U.S. patent application number 12/044388 was filed with the patent office on 2008-12-18 for peptide ligands for prostate specific antigen.
This patent application is currently assigned to LICENTIA LTD.. Invention is credited to Erkki Koivunen, Jari Leinonen, Ale Narvanen, Ulf-Hakan STENMAN.
Application Number | 20080312141 12/044388 |
Document ID | / |
Family ID | 8559201 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080312141 |
Kind Code |
A1 |
STENMAN; Ulf-Hakan ; et
al. |
December 18, 2008 |
PEPTIDE LIGANDS FOR PROSTATE SPECIFIC ANTIGEN
Abstract
The present invention relates to novel peptide ligands for
prostate specific antigen (PSA) binding specifically with it and
enhancing its enzyme activity, to a process for preparation of
these peptides, to diagnostic and pharmaceutical compositions
comprising these peptides, to the use of these peptides for
pharmaceutical and research preparations, to methods using these
peptides in diagnostic assays for determination of the
concentrations of various molecular forms of PSA, to methods for
modulating the PSA enzyme activity and PSA activity dependent
conditions by using these peptides either in vivo or in vitro and
to the use of these peptides in procedures for biochemical
isolation and purification of PSA.
Inventors: |
STENMAN; Ulf-Hakan;
(Kauniainen, FI) ; Koivunen; Erkki; (Helsinki,
FI) ; Leinonen; Jari; (Helsinki, FI) ;
Narvanen; Ale; (Kuopio, FI) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
LICENTIA LTD.
|
Family ID: |
8559201 |
Appl. No.: |
12/044388 |
Filed: |
March 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10363662 |
Sep 26, 2003 |
7414023 |
|
|
PCT/FI01/00856 |
Oct 1, 2001 |
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12044388 |
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Current U.S.
Class: |
514/1.1 ;
530/317 |
Current CPC
Class: |
C07K 7/06 20130101; A61P
13/08 20180101; A61P 35/00 20180101; A61P 43/00 20180101; A61K
38/00 20130101 |
Class at
Publication: |
514/11 ;
530/317 |
International
Class: |
A61K 38/12 20060101
A61K038/12; C07K 7/64 20060101 C07K007/64 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2000 |
FI |
20002159 |
Claims
1. A peptide comprising: at least 6 amino acids bonded together to
form a peptide backbone comprising at least one pair of cysteines
which are spaced apart by at least two amino acids and
interconnected by a disulfide bond to form a cyclic structure
defined by the cysteines, intermediary amino acids and the
disulfide bond, wherein said peptide selectively binds to free
prostate specific antigen.
2. The peptide according to claim 1, wherein the peptide backbone
comprises at least two pairs of cysteines interconnected by
disulfide bonds.
3. The peptide according to claim 2, wherein the peptide enhances
the enzymatic activity of prostate specific antigen.
4. The peptide according to claim 1, wherein said peptide is
represented by formula (I)
CX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8C (I)
wherein X.sup.1 is V or I, X.sup.2 is F, I, W or P, X.sup.6 is Y, N
or L, C is cysteine, and each of X.sup.3, X.sup.4, X.sup.5, X.sup.7
and X.sup.8 is independently selected from an amino acid
residue.
5. The peptide according to claim 4, wherein X.sup.1 is V or I,
X.sup.2 is F or I and X.sup.6 is Y or N.
6. The peptide according to claim 4, wherein X.sup.1 is V, X.sup.2
is F, X.sup.6 is Y and each of X.sup.3, X.sup.4, X.sup.5, X.sup.7
and X.sup.8 is an amino acid residue independently selected from
the group consisting of T, S, D, Y A, F, E, P and L.
7. The peptide according to claim 4, wherein X.sup.1 is V, X.sup.2
is I, X.sup.6 is N and each of X.sup.3, X.sup.4, X.sup.5, X.sup.7
and X.sup.8 is an amino acid residue independently selected from
the group consisting of Y, D, G, H, W, P and V.
8. The peptide according to claim 4, wherein X.sup.1 is I, X.sup.2
is F, X.sup.6 is Y or N, and each of X.sup.3, X.sup.4, X.sup.5,
X.sup.7 and X.sup.8 is an amino acid residue independently selected
from the group consisting of E, P, D, S, Y, G, F, I and L.
9. The peptide according to claim 4, wherein X.sup.1 is V or I,
X.sup.2 is F, I, W or P, X.sup.5 is D or N, X.sup.6 is Y, N or L,
X.sup.7 is A or N, X.sup.8 is F or Y, each of X.sup.3 and X.sup.4
is independently selected from an amino acid residue.
10. The peptide according to claim 1, wherein said peptide is
represented by formula (II)
CX.sub.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7X.sup.8X.sup.9X.sup.10C
(II) wherein X.sup.1 is V, T or R, X.sup.2 is F, X.sup.6 is Y,
X.sup.8 is Y or T X.sup.9 is L X.sup.10 is V or M, C is cysteine,
and each of X.sup.3, X.sup.4, X.sup.5 and X.sup.7 is independently
selected from an amino acid residue.
11. The peptide according to claim 10, wherein X.sup.1 is V,
X.sup.2 is F, X.sup.6 is Y, X.sup.8 is Y, X.sup.9 is L, X.sup.10 is
V and each of X.sup.3, X.sup.4, X.sup.1 and X.sup.7 is
independently selected from the group consisting of A, H, N and
D.
12. The peptide according to claim 11, wherein X.sup.1 is V, T, or
R, X.sup.2 is F, X.sup.6 is Y, X.sup.7 is D or N, X.sup.8 is Y or
T, X.sup.9 is L, X.sup.10 is V or M, and each of X.sup.3, X.sup.4
and X.sup.5 is independently selected from the group consisting of
A, H, N and D.
13. The peptide according to claim 1, wherein said peptide is
represented by formula (IV)
CX.sup.1X.sup.2X.sup.3CX.sup.4X.sup.5X.sup.6CX.sup.7X.sup.8X.sup.9X.sup.1-
0C (IV) wherein X.sup.1 is L, X.sup.3 is T or Y, X.sup.7 is R or W,
C is cysteine, and each of X.sup.2, X.sup.4, X.sup.5, X.sup.6,
X.sup.8, X.sup.9 and X.sup.10 is independently selected from an
amino acid residue.
14. The peptide according to claim 13, wherein X.sup.1 is L and
X.sup.3 is T.
15. A peptide according to claim 1, wherein said peptide is
selected from the group consisting of TABLE-US-00009 CVFTSDYAFC,
(SEQ ID NO 1) CVIYDGNHWC, (SEQ ID NO 2) CIFEPDYSYC, (SEQ ID NO 3)
CVFDDLYSFC, (SEQ ID NO 4) CTFSVDYKYLMC, (SEQ ID NO 5) CVFAHNYDYLVC,
(SEQ ID NO 6) CRFDKEYRTLVC, (SEQ ID NO 7) CVAYCISSLCYYC, (SEQ ID NO
19) CVWYTGNTWC, (SEQ ID NO 20) CVFDALYTFC, (SEQ ID NO 21)
CVIYPGNVWC, (SEQ ID NO 22) CIFDGFYILC, (SEQ ID NO 23) CVPYLGLWLC,
(SEQ ID NO 24) and CMFDPMYMWMTC. (SEQ ID NO 25)
16. A protein comprising the peptide according to any of claims 1
to 15, wherein said protein selectively binds to free prostate
specific antigen, and enhances its enzymatic activity.
17. A diagnostic composition comprising at least one peptide
according to any one of claims 1 to 15, and a diagnostically
acceptable carrier and/or labelling substance.
18. A pharmaceutical composition comprising at least one peptide
according to any one of claims 1 to 15, and a pharmaceutically
acceptable carrier and/or labelling substance.
19. A method for therapeutically treating conditions dependent on
prostate specific antigen (PSA)-producing cells in mammals,
comprising administering to a mammal in need thereof a peptide
according to any one of claims 1 to 15 in an effective amount to
bind to free PSA in said mammal, and enhances its enzymatic
activity in said mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional of U.S. application Ser.
No. 10/363,662, filed Sep. 26, 2003 (now allowed); which is a 371
of PCT/FI01/00856, filed Oct. 1, 2001; the disclosure of each of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel peptide ligands for
prostate specific antigen binding and for enhancing its enzyme
activity. The present invention also relates to a process for the
preparation of these peptides. Further, the present invention
concerns pharmaceutical and diagnostic compositions comprising
these peptides and the use of the peptides for pharmaceutical and
diagnostic preparations. Still further, the present invention
relates to the use of these peptides as medicaments and diagnostic
agents, for the preparation of medicaments and diagnostic agents
and in biochemical isolation and purification procedures.
DESCRIPTION OF RELATED ART
[0003] Prostate specific antigen (PSA) is a highly specific product
of prostatic epithelial cells (1). PSA is a 30 kD serine protease,
the main biological function of which is liquefaction of the
seminal gel formed after ejaculation by proteolytic cleavage of the
semenogelins, which are the major constituents of the seminal clot
(2). PSA in seminal fluid mainly consists of free PSA including
enzymatically active forms and proteolytically cleaved or nicked
forms of PSA (3). In human serum, the major immunoreactive forms of
PSA are the PSA-al-antichymotrypsin complex (PSA-ACT) and free PSA
(PSA-F) (4).
[0004] Measurement of serum PSA is widely used for detection and
management of patients with prostate cancer and it is increasingly
used for screening of this disease. The major problem in the use of
PSA for screening is the high false positive rate caused by benign
prostatic hyperplasia. This can be reduced by assaying the
proportion of either PSA-ACT or free PSA in relation to total PSA
(4) but further improvement in the cancer specificity of PSA is
desirable. This may be accomplished by developing specific assays
for minor variants of PSA. Prostate cancer cells preferentially
secrete the proenzyme form of PSA (5), whereas nicked PSA has been
shown to be formed in benign tissue (6). Thus, assays for these
forms of PSA are of potential clinical utility, but development of
monoclonal antibodies (MAbs) specific for these forms has been
difficult. Another assay with diagnostic utility is that for the
PSA-alpha-2-macroglobulin complex (PSA-A2M) (7). However, in this
complex binding agents with high molecular weight like antibodies
cannot access PSA because the inhibitor encapsulates the
proteinase. Thus, assay of PSA-A2M requires denaturation of the
complex (7). Development of direct assay for PSA-A2M would require
a small binding agent entering the inhibitor and recognizing
specifically PSA.
[0005] PSA may inhibit tumor growth (8) and has recently been shown
to generate angiostatin from plasminogen (9). Because angiostatin
inhibits tumor growth by preventing formation of new blood vessels
in the tumor, generation of angiostatin may retard tumor
development. Thus, agents modulating the enzyme activity of PSA
could be potentially useful in treatment of prostate cancer.
[0006] MAbs are widely used tools for imaging and treatment of
cancer. However, the large size of MAbs limits their ability to
diffuse from circulation into tissues. Furthermore, mouse
antibodies are immunogenic in humans, which limits their use for
therapy. MAbs may also exhibit a prolonged availability in
circulation and interact with Fc receptors in normal tissues,
endangering the patient when toxins or radioisotopes are attached
(10). These problems can be eliminated by using alternative small
molecular weight binding agents.
[0007] An alternative to MAbs in the development of specific
binding agents is to use peptide libraries and phage display
techniques. PSA-binding peptides have been produced before by the
polysome selection method (11), but these peptides did not modulate
the enzyme activity of PSA and were not shown to be useful as part
of assays for PSA. Denmeade et al. have developed peptide
substrates for PSA on the basis of the sequence of semenogelin,
which is the substrate for PSA in seminal fluid (12). However,
these peptides cannot be applied in techniques in which stable
binding to PSA is required.
[0008] Although the above discussion shows that some peptides
reacting with PSA have been developed they have not been shown to
modulate the enzyme activity of PSA or be functional in
applications for detection of PSA.
SUMMARY OF THE INVENTION
[0009] It is an object of the present invention to eliminate the
problems of the prior art and to provide novel PSA binding chemical
agents. In particular, the invention aims at providing novel
peptide ligands and functional equivalents thereof which are
therapeutically and diagnostically useful in particularly for
treatment and diagnosis of conditions involving release of PSA into
serum.
[0010] It is another object to provide a process for the
preparation of novel peptide ligands for prostate specific antigen
which are capable of modulating its enzyme activity.
[0011] It is a third object to provide pharmaceutical and
diagnostic compositions comprising novel chemical compounds capable
of binding to PSA.
[0012] Further, it is a fourth object of the invention to provide
novel diagnostic and therapeutical methods.
[0013] These and other objects, together with the advantages
thereof over known binding agents and processes, are achieved by
the present invention as hereinafter described and claimed.
[0014] The present invention is based on the finding that a group
of novel peptides having a specific structure (specific amino acid
sequences or motifs) are capable of selectively binding to free PSA
or to PSA in complex with A2M and of modulating its enzymatic
activity. The peptides of the invention comprise at least one pair
of cysteines which are spaced apart by a number of at least two
amino acids and which are capable of forming a cyclic structure in
which there is a disulphide bond between said at least one pair of
cysteines. As a result of the disulphide bonding between the
cysteines, the main chain of the peptide is bent and it takes up a
3-dimensional conformation, which can be used as a basis for
development of peptidyl analogues or peptidomimetic compounds
having similar bioactivity as the peptides.
[0015] The novel peptides, which illustrate the present binding
agents, have been found by using phage display libraries of
peptides that were conformationally restrained by designed
disulfide bonds. Surprisingly, and quite contrary to expectations,
the most active peptides enhance the enzyme activity of PSA against
small molecular weight chromogenic substrates and natural protein
substrates. This increased activity of PSA can be utilized in a
number of applications in particular when enhancement of the
enzymatic activity of PSA is used for therapeutic purposes.
[0016] More specifically, the peptide ligands and functional
equivalents thereof according to the present invention are mainly
characterised by what is stated in the characterising parts of
claims 1 to 23
[0017] The diagnostic compositions according to the present
invention are mainly characterised by what is stated in the
characterising parts of claims 26 and 27.
[0018] The pharmaceutical compositions according to the present
invention are mainly characterised by what is stated in the
characterising parts of claims 28 and 29.
[0019] The process for the preparation of the peptide ligands
according to the present invention is mainly characterised by what
is stated in the characterising part of claim 30.
[0020] The therapeutic methods according to the present invention
is mainly characterised by what is stated in the characterising
parts of claims 31 and 32.
[0021] The uses of the present peptide motifs and sequences and
peptidomimetics are mainly characterised by what is stated in the
characterising parts of claims 33 to 40.
[0022] Considerable advantages are obtained with the aid of the
present invention. The present invention provides for the first
time PSA binding ligands which are capable of modulating and even
specifically enhancing the enzyme activity of PSA. The peptides
according to the present invention can bind specifically with
various forms of PSA as part of immuno-peptide assays or
chromatographical matrices. Furthermore, the peptides display
binding specificities which have not been obtained with antibodies.
The present peptides and corresponding peptidomimetic compounds can
be formulated into pharmaceutical compositions for treatment of PSA
secreting cell dependent conditions.
[0023] The peptides developed by Denmeade et al. have been
conjugated to a cytotoxic drug to form a prodrug, which is
activated when cleaved by PSA (13). In an analogous way, the
present peptides showing stable binding with PSA can be potentially
used to selectively deliver cytotoxic drugs, gene therapy vectors
and imaging agents to prostate cancer tissue, without the need for
any activation step.
[0024] Next, the invention will be described in more detail with
the aid of a detailed description and by making reference to the
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the binding of GST-peptides to immobilized
PSA.
[0026] FIG. 2 shows the effect of Zn.sup.2+ on the binding of
GST-peptides to PSA.
[0027] FIG. 3 depicts the effect of PSA in solution on the binding
of GST-C-4 with solid phase PSA.
[0028] FIGS. 4a and 4b present, respectively, the reactivity of
GST-peptides with various proteinases related to PSA and with
PSA-serpin complexes.
[0029] FIG. 5 presents the fractionation of the GST-C-4 by gel
filtration.
[0030] FIG. 6 illustrates the binding of the GST peptides with the
proenzyme form of PSA and with active PSA.
[0031] FIG. 7 depicts the effect of chemically synthesized peptides
on the binding of GST-C-4 to solid phase PSA.
[0032] FIG. 8 shows the effect of GST-C-4 on the enzyme activity of
PSA.
[0033] FIG. 9 depicts the effect of chemically synthesized peptide
C4 on the enzyme activity of PSA.
[0034] FIG. 10 illustrates the effect of chemically synthesized
peptide C-4 and A-1 on the proteolytic activity of PSA towards
IGF-BP-3.
[0035] FIG. 11 presents the combined effect of GST-C-4 and
Zn.sup.2+ on the enzyme activity of PSA.
[0036] FIG. 12 depicts the effect of the chemically synthesized
peptide C-4 on the enzyme activity of PSA in complex with A2M.
[0037] FIG. 13 shows the structure of the derivatized peptide
B2.
[0038] FIG. 14 shows the structure of the derivatized peptide
C4.
[0039] FIG. 15 indicates the effect of SKSKSKS (SEQ ID
NO:47)-tailed peptide C4 on the enzyme activity of PSA.
[0040] FIG. 16 shows the fractionation of an Eu-labelling reaction
mixture of peptide C4.
[0041] FIG. 17 indicates the effect of the various fractions of the
C18 chromatography shown in FIG. 16 on enzyme activity of PSA.
[0042] FIG. 18 shows the effect of technetium labeling on the
binding activity of C4-peptide.
[0043] FIG. 19 shows the effect of iodination on the PSA binding
activity of C4 peptide.
[0044] FIG. 20 shows the fractionation of seminal plasma sample by
B2 peptide affinity chromatography in the presence of 100 .mu.M
Zn.sup.2+.
[0045] FIG. 21 shows the fractionation of seminal fluid as above in
FIG. 20 but without Zn.sup.2+.
[0046] FIG. 22 shows the elution of PSA from a C4-peptide column at
different pH-values.
[0047] FIG. 23 shows the immunoblot analysis after SDS-PAGE under
non-reducing conditions of PSA-containing fractions obtained from
an affinity column of peptide B2.
[0048] FIG. 24 shows the corresponding (cf FIG. 23) immunoblot
analysis after SDS-PAGE under reducing conditions.
[0049] FIG. 25 shows SDS-PAGE under reducing conditions of seminal
fluid fractionated by the B2 peptide column.
[0050] FIGS. 26a to c show plots of the differences between
C.sup..alpha.H chemical shift values in the random coil and values
determined experimentally in DMSO-d.sub.6 for A-1 (a), B-2 (b) and
C-4 (c).
[0051] FIGS. 27a to c show the significant NOE-correlations of
peptides A-1, B-2 and C-4 in DMSO-d.sub.6.
[0052] FIG. 28 shows the three-dimensional structure of the peptide
A-1.
[0053] FIG. 29 shows the three-dimensional structure of the peptide
B-2.
[0054] FIG. 30 shows the three-dimensional structure of the peptide
C-4.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0055] For the purpose of the present invention, the term "ligand"
stands for a chemical compound or a part of a chemical compound
which is capable of binding to the binding domain present on a
large polypeptide molecule, such as a hormone, or on the surface of
a cell. The present peptide ligands bind to PSA, and modulate or
even, in many cases, increase its enzyme activity against synthetic
and natural IGF-BP-3 substrate. In the present context, "ligand" is
used synonymously with "binding agent".
[0056] Generally, the ligand is a chemical compound, e.g. a
peptide, which is soluble in physiological solutions or miscible
with water and serum. The binding of the present ligands to PSA can
be characterized as being "stable" (in contrast to, e.g., an
enzymatic reaction) in the respect that the ligand is attached to a
binding domain of PSA to the extent that its binding can be
measured and determined e.g. by surface plasmon resonance or
immuno-peptide assays (IFMA-assay). Further, the bonded ligand
cannot be washed or rinsed away with physiologically buffered
water. The bonding strength of the present ligands is comparable to
that of peptide agents used for the targeting of breast cancer
(14).
[0057] The binding of the present ligands to PSA is "selective"
which is indicated by the fact that MAbs, which are specific for
free PSA and serine proteinase inhibitors in complex with PSA
prevent the binding of the ligands to PSA. It would appear that the
present ligands bind to a site of PSA located in the vicinity of
the active site, but this is only a theory which we do not wish to
be bound by. Typically, the present peptides and the corresponding
peptidomimetic compounds bind to "free" PSA or PSA in complex with
.alpha.2-macroglobulin. Free PSA is the form of PSA synthesized and
secreted by the prostatic epithelial cells.
[0058] "Peptide" stands for a strand of several amino acids bonded
together by amide bonds to form a peptide backbone. Generally the
peptides comprise molecules with molecular weight lower than 10
kDa, i.e. containing about 90 amino acids or less. Peptides can be
designated as "small peptides" when they consist of about 6-30
amino acid units. As mentioned above, the present peptides
generally comprise at least one cross-link formed by disulfide
bonding between cysteine units. If the peptides contain several
pairs of cysteines, there can be a multiple number of such
cross-links. In addition to disulfide bonds, there can be other
cross-links within the peptides as well. The specific structures of
some exemplary peptides are discussed in more detail below.
[0059] "Peptidyl analogues" are chemical derivatives of the
peptides based on the modification of the peptides by various
chemical reactions, such as cycloadditions, condensation reactions
and nucleophilic additions.
[0060] "Peptidomimetic compounds" are compounds, which resemble the
original peptides mentioned above. They are generally built up of
different chemical building blocks than the amino acids, which form
the original peptides. For example, non-peptidyl compounds like
benzolactam or piperazine based analogues based on the primary
sequence of the original peptides can be used (15, 16). The
resemblance between the peptidomimetic compounds and the original
peptides is based on structural and functional similarities. Thus,
the peptidomimetic compounds mimic the bioactive conformation of
the original peptides and, for the purpose of the present
application, their binding activity with respect to the binding
site of PSA is similar to that of the peptide they resemble. The
peptidomimetic compounds can be made up of amino acids (such as
D-amino acids), which do not appear in the original peptides, or
they can be made from other compounds forming amide bonds or even
ester bonds Examples of synthetic peptidomimetic compounds comprise
poly(ester imide)s, polyesters, N-alkylamino cyclic urea, thiourea,
bicyclic guanidines, imidazol-pyridino-indoles, hydantoins and
thiohydantoins (15, 16). They may contain, e.g., the following
groups: phenyl, cyclopentyl, cyclopentanyl, cyclohexenyl,
cyclohexanyl, naphthyl, indanyl, furyl, thienyl, pyrrolyl,
pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,
pyridyl, imidazoyl, imidazolinyl, imidazolidinyl, morpholinyl,
piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl,
isothiazolidinyl, thiazolyl, thiazolidinyl, isothiazolyl, and
bicyclic rings.
[0061] The peptidomimetic compounds can be characterized as being
"structurally and functionally equivalent" to the peptides.
[0062] Generally, the novel binding agent for prostate specific
antigen (PSA) comprises either
a. a peptide having at least 6 amino acids bonded together to form
a peptide backbone and including at least one pair of cysteines
which are spaced apart by a number of at least two amino acids and
interconnected by a disulfide bond to form a cyclic structure
defined by the cysteines, the intermediary amino acids and the
disulfide bond; or b. a peptidomimetic compound having a spatial
conformation similar to the peptide (mimicking the bioactive
conformation of the original peptide).
[0063] Both of said compounds exhibit selective binding to free
prostate specific antigen and to PSA-A2M (as defined above).
[0064] According to a first preferred embodiment of the present
invention, the novel PSA binding peptide ligands are based on
cyclic structures (disulfide bond between cysteines) of the peptide
motifs according to formula (I)
CX.sup.1X.sup.2XXXX.sup.6XXC (I)
wherein [0065] X.sup.1 is V or I, [0066] X.sup.2 is F, I, W or P,
[0067] X.sup.6 is Y, N or L, [0068] C is cysteine, and [0069] each
X stands independently for an amino acid residue.
[0070] In particular, in formula (I) X.sup.1 is V or I, X.sup.2 is
F or I and X.sup.6 is Y or N. When X.sup.1 is V and X.sup.2 is F,
then X.sup.6 is preferably Y and each X is an amino acid residue
independently selected from the group consisting of T, S, D, Y A,
F, E, P, and L. When X.sup.1 is V and X.sup.2 is I, X.sup.6 is
preferably N and each X is an amino acid residue independently
selected from the group consisting of Y, D, G, H, W, P, and V.
Further, when X.sup.1 is I, X.sup.2 is preferably F, X.sup.6 Y or
N, and each X is an amino acid residue independently selected from
the group consisting of E, P, D, S, Y, G, F, I and L.
[0071] According to another particularly preferred embodiment of
peptides according to formula (I), X.sup.1 is V or I, X.sup.2 is F,
I, W or P, X.sup.5 is D or N, X.sup.6 is Y, N or L, X.sup.7 is A or
N, X.sup.8 is F or Y, the remaining structural units bearing the
same meanings as above.
[0072] Examples of peptides are represented by the sequences
according to SEQ ID NO:s A-1 (SEQ ID NO 1), A-2 (SEQ ID NO 2), A-4
(SEQ ID NO 4), Z-6 (SEQ ID NO 20), Z-7 (SEQ ID NO 21), Z-8 (SEQ ID
NO 22), and Z-10 (SEQ ID NO 24) in Table 1 and SEQ ID NO:s A-3 (SEQ
ID NO 3) and Z-9 in Table 1 (SEQ ID NO 23).
TABLE-US-00001 TABLE 1 SEQ ID Code Library Peptide Sequence No of
isolates 1 A-1* CX.sub.8C C V F T S D Y A F C 2 2 A-2 CX.sub.8C C V
I Y D G N H W C 2 3 A-3 CX.sub.8C C I F E P D Y S Y C 2 4 A-4
CX.sub.8C C V F D D L Y S F C 2 5 B-1 CX.sub.10C C T F S V D Y K Y
L M C 15 6 B-2* CX.sub.10C C V F A H N Y D Y L V C 2 7 B-3
CX.sub.10C C R F D K E Y R T L V C 1 8 C-1
CX.sub.3CX.sub.4CX.sub.2C C V S Y C L F E F C Y V C 2 9 C-2
CX.sub.3CX.sub.4CX.sub.2C C V E Y C W E G S C Y V C 7 10 C-3
CX.sub.3CX.sub.4CX.sub.2C C V A Y C E E W E C Y V C 1 11 C-4*
CX.sub.3CX.sub.4CX.sub.2C C V A Y C I E H H C W T C 3 12 C-5
CX.sub.3CX.sub.4CX.sub.2C C V S Y C D G L Q C W M C 1 13 D-1
CX.sub.3CX.sub.3CX.sub.3C C L S T C A Q S C R I S C 7 14 D-2
CX.sub.3CX.sub.3CX.sub.3C C L L Y C H D A C W W V C 2 15 Z-1
CX.sub.3CX.sub.4CX.sub.2C C V T Y C Y G E V C Y Y C 16 Z-2 C A A Y
C V A G L C Y G C 17 Z-3 C V Q Y C I G G D C W F C 18 Z-4 C V V Y C
D S M K C W T C 19 Z-5 C V A Y C I S S L C Y Y C 20 Z-6 CX8C C V W
Y T G N T W C 21 Z-7 C V F D A L Y T F C 22 Z-8 C V I Y P G N V W C
23 Z-9 C I F D G F Y I L C 24 Z-10 C V P Y L G L W L C 25 Z-11
CX10C C M F D P M Y M W M T C
Table 1. Amino Acid Sequences of PSA-Binding Peptides.
[0073] Single-stranded DNA was purified from phage clones after
three rounds of selection with PSA. Peptide sequences were deduced
from the DNA sequences of the corresponding region of the phage
genome. Consensus residues found in several clones are indicated by
bold font. Peptides expressed as glutathione S-transferase (GST)
fusion proteins are indicated with an asterisk.
[0074] According to a second preferred embodiment of the present
invention the novel PSA binding peptide ligands can further be
based on a cyclic structure of the peptide motifs according to
formula (II)
CX.sup.1X.sup.2XXXX.sup.6XX.sup.8X.sup.9X.sup.10C (II)
wherein [0075] X.sup.1 is V, T or R, [0076] X.sup.2 is F, [0077]
X.sup.6 is Y, [0078] X.sup.8 is Y or T [0079] X.sup.9 is L [0080]
X.sup.10 is V or M, and [0081] X and C have the same meaning as
above.
[0082] Preferably, in formula (II), X.sup.1 is V, X.sup.2 is F,
X.sup.6 is Y, X.sup.8 is Y, X.sup.9 is L, X.sup.10 is V and X is
selected from the group consisting of A, H, N and D. Preferred
sequences are exemplified by SEQ ID NO:s B-1 (SEQ ID NO 5), B-2
(SEQ ID NO 6) and B-3 (SEQ ID NO 7) in Table 1.
[0083] The structural units in formula (II) may also carry the
following meanings: X.sup.1 is V, T, or R, X.sup.2 is F, X.sup.6 is
Y, X.sup.7 is D or N, X.sup.8 is Y, T or A, X.sup.9 is L, X.sup.10
is V or M, and the other units bearing the same meanings as
above.
[0084] According to a third preferred embodiment of the present
invention, the novel PSA binding peptide ligands are based on a
cyclic structure of the peptide motifs according to formula
(III)
CX.sup.1X.sup.2X.sup.3CXXXXCX.sup.8X.sup.9C (III)
wherein [0085] X.sup.1 is V or A, [0086] X.sup.2 is A, S, E, T, V
or Q, [0087] X.sup.3 is Y, [0088] X.sup.8 is Y or W, [0089] X.sup.9
is V, T, M, Y, G or F and [0090] X and C have the same meaning as
above.
[0091] Particularly, the peptide motifs or sequences according to
formula (II) comprise the following amino acid residues: X.sup.1 is
V, X.sup.2 is A or S, X.sup.3 is y, X.sup.8 is Y or W, X.sup.9 is
V, Y or T, and each X is independently selected from the group
consisting of L, F, E, W, G, S, I, H, D, G, L, Q, Y, V, A, M and K.
As will be discussed below in more detail, strong binding to PSA is
achieved by clones containing the CVAYC (SEQ ID NO 11) motif. Thus,
in formula III, X.sup.1 is preferably V, X.sup.2 is A and X.sup.3
is Y.
[0092] X.sup.3 can also have the meaning Y or F and X.sup.5 the
meaning E or Q, the remaining structural units having the same
meanings as above, in particular as in the preceding paragraph.
[0093] Peptides having formula (III) are shown under the following
sequence numbers in Table I: SEQ ID C-1 (SEQ ID NO 8)-C-5 (SEQ ID
NO 12), Z-1 (SEQ ID NO 15)-Z-6 (SEQ ID NO 20) and Z-8 (SEQ ID NO
22).
[0094] According to a fourth preferred embodiment of the present
invention, the novel PSA binding peptide ligands based on a cyclic
structure of the peptide motifs according to formula (IV)
CX.sup.1XX.sup.3CXXXCX.sup.7XXXC (IV)
wherein [0095] X.sup.1 is L, [0096] X.sup.3 is T or Y, [0097]
X.sup.7 is R or W, and [0098] X and C have the same meaning as
above.
[0099] Preferably, X.sup.1 is L and X.sup.3 is T. Corresponding
sequences in Table 1 have been assigned the numbers SEQ ID NO:s D-1
(SEQ ID NO 13) and D-2 (SEQ ID NO 14).
[0100] The present investigation also relates to the use of the
motifs described above as lead sequences for development of binding
agents with alternative characteristics.
[0101] It is another object of the present invention to provide
novel PSA binding peptide ligands based on the sequences listed in
Table 1. The peptides with SEQ ID A-1 (SEQ ID NO 1)-A-4 (SEQ ID NO
4), B-1 (SEQ ID NO 5)-B-3 (SEQ ID NO 7), C-1 (SEQ ID NO 8)-C-5 (SEQ
ID NO 12) and D-1 (SEQ ID NO 13)-D-2 (SEQ ID NO 14) were isolated
as described in methods section below. The peptides Z-1 (SEQ ID NO
15)-Z-11 (SEQ ID NO 25) were isolated as the peptides of A-, B-, C-
and D-series but 200 .mu.M of ZnCl.sub.2 was included in the buffer
used for panning.
[0102] Peptides A1 (CVFTSNYAFC, SEQ ID NO 1), B2 (CVFAHNYDYLVC, SEQ
ID NO 6) and C4 (CVAYCIEHHCWTC, SEQ ID NO 11) exhibit particularly
strong binding and their properties and structures have been
studied in more detail. The results of these studies are given in
Examples 4 and 5.
[0103] The structural analyses discussed in Example 5 show that the
two peptides A-1 and B-2 (corresponding to general formulas I and
II, respectively) have a very similar structure and similar or even
the same biological activity. The preferred side chains in A-1
peptide are Phenylalanine (Phe, F) in position 3 and Tyrosine (Tyr,
Y) in position 7. In the peptide B-2 the preferred amino acids are
the same, viz. Phenylalanine (Phe, F) in position 3 and Tyrosine
(Tyr, Y) in position 7.
[0104] In sequences A-1 and B-2, the rigid .beta.-turn stabilises
the position of aromatic side chains of Phenylalanine (Phe, F) in
position 3 and Tyrosine (Tyr, Y) in position 7. The structure is
stabilised in the peptide A-1 by hydrogen bond between the carbonyl
oxygen of Threonine (Thr, T) in position 4 and the hydrogen of
amide of Tyrosine (Tyr, Y) in position 7. The structure is
stabilised in the peptide B-2 by the hydrogen bond between the
carbonyl oxygen of Alanine (Ala, A) in position 4 and the hydrogen
of the amide of Tyrosine (Tyr, Y) in position 7.
[0105] The preferred amino acids of the peptide C-4 are Tyrosine
(Tyr, Y) in position 4 and Tryptophan (Trp, W) in position 11. The
structure is stabilized by the two disulphide bridges; the first
between positions Cys1 and Cys13 and second between the positions
Cys5 and Cys10.
[0106] It should, however, be pointed out that, in addition to the
above-mentioned preferred amino acids in the indicated positions,
also those alternative amino acids having similar structure and
mentioned in the substituent listings relating to the of formulas
will give rise to the desired activity. The positions mentioned are
calculated from the whole length of the peptides and the cysteins
have also been numbered, which means that, for example, the F in
position 3 of peptide A-1 corresponds to X.sup.2 in formula I.
[0107] Based on the data obtained, the peptidomimetic compounds
according to the invention having an activity similar to that of
the present peptides of formulas I or II will exhibit a basic
structure comprising 10 to 12 structural units and in particular
having in positions 3 and 7 aromatic or alicyclic rigid groups, the
structure being made rigid by a .beta.-turn. The side group
attached to the structural unit in position 7 should further have a
group providing for hydrogen bonding. The peptidomimetic compounds
according to the invention having an activity similar to that of
the present peptides of formula III will exhibit a basic structure
of 13 structural units, which is stabilized by intramolecular
bridges between the units in positions 1 and 13 and 5 and 10,
respectively. Further, in positions 4 and 11, the peptidomimetic
will exhibit aromatic or alicyclic rigid substituents (side
groups), the substituent in position 4 having further an acid
functionality or similar providing for hydrogen bonding
capability.
[0108] The peptidomimetic compounds, as well as the actual
peptides, can have a longer molecular chain than the ones disclosed
above, as long as the motifs (or the corresponding functional
patterns) are present.
[0109] The present invention also relates to diagnostic
compositions comprising an amount of the novel PSA binding
peptides. The diagnostic composition comprising the present
peptides and a diagnostically acceptable carrier may be used as
such or as peptide-conjugate immobilized to a solid phase matrix of
the assay or as labelled peptide-tracer reagent. The diagnostic
composition according to the present invention finds use in assays
based on determination of various molecular forms of PSA. The
diagnostic compositions contain the active component in liquid
phase, preferably aqueous phase, in a concentration of about 0.1 to
500 .mu.g/l. The compositions may contain detergents, such as Tween
or polysorbates, and stabilizing agents. The concentrations of the
other components can be about 0 to 50% of the weight of the
composition.
[0110] As the results of Example 4 show, the present peptides can
be used for purification of PSA and for differentiation between
various forms of free PSA. Because these peptides possess novel
binding specificities towards PSA, i.e. they bind specifically with
the intact form of free PSA, they are potentially useful in
development of assays with improved accuracy for prostate
cancer.
[0111] The present invention also relates to a pharmaceutical
composition comprising an amount of the novel PSA binding peptide
ligands. The pharmaceutical composition comprising the novel PSA
binding peptides according to the invention may be used
systemically, locally and/or topically, and may be administered
e.g. parenterally, intravenously, subcutaneously, intramuscularly,
intranasally, by pulmonary aerosol or in depot form. The
compositions may also include all potential combinations of the
peptides with labelling reagents, imaging reagents, drugs and other
chemicals/-molecules.
[0112] The present invention also relates to the use of the
peptides to mediate gene delivery to PSA expressing cells or to
cells in the vicinity of PSA expressing cells. A general
description of the use of peptides for gene delivery mediation may
be found in reference 17.
[0113] Pharmaceutical compositions suitable for intravenous
infusion or injection are particularly preferred and they comprise
the active component in a concentration of, generally, about 0.1 to
500 g/l, preferably about 1 to 250 g/l. It is preferred to have
somewhat higher concentrations (e.g. about 20 to 200 g/l), to allow
for administration without causing excessive volume load to the
patients. The preparation may be lyophilized and reconstituted
before administration or it may be stored as a solution ready for
administration. The pH of the solution product is in the range of
about 5 to about 8, preferably close to physiological pH. The
osmolality of the solution can be adjusted to a preferred value of
at least 200 mosmol/l using sodium chloride and/or sugars, polyols
or amino acids or similar components. The compositions can further
contain pharmaceutically acceptable excipients and stabilizers,
such as albumin, sugars and various polyols. The amounts of these
components can vary broadly within a range of about 0 to 50 wt-% of
the active component. Liquid formulations provide the additional
advantage of being ready for administration without
reconstitution.
[0114] For oral administration it may be necessary to prepare
derivatives of the present peptides.
[0115] According to a particularly interesting embodiment, the
peptides (and peptidomimetics) are incorporated into a liposome
package containing diagnostic/therapeutic compounds, such as
cytotoxic drugs doxorubicin and methotrexate. Liposome packaging
peptides are disclosed in references 18, 19. Coupling of the
peptides to the surface of the liposome enables the targeting of it
to PSA producing cells.
[0116] The present invention also includes the use of the novel PSA
binding peptides for the manufacture of reagents for PSA based
diagnosis of benign and malignant prostatic disease and diseases
derived from other tissues producing PSA.
[0117] The present invention also includes the use of novel PSA
binding peptides for the manufacture of the above-mentioned
pharmaceutical preparations for the treatment and targeting of
conditions based on the use of present invention. The present
invention also includes the use of novel PSA binding peptides for
treatment of conditions based on regulation of PSA enzyme activity.
Thus, generally the novel peptides and structurally and
functionally equivalent peptidomimetics (=active components) can be
employed in pharmaceutical compositions to treat mammalian cancers
as well as other conditions, by administering an effective dose of
the peptide or peptidomimetic or a therapeutically acceptable acid
or salt or derivative thereof in a pharmaceutical carrier. They can
be administered with a pharmaceutically acceptable carrier at
dosages of from about 1 to about 1,000 micrograms per kg of body
weight daily. As mentioned above the composition may be
administered parenterally, intravenously, subcutaneously,
intramuscularly, intranasally, by pulmonary aerosol or in depot
form.
[0118] The present invention also relates to the use of the novel
PSA binding peptides for biochemical isolation and purification
procedures of various forms of PSA.
[0119] The present invention also relates to a process for the
preparation of these PSA binding peptides by standard solid phase
Merrifield peptide synthesis.
[0120] The peptides according to the present invention can be used
as such or as labelled derivatives as part of quantitative assays
for various molecular forms of PSA, or as compounds to regulate the
activity of PSA or for targeting of PSA producing cells.
[0121] Furthermore, these peptides can be used as lead compounds to
design peptidomimetics for purposes described above. The peptides
can also be used in column chromatographic matrices for biochemical
isolation and purification of various forms of PSA, as already
discussed above and further studied in Example 4. Yet further, the
motifs according to the present invention can be used as lead
sequences for development of binding agents with alternative
characteristics.
[0122] The present peptides can also be used in methods for
modulating the PSA enzyme activity and PSA activity-dependent
conditions by using these peptides either in vivo or in vitro.
Further, the peptides according to the present invention can be
used in procedures for biochemical isolation and purification of
PSA.
[0123] The peptides according to the present invention also
increase the activity of PSA against IGF-BP-3, and, thus, they also
affect the activity of PSA against natural substrates. Thus, the
present peptides are potentially useful for modulation of the
biological function of PSA in prostate pathology and physiology.
Interestingly, none of the peptides identified inhibited enzyme
activity.
[0124] PSA has been shown to inhibit endothelial cell proliferation
and invasion [8, 1999 #626], and to split plasminogen into
fragments corresponding to angiostatin, which inhibits endothelial
cell growth and vessel formation (9). The present peptides bind to
PSA, and increase its enzyme activity against synthetic and natural
substrate. Thus, the peptides could also increase the ability of
PSA to produce angiostatin and to inhibit the growth of blood
vessels associated with cancer progression.
[0125] Because of its highly prostate specific expression, PSA is a
potential target for prostate cancer therapy. Denmeade et al. have
developed peptide substrates (12), which are specifically cleaved
by PSA. These peptides have been conjugated to a cytotoxic drug to
form a prodrug, which is activated when cleaved by PSA (13). In an
analogous way, the peptides according to the present invention can
be used to selectively deliver cytotoxic drugs, gene therapy
vectors and imaging agents to prostate cancer tissue. The small
size of these peptides enables them to penetrate efficiently into
prostatic tissue. Peptides homing selectively to endothelium of
specific target organs have been previously developed by phage
display (20). Likewise, the prostate specificity of our PSA-binding
peptides peptides enables them to be localized specifically to
PSA-producing cells.
[0126] The following non-limiting examples illustrate the invention
further.
EXAMPLE 1
Identification of PSA Binding Phage
[0127] A convenient way to develop novel binding agents for various
targets is to screen libraries of random peptides. Phage display is
a powerful method for selection of novel ligands for various target
proteins (21) including enzymes, antibodies and receptors. Phage
display libraries offer a way to identify specific ligands
possessing binding specificities different from those displayed by
antibodies.
Phage Display Libraries
[0128] The construction of phage display peptide libraries in fUSE
5-phage has been described (21). Libraries with the structures
CX.sub.8C, CX.sub.10C, CX.sub.3CX.sub.3CX.sub.3C and
CX.sub.3CX.sub.4CX.sub.2C, where C is cysteine and X is any of the
20 naturally occurring amino acids, were used.
Antibodies and Proteins
[0129] The development of MAb 5E4 will be described in a separate
report (Leinonen, manuscript in preparation). MAb H117 was obtained
from Abbott Diagnostics (North Chicago, Ill., USA) and 5A10 was
from EG&G-Wallac, Turku, Finland. Anti phage monoclonal
antibody was a kind gift from Dr. Petri Saviranta, University of
Turku, Turku, Finland. A polyclonal anti GST antibody was from
Amersham Pharmacia Biotech. PSA was purified from human seminal
fluid as described previously (3). HK2 was a kind gift from Dr.
Janita Lovgren, University of Turku, Turku, Finland. The
proteinases chymotrypsin and cathepsin G were from Athens Research
and Technology, Athens, Ga., USA, trypsin was purified as described
(22), and kallikrein was from Calbiochem, Calif., USA. The
antibodies against chymotrypsin and cathepsin G were from
Fitzgerald, Mass., USA, an antibody against human plasma kallikrein
from Calbiochem, Calif., USA, and an antibody against trypsin was
prepared as described (23). Anti-ACT and anti-API antibodies were
from Dako, Glostrup, Denmark.
Selection of Phage Peptides
[0130] The screening of phage display peptide libraries was
performed essentially as described by Koivunen et al. (21).
Briefly, each phage library was separately screened with PSA
captured on microtiter wells coated with the monoclonal antibody
5E4, which binds both free and complexed PSA (24). 33 nM PSA in TBS
buffer containing 10 g/L BSA (BSA-TBS) was incubated in wells
coated with MAb 5E4 for 1 h, then the wells were washed to remove
unbound PSA. An aliquot of each phage library (1011-1012 infectious
particles) was added to the wells, and incubated for 3 h at
22.degree. C. during the first round of panning and for 1 h during
subsequent rounds. The phage solution was removed and the wells
were washed with TBS containing 0.5% Tween 20. The panning was also
performed in the presence of 200 .mu.M ZnCl2. The bound phage were
eluted with 0.1 M glycine buffer, pH 2.2 and neutralized with 1 M
Tris base. The eluted phage were amplified by infection of E. coli
K91 kan cells, and purified by precipitation with polyethylene
glycol. After three rounds of selection and amplification single
stranded DNA from individual phage clones was prepared and the
peptide sequences were determined by sequencing the relevant part
of the viral DNA. Sequencing was performed with an ABI 310 Genetic
analyzer and Dye Terminator Cycle Sequencing Core Kit (PE Applied
Biosystems, Foster City, Calif., USA) using the oligonucleotide
5'CCCTCATAGTTAAGCGTAACG (SEQ ID NO:26) 3' as a primer.
[0131] To isolate PSA-binding peptides, we screened four phage
libraries expressing cyclic disulfide-constrained peptides
containing 10, 12 or 13 amino acids at the N-terminus of phage
protein III. The peptides isolated from each library contained
characteristic consensus amino acids in addition to the cysteine
engineered in fixed positions, (Table 1). Most peptides derived
from the CX.sub.8C, CX.sub.10C and CX.sub.3CX.sub.4CX.sub.2C
libraries (9 out of 12) contained valine in the X.sup.1 position.
In addition, phenylalanine frequently occurred at the X.sup.2
position of the CX.sub.8C, and CX.sub.10C libraries. The X.sup.3
position was favored by tyrosine and X.sup.9 position was favored
by V in the CX.sub.3CX.sub.4CX.sub.2C library peptides. In the
phage IFMA (data not shown) and GST-peptide IFMA, the clones
containing the CVAYC (SEQ ID NO 29) motif showed the strongest
binding to PSA (cf. FIG. 1, Table 1).
[0132] FIG. 1 shows the results of experiments in which various
amounts of each GST-peptide were added to wells containing PSA
captured by MAb 5E4. After washing the binding of GST-peptide was
quantified by IFMA using an Eu-labelled anti GST antibody as
tracer. Data represent mean values from duplicate wells.+-.standard
error (SE).
[0133] A tyrosine residue was also enriched in either the position
X.sup.6 or X.sup.8 of the CX.sup.8C and CX.sup.10C peptides and in
position X.sup.8 of the CX.sup.3CX.sup.4CX.sup.2C peptides. After
including Zn.sup.2+ in the panning buffer, several PSA binding
peptides could be isolated (Table 1, peptides with SEQ ID Z-1 (SEQ
ID NO 15)-Z-11 (SEQ ID NO 25)). Most of these peptides contained
similar motifs as the peptides isolated without Zn.sup.2+.
EXAMPLE 2
Characterization of the Peptides
Construction of GST Fusion Protein Containing Selected Peptides
[0134] Single-stranded phage DNA was purified and the insert region
was amplified by PCR with primers upstream (5'AGGCTCGAGGATCCTCGG
CCGACGGGGCT 3') (SEQ ID NO 27) and downstream
(5'AGGTCTAGAATTCGCCCCAGC GGCCCC 3') (SEQ ID NO 28) of the fuse 5
gene III sequence (20). The amplified DNA was isolated and
subcloned between the BamH I and EcoR I sites of the PGEX-2TK
vector (Amersham Pharmacia Biotech, Helsinki, Finland) for
expressing the selected peptides as GST fusion protein.
Recombinants were verified by DNA sequencing. The fusion proteins
were expressed in E. coli BL 21 cells and purified by glutathione
affinity chromatography (Amersham Pharmacia Biotech) as described
(25). The purity of the fusion protein was analyzed by
SDS-polyacrylamide gel electrophoresis in 12.5% homogeneous gels on
the PhastSystem (Amersham Pharmacia Biotech). Their ability of
binding to PSA was measured by an immunofluorometric assay (IFMA)
(see below).
[0135] Peptide was released from the GST fusion partner by thrombin
cleavage. 20 units of thrombin (Amersham Pharmacia Biotech) was
incubated with 1 mg GST-C-4 in PBS buffer at 22.degree. C. for 12
h. Cleaved and intact GST-C-4 fusion proteins were analyzed by gel
filtration on a Superdex 200 HR 10/30 column using 50 mM
sodium-phosphate, pH 7.4 containing 150 mM NaCl and a flow rate of
30 ml/h. Absorbance was monitored at 280 nm and the
protein-containing fractions of 0.5 ml volume were collected and
analyzed by the GST-peptide IFMA as described below.
Immunofluorometric Assays (IFMAs)
[0136] The solid phase antibodies used in the IFMAs were coated
onto microtitration wells at a concentration of 5 .mu.g/mL in TBS
for 16 h at 22.degree. C., the solution was discarded and the wells
were saturated with 10 g/L bovine serum albumin in TBS for 3 h at
22.degree. C. The antibodies used as tracers were labeled with a
Eu.sup.3+ chelate as described previously (26). The assay buffer
was 50 mM Tris-HCL, pH 7.7, 150 mM NaCl, 33.3 .mu.M bovine serum
albumin, 1 .mu.M bovine globulin.
[0137] The binding of individual phage clones to PSA was tested by
IFMA (phage IFMA). In the phage IFMA, about 30 ng of PSA was added
to MAb 5E4 coated wells for 1 h. After washing (Buffer: 150 mM
NaCl, 7.7 mM NaN.sub.3, 0.2 g/L Tween 20), 15 .mu.L of phage and
200 .mu.l assay buffer was added. After incubation for 1 h, the
wells were washed and filled with 200 .mu.l of assay buffer
containing 50 ng europium-labeled anti phage monoclonal antibody
recognizing the M13 coat protein of the phage. After incubation for
60 min, the wells were washed 4 times, and enhancement solution
(EG&G-Wallac) was added. The fluorescence was quantified with a
1234 DELFIA Research fluorometer (EG&G-Wallac).
[0138] Binding of GST-peptide to PSA was determined by an IFMA
(GST-peptide IFMA) similar to the one for phage peptides except
that Eu.sup.3+-labeled antibody to GST was used. Various amounts of
GST-peptide or wild type GST were added to wells containing
captured PSA in 200 .mu.L of assay buffer and incubated for 1 h. To
test the effect of Zn.sup.2+ on the binding between GST-peptide and
PSA, various concentrations of Zn.sup.2+ (1-200 .mu.M) were added
to the buffer in the incubation step of GST-peptide and PSA. In
competition experiments, GST-peptide (167 nM) or wild type GST were
first incubated with increasing amounts of PSA (0-333 nM) in 0.5%
BSA-TBS buffer for 60 min, then added to wells containing PSA
captured by MAb 5E4. After incubation for 1 h the wells were washed
and Eu.sup.3+-labeled anti GST antibody was added. After further
incubation and washing the bound fluorescence was measured as
described above. When assessing the binding of the GST-peptides to
other proteinases, including chymotrypsin, cathepsin G, trypsin,
and kallikrein, these proteinases were captured by their specific
antibodies coated to the microtitration well. Recombinant hK2 was
captured by MAb 5E4, which is known to bind hK2 (24). For assessing
the binding of the GST-peptides with complexes of PSA, including
PSA-ACT and PSA-.alpha.1-protease inhibitor (PSA-API) (28), the
complexes were captured by antibodies against ACT and API,
respectively. After capturing the various proteinases or PSA
complexes, GST-peptide was added and binding was monitored as
described above for the GST-peptide IFMA.
Surface Plasmon Resonance
[0139] The binding kinetics of selected peptides to PSA was studied
by surface plasmon resonance (29) on a BIAcore 2000.TM. instrument
(Biacore AB, Uppsala, Sweden). PSA was captured to the solid phase
by MAb H117 which binds to the same epitope as MAb 5E4 (30). The
capture antibody was covalently coupled onto the surface of the CM5
sensor chip according to the manufacturer's instructions, using
coupling levels of 5000 resonance units (RU). PSA was captured by
injection of 416 nM of PSA in PBS, 4-min contact time (5 .mu.l/min)
and 60 min wash (20 .mu.l/min) providing ligand levels in the range
500-600 RUs. Each analyte (GST-peptide), at various concentrations,
was injected for 1 min at 20 .mu.l/min flow rate. To characterize
the effect of Zn.sup.2+ on the affinity of the peptides, the
sensorgrams were recorded for a constant amount of peptide in the
presence of Zn.sup.2+ both in the running and sample buffer (0-30
.mu.M). PSA-binding to either chemically activated/deactivated
blank surface or MAb H117 alone was subtracted as a non-specific
interaction. Regeneration after each measurement cycle was done
with 3.times.0.5 min injections of 10 mM HCl. Binding data were
analyzed using the BIAEVALUATION software.
[0140] The activity of the peptide cleaved from GST fusion protein
was determined by a competition experiment. After fractionation of
digested fusion protein by gel filtration, the fractions were
incubated in wells containing PSA captured by MAb 5E4 for 60 min.
After emptying of the wells, the GST-peptide (167 nM) was added and
incubated for 1 h. The effect of the cleaved peptide on the binding
between GST-peptide and PSA was monitored by the GST-peptide
IFMA.
[0141] For characterization of the binding site of the peptides on
PSA, monoclonal anti PSA antibodies recognizing various epitopes on
PSA were used (30). Each MAb (33 nM) was incubated in wells
containing PSA captured by MAb 5E4. After washing, 167 nM
GST-peptide was added and binding was monitored by GST-peptide IFMA
as described above.
Reactivity of the Peptides with Various Forms of Free PSA
[0142] The reactivity of the GST-peptides with proPSA and intact
PSA was compared by using active PSA (3) and proPSA (31). 100 ng of
proPSA or intact PSA was reacted with MAb 5E4 coated onto
microtitration wells. After washing, one .mu.g of each GST-peptide
was added to wells containing immobilized PSA. The binding of
GST-peptide was quantified by IFMA using Eu-labelled anti-GST
antibody as tracer.
Binding of Synthetic Peptides with PSA
[0143] Peptides A1 and C4 were synthesized by standard solid phase
Merrifield peptide synthesis using fmoc-chemistry. The peptides
were assayed for binding with PSA by studying the ability of the
synthetic peptide to inhibit the binding of the corresponding GST
peptide with PSA. Synthetic peptide (0-333 nM) and the
corresponding GST peptide (167 nM) were incubated with PSA captured
by MAb 5E4 for 1 h. After this, the wells were washed, after which
Eu.sup.3+-labeled anti GST antibody was added. After further
incubation and washing the bound fluorescence was measured as
described above.
[0144] We constructed GST fusion proteins from four peptides, which
display the strongest binding in the phage IFMA. Among them,
peptide C-4 with the sequence CVAYCIEHHCWTC (SEQ ID NO 11) showed
the strongest binding to PSA both as a GST-peptide (FIG. 1, Table
2) and when expressed on phage (data not shown).
TABLE-US-00002 TABLE 2 Kinetics and affinity for the binding of
GST-peptides to PSA Maximal 50% Peptide Binding ka .times. 10.sup.3
kd .times. 10.sup.-3 K.sub.D stimulation no. (CPS)* (l/Ms) (l/s)
(.mu.M) (.mu.M).sup.# A-1 219000 10.3 80 7.8 2.2 B-2 428000 16.3 57
3.5 1.7 C-4 790000 9.9 28 2.9 0.57 The association rate constat
(ka), dissociation rate constant (kd) and equilibrium dissociation
constant (K.sub.D) for GST-peptides to PSA were measured by surface
plasmon resonance. The values shown are the average ka, kd and
K.sub.D at 3 different concentrations of analyte (GST-peptides).
*Data from GST-peptide IFMA. The experimental procedure is
described on the legend to FIG. 1. .sup.#The peptide concentration
required for half maximal stimulation of the enzyme activity of
PSA. For experimental procedures see legend to FIG. 6.
[0145] Two other selected peptides, B-2 (SEQ ID NO 6)
(CVFAHNYDYLVC) and A-1 (SEQ ID NO 1) (CVFTSDYAFC) bound to PSA but
less efficiently than C-4 (SEQ ID NO 11).
[0146] When Zn.sup.2+, a cation known to bind to PSA, was included
in the assay buffer the binding activity of peptides was increased.
Zn.sup.2+ had a dose dependent effect and the maximal increase in
binding response was detected at a 200 .mu.M concentration (FIG.
2). In the experiments, the results of which are illustrated in
FIG. 2, each GST-peptide was incubated in PSA-containing wells in
the presence of various concentrations of Zn.sup.2+. The binding of
GST-peptide was quantified by IFMA. The control shows the binding
of GST-peptide in the absence of Zn.sup.2+. Data represent mean
values from duplicate wells.+-.SE. However, the effective Zn.sup.2+
concentration is lower because BSA in the buffer contains several
binding sites for Zn.sup.2+. The fourth peptide produced as a GST
fusion protein, D-1 (SEQ ID NO 13) (CLSTCAQSCRISC) did not show
significant binding to PSA even though the corresponding phage
bound to PSA (data not shown). Wild type GST did not bind to PSA
confirming that the binding of the fusion protein was mediated by
the inserted peptide.
[0147] The PSA-binding peptides were further characterized by
estimating the PSA concentration required to reduce the binding of
the peptide to immobilized PSA by 50% (IC50). In this connection,
we refer also to FIG. 3. GST-C-4 was preincubated with increasing
concentrations of PSA in solution for 1 h. Then the samples of the
mixtures were added to wells in which PSA had been captured by anti
PSA MAb. The binding was measured by IFMA. Data represent mean
values from duplicate wells.+-.SE. The corresponding values for the
peptides A-1 (SEQ ID NO I) and B-2 (SEQ ID NO 6) were 3.3 nM and 1
nM, respectively (data not shown). Preincubation of the
GST-peptides with an excess of free PSA inhibited binding to the
solid phase PSA. The binding of peptide C-4 (SEQ ID NO 11) to
captured PSA was reduced by 50% at a PSA concentration of 0.7 nM.
These results confirmed that the selected peptides bind not only to
PSA captured by MAb 5E4 but also to free PSA in solution.
[0148] The specificity of the peptides was studied by reacting them
with various proteinases and PSA complexes captured by antibodies
to microtitration wells. PSA, kallikrein, cathepsin G,
chymotrypsin, trypsin-2, and recombinant hK2 were captured onto
wells coated with antibodies against each proteinase, respectively.
GST-peptide binding was measured by IFMA. The results obtained are
presented in FIG. 4a. None of the proteinases tested showed
significant binding of the GST-peptides. PSA, PSA-ACT and PSA-API
were captured onto wells coated with antibodies against PSA, ACT
and API, respectively. GST-peptide binding to PSA complexes was
measured by IFMA. The results obtained are presented in FIG. 4b.
The peptides did not either bind to PSA-ACT and PSA-API captured by
antibodies to ACT and API, respectively.
[0149] The binding kinetics and affinity of the peptides to PSA was
estimated by surface plasmon resonance. Increasing concentrations
of GST-peptides were injected over PSA captured by MAb H117.
Background-corrected sensorgrams were fitted to single-site
interaction between GST-peptide and PSA using three different
GST-peptide concentrations and the rate equation:
d(GST-peptide:PSA)/dt=ka(GST-peptide).times.(PSA)-kd(GST-peptide:PSA).
[0150] The average equilibrium dissociation constants (KD) for the
peptides A-1, B-2 and C-4 were 2.9-7.8 .mu.M (Table 2). Zn2+ at
concentrations of 1-30 .mu.M reduced both association and
dissociation rates. The affinity increased 3-7-fold because of a
greater decrease in kd than in ka (Table 3). Slow dissociation of
PSA from the capturing MAb caused a decreasing baseline and the
binding constants were corrected for the baseline drift. In gel
filtration, GST-C-4 revealed four peaks (Table 4, FIG. 5).
TABLE-US-00003 TABLE 3 Effect of Zn.sup.2+ on the binding kinetics
and affinity for the binding of GST-peptides to PSA A-1 B-2 C-4
Concentration ka .times. 10.sup.3 kd .times. 10.sup.-3 K.sub.D
increase ka .times. 10.sup.3 kd .times. 10.sup.-3 K.sub.D increase
ka .times. 10.sup.3 kd .times. 10.sup.-3 K.sub.D increase of zinc
(.mu.M) (l/Ms) (l/s) (.mu.M) (fold) (l/Ms) (l/s) (.mu.M) (fold)
(l/Ms) (l/s) (.mu.M) (fold) 0.sup..dagger. 10.3 80 7.8 -- 16.3 57
3.5 -- 9.9 28 2.9 -- [.+-.0.4] [.+-.2.6] [.+-.0.6] -- [.+-.3.5]
[.+-.0.8] [.+-.0.7] [.+-.1.0] [.+-.1.3] [.+-.0.2] 1* 1.8 4.7 2.6 3
1.06 3.5 3.1 -- 4.1 12 2.9 -- [.+-.0.01] [.+-.0.1] [.+-.0.1]
[.+-.0.03] [.+-.0.06] [.+-.0.3] [.+-.0.1] [.+-.0.1] [.+-.0.1] 10*
1.2 1.6 1.4 5.9 2.65 1.6 0.6 6 3.8 2.0 0.53 5.5 [.+-.0.01]
[.+-.0.01] [.+-.0.05] [.+-.0.03] [.+-.0.02] [.+-.0.02] [.+-.0.04]
[.+-.0.2] [.+-.0.05] 30* 1.9 2.0 1.0 7.7 0.83 0.6 0.8 4.7 4.6 4.0
0.86 3.4 [.+-.0.01] [.+-.0.02] [.+-.0.1] [.+-.0.02] [.+-.0.1]
[.+-.0.05] [.+-.0.2] [.+-.0.1] [.+-.0.1] The association rate
constat (ka), dissociation rate constant (kd) and equilibrium
dissociation constant (K.sub.D) for GST-peptides to PSA were
measured by surfact plasmon resonance. .sup..dagger.Evaluations are
based on average of 3 single fits between analyte concentrations
3.4-8.5 .mu.M. Values are given with the standard deviation.
*Evaluations are based on single fits at the given Zn.sup.2+
concentrations using 3.4 .mu.M peptide. Values are given with the
obtained standard errors in each fit.
TABLE-US-00004 TABLE 4 Comparison of the activities of different
molecular size forms of GST-C-4. MW Protein Binding activity Fusion
Protein (kD) (% of total) (% of total) GST-C-4 ~3 3.4 ~30 83.4 53
~100 11.4 26 >500 1.7 21 Thrombin- ~3 17 cleaved ~30 83 5.2*
GST-C-4 In gel filtration, GST-C-4 and thrombin-cleaved GST-C-4
revealed four and two peaks, respectively. The percentage of
protein in each peak was estimated by absorfance at 280 nm. The
binding activity ws calculated from the GST-peptide IFMA assay.
*Denotes binding activity remaining after thrombin-treatment.
[0151] After fractionation of GST-C-4 or thrombin-treated GST-C-4,
5 .mu.l of each fraction was first incubated in wells containing
PSA captured by MAb 5E4. The binding was measured by GST-peptide
IFMA. The arrows show the elution of molecular size standards (669
kD, 150 kD, 43 kD and 1.3 kD).
[0152] The molecular sizes of the peaks were >500 kD, 100 kD, 30
kD and about 3 kD. All of these forms except the 3 kD peak bound to
PSA (Table 4, FIG. 5). This indicates that the fusion protein
exists as a 30 kD monomer, an oligomer (about 100 kD) and a
polymer. The polymeric fusion protein showed the strongest binding
to PSA as indicated by the response in relation to the protein
absorbance at 280 nm. Thrombin treatment of GST-C-4 caused its
nearly complete cleavage into 30 kD and 3 kD components. The latter
consisted of the estimated 31-residue long peptide cleaved from the
GST fusion protein by thrombin. The 30 kD component had only about
5% of the response in the GST-peptide IFMA in comparison to
non-cleaved GST-C-4 (Table 4).
[0153] In inhibition experiment, the 3 kD peak reduced the binding
of GST-peptide to PSA by about 30% (FIG. 5) showing that the
thrombin cleaved peptide retained PSA binding activity. The cross
symbol (x) shows the inhibition of the binding of GST-peptide
induced by peptide cleaved form GST-peptide. 200 .mu.l of each
fraction containing free peptide was first incubated in wells
containing PSA captured by MAb 5E4. After 1 h, the wells were
emptied and GST-C-4 (167 nM) was added. After incubation for 1 h,
the binding of GST-peptide was measured by GST-peptide IFMA. Data
represent average values from duplicate wells.
[0154] To determine the binding site of the peptides on PSA,
inhibition experiments were performed with MAbs binding to various
epitopes on PSA. Antibodies binding to epitope regions, which in
complexed PSA are covered by the serine proteinase inhibitors (so
called free-specific antibodies) inhibited the binding of the
peptides most strongly (>80%). MAbs binding to epitopes distant
from the active site of PSA showed lower degree of inhibition
(Table 5).
TABLE-US-00005 TABLE 5 Inhibition of GST-peptide binding to PSA by
anti PSA MAbs. GST-Peptide ISOBM-MAb Epitope Group A-1 B-2 C-4 25 1
++ ++ ++ 26 1 ++ ++ ++ 40 2-a + + + 90 2-b + + + 57 3-a + + + 89
3-b + + + 86 5 + + + Control 6 - - - PSA-MAbs representing four
epitope groups on PSA were used to compete with the GST-peptides
for binding with PSA. Antibody from group 6, in which MAb 5E4
belongs, was used as a negative control. Each MAb (33.3 nM) was
incubated with PSA captured by 5E4 coated onto microtiter wells.
After washing, GST-Peptide was added. The binding of GST-peptides
was quantified by IFMA. Reduction of binding of GST-peptide to
solid phase PSA (%) ++ = >80% + = 60-80% - = No inhibition
[0155] The GST peptides showed significantly lower binding to
proPSA as compared to active PSA. In the experiments 100 ng of
proPSA or intact PSA was reacted with MAb 5E4 coated onto
microtitration wells. After washing, 1 .mu.g of each GST-peptide
(SEQ ID A-1 (SEQ ID NO 1), B-2 (SEQ ID NO 6) and C-4 (SEQ ID NO
11), Table 1) was added to wells containing immobilized PSA. The
binding of GST-peptide was quantified by IFMA using Eu-labelled
anti-GST antibody as tracer.
[0156] The reactivity with proPSA was 30-60% of that with active
PSA (FIG. 6). After studying 80 MAbs for the ability to
preferentially bind with either form of PSA, no MAb preferentially
recognizing proPSA or intact PSA could be found. Thus, the peptides
may provide novel binding specificities for PSA.
[0157] Peptides synthesized chemically by standard solid phase
Merrifield peptide synthesis inhibited efficiently the binding of
GST peptides to PSA (FIG. 7) which shows that they bind with PSA in
a similar manner as the GST-peptides. The chemically synthesized
peptide C-4 (SEQ ID NO 11) (GACVAYCIEHHCWTCGA) in 20-fold molar
excess inhibited the binding of the corresponding GST-peptide to
PSA by about 70%. Also a shorter derivative of C-4 lacking the
flanking GA-motif in the N- and C-termini of the peptide
efficiently inhibited the binding of GST-peptide with PSA (FIG. 7).
PSA had been captured by anti-PSA MAb in the microtitration wells.
The binding was measured by IFMA.
EXAMPLE 3
Effect of Peptides on the Enzyme Activity of PSA
[0158] The enzyme activity of PSA was studied in the presence of
peptides alone or together with Zn.sup.2+ by using the chymotrypsin
substrate S-2586 (MeO-Suc-Arg-Pro-Tyr-pNA) (Chromogenix, Molndal,
Sweden). Furthermore, the enzyme activity was studied in the
presence of peptides synthesized by standard solid phase Merrifield
peptide synthesis using fmoc-chemistry including a
biotin-conjugated form of the peptide. PSA (333 nM) was incubated
with a 1-100-fold molar excess of GST-peptide or 100-fold excess of
synthetic peptide in TBS buffer, pH 7.8 containing 0.5 g/L BSA for
1 h at 22.degree. C. The effect of Zn.sup.2+ on the enzyme activity
of PSA was studied by including 1-200 .mu.M of ZnCl2 in the
reaction buffer. The combined effect of peptides and Zn2+ on the
enzyme activity of PSA was studied by incubating PSA (333 nM) with
peptide (333 nM) in the buffer containing 1-200 .mu.M Zn.sup.2+ for
1 h. After addition of substrate to a final concentration 0.2 mM,
the absorbance was monitored at 5-min intervals for 2 h at 405 nm
on a Labsystems Multiskan MCC/340 photometer (Labsystems, Helsinki,
Finland). As a control, the effect of wild type GST on the enzyme
activity of PSA was tested.
[0159] The effect of the peptides on the enzyme activity of
chymotrypsin, cathepsin G and trypsin was studied as described
above for PSA. As substrates, S-2586 was used for chymotrypsin and
cathepsin G, and S-2222 (CO-Ile-Glu-(OR)-Gly-Arg-pNA) (Chromogenix)
for trypsin.
[0160] Effect of the peptides on the enzyme activity towards high
molecular weight protein substrates was studied by using PSA to
proteolytically cleave insulin like growth factor-binding protein 3
(IGF-BP-3) (32) alone or in the prescence of synthetic peptide C-4
and A1. The extent of the cleavage of the substrate was quantitated
by monitoring the decrease in IGF-BP-3 immunoreactivity due to
proteolytic cleavage of it. PSA (5 .mu.g/mL) was incubated with
IGF-BP-3 (70 ng/mL) for 16 h at 37.degree. C. with 100-fold molar
excess of peptides to PSA and measured for IGF-BP3 immunoreactivity
by IFMA as described (33). As control, the peptide was also added
to the reaction mixture containing PSA and IGF-BP3 just before
starting the immunoassay to quantify the possible interference of
the peptide in the IFMA for IGF-BP-3.
[0161] The effect of chemically synthesized peptide C-4 on the
enzyme activity of PSA in complex with alpha-2-macroglobulin (A2M)
was analyzed by using purified PSA-A2M complex prepared as
described (7). 1 .mu.g of PSA in complex with A2M was incubated
alone or with 2.5 .mu.g of peptide C4 (SEQ ID NO 11)
(GACVAYCIEHHCWTCGA) synthesized by standard solid phase Merrifield
synthesis for 1 h at 22.degree. C. in 100 .mu.L of TBS buffer
containing 1 g/L of BSA. 10 .mu.g of monoclonal antibody 4G10 was
included to inhibit the activity due to PSA released from the
complex. As a control, the enzyme activity of 1 .mu.g of free PSA
was measured. The enzyme activity was monitored as above by using
S-2586 (Chromogenix) as substrate.
[0162] The enzyme activity of PSA was significantly enhanced by the
GST-peptides and GST-C-4 was the most active one, stimulating PSA
activity against the chromogenic substrate about 5-fold (FIG. 8).
In the experiments, PSA (0.33 .mu.M) was incubated with increasing
concentrations of GST-C-4 (0-100-fold molar excess) for 1 h, after
which the chromogenic substrate S-2586 was added and enzyme
activity was monitored by measuring the absorbance at 405 nm. Data
represent mean values from duplicate wells.+-.SE.
[0163] The effect was dependent on the GST-peptide concentration
and half maximal stimulation was detected with concentrations in
the micromolar range (Table 2). The minimum peptide concentration
affecting the activity varied between 20 nM for C-4 and 300 nM for
A-1 and B-2. Wild type GST did not affect the activity of PSA (data
not shown). The peptides did not have any effect on the enzyme
activity of the other proteinases tested, including chymotrypsin,
cathepsin G and trypsin (data not shown). The peptides prepared by
standard peptide synthesis enhanced the enzyme activity in a
similar manner (FIG. 9). Furthermore, biotin conjugated peptide C-4
also enhanced the enzyme activity in the same way as the
non-conjugated form of C-4 (FIG. 9). In FIG. 9, the derivatives of
C-4 tested include peptides with or without the GA-flanking
residues in the N- and C-termini and a biotin conjugated form of
C-4. PSA (0.33 .mu.M) was incubated with chemically synthesized
peptides (100-fold molar excess) for 1 h, after which the
chromogenic substrate S-2586 was added and enzyme activity was
monitored by measuring the absorbance at 405 nm.
[0164] The effect of the peptides on the enzyme activity towards
high molecular weight protein substrates was assessed by studying
the ability of PSA to proteolytically cleave insulin like growth
factor-binding protein 3 (IGF-BP-3) alone or in the prescence of
synthetic peptides (SEQ ID C-4 (SEQ ID NO 11) and A-1 (SEQ ID NO
1), table 1). In the experiments, PSA (5 .mu.g/mL) in TBS buffer,
pH 7.8 alone or with 100-fold molar excess of peptide was incubated
with IGF-BP-3 (70 ng/mL) for 16 h at 37.degree. C., after which the
concentration of intact IGF-BP-3 was determined by IFMA. As
control, the peptide was also added to the reaction mixture
containing PSA and IGF-BP3 just before starting the immunoassay to
quantify the possible interference of the peptide in the IFMA for
IGF-BP-3 (labeled as BP-3+PSA+(C-4/A-1 for 0 h). PSA alone slowly
cleaved IGF-BP-3 as revealed by an 30% decrease in immunoreactivity
(FIG. 10). The peptides C-4 and A-1 enhanced the activity of PSA
towards IGF-BP-3 leading to about 70% cleavage of IGF-BP-3.
[0165] Because zinc is known to inhibit the enzyme activity of PSA,
we further characterized the effect of GST-peptides on the enzyme
activity of PSA by assessing the combined effect of peptides and
Zn.sup.2+. Zn.sup.2+ negated the enhancement effect of C-4 and
reduced PSA activity in a dose dependent fashion. The effect of
GST-C-4 and Zn.sup.2+ on enzyme activity was determined by
incubating PSA (0.33 .mu.M) with the same molar concentration of
GST-C-4 and various concentrations of Zn.sup.2+. The reaction was
monitored by measuring absorbance at 405 nm after addition of the
chromogenic substrate S-2586. The control shows the activity of PSA
in the absence of peptide and Zn.sup.2+. Data represent mean values
from duplicate wells.+-.SE. At a Zn.sup.2+-concentration of 75
.mu.M the activity was similar to that of PSA in the absence of
zinc and peptide, and at a concentration of 200 .mu.M, almost total
inhibition of enzyme activity was observed (FIG. 11).
[0166] The effect of the chemically synthesized peptide C-4 on the
enzyme activity of PSA in complex was analyzed by using purified
PSA-A2M complex. It has been shown with other proteinases that
after binding with A2M they still can cleave small molecular weight
substrates (34). 1 .mu.g of PSA in complex with A2M was incubated
alone or with 50-fold molar excess of chemically synthesized
peptide C-4 (table 1) for 1 h at 22.degree. C. in 100 .mu.L of TBS
buffer containing 1 g/L of BSA. 10 .mu.g of monoclonal antibody
4G10 was included to inhibit the activity due to PSA released from
the complex. As control, the enzyme activity of 1 .mu.g of free PSA
was measured. The enzyme activity was monitored by measuring
optical density at 405 nm with 0.2 mM S-2586
(MeO-Suc-Arg-Pro-Tyr-pNA) (Chromogenix, Molndal, Sweden) as
substrate. The enzyme activity of PSA in complex with A2M was
reduced about 4-fold compared to that of free PSA, but significant
activity could still be detected (FIG. 12). Thus, the active site
of PSA in complex with A2M is not blocked. The possibility that the
activity could be derived from PSA released from the complex was
ruled out by including a MAb which inhibits the activity of free
PSA completely (FIG. 12). After adding C-4 peptide the enzyme
activity of A2M-complexed PSA was enhanced about 2-fold showing
that the peptide could bind with PSA encapsulated by the inhibitor
and exert the same effect on A2M-complexed PSA as on free PSA (FIG.
12).
[0167] The present invention provides for the first time PSA
binding ligands which can specifically enhance the enzyme activity
of PSA. Several PSA-binding peptides were identified using random
phage-displayed peptide libraries by screening with PSA bound
through a monoclonal antibody. When PSA was initially coated
directly onto microtitration wells, no PSA-binding phage could be
isolated. However, by capturing PSA to a monoclonal solid phase
antibody specific PSA binding phage could be isolated. The
monoclonal antibody used for capturing PSA does not to block the
active site of PSA (27) and thus facilitated isolation of
PSA-binding phage. Apparently, direct coating of PSA onto plastic
changes its structure or causes an unfavorable orientation for
biopanning.
[0168] Some typical amino acid residues could be identified in most
of the selected peptides. The amino acid sequences CVF or CVA were
present in 5 of 12 peptides derived from the degenerate CX.sup.8C,
CX.sup.10C, and CX.sup.3CX.sup.4CX.sup.2C libraries, and tyrosine
was found in 12 of 14 peptides. The peptides with the highest
affinity contained four cysteines. The peptides were expressed and
characterized as GST fusion proteins, which facilitated studies on
the binding affinity and specificity. When expressed as fusion
proteins, the peptides retained their binding activity and the
relative affinities, as estimated by IFMA, were similar on phage
and fusion proteins. The surface plasmon resonance experiments
showed that the peptides bound to PSA with considerable affinity
(Table 2).
[0169] PSA has been shown to bind Zn.sup.2+, and the selected
peptides also contain sequences resembling Zn.sup.2+ binding sites
on zinc finger proteins (35). In the presence of Zn.sup.2+ the
affinity constants of the GST-peptides increased 3-7 fold,
suggesting involvement of Zn.sup.2+ in the binding between PSA and
peptides. The increase in affinity is explained by a stronger
decrease in the dissociation rate than in the association rate of
the complex between peptide and PSA. This may be mediated by
Zn.sup.2+ chelated between amino acid residues of PSA and the
peptide. Interestingly, Zn.sup.2+ has been shown to mediate a high
affinity binding between another serine proteinase, trypsin, and
its small molecule size inhibitor (36). Another possibility is that
Zn.sup.2+ stabilizes the 3-D structure of the peptide in a way
similar to that by which Zn.sup.2+ interacts with DNA-binding zinc
finger proteins.
[0170] PSA-binding peptides have been produced before by the
polysome selection method (11). These peptides are linear rather
than cyclic, and show no similarity with the peptides isolated in
the present study. Furthermore, these peptides were not shown to
effect enzyme activity of PSA and the when conjugated with biotin
the peptides did not show consistent binding with PSA. The affinity
of our peptides is fairly typical of phage display peptides (37)
but lower than those of peptides developed by the polysome
technique (11). However, the differences in affinity may be
accounted for by differences in measuring techniques.
[0171] The phage display peptides were selected against PSA
captured to an anti PSA MAb on the wall of a microtitration well.
The antibody used for capture enhances the enzyme activity of PSA,
apparently by affecting the conformation of the enzyme (30). This
could have contributed to the increase in peptide binding. However,
PSA in solution completely inhibited the binding of GST-peptides
with solid phase PSA. Thus the conformational change induced by the
capture antibody was not necessary for binding of GST-peptides.
[0172] When peptides were expressed as GST fusion proteins, three
molecular size forms were observed: polymer, oligomer and monomer.
The polymer displayed the strongest binding to PSA, and the
oligomer also bound more avidly than the monomer. This result shows
that multivalent binding enhances the binding avidity. Because
phage fUSE 5 expresses three to five copies of peptide inserts with
the same sequence its binding is also multivalent (38). In spite of
the apparent multivalent binding of GST-peptides, the monomeric
peptide cleaved from the GST fusion partner substantially inhibited
the binding of GST-peptide to PSA.
[0173] The peptides did not bind to chymotrypsin and cathepsin G,
although the enzyme specificity of these is similar to that of PSA.
They did not either bind to trypsin and hK2 which cleave C-terminal
to an arginine or lysine residues. HK2 is structurally closely
related to PSA showing 79% identity at the amino acid level. Thus
the peptides we have selected appear to be highly specific for PSA.
The GST-peptides did not either bind to PSA-serpin complexes
occurring in serum, i.e. PSA-ACT and PSA-API, in which the serpins
cover the peptide-binding region on PSA. Peptide-binding was also
blocked by the antibodies that react with free PSA through epitopes
covered in PSA-serpin complexes. These MAbs also inhibit enzyme
activity (30). Some MAbs binding to other epitopes on PSA could
also inhibit the binding, but to a lower degree. Taken together,
these results suggest that the peptides bind close to the active
site of PSA.
[0174] PSA has a restricted chymotrypsin-like enzyme activity
cleaving C-terminally to tyrosine and leucine residues on
semenogelin I, the natural substrate of PSA (39). However, PSA is
more dependent on the sequences surrounding these amino acids than
is chymotrypsin, and several residues surrounding the preferred P1
residues, tyrosine and leucine, play an important role in
determining the substrate specificity and efficiency (40). PSA can
also cleave after glutamine and this type of peptide substrate is
more specific for PSA than those containing tyrosine and leucine at
P1 (12). All the peptides selected by phage display contain
tyrosine or leucine residues, often in combination with other amino
acids (Y--S, Y-A, Y-D, L-V) forming cleavage sites in semenogelin I
(39). However, no peptide contained more than three amino acids
identical to a cleavage site in semenogelin I. All the peptides
contained either two or four cysteines. Thus they probably formed
tight loops, whereas the natural substrate, semenogelin I, does not
contain any disulfide bridge.
[0175] Three of the four GST-peptides studied enhanced the enzyme
activity of PSA against the synthetic peptide substrate S-2586, and
the effect correlated with the affinity. Therefore, the peptides do
not appear to interact with the catalytic triad of PSA, but rather
to bind in the vicinity of the active site changing the
conformation and possibly making the catalytic pocket more
accessible to the synthetic substrate. The effect on enzyme
activity was peptide-specific as wild type GST and GST-D-1 had no
effect. Zn.sup.2+, which inhibits the enzyme activity of PSA,
reversed the stimulating effect of the peptides in a dose dependent
manner.
EXAMPLE 4
Labeling and Use of PSA Binding Peptides in Affinity
Chromatography
Preparation of Labeled Peptide Conjugates
[0176] Peptides were synthesized using solid phase synthesis and
fmoc-chemistry. Biotin was attached in the amino terminus of the
peptides during solid phase synthesis. Labeling of the peptides
with the Eu-chelate was as described in Hemmila et al., 1984 (41).
50-100 .mu.g of peptides were labelled with 3-10-fold molar excess
of Eu-chelate. After Eu-labeling the peptides were purified with
reverse phase chromatography using NovaPak or Sep-Pak C18-column
(Waters, Mass., USA). The columns were equilibrated with 50 mM
triethyl-ammoniumacetate, pH 7 and eluted with acetonitrile
gradient. For 99m-technetium labeling the peptide was first reduced
with 0.2 M mercaptoethanol. After reduction, the peptide was
purified by a Sep-Pak C18 column equilibrated with 0.1 M phosphate
buffer at pH 7.5 and using acetonitrile for elution. Technetium
hydroxylmethylene diphosphonate (HDP)-method was used for peptide
labeling with 99 mTc. 20 .mu.g of the reduced C4-peptide was
labeled with 3 mCi of 99 mTc. After labeling the peptide was
purified by a Sep-Pak C18 column equilibrated with 0.1 M phosphate
buffer, pH 7.5 and using acetonitrile for elution. Peptides were
iodinated by the iodogen method (42).
[0177] As assayed by monitoring the effect of the peptides on the
enzyme activity of PSA, the synthetic peptides could be labeled
with biotin without reduction in PSA binding activity (FIG. 9). To
increase the Eu labeling efficiency of the peptides we added to B2
(CVFARNYDYLVC (SEQ ID NO:6)) and C4 (CVAYCIEHHCWTC (SEQ ID NO:11))
peptides a tail in the amino terminus consisting of serine and
lysine. The structure of the corresponding B2-peptide derivative is
SKSKSKS (SEQ ID NO:47)-amino caproic acid (aca)-CVFAHNYDYLVC (SEQ
ID NO:6) and C4-peptide derivative is SKSKSKS (SEQ ID
NO:47)-aca-CVAYCIEHHCWTC (SEQ ID NO:11). The structures of these
derivatized B2 and C4 peptides are also shown in FIGS. 13 and 14,
respectively.
[0178] FIG. 15 shows the effect of SKSKSKS (SEQ ID NO:47)-tailed
peptide C4 on the enzyme activity of PSA. PSA (0.33 .mu.M) was
incubated with non-tailed and SKSKSKS (SEQ ID NO:47)-tailed C4 (in
30-fold molar excess) for 1 h, after which the chromogenic
substrate S-2586 was added and enzyme activity was monitored by
measuring the absorbance at 405 nm. Data represent mean values from
duplicate wells.+-.SE. This result showed that the addition of the
tail did not affect the binding activity of the peptides.
[0179] After Eu-labeling the peptides were purified by C18
chromatography. FIG. 16 shows the fractionation of Eu-labelling
reaction mixture containing 50 .mu.g C4 peptide on C18 Sep-Pak
column. Acetonitrile (AcN) gradient was used for elution. Flow rate
was 0.5 mL/min and 1 mL fractions were collected. For comparison,
the elution curve of unlabelled C4 peptide in the same column is
shown. This result shows that the Eu-labelled peptide can be
separated from the unlabelled peptide by C18 chromatography.
[0180] FIG. 17 shows the effect of the fractions from the C18
chromatography (FIG. 16) on enzyme activity of PSA. PSA (0.33
.mu.M) was incubated with 100 .mu.L aliquots of the fractions
obtained by C18 chromatography of the Eu-labelling reaction mixture
of C4 (FIG. 16) for 1 h, after which the chromogenic substrate
S-2586 was added and enzyme activity was monitored by measuring the
absorbance at 405 nm. Fractions 26-28 contained the Eu-labelled
C4-peptide because they enhanced the enzyme activity of PSA
strongly (FIG. 17). As control the enzyme activity of PSA alone was
measured. The elution position of Eu-labelled C4 is also indicated
by arrows in FIG. 16. This result shows that Eu-chelate can be
attached to the peptides without affecting their PSA binding
activity.
[0181] FIG. 18 shows the effect of technetium labeling on the
binding activity of C4-peptide. PSA (0.33 .mu.M) was incubated with
Tc99m-labeled C4 (30-fold molar excess) for 1 h, after which the
chromogenic substrate S-2586 was added and enzyme activity was
monitored by measuring the absorbance at 405 nm. Data represent
mean values from duplicate wells.+-.SE. The effect of unlabelled C4
on PSA is shown for comparison. As control, the effect of linear C4
(reduced by mercaptoethanol) on PSA activity and the activity of
PSA alone are shown. This result showed that technetium labeling
between thiol-sulfurs of a C4-peptide reduced the binding by about
50%.
[0182] FIG. 19 shows the effect of iodination on the PSA binding
activity of C4 peptide. 20 .mu.g of the peptide was iodinated by
the Iodogen method. After iodination the peptide was purified by
using C18 Sep-Pak column. PSA (0.33 .mu.M) was incubated with
125I-labeled C4 (30-fold molar excess) for 1 h, after which the
chromogenic substrate S-2586 was added and enzyme activity was
monitored by measuring the absorbance at 405 nm. For comparison,
the effect of unlabelled C4 on PSA activity and the activity of PSA
alone are shown. This result shows that 125-iodination of the
C4-peptide (CVAYCIEHHCWTC (SEQ ID NO:11)) by the iodogen method
almost completely destroyed its binding activity. This suggests
that the single Tyr residue in this peptide is important for
binding.
[0183] These results show that various label groups can be coupled
to the PSA binding peptides without affecting their binding
activity, especially when the coupling is directed aminoterminus or
to a tail added to the original peptide sequence. After chelating
Tc99m between the thiol groups of the peptide the C4 peptide
retained about half of its activity.
PSA Binding Peptides in Affinity Chromatography
[0184] The peptide affinity gels were prepared by coupling the
lysine-serine-tailed derivatives of the peptides B2 and C4 (FIGS.
13 and 14) to activated CH-Sepharose 4B (Amersham-Pharmacia
Biotech) according to the instructions of the manufacturer. The
coupling was performed by using an equimolar concentration of the
peptide as compared to the concentration of the coupling sites in
Sepharose. For peptide affinity chromatography the columns were
equilibrated with Tris-buffered saline, pH 7.8 or with the same
buffer containing 100 .mu.M ZnCl2. After applying the sample the
column was washed with Tris-buffered saline, pH 7.8. The column was
eluted with a linear gradient decreasing pH using 10 mM
ammoniumacetate, pH 6.5 and 10 mM sodium acetate-buffer, pH 3.5, or
stepwise gradient using buffers with pH 6 and 4 for elution.
Binding of PSA to these gels was monitored by PSA immunoassay. The
identity of PSA eluted form the peptide affinity columns was
further evaluated by SDS-PAGE (43) and immunoblotting (44). In
immunoblotting experiments the proteins separated by SDS-PAGE were
transferred to PVDF membranes and immunoreacted with a polyclonal
anti-PSA antibody (Dako, Denmark).
[0185] FIG. 20 shows the fractionation of a seminal plasma sample
by B2 peptide affinity chromatography in the presence of 100 .mu.M
Zn.sup.2+ PSA was precipitated from seminal fluid by ammonium
sulfate, after which it was diluted in equilibration buffer and
applied into the column. After washing the column PSA was eluted
with gradient decreasing pH. The flow rate was 0.5 mL/min and
fractions of 1 mL were collected. About 80% of the applied PSA was
bound to the column and eluted when pH was decreased (FIG. 20).
[0186] In FIG. 21 it is shown the fractionation of seminal fluid as
above in the FIG. 20, except Zn.sup.2+ was omitted from the buffer.
When Zn.sup.2+ was omitted, nearly half of the seminal fluid PSA
applied bound to the column and after eluting with gradient
decreasing pH two peaks were recovered (FIG. 21). Also when
fractionating seminal fluid with C4-peptide-Sepharose in the
presence of Zn.sup.2+, about 80% of PSA bound to the column and
eluted as two peaks with stepwise gradient decreasing pH (FIG. 22).
If Zn was omitted, about 80% of applied PSA was unbound.
[0187] The identity of the PSA forms separated by the B2-peptide
affinity chromatography was evaluated by SDS-PAGE and
immunoblotting using polyclonal anti-PSA antibody. FIG. 23 shows
the immunoblot analysis after SDS-PAGE under non-reducing
conditions of the PSA containing fractions from the B2 peptide
affinity column (FIGS. 20 and 21). The samples were as follows:
lane 1: MW-marker, lane 2: unbound PSA; Zn.sup.2+ included
(fraction no 4 in FIG. 20), lane 3: unbound PSA; Zn.sup.2+ not
included (fraction no. 4 in FIG. 21), lane 5: bound PSA; Zn.sup.2+
included (fraction no 30 in FIG. 20), lane 6: bound PSA; Zn.sup.2+
not included (fraction no 30 in FIG. 21).
[0188] FIG. 24 shows the immunoblot analysis after SDS-PAGE under
reducing conditions of the PSA containing fractions from the B2
peptide affinity column (FIGS. 20 and 21). The samples were as
follows: lane 1: MW-marker, lane 2: unbound PSA; Zn.sup.2+ included
(fraction no. 4 in FIG. 20), lane 3: unbound PSA; Zn.sup.2+ not
included (fraction no 4 in FIG. 21), lane 5: bound PSA; Zn.sup.2+
included (fraction no. 30 in FIG. 20), lane 6: bound PSA; Zn.sup.2+
not included (fraction no. 30 in FIG. 21).
[0189] The immunoblotting analysis under non-reducing conditions of
the fractions from the B2-peptide column revealed band with MW of
about 30 kD both in unbound and bound fractions of PSA
immunoreactivity (FIG. 23). This corresponds to the size of free
PSA. However, immunoblotting after SDS-PAGE under reducing
conditions revealed in the fractions containing the unbound PSA
bands corresponding to molecular weights of about 10 and 20 kD
(FIG. 24). These correspond to the sizes of the fragments of PSA
derived from the proteolytically cleaved or nicked PSA. Some intact
PSA was also detected in the unbound fraction of PSA (FIG. 24). The
bound form of PSA only contained a 30 kD band under reducing
conditions showing that it consists only of intact PSA.
[0190] The SDS-PAGE analysis also showed that the peptide columns
can be used to purify PSA from seminal fluid. FIG. 25 shows
SDS-PAGE under reducing conditions of seminal fluid fractionated by
the B2 peptide column. The samples were as follows: lanes 1, 2 and
3: unbound PSA, lane 4: MW marker, lanes 5, 6 and 7: PSA eluted at
pH 6, 5 and 4, respectively, lane 8: seminal fluid. This result
shows that by fractionating the seminal fluid with PSA binding
peptide column most of the contaminating seminal fluid proteins
could be removed by the one step peptide affinity
chromatography.
[0191] These results show that these peptides can be used for
purification of PSA and for differentiation between various forms
of free PSA. Because these peptides possess novel binding
specificities towards PSA, i.e. they bind specifically with the
intact form of free PSA, they are potentially useful in development
of assays with improved accuracy for prostate cancer.
EXAMPLE 5
Conformational and Biochemical Analysis of the Cyclic Peptides
which Modulate Serine Protease Activity
Materials and Methods
[0192] Peptide synthesis: The peptides were synthesized using
PerSeptive 9050 Plus automated peptide synthesizer, with Fmoc
strategy, TBTU/DIPEA as the coupling reagent and NovaSyn TGA with
4-hydroxymethylphenoxyacetic acid linker as the solid phase
(Novabiochem, Laufelfingen, Switzerland). The side-chain protecting
groups used in synthesis were trityl (Trt) for Asn, Qln and H is,
O-tert-Butyl (OtBu) for Glu and Asp, t-Butyloxycarbonyl (Boc) for
Trp and tert-Butyl (tBu) for Ser, Thr and Tyr. For Cys both
Acetamidomethyl (Acm) and Trt protection groups were used. For
peptides A-1, B-2 and their modifications Cys(Acm) was used in the
Cys position 1: C(Acm)VFTSDYAFC(Trt) (SEQ ID NO: 1) and
C(Acm)VFAHNYDYLVC(Trt) (SEQ ID NO:6), for peptide C-4 and its
modifications Cys(acm) was used in positions 2 and 3:
C(Trt)VAYC(Acm)IEHHC(Acm)WTC(Trt) (SEQ ID NO: 1). During the
cleavage from the resin with 96% TFA, the Acm-protection group
remains in the Cys-side chain. For synthesis of head-to-tail cyclic
C-4 peptide with one cysteine bridge Cys(trt) and Fmoc-Glutamic
acid with .alpha.-allyl ester (Fmoc-Glu-Oall) was used.
Fmoc-Glu-Oall was first attached on the resin via the side chain
carbonyl group. The synthesis continued from Glu(7) to H is(8) (in
parent sequence) NH.sub.2--HHCWTVAYCIE (SEQ ID NO:46)-Oall. The
cyclisation occurred on the resin before cleavage.
[0193] The peptides were purified by HPLC (Shimadzu, Japan) with
C.sub.18 reverse phase column and acetonitrile (ACN) as eluent
(0.1% TFA in H.sub.2O/0-60% ACN gradient for 60 min) and verified
with MALDI-TOF mass spectrometer (Bruker, Germany) and the purity
was determined by analytical HPLC with 240.times.1.4 mm C18 column
0-60% ACN for 30 minutes.
Cyclisation of the peptides: Peptides with cysteins (Acm) were
cyclised by using Iodination method. Lyophilised peptide was
dissolved in 50% acetic acid (AcOH) with the concentration of 2
mg/ml. 1 M HCl (0.1 ml/mg of peptide) was added followed
immediately by 0.1 M iodine solution in 50% AcOH (5 eq./Acm).
Solution was stirred vigorously at room temperature for 30 to 40
minutes. Reaction was stopped with 0.1 M sodium thiosulphate. After
filtering (0.45 .mu.m) peptides were purified with HPLC as
described above. The formation of the sulphur bridges was verified
with MALDI-TOF mass spectrometer (Bruker analytic GMBH, Karlsruhe,
Germany).
[0194] Head-to-tail cyclization was started with the cleavage of
the Oall-group from the carbonyl group of Glu. Three equivalents of
palladium (Pd(PPh.sub.3).sub.4 were dissolved into
ChCl.sub.3-AcOH--N-methylmorpholine under argon. Palladium was
added on the resin and stirred for 2 hours at RT under argon. After
incubation the resin was washed with 0.5% diisopropyl-ethanolamine
DIPEA in DMF and 0.5% sodium diethylthiocarbamate in DMF to remove
the catalyst. After removal of the Allyl-group a head-to-tail
peptide bond was formed with equimolar concentrations of 0.6 M HTBU
and 0.9 M DIPEA in DMF after stirring 2 hours at RT. After coupling
the resin was washed with DMF, DCM and dried. After cleavage the
peptide was purified with HPLC and dissolved in 0.1 M
(NH.sub.4)HCO.sub.3. The sulphur bridge was formed by
air-oxidation. The formation of the peptide bond and the sulphur
bridge was verified with MALDI-TOF.
[0195] PSA activity measurements: The effect of different peptides
on the activity of PSA was studied by using chymotrypsin substrate
S-2586 (MeO-Suc-Arg-Pro-Tyr-pNA) (Chromogenix, Molndal, Sweden). 77
pmoles (2 .mu.g) of PSA, substrate and 10 pmoles of different
peptides were incubated in Tris-buffer (10 mM Tris and 150 mM NaCl
at pH 7.8) with 0.2 mM concentration of the substrate at room
temperature. PSA reaction without any peptide was used as basic
level control. Reaction was measured after 60 minutes incubation at
405 mm using Multiscan RC photometer (Labsystems, Finland). The
effect of the peptides was calculated as a ratio from the OD value
of PSA-peptide complex to PSA alone after 60 minutes
incubation.
[0196] NMR Spectroscopy: NMR samples were prepared by dissolving
purified and lyophilized peptides in 600 .mu.l DMSO-d.sub.6 to 5-10
mM. The pH of the samples were not tested. All spectra were
recorded at 300-320 K on a Bruker Advance 500 NMR spectrometer
(Bruker analytic GMBH, Karlsruhe, Germany) operating at a frequence
of 500 MHz for .sup.1H. All one-dimensional experiments were
recorded at five different temperatures in the range from 300 K to
320 K. The temperature coefficients (d.delta./dT) of the amide
protons were calculated by analysing the chemical shifts at these
five temperatures. All two-dimensional experiments were recorded
either at 305 K or 310 K, depending on the quality and clarity of
the spectra. All chemical shifts are reported with respect to the
DMSO peak at 2.50 ppm.
[0197] For all 2D experiments, standard pulse programs from the
Bruker software library were used. TOCSY spectra were recorded with
mixing times of 80 ms by means of MLEVTP (45) mixing sequence with
TPPI phase cycling. NOESY (48) spectra were mainly recorded with
mixing times of 400 ms for A-1, 420 ms for B-2 and 300 ms for C-4
using TPPI phase cycling. Various mixing times were tested and the
best were decided based on quality and clarity of spectra's.
NOE-build up curves were not determined. As well some COSY (c)
spectra were recorded with mixing times of 30 ms. The data sets
were processed with a phase-shifted sine bell functions. Typically
the data were recorded with a resolution of 1024 points for both
t.sub.1 and t.sub.2.
Structure calculations: Structure calculations were performed by
DYANA software (46). NOE-intensities were approximately calibrated
relative to the .beta.-protons of Tyr7 and Cys10 and aromatic ring
protons of Tyr7 in A-1, the aromatic ring protons of Tyr7 in B-2
and the aromatic ring protons of Trp11 in C-4. NOE correlations
were classified as either strong (1.8 to 2.7 .ANG.), medium (1.8 to
3.5 .ANG.) or weak (1.8 to 5.0 .ANG.). Some pseudo-atom corrections
of 1.5 .ANG. for methyl, 1.0 .ANG. for methylene protons and 2.0
.ANG. for tyrosine ring protons were added when needed (52).
Dihedral angles were not mainly restricted because of fluctuation
of 3-D structures (52).
Results
[0198] The most important data derived from NMR measurements, were
sequential and long range nuclear Overhauser effects,
C.sup..alpha.H conformational shifts and temperature coefficients
of amide protons. The sequential NH(i)/NH(i+1) NOE cross peaks are
always an evidence of a more or less turned conformation. It has
been suggested that the distance between amide protons has to be
less than 3 .ANG. before it is possible to observe NOE arising from
dipolar contact between them. The understanding is that this
dipolar contact primarily arises from right-handed
.alpha.-conformation. The existence of the observed strong
sequential C.sup..alpha.H(i)/NH(i+1) cross peaks were evidence of
.beta.-conformation. When they both exist in the same residue,
there was dynamical equilibration between two conformations.
Intensities of the sequential NH(i)/NH(i+I) and
C.sup..alpha.H(i)/NH(i+1) and intraresidual C.sup..alpha.H(i)/NH(i)
NOE-correlation's were indicative for the main event of backbone
structural rigidity or dynamic behaviour.
[0199] The use of a strong hydrogen-bond-accepting solvent, such as
DMSO-d.sub.6, usually results in a downfield shift of
solvent-exposed NH groups in peptides (50). Most of the NH chemical
shifts lie either at the same location as or downfield from the
random coil chemical shift. There were also significant upfield
shifting among NH chemical shifts. The solvent exposure of NH
groups were also detected by determining the temperature
coefficients of NH groups. Every peptide investigated had an NH
group or groups showing very low .DELTA..delta./.DELTA.T-values
(<3 ppm/K) characteristic for strong solvent shielding
(Raghothama et al. 1996). We also found a couple of moderate
.DELTA..delta./.DELTA.T-values (3-5 ppm/K) which needed to be
discussed. Chemical shifts and .DELTA..delta./.DELTA.T-values of NH
groups for each peptide are listed in Table 6. Part of the NH peaks
were broadened indicating solvent-exposed NH groups or
conformational fluctuation and dynamic behaviour (52, 50). Because
of a broadening of all .sup.3J.sub.NHC.alpha.H could not be
detected very well. Detected coupling constants were usually
between 6.0 and 8.0 Hz, revealing no more information about
structure.
[0200] Because of the low solubility in water, (especially C-4)
spectra were recorded in DMSO-d.sub.6 but significant H.sub.2O-peak
were found in every spectra. It interfered in particular with the
C.sup..alpha.H-peak area and C.sup..alpha.H(i)/C.sup..alpha.H(j)
cross-peaks were usually not found. Second main distraction was
t.sub.1-noise (Wuthrich, 1986, 52) which complicated especially
NH/NH cross peak intensity determination.
[0201] A more detailed approach for each of our peptide will be
discussed in the following sections. All following data were used
with NOE-data as conformational constraint in structure
calculations.
Conformation of the Peptides
A-1:
[0202] In all NMR-measurements of the A-1 mutated peptide, (A-1-4,
Table 7) were used because its even better biological activity. A
low temperature coefficient for amide protons of Tyr7 (3.4 ppb) and
especially Ala8 (1.8 ppb) were detected, proposing that they are
protected from the solvent (Table 6). The low 3.7 ppb/K
(d.delta./dT) (Table 6) of the amide proton of Tyr7 indicate the
existence of .beta.-turn. Tyr7 is a residue i+3 and Ser5 is in
position i+1 of type II .beta.-turn explaining why Ser5 has
C.sup..alpha.H chemical shift in the higher field in respect to
random coil values. Strong NH(i)/NH(i+1) between Asn6 and Tyr7 and
strong intraresidual C.sup..alpha.H(i)/NH(i) for Asn6 and Tyr7
indicates type II .beta.-turn. Conformation with turn residues 4-7
resembles structure of peptide B-2.
[0203] Strong C.sup..alpha.H(i)/NH(i+1) NOE-correlation's were
found between residues 2-6 indicating possible .beta.-type/extended
structure outside of turn area. Chemical shifts of C.sup..alpha.H
are pretty equal with random coil values (FIG. 1) between residues
2-4 and 8-10. Only between in residues Ser5, Asn6 and Tyr7 chemical
shifts of C.sup..alpha.H moved upfield supporting appearance of
.beta.-conformations in this part of peptide (49, 51). Medium
NH(i)/NH(i+1) correlation between residues Phe9 and Cys10 shows
evidence of .alpha.-conformation nearby disulphide bridge.
[0204] Relatively strong downfield shifting for NH was detected for
Val2 and Cys 10 indicating their solvent-exposing conformation.
Strongly upfield shifted amide protons were found for residues 4-9
indicating their solvent-shielding (Table 6).
B-2:
[0205] First of all, our interest was focused on the very low
temperature coefficient for amide proton of Tyr7 (0.7 ppm/K),
indicating its solvent-shielded structure. That revealed hydrogen
bonding between the amide proton of Tyr7 and usually a backbone
carbonyl group. In a classical S-turn, the temperature coefficient
of the fourth residue in the turn should be lowered due to hydrogen
bond formation with the carbonyl of the first residue in the turn.
Also were found moderate temperature coefficient to Tyr9 NH (4.0
ppm/K). Strong downfield shifting for NH were detected for Val2 and
Cys12 indicating their solvent-exposing conformation. Strongly
upfield shifted amide protons were Asn6, Tyr7 and Tyr9 indicating
their solvent-shielding (Table 6).
[0206] The strong NH(i)/NH(i+1) correlation's between residues
His5-Asn6 and weak between Asn6-Tyr7 were found. This data
appropriates to type I S-turn between residues 4-7 (52). Remarkable
strong sequential C.sup..alpha.H(i)/NH(i+1) correlation's between
residues 1-4 and 9-12 and only medium or weak between residues 6-9
were as well detected. This reveals .beta.-sheet type structure
outside of .beta.-turn area. Further, weak NOE-correlation's
between Ala4 C.sup..beta.H.sub.3-protons and Tyr7 and Tyr9 amide
protons indicates type I .beta.-turn (52). Chemical shifts of
C.sup..alpha.H are differs strongly to upfield from random coil
values (FIG. 26) between residues 5-8 indicating turn area
(49).
[0207] However, simultaneous strong or medium NH(i)/NH(i+1) and
strong C.sup..alpha.H(i)/NH(i+1) NOE-correlation's indicated the
existence of conformational changes. NH(i)/NH(i+1)-correlation's
were found between residues 7-8 and 8-9 (medium) and 9-10 and 11-12
(weak). Furthermore strong intraresidual C.sup..alpha.H(i)/NH(i)
correlation's indicates existence of .alpha.-conformation for Asp8
and Tyr9. Long range NOE-correlation's over peptide ring indicates
however structural similarities between conformations. Remarkable
NH(i)--NH(j) NOE-correlation's were found between Val2-Val11 and
Ala4-Tyr9.
C-4:
[0208] The C-4-peptide differs from A-1 and B-2 peptides because of
two disulphide bridges. Very low temperature coefficient for amide
proton of His9 were detected and relatively low for Tyr4, Trp11 and
Cys13. The amide proton of His9 is hydrogen bonded with carbonyl of
Ile6 indicating existence of .beta.-turn. The disulphide bridge
between Cys5 and Cys10 made this part of peptide more rigid. Strong
downfield shifting for NH were detected for Val2, Cys5, Ile6 and
Cys 10 and very strong for Glu7 and Trp11 indicating their
solvent-exposed conformation. Strongly upfield shifted amide
protons were Tyr4, His9 and Cys13 indicating their
solvent-shielding (Table 6).
[0209] Turn type between residues Ile6-His9 were identified as type
II .beta.-turn. Both strong NH(i)/NH(i+1) correlation between
His8-His9 and strong intraresidual C.sup..alpha.H(i) I N H(i)
correlation for His8 indicates type II turn. Very strong downfield
shift for Glu7 NH shows its solvent-exposed conformation, which is
intrinsic for type II turn. The NOE was found between Ile6 NH-Tyr9
NH indicating tight turn. Only conformational disagreement was weak
NH(i)/NH(i+1) correlation between Glu7-His8 indicating fluctuation
in turn area.
[0210] Chemical shifts of C.sup..alpha.H for Glu7 and His8 were
moved strongly upfield indicating turn area. Strong
C.sup..alpha.H(i) I N H(i+1) NOE-correlation's were found between
residues 1-6 and 9-13 indicated .beta.-conformation outside of
.beta.-turn area. Main disagreements were relatively strong
NH(i)/NH(i+1) correlation between Val2-Ala3 and weak NH(i)/NH(i+1)
correlation between Tyr4-Cys5, Ile6-Glu7 and Trp11-Thr12 indicating
dynamic behaviour of backbone.
[0211] Some remarkable medium and long range NOE-correlation's were
found. Remarkable well-defined NOE-cross peaks were detected
between C.sup..alpha.H protons of Ala3 and Thr12 and side chain
protons of Tyr4 and Ile6. Moderated temperature coefficients of
amide protons of Tyr4, Trp11 and Cys 13 are consequences of the
partial hydrogen bonding with carbonyl oxygen's of Trp11, Tyr4 and
Val2 respectively.
[0212] The NMR-data proved bridge formation between Cys1-Cys13 and
Cys5-Cys10. Strong NOE-cross peak were detected between
C.sup..alpha.H protons in residues Cys5 and Cys10. Furthermore, the
chemical shifts of C.sup..alpha.H protons in residues Cys5 and
Cys10 moved interestingly downfield (Table 6). The chemical shift
values can be explained by ring current effect of aromatic ring
nearby these protons (Wuthrich, 1986, 52). Same behaviour can be
detected for amide protons of Ile6 and Trp11. The aromatic ring
which induced these shifts is the indole-ring of Trp11. The NOE
were found between Ile6 HN-Trp11H5.
The Activity of Modified Peptides
[0213] The peptides with a PSA-peptide/PSA ratio above 1.1 was
interpreted to be active, below 1.1 as inactive (Table 7).
[0214] Following modifications did not affect to the activity of
peptides A-1, B-2 and C-4: The removal of the negative charge
D.fwdarw.N in peptides A-1-4 and B-2-2 and E.fwdarw.Q in C-4-3. In
the peptide A-1 the change F(9).fwdarw.Y(9) (A-1-2) and
A(8).fwdarw.N(8) (A-1-3) did not affect to the activity. In the
peptide B-2 the activity remained with the modification of
Y(9).fwdarw.A(9) (B-2-3). The sequences of A-1 and B-2 resemble
each other despite of difference in length. When B-2 was shortened
to 10 amino acids (B-2-4), the difference between the sequences is
A(4)H(5) in B-2-4 vs. T(4)S(5) in A-1-3. In peptide C-4 the peptide
with the modification of Y(4).fwdarw.F(4) (C-4-3) was active.
[0215] The following modifications were inactive: In the peptide
A-1 F(3).fwdarw.Y(3) and both in the peptides A-1 and B-2
modification Y(7).fwdarw.A(7). In the peptide C-4 modifications
Y(4).fwdarw.A(4) and W(1).fwdarw.A(1) were inactive. In addition 10
amino acids sequence C-4-4 with one Cysteine bond and C-4-6,
cyclised by using peptide bonding, were inactive.
CONCLUSIONS
[0216] The sequences and the amino acid positions of the three
peptides A-1, B-2 and C-4 are in Table 8. The two peptides A-1 and
B-2 have similar structure and the same biological activity. The
particularly important side chains in A-1 peptide are Phenylalanine
(Phe) in position 3 and Tyrosine (Tyr) in position 7. In the
peptide B-2 the corresponding amino acids are Phenylalanine (Phe)
in position 3 and Tyrosine (Tyr) in position 7.
[0217] In sequences A-1 and B-2 the rigid .beta.-turn stabilises
the position of aromatic side chains of Phenylalanine (Phe) in
position 3 and Tyrosine (Tyr) in position 7. The structure is
stabilised in the peptide A-1 by hydrogen bond between the carbonyl
oxygen of Threonine (Thr) in position 4 and the hydrogen of amide
of Tyrosine (Tyr) in the position 7. The structure is stabilised in
the peptide B-2 by the hydrogen bond between the carbonyl oxygen of
Alanine (Ala) in position 4 and the hydrogen of the amide of
Tyrosine (Tyr) in position 7.
[0218] The particularly important amino acids of the peptide C-4
are Tyrosine (Tyr) in position 4 and Tryptophan (Trp) in position
11. The structure is stabilised by the two disulphide bridges; the
first between the positions Cys I and Cys 13 and second between the
positions Cys5 and Cys10.
[0219] FIGS. 27a to 27c show the significant NOE-correlations of
peptides A-1, B-2 and C-4 in DMSO-d.sub.6. Relative cross-peak
intensities were estimated from volume integration. Sings and m
mean relatively strong and medium intensities of intraresidual
d.sub.H.alpha.NH(i,i). Sing * mean disturbed NOE-correlation by
some other NOE. NOEs indicated as "other" interaction include long
range main chain-main chain, side chain-main chain or side
chain-side chain interactions.
[0220] FIG. 28 shows a three-dimensional structure of the peptide
A-1, FIG. 29 shows a three-dimensional structure of the peptide B-2
and FIG. 30 shows a three-dimensional structure of the peptide C-4.
The structures have been calculated from the data given above and
in the following tables.
TABLE-US-00006 TABLE 6 .sup.1H chemical shifts and amide proton
temperature coefficients of PSA-binding peptides Peptide
-.DELTA..delta./.DELTA.T Residue NH C.sup..alpha.H C.sup..beta.H
C.sup..chi.H C.sup..delta.H Other (ppb/K) A-1 Cys1 4.20 2.98 Val2
8.52 4.23 1.88 0.81 6.1 Phe3 8.36 4.83 3.08, 2.88 7.18 7.13-7.23
(C.sup..epsilon.H, C.sup..zeta.H) 6.0 Thr4 8.02 4.38 4.18 1.04 7.4
Ser5 8.06 4.14 3.72, 3.65 4.7 Asn6 7.99 4.46 2.46, 2.39 6.93, 7.38
(N.sup..delta.H) 7.2 Tyr7 7.81 4.36 2.86, 2.67 6.94 6.62
(C.sup..epsilon.H) 3.7 Ala8 7.89 4.37 1.09 1.8 Phe9 8.00 4.60 3.06,
2.82 7.13-7.23 (C.sup..epsilon.H, C.sup..zeta.H) 7.0 Cys10 8.48
4.58 3.17, 2.96 7.5 B-2 Cys1 -- 4.39 3.03, 2.82 Val2 8.62 4.42 2.07
0.87, 0.81 4.7 Phe3 8.40 4.77 3.07, 2.73 7.25 7.21-7.24
(C.sup..epsilon.H, C.sup..zeta.H) 9.3 Ala4 8.02 4.25 1.16 7.6 His5
8.42 4.27 3.13, 3.02 7.27 (4H) 8.81 (C.sup..epsilon.H, 2H) 8.7 Asn6
7.99 4.26 2.59, 2.59 6.87, 7.44 (N.sup..delta.H) 5.0 Tyr7 7.85 4.13
2.86, 2.75 6.84 6.58 (C.sup..epsilon.H) 0.7 Asp8 8.16 4.53 2.68,
2.50 7.3 Tyr9 7.53 4.58 2.94, 2.72 6.94 6.57 (C.sup..epsilon.H) 4.0
Leu10 8.30 4.50 1.35, 1.35 1.33 0.55, 0.53 13.1 Val11 8.21 4.29
1.92 0.88, 0.87 10.8 Cys12 8.75 4.67 3.21, 2.89 10.3 C-4 Cys1 --
4.22 3.23 Val2 8.61 4.28 1.98 0.89, 0.88 6.7 Ala3 8.15 4.65 1.20
6.4 Tyr4 7.96 4.71 2.87, 2.66 6.89 6.53 (C.sup..epsilon.H) 4.5 Cys5
8.67 5.38 2.83, 2.72 9.9 Ile6 8.55 4.20 1.81 1.14, 1.55 0.86 6.5
0.92 (C.sup..chi.H.sub.3) Glu7 8.98 3.57 2.17, 1.95 2.28 8.5 His8
8.41 3.93 3.27 7.24 (4H) 8.74 (C.sup..epsilon.H, 2H) 5.9 His9 7.86
4.72 3.04, 2.87 7.18 (4H) 8.63 (C.sup..epsilon.H, 2H) 1.0 Cys10
8.60 5.27 2.74, 2.65 9.1 Trp11 8.58 4.76 3.09, 2.89 7.14 (2H) 7.65
(4H), 6.95 (5H), 4.3 6.98 (6H), 7.26 (7H), 10.62 (NH) Thr12 8.20
4.40 3.94 1.10 10.3 Cys13 7.97 4.50 3.17 4.0
TABLE-US-00007 TABLE 7 The activity of the peptides A-1, B-2, C-4
and their modifications. The peptides with PSA-peptide/PSA ratio
>1.1 is interpreted to be active. PSA-peptide/ Code Peptide
Length Sequence PSA ratio A-1-1 A-1 parent 10 CVFTSDYAFC (SEQ ID
NO: 1) 2.3 A-1-2 A-1 N6Y9 10 CVFTSNYAYC (SEQ ID NO: 32) 2.3 A-1-3
A-1 N6N8Y9 10 CVFTSNYNYC (SEQ ID NO: 33) 1.9 A-1-4 A-1 N6 10
CVFTSNYAFC (SEQ ID NO: 34) 2.4 A-1-5 A-1 Y39 10 CVYTSDYAYC (SEQ ID
NO: 35) 1.0 A-1-6 A1 N6Y7Y9 10 CVFTSNAAYC (SEQ ID NO: 36) 1.0 B-2-1
B-2 parent 12 CVFAHNYDYLVC (SEQ ID NO: 6) 3.0 B-2-2 B-2 N8 12
CVFAHNYNYLVC (SEQ ID NO: 37) 3.1 B-2-3 B2 N8A9 12 CVFAHNYNALVC (SEQ
ID NO: 38) 2.6 B-2-4 B-2 10 N8 10 CVFAHNYNYC (SEQ ID NO: 39) 1.9
B-2-5 B-2 A7 12 CVFAHNANYLVC (SEQ ID NO: 40) 1.1 C-4-1 C4 parent 13
CVAYCIEHHCWTC (SEQ ID NO: 11) 2.1 C-4-2 C-4 F4 13 CVAFCIEHHCWTC
(SEQ ID NO: 41) 2.3 C-4-3 C-4 Q7 13 CVAYCIQHHCWTC (SEQ ID NO: 42)
2.1 C-4-4 C-4 10A5Q7 10 CVAYAIQHHC (SEQ ID NO: 43) 1.1 C-4-5 C-4 A4
13 CVAACIEHHCWTC (SEQ ID NO: 44) 1.0 C-4-6 C-4 A11 13 CVAYCIEHHCATC
(SEQ ID NO: 45) 0.9 C-4-7 Cyclic withpeptide pond 11 ##STR00001##
0.8
TABLE-US-00008 TABLE 8 Synthetic peptides for NMR studies and their
amino acid positions POSITION 1 2 3 4 5 6 7 8 9 10 11 12 13 A-1
(SEQ C V F T S N Y A F C ID NO: 31) B-2 (SEQ C V F A H N Y D Y L V
C ID NO: 6) C-4 (SEQ C V A Y C I E H H C w T C ID NO: 11)
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Sequence CWU 1
1
47110PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 1Cys Val Phe Thr Ser Asp Tyr Ala Phe Cys1 5
10210PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 2Cys Val Ile Tyr Asp Gly Asn His Trp Cys1 5
10310PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 3Cys Ile Phe Glu Pro Asp Tyr Ser Tyr Cys1 5
10410PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 4Cys Val Phe Asp Asp Leu Tyr Ser Phe Cys1 5
10512PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 5Cys Thr Phe Ser Val Asp Tyr Lys Tyr Leu Met Cys1 5
10612PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 6Cys Val Phe Ala His Asn Tyr Asp Tyr Leu Val Cys1 5
10712PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 7Cys Arg Phe Asp Lys Glu Tyr Arg Thr Leu Val Cys1 5
10813PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 8Cys Val Ser Tyr Cys Leu Phe Glu Phe Cys Tyr Val Cys1 5
10913PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 9Cys Val Glu Tyr Cys Trp Glu Gly Ser Cys Tyr Val Cys1 5
101013PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 10Cys Val Ala Tyr Cys Glu Glu Trp Glu Cys Tyr Val Cys1 5
101113PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 11Cys Val Ala Tyr Cys Ile Glu His His Cys Trp Thr Cys1 5
101213PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 12Cys Val Ser Tyr Cys Asp Gly Leu Gln Cys Trp Met Cys1 5
101313PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 13Cys Leu Ser Thr Cys Ala Gln Ser Cys Arg Ile Ser Cys1 5
101413PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 14Cys Leu Leu Tyr Cys His Asp Ala Cys Trp Trp Val Cys1 5
101513PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 15Cys Val Thr Tyr Cys Tyr Gly Glu Val Cys Tyr Tyr Cys1 5
101613PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 16Cys Ala Ala Tyr Cys Val Ala Gly Leu Cys Tyr Gly Cys1 5
101713PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 17Cys Val Gln Tyr Cys Ile Gly Gly Asp Cys Trp Phe Cys1 5
101813PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 18Cys Val Val Tyr Cys Asp Ser Met Lys Cys Trp Thr Cys1 5
101913PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 19Cys Val Ala Tyr Cys Ile Ser Ser Leu Cys Tyr Tyr Cys1 5
102010PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 20Cys Val Trp Tyr Thr Gly Asn Thr Trp Cys1 5
102110PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 21Cys Val Phe Asp Ala Leu Tyr Thr Phe Cys1 5
102210PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 22Cys Val Ile Tyr Pro Gly Asn Val Trp Cys1 5
102310PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 23Cys Ile Phe Asp Gly Phe Trp Ile Leu Cys1 5
102410PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 24Cys Val Pro Tyr Leu Gly Leu Trp Leu Cys1 5
102512PRTArtificial SequenceProstate Specfiic Antigen Binding
Peptide 25Cys Met Phe Asp Pro Met Tyr Met Trp Met Thr Cys1 5
102621DNAArtificial SequenceSequencing Primier 26ccctcatagt
taagcgtaac g 212729DNAArtificial SequencePCR Primer 27aggctcgagg
atcctcggcc gacggggct 292827DNAArtificial SequencePCR Primer
28aggtctagaa ttcgccccag cggcccc 27295PRTArtificial SequenceProstate
Specific Antigen Binding Peptide 29Cys Val Ala Tyr Cys1
53017PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 30Gly Ala Cys Val Ala Tyr Cys Ile Glu His His Cys Trp Thr
Cys Gly1 5 10 15Ala3110PRTArtificial SequenceProstate Specific
Antigen Binding Peptide 31Cys Val Phe Thr Ser Asn Tyr Ala Phe Cys1
5 103210PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 32Cys Val Phe Thr Ser Asn Tyr Ala Tyr Cys1 5
103310PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 33Cys Val Phe Thr Ser Asn Tyr Asn Tyr Cys1 5
103410PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 34Cys Val Phe Thr Ser Asn Tyr Ala Phe Cys1 5
103510PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 35Cys Val Tyr Thr Ser Asp Tyr Ala Tyr Cys1 5
103610PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 36Cys Val Phe Thr Ser Asn Ala Ala Tyr Cys1 5
103712PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 37Cys Val Phe Ala His Asn Tyr Asn Tyr Leu Val Cys1 5
103812PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 38Cys Val Phe Ala His Asn Tyr Asn Ala Leu Val Cys1 5
103910PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 39Cys Val Phe Ala His Asn Tyr Asn Tyr Cys1 5
104012PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 40Cys Val Phe Ala His Asn Ala Asn Tyr Leu Val Cys1 5
104113PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 41Cys Val Ala Phe Cys Ile Glu His His Cys Trp Thr Cys1 5
104213PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 42Cys Val Ala Tyr Cys Ile Gln His His Cys Trp Thr Cys1 5
104310PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 43Cys Val Ala Tyr Ala Ile Gln His His Cys1 5
104413PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 44Cys Val Ala Ala Cys Ile Glu His His Cys Trp Thr Cys1 5
104513PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 45Cys Val Ala Tyr Cys Ile Glu His His Cys Ala Thr Cys1 5
104611PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 46His His Cys Trp Thr Val Ala Tyr Cys Ile Glu1 5
10477PRTArtificial SequenceProstate Specific Antigen Binding
Peptide 47Ser Lys Ser Lys Ser Lys Ser1 5
* * * * *