medium to be tested for antibody activity is present in the assay. More preferred is to combine the culture supernatants from 3-8 microtiter wells and analyze the mixture. If the mixture is positive, the medium from each well can be analyzed independently to identify the secreting hybrid cells. Many assays are known in the art and can detect soluble or insoluble antigens and are shown by Langone, J. and Van Vinakis, H., Methods of Enzymology, 92. Part E (1983).

Regardless of whether the antibody is polyclonal or monoclonal, it is desirable to purify the antibody by standard techniques as known in the art or described by Springer, 1980, Monoclonal Antibodies,: 194, (Eds. Kennett, T. McKearn and K. Bechtol, Plenum Press, New York. Generally this consists of at least one ammonium sulfate precipitation of the antibody using a 50% ammonium sulfate solution. Antibody affinity columns may also be used.

(ii) Human monoclonal antibodies

Peripheral blood lymphocytes are isolated from septic patients and then infected with Epstein-Barr virus and infected lymphocytes, immortalized by fusion to a selectable myeloma cell line, and the hybrid cell lines thus formed are isolated and characterized for antibody production. Mononuclear cells are separated on Ficoll-hypaque (Pharmacia), and monocytes removed from the mixture by adherence to plastic. Standard laboratory techniques are used to accomplish these procedures. Then, non-adherent cells are enriched for antibody producers by antigen-specific panning. Panning is a technique generally known in the art and involves the incubation of a population of antibody-secreting cells on a plastic surface coated with the appropriate antigen. Those cells that express antibody on their surface bind antigen, and therefore adhere to the plastic surface, while cells that do not express cell surface antibodies do not adhere, and can be removed by washing. Specific antibody-secreting cells are enriched for by this technique.

6-well plates (Costar) are coated with a membrane fraction containing convertase prepared from either induced or uninduced HL60 cells as described above, so that 150 pg of membrane-containing material is coated per well in phosphate-buffered saline at 4°C overnight. The wells are blocked after the overnight incubation period with phosphate-buffered saline containing 1% bovine serum albumin for at least 1 hour at 4°C, and subsequent washing with phosphate-buffered saline/BSA. Next, 10<7>lymphocytes in 1 ml of PBS/BSA are added to each well of the six-well dishes. The lymphocytes are incubated on the dishes for 70 minutes, whereby any non-adherent cells are removed by aspiration. The adherent cells are incubated with cell culture medium (MDM, Sigma Chemical Co., St. Louis, Missouri) containing 10# fetal calf serum.

Adherent cells are subjected to Epstein-Barr virus transformation by adding an equal amount of culture medium obtained from the cultivation of Epstein-Barr virus infected marmoset cell line B95-8, thus containing virus, to medium bathing the adherent cells. The cells were cultured in this environment at 37°C for 3 hours, and in this way the lymphocytes in the adherent cell population are exposed to Epstein-Barr infection. After the infection period, the cells are washed and seeded in 96-well microtiter dishes at a density of approximately 10<4->10<5> cells/well in IMDM medium, plus 10% fetal calf serum and 30% conditioned medium. The latter is derived from a lymphoblastoid cell line, preferably JW5. The medium also contains 5 x 10~<5>M2-mercaptoethanol, 50 ug/ml gentamicin sulfate (Sigma) and 600 ng/ml cyclosporin A (Sandimmun, Sandoz, Basel, Switzerland).

After approximately 14 to 21 days of incubation, the cell culture supernatants are combined and screened for convertase neutralizing activity as described above. Positive hybridomas are subcultured at low density, retested for neutralizing antibody, and cultured and condensed into the F3B6 cell line, using polyethylene glycol and the plate fusion technique known in the art. The latter technique is described by Larrick, J.W., (1985) in Human Hybridomas and Monoclonal Antibodies, E.G. Engleman, S.K.H. Foung, J.W., Larrick and A.A. Raubitschek, Editors, Plenum Press, New York, p. 446. F3B6 is a heteromyeloma cell line sensitive to growth in media containing 100 µm hypoxanthine, 5 µg/ml azaserine and 5 µm ouabain. Finally, the resulting hybrids are re-screened to ensure that they produce neutralizing anti-convertase antibody.

B. 26 kd non-cleavable muteins

26 kd TNF muteins have been described as competing for binding to convertase, thereby inhibiting or reducing its activity. The preferred muteins are those having valine at positions 1 and/or 13; or alanine in position -1 and/or proline in position 12, substituted/or deleted. The muteins are constructed using a modification of the site-directed mutagenesis-gap-holding duplex method.

The following solutions/buffers are used to perform the desired procedures: 5 x gapped duplex buffer (GDB) consisting of 0.938 M KCl, 0.063 M Tris, pH 7.5, 10 x PEL consisting of 1.0 M KCl, 0.30 M Tris, 0.15 M MgCl 2 , 0.02 M DTT, pH 7.5; 10 x KB consisting of 0.50 M Tris, 0.10 MgCl2, 0.05 M DTT, 0.001 M EDTA, pH 8.0; a solution containing 0.25 mM dCTP, dATP, dGTP, dTTP, made fresh from 10 mM stock solutions; an ATP solution consisting of 0.1 M ATP prepared by dissolving 60 mg of ATP in 0.80 ml H 2 O and adjusting the pH to 7.0 with 0.1 M NaOH in a final volume of 1.0 ml with H 2 O; 20* PEG/2.5 M NaCl; 3.0 M NaOAc; and TE saturated phenol.

Various bacterial strains and phages are used to provide the desired muteins, and these are BMH 71-18, JM103 for growing phage strains; HB2154; MutL, Su-, rendered competent for DNA transformation; and EB2151; Su-, used as coating cells during transformation; M13 GAP, RF used for formation of gap-containing duplex; and M13mpl9-amber, 26 kd TNF target DNA is cloned into this vector and ssDNA isolated to form gap-containing duplex.

Phage are infected into an appropriate material strain, cultured and titrated as follows. For the creation of a large-scale preparation of either phage for ssDNA or cells for dsDNA or RF DNA, the same infection protocol is used.

Plaque-cleared subjects are produced using standard techniques. This consists of plating phage supernatants on agar plates, followed by careful coating with 4.0 ml soft agar and 100 µl fresh overnight culture of BMH 71-18. Then isolated plaques are picked and incubated with a 1:50 dilution of fresh overnight culture of BMH 71-18 in R26 or R17 + 10 mM MgCl2 with shaking at 37°C for 4.5-6 hours. R17 (N-Z amine power) consisting of 10 g of N^Z amine, type A,. 5 g NaCl with H20 to 1 liter, while R26 consists of 8 g tryptone, 5 g yeast extract, 5 g NaCl with water to 1 liter (YT power). The phage stock is titrated and phage infected into bacteria at a multiplicity of infection (MOI) of 10. After incubating the culture with shaking at 37°C for 5 hours, the cell suspension is pelleted and the supernatant saved for ssDNA isolation, and cells for RF Isolation RF DNA is isolated using established plasmid DNA isolation techniques, while ssDNA is isolated as follows.

250 ml phage supernatant is spun down hard, then 200 ml of the supernatant is decanted, followed by the addition of 50 ml 20* PEG/2.5 M NaCl and incubation overnight at 4°C or on ice for 30 minutes. This mixture is also spun down hard and the supernatant decanted. The bottle is again spun down to pellet the phage precipitate along the side of the bottle, and the remaining fluid is aspirated with a Pasteur pipette. The pellet is resuspended in 5.0 ml of 1 x TE and stored at 4°C, and 0.5 ml is extracted twice with 0.5 ml of TE saturated phenol. To the aqueous layer is added 0.050 ml of 3.0 M NaOAc and 1.0 ml of 95* ethanol. The mixture is placed in a dry ice bath for 10 minutes and centrifuged for 10 minutes in a microfuge at 4°C. The pellet is dried and resuspended in 200 µl of IX TE. This material can be stored in 0.050 ml aliquots at -20°C until use in mutagenesis of 26 kd TNF.

The following oligonucleotides are used to change valine in positions 2 and/or 13, alanine in position -1 and proline in position 12. The oligonucleotides and their corresponding muteins are shown in Table 1.

TABLE 1

1. Mutes

(b) Substitutions 2. Oligonucleotides

The oligonucleotides are kinased using the following reaction solution and conditions: 3 µl 10 x KB buffer, 3 X 10 mM rATP (1:10 dilution of 0.1 M rATP stock solution), 2 X mutagenic oligonucleotide (100 pmol/X), 21 X H 2 O and 1 X polynucleotide kinase (10 units/X). The reaction is run at 37°C for 45 minutes, and then at 65-68°C for 5 minutes. Next, 24 X kinase-treated oligonucleotide is diluted with 56 X H 2 O to provide 2 pmol/X.

The gapped duplex is formed as described below, followed by fusion of the oligonucleotides. The following reagents are combined in a total volume of 40X: 8X 5x GDB buffer, 0.50 pmol ss DNA and 0.10 pmol Hine II linearized M13 GAP RF DNA. 10 X are removed for later use, and the remaining 30 X are processed sequentially as follows: 100°C for 3 minutes, 65°C for 5 minutes followed by cooling to room temperature for 30 minutes, and then placing the reaction mixture on ice. Next, 10X gapped duplex and 10X control ungapped material are subjected to electrophoresis on an agarose gel to examine gapped duplex formation. Assuming the gel shows the presence of a third band, the gap-treated duplex has been formed and the kinase-treated oligonucleotides can be fused to the duplex by combining the 16 X gap-duplex reaction mixture and 4 X diluted kinase-treated oligonucleotide, and heating the mixture to 65°C in 3 minutes, followed by cooling to room temperature for 20 minutes.

The heteroduplex is completed by appropriate extension and ligation reactions consisting of combining the following reagents in a total volume of 40X: 10X gapped duplex and primer, 4X 10X PEL buffer, 4X dNTP (0.25 mM solution prepared from 10 mM stock solutions, 3 X ATP (10 X 0.1 M ATP stock solution + 1490 X H20 = 0.662 mM), 17 X H20, 1 X Klenow (5 p/X) and 1 X T4 DNA ligase (0.6 Weiss p /X, stock diluted with 1 x PEL).The reaction is carried out at 16°C for 2 hours, followed by transformation of 10X extension/ligation mix into 200X, thaw competent HB2154 cells.The cells are kept at 0°C for 30 minutes and then at 42°C for 1.5 minutes, followed by plating different volumes of transformation mix (eg 50X, 10X, etc.) with 100X fresh overnight culture of HB2151 cells + 3.0X soft agar.

The resulting plaques are screened using the plaque hybridization procedure. Various such procedures are known, and a description of the preferred procedure follows. Plates are replicated on duplicate nitrocellulose filter paper (S & S type BA-85) and DNA fixed to the filter by sequential treatment for 5 min with 0.5 N NaOH plus 1.5 M NaCl; 1.0 M NaCl plus 0.5 M Tris-CHl pH 7.4: and 2 x SSC (standard saline citrate). The filters are air-dried and baked at 80°C for 2 hours in a vacuum.

Duplicatef The tubes are prehybridized at 55° C for 2 hours with 10 ml per filter DNA hybridization buffer, 5 x SSC, pH 7.0 x Denhardt's solution (polyvinylpyrrolidone, plus Ficoll and bovine serum albumin; 1 x 0.02* of each), 50 mM sodium phosphate buffer pH 7.0, 5 mM EDTA, 0.1 * SDS and 100 pg/ml yeast RNA. The prehybridization buffer is removed and the samples hybridized with an appropriate kinase-treated probe, i.e. kinase-treated oligonucleotides as shown above, under conditions that depend on the desired stringency. About 2 x 10<*>>cpm/ml in total is used. Common, moderate, stringent conditions using a temperature of 42 °C plus 50* formamide for 24-36 hours with 1-5 ml/filter DNA hybridization buffer containing probe. For higher stringency, higher temperatures and shorter times are used. Preferred hybridization conditions consist of hybridizing the probes to the filters in 5 x SSC, Denhardt's solution, 50 mM NaPO 4 , pH 7.0, 5 mM EDTA, 0.1* SDS and 100 mg/ml yeast RNA at 10° under Trø to the oligonucleotide used to perform the screening. Then the filters are washed twice, 30 minutes each wash, at room temperature with 2 x SSC, 0.1* SDS, then washed once with 2 x SSC and 0.1* SDS at 5°C under Tjyj to the oligonucleotide used for to screen, and air dried. Finally, the filters are autoradiographed at -70°C for 36 hours. Autoradiography shows the plaques containing the virus containing the muteins of interest. In addition to constructing muteins in which valine at position 2 and/or 13 has been deleted or substituted, large deletion muteins can be produced that include the two major cleavage sites of 26 kd TNF. A preferred mutein lacks the amino acids covering the region -9 to +14, as shown in Figure 1. This mutein was constructed using the materials and methods described above and the oligonucleotide, CP375 having the following sequence:

C. Protein/peptide inhibitors

Peptides with the following amino acid sequences are synthesized by the solid phase method described in detail by Merrifleid, R.B. (1985) in Science, 232:341-347: Gln-Ala-Val-Arg-Ser-Ser-Ser, Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp- Lys-Pro-Val-Ala; Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala; Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala; and Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro. A Blosearch 9500 automated peptide machine is used with hydrogen fluoride cleavage and purification by preparative HPLC using a Waters Delta prep 3000 instrument, on a 15-20 pm Vydac C4 PrepPAK column.

That these peptides inhibit the convertase activity has been shown by performing the analysis described above in the presence of varying amounts of each peptide. Gel electrophoresis and Western blotting of the reaction mixture show an inhibition of the conversion of 26 kd TNF to the 17 kd form.

EXAMPLE 4

Protective effect of the convertase inhibitors

for the treatment of sepsis

Compounds that are effective inhibitors of convertase activity have been shown to prevent sepsis in a baboon model system as follows. Anti-convertase antibody, murine, human or recombinant, at a concentration of 5 mg/kg is administered in a single I.V. bolus 60 minutes before the animals are treated with a lethal dose of E.coli and 2 mg/kg at the same time as the E.coli treatment. The antibody is administered in a physiological balanced salt solution, and approximately 4 x 10 2 E.coli organisms are used. The E.coli dose is infused over a period of two hours. The animals receiving the antibody are protected for at least 7 days, while the control animals receiving only balanced saline expire within 16 to 32 hours.

Similar protection by the TNF mutein convertase inhibitors is shown in Example 3. The muteins are administered at a concentration of 5 mg/kg in a single I.V. bolus 60 minutes before the animals are exposed to 4 x 10<10>E.coli organisms. The baboons also receive 2 mg/kg of the muteins at the same time as the E. coli supply.

Finally, the peptides shown in Example 3, i.e. Gln-Ala-Val-Arg-Ser-Ser-Ser and Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys -Pro-Val-Ala, tested as described above and provides similar protective effects.

The present invention has been described with reference to specific embodiments. The invention shall also cover changes and substitutions which can be carried out by professionals without deviating from the scope of the invention and the attached claims.

Claims (8)

1. Method for screening prophylactic agents or therapeutic agents for diseases caused by, exacerbated by or associated with the production of mature tumor necrosis factor (TNF) hormone, or one or more lower molecular weight species of said mature protein hormone, which is produced from a TNF prohormone by convertase cleavage of said prohormone, characterized in that it comprises the following steps: a) contacting said TNF prohormone with an effective amount of said convertase; b) measuring the conversion of said TNF prohormone to said TNF mature hormone or one or more lower molecular weight species of said mature hormone; c) repeating steps (a) and (b) above, further comprising a molecule believed to be identified as a prophylactic or therapeutic disease caused by, aggravated by, or associated with production of said mature TNF hormone; d) measuring conversion of said TNF prohormone to said mature TNF hormone or lower molecular weight species in step (c); and e) comparing the amount of conversion of said prohormone from steps (b) and (d), wherein a smaller cleavage in step (d) than in step (b) indicates that the molecule of step (c) is potentially a prophylactic agent or therapeutic agent for a disease caused by, aggravated by, or associated with the production of mature TNF hormone or one or more lower molecular weight species of mature TNF hormone. 2. Process for the production of therapeutic agents or prophylactic agents for the treatment of disease, characterized in that the disease being treated is caused by a mature TNF hormone or a species with a lower molecular weight thereof, identified according to the method in claim 1. 3. Method for identifying prophylactic agents or therapeutic agents for sepsis, characterized in that it comprises the following steps: a) contacting TNF prohormone having a molecular weight of about 26,000 with an effective amount of TNF convertase; b) measuring the conversion of TNF prohormone having a molecular weight of about 26,000 to one or more TNF species of lower molecular weight; c) repeating steps (a) and (b) above, and further including a molecule believed to be identified as a prophylactic agent or therapeutic agent for sepsis; and d) measuring inhibition of said conversion of said TNF having a molecular weight of approximately 26,000 to one or more TNF species with a lower molecular weight. 4. Method According to claim 3, characterized in that measurement of inhibition of said convertase comprises the following steps: a) labeling of said TNF prohormone with a suitable marker; b) treating said labeled TNF prohormone with said convertase to convert said labeled prohormone TNF to said lower molecular weight TNF having a molecular weight of about 17,000; c) separating said labeled TNF prohormone from said 17,000 molecular weight TNF; and d) measurement of said marker present in said 17,000 molecular weight TNF or measurement of reduction of said marker in the labeled TNF prohormone. 5. Process for producing prophylactic agents or therapeutic agents according to claim 1, characterized in that said prophylactic agents or therapeutic agents comprise anti-convertase antibody. 6. Process for the production of prophylactic agents or therapeutic agents according to claim 1, characterized in that said prophylactic agents or therapeutic agents comprise non-hydrolyzable muteins of the TNF prohormone which have a molecular weight of approximately 26,000. 7. Process for the production of prophylactic agents or therapeutic agents according to claim 6, characterized in that said muteins have valine in position 2 and valine in position 13 is substituted or deleted. 8. Process for producing prophylactic agents or therapeutic agents for sepsis according to claim 4, characterized in that said prophylactic agents or therapeutic agents comprise a peptide or protein which has an amino acid sequence substantially similar to an amino acid sequence present on said 26 kD TNF prohormone to which said convertase binds.