Method for promoting cell growth and immunosuppression using chaperonin

A process for the detection of cpn10 in serum or other biological fluids including the steps of (i) raising antibody to cpn10; (ii) reacting said antibody with a sample of biological fluid suspected of containing cpn10; and (iii) detecting the presence of cpn10 in said sample by a signal amplification resulting from production of a cpn10-antibody complex. There is also provided a process for promotion of cell growth or immunosuppression including the step of administration of cpn10 to a mammalian subject. There is also provided recombinant cpn10.

FIELD OF THE INVENTION 
This invention relates to chaperonin 10 otherwise known as cpn10. 
BACKGROUND OF THE INVENTION 
Chaperonins belong to a wider class of molecular chaperones, molecules 
involved in post-translational folding, targeting and assembly of other 
proteins, but which do not themselves form part of the final assembled 
structure as discussed by Ellis et al., 1991, Annu. Rev. Biochem. 60 
321-347. Most molecular chaperones are "heat shock" or "stress" proteins 
(hsp); i.e. their production is induced or increased by a variety of 
cellular insults (such as metabolic disruption, oxygen radicals, 
inflammation, infection and transformation), heat being only one of the 
better studies stresses as reviewed by Lindquist et al., 1988, Annu. Rev. 
Genet. 22 631-677. As well as these quantitative changes in specific 
protein levels, stress can induce the movement of constitutively produced 
stress proteins to different cellular compartments as referred to in the 
Lindquist reference mentioned above. The heat shock response is one of the 
most highly conserved genetic system known and the various heat shock 
protein families are among the most evolutionarily stable proteins in 
existence. As well as enabling cells to cope under adverse conditions, 
members of these families perform essential functions in normal cells. 
There are two types of cpn molecules, cpn60 (monomeric M.sub.r .about.60 
000) and cpn10 (monomeric M.sub.r .about.10 000). Cpn60 has been studied 
extensively. It has been identified in all bacteria, mitochondria and 
plastids examined, and a cytoplasmic form, TCP-1, has been identified in 
eukaryotic cells; its presence on the surface of some cells has been 
reported, although this has been questioned in the Ellis reference 
referred to above and also in van Eden, 1991, Immunol. Reviews 121 5-28. 
Until very recently, cpn10 had been identified only in bacteria but 
structural and functional equivalents have now been found in chloroplasts 
(Bertsch et al., 1992, Proceedings of the National Academy of Sciences USA 
89 8696-8700) and in rat (Hartman et al., 1992, Proceedings of the 
National Academy of Sciences USA 89 3394-3398) and bovine liver 
mitochondria (Lubben et al., 1990, Proceedings of the National Academy of 
Sciences USA 87 7683-7687). 
Cpn60 and cpn10 interact functionally, in the presence of ATP, to mediate 
protein assembly. Instances of cpn10 acting independently of cpn60 have 
not yet been reported but cpn60, apparently acting alone, has been 
implicated in quite disparate events. For example, it is an 
immuno-dominant target of both antibody and T-cell responses during 
bacterial infections but, because the protein is so highly conserved, self 
reactivity is generated. Healthy individuals may use this self-recognition 
to eliminate transformed and infected autologous cells but defects in 
control of such recognition may lead to autoimmune disease as discussed by 
van Eden, 1991, Immunol. Reviews 121 5-28. Not surprisingly, cpn60 has 
been associated with conditions such as rheumatoid arthritis. There is 
thus a growing awareness that molecular chaperones, with their capacity to 
bind to and alter the conformation of a wide variety of polypeptides, may 
occupy key roles in cellular functions other than protein biogenesis. 
Reference may also be made to Hartman et al., 1993, Proceedings of the 
National Academy of Sciences USA 90 2276-2280 which describes the 
stabilization of protein molecules using cpn10 and cpn60. 
It can also be established that for mammalian cpn10's, there is a very 
close sequence homology. Thus, for example, the rat cpn10 molecule 
(Hartman et al., 1992, Proceedings of the National Academy of Sciences USA 
80 3394-3398) has greater than 99% homology with the structure of bovine 
cpn10 reported in EMBL Data Base Directory under MT BTC PN10 which was 
submitted by J. E. Walker, MRC Lab. of Molecular Biology, Hills Road, 
Cambridge, UK. This has to be contrasted with bacterial cpn10's which have 
an average degree of homology with rat chaperonin 10 of only 34% (Hartman 
et al., 1992). 
Early Pregnancy Factor (EPF) 
EPF was first described as a pregnancy associated substance (Morton et al., 
1976, Proc. R. Soc. B. 193 413-419) and its discovery created considerable 
interest as it enabled the detection of a potential pregnancy within 6-24 
hours of fertilisation. Initially EPF was assigned a role as an 
immuno-suppressant by virtue of its ability to release suppressor factors 
from lymphocytes (Rolfe et al., 1988, Clin. exp. Immunol. 73 219-225). 
These suppressor factors depress the delayed type hypersensitivity 
reaction in mice and therefore might suppress a possible maternal immune 
response against the antigenically alien fetus. More recent studies have 
shown that production of EPF is not confined to pregnancy. It is a product 
of primary and neoplastic cell proliferation and under these conditions 
acts as a growth factor (Quinn et al., 1990, Clin. exp. Immunol. 80 
100-108; Cancer Immunol. Immunother, 1992, 34 265-271). EPF is also a 
product of platelet activation and it is proposed therefore that it may 
play a part in wound healing and skin repair (Cavanagh et al., 1991, 
Journal Reproduction and Fertility 93, 355-365). 
To date, the rosette inhibition test remains the only means of detecting 
EPF in complex biological mixtures (Morton et al., 1976, Proc R Soc B 
413-419). This assay is dependent on the original finding of Bach and 
Antoine, 1968, Nature (Lond) 217 658-659 that an immunosuppressive 
anti-lymphocyte serum (ALS) can inhibit spontaneous rosette formation in 
vitro between lymphocytes and heterologous red blood cells. A modification 
of the assay was introduced to detect EPF after it was demonstrated that 
lymphocytes, preincubated in EPF, give a significantly higher rosette 
inhibition titre (RIT) with an ALS than do lymphocytes from the same donor 
without EPF as described in the 1976 reference above. This test has been 
described in detail in the above 1976 reference as well as in Morton et 
al., 1987, in "In Current Topics in Developmental Biology" Vol 23 73-92, 
Academic Press, San Diego, but briefly it involves a cascade of events 
with EPF binding to lymphocytes and sequentially inducing the release of 
suppressor factors (Rolfe et al., 1988, Clin. exp. Immunol. 73 219-225); 
(Rolfe et al., 1989, Immunol. Cell Biol. 67 205-208). 
In Athanasas-Platsis et al., 1989, Journal Reproduction and Fertility 87 
495-502 and Athanasas-Platsis et al., 1991, Journal Reproduction and 
Fertility 92 443-451, there is described the production of monoclonal and 
polyclonal antibodies to EPF and passive immunization of pregnant mice 
with these antibodies which causes loss of embryonic viability. These 
studies established that EPF is necessary for the successful establishment 
of pregnancy. 
In Quinn et al., 1990, Clin. exp. Immunol. 80 100-108, it is proposed that 
EPF is a growth regulated product of cultured tumour and transformed 
cells. These cells are also dependent upon EPF for continued growth i.e. 
EPF acts in an autocrine mode. 
It has been established that EPF plays a role in promoting tumour growth 
since the growth of tumour cells can be significantly retarded by anti-EPF 
mAbs. In addition this reference suggests that hybridomas producing high 
affinity anti-EPF antibodies may be inherently unstable. 
In Quinn et al., 1992, Cancer Immunol. Immunother, 34 265-271, there is 
also described the effect of monoclonal antibodies (mAbs) to EPF on the in 
vivo growth of transplantable murine tumours. The main thrust of this 
reference is that neutralisation of EPF retards tumour growth in vivo. 
It is clear from the above Quinn et al. 1992 reference that when cancer is 
in the very early stage of growth, neutralisation of EPF by anti-EPF mAb 
will prevent its development. However, once the cancer becomes 
established, treatment with these mAbs will retard but not entirely 
destroy the tumour. 
Other references in regard to the role of EPF in tumour growth include 
Quinn, 1991, Immunol. Cell Biol. 69 1-6 and Quinn, K. A. in a PhD thesis 
entitled "Early pregnancy factor: a novel factor involved in cell 
proliferation.sup.11 from the University of Queensland in Australia in 
1991. 
EPF is reviewed in detail by Morton et al., 1992, Early Pregnancy Factor, 
Seminars in Reproductive Endocrinology 10 72-82. The site and regulation 
of EPF production is described, followed by the purification of EPF from 
platelets and the relationship of the purified product to EPF derived from 
other sources. This review also discusses certain aspects of the bioassay 
for EPF (i.e. the rosette inhibition test) including monitoring 
purification procedures and investigating sources of production. The 
biological activity of EPF is discussed and possible clinical applications 
of EPF and its antagonists are described. 
Morton et al., 1992, Reprod. Fertil Dev. 4 411-422 reviews previous 
publications describing the immuno suppressive and growth factor 
properties of EPF. The role of EPF in maintaining the pre-embryo is also 
discussed in this reference. 
Both of the abovementioned references, which are essentially review 
articles, describe the preparation of purified EPF for blood platelets 
which included the initial sequential steps of heat extraction of the 
platelets, cation exchange chromatography on SP-SEPHADEX, crosslinked 
dextran beads C-25, affinity chromatography on Heparin-SEPHAROSE, 
crosslinked agarose beads CL-6B and Concanavalin-A-Sepharose 4B. The final 
purification of EPF was achieved by high performance hydrophobic 
interaction chromatography, followed by three reversed phase (RP)-HPLC 
steps. After the final RP-HPLC step, EPF was isolated as single UV 
absorbing peak coincident with biological activity, well separated from a 
number of minor contaminants. The biological and radioactivity of an 
iodinated sample of this material eluted with identical retention time 
when fractionated under the same conditions. When analysed by SDS-PAGE and 
visualised by autoradiography, the iodinated material ran as a single band 
of approximate Mr 10,000, again coincident with biological activity. The 
approximate yield of EPF by this method was 5 .mu.g per 100 platelet 
units. 
This demonstrates that it was necessary to use this complex purification 
procedure to obtain only a small amount of native EPF and thus this method 
could not be used on a commercial scale. In this regard, the only 
practical sources known for obtaining native EPF at this time were 
platelets and regenerating liver. 
Surprisingly, in accordance with the present invention, the final 
fractionated EPF when subjected to sequencing as more fully described 
hereinafter found that the structure of native EPF corresponded to 
chaperonin 10 which could not have been predicted from the aforementioned 
prior art. 
This unexpected discovery as will be apparent from the disclosure 
hereinafter has now been reduced to practice in that recombinant 
chaperonin 10 has been found to have all the biological activity 
previously associated with EPF and thus EPF can now be produced 
commercially which was not the case previously using suitable techniques 
for producing recombinant cpn10. It will also be apparent that EPF can now 
be produced synthetically. 
SUMMARY OF THE INVENTION 
In one aspect, the invention resides in the discovery that cpn10 is EPF and 
has the hitherto unknown or unsuspected properties demonstrated by EPF. 
The unknown or unsuspected properties of cpn10 include extracellular 
activities such as the ability to act as a growth factor and an 
immunosuppressive factor. In another aspect the invention provides one or 
more methods for using cpn10 to exploit the unknown or unsuspected 
properties of cpn10. The one or more methods includes a method of using 
cpn10 to promote growth and a method of using cpn10 to suppress 
immunological activity. 
The term "cpn10" as used herein, insofar as methods of promotion of cell 
growth and immunosuppression are concerned, includes within its scope 
recombinant cpn10 as well as cpn10 which is produced synthetically. The 
term "cpn10" also includes eucaryotic cpn10 as well as procaryotic cpn10 
inclusive of groES or derivatives of recombinant cpn10. The recombinant 
cpn10 may be produced by recombinant DNA technology as described 
hereinafter. The term also includes biological fragments. 
The present invention also includes within its scope a modified recombinant 
cpn10 as well as derivatives and peptide fragments derived therefrom. 
The invention in another aspect refers to an assay for detection of cpn10 
which includes the detection of native cpn10.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention includes within its scope the following. 
Assay For cpn10 
The detection of cpn10 in serum or other biological fluids using monoclonal 
or polyclonal antibodies against recombinant or synthetic cpn10or against 
modifications or fragments thereof alone or in combination with each other 
or with cpn60 (in the presence of ATP or other nucleotide triphosphates) 
for the purpose of: 
(a) pregnancy diagnosis in any mammalian species; 
(b) monitoring embryonic well-being in "at-risk" pregnancies; 
(c) diagnosis of tumours; and 
(d) monitoring patients after surgical removal of tumours. 
Treatment With CPM10 
The use of recombinant cpn10 as a growth factor or immunosuppressant in the 
treatment of: 
(a) skin or organ grafts; 
(b) wound healing, tissue repair or regeneration of tissue; 
(c) autoimmune disease; 
(d) infertility/miscarriage; 
(e) allergic disease; and 
(f) inflammatory conditions. 
Experimental 
Purification of cpn10 
(a) Purification of Human EPF from Human Blood Platelets (FIG. 1a, 1b, 1c, 
1d) 
Extraction 
Platelet concentrates (from the Blood Bank), up to 7 days clinically 
outdated, were washed with Tyrodes buffer, following the techniques 
described in Methods in Enzymology, 1989, 169 7-11, snap frozen in liquid 
N.sub.2 and stored at -70.degree. C. 
Immediately prior to purification, approximately 100 washed platelet units 
were thawed in a boiling water bath, then held at 75-85.degree. C for 15 
min with continuous, gentle stirring. After cooling on ice, cellular 
debris was removed by centrifugation (8000 g, 20 min, 4.degree. C.) and 
the pellet extracted twice by homogenisation in 0.05 M-acetic acid/0.1 
M-NaCl/0.1 mg/ml sodium azide pH 3.0 followed by centrifugation (8 000 g, 
15 min 4.degree. C.). The three supernatants were pooled giving a total 
extract volume of 500-600ml. 
Ion -Exchange Chromatography 
This extract from 100 platelet units was adjusted to pH 3.0 with conc. HCl 
and stirred gently, overnight, 4.degree. C., with 250ml SP-SEPHADEX, 
crosslinked dextran beads C-25 (Pharmacia-LKB), previously swollen and 
equilibrated with 0.05 M-acetic acid/0.1 M-NaClpH 3.0. The gel was then 
packed into a column washed with 20 vol of the same buffer and eluted with 
5 vol 0.5 M-sodium phosphate buffer/0.05 M-NaClpH 7.5. The gel was then 
discarded. 
Affinity Chromatography 
The SP-SEPHADEX, crosslinked dextran beads; eluate was adjusted to pH 
6.3-6.4 with conc. HCl and applied to a column of Heparin-SEPHAROSE, 
crosslinked agarose beads CL-6B (2.5.times.7.5 cm; Pharmacia-LKB) 
previously equilibrated with 0.05 M-sodium phosphate buffer 0.05 M-NaCl pH 
6.3. The column was then washed with 5 vol of the same buffer and eluted 
with 5 vol 0.05 M-Tris-HCl/5 mM-CaCl.sub.2 /0.2 M-NaCl pH 7.5, applied in 
the reverse direction to that used for sample application. 
High Performance hydrophobic Interaction Chromatography (HIC-h.p.l.c.) 
Solid (NH.sub.4),.sub.2 SO.sub.4 was added to the Heparin-SEPHAROSE, 
crosslinked agarose beads eluate to a final concentration of 2 M and, 
after passage through an 0.45 .mu.m filter, the sample was pumped through 
a dedicated solvent line onto a TSK Phenyl 5PW column (7.5.times.75 mm, 
Pharmacia-LKB), previously equilibrated with 0.1 M-Tris-HCl pH 7.0/5mM 
CaCl.sub.2 /2 M-(NH.sub.4).sub.2 SO.sub.4. The column was washed with 10 
vol of the same buffer and eluted with a 50 min linear Gradient from this 
buffer to 0.1 M-Tris-HCl pH 7.015 mM-CaCl.sub.2 /10% acetonitrile. (FIG. 
1a) 
RP-h.p.l.c. 
Active HIC-h.p.l.c. fractions were pooled, then fractionated on a C.sub.3 
column (Ultrapore RPSC. Beckman Instruments) using a solvent system 
consisting of A, 0.04 M Tris/HCl pH 7.0/5 mM-CaCl.sub.2 and B, 0.04 
M-Tris/HCl pH 7.0/5 mM-CaCl.sub.2 /80% (v/v) acetonitrile. The column was 
equilibrated with Solvent A prior to sample application, after which it 
was washed with 5 vol solvent A and eluted with a 30 min linear gradient 
from this solvent to 75% solvent B. (FIG. 1b) 
RP-h.p.l.c.2 
Active fractions from RP-h.p.l.c.-1 of several 100 unit platelet 
preparations were pooled, EDTA and DTT added to a final concentration of 
20mM and 1mM respectively and the mixture allowed to stand for 0.5-1 h, 
4.degree. C. Following dilution with 2 vol solvent A, it was applied to a 
C.sub.3 column, dedicated to this and subsequent steps, and fractionated 
as described for RP-h.p.l.c.-1, but omitting CaCl.sub.2. (FIG. 1c) 
Rph.p.l.c.3 
Active fractions from RP-h.p.l.c.-2 were pooled, trifluoroacetic acid (TFA) 
added to a final concentration of 0.1% and, following dilution with 2 vol 
0.1% TFA, the mixture was applied to the C.sub.3 column, which had been 
equilibrated previously with 0.1% TFA. The column was then eluted with a 
30 min linear gradient from this solvent to 60% (v/v) acetonitrile/0. 1% 
TFA, followed by a 3 min linear gradient to 90% (v/v) acetonitrile/0. 1% 
TFA. Active fractions were pooled. (FIG. 1d) 
One unit represents platelets from a single blood donation which is 
approximately 500 ml. The "active fractions" were fractions active in the 
rosette inhibition test. 
Purification of EPF from other sources 
EPF has been purified from various sources as discussed in Cavanagh & 
Morton, 1994, Eur. J. Biochem. 222 551-560; Quinn et al., 1994, Hepatology 
20 No 5 1294-1302. 
In all instances, biological activity followed the same pattern throughout 
the complex purification scheme described above for human platelets. 
Furthermore the final active fraction from all sources was bound 
specifically by an immobilised monoclonal anti-EPF and could be recovered 
virtually quantitatively (see FIG. 1e). 
These studies are important for several reasons: 
A. The biochemical and immunological similarity observed with all these 
materials provides strong evidence that the bioassay is detecting a single 
substance or closely related family of substances acting in diverse 
biological situations. 
B. The active agents purified from all of these materials are from several 
to many orders of magnitude more potent than virtually all of the 
substances previously reported to be EPF. This confirms our surmise, based 
on detailed analysis of the EPF bioassay as discussed above, that activity 
associated with most putative EPF preparations must reflect the presence 
of a very minor contaminant. 
C. The only source materials providing sufficient EPF to study at the 
protein (as opposed to activity) level were platelets and regenerating 
liver, yielding, respectively, an average of 15 .mu.g per 100 units 
(equivalent to .about.50 liter blood) and 5 .mu.g per 40 g tissue (liver 
remnant from 6 rats). It is immediately apparent that far more EPF is 
present within the cell than appears in the extracellular space; 
nevertheless, accumulated knowledge of the biology of EPF (reviewed 
recently in the abovementioned Morton et al. 1992 reference) indicates 
that this extracellular appearance is not fortuitous. 
Human platelet-derived EPF, being most abundant, has been studied in some 
detail. On SDS-PAGE, it ran as a single band of Mr approx, 8.500, 
coincident with biological activity (see FIG. 2a); EPF from regenerating 
rat liver exhibited identical behaviour. Mass spectometry of the platelet 
material provided an accurate and precise determination of molecular mass 
10 843.5.+-.2 Da, along with definitive evidence of the high degree of 
homogeneity of the preparation (see FIG. 2b). Following attempts at Edman 
degradation, which indicated that the molecule is N-blocked, proteolytic 
cleavage of approx. 4 nmol EPF was undertaken. Resultant peptide fragments 
were separated by reversed-phase HPLC and subjected to sequencing by Edman 
degradation. Three areas of sequence containing 12 (fragment 1), 27 
(fragment 2) and 33 (fragment 3) residues were found to correspond with 
residues 7 to 18-27-53 and 69 -101 (the C-terminus) in rat mitochondrial 
cpn10. In fragment 2, residue 52 was different (S in cpn10, G in rat 
cpn10, this change alone could account for human cpn10 being 30 Da larger 
than rat cpn10). All other residues were identical, consistent with the 
highly conserved nature of chaperonins (see FIG. 2c). 
Since confirming sequence identity between EPF and cpn10 several studies of 
functional relationship have been performed, using rat mitochondrial cpn10 
E. coli cpn10 (known as groES) and E. coli cpn60 (groEL). First it has 
been demonstrated that cpn10 can act as EPF. Rat cpn10 was tested in the 
EPF bioassay and found to be positive over the range of dilutions 
expected; this activity could be neutralised by monoclonal antibodies to 
EPF (see TABLE 1). Interestingly, E. coli cpn10, which is .about. 40% 
homologous with rat cpn10, exhibited no activity in the bioassay (see 
TABLE 1): this is consistent with the observation that E. coli conditioned 
medium is not active in the EPF bioassay, while medium conditioned by all 
mammalian cell lines tested, as well as by yeast cells is active. Cpn60 
was inactive in the bioassay and had no effect upon the activity of EPF. 
It was then shown that EPF can act as cpn10. EPF was mixed with cpn60 , in 
the presence or absence of ATP, and the mixture fractionated on a TSK 
G3000SW gel permeation column: resultant fractions were analysed by 
SDS-PAGE. Cpn60 is a decatetramer and elutes in the excluded volume of 
this column (exclusion limit 300 000). In the presence of ATP, but not in 
its absence, EPF also appears in this fraction, demonstrating formation of 
a stable complex with cpn60. This fraction was active in the EPF bioassay 
but the equivalent fraction from the experiment without ATP (where EPF did 
not associate with cpn60) was not (see FIG. 3a). Thus EPF and cpn10 
activity reside in the same molecule. 
These investigations provide unequivocal evidence that platelet-derived EPF 
is a structural and functional homologue of cpn10the relationship between 
cpn10 and activity in the rosette inhibition test was then examined (FIG. 
3b). In the presence, but not in the absence of ATP, immobilised cpn60 
could remove all activity from the archetypal source material, pregnancy 
serum and activity could be recovered by removing ATP from the immobilised 
complex. As with the experiment described in FIG. 3a, this requirement for 
ATP demonstrates the specificity of the interaction between cpn60 and the 
active moiety; cpn10 is thus the molecular entity initiating response in 
the EPF bioassay. 
Identification of EPF as a cpn10 has been a major step forward in research 
on this subject and helps to explain many of the findings that have been 
made to date. Criticism has been raised against claims that EPF production 
occurs in such a wide variety of biological situations e.g. pre- and 
post-implantation pregnancy, primary and tumour cell proliferation and 
platelet activation. In its role as a hsp (heat stress protein) following 
the advent of the present invention, these are all conditions in which the 
rapid onset of EPF production would now be expected. Functions of hsp's 
that are vital to the survival of cells are intracellular as shown in the 
Linquist et al. reference above. In contrast, the activity of EPF 
described to date is extracellular; for example, it appears in serum of 
mice within 4 to 6 hours after mating as discussed in Morton et al., 1987, 
Current Topics in Development Biology, Vol 23 73-92 and 4 to 8 hours after 
partial hepatectomy in rats as shown in the Quinn PhD thesis (1991), 
available from the Biological Sciences Library, University of Queensland 
Australia, catalogued under both author and title. We have shown that EPF 
can act in an autocrine mode as discussed in the Quinn et al., 1990 
reference referred to above or exocrine mode as discussed in the Rolfe et 
al. 1988 referred to above; these are not roles previously described for 
hsp's. 
It will also be appreciated that since the structure of EPF is now known, 
it can be produced in commercial quantities by any suitable technique of 
recombinant DNA technology. 
(b) Cloning of Human cDNA Encoding cpn10 and Production of cpn10 
Production for commercial use may be obtained by inserting a mammalian 
cpn10 gene, preferably a human cDNA cpn10 gene, into a suitable vector 
such as plasmids from the pGEX system, and pET system expressing the 
encoded mammalian cpn10 and purifying the recombinant cpn10. 
______________________________________ 
Abbreviations 
______________________________________ 
ANGIS Australian National Genomic Information Service 
bp base pair 
BSA bovine serum albumin 
cDNA complementary DNA 
cpn 10 Chaperonin 10 
DNA deoxyribonucleic acid 
E. coli Escherichia coli 
GSH glutathione (reduced form) 
GST glutathione-S-transferase 
LB Luria-Bertani Broth 
M Molar 
ORF open reading frame 
PCR polymerase chain reaction 
rEPF recombinant Early Pregnancy Factor 
RSP reverse sequencing primer 
SDS sodium dodecyl sulphate 
SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel 
electrophoresis 
Tris Tris(hydroxymethyl)aminomethane 
USP universal sequencing primer 
______________________________________ 
Cloning of Human cpn10 Open Reading Frame 
Polymerase chain reaction (PCR) was used to initially amplify part of the 
ORF (274 bp) of the human cpn10 cDNA from a melanoma cell line A2058 cDNA 
lambda library (Stratagene). A degenerate cpn10 amplimer (P1) was designed 
from the amino acid sequence VLDDKDYFL (SEQ ID NO:1) corresponding to 
amino acid residues 83-91 of human cpn10. The primer P1 has the sequence 
5' ARRAARTARTCYTTRTCRTC 3' (SEQ ID NO:2) where R is A or G and Y is C or 
T. The reverse sequencing primer (RSP) was used for PCR amplification (the 
non-specific primer) as well as for sequencing DNA constructs and has the 
sequence 5' CAGGAAACAGCTATGAC 3' (SEQ ID NO:3). The universal sequencing 
printer has the sequence 5' GTAAAACGACGGCCAGT 3' (SEQ ID NO:4). PCR 
amplification of the phage library was achieved using a non-specific 
upstream amplimer (RSP) and P1, each at 0.5 .mu.M final concentration, 1.5 
mM MgCl.sub.2 (Pharmacia Biotech), 1 .times. polymerase buffer (Boehringer 
Mannheim) and 5 units of Thermus aquaticus DNA polymerase (Boehringer 
Mannheim) in a final volume of 50 .mu.L. For 30 cycles, the parameters 
were: denaturation at 94.degree. C. for 1 min. annealing at 40.degree. C. 
for 30 sec and extension at 72.degree. C for 3 min. A final extension at 
72.degree. C. for 7 min was followed by a soak cycle at 4.degree. C. for 
10 min. An aliquot of 1 .mu.L was reamplified under the same conditions to 
increase the cop) number. 
Two cpn10 specific amplimers encompassing the open reading frame were 
designed. The upstream primer P2, 5'-GCGCGGATCCATGGCAGGACAAGCGTTTAG-3' 
(SEQ ID NO:5) was designed from the sequence of the initial PCR fragment. 
The downstream primer P3, 5' ATATGAATTCAGTCTACGTACTTTCC-3' (SEQ ID NO:6) 
was designed from sequence obtained from the Expressed Sequence Tag 
database via ANGIS (Accession No. HUM00TB037). A 319 bp fragment was 
amplified from the phage library using the same reaction and cycling 
conditions as above except the annealing temperature was 50.degree. C. 
DNA Constructs and Analysis 
All restriction enzyme digests of PCR products and vectors were performed 
according to Sambrook et al. (Sambrook et al., 1989, Molecular Cloning: A 
Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, 
N.Y.) using restriction enzymes and their buffers obtained from Boehringer 
Mannheim. The initial PCR fragment was digested with Eco R1 and ligated 
(Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. 2nd Ed. 
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) into the Eco RI and 
Sina I sites of pBluescript KS(+) (Stratagene) creating the plasmid pRMI 
(FIG. 4: partial cpn10 insert 274 bp). The 319 bp product was digested 
with Bam HI and ECo RI and initially cloned into the expression plasmid 
pGEX-2T (Pharmacia Biotech) creating the plasmid pRM2 (FIG. 5). To confirm 
its identity, the Bum HI-Eco R1 fragment was subcloned into pBluescript 
(SK+) (pRM3; FIG. 6) and sequenced. DNA was analysed on 0.8-1.0% (w/v) 
agarose gels containing ethidium bromide and after electrophoresis was 
viewed under UV illumination. 
Transformation of E. Coli 
Competent E. coil DH5.alpha. cells (100 .mu.L ) were transformed with the 
plasmids by the heat pulse method (Sambrook et al., 1989, Molecular 
Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold 
Spring Harbor, N.Y). The mixture of cells and DNA (10-100 ng) was placed 
on ice for 30 min and heat pulsed for exactly 2 min at 42.degree. C. and 
placed back on ice for 2 min. The cells were allowed to recover at 
37.degree. C. with shaking for 1 hr after the addition of 0.9 mL of LB. A 
100 .mu.L aliquot was plated onto LB agar plates supplemented with 
Ampicillin at a final concentration of 100 .mu.g/mL. After incubation 
overnight at 37.degree. C. random colonies were selected for further 
investigation. 
DNA Sequence Determination 
Restriction fragments of the PCR products were cloned into pBluescript and 
sequenced in both orientations by the dideoxy chain-termination method 
using the T7 Polymerase Kit according to the manufacturer's instructions 
(Pharmacia Biotech). Approximately 2 .mu.g of plasmid DNA was denatured, 
ethanol precipitated and annealed to either the USP, RSP or P3. The 
sequencing reactions were electrophoresed on a 8% acrylamide/46/% urea 
gel. After fixing and drying, X-ray film was exposed to the gel overnight 
and developed. 
Expression and Purification of recombinant cpn10 E. coli 
Clones transformed with pRM2 were screened for expression of the 
Glutathione-S-transferase fusion protein on a small culture scale (2 ml) 
according to methods described by Smith et al. (Smith et al., 1988, Gene 
67 (1) 31-40). An overnight culture was diluted, induced to express the 
fusion protein by the addition of IPTG to 0. mM and grown at 37.degree. C. 
for several hours. The cells were pelleted, lysed in PBS/0.1% Triton X-100 
and the lysate mixed with 50% Glutathione-Agarose beads (Sigma Chemical 
Company). The recombinant fusion protein was eluted from the affinity 
beads by boiling in SDS loading buffer. An aliquot of the sample was run 
on a 10% SDS-PAGE gel. The gel was fixed and then stained with Coomassie 
blue. After confirming the expression of the fusion protein the 
purification of rcpn10 from the GST moiety was undertaken on a larger 
scale. 
Cells were grown and induced as above, the cell pellet resuspended in PBS, 
sonicated (output level 4, 50% duty cycle, 2.times.30 sec) and the cell 
lysate stored at -30.degree. C. Lysate from 10 liter cell culture was 
thawed and rcpn10 isolated by similar techniques to those used by Gearing 
et al. (Gearing et al., 1989, Biotechnology 7 1157-1161) for isolation of 
rLIF. Briefly, TRITON X-100, a non-ionic surfactant; was added to a final 
concentration of 0.1% and cellular debris removed by centrifugation (15 
min, 15000 rpm, 4.degree. C.). Ten ml glutathione-SEPHAROSE, cross linked 
agarose beads 4 gel (Pharmacia - LKB Biotechnlogy) was added to the 
supernatant and the slurry mixed for 2 hr, 4.degree. C. The gel was 
pelleted, washed .times. 5 with 50 ml PBS/0.1% Triton X-100 once with 50 
ml 0.05 M Tris-HCl pH 8.0/0.15 M NaCl and once with 0.05M Tris-HCl pH 
8.0/0.15 M NaCl/2.5 mM CaCl.sub.2. The gel was resuspended in 4 ml of 0.05 
M Tris-HCl pH 8.0/0.15 M NaCl/2.5 mM CaCl.sub.2 buffer, 1000 units 
thrombin (Sigma T6884) added and the slurry was mixed in a shaking 
waterbath for 1 hr, 37.degree. C. The gel was pelleted, the supernatant 
retained, and the gel was then washed with 3.times.4 ml 0.05 M Tris-HCl pH 
8.0/0.15 M NaCl. These washes and the first supernatant, which contain the 
rcpn10, were pooled, yielding 4-5 mg recombinant protein. Additional 
rcpn10, which was non-specifically bound to the gel, was recovered as 
follows. Four ml 0.05 M Tris-HCl pH 8.0/2 M NaCl was added and the slurry 
mixed for 2 hr, 4.degree. C. 
After pelleting, the gel was washed with 3.times.2 ml of this 0.05 M 
Tris-HCl pH 8.0/2 M NaCl buffer, the washes pooled with the first 
supernatant, yielding a further approximately 1 mg rcpn10. Protein 
concentrations were estimated by the method of Lowry et al. (Lowry et al., 
1951, J. Biol. Chem. 193 265-275); proteins were analysed by SDS-PAGE 
using 15% Tris-Tricine gels (Schagger et al., 1987, Anal. Biochem. 166 
368-379). 
The recombinant cpn10 has two additional amino acids at the N terminus. The 
N terminus of the recombinant protein is Gly-Ser-Methionine-Ala whereas 
the N-terminus of native protein is Ac-Ala. The amino acid sequence of the 
recombinant cpn10 is as follows: 
GSMAGQAFRKFLPLFDRVLVERSAAETVTKGGIMLPEKSQGKVLQATVVA 
VGSGSKGKGGEIQPVSVKVGDKVLLPEYGGTKVVLDDKDYFLFRDGDILGKYVD (SEQ ID NO:9) 
2. Application of Mammalian cpn10 16 
(a) Assay for cpn10 
Antigen 
A bacterial fusion protein, GST/cpn10, was expressed and isolated with 
glutathione-Sepharose, as described for preparation of cpn10. The fusion 
protein was eluted from the gel by application of 50 mM reduced 
glutathione in Tris-buffered saline. Eluted fractions were analysed by 
SDS-PAGE and those containing the most fusion protein were pooled. Protein 
concentration was determined by the method of Lowry et al., 1951, J. Biol. 
Chem. 193 265-275. 
Antibody 
Antibodies against the fusion protein were raised in rabbits using an 
immunisation schedule consisting of 4.times. weekly injections followed by 
at least 4.times. monthly boosts. Approximately 10 pg protein, emulsified 
in Freund's Complete Adjuvant for the first injection and in Incomplete 
Adjuvant thereafter, was used for each injection. Rabbit serum was 
screened for anti-cpn10 antibodies by ELISA using plates coated initially 
with cpn10 (5 .mu.g/ml) and a streptavidin-biotin detection system 
(Amersham). The antibody (Ab) titres against cpn10 and against the whole 
fusion protein (in this case GST/cpn10, 5 .mu.g/ml, was bound to the 
plate) in serum of rabbit #42 are shown in FIG. 7. Titration of a serum 
sample against cpn10, taken from this rabbit after the 4th booster dose, 
is illustrated in FIG. 8. 
Immunoassay 
This antibody was then used in a competitive binding assay for detection of 
cpn10, performed as follows. Anti-serum, diluted 1:32000 and 1:64000 
(final dilution: diluent 50 mM sodium phosphate buffer, pH 7.4, containing 
0.2% w/v zelatin) %was incubated, separately (overnight, 4.degree. C.) 
with various concentrations of cpn10. These mixtures were then tested by 
ELISA as described above, in plates coated with 5 .mu.g/ml cpn10, as 
illustrated in FIG. 9. Absorbance values for each antibody/cpn10 mixture 
are compared with values obtained for the same antibody dilution incubated 
without cpn10. The degree of inhibition of binding of antibody to the 
plate is proportional to the amount of cpn10 in the original 
antibody-cpn10 mixture; from this, a standard curve can be constructed, as 
shown in FIG. 10. 
While rabbit #42 is not sensitive enough to detect the very low 
concentration of cpn10 present in serum, we have established techniques 
which can: 
(1) produce an anti-cpn10 antibody which displays normal hyperimmunisation 
properties: thus with known techniques for enhancement of the immune 
response, an antibody with greater avidity could be produced, and 
(2) produce a response in a standard immunoassay technique. 
With improved antibodies, application of known methods for enhancement of 
the detection system and pretreatment of serum to both concentrate and 
partially purify cpn10 (e.g. by application to CI.sub.18 Sep-pak cartridge 
[Waters] and elution with 80% acetonitrile in Tris-buffered saline, or to 
immobilised cpn60 in the presence of ATP and elution with EDTA), this 
technique, alone or in combination with other immunoassay techniques, 
could be developed for detection of cpn10 in serum. 
Sensitivity of the Rosette Inhibition Test, the EPF Bioassay 
The rosette inhibition test is non-quantitative and cannot be used to 
determine the cpn10 concentration in serum with accuracy. The assay may be 
used semi-quantitatively by comparing the limiting dose of samples, i.e. 
the highest dilution of sample giving a position response in the bioassay. 
Caution must be exercised with this approach since other substances in 
complex biological fluids, themselves inactive in the bioassay, can 
influence the response of active materials. 
We have determined that the bioassay can detect as little as 5 -50 .mu.m/ml 
pure cpn10 (Cavanagh et a/l., 1994, Eur. J. Biochem. 202 551-560). Based 
on the observed limiting dose of serum from pregnant women (known 
inhibitory substances having been removed from early pregnancy serum) and 
tumour-bearing animals and individuals as well as from rats 24 hr 
post-partial hepatectomy, the cpn10 concentration of serum is likely to be 
in the range 0.1-100 pg/ml. 
Treatment with cpn10 
(a) Organ/Skin Grafts 
The Effect of Recombinant cpn10 on The Survival of Allogenic Skin Grafts In 
Rats 
Skin grafting 
Skin grafts were exchanged between inbred Lewis and DA rats (.about.100 g) 
using the following protocol. Abdominal full thickness skin was sutured 
onto a similar sized defect created on the lateral thoracic region using 
standard techniques. A group of six rats were grafted in one session, with 
each rat receiving one autograft and one allograft. Two Lewis and 2 DA 
rats received daily .times. 2 injections of recombinant cpn10 and one 
Lewis and one DA received buffer, injected around the site of the grafts. 
Different groups received different doses of cpn10. Injections were 
continued for 14 days. The grafts were covered with Vaseline gauze, 
Melolin dressing, plastic wrap and Co-flex elastic bandage. After 7 days, 
the grafts were examined daily for signs of necrosis. The day of rejection 
was taken as that on which 50% of the transplanted skin had undergone 
necrotic degradation. 
Life Span of cpn10 Activity In Serum Following Injection of Recombinant 
cpn10 into Mice 
Various doses of recombinant cpn10 (see FIG. 11) were injected i.p. into 
BALB/c mice (.about.20 g) and the mice bled at various times after 
administration, commencing at 15 minutes (zero time). Serum was tested for 
cpn10 activity in the rosette inhibition test (see Morton et al., 1987, 
Current Topics in Developmental Biology 23 73-92) with spleen cells from 
C57BL/6 mice. Mice receiving platelet cpn10 were tested in parallel. The 
half life of cpn10 activity in serum was determined. 
Results 
The results are shown in TABLE 2 and FIG. 11 
There was a significant prolongation of graft survival time following 
injection of recombinant cpn10 (p &lt;0.001, Student's t test). The results 
showed a bell-shaped dose response curve, with the most effective doses 
being in the range of 2 to 20 .mu.g cpn10.times.2/rat/day. The experiments 
in mice suggest that this recombinant cpn10 has a shorter than expected 
half life in serum, when compared with platelet cpn10; the half life of 1 
.mu.g and 15 .mu.g recombinant cpn10 in serum of mice was only 3 hours and 
7 hours respectively, compared with platelet cpn10 (5 .mu.g), which had a 
half life of 4 days. However, these results have shown that cpn10 can 
significantly prolong the viability of allogenic skin grafts in rats. (See 
TABLE 2). 
(b) Treatment of Mammals Including Humans With cpn10 To Promote Wound 
Healing 
The Involvement of cpn10 In Tissue Repair 
Growth factors are likely to be involved in the healing process,as their 
initial release from platelets is of fundamental importance in wound 
repair (Falange, 1993, J. Dermatol. Surg. Oncol. 19 716-720). Platelets 
have been shown to be a rich source of cpn10 (cpn10; Cavanagh et al., 
1994, Eur. J. Biochem 222 551-560) and therefore may be one of the growth 
factors intimately involved in wound healing. Studies have been carried 
out to determine the effect of topically-applied recombinant cpn10 
(rcpn10) on the healing of full-thickness skin defects created in mice. 
Methods 
Outbred, male Quackenbush mice (aged 8 weeks) were anaesthetized with 
Nembutal, shaved, skin sterilized with 70% v/v ethyl alcohol and a full 
thickness defect (8 mm diameter) created in the lateral thoracic region. 
One .mu.g rcpn10 in 5 .mu.l Tris-buffered 0.9% w/v sodium chloride 
(saline) pH 7.4, Tris-buffered saline alone (5 .mu.l) or saline alone (5 
.mu.l) was applied directly to the wound, which was then covered with 
Vaseline gauze, Melolin non-adherent dressing and held in place with 
Co-flex elastic bandage. Twice daily, the mice were lightly anaesthetized 
with halothane (Fluothane, ICI), the dressings removed, 5 .mu.l of the 
appropriate solution applied and the wound redressed. At various 
intervals, i.e. 24 hr, 48 hr, 3 d, 4 d, 5 d, 6 d and 7 d, groups of mice 
were euthanased with halothane, the would and surrounding tissue removed 
and the area of the wound measured. 
Results 
Following treatment with rcpn10, there was a significant accceleration of 
wound contraction, when compared with wounds treated with buffer or saline 
(FIG. 12). In the wounds treated with cpn10, wound contraction commenced 
within the first 24 hrs, whereas the control wounds, contraction commenced 
after 2 days (FIG. 12). From 3 days, there was no significant difference 
in wound size. 
Conclusions 
Cpn10 applied topically to full thickness wounds in mice, accelerates 
contraction and healing, with the process appearing to commence directly 
after wounding. Wound contraction in the control mice did not become 
evident until at least 48 hrs later. 
Normally, wound healing takes place in three phases. Phase 1, the 
inflammatory phase (0-48 hrs), begins immediately after injury and is the 
time during which activated platelets secrete growth factors into the 
defect, facilitating fibroblast activation and increasing the activity of 
cells, e.g. macrophages, involved in the subsequent stages of wound 
healing. Phase 2, the proliferative phase (2-6 days), begins as the first 
fibroblasts appear and epidermal cells multiply and migrate to the wound 
site. Phase 3 is the maturation phase. 
Wound contraction does not normally commence in phase 1, also known as the 
lag phase. During this phase, the shape and size of the excised wound is 
influenced by elastic forces in the neighbouring skin. These forces 
increase the initial size of the defect and give it a different shape 
corresponding to the tension lines present in the skin. As we have shown 
in the groups of mice treated with buffer or saline, the wounds were 
enlarged during the first 48 hrs. In contrast, the wounds in mice treated 
with rcpn10 contracted during this time suggesting that administration of 
rcpn10 directly to the wound accelerated migration of fibroblasts and 
deposition of collagen to the wound area. This finding will have enormous 
significance in the treatment of wounds including bums, as accelerated 
would contraction will greatly decrease fluid loss and risk of infection. 
(c) Autoimmune Disease 
The Effect of cpn10 On The Development of Experimental Allergic 
Encephalomyelitis In Rats, An Animal Model of Autoimmune Disease 
Introduction 
Experimental allergic encephalomyelitis (EAE) is an autoimmune 
demyelinating disease of the nervous system, induced by inoculation of 
animals with central nervous system myelin basic protein (MBP) in 
adjuvant, and widely studied as an animal model of multiple sclerosis 
(Raine, 1984, Laboratory Investigation 50 608-635). The clinical features 
of EAE in the rat, a commonly studied species, are dramatic overnight 
weight loss from day 10 after inoculation, followed by tail weakness and 
paralysis, hindlimb weakness and sometimes paralysis. Forelimb weakness 
and paralysis sometimes occur (Pender. 1986, Journal of Neurological 
Sciences 75 317-328). Experiments were undertaken to determine if 
administration of rcpn10 to rats following inoculation, would influence 
progress of the disease. 
Methods 
EAE Model 
EAE was induced in inbred female Lewis rats (aged .about.10 weeks) 
following inoculation with MBP in Freund's adjuvant into one footpad. 
Three groups of rats were included in the study. All were inoculated on 
day 0. Group 1 (n=4) received no treatment and animals were not handled 
during the incubation period of the disease (day 0 to day 8). Group 2 
(Control group; n=5) received Tris-buffered saline (0.1 ml) i.p..times.2 
daily from day 0 to day 20. Group 3 (Test group; n=5) received 15 ug 
rcpn10 in Tris-buffered saline (0.1 ml) i.p..times.2 daily from day 0 to 
day 20. From day 8, all rats were weighed and examined daily for 30 days. 
(I) Tail weakness was graded as follows:- 
0=no weakness; 
1=weakness of distal part of the tail only, the distal tail failing to curl 
round the examiner's finger; 
2=weakness of the whole tail but the proximal tail still being able to be 
erected vertically against gravity: 
3=severe weakness with only a flicker of tail movement; 
4=complete flaccid paralysis of the tail. 
(II) Hindlimb weakness was graded thus: 
0=no weakness; 
1=slight dragging of the toes of both hindfeet; 
2=severe dragging of both hindfeet but not of the rest of the hindlimbs; 
3=severe dragging of both hindlimbs, often with both hindlimbs displaced to 
one side of the body; 
4=total flaccid paralysis of the hindlimbs. 
(III) The forelimbs were assessed in a similar way to the hindlimbs. 
Total score was the sum of the scores in (I). (II) and (III). 
Result 
The time of onset of weight loss and period of maximum weight loss in the 
groups receiving no treatment or receiving injections of buffer alone 
(Control group) did not differ significantly (Table 3). However, initial 
weight loss was delayed in the group receiving cpn10 (Test group), 
compared with the group receiving no treatment, as also was the period of 
maximum weight loss (Table 3; p&lt;0.001 .chi..sup.2 distribution). There was 
no significant difference between the means of maximum weight loss in the 
three groups (Table 3). 
Administration of i.p. injections 2 .times. daily to the rats with the 
necessary handling involved did not affect the time of onset or the 
severity of the disease but did prolong the course of the disease for 
several days (day 17 to day 18 as shown in FIG. 13). However, one marked 
difference between these two groups was the recurrence of severe disease 
in the Control group at day 22, persisting until day 30. In the group 
receiving no treatment, mild disease only recurred in 3 rats on days 27 
and 28. 
As with early weight loss, weakness and paralysis of the tail and limbs 
were delayed in the Test group, receiving rcpn10, compared with the 
Control group (FIG. 14: day 12, p&lt;0.01: day 13, p&lt;0.05, Heteroscedastic t 
test). In the Test group over the period from 14 to 16 days, only one rat 
developed severe disease, similar to that developed by the Control rats. 
The remaining 4 rats only developed mild disease during this period (FIG. 
15, p&lt;0.95, Heteroscedastic t test). Severe disease did not recur in the 
Test group, during the examination period; one rat developed mild disease 
at day 22 and the remaining rats from day 27 to day 30. 
Conclusions 
Treatment itself, i.e. administering fluid i.p..times.2 daily to rats, did 
not effect the onset or severity of the disease but did marginally extend 
its time course. Furthermore, severe disease did recur during the 
observation period in the rats receiving daily injections of Tris-buffered 
saline but not in the rats receiving no treatment. 
The most interesting observation made in the this study, was that treatment 
of the rats with cpn10 did significantly delay the onset and modify the 
clinical features of the disease in 4 out of 5 rats. It also prevented the 
recurrence of severe disease during the time these animals were under 
observation. 
(d) Infertility and Miscarriage 
A further aspect of the invention is the treatment of fertility and/or 
miscarriages with the administration of cpn10. This is of importance where 
the problem arises from the lack of cpn10. Experimental support below 
demonstrates the requirement for cpn10 during embryo development. 
To create the situation of reduced cpn10 concentration, anti-cpn10 
antibodies were developed and used. There is no animal model system 
available. It follows therefore from the experimental support that 
administration of cpn10 to increase the concentration of cpn10 during 
pregnancy will overcome the aforementioned problems of infertility and 
miscarriage. 
Synthesis of cpn10 Derived Peptides 
Peptides were synthesized to correspond with an N-terminal fragment 
(N-peptide i.e. Ac-AGQAFRKFLPLC) and an internal fragment (I-peptide i.e. 
EKSQGKVLQATC SEQ ID NO:8) of cpn10. 
Conjugation of Peptides to Ovalbumin 
Peptides were conjugated to ovalbumin by the hetero-bifunctional reagent 
SPDP, following manufacturer's instructions (Pharmacia-LKB Biotechnolkog, 
Uppsala, Sweden). 
Immunisation Schedules 
Adult outbred New Zealand rabbits were immunised with one of the conjugates 
in 4 .times. weekly injections followed by several monthly boosts. 
For injection, the antigen was dialysed into 0.9% saline (Mr 12-15000 cut 
off dialysis tubing, Visking, Union Carbide, IL, USA) and emulsified with 
an equal volume of Freund's adjuvant (complete for the first injection, 
incomplete thereafter). Immunisations were via the s.c. route. 
Screening of Anti-Serum 
Antisera were tested in an ELISA against the relevant antigens (viz. 
I-peptide or N-peptide; ovalbumin) (5 mg/ml). Bound IgG was detected by 
the biotin-streptavidin system (Amersham) with o-phenylene diamine as 
substrate. Absorbance %%as read at 492 nm. 
IgG was precipitated from anti-serum by 45% ammonium sulphate and the 
concentration determined by Lowry and gel electrophoresis. The IgG 
preparations were tested in an ELISA (Table 4) against the immunising 
peptide, conjugated to bovine serum albumin. The preparations were also 
tested for their ability to neutralise activity of mouse pregnancy serum 
in the rosette inhibition test. Various concentrations of antibody were 
incubated with an equal volume of serum, then the mixtures tested for 
activity in the rosette inhibition test. The lowest concentration of 
antibody that could completely neutralise activity was determined (see 
Cavanagh et al., 1994, Eur. J. Biochem. 222 551-560). Ten pg of 
anti-N-peptide Ab neutralised the activity of 1 ml pregnancy serum while 4 
ng anti-I-peptide Ab was needed for complete neutralisation. 
Passive Immunisation 
Mature outbred male and female Quackenbush mice were caged in pairs at 7.30 
a.m. and separated at 8.30 a.m. Female mice with vaginal plugs were 
injected with anti-N-peptide/ovalbumin, anti-I-peptide/ovalbumin or 
anti-ovalbumin lgG preparations at 9.00 a.m. and 5.00 p.m. on days 1 (day 
of mating) and 2 of pregnancy. The dose of specific IgG injected in the 2 
dose regimen was estimated as approximately 1 mg/mouse/day. On day 7, mice 
were euthanased with CO.sub.2, uteri examined for implanted embryos and 
the number of corpora lutea (CL) counted. In each group, the number of 
embryos/CL in the mice treated with the test IgG was compared with the 
number receiving the same dose of control IgG (.chi..sup.2 test). 
Results 
The results, shown in Table 5 clearly demonstrate that neutralisation of 
cpn10 in pregnancy serum can adversely affect embryonic viability in the 
early stages of pregnancy. The ability of antibodies to neutralise 
activity in the rosette inhibition test is an in vitro monitor of their 
ability in vivo to adversely affect pregnancy. 
Other Aspects of The Invention 
In another aspect of the invention, further work has now elucidated two 
regions of the molecule with biological activity, corresponding with 
residues 1-11 and 34-44 in rat and human cpn10. 
A peptide having the amino acid sequence Ac-AGQAFRKFLPL (SEQ ID NO:10) as 
well as a peptide having the sequence EKSQGKVLQAT (SEQ ID NO:11)have been 
found to be active in the rosette inhibition assay. Antibodies raised 
against both of these peptides are active as antagonists of cpn10 as 
described in detail in International Application PCT/AU94/00742 
(WO95/15339). Both these peptides are prepared synthetically. 
The invention therefore includes within its scope amino acid sequences: - 
(i) AGQAFRKFLPL (SEQ ID NO:12); 
(ii) Ac-AGQAFRKFLPL (SEQ ID NO:10) where Ac is acetyl; 
(iii) EKSQGKVLQAT (SEQ ID NO:11) which may function as active centres of 
the cpn 10 molecule. 
The invention also includes within its scope molecules (i), (ii) and (iii) 
having one or more end sequences A.sub.1 and A.sub.2 ie. 
(iv) A.sub.1 AGQAFRKFLPLA.sub.2 ;(SEQ ID NO:13) 
(v) AGQAFRKFLPLA.sub.2 ; (SEQ ID NO:14) 
(vi) A.sub.1 AGQAFRKFLPL; (SEQ ID NO:15) 
(vii) Ac-A.sub.1 AGQAFRKFLPLA.sub.2 ; (SEQ ID NO:16) 
(viii) Ac-AGQAFRKFLPLA.sub.2 ; (SEQ ID NO:12) 
(ix) Ac-A.sub.1 AGQAFRKFLPL; (SEQ ID NO:18) 
(x) A.sub.1 EKSQGKVLQATA.sub.2 ; (SEQ ID NO:19) 
(xi) EKSQGKVLQATA.sub.2 ; (SEQ ID NO:20) 
(xii) AIEKSQGKVLQAT; (SEQ ID NO:21) 
wherein A.sub.1 and A.sub.2 are amino acid sequences which may be added to 
one or each end of molecules (i) through (xii) and wherein Ac is acetyl. 
In the above molecules (i) through (xii), it will be appreciated such 
molecules also include within their scope a single amino acid addition, 
deletion or substitution. 
In regard to the use of cpn10 in regard to treatment of autoimmune disease, 
relevant diseases that may be treated by administration of cpn10 include 
insulin dependent diabetes mellitus, rheumatoid arthritis, systemic lupus 
erythematosis, Sjogren's syndrome, Graves disease and multiple sclerosis. 
This is evident from the relevant supporting data in regard to the EAE rat 
model provided herein. 
In relation to the use of cpn10 in relation to treatment of organ 
transplants, skin grafts, the relevant supporting data given herein refers 
to the rat skin graft model described herein. 
In relation to the use of cpn10 in relation to infertility treatment or 
prevention of miscarriage, the relevant supporting data refer to the 
effect of cpn10 antibody on embryonic development and implantation in 
mice. 
In relation to the use of cpn10 in relation to wound healing and tissue 
repair or regeneration of tissue, this means that cpn10 can be used in 
treatment of burns, surgery, trauma, skin ulcers including bed sore and 
diabetic ulcers, infectious diseases involving tissue and organ damage 
(e.g. hepatitis), metabolic disease involving tissues and organ damage 
(e.g. liver cirrhosis) and degenerative disease involving tissue or organ 
damage. The support for these conclusions is given in the mouse wound 
model referred to herein and the liver regeneration data after partial 
hepactectomy in rats discussed in Quinn et al., 1994, Hepatology 20 No 5 
1294-1302. 
The data referred to herein also provides clear support for the use of 
cpn10 in treatment of inflammatory conditions including inflammatory bowel 
disease and infectious disease. Such data as described herein includes 
references drawn from the immunosuppressive effect of cpn10 in the rat EAE 
and skin graft models. This is also supported by Rolfe et al., 1983, Clin. 
exp. Immunol. 51 45-52 and Nature 278 No. 5705 649-651 showing that EPF 
can reduce delayed type hypersensitivity in mice. 
The use of cpn10 in treatment of allergic disease including allergic 
rhinitis, asthma, atopic dermatitis, acute urticaria and drug 
hypersensitivity is also fully supported by the immunosuppressive effect 
of cpn10 in the rat EAE and skin graft models. This conclusion can also be 
drawn from Rolfe et al., 1983, Clin. exp. Immunol. 51 45-52 and Noonan et 
al., 1979, Nature 278 No. 5705 649-651 showing effect of EPF in reducing 
delayed type hypersensitivity in mice. 
The use of cpn10 in relation to diagnosis of tumours and/or monitoring 
patients after surgical removal of tumours is supported by the reference 
Quinn et al., 1992, Cancer Immunol. Immunother. 34 265-271. 
In regard to dosages that may be employed concerning administration of 
cpn10, a convenient dosage would be of the order of 1-1000 .mu.g/kg of 
body weight and more preferably 50-200 .mu.g/kg of body weight. 
TABLE 1 
______________________________________ 
Limiting Dose (log reciprocal) 
Sample Untreated + 5/341 
______________________________________ 
Human platelet EPF 
13 &lt;2 
(50 .mu.g/ml) 
Rat liver cpn 10 (50 
13 &lt;2 
.mu.g/ml) 
E. coli cpn 10 (groES) 
NA NT 
(50 .mu.g/ml) 
______________________________________ 
TABLE 2 
______________________________________ 
TREATMENT SKIN GRAFT SURVIVAL TIME 
rEPF/cpn 10 (dose 
Lewis .fwdarw. DA 
DA .fwdarw. Lewis 
x 2/rat/day) Days .+-. SD (n) 
Days .+-. SD (N) 
______________________________________ 
buffer alone 8.7 .+-. 0.75 (7) 
9.1 .+-. 0.83 (8) .sup. 
1 .mu.g -- 9.0 .+-. 1.0 (3) (NS) 
5 .mu.g 14.0 .+-. 1.6 (4)* 
14.5 (2) 
20 .mu.g 15.2 .+-. 0.92 (4)* 
12.5 (2) 
70 .mu.g 10.0 (2) 11.0 (2) 
______________________________________ 
TABLE 3 
______________________________________ 
Max. Max. 
Onset period weight 
of of loss 
weight weight (% wt. 
Treatment loss loss at 
Group n (day) (day) p' d 10) p* 
______________________________________ 
No 4 11 14-16 15.4 
treatment 
Buffer 5 11 15-17 NS 13.9 NS 
(Control) 
cpn 10 5 12-14 17-19 p &lt; 13.9 NS 
(Test) 0.001 
______________________________________ 
TABLE 4 
______________________________________ 
Titre (reciprocal serum dilution 
Antibodies (mg/ml) 
N-peptide (5 .mu.g/ml) 
I-peptide (5 .mu.g/ml) 
______________________________________ 
Anti-N-peptide 
128000 &lt;1000* 
Anti-I-peptide 
&lt;1000* 32000 
Anti-ovalbumin 
&lt;1000* &lt;1000* 
______________________________________ 
TABLE 5 
______________________________________ 
Antibody No. of Corpora 
(total dose 
animals lutea/mouse 
2 in (mean .+-. Embryo/mouse 
mg/mouse) 
group sem) (mean .+-. sem) 
p* 
______________________________________ 
Anti-N- 6 19.1 .+-. 1.2 
10.6 .+-. 3.8 
&lt; 
peptide- 0.05 
ovalbumin 
Anti-I- 6 20.8 .+-. 0.8 
17.1 .+-. 1.1 
&lt; 
peptide- 0.02 
ovalbumin 
Anti- 5 17.8 .+-. 1.0 
16.8 .+-. 0.5 
NS 
ovalbumin 
______________________________________ 
TABLE LEGENDS 
FNT TABLE 1 
FNT Activity of cpn 10 in the rosette inhibition test 
FNT TABLE 2 
FNT Skin graft survival time. 
FNT p value when compared with control group receiving buffer alone 
FNT *p&lt;0.001 
FNT TABLE 3 
FNT Time of Initial Weight Loss and Maximum Weight Loss in the Three 
FNT Treatment Groups during the Course of EAE. 
FNT .chi..sup.2 distribution 
FNT Student's t test 
FNT TABLE 4 
FNT Anti-N-peptide, anti-I-peptide and control anti-ovalbumin antibodies tested 
in an ELISA against N-peptide and I-peptide. 
FNT * 1 in 1000 was the lowest dilution tested. 
FNT TABLE 5 
FNT Effect of passive immunization of confirmed-mated mice at days 1 and 2 
p.c., with antibodies to cpn10-derived peptides, on the number of of 
implanted embryos and corpora lutea present at day 7 p. c. 
FNT * (Heteroscedastic t-test). 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 25 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 9 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Val Leu Asp Asp Lys Asp Tyr Phe Leu 
1 5 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 20 CRTC 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 17 C 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 17 T 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 30 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 30 GGAC AAGCGTTTAG 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 26 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 26 CGTA CTTTCC 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 104 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- Gly Ser Met Ala Gly Gln Ala Phe Arg Lys Ph - #e Leu Pro Leu Phe Asp 
# 15 
- Arg Val Leu Val Glu Arg Ser Ala Ala Glu Th - #r Val Thr Lys Gly Gly 
# 30 
- Ile Met Leu Pro Glu Lys Ser Gln Gly Lys Va - #l Leu Gln Ala Thr Val 
# 45 
- Val Ala Val Gly Ser Gly Ser Lys Gly Lys Gl - #y Gly Glu Ile Gln Pro 
# 60 
- Val Ser Val Lys Val Gly Asp Lys Val Leu Le - #u Pro Glu Tyr Gly Gly 
#80 
- Thr Lys Val Val Leu Asp Asp Lys Asp Tyr Ph - #e Leu Phe Arg Asp Gly 
# 95 
- Asp Ile Leu Gly Lys Tyr Val Asp 
100 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Cys 
# 10 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r Cys 
# 10 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu 
# 10 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r 
# 10 
- (2) INFORMATION FOR SEQ ID NO:12: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 11 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- Ala Gly Gln Ala Phe Arg Lys Phe Leu Pro Le - #u 
# 10 
- (2) INFORMATION FOR SEQ ID NO:13: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:14: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- Ala Gly Gln Ala Phe Arg Lys Phe Leu Pro Le - #u Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:15: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu 
# 10 
- (2) INFORMATION FOR SEQ ID NO:16: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 14 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- Xaa Xaa Ala Gly Gln Ala Phe Arg Lys Phe Le - #u Pro Leu Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:17: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- Xaa Ala Gly Gln Ala Phe Arg Lys Phe Leu Pr - #o Leu Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:18: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- Xaa Xaa Ala Gly Gln Ala Phe Arg Lys Phe Le - #u Pro Leu 
# 10 
- (2) INFORMATION FOR SEQ ID NO:19: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 13 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- Xaa Glu Lys Ser Gln Gly Lys Val Leu Gln Al - #a Thr Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:20: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- Glu Lys Ser Gln Gly Lys Val Leu Gln Ala Th - #r Xaa 
# 10 
- (2) INFORMATION FOR SEQ ID NO:21: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- Xaa Glu Lys Ser Gln Gly Lys Val Leu Gln Al - #a Thr 
# 10 
- (2) INFORMATION FOR SEQ ID NO:22: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 43 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- Lys Val Leu Xaa Ala Thr Val Val Ala Val Gl - #y Ser Gly Ser Lys Glu 
# 15 
- Tyr Gly Gly Thr Lys Val Val Xaa Xaa Xaa Xa - #a Asp Xaa Phe Leu Phe 
# 30 
- Arg Asp Gly Asp Ile Leu Gly Lys Tyr Val As - #p 
# 40 
- (2) INFORMATION FOR SEQ ID NO:23: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 28 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- Lys Ser Gln Gly Lys Val Leu Gln Ala Thr Va - #l Val Ala Val Gly Xaa 
# 15 
- Gly Xaa Lys Val Leu Leu Pro Glu Tyr Gly Gl - #y Thr 
# 25 
- (2) INFORMATION FOR SEQ ID NO:24: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 47 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- Lys Phe Leu Pro Leu Phe Asp Arg Val Leu Va - #l Glu Lys Gly Gly Ile 
# 15 
- Met Leu Pro Glu Lys Xaa Gln Gly Lys Val Va - #l Leu Asp Asp Lys Asp 
# 30 
- Tyr Phe Leu Phe Arg Asp Gly Asp Ile Leu Gl - #y Lys Tyr Val Asp 
# 45 
- (2) INFORMATION FOR SEQ ID NO:25: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 102 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ix) FEATURE: 
(A) NAME/KEY: Modified-sit - #e 
(B) LOCATION: 1 
#/product= "Other"R INFORMATION: 
#"The Xaa at position 1 is acetyl." 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- Xaa Ala Gln Ala Gly Phe Arg Lys Phe Leu Pr - #o Leu Phe Asp Arg Val 
# 15 
- Leu Val Glu Arg Ser Ala Ala Glu Thr Val Th - #r Lys Gly Gly Ile Met 
# 30 
- Pro Leu Glu Lys Ser Gln Gly Lys Val Leu Gl - #n Ala Thr Val Val Ala 
# 45 
- Val Gly Ser Gly Gly Lys Gly Lys Gly Gly Gl - #u Ile Gln Pro Val Xaa 
# 60 
- Xaa Lys Xaa Gly Xaa Xaa Val Leu Leu Pro Gl - #u Tyr Gly Gly Thr Lys 
#80 
- Val Val Leu Asp Asp Lys Asp Tyr Phe Leu Ph - #e Arg Asp Gly Asp Ile 
# 95 
- Leu Gly Lys Tyr Val Asp 
100 
__________________________________________________________________________