CA2139127A1 - Compositions for the inhibition of protein hormone formation and uses thereof - Google Patents

Abstract

Compounds having proteolytic inhibitory activity useful for treating diseases, particularly as applied to the treatment of sepsis, AIDS or autoimmune diseases, resulting from a decrease in the circulating level of mature protein hormones derived from the proteolytic cleavage of prohormone precursors such as 26 kD TNF, and methods for identifying compounds having the de-sired inhibitory activity are provided.

Description

WO 94/00555 PCr/US93/06120 2 1 ~ 7 , COMPOSmONS FOR THE INHIBITION OF PROTEIN HORMONE
FORMATION AND USES THEREOF
This is a continuation-in-part of U.S. Serial No. 07/395,253, filed August 16, 1989, which is p~nr~ing.
Field of the Invention This invention is in the area of immllnology/bjorllPmi~try, and particularly concerns the development of co",posilions and methori~ for identifying inhibitors of protein hormone release, and prophylactic and theld~)eulic uses of the inhibitors for treating ~ ces ~ tf~d with elevated levels of the hormones. More specifir~lly, the invention f~rilit~tPs the identification of co",posi~ions and methods for identifying inhibitors of a TNF convertase. These inhibitors may be used to treat a variety of (~ cPS~ particularly sepsis, ll,eli"~ts)id arthritis, c~rh~Yi~
AIDS and autoimmune ~ ~s, and thus affords the physician alternate ll~al",ent regim~os.

Background of the Invention In the United States alone nosoco"~ial ba;~ ;",ia develops in about 194,000 patients per year, and of these about 75,000 die. Maki, D.G., 1981, Nosocomial Infect., (Dikson, R.E., Ed.), page 183, Yrke Medical Books, U.S.A.. Most of these deaths are attributable to six major gram-negative bacilli -- Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsie~la, Enterobacter and Serratia. The current tre~tment for bacteremia is the ~dminictration of antibiotics which, have limited effectiveness in tleal,--el-t of septic shock.
25 The precise pathology of bacteremia is not completely elucid~ted. Nevertheless, it is known that certain bacterial endotoxins called lipopolysaccharides (LPS), are the primary causative agent. LPS consists of at least three ~ignifi~nt antigenic regions: lipid A; core pol~cchalide; and O-specific polysaccharide. The latter is also r~felled to as O-specific chain or simply O-antigen. The O-specific chain 30 region is a long-chain polysaccharide built up from repeating polysaccharide units.
The number of polysaccharide units differs among dirrerent bacterial species and WO 94/00s5s Pcr/US93/06120 ~39~ 2-may vary from one to as many as six or seven monoca~h~ri(lP units. While the O-specific chain varies among dirr. .cnt gram-negative b~cter ~ the lipid A and core poly~r~h~rides are similar if not id~Pntie~l.
Since LPS plays a key role in sepsis, many appluaches have been pursued to 5 neutralize its activity. ~ntly, there is conci~lp-rable work which suggest that antiLPS antibody will soon be a valuable clinical adjunct to the standard antibiotic therapy.
LPS inilialcs a r~C~ e of bior-h~P-mi~l events that eventually causes the death of the patient. It is widely believed that an early result of LPS introduction is the ~tim~ tion of ~l~acn~phage cells and the production of tumor necrosis factor (TNF) as a result of LPS. Thus, considerable effort has been eYrPn~led to produce neutralizing antibody to TNF, or other molecllles that could inhibit its effects. It is likely that anlibo Iy to TNF will have valuable clinical appli~tion~. Tracey, et al., 1987, Nature, ~:662.
TNF has been shown to exist in both membrane-bound and soluble s~l~;led forms.
Decker, et ak, 1987, J. of Immunol., 138:957; Kriegler, et al., 1988, Ce11, 53:45.
Human TNF has been cloned and shown to consist of a 17 kD polypeptide, plus an Im~sll~lly long 76 amino acid putative signal leader sequence. The 17 kD
moleeule is a key agent involved in init;~;ilg the biochpmi~l cascade responsible for sepsis. It has been propo~ by Kriegler, _ a1., 1988, Cell, 53:45, that TNF
may exist as both a me",bldlle bound 26 kD form, and a soluble form coll~l,onding to the 17 kD c~ci~s. The 26 kD form is the pl~;ul~or, or prohormone, of the mature 17 kD molecule. It has further been proposed by Kriegler, et al. above, that the two forms of TNF may have dirr. I~nt biologicaleffects, primarily as a result of dir~rences in tissue distribution.
It will be appl~iated that be~ se TNF plays a key role in causing sepsis and other ~licP~es that there is a need to identify and develop anti-TNF
prophylactics/thel~u~;rs. As mentioned above, anti-TNF antibody appears to be promising, and has been shown to be effective in baboons. However, these studies have involved the use of non-human TNF and non-human TNF antibody.

WO 94/00555 2 1 3 9 1 2 7 Pcr/US93/06120 From a practical st~n~lpoint non-human anti-TNF antibody will have limited the~ ;c application because of immunologic rejection of the antibody by a patient's immune system. Consequently, a human antibody, or a gçnPtir~lly çng;n~F.cd anlibody concicting of the human con~nt region and the mouse S variable region ("hum~ni7~ antibody") is plefe.-~d.
TNF, in ^~ ition to playing a critical role in sepsis, has recently been shown to be involved in i~ ;st;ng the e Ap~ssion of human i.. -.nocle~riency virus in human cells that carry latent virus. Folks et al., 1989, PNAS (USA), 86:2365. Thus, preventing or inhibiting the formation of the 17 kD, or lower molecular weight forrns of TNF might be a valuable prophylactic for the tre~tmPnt of AIDS patients by preventing the cAplession of virus that is latent in the patient.
TNF also plays a role in various autoimmune iicP~cp~s~ particularly rhe~ oid arthritis. Duff, et 1 1987, International Conference on Tumor Necrosis Factor and Related Cvtotoxins, 175:10. Thus, colllpounds or methods for inhibiting TNF
action will have con~i~çrable application for the ~r~atlllel~t of a variety of ~1icP~cPs of illllllunologic origin.
In addition to antibody, other molecules with TNF inhibitory activity are being sought . Non-anLibody TNF inhibitors are described by Se~inger, et al., 1988, L
Exp. Med., 157:151, and Se~inger, et al, 1989, J. Biol. Chem,. 264:11966, and in Eulo~l Patent Application No. 88830365.8, inventors Wallach, et ak The inhibitors are present in the urine of febrile patients, and have been purified and shown to have mol~cul~r weights of about 27,000-33,000. These inhibitors are now known to be soluble forms of the TNF receptor. Although these molecules exhibit TNF-inhibitory activity, neither of the inhibitors has yet been shown to be effective in the lr~t--,Pnt of sepsis in humans.
From the Çoregoil~g tliccussion it is appar~nt that there is a need to identify and develop ~ ition~l anti-TNF inhibitors, both antibody based or otherwise, that are çffic~cious in the tre~tn Pnt of sepsis.

Sullllllal~ of the Invention WO 94/00555 PCr/US93/06120 ?~39~ 4-In its most general form, the invention de~cl~ribe~ herein pr~nts metllods and co~..rûc;l;~n~ for inhibiting the pr~llc!;on of a mature form of TNF, from its prohormone pr~ul~r, proTNF. These co,,,pûsilions are useful for preventing or treating ~ cPs in p~ti~ont~ d with elevated circulAtin~ levels of mature 5 TNF. The invention also relates to a method for identifying molecules that inhibit the production of a mature form of TNF. Such inhibitors are distinguishable fromantiTNF antibody, which neutralizes TNF. This method can be used to identify m~i~A...~ such as prophylactics and/or thc.Apeul;cs for the treatment of sepsis and other ~ cP~s caused by the productio~ of mature TNF. These m~dicAm.ontc 10 are able to ill~lre~G with the cleavage of the 26 kD proTNF prohorrnone by enzymes termed conve.t~s. Thus, these m~il~z...r.~l~ inhibit the production of lower m~eculAr weight sepsis-induçing mollecules (i.e., 17 kD TNF).
S~ifir~lly, the plGre.lGd inhibitors as described herein inte.rGlG with the activity of a TNF convertase to prevent removal of the N t~i",inal portion of the 26 kD
molocule including at least the 76-Amino ^id signal se~uence to produce a matureform of TNF such as the 17 IcD TNF. The invention also includes a class of co"")ounds that are both inhibitors of a TNF convertase and effective in the prevention and/or tr~Atm~nt of sepsis. Co",~unds in this class include anti-convertase antibody, muteins of the prohormone form, and proteins or 20 peptides that CUIIIPelG with the 26 kD form of TNF for binding to the convertase.
Also c1~ime~ are small molecular weight co...l)ou~lds that sperifiçAlly inhibit the class of proteases that inçludes TNF convertases, or prere.dbly, show selective ificity for inhibition of TNF converta e.
A(l/litionAlly, the present inventors have purified a TNF convertase to 25 nearhomogeneity, discovered its amino acid se~lucnce, and cû,,,parGd it to known serine proteases. The purified TNF convertase contains an N-terminal amino acid sequence eC~ ;A11Y identirAl to PR-3, a known n~ul,uphil protease having the same mol~ul~r weight. They have also identified various inhibitors of TNF
convertase and have tested them in in vitro and in vivo assays.

WO 94/00555 213 9 1 2 7 PCr/USs3/06120 Spe~ifi~11y, an object of the present invention is to provide small mo1e~1-1Ps that spe~ifi~l1y inhibit TNF convel~s.
Another object of the invention is a method for treating ~1icP~cP~s such as septirerni~ septic shock, cerebral malaria, ~ oid arthritis, AIDS, c~c-hP~Yi~
5 and graft-versus-host disease by ~dminictPring a PR-3 inhibitor.
In one aspect of this invention, a method for identifying a prophylactic or thelai~eulic of a disease caused by a mature tumor necrosis factor (lNF) produced from a proTNF by cleavage of said proTNF by a TNF convertase is provided, the method comprising the steps of:(a) c~llt~-ting the proTNF with an amount of the 10 TNF convertase effective for cleaving the proTNF;(b) Il,easuring the conversion of the proTNF to the mature TNF in step (a); (c) ~PAI;ng steps (a) and (b) further including a molecule sought to be id~PntifiP~I as a prophylactic or theld~ulic of .1i~P~cps caused by the mature TNF; (d) measuring the conversion of the proTNF
to the mature ~NF in step (c); and (e) cGIlll)~ing the conversion ",~.lred in step 15 (b) with the conversion measured in step (c) to del~l",ine whether the molecule is a suitable prophylactic or th~ldpeulic of ~isP~Cps caused by mature TNF.
In another aspect of the invention, a theld~eulic or prophylactic compound for treating a disease caused by a mature TNF produced from a proTNF by cleavage of said proTNF by a TNF convertase is provided, the theld~ulic or prophylactic 20 identifiPd by a method comprising the steps of: (a) contacting the proTNF with an ~mol1nt of the TNF convertase effective for cleaving the proTNF; (b) mP~cufing the conversion of the proTNF to the mature TNF in step (a); (c) ~e~ling steps (a) and (b) further including a molecule sought to be identifi~P~ as a prophylactic or the~ulic of ~ cPS caused by the mature TNF; (d) measuring the conversion of 25 the proTNF to the mature TNF in step (c); and (e) co,,,~uing the conversion - measured in step (b) with the conversion measured in step (c) to identify whether the molecule is a suitable prophylactic or theldpeulic of diC~p~cps caused by mature TNF.
In yet another aspect of the invention, a method for treating a patient having a30 disease or susceptible to a disease caused by a mature TNF produced from a WO 94/00s55 Pcr/US93/06120 ?,~39~ 6-proTNF by cleavage of said proTNF by a TNF convertase is provided, the method comprising ~mini~ring to a patient in need of such Llr~ -nt an effective ~mount of an inhibitor of a TNF convertase. In a p~rel~d embo~impnt> the disease is selPcte~d from the group concicting of sepsis, rhru~A~oid arthritis, c~rh~Y~i~ cerebral malaria, AIDS, and graft-versus-host ~
In a further aspect of this invention, a IJh~..~ utirql co,..pos;~iQn for the tre~tm~nt of a disease caused by a mature TNF produced from a proTNF by cleavage of said proTNF by a TNF convertase is provided, the co...posiLion comprising an effective amount of an inhibitor of a TNF-convertase and a 10 pharm~ eutir~lly ~cceptq~-le eYciE~ cnt Brief Description of the Drawin~
Figure 1, panel A, shows the rçstriction map of the DNA s~u~nce that encodes 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.
15 Figure 2 shows the predicted amino acid sequence of the unpn~cessed pr~;ul~or of human PR-3, derived from the DNA sequence of the cDNA clone.
Figure 3 shows the conversion of 26 kD TNF by TNF convertase. Lanes A, B, and C show various controls: TNF 6.8 cell Iysate (A), 26 kD
transcription/translation (B) and incub~tion (C) controls. Lanes D, E, and F show 20 the conversion of transcription/translation gen~.dLed 26 kD TNF to predominately 17 kD TNF by convertase present in either HL60 S-l cytosol uninduce~ (D) and induced (E) fractions, or a P-l pellet fraction plcpared from ind~ced ce!ls. G is a blank lane.
Figure 4 shows the effect of convertase inhibitors on the conversion of 26 kD
25 TNF to its lowa molecular weight forms as deterrnined by gel ele~;lrl)l)horesis.
Lanes A,B,C,and D of panel 1 show, ~pe~ ely; i~ unp~ipi~lion of a cell lysate of the pFVXM-TNF6 transfected cell line TNF 6.8 (Kriegler, et al., 1988, in ~, 53:45), imm~np,G~ipitalion of in vitro transcribed/tr~n~ P.d 26 kD TNF, the effect of (1-((3((acetyloxyl)-7-methoxy-8-oxy-8-oxo-5-thio-1-azabicyclo [4.2.0]
30 oct-2-en- 2-yl) carbonyl) morpholine, S,S-dioxide, (6R-cis) on the conversion of WO 94/00555 Pcr/US93/06120 21~9127 26 kD TNF, and the conversion of 26 kD TNF in the absence of (1-((3-((acetyloxyl)-7-methoxy-8-oxy-8Oxo-5-thio-1-azabicyclo[4.2.0] oct-2-en-2-yl) carbonyl) morpholin~, S,S~lio~ide, (6R-cis). Lanes A and B of panel 2 show, r~ ely; i.. unp.~;pi~ion of a cell lysate of the pFVXM-TNF6 S transfected cell line TNF 6.8 (Kriegler, et al., 1988, in Cell, 53:45), and im".unpr~;~ ;on of in vitro transcribed/l-~n~ d 26 kD TNF. Lanes C and D
show the conversion of 26 kD TNF in the presence and ~hS~nce of 3,4dichloro-isoco~l,..~in, r~ rely. Lanes E and F, show the conversion of 26 kD TNF in the presence and absence of pl~tin~l, r~specli~ely.
Figure 5 shows additional gel eleclrûphoretic assays on 26 kD TNF, demonstratinginhibition of purified human PR-3 from HL-60 cells by various serine protease inhibitors.
Figure 6A shows gel electrophoretic analysis of purified human neulluphil PR3 activity on 26 kD TNF, showing dirr~aenLial inhibitory activity of poten~ial serine protease inhibitors.
Figure 6B shows similar results obtained using a colorimetric assay testing the same co---pounds.
Figure 7 shows the effect of prophylactic llt ~n..~nt of mice with a TNF convertase inhibitor prior to lethal injection with LPS: circul~ting serum TNF levels are 20 decreased.
Figure 8 shows the effect of prophylactic tr~tm~-nt of mice with a TNF convertase inhibitor prior to lethal injection with LPS: survival is prolonged.

Detailed Desc,i~)lion of the Invention 25 Definitiûns - To facilitate underst~n~ling the nature and scope of applicant's invention, several definitions r~arding various aspects of the invention are presenled below. It will be understood, however, that these definitions are general in nature, and encomp~c~ within the definitions are m~ning.~ well known to those skilled in the30 art.

7~l 39~ -8-Sepsis" is herein defined to mean a disease rçsl~1ting from gram positive or gram negative b~rPri~l infection, the latter primarily due to the bact~pr~ ndo~in, lipopolysaccharide (LPS). It can be inducPd by at least the six major gram-negative bacilli and these are Pseudomonas aeruginosa, Escherichia coli, 5 Proteus, K~bsie1~(7, Enterobacter and Serratia.
The terms "prohormone" and "mature" hormone have the following mP~ning~.
UProhormone" is intended to cover pr~tcins that contain a peptide seg,..cl-l which is removed during the in vivo production of the Umature" form of the hormone.
Preferably, these are prl~teins produced at least in part by cells of the immunesystem, such as T-cells or macrophages. The ~Jlcfe.lcd embo~limPnt of the invention is the 26 kD TNF pr~hol",one, or UproTNF" as ~ cu~ed in detail below. ProTNF is cleaved primarily to a 17 kD mature form, p~cfe~bly having the N t~l""nal s~u~ncc of amature TNF", Val-Arg-Ser-Ser. However, Umature TNF" is in~ended to include other cleavage products also formed from the prohormone. These cleavage products will retain the biological char~rtpri~tirs of the 17 kD form of mature TNF, and are truncated (i.e., cleaved) forms of proTNF
wherein at least the N-terminal 76-amino acid leader sequence is removed.
As used herein, UproTNF" refers to TNF having a molecular weight of about 26,000, which is the prohormone form of TNF-a (reference cloned sequence of 20 Figure 1). It is known that the pn~l)cl)tide se~."Pnl of a prohormone varies in length depPnding on the species from which it is derived, but the amino acid sequence of this segment is highly conserved. Indeed, in the mouse, approximately 86% of the 79amino acids that make up the putative leader sequence of the prohormone are i~Pntir~l to the 76 known amino acids that 25 comprise the putative leader of human TNF. Thus, it will be appreciated by those skilled in the art that when rererence is made to proTNF it is intend~P~ that the molecule can be derived from any particular species so that it may have a slightly altered leader sequence collll)~ed to the human sequence as is known in the art.The term "convertase" or "TNF convertase", as used herein, refers to one or more30 enzymes normally present in an animal that are capable of cleaving 26 kD TNF to WO 94/00~55 Pcr/US93/06120 g a mature TNF having TNF biological activity in trimeric form in a TNF bioassay.
In unstimulated cells, a convertase is recovered largely in fractions concictingsubst~nti~lly of ",e",bl~dncs, although some activity is located in the cytosol. A
TNF convertase is normally ~C~ t*~ with cells that produce TNF. One TNF
S convertase is now known to be the serine protease ~Lpr~in~ 3n~ also callcd PR-3n, aP-29Bn or Umyeloblastinn.
The phrase ''~c~b~dne-~cc~~ dll as applied to TNF convertase in~ tes a form of the convertase that is initially isolated in ~ulJsl~.-ti~lly insoluble form, as in~ic~ted by the pr~ ~ncc of much of the cQnvertase activity in a 30,000 x g pellet fraction. However, some TNF convertase is soluble when isolated from neullophil granules.
~Reco",binanl antibody" refers to antibody wnerein one portion of each of the amino acid sequences of heavy and light chain is homologous to collesponding sequences in antibody derived from a particular species or belonging to a particular class, while the rc~.. Aining sPgm~-nt of the chains is homologous to collc~ollding sequences in another. Most commonly, in a recombinant antibody the variable region of both light and heavy chain copies the variable regions ofantibody derived from one species of ,.,A.",.,~l, while the constant regions arehomologous to the sequences in antibody derived from another. One example is 20 "h--."~n;~d" mouse antibody where the cQn~ nl regions of the mouse antibody are replaced with a human coris~nt region.
In its most general form, the instant invention concerns methods and compo~itions for identifying inhibitors of f~ ces ~soci~ with the production of mature hormones from their prohormone forms. The plcre.l~,d embo ~iment of a 25 I"~hGl",one is 26 kD TNF, which is then cleaved to a lower molecular weight "mature" form, prcrcldbly 17 lcD, which, in its mllltim~ric (usually trimeric) form, is substantially involved in producing life~ Pning physiological changes ~o~i~tP.d with sepsis. Thus, molecules which are capable of inlelrcling with theconversion of the 26 kD TNF to the mature form are useful for preventing or 30 treating sepsis.

WO 94/Oosss I Pcr/US93/06120 ?.~39~'~

The assays desrribe~ herein detect the conversion of a prohormone to its mature hormone form, with the plefc.,~d çmbotiimtont being the enzymatic conversion of the 26 kD mol~~ r weight form of TNF to, plcf~ably, a 17,000 moleclll~r weight form. The enzyme ~nsible for the conversion is termed TNF
5 convertasen. Thus, the invention is most readily ~,esenlcd in four parts. Part one shows the materials and methods for re~li7ing proTNF, the 26 kD form of TNF.
Part two identifi~-s sources of TNF-convertase, and metho~s for purifying the enzyme. Part three describes the identific~tion of various convertase inhibitors.
Finally, part four of the invention pn sent~ a desc,;~ltion of ways of using theinhibitors to treat palie~ suffering from sepsis or other ~ s. Each of these s~tion~ will now be addressed sep~lely.
Several patents/patent applie~tion~ and scientific ,~ felcnces are efcllcd to below. The instant invention draws on some of the material and methods shown in these rcfelcnces, and thus it is intende~ that all of the r~fer~nces, in their 15 entirety, be inco,~,~ted by reference.

I.26 kD TNF
The TNF and proTNF of the current invention may be obtained in native, synthetic or recombinant forms by metho ls known in the art. While the 20 recombinant systems described below render the 26 kD proTNF obtainable in considerable amounts and f~cili~t~ the assay procedures for TNF inhibitors, it will be appreciated that nonr~",binant systems may also be used. For in~t~nce~ it has been shown that the 26 kD moko~llle can be identified in stimulated monocytes. Kriegl~.r, et al., 1988, Cell, 53:45. Thus, a suitable assay procedure 25 is to stimulate monocytes to produce the 26 kD proTNF molecule, and then to measure the disa~dnce of the 26 kD molecule as a result of action by the convertase. Preferably the 17,000 molecular weight mature TNF is gencl~ted.
The 26 kD proTNF is cleaved by convertase at one or more internal sites to generate mature TNF". The major site is at the junction which separates the 30 secleted form of TNF (the 17 kD species) from the leader sequence. The Wo 94/00555 Pcr/uS93/06120 21391~7 sequence at this junction is Gln-Ala-Val-Arg-Ser-Ser-. The major cleavage site lies between alanine and valine, since valine is known to be the amino-terminal amino acid of the 17 kD molecule (the primary mature form). Several other species of TNF may be produced by the convertase, and these are the products of S minor or s~con-l~ry cleavage sites: for e-~."ple, between the Val and the Arg in the se luel~ce above, or bet veen Pro and the Val located at + 12 and + 13 in the arnino acid sequence. The assays desclibed herein can monitor the inhibition of the conversion of the 26 kD proTNF speiciP-s or the a~pe~lce of a mature TNF
form i~l-;.yecti~e of its cleavage site.
The proTNF form and mature TNF form have been cloned and ~;A~ ssed in a number of systems. For in~;.nce, the cloning of rabbit TNF is licrlo~P~d in EP
146,026, published June 26, 1985 (Dainippon Pl~ ir~l Co., Ltd.) and EP
148,311, published July 17, 1985 (Asahi Kasei Kogyo Kabushiki). The cloning of human TNF having 151 and 155 amino acids (2 and 6 less than the native mature form) is ~icrlos~P~ in EP 155,549, published Septk~.. ber 25, 1985 (Dainip~on Phal~ tir~l Co., Ltd.), and human TNF having 155 amino acids is ~licclosPd in EP 158,286, published October 16, 1985 (Asahi Kasei Kogyo Kabushiki Kaisha) and coll~;,~nding GB 1,158,829A, published November 20, 1985. The cloning of mature TNF (157 amino acids) and various modifiP~ forms (muteins) thereof is ~ os~P~ in EP 168,214, published January 15, 1986 (Genentech) and PCT US 85/01921, filed October 3, 1985, (Cetus Col~ldlion).
In addition, U.S. Patent Nos. 4,677,063 and 4,677,064 show cDNA sequences that encode the 26,000 and 17,000 forms of TNF, as well as muteins of these molecules.
The cDNA sequence that Pnc~es the 26 kD TNF species is preferably obtained from the pl~cmid, pB11, described in commonly owned co-pending application, U.S. Serial No. 670,360, filed November 9,1984; and U.S. Patent Nos. 4,677,063 and 4,677,064. The pl~cmid pB11 cont~inc the SV40 ~r~lllolel in operable linkage to the TNF coding sequence, and thus is useful for eA~ s~ing the 26 kD
TNF species in eukaryotic host cells. Additionally, a second plasmid which WO 94/00555 ~ PCr/US93/06120 9~

cQnt~inc the entire s~4uence which enrodes the 26 kD TNF species is described inthe forgoing U.S. patent appli~ Q~ and patents. It is dçcign~t~d pE4. The pl~cmi~ pE4 is on deposit with the American Type Culture Collection, Accession No. 39894.
The cDNA sequence that enc~es the 26 kD TNF species is present in the plasmid pBll as a Pstl fr~mP-nt Thus, it is readily removed and inserted into any one ofa n,J",ber of suitable ~ ~pression systems. The pre~.~ e~p~ssion system is the pl~cmid pFVXM, which is ~escrihed in co-pending U.S. Serial No. 855,865, entitled Infective Drug Delivery System, inventor Kri.o.gl.o.r, et al. (~ ndon~.d in favor of U.S. Serial No. 571,017, filed August 22, 1990). pFVXM is on deposit with the A",el;ca~ Type Culture Crll~ction and has Acc~ccion No. 67,103.
pFVXM is a retroviral vector that was derived from the plasmid pEVX described by Kri~.gl.o.r, et al., 1984, in Cell. 38:483. pEVX has a Moloney murine leukPmi~
virus derived splice donor site 3' to the 5' - long terminal repeat. It was previously shown that this splice donor sequence decreases the yield of correctly spliced ~ncl~tional ~mpl~t~.s of retroviral constructions. Thus, pEVX was çngin~.ed to remove the splice donor site, and replaced with an analogous SmaI
fragment of the Harvey murine ~",a virus genome, which lacks the Moloney murine leuk.orni~ virus splice donor sequence. The rçslllting vector, pFVXM, lacks the Moloney murine le~-k~mi~ virus spliced donor sequence and carries a viral p~ ging sequence. pFVXM has a convenient PstI site in which the DNA
sequences that encodec the 26 kD TNF species can be inserted.

II.TNF Convertase TNF convertase activity arises from the proteolytic action of one or more enzymes. A variety of biological materials are available as sources of TNF
convertase activity. These include tissues, cells, or extracts, or fluids ~csoci~ted therewith that are preferably, but not ne~es~.ily, of immunologic origin.
Moreover, estab!ished cell lines may also be utili7~d. Suitable sources would include human peripheral blood mononuclear cells, such as leukocytes or cell lines WO 94/00555 PCr/US93/06120 of leukocyte origin, preferably macrophages and monocytes. NeuL,~,phils are a particularly useful source of TNFconvertase. Re~ c~ of the ease of manipulating established cell lines, one plefe,l~ cell source of TNF convertase is HL60.
Thus, the conversion of the 26 kD proTNF species to mature TNF can be affected by combining the 26 kD species with either intact HL60 cells, extracts derived lllerGrlulll, or media in which the HL60 cells were grown and thus co~t~inc TNF
convertase activity. In some cell types, TNF convertase activity is present in the culture medium after the appr~pliate stimulation, which is ~1iccucced more below.
Further, ber~lse the TNF convertase activity is partially membrane-~c~ ted under certain con~litionc) it is possible to obtain a membrane fraction that may be utilized.
The procedures for i~l~ting monocytes are well known in the art, as are other methods for culturing cell lines such as HL60. Briefly, monocytes may be ~re~aled from p~ liphGl~l blood by centrifugation first through Ficoll-hypaque and Percoll (49.2%) using ~landald procedures. This yieids an enriched population ofmonocytes and lymphocytes, and the monocytes can be further enriched by plating the Illi~lur~ of cells onto tissue culture dishes and incub~ting the cells for a time s--ffiçiont to permit the monocytes to adhere to the surface of the dishes. The lymphocytes are then washed off of the plates leaving primarily adherent monocytes. These cells may then be used as is, or can be stimulated to produce çnh~nce~ levels of TNF convertase using known monocyte activators, preferably lipopolysaccharide and phorbol myristate acetate. The cells may be fractionated,and either an extract or a membrane fraction plGpar~d there~lol-l and employed in the assays described below.
We have isol~ted TNF convertase from 12 liters of HL60 culture by isolating the - cell membrane fraction, solubilizing it in a 0.5% Nonidet P-40 detergent, subjecting the solution to anion eYçh~nge chr~lllatogldl)hy, cation e~ch~nge-HPLC, anion eYc-h~nge-HPLC, and reverse-phase HPLC to yield 20 mg of 1,000-fold purified TNF convertase, which is equivalent to ~320 Units at an 18% yield.
The convertase was found to have a molecular weight of approximately 29-30 kD

~ - 14-by SDS-PAGE analysis (silver-stained). The convertase was sequenced, and the first amino acids were found to be identi~l, within experimental error, to the mature N-terminal sequence of a known neuL,ophil prole;,-~ce, PR-3 (Campanelli et _1., 1990, J. Exp. Med., 172:17091715). The purified convertase was shown S to cleave the 26 kD proTNF to the 17 kD mature form.
As ~les~ribe~ more fully below, the amino acid sequence for PR-3 has been elu~ tP~, as predicted from the sequence of the cDNA clone is shown in Figure 2. PR-3 is known in the art as a protease having activities lmlelated to TNF
pr~cecc;ng. It is clqccifi~d as a human polymol~honuclear leukocyte serine ~llo~inase that de~ra~es elastin, fibronPctin, l~minin, vitronectin, and collagen type IV; sPe Rao et al., 1991, J. Biol. Chem.. 266:954~9548. By SDS-PAGE
analysis purified PR-3 has been reported to have a major band at 26.8 kD with two smaller bands having slightly larger molecular masses, possibly ~e~ sc ~ g dirrer~nt glycosylated spel ies, see Rao et al., supra. PR-3 is structurally similar to other serine pr~leases, such as e1~ct~Ce, c~thPpcin G, mouse granzyme B, rat mast cell prolease II, human Iymphocyte p,~t~se, and chymotrypsin; see C~mp~n~Plli et al., 1990, J. Exp. Med., 172:1709-1715. PR-3 is inhibited by a2-macroglobulin, phenylmethyl-sulfonyl fluoride (PMSF), and alantitrypsin.
SequP-ncing of the PR-3 digPstion products of r~lio~ Pllpcl 26 kD TNF show that PR-3 prefers to cleave the proTNF to produce an N-terminal Val-Arg-Ser sequence (amino acids 1-3 of the 17 kD mature form) although cleavage may occur to produce an N-terminal Arg-Ser-Ser (amino acids 2~) or Val-Ala-His (amino acids 1315) sequences. Rao et al., supra, reports that PR-3 prefers small~lirh7.ti~ amino acids in the substrate cleavage site. Serine proteases such as Pl~ct~ce, c~thepcin G, and plasmin do not efficiently convert the 26 kD proTNF to the 17 kD mature form.
PR-3 may also be i~1~tP~ from neutrophils. Neul~ophils are sel)~d~d from human blood, then granules and membranes are isolated, and the Illixlulc is fraction~t~P~ on RP-HPLC, as described below.

WO 94/00555 2 1 3 9 1 2 ~ PCr/US93/06120 As shown below, PR-3 is inhibited by peptide diphenyl phosphon~te inhibitors, el~tin~l, and dichloro-i~ (DCI). The peptide diphenyl phosphonate inhibitors include Boc-Val-Pro-Val-p(OPh)2 and Boc-Ala-Pro-Val-p(OPh)2.
Boc-Ala-Gln-Alap(OPh)2 and Boc-Leu-Ala-Gln-Ala-p(OPh)2, have also been tested 5 and have much less inhibitory activity. UBoc" means tert-butyloxycarbonyl and p(OPh)2" epf~.ll~ the diphenyl phos~hon~tP moiety, wherein the formula -COOH group is replaced with ~P(=O)(O-phenyl)2. See Oleksyszyn et 1, 1991, Biochem.. 30:485. It will be a~pf~;aled that other peptide diphenyl phosphon~te molecules may inhibit PR-3.
Potential inhibitors may be constructed using the procedures shown in Oleksyszynet 1, supra, using small ~liph~tic peptides~ for an eY~mple. Once the pol~
inhibitors are made, they may be tested in the assays shown below. Modeling studies predict that Boc-Val-Pro-His-p(OPh)2 will be a potent PR-3 inhibitor.

n~.~nhibitors of TNF convertase Activity-Prophylactics or The,ayeulics of SepsisInhiSitors of convertase activity will also be prophylactics or therapeutics that may be used in the ~ nt of sepsis and certain other ~ ces in which circul~ting TNF has been implicated, inclulling rh~.J...~toid arthritis and cachexia. Inhibitors of TNF convertase can be identifi~d by pr~lul~s that enable one to measure the conversion of proTNF to mature TNF. Several such assay procedures are desrribed herein, and in Exal.lple 4 below. A suitable assay would consist of colllbining 26 IcD proTNF, a TNF convertase, and a putative inhibitor. It will be understood by those skilled in the art that the inhibitory material may be added to the convertase before the convertase is added to TNF, or it can be added to TNF
prior to, or immeAi~tely after adding the convertase. The order of addition may - f;~çilit~t~ id~-ntific~tion of inhibitors, but it is not de~llninative. If a subst~nce has inhibitory activity, this can be revealed by electrophoretic analysis of the solution which will reveal, relative to control reaction, an increase in the amount of the 26 kD spe.cies, and concolllilalltly a decrease in mature TNF s~ies. Applicants have also identified a colorimetric assay to detect convertase inhibitors. The assay WO 94/00555 PCr/US93/06120 7~ 1 is convenient and col.~lates with the autoradiographic assay for cleavage of 26 kD
TNF. The colorimPtic assay is dçcn,rihed in detail in Example 4. Also see Kam etal., 1992, FEBS, 297(1.2):119-123.
Other co...pounds with anti-convertase activity include anti-convertase antibody, S either polyclonal or monoclon~l, or recombinant antibody. Preferably these ~ntiho liPs will be hum~ni7Pd antibodies. l~onoclon~l antibody to the convertasemay be produced using the general pr~cedur,_s described by Kohler, G. and ~ilctein, C., 1975, Nature ~:495, which have been m~ifiPd over the years as is known in the art. These initial studies involved fusing murine Iymphocytes and 10 drug ~çkP~t~ le pl~cm~ytomas to produce hybridomas. Subsequently, the technique has been applied to produce hybrid cell lines that secrete human monoc1Onal antibodies. The latter pr~lules are generally described in Abrams, P., 1986, Methods in Enzymolo~y, 121:107, but other mo~ifit~tions are known to those skilled in the art. Regardless of whether murine or human antibody is 15 procluce~, the antibody S~XIc~ g cells are co---bined with the fusion partner and the cells fused with a suitable fusing agent, ~Jlcfclably polyethylene glycol, and more preferably polyethylene glycol 1000. The latter is added to a cell pellet con~ ing the antibody-sec,cting cells and the fusion partner in small amounts over a short period of time acco...p~niP~ with gentle agitation. After the addition 20 of the fusing agent, the cell ...ixlu,~ is washed to remove the fusing agent and any cellular debris, and the cell ".i~lu,c con~ ing of fused and unfused cells seeded into appr~p,iate cell culture c~mbPrs cont~ining selective growth media. After aperiod of several weeks, hybrid cells are appd-c,-t, and may be idçntifi~pd as to antibody production and subcloned to ensure the availability of a stable hybrid cell 25 line.
A p,erc"cd antibody is human monorlonal antibody which can be produced from - lymphocytes sP-n~iti7Pd with convertase either in vivo or in vitro and immortalized as antibody-prod~c-ing hybrid cell lines, thereby making available a renewable source of the desired antibody. In vitro immuni_ation techniques are well known 30 in the art, while in vitro techniques are generally described by Luben, R. and Wo 94/00555 ~ 1 3 9 1 % ~ PCr/USs3/06120 -Mohler, M., 1980, Molecular Immunolo~y, 17:635, Reading, C. Methods in Enzymolo~y, 121 (Part One):18, or Voss, B., 1986, Methods in Enzymolo~y, 121:27. A number of in vitro immunization systems have been shown to be effective for s~c;l;~;ng human B-cells. Reading, C., 1982, J. of Immun.
5 Methods, 53:261.
It will be a~a~cnt to those skilled in the art, that in lieu of immunizing individuals directly with TNF convertase, lymphocytes may be isolated from individuals that are c~ iencing~ or have eA~.ienced a bacteremic attack. For example, human p~tiPntc having Wegener's granulomatosis are natural source of antiPR-3 10 antibodies and also contain human cells suitable for deriving human monoclonals.
A fraction of these ly.,.l,hocytes will be s~ d to the convertase and may be used to produce pel---anent antibody-secreting hybrid cell lines. For e~mpl~, immunoco...pro...ised human pa~ient~ are generally susceptible to bacterial infections, particularly those suffering from various m~lign~ncies, extensive burns, 15 etc., and lymphocytes ico!~tp~ th~.crlom may be a sour e of antibody s~reling cells.
Senciti7~Pd lymphocytes can be immortalized by viral tran~folmation. The prcfe~cd vi al tran~fo~ ation technique for human lymphocytes involves the use of Epstein-Barr virus. The virus is capable of tran~fo~,lling human B-cells, and20 has been used to geneldte 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 procedure whereby s~nciti7pd lymphocytes may be immortalized consists of a combination of the above two techniques, that is viral tran~follllalion and cell 25 fusion. The prcrcllcd co",bindtion consists of transr~,l.l~ing antibody-secreting cells with Epstein-Barr virus, and subse luenlly fusing the transformed cells to a suitable fusion partner. The fusion partner may be a mouse myeloma cell line, a helcro,l,yeloma line, or a human myeloma line, or olher immortalized cell line.
PCT No. 81/00957; Schlom et al., 1980, PNAS (USA), 77:6841; Croce _ al., 1980, Nature, 288:488. The prcfcllcd fusion partner is a mouse-human WO 94/00555 Pcr/US93/06120 ?,~3~

hetero-hybrid, and more prGf~l~d is the cell line decign~t~ F3B6. This cell lineis on deposit with the A...ç. ;ç~,l Type Culture Coll~tion, Accession No. HB8785.
It was depoc;l~d April 18, 1985. The pr~lu~Gs for genG.~ting F3B6 are dec-~rihed in EPA No. 174,204.
S Techniques applicable to the use of Epstein-Barr virus tran~r(",..a~ion and the production of immortal antibody s~lGIing cell lines are p~sented by Roder, J. etal., 1986, Methods in Enzymolo~y, 121:140. R~cir~lly, the ~l~lulG con~i~tc of icol~tine Epstein-Barr virus from a suitable source, generally an infected cell line, and exposing the target antibody-secreting cells to s~l~,-,atant~ c4nl~ining the10 virus. The cells are washed and cultured in an a~,op,iate cell culture medium.
Subs~uen~ly, virally transformed cells present in the cell culture can be idP-ntifi~d by the presence of the Epstein-Barr viral nuclear antigen, and transformed antibody-sec,c;ling cells can be idçn~ifi~ using standard metho~s known in the art.
It will be appa~nt to those skilled in the art that while a plefelled embodiment of 15 the instant invention is a neutralizing anti-TNF convertase monoclonal antibody, singly or in combination, that the antibody(s) may be altered and still ~ inl~;nbiological activity. Thus, enco...p~c~d within the scope of the invention is antibody mo~ifie~d by reduction to various size fragments, such as F(ab')2, Fab,Fv, or the like. Also, the hybrid cell lines that produce the antibody may be considered to be a source of the DNA that encodes the desired antibody, which may be isolated and transferred to cells by known genetic techniques to produce genetic~lly en~in~ d antibody. An example of the latter would be the pr~duction of single-chain antibody having the antibody combining site of the hybridomas described herein. Single-chain antibodies are described in U.S. Patent No.

4,704,692. A second example of gçnetir~lly engin~Pred antibody is recombinant, or chimeric antibody. Methods for produçing recombinant antibody are shown in U.S. Patent No. 4,816,567, to Cabilly, et al.; J~p~nese Patent Application No.
84169370, filed August 15, 1984; U.S. Serial No. 644,473, filed August 27, 1984; British Patent Application No. 8422238, filed on September 3, 1984;
J~p~nese Patent Application, No. 85239543, filed October 28, 1985; U.S. Serial 5 2 1~ 7 PCr/US93/06120 No. 793,980 on November 1, 1985; U.S. Serial No. 77,528, filed July 24, 1987.
Also, British Patent Appl~ tion No. 867679, filed March 27, 1986 describes metho~ls for producing an altered antibody in which at least parts of the compl~-n~nt~ry detel.l.ining regions (CDRs) in the light or heavy chain variable5 domains have been lci~laccd by analogous parts of CDRs from an antibody of dirr~ t ~peçificity. Using the pr~cedu~s described therein, it is feasible to construct lcco--lbinant antibody having the CDR region of one species grafted onto antibody from a second species that has its CDR region re~l~^P~l. The prcÇellcd e--lbo~ nl in this in~t~nre is a murine anti-convertase antibody CDR region that replaces the CDR region of human antibody.
In addition to antibodies, co...l~o~ s that colllpcle with 26 kD proTNF for binding to the convertase will inhibit or reduce the conversion of 26 kD proTNF to the mature form, and may thus be useful m~irs~ "t~ for treating sepsis and other ces. One such class of reagents consists of peptides, polypeptides, or proteins, or other colll~unds synthetic, or naturally occul~ing, that have TNF
convertase-binding activity similar to or better than the 26 kD proTNF. ~cfcl~cdpeptides or proteins are those that contain amino acid sequences similar to thatfound at the junction between the 76 amino acid leader sequence of proTNF and the 17 kD mature form. On such sequence is Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser, where the second Ala is the residue presenton the leader r~ ining in the Illelllblane after the cleavage event, and Val is the N-terminal amino acid of the matl re TNF. It is ilu~~ l to note that while the se~uence is shown to consist of nine amino acids, that what is minim~lly intended is a peptide con~ining at least the dipeptide sequence Ala-Val that is recogniædby the convertase.
An alternate embo iimçnt of a peptide/protein convertase inhibitor is one that has the amino acid sequence, a sequence that is function~lly similar to (SEQ ID NO:
1). This peptide spans two TNF convertase cleavage sites, and thus would preventthe formation of the 17 kD mature TNF, among others. The first and dominant cleavage site is between alanine and valine at positions -1 and +1; and secondary WO 94/OOS55 PCr/US93/06120 ~ 39~?q 20-sites are between valine and ~inine at pocitionc + 1 and +2, and proline and valine at pocitionc + 12 and + 13. These pOSitir~nc CGll~ ~.pond to the amino acid s~tu~.-ce shown in Figure 1.
S A second class of cc.~ e inhibitors concictc of cc,---~.unds including thesequence shown above, that is (SEQ ID NO: 1), but wherein certain amino acids have been altered or deleted to yield a non-cleavable substrate. A prerellc;d embolim~nt of this peptide is a 26 kD proTNF mutein produced by standard site-specific mutagenesis techniques. Most p~fe.led is a mutein wherein the (-1)10 alanine or (+ 1) valine or both are ~ l.sl;tuled or deleted.
The peptides described above can be made by techniques well known in the art, such as, for example, the Merrifield solid-phase method described in Science, 232:341347 (1985). The plucedure may use commercially available synthPci7Prs such as a Biosearch 9500 aulu-l.at~d peptide m-~hine, with cleavage of the blocked 15 amino acids being achieved with hydrogen fluoride, and the peptides purified by prepa~li~e HPLC using a Waters Delta Prep 3000 instrument, on a 15-20_m Vydac C4 PrepPAK column.
The peptide diphenyl phosphonates described above are also used as inhibitors.
Useful peptides may be ~tt~h~d to Boc and the diphenyl phosphonate moiety (see Oleksyszyn et al., 1991, Biochem.. 30:485) and tested in a convertase inhibition assay. Pl~felled peptides are Boc-Val-Pro-Val-p(OPh)2, Boc-Ala-Pro-Val-p(OPh)2, and BocVal-Pro-His-p(OPh)2. However, it will be seen that other peptide diphenyl phc.s~hona~s may be used in the inhibition assays described below to identify further TNF convertase inhibitors. Examples are disclosed below and are shown in Oleksyszyn et a1., 1991, Biochem., 30:485.
The C~ificity of the i(lentified TNF convertase PR-3 is similar to enzymes such as ~l~ct~ce~ which typically cleave immedi~ly following certain neutrally charged amino acids, such as between valine, proline, and alanine re~id~lec. Thus, in addition to the peptide inhibitors mentioned above, a variety of other inhibitors known to inhibit el~ct~oe may also generally inhibit an enzyme that cleaves the 26 WO 94/00555 2 f 3 9 I 2 7 Pcr/US93/06120 kD proTNF. Those coll,pounds that inhibit TNF convertase can be identified using the assays ~esr-ribed below. A variety of el~ct~cP inhibitors are commercially available from suppliers such as Roehrin~or ~nnh~im R~ mic~ls, or are known in the art. Doherty, et al., 1986, Nature, ~:192;
U.S. Patent Nos. 4,711,886; 4,797,396; 4,717,722; and 4,699,904. The pl~Çelled el~ct~e inhibitors are mo~ifi~d ce~h~lo~orin antibiotics, such as those shown byDoherty, et 1, above. More p~-e.l~d is (1 -((3-((acetyloxyl)-7-methoxy-8Oxy-8-oxo-5-thio- 1 -azabicyclo t4.2.0] oct-2-en-2-yl) carbonyl) morpholine, S,SdiQxi~e~ (6R-cis). Also, Stetler, et al., 1986, Nucleic Acids Research. 14:7883, describe a cDNA clone that codes for an inhibitor of neull~hil el~ct~ce. However, p~f~.~c;d inhibitors are those which inhibit PR-3 more effectively than el~ct~ce~ since el~ct~ce activity may help ameliorate septic shock by, for example, degrading circul~ting TNF or rel~cing soluble TNF lcceptol~ which, in turn, inhibit circul~tin~ TNF. (See Scuderi, 1991, Cellular Immunolo~y~ 135:299-313).
Additionally, inhibitors may be found by modeling the crystal structure for PR3 by adapting the known structure for the closely homologous el~ct~ce molecule.
Co~ )uler models known in the art may be constructed to establish il"~,~nt contact points in the substrate-binding site of PR-3. Potential inhibitors may be de-si~n~.d based on this infolllldLion and then tested in the present assay systems, as well as in relevant animal models for septic shock.
Reco."binallt techniques may be used to obtain the inhibitors, the proTNFs, mature TNFs or TNF convertases described herein. Most of the recombinant techniques that are described herein that may be used to transform cells, fabricate vectors, extract .. ~ s~nger RNA, and the like are widely practiced in biotechnology and most practitioners are f~mili~r with the standard m~t~ri~lc and methods employed. However, for convenience, the following paragraphs are offered as a guideline.

30 A.General Clonin~ Techniques WO 94/005~5 PCr/US93/06120 39~ - 22-Construction of suitable vectors COI~A;n;I-g the desired TNF coding s~uence employs standard ligation and restriction techniques which are well understood in the art. T~9lqtP~ vectors, DNA se~u~nces, or syntheci7pd oligonucl~P~tides are cleaved, tailored, and rçlig~qtpd in the form desired.
S Site-specific DNA cleavage is pelrul"~ed by treating with suitable restrictionen_yme(s) under cQIlAitionc which are generally understood in the art, and the particulars of which are spe~ifiPd by the mqnufqc~rer of these co",.,.crcially available restriction enzymes. See, e.g., New F.nglqnd Biolabs, Product Catalog.In general, about 1 g of plqcmid or DNA se~luence is cleaved by one unit of 10 enzyme in about 20 _1 of buffer solution. In the examples herein, typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA
substrate. Tncll~qtiQn times of about 1-2 hours at about 37 C are workable, qlthough variations can be tr'er~q-~Pd. After each inrubqtion, protein is removed by extraction with phenol/chlororol..., and may be followed by ether extraction, and 15 the nucleic acid recovered from aqueous fractions by pr~ipildtion with ethanol followed by chlulllalogld~hy using a Sephq-~eY G-50 spin column. If desired, size separation of the cleaved fragments may be ~lro~---ed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymolû~y, 1980, 65:499560.
20 Restriction cleaved r,Ag... ~ may be blunt-ended by treating with the large fragment of E. coli DNA polymerase I, that is, the Klenow frAgmPnt in the presence of the four deoxynucleotide tripho~hates (dNTPs) using incubAtion timesof about 15-25 ~..inul~s at 20-25 C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM
MgC12, 6 mM DTT and 10 mM dNTPs. After ~ ..æl-t with Klenow, the Illixlulc is extracted with phenol/chlon~folln and ethanol l~lc~;piL~ d. Treatment under a~r~liate conditions with S1 nuclease results in hydrolysis of single-stranded portions.
Ligations are pelrul,--ed in 15-30 1 volumes under the following standard conditions and te-~ ures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM
DTT, 33 _g/ml BSA, lO mM-50 mM NaCl, and 1 mM ATP, 0.3-0.6 (Weiss) WO 94/00555 2 1 3 g :~ 2 7 Pcr/US93/06120 units T4 DNA ligase at 4-C for "sticky end" lig~tion, or for "blunt-end" ligations.
Intermoloc~ r "sticky end" lig~tiQnc are usually ~ ro~ ed at 33-100 g/ml total DNA conc~ntration. In blunt-end lig~tionc, the total DNA concentldtion of the ends is about 1 M.
S In vector construction employing "vector fr~gm~ntc," the vector fragment is commonly treated with bacterial ~ linP phosph~t~e (BAP) in order to remove the 5' phosph~te and prevent religation of the vector. BAP digestions are cQndllct~ at pH^ 8 in appr~ y 150 mM Tris, in the p~nce of Na+ and Mg~2 using about 1 Unit of BAP per g of vector at 60 C for about 1 hour.
Nucleic acid fra~m~ntc are recovered by extracting the prep~d~ion with phenol/chlolofo~l,l, followed by ethanol precipi~tion. Alternatively, religation can be prevented in vectors which have been double-digested by additional restriction enzyme digestion of the unwanted fragm~ntc.
In the constructions set forth below, correct lig~tionc are confirmed by first transforming the a~,~p,iate E. coli strain with the ligation Il~ u~c. SuccessfulL,ansru",lants are s~l~ct~d by rçcict~nce to ~mpicillin, tetracycline or other antibiotics, or using other Ill~kel~ depen~ing on the mode of plasmid construction, as is understood in the art. Miniprep DNA can be prcp~cd from the tran~Ço""anls by the method of Ish-Howowicz et al., 1981, Nucleic Acids Res., 2:2989, and analyzed by restriction and/or sequenced by the dideoxy method of Sanger et al., 1977, PNAS (IJSA), 74:5463 as further described by Messing et al., 1981, Nucleic Acids Res., 2:309, or by the method of Maxam al., 1980 Methods in EnzymoloFy, 65:499.
Host strains used in cloning in M13 consist of E. coli strains susceptible to phage infection, such as E. coli K12 strain DG98. The DG98 strain has been deposited with ATCC July 13, 1984 and has Accession No. 1965.
Depe-n-ling on the host cell used, tran~Çu,lllation is done using standard techniques appç~p,iate to such cells. C~kil~m treatment employing calcium chloride, as described by Cohen, 1972, PNAS (USA) 62:2110, or the RbCl2 method described by Maniatis et al., 1984, Molecular Clonin~: A Labo,~to,y Manual, Cold Spring Wo 94/00555 Pcr/US93/06120 9~ 24-Harbor Press, p. 254, may be used for procaryotes. Transfection may also achieved using a mo lific~tion of the calcium phosph~te pl~ ip;~ ion technique of Graham et al., 1973, Virolo~y, 52:456 or Wigler et al., 1978, Cell, 14:725.

5 B.Oli~onucleotide Probes Synthetic oligonucleotides are p.c~cd by the triester method of M~tteucci et al., 1981, J. Am Chem. Soc., 103:3185 or using co....ner~;ally available au~o.l.a~ed oligonucl~tide syntheci7prs. Kin~cing of single strands prior to ~nnP~ling or for eling is achieved using an excess, e.g., approyim~t~ly 10 Units of 10 polynucleotide kinase to 0.1 nmole s~lbsl,dte in the presence of 50 mM Tris, pH
7.6, 10 mM MgCI2, 5 mM dithiothreitol, 1-2 mM ATP, 1.7 pmoles g32P-ATP
(2.9A mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.

C.Muta~enesis 15 Mutagenesis can be carried out using any numba of procedures known in the art.
These techniques are described by Smith, 1985, Annual Review of Genetics, 19:423, and morlifir~tions of some of the techniques are described in Methods inEnzymolo~y, 154. part E, (eds.) Wu and Gn~ss.-lal (1987), cha~lel~ 17, 18, 19, and 20. The plcfcll~d procedure is a mo~ifir~tion of the gapped-duplex 20 site-directed mutagenesis metho~. The general procedure is described by Kramer et al., in chapter 17 of the Methods in Enzymolo~y, above.
Conventional M13 mutagenesis metho~c involve ~nn~ling a short synthetic oligonucleotide to single stranded M13 DNA having a cloned target coding sequence that is sought to be mutagenized. The oligonucleotide is almost, but not entirely comple .. ti1. y to the target sequence and has at least one mispaired nucleotide. After the ~nne~ling reaction, the r~ ini~-g portion of the single stranded DNA must be filled in to give heler~xlul.lex DNA that can be transfected into a suitable host cell which allows for the eAplession of the mutation. In the gapped-duplex method, a partial DNA duplex is constructe~d that has only the 30 target region exposed, unlike the conventional methods which have the target WO 94/00555 Pcr/US93/06120 2l39l27 region and the rest of the single-stranded M13 DNA exposed. Like the convehlional m~th~c, a short oligonllcl~tide is ~nne~led to the target region, and eYt~-nded and ligated to produce a heterodupleY. However, be~..cf only a small portion of single-stranded DNA is available for hybri.li7~tion in the gappedduplex 5 method7 the oligonucleotide does not anneal to undesired sites within the M13 genome. Further, this method has the ad-1ition~l advantage of introducing fewer errors during the formation of the heteroduplex since only a very small region of DNA on either side of the target region has to be filled in.
More sperifir~lly, the gapped-duplex method involves cloning the target DNA
sequence into an al)pn~pliate M13 phage that carries sPle~t~hle ~alk~l~, such asfor example the stop codon amber mutation. The latter allows for negative selection in a host cell that cannot ~upprcSS the effects of the mutation. Preferably the phage is M13mp9 which c~nt;~ two amber codons in critical phage genes.
Thus, the sequence that encodes 26 kD TNF is cloned into M13mp9 amber+, and single-stranded DNA is plC~)alCId til~rÇUIll using standard techniques. Next, double-stranded replicative form DNA from M13 GAP, a g~netic~lly engine~red M13 derivative that lacks the amber codons is cleaved with HincII restriction enzyme. The base sequel-ce of M13 GAP is similar to M13mpl8, which lacks both the amber codons and the sequence between base pairs 6172 and 6323. This 20 deletion flanks the multiple cloning sites of the M13mp series and gencl~tes a unique HincII site. Gapped-duplex DNA is formed, using standard DNA/DNA
hybridization techniques, concicting of cinglestranded DNA having the amber codons, and a second strand of DNA from HincII digested M13 GAP lacking both the amber codons and the TNF coding sequences. Thus, the only portion of the 25 gapped-duplex that is exposed is the 26 kD TNF target sequence. The desired oligonucleotide is ~nne~1ed to the gapped-duplex DNA, and any r~ ining gaps filled in with DNA polymerase and the nicks sealed with DNA ligase to produce a heler~lu~lex. The latter is transfected, preferably ir.to a micm~t~h repair deficient host, and mixed phage produced. From the mixed phage population, phage 30 carrying unmutated 26 kD TNF DNA, which also have the amber mutations, can WO 94/00555 9 ~ PCr/US93/06120 be se~ted against by infecting the mixed phage population into a host cell that cannot ~uppless the amber mut~tion. Clones can then be screened for phage that carry the desired TNF mutation.

WO 94/00555 Pcr/uS93/06120 2139~7 IV.Methods of Use of TNF Convertase Inhibitors Co,.,pounds idPntifiP~ as having TNF convertase-inhibitory activit,v will also have prophylactic or Ihcld~cu~ic appli~tionc in the l,e~ n~ of sepsis. Rec~llse the onset of sepsis is ~oe:~ with an incf~ in circ--1~ting mature TNF, these 5 inhibitors may be used prophyl~-ti~lly in those in~l~n~es where there is a risk of b~tPri~l infection, particularly in a pre-operative setting. Similarly, when there is an early ~i~nosic of sepsis, the inhibitors will have benPfi~i~l thc~dpculic effects in ~ulJs~ lly reduçing the amount of the soluble, 17 kD form of TNF that is produc~d.
Incç~sRs in circul~ting mature TNF are also ~ccoci~,~d with the 1iCP~CP,c rh~.. ~o;(l arthritis, c~chPxi~ cerebral malaria and graft-versus-host ~licp~ceThus, the inhibitors of this invention will also have useful prophylactic or ~u~;c applications in the l~ of these licp~ces~
Another mP~ l applic~tion for inhibitors of convertase is for the l,e~ -t of AIDS. It has been shown that TNF causes the activation of latent human immunodçficiPncy virus. Folks et al., 1989, PNAS (USA). 86:2365. Thus, pfe~enting or inhibiting the formation of mature TNF, by inhibition of TNF
convertase would be a valuable tre~tm~-nt for AIDS, and would preferably be usedto treat p~ti~nt~ that are infected with the virus that is in a latent phase.
20 The inhibitors of this invention may be ~rlmini~tered at concerlldlions that are thel .peuli~lly effective for prevention of sepsis, AIDS, etc. To accomplish these goals, the peptides and chemic-~l compounds are ~minict~red pa~ntel~lly (i.e., via intravascular [intld~lial or intravenous], intramuscular, or subcut~n~us routes). Metho~s to accomplish this ~rlminictration are known to those of ordinary 25 skill in the art.
- Before ~iminictration to ~l;. ~.t~, formul~ntc or pharm~r~utic~lly acceptable excipients may be added to the peptides and çhernic~l coll.pounds. A liquid formulation is ple~lled. For example, these formulants may include oils, polymers, vit~min~, carbohydrates, amino acids, buffers, albumin, surfactants, or 30 bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as WO 94/00555 Pcr/us93/06120 ~9 28-mono-, di-, or polysaccharides, or water soluble gluc~n~. The saccharides or glucans can include fructose, dcA~ose, lactose, glucose, mAnnose~ sorbose, xylose, m~ltos,e" sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcelloluQse, or ~ lules thereof.
S Sugar alcohol is defined as a C4 to C8 hydlu~l,on having an -OH group and includes gAl~ctitol, inositol, ...Ann;lol, xylitol, sorbitol, glycerol, and arabitol.
~nnitol is most p~crcllcd. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous ~lc~dtion.
Prcr~.dbly, the sugar or sugar alcohol concentration is between 1.0 w/v% and 7.0w/v%, more preferable between 2.0 and 6.0 w/v%. Plc-e.dbly amino acids include levorotary (L) forms of carnitine, a,~inil-e, and betaine; however, other amino acids may be added. Plc~cllcd polymers include polyvinylpyrrolidone (PVP) with an average mol~ulAr weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also p~c~cllcd to use a buffer in the cGIllposilion to minimi7e pH
changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used, but citrate, phosphate, succinAte~ and glutamate buffers or Il~ lurcs thereof are pmcfcllcd. Most ~lc-cllcd is a citrate buffer.
Preferably, the concentration is from 0.01 to 0.3 molar. Surfactants that can beadded to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, the present peptides and c~Pmiç~l co.,.pol...ds can be c-hemir~llymo lified by covalent conjugalion to a polymer to increase their circul~ting half-life, for example. P~c~cllcd polymers, and methods to attach them to peptides, are shown in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546 which are all hereby incol~ldted by reference in their entireties.
P~cfe~fcd polymers are polyoxyethylated polyols and polyethylene glycol (PEG).
PEG is soluble in water at room ~",peldlule and has the general formula:
R(O-CH2-CH2)"O-R where R can be hydrogen, or a pn)tecli~e group such as an alkyl or alkanol group. Preferably, the plotecli~e group has between 1 and 8 WO 94/00555 2 1 3 9 1 ~ 7 Pcr~US93/06120 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably bclween 1 and 1,000, more preferably between 2 and 500. The PEG
has a p~cfellcd average mo1~cul~r weight between 1000 and 40,000, more preferably between 2000 and 20,000, most plcreldbly between 3,000 and 12,000.
5 Preferably, PEG has at least one hydroxy group, more l"cfeldbly it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor.
Water-soluble polyoxyethylated polyols are also useful in the present invention.They include polyoxyethylated sorbitol, polyoxyethylated glucose, 10 polyoxyethylated glycerol (POG), etc. POG is p,cfc"cd. One reason is because the glycerol backbone of polyoAycLhylated glycerol is the same backbone occurring naturally in, for el~ml)le, ~nim~lc and humans in mono-, di-, triglycerides.
Thel~fo~e, this br~nching would not l-~sc~.ily be seen as a foreign agent in thebody. The POG has a pre~ d molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, I. Bio. Chem.
263:15064-15070, and a ~liccuccion of POG conjugates is found in U.S. Patent No.4,766,106, both of which are hereby inco~ ted by reference in their entireties.
After the liquid pharm ~xuti~ ~l co""~o~ilion is p~pa~ed, it is preferably lyophili7ed to prevent degradation and to preserve sterility. Methods for 20 lyophili7ing liquid co"~posilions are known to those of o~in~ y skill in the art.
Just prior to use, the composition may be reconctituted with a sterile diluent (Ringer's solution or sterile saline, for example) which may include additional ingredients. Upon reconstit~tion~ the composition is preferably ~minictered to subjects using those metho ls that are known to those skilled in the art.
25 Insoluble inhibitors can be formulated by co"lbination with one or more solubili_ers. Prere.led solubilizers include: ethanol; oils, such as corn oil; PEG;
propylene glycol; and non-ionic surfactants. Preferred co-solvents have a molecular weight between 50 and 1,000, more preferably between 100 and 600.
Preferably their concentration is between 1 and 75% w/w, more plcr~ldbly between 10 and 50%. The concentration of ethanol is preferably between 0.I%

Wo 94/00555 Pcr/uS93/06120 and 20%, more p c;r~ldbly bc;lween 1 and 5%. Prefe,lcd non-ionic surfactants have a hydrophile-lipophile balance between 14 and 40, more preferably belween 15 and 20, most p~ere dbly between 17 and 19. Preferably, the non-ionic surf~ct~ntc have a mo!ccul~r weight in the range between 100 and 250,000, more preferably between 4,000 and 200,000, most preferably between 6,000 and 150,000. ~ef~dbly, the non-ionic surf~ t~ntC are effective in the concentration range of 0.005% to 10% w/v, more preferably in the range of 0.01 to 5% w/v, most plerc,dbly in the range of 5 to 2.5% w/v. P~cfe,dbly, the non-ionic surf~ t~ntc include those ~"~I"only used in the pharrn~r~uti~l, food, and cosmetic industries ~lef~lled non-ionic surf~t~ntc include: polyoxyethylene sûlbi~l fattyacid esters (i.e., Tweens), polyethylene glycol esters, polyethylene fatty acid esters, block copolymers of ethylene oxide and propylene oxide (i.e., Pluronics), ethylated fatty alcohol ethers (i.e., laureth-12), octylphenoxy polyethyoxy ethanol co...l)ou,-dc (i.e., Tritons), and polyoxyethylated castor oil (i.e., Cremophor).
15 These non-ionic surf~t~ntc can be produced by means known in the art or purchased from commercial suppliers.
Other non-ionic surfactants can be de~l~incd by using the present scr~ning m.othod. In this method a non-ionic surfactant is added to an insoluble inhibitor.
The res--lting solution is mixed or homogenized and allowed to stand for 24 hours 20 at room ~",peldlure. lf the inhibitor remains in solution, as measured by RP-HPLC, GC, or visual or ~ ,upllotol~cllic clarity, then the surfactant is useful to solubiliæ the inhibitor.
Having generally described what the applicants believe their invention to be, prese.lled below are examples that are illustrative of the scope of the invention. It 25 will be appreciated by those skilled in the art that the examples are nût intended to be construed as limiting the invention to the materials and methods shown as there are nu",eluus substitutions that can be made therein without departing from the scope of the invention.
Example 1 30 Isolation and Identification of a TNF Convertase WO 94/00555 PCr/US93/06120 213~1Z7 HL60 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in T-175 flasks con~ ing RPMI 1640 medium supple-nçntçd with 20% fetal bovine serum (GIBCO) and L-glut~mine R~t~hP~s totalling 3 liters of HL60 cells were grown to c~nfl~Jency and harvested. The cells were S resuspendçd in a~r~ Ply 120 ml of a hypotonic buffer and lysed by ni~loge cdvitation (400 psi, 30 ...in~l~S at 4 C). The ho,.,ogendle was centrifuged at 10,000 x g for 10 minutp~s~ and both the ~LIpellld~l and the cell debris pellet were stored at -20 C.
HL60 cell debris from 3 batches of HL60 cell cultures were thawed in 250 ml of 10 mM Tris pH 8.5 collt~ining 0.5% NP-40, 5 mM EDTA, and 2 mg/ml l~upep~ (DEAE buffer) and dialyzed for 4 hours in the same buffer. The protease inhibitors used during purification were shown to have no effect on theconvertase activity detec~ed in HL60 lysdtes. Particul~tes were removed by centlifugation (10,000 x g, 10 IllillU~S) and the sample fractionated by anion 15 ~xch~nge chromatogldphy on a DE~AF~e~h~.use column (2.6 x 21 cm, Pharmacia) eluted with a 680 ml, NaCl gradient from 0-0.8 M. Fractions contdil-ing TNF
convertase activity were identified throughout the purification using the 35S-proTNF convertase assay. Pooled DEA~ fractions were dialyzed into 20 mM
sodium phosph~tto- buffer, pH 6.5, conl;~;ning 01 % NP-40, 1 mM EDTA, and 1 20 mg/ml leupeplin, divided into three equal portions and each subjected to cation excl~nge HPLC on a TSK-SP-SPW column (7.5 x 75 mm, BioRad) eluted with a 45-minute, sodium chloride gr~dient from 0-0.6 M. Fractions enriched in convertase activity were pooled and dialyzed into DEAE buffer CO~t;~;nillg 0.1 %NP-40. The pooled m~t~ri~l from the SP column was divided into three portions, 25 and each was subjected to anion exchange HPLC on a TSK-DEAE-5PW column (7.5 x 75 mm, BioRad) eluted with a 45-minute, sodium chloride gradient from 0-0.6 M. The pool of convertase activity was further purified by RP-HPLC on a Vydac C4 column using an ~eton;t.ile/0.1% TFA mobilephase.
This ~ ellt provided a 1,000 fold purification, resl-lting in 20 mg of convertase (approximately 320 Units) at an 18% yield. Fractions from the RP-HPLC were WO 94/00555 PCr/US93/06120 2~9~ 32-tested for c4nvertase activity and sized on SDS-PAGE. The fraction that c4~ I;.;n~d convertase activity conl~ined a protein having a molecular mass of appnJ~ P~y 28-31 kD. The convertase was s4u~ nced, and an 18-amino-acid sequence at the N-terminus proved to be idçnti~l to that of the serine protease 5 PR-3. PR-3 was subsequently i~l~t~d from human ncuLIopllils, essf3-~;~11y using published procedures, and it was found to have the same activity as TNF
convertase in the 35SproTNF assay.
The idçntifil~tion of PR-3 as a TNF convertase was further strengthened by Nlc~ al sequçnring of cyanogen bromide cleavage fr~gm~nt~, as well as amino acid co",position of PR-3 both of which agreed (within ~ error) with the published amino acid sequence of mature, active PR-3 (C~ nçlli et al., 1990, J. Exp. Med., 172:1709-1715).

E~alllple 2 Clonin~ and Recombinant Expression of Human PR-3 RNA was purified from HL60 cells and a cDNA library was constructed in the plasmid pGEM. Construction of the cDNA used C tailing of cDNA and G tailing of the vector, then ligation into the plasmid (Gene Transfer and Expression, 1990, pgs 114135). Clones were scrccned using a unique oligonucleotide probe derived from the known sequence of myeloblastin (Bories et al., 1989, Cell, 59:959-968).Sequçn~ing of one clone MY17 was pc,rol",ed using plasmid double-strand sequen~ing and the Sequenase kit and an aulo",ated ABI sequencer. Sequence for MY17 is shown in Figure 2. Novel fcalulcs for the sequence include 5 nucleotide dirrtlc"ces from the original publication by Bories et al., 1989, Cell, 59:959-968, and three nucleotide differences from the C~mr~nçlli et al., 1990, J. Exp. Med.,172:17091715. Ad~lition~l 5' sequence and an ;~-lition~l 5' methionine coding sequence was found. The two carboxyl terminal amino acids in PR-3, arginine and proline, are similar to that of Bories et al., supra, but differ from the glycine and proline sequence from C~mp~n~ et al., supra.

WO 94/00555 2~ 3~1 2 7 Pcr/US93/06120 Transient mqmmqliqn eAp~ssion of PR-3 was pclr~ ed by cloning the l.0 Kb HinIII-EcoRI PR-3 fragment from MY17 into the PstI site of SR-a vector. COS
cells were ~ e~ Y transfected using the DEAE/Dextran method Kri~ler, l990, Gene Transfer and Expression, pp. 99-lO0, Stockton Press. Transient eAylcssion revealed low levels of PR-3 eAy~ssion by Western blot analysis. PR-3 was mutagenized to oylillliLe its eAp-ession in ",q~ ,qliqn, bacterial and insect c Ayl~ssion systems. The PR3 gene in the pGEM vector was mutagenized using oligonuç1e~tide difec~d mutagenesis. Two constructs were made; A) delta ogen PR-3 and B) delta signal peptide PR-3.
A)Delta zymogen PR-3 was made using an oligonucleotide that deletes the codons for amino acids at position -1 and -2 (gllltqmic acid and qlqnine, ~i,ye.~ ely).This gene c. n be removed from pGEM by EcoRI digestion, and the gene transferred to SR-a for transient m~mm~ n eAyl~ sion and pcDNA I for production of stable tl~re~nls.
lS B)Delta signal PR-3 was made using an oligonucleotide that deleted the leader and added an ATG prior to the position l isohPuçine of the mature protein. This genecan be removed from pGEM by EcoRI digestion, and transferred to SR-a and pcDNA I for transient and stable m~mm~ n cAyn~ssion. In addition, this construct was placed in DG160 a I Pl based bacterial eAyue~ion vector at 8-12 nucleotides from the Shine-Dalgarno ribosomal binding site.
Another construct, the cec-opin B PR-3 construct, was made so that the insect leader for cec-opin B was placed before the position 1 isoleucine of the mature PR-3 protein. This was placed in the insect vector, pAcC13.
D)For optimization of bacterial cAylc~sion~ mutagenesis of the third nucleotide from a purine to pyrimidine in the codons for the first 2-8 amino acids of deltasignal PR-3 was pelÇol",ed using ovcllapying synthetic oligonucleotides and polymerase chain reaction amplification of the synthetic fragment. This fragmentwill be cloned into the 5' smaI site of PR-3, to decrcase the GC content of the 5' RNA and facilitate CAyl~ ssion.
Example 3 WO 94/00555 Pcr/US93/06120 g~ 34-Conversion of 26 kD proTNF to Mature TNF
The vector pFVXM, on deposit with the ~m~ n Type Culture Collection, Accecsion No. 67,103, was used to produce a vector pFVXM-TNF6, which co~.~;nC the DNA sequence that enc~les the 26 kD TNF species To produce the S latter vector, the p1~mid Bl l which cont~inc the cDNA sequence that encodes the 26 kD TNF species was treated with ~I, which excises the coding s~u~nce.
The f~gment was purified using standard ele;~hor~tic techniques. Next, the vector pFVXM was treated with PstI, and the PstI fragment from pBl l cont;~ g the 26 ld~ coding sequence was insel ~d into the polylinker region of the vectorusing 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 1, 1988, above, or as described in U.S. Serial No. 395,254, entitled "Cleavage Site Rloc~ing Antibody to Pluh~ one Proteins and Uses Thereof,"
filed August 16, 1989.
TNF 6.8 eAp~sses both 26 kD and 17 kD TNF. Figure 3 shows the conversion of 26 kD TNF by convertase activity present in HL60 cells. The production of labelled 26 kD TNF by in vitro transcription/translation, and analysis by gel elecLI~horesis is described below in Example 4. Note that the S-l cytosol or pellet fractions cause the near complete conversion of 26 kD TNF to a 17 kD
speries. Figure 3 also shows, for co~p~ e pul~,oses, 26 kD and 17 kD TNF
in a lysate of TNF 6.8 cells.
pFVXM and the plasmid pBl l were both amplified in E. coli strain HB101.
.ig~tion of the fragment~ was carried out using standard conditions. Plasmid DNA was isolated after the ligation procedure and the correct orientation of theTNF encoding s~lences was established by restriction analysis.
Plasmid DNA was ple~ed according to the procedure of Birnboim and Doly, as described in Nucleic Acid Research 7:1513 (1979). The plasmid DNA was banded twice in cesium chloride, and exh~llstively dialyzed against TE buffer consisting of 10 mM Tris, pH 8.0, and 1 mM EDTA.

WO 94/00555 PCr/US93/06120 Example 4 TNF Convertase Assays A.In Vitro Transcription/Translation Assay A plcÇc.lcd assay pf~lulc conlciC-t~ of in vitro tr~nC~riptinnltr~ncl~tion to produce S the 26 kD molecllle, followed by tr~tm~nt with convertase in the presence or ?~S~on~ of cû---~unds being tested for convertase inhibitory activity. The pf~lur~ entails in vitro ~ s~;liplion/translation of the TNF cDNA present in theplasmid Bl 1. Thus, the sequence was removed from pBl 1 by PstI digestion and was ins~llcd into the PstI site of pGEM-3 (obtainable from Promega Biotec). The res~ltin~ plasmid, termed pGEM-TNF14, was amplified in E. coli using established techniques, and p1~mi.1 DNA was pl~pal~d according to the ~lucedule of Birnboim and Doly, described above. Plasmid DNA was in vitro transcribed by line~i~ing it with HindIII, and the lin~i~ed plasmid tc~plates used to prepare capped tldl~s;liplS with T7 RNA polymerase and an in vitro ll~u scli~lion kit 15 supplied by ~u",cga Biotec. Transcription was pclrull,led using standard techniques as suggei.t~d by the m~n~lf~tllrer's instnlctionc.
The mRNA was tr~ns~ted in vitro in the presence of 35S-cysteine to produce 35S-cysteine-l~helled 26 kD TNF. A rabbit reticulocyte lysate translation kit was used, also supplied by Plu~,,ega Biotec, and the conditions fcco..~ -n~ed by the manufacturer were followed.
35S-cysteine-labelled 26 kD TNF was uscd to assay for convertase inhibitors as follows. 25 _1 of in vitro tr~ncl~t~ material was combined with 250 1 of convertase activity partially purified from llnin~uced HL60 cells, plus co",pounds to be assayed for inhibitory activity. The convertase was produced by harvesting2 x 109 HL60 cells, and isolating S-l and P-30 fractions totalling 18 and 6 ml, especli~/ely. 250 l of the P-30 fraction was used, although the S-l fraction mayalso be used. The assay was carried out at 30_C for 1 hour, essentially as described above. Next, the reaction mixture was immunoprecipilatcd with anti-TNF polyclonal antisera and protein A Sephan~se, pelleted and washed. The bound protein was eluted and ele~luphoresed using SDS-PAGE. The gel was WO 94/00555 PCr/US93/06120 ~39~ 36-fixed in 40% meth~nol, 10% acetic acid, soaked in F.nlight~ning (Dupont), dried,and eYpo~d to x-ray film which was subsequently developed. The gel elec~holelic profiles of 26 kD TNF treated with HL60 convertase and varying ~lilutiQn~ of the potential inhibitory col.lpound, revealed those co.llpounds with 5 inhibitory activity.
Using the above assay, it was dclc Illin~d that 3,4-dichloro-isocoulll~iil and el~tin~l at conce~ tions of 100 _g/ml and 5 mg/ml"~pec~-/ely, inhibit the convertase. It was also shown that (1-((3-((acetyloxyl)-7-methoxy-8-oxy-8-oxo-5-thiol-azabicyclo[4.2.0] oct-2-en-2-yl) carbonyl) morpholine, S~s~lio~yide7 (6R-cis) at a concentration of 1 mM
inhibits convertase activity. These results are shown in Figure 4.
The above assay was also used with pure HL60 cell PR-3 to test a variety of p~leinase inhibitors for TNF convertase inhibitory activity, as shown in Figure 5.
Pure PR-3 (0.3 mg/ml) was plci~cub~ed for 30 ~I~inu~s with the following inhibitors prior to addition of 35S-l~helled 26 kD-TNF and assaying as describedabove: DCI (45 mM), a-2-macroglobulin (1 mg/ml), PMSF (20 mM), leul)e~lin (2 mg/ml), EDTA (10 mM), or ~ in ( 2 mg/ml). The first three of these inhibitors showed significant inhibitory activity.

WO 94/00555 Pcr/US93/06120 213912~

B.Monocyte Assay The 26 kD form of TNF can also be produced by stimul-q-tP~ monocytes which produce 26 kD TNF, as described by Kriegler, et al., 1988, Cell, 53:45.
Briefly, human monocytes are purified from human blood by cenl,irugation, and 5 ~ubs~uently ennchP~ based on the adherence of monocytes to cell culture dishes.
Centrifugation CQnCictc of purifying the monocytes through Ficoll-hypaque and percoll (49.2%), oblainable from Pharmacia. The manufacturers recommen~e~
procedures followed. Next, the mixture of cells res--lting from the centrifugation step, concictin~ of monocytes and lymphocytes, are plated onto tissue culture dishes conl~ining RPMI media supplemented with 20% fetal calf serum. The dishes are incubqtP~ for 30 ~ Jte5 at 37 C after which they are extensively rinsed with the same media. This llæ~ nt removes non-adherent lymphocytes and leaves only adherent monocytes.
Monocyte 26 kD TNF is r~qdiolqhPllP~ as follows. The monocytes are inc~l~qtp~
for 3 hours at 37_C in RPMI media supple.. ~-led with 20% fetal calf serum, 100 ng/ml lipopolysaccharide, and 10 g/ml phorbol myristate acetate for 30 ~h~ules at 37_C. The latter two colllpounds induce the t;A~ s~ion of TNF. The RPMI
media is cysteinerninus~ and the fetal calf serum present at a final concentration of 5 % . The serum is dialyzed prior to use to remove any cysteine present. After the 30-minute incubqtion period, 100 uCi 35S-cysteine is added, and the cells are r. liolqhp~lp~ for 3 hours at 37 C, after which they are lysed and used to assay for convertase activity. The steps for carrying out the assay, as well as identifying inhibitors of the convertase, are similar to those described above.

C.Colorimetric Assay for Convertase Inhibition TNF convertase inhibition can also be measured by a colorimetric assay. In this type of assay, the actual conversion of proTNF to mature TNF is measured indirectly using a colorimetric TNF convertase subs~rate. By Ucolorimetric TNF
convertase substrate" is meant a compound that is cleaved by a TNF convertase torelease a compound that absorbs light of a certain frequency. One such substrate Wo 94/00555 Pcr/US93/06120 is Boc -AlaONp (R~rhPm Ri~sciPnce, Inc., Phil~ ~Plphi~ PA). Other such S~SlldteS can be discerned from the structure of TNF convertase and other serineprote~Ps Although the eY~mrlc herein uses purified native PR-3 as the TNF
convertase, it is cont~ ..pl~ted that ,cco...binant PR-3 or other TNF convertases 5 can be used in this assay as well.
Peptide diphenyl phosphon~tP inhibitors were synth-P-~i7~ and stored as lyophilized solids as described in Oleksyszyn and Powers, 1991, Biochem., 30:485493.
Inhibitor solutions (10 mg/ml) were plcpared in 100% dimethyl sulfoxide (DMSO) and diluted into aqueous buffers upon illiti~tiS~n of the ~ ~.i.l.enls. 3,4, dichloro-i~ocu.. ~.in was purchas_d from CalBiochem. Purified PR-3 (10 ml, 0.1 mg/ml) was mixed with varying conc~ dlions of protease inhibitor (400 ml final volume) in 20 mM sodium phosphate buffer, pH 7.0, conl~;nil~g 0.1 M sodium chloride. Aliquots (40 ml) were removed at ~l~t~d times and diluted 1/10 into a colorimetric assay for convertase, contdining 0.5-1 mM BOC-Ala-ONp (prepared lS fresh from a 50 mM stock in 100% methanol) in 0.02 M sodium phosphate buffer, pH 7.0, 0.1 M sodium chloride. The increase in absorbance was monitored at 347 nm on a Hewlett Packard 8450A spectrophotometer, and using an extinction co-effirient of 5.5 x 103 M~lcm~l.
Example 5 20 Peptide Diphenyl phosphonate Inhibitors of TNF Convertase Several peptide diphenyl pho~honales were tested for inhibitory activity:
BocVal-Pro-Val-p(OPh)2 (VPV), Boc-Ala-Pro-Val-p(OPh)2 (APV), Boc-Ala-Gln-Alap(OPh)2 (AQA), and Boc-Leu-Ala-Gln-Ala-p(OPh)2 (LAQA).
The peptides were plepa-ed by çh~mirz~l synthesis using the Merrifield method and 25 the diphenyl phosphonates were p~ep~red according to the method similar to the one shown in Oleksyszyn et al., supra.
The peptide diphenyl phosphonates were tested in the colorimetric assay described in Example 3, for inhibition of TNF convertase/PR-3 activity. The results are shown in Figure 6. VPV and APV demonstrated inhibitory activity. AQA and WO 94/00555 2 1 ~ Y 1 ~ ~ PCr/USs3/06120 LAQA showed ...a,~,inal, if any inhibition at the concf ~ dlions tested. Dichloro isocoL..n~in (DCI) showed 100% inhibition in the assay.

Example 6 TNF Mutein/Antibody/Peptide Inhibitors of Convertase Activity The following compounds will have convertase inhibitory activity and can be p,~ a c;d as follows. These co,--?ounds may be tested for inhibitory activity asdescribed in Example 4 above.

A.Anti-Convertase Antibody Monoclon~l or polyclonal antibody is pr~a~ed that binds to the convertase and thereby stenc~lly prevents the convertase from binding to 26 kD TNF or otherwiseneutraliæs the enzymatic activity of the convertase. The procedure consists of immunizing an a~propliate host animal with a membranous fraction of HL60 cells produ~ing TNF convertase. Alternatively, purified TNF convertase may be used from native or leco",binant sources. For eY~mr1e, PR-3 from human neullophils may elicit anti-TNF convertase antibodies. A sufficient amount of material should be used to elicit an immune respor-se, and usually this will consist of between 10 _g to lO mg per kilogram of body weight. Immunization may be conductçd with adjuvant in a biologically acc~pt~l~,le buffer, as is known in the art. The bestimmunization route can be deterrnined e,~.i",er,~lly, and the primary immunization may be followed by one or more S~Qn~l~ry immunizations depen-ling on the strength of the immune response to the initial immuni7~fion.
The presence of neutralizing anti-c,onvertase antibody in the sera may be cletrcted using the convertase assay desc,ribed above wherein antisera is present in the assay - "~luie. Inhibition of the conversion of the 26 kD TNF species to species having the molecular weight of mature TNF indir~tes the presenc,e of a neutralizing antibody. It is, of course, ~c~umed that the proper controls are c,onduct~ to insure that anti-sera from non-immunized ~nim~l~ is not inhibitory. Polyclonal antibody may be purified as described below.

WO 94/00555 PCr/USs3/06l20 ~,~39~ 40-Monoclonal antibody to the convertase may be produced using either in vivo or invitro immunization techniques, and senciti7pd ly...phocytes resulting the,Gr,u-" can be used to prGpafe hybrid cell lines that secrete the appr~liate monoclonal antibody. Rodent, preferably of murine origin, or human antibody is most p~erGllGd. The in vitro immlmi7~tiQn pÇ~)CedUlG involves senciti7ing lymphocytesto the convertase by i~ unizing either mice or humans, and icol~ting theçGr,u--lthe antibody-sec~Gling cell fraction and immortalizing the cells therein by one of several pr~lulGs. An altemate embodiment is to isolate lymphocytes that have already been sensitized to the convertase from septic patients or Wegener's granulo---atosis p~tiPntc as described above.

(i)Murine Antibody For in vivo i.. unization of mice, the procedure of Kohler and Mil~tein desçribed in Nature, 256:495 (1975) may be followed, or modifi~d l~ucedu~s such as those shown by Fendly et al., 1987, Hybridoma, _:359; Buck,et al., 1988, In Vitro~
18:377. In vitro techniques are generally describe~ by Luben, R. and Mohler, M., 1980, Molecular Immunolo~y, 17:635, Reading, Methods in Enzymolo~y, 121 (Part One):18, or Voss, 1986, Methods in Enzymolo~y, 121:27.
Mice are immunized with 1 mg/ml of a membranous fraction of HL60 cells 20 previously shown to be positive for convertase activity. Alternatively, a smaller amount of purified TNF convertase may be employed. The immunization is carried out in complete Freund's adjuvant. Two additional immunizations, or boosts, are ~,~,-ned at monthly intervals without adjuvant, and one month after the last boost the mice are given an I.V. boost of 10 g of membranous m~tPri~l.
25 Three days after the I.V. boost, mice are sacrificed, their spleens removed, and the spleenocytes isolated and fused to an i.. o,lalized drug select~hle myeloma partner cell line. Numerous such myeloma lines are known in the art, most of which are inc~r~hle of growth in HAT supplemented cell culture media. A typical myeloma cell line is SP-2/OAg 14. Thus, the hybridomas are formed by 30 combining splenocytes and myeloma cells in a 5:1 ratio, which generally consists WO 94/005ss 2 I ~ 91 2 7 Pcr/US93/06120 of 2 x 106 myeloma cells to 1 x 107 splenocytes. The cell mixture is pçllpt~l~
media removed and fusion affected by the addition of 1.0 ml of 40% (v/v) solution of polyethylene glycol 1500 by dropwise ~ ition over 60 sçcQnds at room ~ peldtu~e~ followed by a 60 second incub~tion at 37_C. To the cell suspçn~ion S with gentle agitation is added 9 ml of Dulbecco's ~o lifi~.d Eagles medium over S
minutes. Cell clumps in the ~llixlule are gently resuspended, the cells washed to remove any residual PEG and plated in microtiter plates at about 2 x 105 cells/well in DMEM supple-m~nt~.d with 20% fetal calf serum. After 24 hours, the cells are fed a 2 x solution of hypox~nthine and azaserine selection ",~Ai~l.".
Media from wells that exhibit positive cell growth may be screened for neutralizing monoclQn~l antibody to the convertase. The pl~ fe.led assay is the convertase assay des; ~ ;bed in Ex~ le 2, above, wherein media sought to be te~sted for antibody activity is present in the assay. More pl~lled is to combine culture supernatants from 3-8 microtiter wells, and assay the ...i~clu,e. If the15 Illi~clule is positive, then media from each well may be assayed independçntly to identify the sec,eling hybridoma(s). Many assays are known in the art and can detect soluble, or non-soluble antigens, and are shown by Langone, J. and Van Vinakis, H., Methods of Enzymolo y, 92. Part E (1983).
Regardless of whether the antibody is polyclonal or monoclonal it is desirable to 20 purify the antibody by standard techniques as is known in the art, or described by Springer, 1980, Monoclonal Antibodies,:194, (Eds. KPnnett T. McKearn and K.
Bechtol, Plenum Press, New York. Generally this consists of at least one ammonium sulfate pr~cipit~lion of the antibody using a 50% ammonium sulfate solution. Antibody affinity columns may also be used.

Wo 94/00555 PCr/USs3/06120 2~9~

(ii)Human Monoclonal Antibody Peliph~dl blood lymphocytes are icQl~t~d from septic p~tiPntc~ and then infectedwith Epstein-Barr virus and the infe~ed lymphocytes immortalized by fusion to a select~hle myeloma cell line, and the hybrid cell lines so gen~ted icol~t~d and 5 characterized as to antibody productiQm More sr~er-ific~lly~ mononu~ r cells are se~dted on Ficoll-hypaque (Pharmacia), and monocytes d~leted from the mixture by adherence to plastic. Standard labolat~ly techniques were utilized to effect these procedures. Next, non~-lherent cells are enriched for antibody producers by antigen-specific p~nning. Panning is a technique generally known in the art, and involves incub~;on of a population of antibody secleling cells on a plastic surface coated with the appr~")l;ale antigen.
Those cells that express antibody on their surface bind antigen, and consequently adhere to the plastic surface, whereas cells that do not express cell surface antibody, do not adhere and can be removed by washing. Thus, specific 15 antibodysecreLing cells are enriched for by this technique.
More sperific~lly~ ~well plates (Costar) are coated with purified TNFconvertase or a membrane fraction cont~ ing convertase prepared from either induced or uninduced HL60 cells, as described above, such that 150_g of membranous material is coated per well in phosphate buffered saline at 4_C overnight. The 20 wells are blocked after the overnight incub~tion period with phosphate buffered saline conl;1ining 1% bovine serum albumin for at least 1 hour at 4_C, and sul)s~uently washed with phosph~t~ buffered saline/BSA. Next, 107 lymphocytes in 1 ml of PBS/BSA are added to each well of the six well plates. The lymphocytes are allowed to incub~te on the plates for 70 minutes, after which any 25 nonadherent cells are removed by aspiration. The adherent cells are incubatedwith cell culture medium (IMDM, Sigma ChPrni~l Co., St. Louis, Missouri) conl;.inil-g 10% fetal calf serum.
The adherent cells are subjected to Epstein-Barr virus transformation by adding an equal amount of culture media obtained from growing the Epstein-Barr virus 30 infected marmoset cell line, B95-8, and thus cont~inil-g the virus, to media bathing WO 94/00555 Pcr/US93/06120 ~1~912~

the adherent cells. The cells were cultured in this envilunlllent at 37_C for 3 hours, and in this way the ly~n~hocytes in the adherent cell population are subjected to Epstein-Barr infection. Following the infection period, the cells are washed and plated onto 96 well microtitre plates at a density of about 10~ - 105cells/well in IMDM ".P.lit.. ", plus 10% fetal calf serum, and 30% conditioned mPAillm. The latter is derived from a lymphoblastoid cell line, preferably JW5.
The -.ediu-., also cQntztinc 5 x 10-5 M 2l,lcl~5t~ 1,q~ol, 50 g/ml genta."ycin sulfate (Sigma), and 600 ng/ml cyclosporine A (S~n-limmun, Sztn-loz, Basel, Swi~ -d) After about 14 to 21 days of inc~ ;on, cell culture ~U~IId~ i are combined and screened for TNF conv~a3e neutralizing activity as described above.
Positive hybridomas are subcultured at low density, ~ ~d for neutralizing antibody, and grown up and fused to the cell line F3B6 using polyethylene glycoland the plate fusion technique known in the art. The latter technique is described 15 by Larrick, 1985, in Human Hybridomas and Monoclonal Antibodies, E.G.
F.nglPrnzln, S.K.H. Foung, J.W., Larrick, and A.A. Raul)iL~chP~, Editors, PlenumPress, New York, page 446. F3B6 is a heteromyeloma cell line that is sensitive to growth in media containing 100 M h~z~n~ P, 5 g/ml azaserine and 5 M
ollz~bz~in. Finally, the res~lting hybrids are again screened to insure that they 20 produce neutralizing anti-convertase antibody.

B.26 kD Muteins 26 kD TNF muteins are described that co"-~le for binding to the convertase, thereby inhibiting or reducing its activity. The ~lefell~d mutein embodiments are 25 thûse having valine at positions 1 and/or 13; or alanine at position -1 and/or proline at position 12, replaced or deleted. The m~ltPin~ are constructed using a mo lifi. ~tion of the site-directed mutagenesis gapped-duplex mPthod The following solutions/buffers are used to ~ClÇol... the desired procedures: S x gapped-duplex buffer (GDB) con~icting of 0.938 M KCl, 0.063 M Tris, pH 7.5;
10 x PEL con~i~ting of 1.0 M KCL, 0.30 M Tris, 0.15 M MgC12, 0.02 M DTT, WO 94/005ss Pcr/us93/06120 ~39~ 44-pH 7.5; 10 x KB concicting of 0.50 M Tris, 0.10 M MgCl2, 0.05 M DTT, 0.001 M EDTA, pH 8.0; a solution co~ ning 0.25 mM dCTP, dATP, dGTP, dl~
made fresh from 10 mM stocks; an ATP sQlutiQI- cQnCicting of 0.1 M ATP made by dissolving 60 mg of ATP in 0.80 ml of H2O and adjusting the pH to 7.0 with 0.1 M NaOH in a final volume of 1.0 ml with H2O; 20% PEG/2.5 M NaCl; 3.0 M NaOAc; and TE-sa~ul~led phenol.
Various bacterial strains and phage are employed to yield the desired mut~inc and these are BMH 71-18, JM103 for growing phage strains; HB2154: MutL, Su~, made co~ ent for DNA tran~Ço""alion; and HB2151: Su~, used as lawn cells during tran~Ç(""~ation; M13 GAP, the RF is used for the formation of tne gapped-duplex; and M13mpl9amber, the 26 kD TNF target DNA is cloned in this vector, and ssDNA icn1~t~ for the formation of gapped-duplex.
Phage are infected into an approp,iale bacterial strain, grown up, and titered as follows. In making a large-scale p~el)~alion of either phage for ssDNA or cells for dsDNA, or RF DNA, the same infection l.ro~ocol is used.
Plaque-purified phage is produced using standard techniques. Briefly, this consists of streaking phage s. ~,~;"~a~t~ on agar plates, followed by careful overlay with 4.0 ml of soft agar and 100 1 of fresh overnight culture of BMH 71-18. Next, isolated plaques are picked and incub~t~d with a 1:50 dilution of fresh overnight culture of BMH 71-18 in R26 or R17 + 10 mM MgCL with ch~king at 37_C for 4.5-6 hours. R17 (N-Z amine broth) consist of 10 g N-Z amine, type A, 5 g NaCl with H2O to 1 liter, while R26 consist of 8 g tryptone, 5 g yeast extract, 5 g NaCl, with water to 1 liter (YT broth). The phage stock is titered, and phage infected into bacteria at a m--ltipli~ity of infection (MOI) of 10. After in-ub~tin~
the culture with sh~king at 37_C for 5 hours the cell sUcpe~cion is pelleted, and the supe,natant saved for ssDNA isolation, and the cells for RF isolation. RF
DNA is icol~ted using established plasmid DNA isolation techniques, while ssDNA is isolated as follows.
250 ml of phage .,.lpe",a~nt is spun down hard, after which 200 ml of the sL~.,-atant is dec~nt~d, followed by adding 50 ml of 20% PEG/2.5 M NaCI, and WO 94/00s5~ Pcr/us93/06120 213~

incub~tion overnight at 4_C, or on ice for 30 I,linules. This ~ ure is also spundown hard, and the su~clnalant le~nt~d. The bottle is spun again to pellet the phage pr~ipilate along the sides of the bottle, and the r~ ining fluid aspiratedwith a Pasteur pipette. The pellet is resuspended in 5.0 ml of 1 x TE, and stored S at 4_C, after which 0.5 ml of is extracted twice with 0.5 ml of TE satul~ed phenol. To the aqueous layer is added 0.050 ml of 3.0 M NaOAc and 1.0 ml 95% ethanol. The Illixlul~ is placed in a dry ice bath for 10 minutes, and centrifuged for 10 ,llinutes in a microfuge at 4 C. The pellet is dried, and resuspe-nded in 200 1 of 1 x TE. This material may be stored in 0.050 ml aliquots at -20 C unhl used in the mutagenesis of 26 kD TNF.
The following deletions and substitutions in Table 1 are ~erelled proTNF
muteinc. These muteins can be ~ ,~ed using appr~liate oligonucleotides by methods known in the art.

WO 94/00555 PCr/US93/06120 i ?~39~ - 46 -Table 1 Deletions VAL 1 + PRO 12 VAL 1 + VAL 13 SubsLilulions (VAL 1 ALA 1) + (VAL 13 ALA 13) (VAL 1 GLY 1) + (VAL 13 GLY 13) (VAL 1 LEU 1) + (VAL 13 LEU 13) (VAL 1 MET 1) + (VAL 13 MET 13) (VAL 1 PHE 1) + (VAL 13 PHE 13) (VAL 1 HIS 1) +-(VAL 13 HIS 13) (VAL 1 THR 1) + (VAL 13 THR 13) (ALA 1, VAL 1 GLN 1, HIS1) + (PRO 12, VAL 13 GLN 12, HIS 13) (ALA 1, VAL 1 _ GLN 1, HIS 1) + (PRO 12, VAL 13 _ SER 12, THR 13) The oligonucleotides are kinased using the following reaction solution and conditions: 3 ml 10 x KB buffer, 3 1 10 mM rATP (1:10 dilution of 0.1 M rATP
stock), 2 I mutagenic oligonucleotide (100 pmole/l), 21 I H2O, and 1 1 polynucleotide kinase (10 Units/l). The reaction is run at 37 C for 45 minutes, and then at 65-68 C for 5 ,..inules. Next, 24 1 of the kinased oligonucleotide is diluted with 561 of H2O to give 2 pmole/l.
The gapped-duplex is formed as described below, followed by ~Tln~ling the oligonucleotides. The following reagents are combined in a total volume of 40 1:8 1 5 x GDB buffer, 0.50 pmole ssDNA, and 0.10 pmole HincII linearized M13 GAP RF DNA. 10 l is removed for future use, and the fem~ illg 30 1 is treated sequentially as follows: lOO_C for 3 minutes, 65_C for 5 minutes, followed by cooling to room tel,-l)eldtule for 30 minutes, and then placing the reaction ",ixlu,e on ice. Next, 10 l of gapped-duplex and 10 l of control ungapped material is subject to elecl~ophoresis on agarose gel to check gapped-duplex formation. If the gel shows the presence of a third band, the gapped-duplex has formed, and the kinased oligonucleotides can be ~nn~led to the duplex by combining 161 of gapped-duplex reaction ",ixlure and 4 1 of diluted kinased oligonucleotide and WO 94/00555 2 1 3 9 1 2 ~ Pcr/US93/06120 heating the ~ clure to 65 C for 3 .llinu~s, followed by cooling to room telllp~.dlul~ for 20 ...;n~ s The het~.~lu~,lex is completed by the appr~pliate eYten~i~m and lig~tion reactions c~n~isting of colllbining the following reagents in a total volume of 40 l:10 l gapped-duplex and primer, 4 1 10 x PEL buffer, 4 I dNTP's (0.25 mM solution made from 10 mM stocks, 3 1 ATP (10 1 of 0.1 M ATP stock + 1490 I H2O =
0.662 mM), 17 I H20, 1 I Klenow (5 u/l), and 1 I T4 DNA ligase (0.6 Weiss u/l, diluted stock with 1 x PEL). The reaction is conduct~ at 16_C for 2 hours, followed by tran~Ç~llnalion of 10 l of the extension/ligation Illi~lu~e into 200 l of thawed co.. p~tent HB2154 cells. The cells are kept at 0_C for 30 minutes, and then 42 C for 1.5 ...in~ s, followed by plating various volumes of the tran~rollllalion mix (e.g., 50 I, 10 l, etc.) with 100 1 of fresh overnight culture of HB2151 cells + 3.0 l of soft agar.
The resultin~ plaques are scr~.~ed using the plaque hybridization pr~lule.
While a variety of such proce lu,l s are known, a des~liplion of the p~ere,l~d prucedul'e follows. Plates are replicated onto duplicate nitrocellulose filter papers (S & S type BA85) and the DNA fixed to the filter by sequential treatment for 5 ...;nu~eS with 0.5^ N NaOH plus 1.5 M NaCl; 1.0 M NaCl plus 0.5 M Tris-HCl pH 7.4; and 2 x SSC (~tandar~ saline citrate). Filters are air-dried and baked at 80 C for 2 hours, in vacuo.
The duplicate filters are prehybridized at 55 C for 2 hours with 10 ml per filter of DNA hybridization buffer, 5 x SSC, pH 7.0, 5 x Denhardt's solution (polyvinylpyrrolidone, plus Ficoll and bovine serum albumin; 1 x 0.02% of each),50^ mM sodium phosphate buffer at pH 7.0, 5 mM EDTA, 0.1% SDS, and 100 _g/ml yeast RNA. The prehybridization buffer is removed and the samples hybridized with the appr~plidle kinased probe, specifically, kin~ced oligonuclc~tides as shown above, under conditions which depend on the stringencydesired. About 2 x 106 cpm/ml total is used. Typical moderately stringent conditions employ a telllpeldlu~ of 42_C plus 50% form~mitle for 24-36 hours with 1-5 ml/filter of DNA hybridization buffer con~inil-g probe. For higher WO 94/OOS5S Pcr/US93/06120 ~9~ - 48 -s~ gf ncies high lc~pc~alurl s and shorter times are employed. The plcrellcd hybridization conditions consists of hybridizing the probes to the filters in 5 x SSC, Denhardt's solution, 50 mM NaPO4, pH 7.0, 5 mM EDTA, 0.1% SDS, and 100 mg/ml yeast RNA at lO_C below the TM of the oligonucleQti~e used to do the S scl~nillg. Next, the filters are washed twice, 30 minutes each wash, at room ~".pcldlu,e with 2 x SSC, 0.1% SDS, then washed once with 2 x SSC and 0.1%
SDS at 5_C below the TM of the oligonucleotide used to screen, and air-dried.
Finally, the filters are autoradiographed at -70-C for 36 hours. Autoradiographyreveals those plaques col-t~ining the virus that carries the muteins of interest.
In addition to constructing ~ulcins wherein valine at position 2 and/or 13 have been deleted or sub;,l;lu~d, large deletion muteins may be produced that encompass the two predo"-inate cleavage sites of 26 kD TNF. A pr~relled embo-ii...æl-t mutein lacks the amino acids ~p~nning the region -9 to + 14, as shown in Figure 1. This mutein was constructed using the m~t~ri~l~ and metho l~
described above and the oligom~leQti~le, CP375 which has the following sequence (SEQ ID NO: 2).

C.Protein/Peptide Inhibitors Peptides having the following amino acid sequences are synth~si7~d by the solid-phase method, described in detail by Merrifield, 1985), Science, ~:341-347: Gln-Ala-Val-Arg-Ser-Ser-Ser; (SEQ ID NO: l); (SEQ ID NO: 3 );
(SEQ ID NO: 4); and (SEQ ID NO: 5). A Biosearch 9500 automated peptide m~chine is used with hydrogen fluoride cleavage, and purification by prepal~ti~eHPLC using a Waters Delta Prep 3000 instrument, on a 15-20_m Vydac C4 PrepPAK column.
TNF convertase inhibitory activity of these peptides is shown by pe,~l",ing the assay described above in the pre~ence of varying amounts of each peptide. Gel electrophoresis and Western blotting of the reaction ",i~clu,c shows an inhibition of conversion of the 26 kD proTNF to the 17 kD mature form.

WO 94/00555 213 91 2 ~ PCr/US93/06120 E~a",~le 7 TNF Convertase Inhibitory Activity of DCI in L929 Mice DCI sperifi~lly sllppl~sses the release of TNF but not IL-6 from mouse ~I~acrùphages as shown below.
Release of TNF by mac~phages after stim~ tion by LPS is a major source of TNF. In these studies pelitone~ ~-u~)hages were purified by adhesion, cultured in 24 well plates and LPS was added to induce secretion of TNF. Analysis of the kinPtics of TNF release showed a m~l~im~l peak at 3 hours. DCI was then added in dimethyl sulfoxide excipient to cultures. The control cultures had DMSO aloneadded in equivalent concentrations. Su~lllat~ls were c~llPctP~ and assayed for TNF and IL-6. Results show that TNF secretion is markedly ~ ssed with DCI
but not control excipient. In contrast the IL-6 response was not significantly altered, thus ruling out a noncpP~ific toxic effect (see Table 2).
Since DCI was able to sper-ifi~lly suppress LPS induced TNF secretion in murine macrophages the the-dl)eulic effect of ~;lmini~tration of DCI to mice injected with LPS was e.~...;n~d.
Stability and formulation studies showed that DCI when dissolved in corn oil wasstable and retained serine protease inhibitor activity. Infection of DCI/oil into mice showed an LD 50% at a dose of 1 mg/ml. This lep.esented a maximal tolerated dose of DCI that could be a~lmini~tered.
The kinetics of TNF and IL-6 in mice injected with a lethal dose of LPS was studied. TNF showed a sharp peak at 2 hours with return to b~celine. IL-6 showed a slower gradual increase. Injection of DCI 1 hour before the LPS dose resulted in a marked inhibition of serum TNF secretion (see Figure 7). Also, there was no delayed increase in TNF measured up to the 6-hour time point. This was true for both immunoreactive mouse TNF measured by ELISA and bioactive TNF measured by lysis of L929 cells. IL-6 levels were not reduced by this therapy.
The effect of DCI on survival of mice injected with a dose of LPS that results in 100% death of ~nim~ls by 24 hours was also investi~ted. Results show that WO 94/0055s PCr/US93/06120 C?~
~,~3~ so-prophylactic therapy with DCI could prolong survival of mice (see Figure 8).
There was a dose r~spon~, r~l~tiorshir noted by 0.75 mg being more effective than 0.5 mg.
In ~,~.. ~. y, these studies show that DCI is able to specifir~lly inhibit LPS
S induced TNF pro~hlction by murine macrophages. This spff~ificity of inhibition of TNF could also be seen in ~nim~l~ injected with a lethal dose of LPS.
Furthermore, the survival of ~nim~l~ was prolonged with DCI therapy in a dose related manner. These studies show that DCI (a serine protease inhibitor) may bebeneficial in a sepsis model in prolonging survival by ~. ppression of the systemic release of TNF.

Table 2 SampleTNF (ng/ml)IL-6 (pg/ml) DMSO control6.9299 DCI 20 (mg/ml)0.05189 Adherent peritoneal macrophages (106/ml) were cultured with LPS and either DMSO or DCI DMSO. Cells were cultured for 3 hours and "~lls were collected. TNF was measured by ELISA and IL-6 by B9 bioassay.

WO 94/005S5 2 1 3 ~ PCr/USs3/06120 Example 8 P~ole~ e Effect of TNF Convertase Inhibitors in the T~at.l.ent of Sepsis CG..-POUndS that are effective inhibitors of convertase activity are shown to prevent sepsis in a baboon model system as follows. Anti-TNF convertase antibody, S murine, human, or r~col"binant, at a con~Pntration of S mg/kg is ~rlminist~red in a single I.V. bolus 60 ...inub s before the ~nimql~ are çh~llPn~Pcl with a lethal dose of E. coli, and 2 mg/kg ~im~ n~oucly with the E. coli çh~llPnge. The antibody is ~-lmini~tPred in a physiologically b~l~nce~ salt solution, and about 4 x 101 E.
coli org~ni~mc are used. The E. coli dose is infused over a 2 hour period.
Animals that receive the antibody are p.~tecled for at least 7 days, whereas control ~nim~l~ that are ~1mini~red only the b~l~nce~ salt solution expire within 16 to 32 hours.
Similar plot~lion is attributable to the TNF mutein convertase inhibitors shown in Fy~mplc 5. The muteins are ~lmini~t~Pred at a concentration of 5 mg/kg in a single I.V. bolus 60 minutes before the ~nim~l~ are ch~llPnged with 4 x 101 E.
coli org~ni~m~. The baboons also receive 2 mg/kg of the muteins simultaneously with the E. coli ch~llPn~e.
Finally, the peptides shown in Example 5, that is, Gln-Ala-Val-Arg-Ser-Ser-Ser and, (SEQ ID NO: 1) are tested as described above and yield similar pr~tec~ e 20 effects.

Example 9 Modelling of Human PR-3 Onto Human Fl~ct~e Crystal Structure and Use of Inhibitor-Enzyme Complex Models to Predict Novel PR-3 Inhibitors 25 A model for the FNF convertase PR-3 was constructed by de~ll"ining structuralsimilarities shared between PR-3 and other serine pr~tein~ces. A final 3-D modelof the enzyme was generated by first delel."ining that the PR-3 sequence shared a highest degree of sequence homology with human neulç~phil el~t~ce (HNE). The crystal structure HNE (Navia et al., 1989, PNAS (USA), 86:7) was used as a 30 scaffold to build a three ~limPncional lel l~;sentalion of the PR-3 protein using the WO 94/00555 Pcr/uS93/06120 ~39~1 computer plUgl~lll Homûlûgy (Biosym, San Diego). The model was further refined by two rounds of minimi7~ion using the computer plU~ldlll Discover (Biosym, San Diego). The design of poter,lial inhibitors that dirÇGrGnliate between HNE and PR-3 is dete""ined by the unique and similar amino acids found in the active sites of these en_ymes. Most notably, the catalytic triad common to this class of plOle;~ eS iS spatially conserved. Within the binding pocket of the Pl residue (Sl site) several .~ignific~nt dirrcLe.~ces in amino acid side chains are pr~posGd by the model. The following described object compound of the present invention takes into account the unique aspartic acid and leucine amino acids found within the Sl pocket of the PR-3 model and can be lG~ ~nl~;d by the following general formula.

~JI`N1R4 H

in which Rl, R2 are lower alkyl, optionally substituted ar(lower)alkyl, cyclo(lower)alkyl(lower)alkyl or optionally substituted heterocyclic(lower)alkyl, natural amino acids, -OH, -NH2, lower alkylimino or lower alkylene;
R3 is pyroyl, imi~7oyl, butylamine, or ethyl-epoxide; and R4 is aldehyde, diphosphonylate, ethoxycoul"~a,inyl, chloromethyl and difluûrc,,.,Gtllyl ketonyl.
An example of a PR-3 inhibitor based on this model is Boc-Val-Pro-Hisp(OPh)2.
Inhibition of PR-3 activity by such a compound is unexpected in light of the generally accepted belief that elastase and PR-3 selectively bind and cut after WO 94/00s55 2I 3 g 1 Z 7 PCr/USs3/06120 residues quite different from hi~ti-line, namely those with short aliphatic sidechains such as ~l~nine-The present invention has been described with reference to specific embo liment~.
However, this application is inttonded to cover those changes and substitutions 5 which may be made by those skilled in the art without departing from the spirit and the scope of the ~ppended claims.

WO 94/005s5 PCr/US93/06120 9 ~'1 Sequence Listing Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala (SEQ ID NO:
1) s GTTTGCTACA ACATGGAGGT CCCTGGGGGA (SEQ ID NO: 2) Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-HisVal-Val-Ala (SEQ ID NO: 3) Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala (SEQ ID NO: 4) Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro (SEQ ID NO: 5)

Claims (38)

WE CLAIM:

1. A method for identifying a prophylactic or therapeutic of a disease caused bya mature tumor necrosis factor (TNF) produced from a proTNF by cleavage of said proTNF by a TNF convertase, the method comprising the steps of:
(a) contacting the proTNF with an amount of the TNF convertase effective for cleaving the proTNF;
(b) measuring the conversion of the proTNF to the mature TNF in step (a);
(c) repeating steps (a) and (b) further including a molecule sought to be identified as a prophylactic or therapeutic of diseases caused by the mature TNF;
(d) measuring the conversion of the proTNF to the mature TNF in step (c); and (e) comparing the conversion measured in step (b) with the conversion measured in step (c) to determine whether the molecule is a suitable prophylactic or therapeutic of diseases caused by mature TNF.

2. The method of claim 1, wherein the proTNF is 26 kD TNF.

3. The method of claim 1, wherein the disease is selected from the group consisting of sepsis, rheumatoid arthritis, cachexia, cerebral malaria, AIDS, and graftversus-host disease.

4. The method of claim 3, wherein the disease is sepsis.

5. The method of claim 1, wherein the TNF convertase is proteinase-3 (PR-3).

6. The method of claim 5, wherein the PR-3 is native PR-3.

7. The method of claim 5, wherein the PR-3 is recombinant PR-3.

8. The method of claim 1, wherein the conversion of proTNF to mature TNF
in steps (b) and (d) is approximated by a colorimetric assay measuring TNF
convertase cleavage of a colorimetric TNF convertase substrate.

9. A therapeutic or prophylactic compound for treating a disease caused by a mature TNF produced from a proTNF by cleavage of said proTNF by a TNF
convertase, the therapeutic or prophylactic identified by a method comprising the steps of:
(a) contacting the proTNF with an amount of the TNF convertase effective for cleaving the proTNF;
(b) measuring the conversion of the proTNF to the mature TNF in step (a);
(c) repeating steps (a) and (b) further including a molecule sought to be identified as a prophylactic or therapeutic of diseases caused by the mature TNF;
(d) measuring the conversion of the proTNF to the mature TNF in step (c); and (e) comparing the conversion measured in step (b) with the conversion measured in step (c) to identify whether the molecule is a suitable prophylactic or therapeutic of diseases caused by mature TNF.

10. The therapeutic or prophylactic of claim 9, wherein the proTNF is 26 kD
TNF.

11. The therapeutic or prophylactic of claim 9, wherein the disease is selected from the group consisting of sepsis, rheumatoid arthritis, cachexia , cerebral malaria, AIDS, and graft-versus-host disease.

12. The therapeutic or prophylactic of claim 11, wherein the disease is sepsis.

13. The therapeutic or prophylactic of claim 9, wherein the TNF convertase is proteinase-3 (PR-3).

14. The therapeutic or prophylactic of claim 13, wherein the PR-3 is native PR-3.

15. The therapeutic or prophylactic of claim 13, wherein the PR-3 is recombinant PR-3.

16. A therapeutic compound for treating a disease caused by a mature TNF
produced from a proTNF by cleavage of said proTNF by a TNF convertase, the compound being a non-cleavable mutein of TNF.

17. The therapeutic compound of claim 16, wherein the non-cleavable mutein includes a substitution or deletion at amino acid 1 (valine) or amino acid 13 (valine).

18. A method for treating a patient having a disease or susceptible to a diseasecaused by a mature TNF produced from a proTNF by cleavage of said proTNF by a TNF convertase, the method comprising administering to a patient in need of such treatment an effective amount of an inhibitor of a TNF convertase.

19. The method of claim 18, wherein the proTNF is 26 kD TNF.

20. The method of claim 18, wherein the disease is selected from the group consisting of sepsis, rheumatoid arthritis, cachexia, cerebral malaria, AIDS, and graftversus-host disease.

21. The method of claim 20, wherein the disease is sepsis.

22. The method of claim 18, wherein the TNF convertase is proteinase-3 (PR-3).

23. The method of claim 22, wherein the PR-3 is native PR-3.

24. The method of claim 22, wherein the PR-3 is recombinant PR-3.

25. The method of claim 18, wherein the inhibitor is a peptide diphenyl phosphonate having the formula Boc- X- p(OPh)2, wherein X is an oligopeptide selected from the group consisting of Val-Pro-Val, Ala-Pro-Val and Val-Pro-His.

26. The method of claim 25, wherein X is Val-Pro-His.

27. The method of claim 18, wherein the inhibitor is a neutralizing antibody to PR-3.

28. A pharmaceutical composition for the treatment of a disease caused by a mature TNF produced from a proTNF by cleavage of said proTNF by a TNF
convertase, the composition comprising an effective amount of an inhibitor of a TNFconvertase and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition of claim 28, wherein the disease is selected from the group consisting of sepsis, rheumatoid arthritis, cachexia, cerebral malaria, AIDS, and graft-versus-host disease.

30. The pharmaceutical composition of claim 29, wherein the disease is sepsis.

31. The pharmaceutical composition of claim 28, wherein the TNF convertase is proteinase-3 (PR-3).

32. The pharmaceutical composition of claim 31, wherein the PR-3 is native PR-3.

33. The pharmaceutical composition of claim 31, wherein the PR-3 is recombinant PR-3.

34. The pharmaceutical composition of claim 28, wherein the inhibitor is a peptide diphenyl phosphonate having the formula Boc- X- p(OPh)2, wherein X is an oligopeptide selected from the group consisting of Val-Pro-Val, Ala-Pro-Val and ValPro-His.

35. The pharmaceutical composition of claim 34, wherein X is Val-Pro-His.

36. A peptide diphenyl phosphonate having the formula Boc- X- P(OPh)2, wherein X is an oligopeptide selected from the group concicting of Val-Pro-Val, AlaPro-Val and Val-Pro-His.

37. The peptide diphenyl phosphonate of claim 36, wherein X is Val-ProHis.

38. A method for treating a patient for autoimmune diseases, the method comprising administering to a patient in need of such treatment an effective amount of an inhibitor of a TNF convertase.