Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
`91/06650 2 0 7 1 8 7 1 PC~r/US90/06072
Cl INHIBITOR MUTEINS AND USES THEREOF
This invenlion is in Ihe area of molecular biology/immunology, and presenls
genetically engineered conslrucls of C1 inhibitor, lermed C1 inhibilor muleins, Ihat are
resistant IO proteolylic altacL;. The muleins have considerable applications, preferabl~
as anti-inflammalo.~y agenls and more preferably for the prophylaclic or Iherapeutic
treatment of sepsis.
In the United States alone nosocomial bacteremia develops in about 194,000
patients, 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, and these are Pseudomonas
aeruginosa, Escherichia CO'li, Proteus, Klebsiella, Enterobacter and Serratia. The
current treatment for bacteremia is the administration of antibiotics which,
unfortunalely, have lirniled effectiveness.
The precise pathology of bacleremia is nol completely elucidated, nevertheless,
1, it is known that bacterial endoloxins, lipopolysaccharides (LPS), are the primary
causative agent. LPS consist of at least three significant antigenic regions, the lipid A,
core polysacchaIide, and O-specific polysacchaIide. The latter is also referred to as O-
specific chain or simply O-antigen. The O-specific chain region is a long-chain
polysaccharide built up from repeating polysaccharide units. The number of
polysaccharide units differs among different bacterial species and may vary from one to
as many as six or seven monosaccharide units. While the O-specific chain varies
among different gram-negative bacteria, the lipid A and core polysaccharides are similar
if not identical.
Since LPS plays a key role in sepsis, a variety of approaches has been pursued
to neutralize its activity. Presently, there is considerable work which suggest that
antibody to LPS will soon be a valuable clinical adjunct to the standard antibiotic
therapy.
LPS initiates a cascade of biochemical events that eventually causes the death of
the patient. It is widely believed that the second event, after the introduction of LPS, is
the production of tumor necrosis factor (TNF) as a result of LPS stimulation of
macrophage cells. Thus, considerable effort has been expended to produce
neutralizing antibody to TNF, or other molecules that could inhibit its septi-c effects. I
is likely that antibody to TNF will have valuable clinical applications. Tracey, et al.,
1987, Nature, 330:66''.
Sepsis caused by gram-negative bacteria is thought to involve activation of the
complement system and causes a depletion of various complement component. One
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component of a complement system, C~a, causes the a~gregation of neutrophils andthe aggregates are thought to embolize and cause ischemia. Siegel, J., 1981, Ann.
Rev. Med., 32:175. It has been proposed that CSa is thus responsible for the observed
organ failure phenomena in sepsis.
Cl is a plasma glycoprotein with a molecuiar weight of about 105,000 and is a
member of the super family of serine protease inhibitors which include such members
as al-antitrypsin, al-antiplasmin, antithrombin III, and plasrninogen activator
inhibitor types I and II. One mechanism by which the activator components of thecomplement system are controlled is by the C1 inhibitor. The C1 inhibitor is known to
inhibit activating components of the classical pathway of complement (Clr and Cls)
and the intrinsic coagulation system (Factor XIa, Factor XIIa, and Kallikrein).
Further, C1 inhibitor has been shown to interact with the fibrinolytic components
plasmin and tissue plasminogen activator.
C1 inhibitor is susceptible to proteolytic cleavage by so called non-target
proteases, particularly lysosomal serine protease elastase. Browere, M. and Harpel,
P., 1982, J. Biol. Chem., 257:9849. This enzyme is released from
polymorphonuclear leukocytes and is present in the circulation of septaremic patients.
It is thought that the decrease in the concentration of coagulation factors observed in
these patients may, in part, be the result of proteolysis by leukocyte elastase of C1
inhibitor. It will be appreciated, that a possible prophylactic/therapeutic approach to
treating sepsis would be to genetically engineer C1 inhibitors that are resistant to
proteolytic cleavage and administer these to patients that are at risk of contracting
sepsis, or that are already septic.
The life threatening nature of sepsis mandates the identification and
development of additional therapeutics or prophylactics, both antibody based or
otherwise, that may be efficaciously applied in the treatment of sepsis.
In its most general form, the invention described herein presents C1 inhibitor
muteins, methods of constructing the muteins, and applications of the muteins,
preferably as anti-inflammatory agents and more preferably for the prophylactic or
therapeutic treatment of sepsis.
A second object of the invention described herein relates to C1 inhibitor muteins
that are both elastase resistant, and that maintain the capacity to covalently bind to, and
inactivate components of the complement system.
A third object of the invention is a description of C1 inhibitor muteins that have
amino acids at positions 440 and/or 442 mutated to another suitable amino acid, or
, 91/06650 2 0 ~ 1 8 7 1 Pcr/us90/06072
deleted, that are both elastase resistant, and that maintain the capacity to covalently bind
to, and inactivate components of the complement system.
A fourth object of the invention is a description of C1 inhibitor muteins that
display differential sensitivity to proteases and inhibitory activity depending on the type
of amino acid that is substituted for amino acids at positions 440 and!or 442.
A fifth object of the invention is a description of C1 inhibitor muteins that
exhibit differential inhibitory activity towards various substrates depending on the type
of amino acid that is substituted for amino acids at positions 440 and/or 442.
Further, the invention concerns the prophylactic or therapeutic use of C1
inhibitor muteins as anti-inflammatory medicaments, and preferably for the
prophylactic or therapeutic treatment of sepsis.
These and further objects of the invention will become apparent after a
consideration of the detailed description of the invention shown below.
Figure 1 shows the cDNA sequence corresponding to recombinant C1 inhibitor.
Figure 2 schematically presents a generalized assay procedure for deterrnining
the inhibitory or protease sensitivity of the C1 muteins.
Figure 3 shows the degree of inhibitory activity (complex formation) and
protease sensitivity (inactivation) of C1 muteins to Cls and Kallikrein.
Fig,ure 4 shows the degree of inhibitory activity ~complex formation) and
protease sensitivity (inactivation) of C1 muteins to B-12a and plasmin.
Figure S shows the amount of neutrophil elastase needed for 50% inhibition of
several Cls inhibitor muteins.
1. Dçfinitions
To facilitate understanding the nature and scope of applicant's invention, several
definitions regarding various aspects of the invention are presented below. It will be
understood, however, that these definitions are general in nature, and encompassed
within the definitions are meanings well known to those skilled in the art.
Sepsis is herein defined to mean a disease resulting from gram-positive or gram-negative bacterial infection, the latter primarily due to the bacterial endotoxin,
lipopolysaccharide (LPS). It can be induced by at least the six major gram-negative
bacilli and these are Pseudomonas aeruginosa, Escherichia coli, Proteus, Klebsiella,
Enterobacter and Se~ratia.
By Cl inhibitor is meant a plasma glycoprotein with a molecular weight of
about 105,000 that belongs to the super family of serine protease inhibitors. It inhibits
activated components of the classical pathway of complement, Clr and Cls, and the
intrinsic coagulation system, factor XIa, factor XIIa, and Kallikrein. C1 also interacts
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with plasmin and tissue plasminogen activator. Cl has the further property of itself
being inactivated by pro~eases, notably elastase. It will, or course, be understood that
intended to come within the scope of the definition of the C1 inhibitor are fragments of
the molecule that maintain biologically acuvity.
By Cl inhibitor mutein is meant a molecule that has the biological ac~ivitv of
Cl inhibitor, although to different degrees as exemplified by the data of the in~ention,
and is particularly resistant to proteolytic attack.
Several patents/patent applications and scientific references are referred to
below. The instant invention draws on some of the material and methods shown in
these references, and thus it is intended that all of the references, in their entirety, be
incorporated by reference.
2 . Cl Inhibitor M~lteins
C1 inhibitor has been cloned and expressed and thus is readily available to the
practitioner to perform the herein described mutagenesis. For example, cloning and
expression is described by Bock, ç~ al., 1986, Biochemistrv~ 25:4292. The cDNA
sequence is shown in Figure 1. Further, Eldering, et al., 1988, J. Biol. Chem.,
263:11776, show a Aat II-HaeII C1 inhibitor cDNA fragment that encodes the entire
molecule. This fragment can be manipulated using the procedures described below to
generate the C1 inhibitor muteins.
A. Mutein Construction--General Proce~ures
Construction of suitable vectors containing the desired coding and control
sequences for the Aat II-HaeII (:~1 inhibitor cDNA fragment employs standard ligation
and restriction techniques which are understood in the art. Isolated plasmids, DNA se-
quences, or synthesized oligonucleotides are cleaved, tailored, and religated in the forrn
desired.
More specifically, site specific DNA cleavage is performed by treating with the
suitable restriction enzyrne (or enzymes) under conditions which are generally
understood in the art, and the particulars of which are specified by the manufacturer of
these commercially available restriction enzymes. See, e.g., New England Biolabs,
Product Catalog. In general, about l ~lg of plasmid or DNA sequence is cleaved by
one unit of enzyme in about 20 ~ of buffer solution; in the exarnples herein, typically,
an excess of restriction enzyme is used to insure complete digestion of the DNA
substrate. Incubation times of about one hour to two hours at about 37C are workable,
although variations can be tolerated. After each incubation, protein is removed by
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s
extTaction with phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered fTom aqueous fractions by precipitation with ethanol and
resuspension in 10 mM Tris~ I mM EDTA, pH 7.5. If desired, size separation of the
cleaved fragments mav be performed by polyacrvlamide gel or agarose gel electro-phoresis using standard techniques. A general description of size separations is found
in Methods in Enzvmologv, 1980, 6~:499-560.
Restriction cleaved fragments may be blunt ended by treating with the large
fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxy-nucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 minutes at
20 to 25C in 50 mM Tris pH 7.6,50 mM NaCl, 6 rnM MgCl2, 6 mM DTT and 5- l O
~LM dNTPs. The Klenow fragment fills in at 5' sticky ends but chews back protruding
3' single strands, even though the four dNTPs are present. If desired, selective repair
can be performed by supplying only one of thej or selected, dNTPs within the
limitations dictated by the nature of the sticky ends. After treatrnent with Klenow, the
15 rnixture is extTacted with phenol/chloroform and ethanol precipitated followed by
running over a Sephadex G-50 spin colurnn. Treatment under appropriate conditions
with S 1 nuclease results in hydTolysis of any single-stranded portion.
Synthetic oligonucleotides are prepared by the triester method of Matteucci et
ak, 1981, J. Am. Chem. Soc., 103:3185, or using commercially available automated20 oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for
labelling is achieved using an excess, e.g., approximately 10 units of polynucleotide
kinase to 0.1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2,5
mM dithiothreitol, 1-2 mM ATP,1.7 pmoles ~32P-ATP (2.9 mCi/mmole), 0.1 mM
spermidine,0.1 mM EDTA.
Ligations are performed in 15-30 ~ volumes under the following standard
conditions and temperatures: 20 mM Tris-CI pH 7.5, 10 mM MgCl2,10 mM DTT, 33
~g/ml BSA, 10 mM-50 mM NaCl, and either 40 ~LM ATP, 0.01-0.02 (Weiss) units T4
DNA ligase at 0C (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4
DNA ligase at 14C (for "blunt end" ligation). Intermolecular "sticky end" ligations are
usually performed at 33-100 ~lg/ml total DNA concentrations (5-100 nM total end
conce ~ -ation). Intermolecular blunt end ligations (usually employinr a 10-30 fold
molar ~xcess of linkers) are perfotmed at 1 IlM total ends concentrati~,n.
In vector construction employing "vector fragments", the vector fragment is
~mmonly treated with bacterial alkaline phosphatase (BAP) in order to remove the 5'
3; phosphate and prevent religation of the vector. BAP digestions are conducted at pH 8
in approximately 150 mM Tris, in the presence of Na+ and Mg+2 using about 1 unit of
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BAP per llg of vector at 60C for about l hour. In order to recover the nucleic acid frag-
ments, the preparation is extracted with phenol/chloroform and ethanol precipitated and
desalted by application to a Sephadex G-50 spin column. Alternatively, religation can
be prevented in vectors which have been double digested by additional restriction
enzyme digestion of the unwanted fragmen~s.
For portions of vectors derived from cDNA or genomic DNA which require
sequence modifications, site specific primer directed mutagenesis is used. This is
conducted using a synthetic prirner oligonul~leotide complementary to a single stranded
phage DNA to be mutagenized except for lirnited mismatching, representing the desired
o mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis
of a strand complementary to the phage, and the resulting double-stranded DNA istransformed into a phage-supporting host bacterium. Cultures of the transformed
bacteria are plated in top agar, permitting plaque formation from single cells which
harbor the phage.
Theoretically, 50% of the new plaques will contain the phage having, as a
single strand, the mutated form; 50% will have the original sequence. The resulting
plaques are hybridized with kinased synthetic pritner at a temperature which permits
hybridization of an exact match, but at which the rnisrnatches with the original strand
are sufficient to prevent hybridization. Plaques which hybridize with the probe are then
20 picked, cultured, and the DNA recovered. Details of site specific mutation procedures
are described below in specific examples.
Correct ligations for plasmid construction are confirrned by firs~ transforming
E. ~Qli strail1 MM294 obtained from E. ~ Genetic Stock Center, CGSC #6135, or
other suitable host with the ligation mixture. Successful transformants are selected by
25 ampicillin, tetracycline or other antibiotic resistance or using other markers depending
on the mode of plasmid construction, as is understood in the art. Plasmids from the
transformants are then prepared according to the method of Clewell, D.B., ~.t al., 1969,
Proc. Natl. Acad. Sci. (USA), 62:1159, optionally following chloramphenicol amplifi-
cation tClewell, D.B., 1972, J. Bacteriol., 110:667). The isolated DNA is analyzed by
30 restriction and/or sequenced by the dideoxy method of Sanger, F., I ~1-. 1977, Proc.
Natl. Acad. Sci. (USA), ~:5463 as further described by Messing ç~ al., 1981, Nucleic
Acids Res., 9:309, or by the method of Maxam et al., 1980, Methods in Enzvmolog~,
65:499.
Depending on the host cell used, transformation is done using standard
35 techniques appropriate to such cells. The calcium treatment employing calciumchloride, as described by Cohen, S.N., Proc. Natl. Acad. Sci. (USA) (1972) 69:2110,
or the RbC12 method described in Maniatis et al., Molecular CloQin ~n A Laboratorv
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Manual (1982) Cold Spring Harbor Press, p. 2~4 was used for procaryotes or othercells which contain substantial cell wall barriers~ For mamrnalian cells without such
cell walls, the calcium phosphate precipitation method of Graham and Van der Eb,Virolo~v, 1978, 5~:~46 is preferred.
Host strains used in cloning and expression herein are as follows. For cloning
and sequencing, E. coli strain HB 101 may be used as the host. For Ml3 phage
recombinants, E. ~Qli strains susceptible to phage infection, such as E.~ K12 strain
DG98 are employed. The DG98 strain has been deposited with ATCC 13 July 1984
and has accession number 1965. A preferred expression system for the C1 inhibitor
10 muteins is the COS-lcell /pSVL vector system shown by Eldering et al., 1988, Journal
of Biolo~cal Chemistrv, 263:11776. pSVL (Pharmacia, Uppsala, Sweden) consists
of the SV40 origin of replication, the SV40 late promoter, and the VP1 intron in front
of a polylinker followed by the SV40 late polyadenylation signal, fused to a pBR32
fragment containing the origin of replication and ampicillin resistance gene.
Mutagenesis can be carried out using any number of procedures known in the
art. These techniques are described by Smith, 198~, Annual Review of Genetics,
19:423, and modifications of some of the techniques are described in ~lethods inEnzvmolo~v,154~ part E, (eds.) Wu and Grossman (1987), chapters 17, 18, 19, and
20~ The preferred procedure is a modification of the Gapped Duplex site-directed20 mutagenesis method which is described by Kramer, _t al., in chapter 17 of volume 154
of Methods in Enzvmolo~v, above; and by Kramer et al., 1984, Nuçleic Acids
Research, 12:9441.
B. Mutein CQnstruction - Preferred Procedures
Conventional M13 mutagenesis methods involve annealing a short synthetic
25 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
complementary to the target sequence and has at least one mispaired nucleotide. After
the annealing reaction, the remaining portion of the single stranded DNA must be filled
in to give heteroduplex DNA that can be transfected into a suitable host cell which
30 allows for the expression of the mutation. In the gapped duplex method, as described
by Kramer, et al., in chapter 17 of the Methods in Enzvmolo,,v, a partial DNA duplex
is constructed that has only the target region exposed, unlike the conventional methods
which have the target region and the rest of the single stranded M13 DNA exposed.
Like the conventional methods, a short oligonucleotide is annealed to the target region,
35 and extended and ligated to produce a heteroduplex. However, because only a small
portion of single-stranded DNA is avallable for hybridization in the gapped duplex
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method, the oligonucleotide does not anneal to undesired sites within the M13 genome.
This method has the additional advantage of introducin~ fewer errors during the
formation of the heteroduplex since onlv a very small region of DNA on either side of
the target region has to be filled in.
More specifically, the gapped duplex melhod involves cloning the larget Aa~
HaeII C1 inhibi~or cDNA fragment into an appropriate M13 phage that carries
selectable markers, such as, for exarnple, the stop codon amber mutation. The latter
allows for negative selection in a host cell that cannot suppress the effects of the
mutation. Preferably the phage is M13mp9 which contains two amber codons in
10 critical phage genes. Thus, the sequence that encodes C1 is cloned into M13mpg
amber+, and single stranded DNA is prepared therefrom using standard techniques.Next, double strandedreplicative form DNA from M13 GAP, a genetically engineeredM13 derivative that lacks the amber codons is cleaved with the appropriate restriction
enzyme. The base sequence of M13 GAP is similar to M13mpl8, which lacks both thel S amber codons and the sequence between base pairs 6172 and 6323. This deletion
flanks the multiple cloning sites of the M13mp series and generates a unique restriction
site. Gapped duplex DNA is formed, using standard DNA/DNA hybridization
techniques, consisting of single stranded DNA having the amber codons, and a second
strand of DNA from digested M13 GAP lacking both the amber codons and the C1
coding sequences. Thus, the only portion of the gapped duplex that is exposed is the
C1 target sequence. The desired oligonucleotide(s) is annealed to the gapped duplex
DNA, and any remaining gaps filled in with DNA polymerase and the nicks sealed
with DNA ligase to produce a heteroduplex. As applied to the instant invention
mutagenesis was performed with a mixture of oligonucleotides that code for 16
different muteins. The sequence of the degenerate oligonucleotide used to isolate PS
and P3 double mutants and the PS leucine and valine single muteins is:
S' GCG GGC C(AG) (GC) AGA G (AG) (GC) GGC GGA G 3'
The oligonucleotide is complementary to nucleotides 1412-1433 of Bock et al., above.
In addition to the above, several other single P3 muteins were obtained using
the following oligonucleotides:
P3-ala 5' GGTGOGGGOGGCAGAGATGG 3'
P3-gly S' GGTGOGGGCTCCAGAGATGG 3'
P3-arg 5' GGGTGOGGGCTCTAGAGATGGCG 3'
P3-leu 5' GOGGGCCAGAGAGATGG 3'
P3-thr S' GGGTGOGGGOGGTAGAGATGGCG 3'
The heteroduplex is transfected, preferably into a mismatch repair deficient
host, and mixed phage produced. From the mixed phage population, phage carlving
~91/066~0 2 0 7 1 ~ 7 1 PC~r/US90/06072
unmutated C1 DNA, which also have the amber mutations, can be selected a_ainst bv
infec~ing the mixed phage population into a host cell that cannot suppress the amber
mutation. Clones can then be screened for phage that carry a Cl mutation, and the
molecules sequenced to determine the position of the mutation. Clones were screened
5 using a mixture of kinased degenerale oligonucleotides. Using the foregoing method.
11 C1 inhibitor muteins were produced.
C1 inhibitor mutein DNA fragments were excised from M13 with the
appropriate restriction enzymes and cloned into a vector suitable for expression in COS
cells. The vector is pSVL and is shown in Bock et ah, above. Alternatively pC1-INH,
10 also described by Bock et al., could be used.
SV40-transformed COS-1 monkey cells were grown in Iscove's Modified
Dulbecco Cell Culture Mediurn containing penicillin and streptomycin. The media was
supplemented with 10% v/v) heat-inactivated fetal calf serum. Transfection of the cells
was performed essentially as described by Luthmann, and Magnusson, 1983, NucleicAcids Research,5:1295. This consisted of incubating subconfluent COS-1 cells in the
cell culture media for 90 minutes with supercoiled vector encoding C1 inhibitor mutein
DNA (5-7.5 ~Lg/ml) and DEAE-dextran (200 ~lg/rnl). After a 90 minute incubation
period, the cells were washed twice with cell culture media, and incubated for an
additional 2 hours with cell culture media containing 80 ~lg/ml chloroquine. Twofurther washes were performed, and the cells incubated with cell culture media
supplemented with 10% fetal calf serum. This media was replaced 24 hours later with
serum free media. The latter media was harvested after 72 hours, centrifuged to
remove cells and debris, and assayed for C1 inhibitor activity, as described below.
C. Assav for C1 ~nhibitor Mutein Activities
Figure 2 presents a generalized assay screening format for determining both the
inhibitory (complex formation) or protease sensitivity (inactivation) of the C1 muteins.
The latter measures inactivation of the C1 inhibitor muteins by non-target proteases.
This procedure is also described by Eldering ç~ al., 1988, in J. of Biolo,,ical Chem.
~: 11776. Modifications of these methods are also known in the art.
Generally, inhibitor activity of C1 muteins may be determined by measuring
complex formation between the C1 inhibitor and a substrate. The preferred substrates
are, of course, Cls, Kallikrein, B-12a, and plasmin. The C1 inhibitor, or C1 inhibitor
muteins, inhibit the protease activity of the substrate by forrning a covalent bond with
the substrate. These molecules were purified by techniques known in the art, or as
3~ described by Liebermann, H., et al., 1984, J. Mol. Biol.,177:531 and Nuijens, J., et
ak, 1987, Immunologv, 61:387.
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Many procedures are available for measuring the complex resulting from the
interaction of the C1 inhibitor mutein and its target proteinase substrate. and include
standard immunochemical, radiochemical, or elisa assays. Levin~ M., et ah, 1983, J
Biol. Chem., ~58:6~15, Nuijens, J., et ah, 1987, Thromb. Haemost., :-8:778, de
Agostini, 1985, PNAS, 8~:~190. The procedures generally consist of contacting a Cl
inhibitor mutein with a target substrate in solution to permit comple~; formation to
occur, then separating the complex from uncomplexed reactants, and detecting theamount of complex formed. Other assays may be employed whereby the amount of
reactants, that is, free C1 inhibitor mutein or target molecule, remaining in the reaction
10 solution are measured after complex formation has occurred.
The preferred assay is a radioirnmune assay based on the observation that
functional C1 inhibitor muteins bind to activated target substrate, such as Cls. The
assay can be performed in several ways, but preferably purified activated Cls iscoupled to a solid matrix, preferably Sepharose 4B via cyanogen bromide as is known
1; in the art. Coupled Cls is incubated in an appropriately buffered solution containing, if
desired, a small amount of detergent, preferably Tween-20. The latter is used at a
concentration of about 0.1% (w/v). The capacity of the C1 inhibitor muleins to
complex with the target substrate can be detected using anti-C1 inhibitor muteinantibody. Alternatively, a radiolabelled second antibody can be used to detect the
20 bound first antibody. The antibody may be polyclonal or monoclonal and is incubated
for a sufficient time to permit detectible binding of the antibody to the C1 inhibitor to
occur. After the appropriate washing steps are conducted whereby non-specifically
bound radiolabelled antibody is separated from antibody bound to C1 inhibitor muteins,
the amount of radioactivity associated with the latter is determined. In this way, and
25 incorporating the appropriate controls in the assay scheme, the inhibitory capacity of the
C1 muteins is determined.
Using similar approaches, the non-target protease sensitivity of the C1 inhibitor
muteins may be determined. Numerous assays may be employed that measure the
amount of intact C1 inhibitor mutein, or fragments that are derived from the muteins,
30 remaining after exposure to protease. Many procedures are available for measuring the
protease sensitivity of the C1 inhibitor muteins, and include standard immunochemical,
radiochemical, or elisa assays. A solid phase assay is preferred whereby C1 inhibitor
muteins are reacted with non-target protease bound to a solid matrix and thè amount of
proteolysis of the muteins measured by determining the amount of intact mutein
35 remaining, or preferably by detecting fragments of the mutein. Alternatively, the C1
inhibitor mutein may be bound to a solid support matrix and this material subjected to
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`91/06650 P ~ /US90/06072
proteolysis provided attachment of the mutein does not stericly interfere with protease
accessability to the mutein .
An exemplary non-target protease that cleaves C1 is neutrophil elastase. Thus.
this enzyme may be coupled to CNBr treated Sepharose 4B, and incubated with a C1inhibitor mutein, and the presence of proteolytically cleaved C1 mutein monitored using
a number of techniques. The preferred procedure is to pellet the Sepharose-coupled
elastase, and measure the presence of cleaved C1 inhibitor in the supernatant using an
antibody that recognizes this molecule. Such antibodies are available and are described
by Nuijens, et al., I988, lood, 22:1841. They may be attached to a solid matrix to
o facilitate separating the cleaved C1 inhibitor mutein from the other reactants. The
Sepharose beads containing antibody having bound cleaved C1 mutein inhibitor arespun down, washed and separated from the supernatant containing uncleaved C1
inhibitor or cleaved but unbound fragments. Subsequently, the amount of bound
cleaved C1 inhibitor can be determined using a second radiolabelled antibody that
recognizes the cleaved molecule. Finally, the amount of radioactivity adherent to the
Sepharose beads may be determined as an indication of the amount of cleavage
resulting from elastase.
The general procedures for determining both the inhibitory or non-target
protease sensitivity of the C1 inhibitor muteins are shown in Figure 1. The procedures
involving Cls will be described briefly, the procedures for the other substrates is
similar with modifications as noted by Eldering, 1988, J. Biolo~ical Chem.,
~:11776, and as shown by Nuijens et al., above.
As mentioned above, the radioimmune assay to determine the C1 inhibitor
activity of the various muteins is based on tne observation that functional C1 inhibitor
mutein will bind to activated Cls. Thus, purified activated Cls is coupled to CNBr
Sepharose 4B and suspended in phosphate-buffered saline, pH 7.4 10 mM EDTA, and
0.1% (w/v) Tween-20. Next, 0.3 ml of this mixture containing 1.5 ~g of activatedCls is incubated with various dilutions of the C1 inhibitor muteins for 5 hours. The
Sepharose 4B beads are collected, extensively washed, and complexed C1 inhibitormutein ipcubated (>4 hours) with 125I-polyclonal anti-C1 antibody. The antibody is
described by Hack, et al., above. The Sepharose beads are washed, and bound
radioactivity determined using a LKB 1260 multigamma II gamma counter.
Inactivation of t~. C1 inhibitor muteins is determined as follows. Porcine
pancreatic elastase is coupled to Sep rose 4B such that about 3.75 mg of elastase is
coupled to 300 mg of Sepharose. The beads are suspended in phosphate buffered
saline containing, 10 mM EDTA, and 0.1% w/v Tween-20. Various amounts of C1
inhibitor muteins are incubated with 150 ~1 of Sepharose 4B suspension containing 5.6
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mg of elastase in 500 lal of volume for l hour at room temperature. Subsequently, the
mixture is centrifuged~ and the supernatant assayed for the presence of inactivated C1
inhibitor mutein using KOK12 monoclonal antibody bound to Sepharose, and
polyclonal l2sI-anti-C1-inhibitor antibodies, as described above.
Figure 3 shows the degree of inhibitory activity (complex formation) and
protease sensitivity (inactivation) of 11 C1 inhibi~or muteins as assessed using Cls and
Kallikrein as substrates and neutrophil elastase as the source of protease. Figure 4
shows the same data with the exception that the substrates were B-12a and plasmin.
It is noteworthy that the 11 C1 inhibitor muteins exhibit considerable vafiationin inhibitor activity, and protease sensitive. This was a function of both the type of
amino acid used to substitute for the wild type arnino acid, and the substrates used to
test for inhibitory activity.
The wild type Cl inhibitor has isoleucine and valine at positions 440 and 4~.
respectively. Mutations at position 440 and 442 are termed Ps and P3 muteins,
respectively. The figures show that muteins altered only at P3 (Ala, Gly, Arg, Leu or
Thr substituted for Val) display different properties depending on the target protease
used. When solid phase Cls is used, complex forrnation occurs in the order Arg =Gly~Ala<Leu~wild-type = Thr.
Further, it appears that two variants, Ps-leu:P3-ala; and Ps-leu:P3-leu show
significantly reduced susceptibility to inactivation. The amount of HNE needed for
50% inactivation, as determined by residual functional activity towards Cls, is
increased by a factor 5 to 8 compared to wild type C1 inhibitor (Figure 5).
D. Cl Pe~vlated Muteins
The preferred embodiment Clmuteins consists of modified muteins that have a
substantially longer in vivo circulating lifetime than the unmodified molecules. Favored
modified C1 inhibitor muteins are those having a water soluble polymer bound to the
muteins. Exemplary of such water soluble polymers are polyacrylic acid, and
derivatives thereof, dextran, carboxymethylcellulose, polyethylene glycol, and
polyoxyethylated glycerol. .The preferred embodiment water soluble polymer is
polyethylene glycol. It is disclosed in U.S. Patent No. 4,179,337, along with methods
for binding polyethylene glycol to proteins. Polyethylene glycol modified IL-2 is
shown in U.S. Patent No. 4,766,106. Using the compositions and procedures
described in these two patents, polyethylene glycol modified C1 muteins are readily
produced by those skilled in the art.
2071871
9~/06650 PCI/US90/06072
E. Administration of Cl Muteins
It will be appreciated by those skilled in the art that the C1 muteins describedherein can be administered to mamrnals, including humans, either alone or in
combination with other anti-inflammatory agents, or they may be combined with
5 various pharmaceutically acceptable diluents or carriers. Such are widelv known to
those skilled in the art and are formulated according to standard pharmaceuticalpractices.
Exemplary diluents include physiologic saline, or buffered saline, as well as
Ringer's and dextrose injection fluid, and dextrose saline and lactated Ringer's injection
10 or diluent solutions containing additional therapeu~ic agents, preferably antibiotics or
antibody known to be efficacious in the treatrnent of sepsis. Such antibody would
include those known to be beneficial for the therapeutic treatrnent of sepsis caused bv
different strains of bacteria, preferably Pseudomonas aeruginosa, Escherichia coli,
Proteus, Klebsiella, Enterobacter and Serratia.
15 F. Therapeutic Applicanon of Cl Inhibitor Muteins
One embodiment of the invention is the adrninistration of an effective arnount of
the subject C1 inhibitor muteins to individuals that are at a high risk of developing
sepsis or that have developed sepsis. An example of the former category are patients
about to undergo surgery. W .e the mode of adrninistration is not particularly
20 irnportant, parenteral administration is preferred because of the rapid progression of
sepsis, and thus, the need to have the C1 inhibitor muteins compositions disseminate
quickly throughout the body. The preferred mode of adminis~ation is to deliver an
I.V. bolus slightly before, during, or after surgery. The dosage of the C1 inhibitors
will normally be determined by the prescribing physician. It is to be expected that the
25 dosage will vary according to the age, weight and response of the individual patient.
Having generally described what the applicants believe their invention to be,
presented below are examples that are illustradve of the scope of the invention. It will
be appreciated by those skilled in the art that the examples are not intended to be
construed as limiting the invention to the materials and methods shown as there are
30 numerous substitutions that can be made therein without departing from the scope of
the invention.
The present invention has been described with re~erence to specific
embodiments. However, this application is intended to cover those changes and
substitutions which may be made by those skilled in the art without departing from the
35 spirit and the scope of the appended claims.
