Interruption of binding of MDM2 and P53 protein and therapeutic application thereof

A method for interfering with the binding between p53 and MDM2 or a protein having a p53 binding site analogous to that of MDM2, which method comprises administering a effective amount of a compound, selected from the group consisting of a peptide having up to twenty eight amino acids which is able to disrupt or prevent binding between p53 and MDM2, or a functional peptide analogue thereof. Compounds for use in the method, methods for detecting such compounds and their application in the diagnosis and treatment of tumours is also described and claimed.

FIELD OF THE INVENTION 
The invention relates to the area of cancer detection and therapeutics. 
More particularly it relates to the prevention or disruption of the 
inactivation of the p53 tumour suppressor which occurs as a result of the 
binding of a protein through the amino acid motif within the region of p53 
represented by amino acids 16-30 QETFSDLWKLLPENN (SEQ ID NO:1) of the 
human p53 protein. An example of such a protein is the oncogene protein 
MDM2 (human MDM2). 
BACKGROUND OF THE INVENTION 
Inactivation of the p53 tumour suppressor is a frequent event in human 
neoplasia. The inactivation can occur by mutation of the p53 gene or 
through binding to viral or cellular oncogene proteins, such as the SV40 
large T antigen and MDM2. While the mechanism through which wild-type p53 
suppresses tumour cell growth is as yet poorly defined it is clear that 
one key feature of the growth suppression is the property of p53 to act as 
a transcription factor (Farmer, G., et al. (1992). Nature, 358, 83-86; 
Funk, W. D. et al. (1992). Mol. Cell. Biol., 12, 2866-2871; Kern, S. E., 
et al. (1992). Science, 256, 827-830). Currently, considerable effort is 
being made to identify growth control genes that are regulated by p53 
binding to sequence elements near or within these genes. A number of such 
genes have been identified. In cases such as the muscle creatine kinase 
gene (Weintraub, H., et al. (1991). Proc. Natl. Acad. Sci. U.S.A., 88, 
4570-4571; Zambetti, G. P., et al. (1992). Genes Dev., 6, 1143-1152) and a 
GLN retroviral element (Zauberman, A., et al. (1993). Embo J., 12, 
2799-2808) the role these genes might play in the suppression of growth 
control is unclear. Yet there are other examples, namely mdm2 (Barak, Y., 
et al. (1993). Embo J., 12, 461-468; Wu, X., et al. (1993). Genes Dev., 7, 
1126-1132) GADD 45 (Kastan, M. B., et al. (1992). Cell, 71, 587-597) and 
WAF1 or CIP1 (El-Beiry, W. S., et al. (1993). Cell, 75, 817-825; Harper, 
J. W., et al. (1993). Cell., 75, 805-816) where their involvement in the 
regulation of cell growth is better understood. 
In the present text "mdm2" refers to the oncogene and "MDM2" refers to the 
protein obtained as a result of expression of that gene. 
Mdm2, a known oncogene, was originally found on mouse double minute 
chromosomes (Cahilly-Snyder., L., et al. (1987) Somatic Cell Mol. Genet. 
13, 235-244). Its protein product was subsequently found to form a complex 
with p53, which was first observed in a rat fibroblast cell line (Clone 6) 
previously transfected with a temperature sensitive mouse p53 gene 
(Michalovitz, D., et al. (1990). Cell, 62, 671-680). The rat cell line 
grew well at 37.degree. C. but exhibited a G1 arrest when shifted down to 
32.degree. C., which was entirely consistent with an observed temperature 
dependent switch in p53 conformation and activity. However, the p53-MDM2 
complex was only observed in abundance at 32.degree. C., at which 
temperature p53 was predominantly in a functional or "wild-type" form 
(Barak, Y. et al. (1992). Embo J., 11, 2115-2121 and Oren, 1992; Momand, 
J., et al. (1992). Cell, 69, 1237-1245). By shifting the rat cell line 
down to 32.degree. C. and blocking de novo protein synthesis it was shown 
that only "wild-type" p53 induced expression of the mdm2 gene, thereby 
accounting for the differential abundance of the complex in terms of p53 
transcriptional activity (Barak, Y., et al. (1993). Embo J., 12, 461-468) 
The explanation was further developed by the identification of a DNA 
binding site for wild-type p53 within the first intron of the mdm2 gene 
(Wu, X., et al. (1993). Genes Dev., 7, 1126-1132). Reporter constructs 
employing this p53 DNA binding site revealed that they were inactivated 
when wild-type p53 was co-expressed with MDM2. 
This inhibition of the transcriptional activity of p53 may be caused by 
MDM2 blocking the activation domain of p53 and/or the DNA binding site. 
Consequently, it was proposed that mdm2 expression is autoregulated, via 
the inhibitory effect of MDM2 protein on the transcriptional activity of 
wild-type p53. This p53-mdm2 autoregulatory feedback loop provided a novel 
insight as to how cell growth might be regulated by p53. Up to a third of 
human sarcomas are considered to overcome p53-regulated growth control by 
amplification of the mdm2 gene (Oliner, J. D., et al. (1992). Nature, 358, 
80-83). Hence the interaction between p53 and MDM2 represents a key 
potential therapeutic target. 
The cDNA sequence encoding the human MDM2 protein (which is also referred 
to as "HDM2" in the art) is known from WO93/20238. This application also 
discloses that human MDM2 protein binds with human p53 and it has been 
suggested that molecules which inhibit the binding of MDM2 to p53 would be 
therapeutic by alleviating the sequestration of p53. However it is also 
suggested that the p53 and MDM2 binding site is extensive, including amino 
acid residues 13-41 of p53 as well an additional nine to thirteen residues 
at either the amino or carboxyl terminal side of the peptide are also 
involved. This would indicate that a large polypeptide or other large 
molecule would be required in order to significantly interfere with the 
binding. 
The applicants have therefore sought to immunochemically characterize the 
p53-MDM2 complex, and also determine in fine detail the MDM2 binding site 
on p53. 
Surprisingly, it has been found that only a relatively small number of 
amino acids within the p53 protein are involved in binding to MDM2. 
SUMMARY OF THE INVENTION 
The precise identification of this binding site is vital to allow the 
rational design of molecules which will disrupt or prevent binding between 
p53 and MDM2 or proteins containing analogous p53 binding sites. In 
addition it allows for the design of screening procedures which will 
enable compounds which can disrupt or prevent the binding interaction to 
be accurately and rapidly identified. 
The applicants have found that the site on the p53 protein which is 
responsible for binding to MDM2 is a small sequence of only six amino 
acids, of which three amino acids have been found to be critical. This 
sequence is represented by the sequence TFSDLW (SEQ ID NO:2) in human 
(amino acids 18-23 in the sequence) and TFSGLW (SEQ ID NO:3) (amino acids 
18-23) in mouse, of which the critical amino acids appear to be F--LW (SEQ 
ID NO:4). By disrupting or preventing p53 from binding in this specific 
region, the deleterious effects of binding to MDM2 or proteins having an 
analogous p53 binding site can be avoided. Proteins having a p53 binding 
site which is analogous to that of MDM2 will generally comprise oncogene 
proteins which bind to p53 through the amino acid motif within the region 
of p53 represented by amino acids 16-30 (QETFSDLWKLLPENN) (SEQ ID NO:1) of 
the human p53 protein. 
This finding has recently been reinforced by a report that two components 
of the transcriptional machinery, namely TAF.PI.40 and TAF.PI.60, require 
Leu-22 and Trp-23 for them to bind to p53 and mediate p53 transcriptional 
activity (Thut- C. J., et al., 1995, Science 267:100-4). The same two 
amino acids of p53 are critical for the binding of MDM2 as disclosed above 
as well as Ad E1b, thus strengthening the hypothesis that MDM2 and E1b act 
by binding to p53 and blocking the transcriptional activity of p53. 
Hence the present invention provides a method for interfering with the 
binding between p53 and MDM2 or an oncogene protein having an analogous 
p53 binding site, which method comprises administering a effective amount 
of a compound, selected from the group consisting of a peptide having up 
to twenty eight amino acids which is able to disrupt or prevent the 
binding between p53 and MDM2, or a functional peptide analogue thereof. 
It may be expected that small peptides, for example of from 4 to 10 amino 
acids, suitably from five to 10 amino acids, or peptide analogues thereof 
would be particularly suitable in such a process. Peptides which would be 
of particular interest are those which show a consensus with the fragment 
of p53 which has been found to be crucial for binding. Such peptides 
include fragments of p53 protein which includes at least some of amino 
acids 18-23 within the sequence of human p53, as identified in WO93/20238 
or a peptide analogue thereof. Suitably these peptides are those which are 
circular, linear or derivatised to achieve better penetration of 
membranes. 
Novel peptides or peptide analogues of this type form a further aspect of 
the invention. 
Hence preferred peptides include the sequence FxxLW (SEQ ID NO:4) such as 
TFSDLW (SEQ ID NO:2) or a portion thereof. As used herein, `x` refers to 
any amino acid. In a preferred embodiment, an aspartate residue in the 
sequence is replaced by a glutamate residue so that the sequence is FxELW 
(SEQ ID NO:5) such as TFSELW (SEQ ID NO:6). 
Other compounds which may interfere with the binding include organic 
compounds which are modelled to achieve the same three dimensional 
structure as the said region of the p53 peptide. Hence in an alternative 
embodiment the invention provides an organic compound which is modelled to 
resemble the three dimensional structure of the amino acids represented by 
the sequence F--LW (SEQ ID NO:4) as it appears in human p53 in the region 
of amino acids 19-23 and which binds to human MDM2. In particular the 
organic compound may be modelled to resemble the three dimensional 
structure of the sequence TFSDLW (SEQ ID NO:2) as it appears in the region 
of amino acides 18-23 of human p53. 
A suitable oncogene protein is MDM2 but the disruption of binding of p53 to 
other oncogene proteins containing a p53 binding site analogous to that of 
MDM2 are included within the scope of the present invention. Examples of 
other such oncogene proteins include the adenovirus EIB 58 kD protein, the 
Tata box binding protein TBP and the transcription factor of the E2F 
family. 
As used herein the expression `peptide analogue` refers to peptide variants 
or organic compounds having the same functional activity as the peptide in 
question, in particular which interfere with the binding between p53 and 
MDM2. Examples of such analogues will include chemical compounds which are 
modelled to resemble the three dimensional structure of the sequence 
TFSDLW (SEQ ID NO:2), and in particular the arrangement of the F--LW (SEQ 
ID NO:4) amino acids as they appear in human p53, which compounds bind to 
human MDM2. 
Suitable modelling techniques are known in the art. This includes the 
design of so-called `mimetics` which involves the study of the functional 
interactions of the molecules and the design of compounds which contain 
functional groups arranged in such a manner that they could reproduce 
those interactions. 
The designing of mimetics to a known pharmaceutically active compound is a 
known approach to the development of pharmaceuticals based on a "lead" 
compound. This might be desirable where the active compound is difficult 
or expensive to synthesise or where it is unsuitable for a particular 
method of administration, eg peptides are unsuitable active agents for 
oral compositions as they tend to be quickly degraded by proteases in the 
alimentary canal. Mimetic design, synthesis and testing is generally used 
to avoid randomly screening large number of molecules for a target 
property. 
There are several steps commonly taken in the design of a mimetic from a 
compound having a given target property. Firstly, the particular parts of 
the compound that are critical and/or important in determining the target 
property are determined. In the case of a peptide, this can be done by 
systematically varying the amino acid residues in the peptide, eg by 
substituting each residue in turn. These parts or residues constituting 
the active region of the compound are known as its "pharmacophore". 
Once the pharmacophore has been found, its structure is modelled to 
according its physical properties, eg stereochemistry, bonding, size 
and/or charge, using data from a range of sources, eg spectroscopic 
techniques, X-ray diffraction data and NMR. Computational analysis, 
similarity mapping (which models the charge and/or volume of a 
pharmacophore, rather than the bonding between atoms) and other techniques 
can be used in this modelling process. 
In a variant of this approach, the three-dimensional structure of the 
ligand and its binding partner are modelled. This can be especially useful 
where the ligand and/or binding partner change conformation on binding, 
allowing the model to take account of this the design of the mimetic. 
A template molecule is then selected onto which chemical groups which mimic 
the pharmacophore can be grafted. The template molecule and the chemical 
groups grafted on to it can conveniently be selected so that the mimetic 
is easy to synthesise, is likely to be pharmacologically acceptable, and 
does not degrade in viva, while retaining the biological activity of the 
lead compound. The mimetic or mimetics found by this approach can then be 
screened to see whether they have the target property, or to what extent 
they exhibit it. Further optimisation or modification can then be carried 
out to arrive at one or more final mimetics for in vivo or clinical 
testing. 
In order to identify compounds which are useful in the above described 
methods, compounds may be screened for interference of the MDM2/p53 
interaction. Suitably screening methods would be based upon observations 
with regard to compounds which interfere with the binding between peptides 
which comprise or represent the binding site of an oncogene protein such 
as MDM2 and p53, based upon the information regarding said binding site 
given herein. Such methods include immunoassay techniques such as 
radioimmunoassay (RIA) and enzyme linked immunoabsorbent assay (ELISA) 
which are well known in the art. Particularly suitable techniques are 
competitive assay techniques where a peptide or reagent which either is or 
represents one of the peptides or reagents which either is or represents 
either p53 or the oncogene protein is exposed to a compound under test and 
the other one of the peptides or reagents which either is or represents 
either p53 or the oncogene protein. The presence of bound complex is then 
detected. This may be achieved either by labelling of the said one of said 
peptides or reagent for example using a gold or other visible label, or by 
administering a labelled antibody or sequence of antibodies, one of which 
includes a label, in a conventional manner. Suitable antibodies for 
example for MDM2 and p53 are described herein. Suitably one of the 
peptides or reagents representing p53 or the oncogene protein is 
immobilised on a support. 
Hence the invention further provides a method of identifying compounds 
which interfere with the binding of human MDM2 to human p53, said method 
comprising 
immobilising a first peptide molecule, 
adding a compound to be tested and a second peptide molecule; detecting the 
presence of bound second peptide at the immobilisation site; wherein one 
of the first peptide or second peptide is MDM2 or an oncogene protein 
having a p53 binding site analogous to that of MDM2 or a fragment thereof 
which includes said binding site, and the other is a fragment of human p53 
of from five to twenty eight amino acids including the amino acid residues 
FxxLW (SEQ ID NO: 4) or a peptide analogue thereof. 
A immunoassay for detecting binding is illustrated hereinafter. 
A biotinylated peptide containing the MDM2 binding site fo p53 from amino 
acids 16-25 (or smaller peptides as described above containing for example 
TFSDLW (SEQ ID NO:2)) would be immobilised on a streptavidin coated ELISA 
plate. Recombinant MDM2 protein would be added to these ELISA plates in 
the presence of absence of test compounds or agents. This would then be 
incubated for 2 hours at 4.degree. C. Bound MDM2 would be detected by a 
standard ELISA procedure. The inhibitory or stimulatory effect of these 
reagents would be determined by reference to control wells in which no 
such test compounds were included. Further experimental details are as 
described hereinafter and the binding assay is illustrated in FIG. 7. 
The invention includes quantitative assays. For example there is provided a 
method of identifying compounds which interfere with the binding of human 
MDM2 to human p53, said method comprising binding a predetermined quantity 
of a first peptide which is detectably labelled to a peptide, adding a 
compound to be tested; and determining the quantity of the first protein 
which is displaced from or prevented from binding to the second peptide, 
wherein one of the first peptide or the second peptide is MDM2 or a 
peptide having a p53 binding site analogous to that of MDM2, and the other 
is a fragment of human p53 of from six to twenty eight amino acids 
including the amino acid residues 18-23 in the sequence of human p53 as 
set out in WO93/20238, or a peptide analogue thereof. 
Assays which include fragments of p53 including the MDM2 binding site as 
characterised above, or peptide analogues thereof form a further part of 
the invention. The assay may be formulated as a kit which also forms part 
of the invention. A particularly useful form of the assay would be one 
which was adapted to test levels of oncogene protein in biological 
samples. Such a kit would comprise a fragment of p53 or a peptide analogue 
as binding agent thereof together with an antibody which is specific for 
the oncogene protein such as MDM2. Alternatively antibodie(s) able to 
detect bound complex may be included in the kit. 
This could be used in diagnosis to measure the levels of oncogene protein 
or MDM2 in blood samples in the case of leukaemias or solid carcinomas 
such as sarcomas and glioblastomas. 
Suitably in the above described assay methods, the oncogene protein is 
human MDM2 and the other protein comprises a fragment of human p53 of from 
12 to 28 amino acids including the sequence TFSDLW (SEQ ID NO:2). 
The methods can be readily adapted to provide a high throughput screen, for 
example by carrying out the process in a 96-well format. Automated 
screening techniques can be applied in these circumstances as would be 
understood in the art. Compounds from various sources can be screened in 
large numbers. One potential source of compounds are the available 
synthetic combinatorial peptide libraries. 
The use of compounds identified by this screening method in the treatment 
of tumours forms a further aspect of the invention. 
Methods of treatment of conditions such as cancer and other malignancies 
are envisaged by the administration of the compounds of the invention. 
Hence the invention also provides a method for inhibiting the growth of 
tumour cells which contain a human MDM2 gene amplification which method 
comprises administering a effective amount of a compound which interferes 
with the binding between p53 and an MDM2, said compound being selected 
from the group consisting of a peptide having up to twenty eight amino 
acids which is able to disrupt or prevent the binding between p53 and 
MDM2, or a functional peptide analogue thereof. 
Preferably in the above-described method of treatment, the compound is a 
peptide of from six to twenty eight amino acids which has a consensus with 
a region of human p53 and includes the sequence FxxLW (SEQ ID NO:4) for 
example TFSDLW (SEQ ID NO:2). 
Alternatively, the compound used in the method is peptide analogue such as 
an organic compound which binds to the same site on MDM2 as the sequence 
TFSDLW (SEQ ID NO:2). 
For use in these applications, the compounds are suitably applied in the 
form of compositions with pharmaceutically acceptable carriers. These may 
be solid or liquid for carriers and the compositions suitable for oral or 
parenteral application as would be understood in the art. Dosages of the 
compounds will depend upon the patient, the particular condition and the 
nature of the specific compound chosen. For example, when the compound is 
a peptide fragment dosages of from 0.1 to 10 mg/Kg may be effective. 
It has been suggested (Picksley and Lane. (1993). Bioessays. 15, 10, 
689-690) that mdm2 expression is autoregulated in a feedback loop, via the 
inhibitory effect of MDM2 protein on the transcriptional activity of 
wild-type p53. Any interference with the binding between p53 and MDM2 in 
accordance with the present invention, will affect the p53-MDM2 
autoregulatory loop. Given p53's role as guardian of the genome, compounds 
which have such an effect could enhance the activity of other therapeutic 
agents. 
Hence in a further aspect the invention comprises a pharmaceutical 
composition comprising synergistic amount of a compound of the invention 
in combination with another anticancer therapeutic agent. 
DNA encoding an MDM2-binding, p53 derived peptide, or multiple copies 
thereof may also be administered to tumour cells as a mode of 
administering the peptide. Hence the invention provides a method for 
inhibiting the growth of tumour cells which contain a human MDM2 gene 
amplification, the method comprising applying to said tumour cells a DNA 
molecule which expresses a polypeptide comprising a portion of p53 or a 
variant thereof, said portion comprising amino acids 18-23 of p53, said 
polypeptide being capable of binding to human MDM2. 
The DNA will typically be in an expression construct, such as a retrovirus, 
DNA virus, or plasmid-vector, which has the DNA elements necessary for 
expression properly positioned to achieve expression of MDM2-binding 
peptide. The DNA can be administered inter alia encapsulated in liposomes, 
or in any other form known to the art to achieve efficient uptake by 
cells. 
By identifying the binding site so specifically, the applicants,have opened 
up the possibility of making small therapeutic compounds which will target 
this site specifically. This is advantageous since small molecules are 
more likely to be able to penetrate into a cell and hence be 
therapeutically active. Furthermore the diagnostic process can be effected 
more accurately and using simpler molecules as a result of this discovery.

DETAILED DESCRIPTION OF THE INVENTION 
The first indication of an interaction between MDM2 protein and p53 protein 
emerged from work on a rat cell line, Clone 6, which expressed a 
temperature sensitive mutant form of mouse p53 (Barak and Oren, 1992; 
Michalovitz, D., et al. (1990). Cell, 62, 671-680; Momand, J., et al. 
(1992). Cell, 69, 1237-1245). MDM2 was readily observed to form a complex 
at 32.degree. C. with p53 but was just detectable when cells were grown at 
37.degree. C. 
The formation of a p53-MDM2 complex in Clone 6 cells at 32.degree. C. and 
37.degree. C. was re-examined in a quantitative manner. The results 
confirm previous immunoprecipitation observations that the level of MDM2 
at the lower temperature is significantly elevated, approximately 10-30 
fold greater than that at 37.degree. C., at which temperature MDM2 is only 
just detectable. Consequently, the p53-MDM2 complex is readily observed at 
32.degree. C. and not at 37.degree. C. The levels of p53 also vary at the 
two different temperatures. However, the p53 levels are elevated 
approximately five fold at 37.degree. C. as compared with that at 
32.degree. C., -the opposite behaviour to that of MDM2. Accordingly, the 
difference in the levels of p53 and MDM2 are likely to have alternative 
explanations. In the case of MDM2 other groups have established that the 
increase of MDM2 at 32.degree. C. is due to increased transcription of 
MDM2 due to a conformational change in p53 to a presumed transcriptional 
active form (Barak, Y., et al. (1993). Embo J., 12, 461-468; Wu, X., et 
al. (1993). Genes Dev., 7, 1126-1132). The same explanation does not apply 
for p53 even though wild type p53 is required for p53 expression (Deffie, 
A., et al. G. (1993). Mol. Cell. Biol., 13, 3415-3423), and is probably 
explained by the increased half life of the mutant conformation of p53 at 
37.degree. C. (Gannon J. V. et al (1991) Nature, 349, 802-806). Data 
described herein after using both direct observation of the p53-MDM2 
complex by ELISA and immunoprecipitation combined with the indirect 
inference of the loss of the Bp53-19 epitope suggested that nearly all p53 
molecules are complexed to excess MDM2 protein in C6 cells at 32.degree. 
C. This is not consistent with the powerful p53 dependant transcriptional 
response seen in these cells at this temperature and suggests that either 
that complexing to MDM2 is unable to completely inactivate p53 in vivo or 
that small amounts of "free" p53 may be very active. The complex between 
p53 and MDM2 may be regulated in cells to release functional p53 at the 
individual cell level perhaps as a cell cycle dependant response. 
The present invention is based upon the identification of the minimal MDM2 
binding site to be TFSD/GLW (SEQ ID NOS:2 and 3). This site is in a 
location broadly reported by other groups to be the MDM2 binding domain of 
p53, specifically aa1-41 and 13-57 (Oliner, J. D., et al. (1993). Nature, 
362, 857-860), aa1-52 (Chen, J., et al. (1993). Mol Cell Biol, 13, 
4107-14) and aa1-159 (Brown, D. R., et al. (1993). Mol. Cell. Biol., 13, 
6849-57.) Notably, a construct generated by Oliner and co-workers 
encompassing aa13-41 of p53 was not sufficient for MDM2 binding in a three 
hybrid protein system, and differs from our observations. The disparity 
might be explained by the close proximity of the fusion protein sequence 
adjacent to the TFSDGLW (SEQ ID NO:7) sequence at aa18-23 as the present 
data does show that flanking sequences do contribute in a minor way to 
MDM2 binding. The TFSD/GLW (SEQ ID NOS:2 and 3) sequence is very closely 
adjacent to the transactivation domain aa20-42 (Unger, T., et al. (1992). 
Embo J., 11, 1383-1390), and as shown by others the binding of MDM2 to 
this site interferes with the transcriptional activity of p53 (Oliner, J. 
D., et al. (1993). Nature, 362, 857-860). While substitution analysis of 
the MDM2 binding site on p53 identified the TFSD/GLW (SEQ ID NOS:2 and 3) 
sequence to be the key region required for MDM2 to bind p53, other 
residues flanking this site also contribute in a minor way to MDM2 
binding, but clearly the TFSD/GLW (SEQ ID NOS:2 and 3) sequence is a 
minimal target for agents that might disrupt complex formation without 
effecting the transactivation activity (for which as yet the key residues 
are undetermined). The first two residues TF are part of the conserved box 
I, and the latter four SD/GLW (SEQ ID NOS:8 and 9) are outside but are 
also part of a region of p53 that is conserved from Xenopus to man. 
The corresponding binding site on MDM2 for p53 has variously been reported 
to be between aa1-121, 19-102 (Chen, J., et al. (1993). Mol Cell Biol, 13, 
4107-14) together with aa102-294 or 249-491, and also 1-221 (Brown, D. R., 
et al. (1993). Mol. Cell. Biol., 13, 6849-57). Notably, a monoclonal 
antibody against the N-terminal region of human MDM2, 3G5 (maps at 
aa59-89) is able to immunoprecipitate MDM2 but not co-immunoprecipitate 
p53 (Chen, J., et al. (1993). Mol Cell Biol, 13, 4107-14), an analogous 
observation to our findings with antibody Bp53-19. 
The binding of MDM2 to p53 peptides has obvious parallels to a similar 
study that used small peptides to identify the binding sites of Adenovirus 
E1A and human papilloma virus E7 for a range of proteins including 
retinoblastoma protein, p107, cyclin A and p130 (Dyson, N., et al. 
(1992a). J. Virol., 66, 4606-4611). The MDM2 binding site on p53, appears 
to be a single domain rather than two domains as in the case of E1A and 
E7. The MDM2 binding site on p53 overlaps precisely with a highly 
immunogenic epitope on the protein; many independently isolated monoclonal 
antibodies to p53 recognise the site, and antibodies to it are present in 
the sera of cancer patients (Schlichtholtz, B., et al. (1993). Cancer 
Res., 52, 6380-6384). This suggests that it has an exposed and defined 
structure. It is possible that the amino acid sequence of the 
complementarity determining regions of these antibodies will show homology 
to the p53 binding site of MDM2. It also suggests that anti-p53 antibodies 
used to examine p53 levels where high levels of MDM2 are present must be 
chosen with care. Binding of MDM2 to this site may be regulated by 
phosphorylation since there is a DNA-dependent kinase site at serine 20 
(Less-Miller, S. P. et al. (1990). Mol. Cell. Biol., 10, 6472-6481) and 
other phosphorylation sites at serine 6, 9 and 15 (Samad, A., et al. 
(1986). Proc. Natl. Acad. Sci. U.S.A., 83, 897-901; Meek, D. W. et al. 
(1988). Mol. Cell. Biol., 8, 461-465; Meek and Eckhardt, 1988). 
The following examples are provided to exemplify various aspects of the 
invention and are not intended to limit the scope of the invention. 
In these examples, the following materials and methods were used. 
Materials and methods 
Cell culture 
Clone 6 cells (Michalovitz et al., 1990) were grown in Dulbecco's Modified 
Eagle Medium (DMEM) supplemented with 10% FCS at either 32.degree. or 
37.degree. C. The Spododoptera frugiperda cell line, SF9, was grown at 
27.degree. C. in ExCell 400 medium (J.R. H. Biosciences, Sera-Lab, UK) 
supplemented with 5% FCS and glutamine. 
Expression of MDM2 in insect cells 
The mouse mdm2 gene was obtained from a mouse prostate cell line (Lu et 
al., 1992) by polymerase chain reaction and then cloned into a 
Spododoptera frugiperda expression vector pVL1393 using standard DNA and 
baculovirus expression techniques. An expression clone was identified by 
the production of a 90-95 kDa protein that was recognized by anti-MDM2 
antibodies. 
Antibodies 
p53 protein was detected using the polyclonal sera CM1 (Midgley, C. A., et 
al. (1992). J. Cell. Sci., 101, 183-189), or monoclonal antibodies PAb421 
(Harlow E. et al., (1981) J. Virol., 39, 861-869) and Bp53-19 (Bartek J., 
et al (1993). J. Pathol., 169, 27-34). MDM2 was detected using rabbit 
anti-MDM2 polyclonal sera (Barak, Y., et al. (1993). Embo J., 12, 461-468) 
or monoclonal antibody 4B2 (Chen et al., 1993) and SMP14 (a previously 
unreported monoclonal antibody raised by us against a peptide, 
CSRPSTSSRRRAISE (SEQ ID NO:10), containing part of the human MDM2 sequence 
from aa154 to 167 (Oliner, J. D., et al. (1992). Nature, 358, 80-83) the 
first cysteine is not part of the MDM2 sequence but was added to provide 
an extra coupling option). An antibody, PAb419, raised against SV40 large 
T antigen (Harlow, E., et al. (1981). J. Virol., 39, 861-869) was used as 
an irrelevant control for immunoprecipitations. 
Immunoprecipitation 
Cells were lysed in ice-cold NET buffer (50 mM Tris-HCI, pH8.0, 150 mM 
NaCl, 5 mM EDTA, 1% NP40) containing 1 mM phenylmethylsulphonyl fluoride, 
for 30 min at 4.degree. C. Debris was removed from the cell extract by 
centrifugation at 14,000 rpm in a refrigerated Eppendorf centrifuge. The 
immunoprecipitation procedure was essentially as previously described 
(Gannon, J. V., et al. (1990). Embo J., 9, 1595-1602) using 1 .mu.g of 
purified mouse monoclonal antibody, and Protein G Sepharose beads 
(Pharmacia) for both pre-absorption of the cell extracts and subsequent 
isolation of the antibody-protein complex. 
Screening of p53 peptide library 
Peptide libraries of the entire human p53 protein and a partial N-terminal 
region of the mouse p53 protein was obtained from Chiron Mimotopes P/L 
(Victoria, Australia). The libraries were in the form of 15 mer peptides 
linked to biotin via an additional peptide spacer region of 
serine-glycine-serine-glycine, and each peptide shared a 5 amino acid 
overlap with the previous peptide in the primary sequence. ELISA plates 
were coated with 100 .mu.l of 5 .mu.g/ml streptavidin (Vector labs) per 
well and incubated overnight at 37.degree. C. and then blocked with 
phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA) 
for 1 hour at room temperature. The stock biotinylated peptides were 
diluted to 5 .mu.g/ml in PBS containing 0.1% BSA and 50 .mu.l of each were 
plated into designed wells and then incubated at room temperature for 1 
hour. The plates were washed four times with PBS containing 0.1% Tween 20 
before addition of the cell extract (50 .mu.l of 1-4 mg/ml per well) or 
purified protein. The plates were incubated at 4.degree. C. for 2-3 hours, 
before washing four times with PBS containing 0.1% Tween 20 to remove 
unbound protein. In the case of cell extracts bound protein was detected 
with the appropriate primary antibody at 1-3 .mu.g/ml, and followed by an 
anti-mouse horse radish peroxidase conjugate and 3'3'4'4'-tetramethyl 
benzidine (TMB) substrate as in the standard ELISA assay (Harlow, E., et 
al (1988). Antibodies: a laboratory manual. New York. Cold Spring Harbor 
Laboratory Press and Lane, 1988). 
The levels of p53, MDM2, and complexes thereof were determined by a two 
site immunoassay using stated antibodies. Mouse monoclonal antibodies were 
used as the solid phase by incubating Falcon microtitre dish wells with 50 
.mu.l of a 30 .mu.g/ml solution of purified antibody overnight at 
4.degree. C. The plates were blocked with 2% bovine serum albumin in PBS 
for 2 h at room temperature, and washed with PBS. Cell extracts were 
prepared as described for immunoprecipitations and then serially two-fold 
diluted before adding 50 .mu.l per well and incubating at 4.degree. C. for 
two hours. The plates were then washed with 0.1% NP-40 in PBS, before 
addition of 50 .mu.l of detecting polyclonal antisera at 1/1000 dilution. 
The plates were washed again with 0.1% NP-40 in PBS and 50 .mu.l of 1/1000 
dilution peroxidase conjugated swine anti-rabbit Ig serum (DAKO) was added 
for 2 h, then visualised by the TMB reaction. 
EXAMPLES 
Example 1 
Immunoprecipitation of MDM2, p53 and the MDM2-p53 complex 
The observation that the rat cell line, Clone 6 expressed a temperature 
sensitive mutant form of mouse p53 was reexamined using a panel of p53 
monoclonal antibodies. 
Western blots were obtained of immunoprecipitates of MDM2, p53 and the 
MDM2-p53 complex from Clone 6 cells grown at 32.degree. C. for 24 hrs 
(FIG. 1A) or continuously at 37.degree. C. (FIG. 1B). The 
immunoprecipitates were obtained using 1 .mu.g of purified antibody which 
were as follows: in lanes 1 and 4, - PAb421; in lanes 2 and 5, - Bp53-19; 
and in lanes 3 and 6, - 4B2. MDM2 was detected in lanes 1, 2 and 3 using 
SMP14 antibody supernatant and rabbit anti-mouse horse radish peroxidase 
conjugate; and p53 detected in lanes 4, 5 and 6 using a 1 in 200 dilution 
of DM-1 and swine anti-rabbit horse radish peroxidase conjugate. An 
irrelevant antibody, PAb419, did not immunoprecipitate either MDM2 or p53 
from cell extracts prepared at either 32.degree. C. or 37.degree. C. (data 
not shown). The molecular weight of the markers are given in kDa. 
It was surprisingly found that one of the antibodies, Bp53-19, failed to 
immunoprecipitate p53 from Clone C6 cells grown at 32.degree. C. for 24 
hours, but efficiently precipitated p53 from cells grown continuously at 
37.degree. C. (compare FIG. 1A track 5 with FIG. 1B track 5), whereas 
PAb421 precipitated p53 at both temperatures (FIG. 1A track 4 and 1B track 
4). Investigations were then carried out to determine whether Bp53-19 
would co-immunoprecipitate MDM2 with p53. From the immunoprecipitation 
western data in FIGS. 1A and 1B it is clear that Bp53-19 does not 
co-immunoprecipitate MDM2 from cell extracts grown at 32.degree. or 
37.degree. C. (track 2 in FIGS. 1A and B). Other p53 antibodies such as 
PAb421 do however co-immunoprecipitate MDM2 with p53 at 32.degree. C. but 
not at 37.degree. C. (track 1 FIGS. 1A and B). Conversely, antibodies 
against MDM2 such as 4B2, FIG. 1, and SMP14 (data not shown) 
co-immunoprecipitate p53 at 32.degree. C. but not at 37.degree. C. (track 
6 FIGS. 1A and B). The two bands recognized by 4B2 (and SMP14) at just 
below 80 kDa are truncated forms of rat MDM2, as full length migrates on 
an SDS-PAGE gel with an apparent relative molecular mass of 90 kDa, 
multiple forms of MDM2 are often observed (Chen, J., et al. (1993). Mol 
Cell Biol, 13, 4107-14). 
Example 2 
Two-site immunoassay to determine levels of MDM2, p53 and MDM2-p53 complex 
Two-site immunoassays were carried out to determine the levels of MDM2, p53 
and MDM2-p53 complex in Clone 6 cells grown at 32.degree. C. for 24 hrs 
(FIG. 2A) or continuously at 37.degree. C. (FIG. 2B). In FIG. 2A the 
coating antibodies were one of the following purified antibodies as stated 
in the figure legends: 4B2, 421 and Bp53-19, probed with rabbit anti-p53 
serum CM1 or rabbit anti-MDM2 serum, and then detected using swine 
anti-rabbit horse radish peroxidase conjugate and TMB as substrate. At 
37.degree. C. the MDM2-p53 complex was undetectable by any combination of 
antibodies. 
The two-site immunoassays of the levels of MDM2, p53 and MDM2-p53 complex 
at 32.degree. C. and 37.degree. C. are consistent with the 
immunoprecipitation results of Example 1. A striking feature apparent from 
the data in FIG. 2A is that the levels of p53 and p53-MDM2 complex are 
very similar suggesting that most, but not all, p53 is in complex with 
MDM2 at 32.degree. C. The inability of Bp53-19 to detect a p53-MDM2 
complex at 32.degree. C. is again notable since other combinations of 
antibodies are able to do so. 
From comparison of the two-site immunoassays at 32.degree. C. and 
37.degree. C. it is clear why MDM2 is not immunoprecipitated at 37.degree. 
C., as the levels of MDM2 protein are very much lower and are only just 
detectable. No MDM2-p53 complex could be detected by the two-site 
immunoassay of cell extracts prepared at 37.degree. C., see FIG. 2B, where 
the data for the 4B2 (as the capture antibody) and CM1 (as the detecting 
antibody) combination of antibodies is shown (similarly antibodies PAb421 
or Bp53-19 and rabbit anti-MDM2 polyclonal did not detect the complex). 
The diminished level of MDM2 at 37.degree. C., less than 10% of that at 
32.degree. C., is in contrast to the situation with p53 which is elevated 
approximately 5 fold relative to the levels at 32.degree. C. 
The explanation for the ability of PAb421 and 4B2 only being able to 
coprecipitate p53 and MDM2 together at 32.degree. C., but not at 
37.degree. C. is consistent with difference in levels of MDM2 at the two 
temperatures, and also with the published observations that mdm2 
expression is dependent on the "wild-type" form of p53 predominantly 
present at 32.degree. C. 
The failure of Bp53-19 to co-immunoprecipitate MDM2 or detect the p53-MDM2 
complex at 32.degree. C. is unexpected for two reasons. Firstly, the 
two-site assay suggests there is MDM2 protein in excess, which is able to 
form complexes with p53 as detected by the capturing antibodies PAb421 and 
4B2. Secondly, the two-site immunoassay at 37.degree. C. suggests that 
Bp53-19 is almost as efficient as PAb421 at recognizing p53 in the cell 
extracts. The simplest interpretation for this observation is that Bp53-19 
recognizes the same region on p53 that MDM2 binds to. 
Example 3 
Identification of MDM2-p53 binding site 
It has previously been shown that Bp53-19 and MDM2 interact with the amino 
acid terminal end of p53 (Stephen et al manuscript in preparation; Oliner, 
J. D., et al. (1993). Nature, 362, 857-860). A complete peptide library of 
the human p53 protein, and a partial peptide library of the mouse p53 
protein were available to identify the region to which MDM2 binds. The 
human p53 sequence starts at peptide number 3 and ends at peptide 79, and 
each peptide consists of 15 amino acids, with the last five amino acids 
being present in the next peptide along. The mouse p53 sequence is partial 
and consists of the N-terminal sequence from amino acid 1-92, again each 
overlapping the next and previous peptide by five amino acids. 
These libraries consisted of 15 amino acid long sections of the p53 primary 
amino acid sequence, that consecutively overlapped by 5 amino acids, and 
were each attached to biotin via a 4 amino acid long spacer. By 
immobilizing the biotinylated peptides on streptavidin coated ELISA plates 
the MDM2 binding site on p53 could be quickly identified if it was 
encompassed within a stretch of fifteen amino acids or less. Extract 
containing MDM2 was added to an ELISA plate with the peptide library bound 
to it, and the bound MDM2 protein was later detected using monoclonal 
antibody 4B2 and the standard ELISA assay. Several sources of recombinant 
MDM2 protein were used to challenge the p53 library, these included crude 
extracts and partially purified preparations of human and mouse MDM2 
expressed in E.coli and also mouse MDM2 expressed in insect cells;--all 
forms identified the same peptides in the p53 library. The results using 
the mouse MDM2 expressed in insect cells are shown in FIGS. 3A and B. The 
peptide library was challenged with insect cell extract alone, SF9, and 
insect cell extract expressing mouse MDM2, SF9 Mus MDM2. Binding of MDM2 
to the peptides was determined by an ELISA assay using monoclonal antibody 
4B2, and then detecting bound antibody with rabbit anti-mouse Ig 
conjugated horse radish peroxidase and TMB substrate. In FIG. 3C is shown 
the results from a control experiment using peptides 59, 71, 83 and 95, as 
used in FIG. 3B but conducted in the presence or absence of extract to 
verify the specificity of the detecting antibody, 4B2. The results are 
presented alongside the ELISA readings for extract of insect cells alone 
not expressing mouse MDM2. The specificity is remarkable,--suggesting a 
strong interaction between MDM2 and p53 derived peptides. From the 
controls shown in FIG. 3C it can be seen that the binding is only observed 
in the presence of extract expressing MDM2, and is not due to the antibody 
recognizing the peptide alone, moreover identical results were obtained 
using SMP14 as the primary detecting antibody (data not shown). 
The four peptides that bind MDM2 are shown in FIG. 4. Peptides 5 and 6 
identify a site at the N-terminal end of human p53, whereas peptides 83 
and 84 identify the corresponding region in the N-terminal end of mouse 
p53. Collectively, these four peptides define the consensus MDM2 binding 
site on p53 to be -QETFSD/GLWKL-(SEQ ID NOS:11 and 12), the aspartate to 
glycine being the only amino acid difference between the human and mouse 
sequence. The peptides involved in binding MDM2 are also those recognized 
by the p53 antibodies DO-1 and Bp53-19 (Stephen et al, manuscript in 
preparation). 
To define key residues on p53 that are involved in the interaction with 
MDM2 a form of the consensus binding site sequence -QETFSDLWKL(SEQ ID 
NO:11)- was modified by substituting alanine at each position in the 
sequence and determining what effect this had on the binding of MDM2 from 
the insect cell extract expressing MDM2. This experiment was conducted in 
concert with examining the effect on binding of the antibodies DO-1 and 
Bp53-19. The results are presented in FIG. 5. The amino acid sequences are 
as stated. In A the sequence QETFSDLWKLLPENN (SEQ ID NO:1) represents the 
sequence of peptide 6 from FIG. 3 and SPDDIEQWFTEDPGP (SEQ ID NO:13) is an 
irrelevant peptide control. Formally the first serine residue on the 
stated peptide is part of the spacer coupling the consensus peptide to 
biotin, since serin also precedes the consensus p53 sequence this residue 
was also substituted with alanine. With regard to MDM2 binding all alanine 
substitutions in the consensus binding site reduce the level of binding as 
measured by ELISA, however, the key residues would appear to be TFSDLW 
(SEQ ID NO:2) as substitutions in these positions reduce the amount of 
MDM2 binding to less than 15% of that seen with the unchanged consensus 
sequence. Interestingly, a higher level of binding of MDM2 is observed to 
the smaller consensus peptide rather than to peptide 6 (QETFSDLWKLLPENN) 
(SEQ ID NO:1) of the p53 peptide library reaffirming the definition of the 
binding site. In the case of monoclonal antibody DO-1 binding to the 
consensus sequence the key residues are ETFSDLK (SEQ ID NO:14), with D and 
K being the most crucial. The importance of the aspartate residue to the 
DO-1 epitope is consistent with the report that DO-1 only recognizes human 
p53 and not mouse p53,--the only difference being an aspartate to glycine 
change. While this difference has a critical affect on DO-1 binding it 
does not grossly affect the interaction of MDM2 .DELTA. with the protein 
or peptides. However substitution of alanine for aspartate at this 
position blocks binding of all three protein ligands. The ability of MDM2 
to distinguish alanine from either glycine or aspartate at this position 
may imply that the polar environment of this region of the binding site is 
critical for the interaction. It has also been established from phage 
display libraries that the epitope of DO-1 is FSDLWKL (SEQ ID NO:15) 
(Stephen et al, manuscript in preparation), which is in agreement with our 
observations on key residues. For the antibody Bp53-19 the alanine 
substitution series identifies the key residues to be F-DLW-(SEQ ID NO:16) 
with the latter three residues being the most crucial, and is similar to 
the requirements for MDM2 binding to the consensus binding site. Not 
surprisingly, it was found that the pre-binding of antibody Bp53-19 onto 
he SQETFSDLWKL (SEQ ID NO:17) biotinylated peptide blocked binding of MDM2 
to the peptide when added later (data not shown). 
Example 4 
Further characterisation of the p53-MDM2 binding site 
The process of Example 3 was repeated but using insect cells infected with 
baculovirus expressing Mus MDM2 from SF9 cells in two 180 ml.sup.2 tissue 
culture flasks. The extract was prepared in approximately three mils of 
lysisi buffer to give a concentrated supernatant of 13 mg/ml. 
As before, the MDM2 binding site was defined by alanine substitution of the 
p53 derived peptide, SQETFSDLWL (SEQ ID NO:18) and the results are shown 
in FIG. 6. Additionally other conserved substitutions were tested (i.e 
those commonly seen in highly conserved proteins of identical function) 
and the results are also shown in FIG. 6. With a higher protein 
concentration, (13 mg/ml as opposed to 1-4 mg/ml) the alanine substitution 
experiment reveals the same six amino acids are important (.sup.18 
TFSDLW.sup.23) (SEQ ID NO:2) in addition to glutamate (ETFSDLW) (SEQ ID 
NO:19). This data however establishes that the most critical residues are 
F--LW (SEQ ID NO:4) as these are intolerant of both alanine substitutions 
and some, if not all of the conserved substitutions. 
A further interesting observation relates to the fact that substitution of 
the aspartate residue for the glutamate enhances binding of MDM2, 
indicating that such a residue may usefully be included in the therapeutic 
peptides of the invention. This also illustrates that this approach can 
lead to the discovery of agents that have enhanced binding to MDM2. 
Additional References 
Dyson, N., et al. (1992b). J. Virol., 66, 6893-6902. 
Houghten, R. A., et al. (1991). Nature, 354, 84-86. 
Lu, X., et al (1992). Cell, 70, 153-161. 
__________________________________________________________________________ 
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(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
SerGlnGluThrPheSerAspLeuTrpLysAla 
1510 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
AspGlnGluThrPheSerAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
SerGlnGluThrPheAspAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
SerGlnGluSerPheSerAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
SerGlnGluThrIleSerAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
SerGlnGluThrLeuSerAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
SerGlnGluThrMetSerAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
SerGlnGluThrPheThrAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
SerGlnGluThrPheProAspLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
SerGlnGluThrPheSerGluLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
SerGlnGluThrPheSerGlnLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
SerGlnGluThrPheSerAsnLeuTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
SerGlnGluThrPheSerAspMetTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
SerGlnGluThrPheSerAspIleTrpLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
SerGlnGluThrPheSerAspLeuArgLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
SerGlnGluThrPheSerAspLeuPheLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
SerGlnGluThrPheSerAspLeuTyrLysLeu 
1510 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
SerGlnGluThrPheSerPheLeuIleLysLeu 
1510 
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