Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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,
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
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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.
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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
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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.
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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
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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
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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.
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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
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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.
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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
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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
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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
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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
WO 94/00555 PCI/US93/06120
~ - 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
-
- 17-
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~
- 18 -
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
WO 94/00555 2 1~ 7 PCr/US93/06120
- 19 -
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
- 23 -
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
- 25 -
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
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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.
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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
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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
30-
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
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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
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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
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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.
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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.
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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
38-
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
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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
- 41 -
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
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(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
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- 43 -
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
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- 45 -
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
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Table 1
Deletions
VAL 1
VAL 13
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
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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
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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
- 49 -
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
- 52 -
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.
R2
~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
- 53 -
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)
