U.S. patent application number 10/461493 was filed with the patent office on 2003-10-23 for antigenic peptides reacting with anti-ovary antibodies.
This patent application is currently assigned to BIO MERIEUX. Invention is credited to Bene, Marie-Christine, Dalbon, Pascal, Faure, Gilbert G., Jolivet, Michel, Jolivet-Reynaud, Colette.
Application Number | 20030199452 10/461493 |
Document ID | / |
Family ID | 9499441 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030199452 |
Kind Code |
A1 |
Jolivet-Reynaud, Colette ;
et al. |
October 23, 2003 |
Antigenic peptides reacting with anti-ovary antibodies
Abstract
An oligopeptides comprising the following sequence Thr Gln Cys
His Cys Gly Lys Cys (SEQ ID NO: 5) may be used as an antigenic
compound capable of being recognized by anti-ovary antibodies and
as an immunogenic compound capable of inducing the production of
anti-ovary antibodies. In addition, the oligopeptides may be used
to detect and/or quantify anti-ovary antibodies.
Inventors: |
Jolivet-Reynaud, Colette;
(Bron, FR) ; Faure, Gilbert G.; (Nancy, FR)
; Dalbon, Pascal; (Lyon, FR) ; Jolivet,
Michel; (Bron, FR) ; Bene, Marie-Christine;
(Nancy, FR) |
Correspondence
Address: |
OLIFF & BERRIDGE, PLC
P.O. BOX 19928
ALEXANDRIA
VA
22320
US
|
Assignee: |
BIO MERIEUX
MARCY L'ETOILE
FR
|
Family ID: |
9499441 |
Appl. No.: |
10/461493 |
Filed: |
June 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10461493 |
Jun 16, 2003 |
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09331872 |
Sep 24, 1999 |
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6602853 |
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09331872 |
Sep 24, 1999 |
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PCT/IB97/01611 |
Dec 31, 1997 |
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Current U.S.
Class: |
424/192.1 ;
435/7.2; 514/9.9; 530/326 |
Current CPC
Class: |
G01N 2800/36 20130101;
C07K 14/59 20130101; A61K 38/00 20130101; C07K 16/18 20130101; G01N
33/689 20130101 |
Class at
Publication: |
514/13 ; 530/326;
435/7.2 |
International
Class: |
C07K 014/59; G01N
033/53; G01N 033/567; A61K 038/24 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 31, 1996 |
FR |
96 16385 |
Claims
What is claimed is:
1. An oligopeptide consisting of: a sequence selected from the
group consisting of a reference peptide sequence SEQ ID NO: 5, SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or any
peptide sequence that is a functional variant of said reference
sequence SEQ ID NO: 5 in that it displays an antigenicity and/or an
immunogenicity toward anti-ovary antibodies identical or similar to
the antigenicity and/or the immunogenicity of the reference
peptide, wherein the functional variant differs from said reference
sequence by modification of the hydrocarbon residue of at least one
amino acid of said reference sequence.
2. An antigenic compound capable of being recognized by anti-ovary
antibodies, having a peptide sequence including the sequence of the
oligopeptide according to claim 1.
3. An immunogenic compound capable of inducing the production of
anti-ovary antibodies, having a peptide sequence including the
sequence of the oligopeptide according to claim 1.
4. A process for detection and/or quantification of anti-ovary
antibodies in a biological sample, comprising: placing said
biological sample in contact with at least one oligopeptide
comprising a reference peptide sequence SEQ ID NO: 5, or any
peptide sequence that is a functional variant of said reference
sequence in that it displays an antigenicity and/or an
immunogenicity toward anti-ovary antibodies identical or similar to
the antigenicity and/or the immunogenicity of the reference
peptide, and observing the formation of an antigen-antibody complex
between said oligopeptide and an anti-ovary antibody.
5. The process according to claim 4, wherein the functional variant
differs from said reference sequence by modification of the
hydrocarbon residue of at least one amino acid of said reference
sequence.
6. The process according to claim 5, wherein the modification
substantially conserves the hydrophilic or hydrophobic character of
the hydrocarbon residue of the at least one amino acid.
7. The process according to claim 4, wherein the oligopeptide
comprises a sequence selected from the group consisting of SEQ ID
NO: 1 to SEQ ID NO: 5.
8. The process according to claim 7, wherein the oligopeptide
consists of a sequence selected from the group consisting of SEQ ID
NO: 1 to SEQ ID NO: 5.
9. The process according to claim 4, wherein the biological sample
is selected from the group consisting of blood serum, follicular
liquid, peritoneal liquid, cervical mucus and saliva.
10. The process according to claim 4, wherein the formation of an
antigen-antibody complex is observed by an immunoenzymatic
technique.
11. The process according to claim 4, wherein said oligopeptide
consists of FSH, a functional variant of FSH or the .beta.-chain of
FSH.
12. The process according to claim 4, wherein said oligopeptide
comprises a peptide sequence corresponding to the sequence of amino
acids 78-93 of the .beta.-chain of FSH.
13. A process for inducing the production of anti-ovary antibodies,
comprising administering an immunogenic compound according to claim
3 to a patient.
14. The process according to claim 13, wherein the functional
variant differs from said reference sequence by modification of the
hydrocarbon residue of at least one amino acid of said reference
sequence.
15. The process according to claim 14, wherein the modification
substantially conserves the hydrophilic or hydrophobic character of
the hydrocarbon residue of the at least one amino acid.
16. The process according to claim 13, wherein the immunogenic
compound comprises a sequence selected from the group consisting of
SEQ ID NO: 1 to SEQ ID NO: 5.
17. The process according to claim 13, wherein the immunogenic
compound consists of FSH, a functional variant of FSH or the
.beta.-chain of FSH.
18. The process according to claim 13, wherein said immunogenic
compound includes a peptide sequence corresponding to the sequence
of amino acids 78-93 of the .beta.-chain of FSH.
19. A reagent for detection and/or quantification of anti-ovary
antibodies containing: (a) at least one oligopeptide comprising a
reference peptide sequence SEQ ID NO: 5, or any peptide sequence
that is a functional variant of said reference sequence in that it
displays an antigenicity and/or an immunogenicity toward anti-ovary
antibodies identical or similar to the antigenicity and/or the
immunogenicity of the reference peptide, and (b) a solid
support.
20. The reagent according to claim 19, wherein the functional
variant differs from said reference sequence by modification of the
hydrocarbon residue of at least one amino acid of said reference
sequence.
Description
[0001] The present invention concerns immunology and its direct
impact in endocrinology, both in man and in animals. More
particularly, this impact concerns the reproductive system. It is
in fact through pathologies such as repeated spontaneous abortions,
antiphospholipid antibody syndrome and preeclampsia that dysimmune
mechanisms have been observed in gynaecology and obstetrics.
[0002] The development of in vitro fertilization (IVF) techniques
has greatly contributed to improving understanding of the
mechanisms of human fertilization. However, this technique
sometimes encounters unexpected failures. Involvement of
unfavorable immune reactions generated by the technique itself has
been envisaged, on account of the considerable traumas inflicted on
the ovary in the course of the hormonal stimulation and follicular
punctures, which can liberate substantial quantities of potentially
antigenic material into the peritoneal cavity.
[0003] Previous studies have revealed antigenic components in the
thecae, constituents of the ovary, and the presence of serum
antibodies directed against the cells of the ovarian thecae or
against the corpus luteum has been reported in different
pathological contexts (adrenal insufficiency, ovarian
insufficiency, to a greater or lesser extent associated with
polyendocrinopathy) (6). Furthermore, SHIVERS et al. (1) have shown
that antibodies produced against ovarian antibodies, called
anti-ovary antibodies (AOA) inhibited in vitro fertilization in the
hamster.
[0004] The publication by SAXENA and RATHNAM ("Chemical synthetesis
[sic] of peptide fragments of the hormone-specific beta-subunit of
human follicule-[sic]-stimulating hormone", Biochemistry, Vol.24,
No.3, 1985, pp 813-816) discloses the chemical synthesis of
peptides in order to determine the specific antigenic determinants
of the human FSH hormone. These are the peptides V3+1/2C2
(corresponding to the amino acids 76-118 of the .beta.-subunit of
FSH) and V1+C1 (corresponding to the amino acids 1-33 of the
.beta.-subunit of FSH) which are presented as being the antigenic
determinants of the hormone FSH which are reactive toward
antibodies to the hormones LH and TSH, which proves a homology of
the antigenic sites for the three hormones FSH, LH and TSH.
[0005] The publication by SANTA-COLOMA et al. ("Serine analogues of
hFSH-beta-(33-53) and hFSH-beta-(81-95) inhibit hFSH binding to
receptor", Biochemical and Biophysical Research Communications,
Vol.184, No.3, 15 May 1992, pp 1273-1279) discloses synthetic
peptides corresponding to the amino acids 33-53 and 81-95 of the
.beta.-subunit of FSH, containing free sulfydryl groups, which make
it possible to inhibit the binding of the hormone FSH to its
receptor and which also display a partial agonist activity on the
said FSH receptor. It is stated that the 4 cysteine residues are
not essential to the binding to the receptor but are important for
the partial agonist activity of these peptides on the receptor.
[0006] The publication by TANG et al. ("Premature ovarian failure:
a search for circulating factors against gonadotrophin receptors,
American Journal of Obstretics [sic] and Gynecology, Vol.146, No.7,
1 August 1983, pp 813-821) presents a study performed on nine
patients displaying early menopause. This study suggests that
factors active against the gonadotrophin receptor seem not to be
involved in the majority of patients displaying premature failure
of the ovaries. Instead, a factor active against the FSH receptor,
which may be an antibody, is suggested as being possibly present in
patients displaying early menopause and auto-immunity.
[0007] The inventors of the present invention have surprisingly
discovered that a well defined peptide sequence, which corresponds
in its primary structure to a part of the .beta.-subunit of FSH,
displayed an antigenic and immunogenic character toward anti-ovary
antibodies such as previously defined.
[0008] In the course of previous studies, the authors of the
present invention demonstrated the existence of a correlation
between the level of anti-ovary antibodies observed and the
failures of IVF attempts (2).
[0009] The authors then became interested in the antigens
potentially involved in the generation of these auto-immune
responses, and firstly isolated and characterized an immunodominant
peptide reacting with the anti-ovary antibodies, then demonstrated
the value of this peptide in the detection of AOAs.
[0010] The importance of this detection has been demonstrated, in
different pathologies resulting in sterility, by the authors of the
present invention.
[0011] In examining the AOA levels before and after follicular
puncture, they observed, in women undergoing a first IVF attempt, a
level of AOA higher than the level in control women, even when for
these women an IVF protocol had been recommended for different
types of sterility, namely sterility of tubal origin, of idiopathic
origin, resulting from endometriosis or linked with severe
ovulation disorders.
[0012] The detection of anti-ovary antibodies in a sterility
pathology, after one or several vain IVF attempts, can become
decisive to the policy to be followed concerning these failures.
The authors have in fact demonstrated the efficacy of
corticotherapy on the favorable outcome of an IVF attempt (3).
[0013] The study of ovarian auto-immunity is nowadays essentially
addressed using animal tissue substrates or animal ovarian extracts
(4). The ovarian extract used by MONCAYO et al. was of animal,
namely bovine, origin. It has not been demonstrated that the
antigens recognized were actually the same in the human species and
in cattle.
[0014] According to the present invention, an antigenic peptide
capable of replacing ovarian extracts, and follicle stimulating
hormone (FSH or gonadotrophin A) which remains a product difficult
to purify, and only on the market in recombinant forms, is
provided.
[0015] The immunochemical studies performed by the authors have
thus made it possible to detect an oligopeptide immunodominant
toward the AOAs, belonging to the .beta.-chain of FSH.
[0016] Thus, the first object of the invention is an oligopeptide
consisting of the reference peptide sequence Thr Gln Cys His Cys
Gly Lys Cys (SEQ ID NO: 5), or any peptide sequence, functional
variant of the said reference sequence, in the sense that the first
displays an antigenicity, and/or an immunogenicity toward
anti-ovary antibodies equivalent to the antigenicity and/or the
immunogenicity of the second.
[0017] A functional variant of the said reference sequence is
advantageously obtained by modifying the hydrocarbon residue of at
least one of the amino acids of the said reference sequence, while
conserving an antigenicity identical or similar to that of the said
reference sequence, or else by modifying the hydrocarbon residue of
at least one of the amino acids of the said reference sequence,
while substantially retaining the hydrophilic/hydrophobic character
of the said hydrocarbon residue.
[0018] By equivalent activity is understood an identical or similar
antigenic and/or immunogenic activity, even if differences of
degree may exist in the antigenic and/or immunogenic responses.
[0019] An oligopeptide of the invention can thus consist of a
peptide sequence corresponding to the formula:
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8,
[0020] in which formula:
[0021] Xaa1 represents threonine,
[0022] Xaa2 represents glutamine,
[0023] Xaa3 represents cysteine,
[0024] Xaa4 represents histidine,
[0025] Xaa5 represents cysteine,
[0026] Xaa6 represents glycine,
[0027] Xaa7 represents lysine, and
[0028] Xaa8 represents cysteine,
[0029] the said sequence being identified by SEQ ID NO: 5, or a
peptide sequence which is a functional variant of SEQ ID NO: 5.
[0030] Preferably, the said functional variant sequence corresponds
to the formula
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8,
[0031] in which formula:
[0032] Xaa1 represents threonine or the unit NH.sub.2--CH(R1)COOH
where R1 is an oxygenated hydrocarbon radical conferring on the
said variant sequence an antigenicity identical or similar to that
of SEQ ID NO: 5,
[0033] Xaa2 represents glutamine or the unit NH.sub.2--CH(R2)COOH
where R2 is an oxygenated and/or aminated hydrocarbon radical
conferring on the said variant sequence an antigenicity identical
or similar to that of SEQ ID NO: 5,
[0034] Xaa3 represents cysteine or the unit NH.sub.2--CH(R3)COOH
where R3 is a sulfurated hydrocarbon radical conferring on the said
variant sequence an antigenicity identical or similar to that of
SEQ ID NO: 5,
[0035] Xaa4 represents histidine or the unit NH.sub.2-CH(R4)COOH
where R4 is an aminated hydrocarbon radical conferring on the said
variant sequence an antigenicity identical or similar to that of
SEQ ID NO: 5,
[0036] Xaa5 represents cysteine or the unit NH.sub.2--CH(R5)COOH
where R5 is a sulfurated hydrocarbon radical conferring on the said
variant sequence an antigenicity identical or similar to that of
SEQ ID NO: 5,
[0037] Xaa6 represents glycine or the unit NH.sub.2--CH(R6)COOH
where R6 is a hydrocarbon radical conferring on the said variant
sequence an antigenicity identical or similar to that of SEQ ID NO:
5,
[0038] Xaa7 represents lysine or the unit NH.sub.2--CH(R7)COOH
where R7 is an aminated hydrocarbon radical conferring on the said
variant sequence an antigenicity identical or similar to that of
SEQ ID NO: 5,
[0039] Xaa8 represents cysteine or the unit NH.sub.2--CH(R8)COOH
where R8 is a sulfurated hydrocarbon radical conferring on the said
variant sequence an antigenicity identical or similar to that of
SEQ ID NO: 5.
[0040] Advantageously, the constituent radicals R1 and/or R2 and/or
R3 and/or R4 and/or R5 and/or R6 and/or R7 and/or R8 of the said
units are respectively selected among radicals conferring on the
said units a hydrophilicity/hydrophobicity identical or similar to
that of the respectively corresponding amino acids.
[0041] It is evident from the general knowledge of the person
skilled in immunology and peptide synthesis that the
hydrophilic/hydrophobic character of the constituent units of
proteins/peptides is of importance to the antigenic power of the
latter. Moreover, many works on general biochemistry (5) have
classified the 20 amino acids on the basis of their greater or
lesser hydrophobic/hydrophilic character. According to the present
invention, it is considered that the selection of the radicals R1
to R8 in the above formula can be made by the aforesaid skilled
person with the aid of the said works, and that he in addition has
routine experimental protocols available for determining the
hydrophilic/hydrophobic character of the constituent unit that he
has selected.
[0042] Of course, other physicochemical parameters of the amino
acids can be used to determine variant sequences, such as for
example the pKa constant.
[0043] As functional variants of the peptide sequence SEQ ID No.5,
the oligopeptide consisting of the peptide sequence SEQ ID No.1 for
example, may be cited. Example 2 below in fact demonstrates
identical activity for the oligopeptides SEQ ID No.1 and 5. The
oligopeptides SEQ ID No.2, 3 and 4, having weaker activity than the
oligopeptides cited above, are considered in the present invention
as functional variants. In fact, inasmuch as their antigenic or
immunogenic activity toward anti-ovary antibodies is weaker, but
useful all the same, for example mixed with other oligopeptides of
the same activity, in a diagnostic kit, these oligopeptides are
functional variants of SEQ ID No.5.
[0044] The oligopeptides described at the end of the specification
are:
[0045] SEQ ID No.1: linear sequence of 16 amino acids corresponding
to the sequence of amino acids 78-93 of the .beta.-chain of
FSH,
[0046] SEQ ID No.2: sequence of 16 amino acids, bridged by a
disulfide bond in position 5 and 7, corresponding to the sequence
of amino acids 78-93 of the .beta.-chain of FSH,
[0047] SEQ ID No.3: sequence of 16 amino acids, bridged by a
disulfide bond in position 5 and 10, corresponding to the sequence
of amino acids 78-93 of the .beta.-chain of FSH,
[0048] SEQ ID No.4: sequence of 16 amino acids, bridged by a
disulfide bond in position 7 and 10, corresponding to the sequence
of amino acids 78-93 of the .beta.-chain of FSH,
[0049] SEQ ID No.5: linear sequence of 8 amino acids corresponding
to the sequence of amino acids 80-87 of the .beta.-chain of
FSH.
[0050] The surprising feature of the invention is that the
oligopeptides of the invention, such as those represented by SEQ ID
No.1 and 5, the primary amino acid structure of which corresponds
respectively to the amino acids 78-93 and 80-87 of the .beta.-chain
of FSH, although probably having a three-dimensional structure
different from that of the .beta.-chain of FSH, display an
antigenic and/or immunogenic activity toward anti-ovary
antibodies.
[0051] In a preferred mode of implementation according to the
invention, the oligopeptide has the peptide sequence identified by
SEQ ID No.5.
[0052] In another preferred mode of implementation according to the
invention, the oligopeptide has the peptide sequence identified by
SEQ ID No.1.
[0053] In yet another mode of implementation according to the
invention, the oligopeptide has the peptide sequence identified by
SEQ ID No.2 or 3 or 4, as identified at the end of the
specification.
[0054] A second object of the present invention is an antigenic
compound capable of being recognized by the anti-ovary antibodies,
or an immunogenic compound capable of inducing the production of
anti-ovary antibodies, the said compound corresponding to any one
at least of the following definitions:
[0055] a compound having a peptide sequence including the sequence
of the oligopeptide described above, and preferably including the
sequence SEQ ID No.5; in particular, such a compound has a peptide
sequence which includes or which consists of SEQ ID No.1, as
identified at the end of the specification;
[0056] a compound consisting of FSH or a functional variant of
FSH;
[0057] a compound consisting of the .beta.-chain of FSH;
[0058] a compound whose antigenicity or immunogenicity is
representative of the anti-genicity or immunogenicity of the crude
ovarian antigens, the primary structure of the said compound
including a peptide sequence corresponding to the sequence of amino
acids 78-93 of the .beta.-chain of the FSH of the human race or of
any animal species, the said sequence being immunodominant and
being recognized by the anti-ovary antibodies of the said human
race or of the said species.
[0059] Another object of the invention is a composition antigenic
or immunogenic toward anti-ovary antibodies, including an antigenic
or immunogenic compound such as defined above.
[0060] One application of an oligopeptide or of a compound of the
invention lies in the detection and/or quantification of anti-ovary
antibodies. Thus a third object of the invention is a process for
detection and/or quantification of anti-ovary antibodies in a
biological sample, including the stage consisting in placing the
said biological sample in contact with at least one peptide or one
compound of the invention, and in observing the formation of an
antigen-antibody complex between the said peptide or compound and
an anti-ovary antibody.
[0061] The formation of the aforesaid complex can be detected by
different classical bioassay formats, known to the skilled person,
such as immunocytochemical and immunoenzymatic (ELISA) techniques,
competition techniques and "sandwich" techniques.
[0062] In accordance with this process, the biological sample is
advantageously selected among the blood serum, the follicular
liquid, the peritoneal liquid, the cervical mucus and the
saliva.
[0063] The stage of observation of the formation of an
antigen-antibody complex is preferably effected by the
immunoenzymatic technique called ELISA, with which it has been
possible to obtain results perfectly correlated those [sic]
obtained by the reference technique for the skilled person, namely
indirect immunofluorescence.
[0064] Finally, the last objects of the invention are firstly a
reagent for detection and/or quantification of anti-ovary
antibodies including at least one peptide or one compound of the
invention, and secondly a kit for detection and/or quantification
of anti-ovary antibodies including at least the said reagent.
[0065] Before presenting the benefits of the invention and
describing it in more detail, certain terms used in the present
text are defined below.
[0066] A peptide sequence, functional variant of the reference
peptide sequence (SEQ ID NO: 5) is a sequence of amino acids,
called modified, differing from the sequence (SEQ ID NO: 5) by
modifications which confer on the said functional variant sequence
an antigenicity identical or similar to that of the reference
sequence. The modifications of the said sequence can concern its
size, and each of the amino acids that constitute it. Such
modifications are in particular selected among substitutions,
deletions and additions of amino acids in the sequence of the first
oligopeptide, but also among replacement of an amino acid of the L
series by an amino acid of the D series, modification of the
side-chains of the amino acids, modification of the peptide bonds
such as carba, retro, inverso, retro-inverso, reduced or
methylene-oxy bonds, subject to the condition that these
modifications do not result in any significant alteration of the
antigenic properties of the said sequence toward anti-ovary
antibodies.
[0067] As an example of substitution, and as previously mentioned,
an amino acid of SEQ ID No: 5 can be replaced by another amino acid
of the same hydrophilicity/hydrophobicity or of comparable
hydrophilicity/hydrophobicity.
[0068] The competent skilled person, who routinely utilizes the
techniques at his disposal, in particular the techniques of
automated peptide synthesis and the techniques of determination of
the antigenicity of a peptide such as ELISA tests, is capable, in a
first stage, of synthesizing a modified sequence such as defined
above, and, in a second stage, of testing the antigenicity of the
peptide obtained. If the latter displays an antigenicity at least
substantially equivalent to that of the said reference sequence, it
corresponds to the definition of functional variant sequence of the
said reference sequence. The skilled person can in particular refer
to the examples 2 and 3 below, which describe a protocol to be
followed for comparing the antigenicity of two peptides toward
AOAs.
[0069] A compound of the invention includes a sequence of a
variable number of amino acids, including at least the sequence of
one oligopeptide of the invention. It can in particular consist of
a protein, a glycoprotein, a fusion protein or a fusion peptide. It
can moreover be bound on a solid support.
[0070] An oligopeptide or a polypeptide of the invention may be
obtained by any obtention route and in particular chemical
synthesis or genetic recombination.
[0071] The objects of the invention are also of value outside the
study of physiopathogenic hypotheses relating to certain clinical
situations, such as mentioned previously. In fact, the provision of
a reliable test for estimation of anti-ovary antibodies can have
other diagnostic applications and therapeutic applications.
[0072] The gonadic system is the most fragile of the endocrine
systems. In various polyendocrinopathies, impairment of gonadic
function may be the first sign of the illness. In the face of
irregularities in the menstrual cycle, an investigation of the
anti-ovary antibodies can be the first sign of an incipient
autoimmune ovaritis.
[0073] Two other groups of pathology are directly concerned by this
type of investigation. These are the group of early menopauses and
the group of ovarian dystrophies.
[0074] These groups are very heterogeneous in their mechanisms and
the detection and/or quantification of anti-SEQ ID NO:1 antibodies
makes it possible to identify more homogeneous subgroups with
possible therapeutic implications.
[0075] In the sterility field, patients suffering from so-called
"idiopathic" sterility can benefit from this type of analysis, in
the same way as the search for other autoantibodies.
[0076] Endometriosis, a disease whose mechanisms are still unknown
today, obviously occurs in patients in this particular field. Many
autoantibodies have been identified in patients suffering from this
disease. The mechanism of the sterility is not always clear in
these patients. The search for anti-peptide SEQ ID NO: 1 antibodies
is of great interest here both on the physiopathological level and
on the therapeutic level.
[0077] Indeed, the search for such autoantibodies can also have
direct therapeutic implications.
[0078] The characteristics and advantages of the objects of the
present invention will be evident from the following Examples 1 to
4, supported by FIGS. 1 to 4, among which:
[0079] FIG. 1 shows the increase in the levels of anti-peptide SEQ
ID NO: 1 antibodies,
[0080] FIG. 2 shows the increase in the levels of anti-peptide SEQ
ID NO: 1 antibodies, after the first IVF attempt and the increase
in the IgM SEQ ID NO: 1 after the subsequent IVF attempts,
[0081] FIG. 3 shows a comparison of the change in the anti-ovary
antibodies and anti-peptide SEQ ID NO: 1, before and after puncture
in a population of women suffering from tubal sterility, and
[0082] FIG. 4 demonstrates the higher levels of anti-peptide SEQ ID
NO: 1 antibodies, in the absence of biological pregnancy measured
from the increase in the levels of HCG (solid columns).
EXAMPLE 1
Immunoenzymatic Technique for Estimation of Anti-Ovary
Antibodies
[0083] a) Reagents
[0084] Antigen:
[0085] Ovary
[0086] Corpus luteum
[0087] FSH (Metrodine.RTM., Serono, Levallois-Perret, France)
Peptides SEQ ID Nos: 1 to 5
[0088] Carbonate-bicarbonate buffer 0.1 M, pH 9.6
[0089] Phosphate buffer saline (PBS) 0.001 M, pH 7.4 (PBS)
[0090] Tween 20 (Sigma, St Louis, Mo., USA)
[0091] Gloria powdered skimmed milk
[0092] Bovine serum albumin (BSA) (Sigma)
[0093] Peroxidase-conjugated anti-human immunoglobulins (IgG, IgA,
IgM) (Miles, Napperville, Mass., USA)
[0094] Phosphate-citric acid buffer 0.1 M, pH 5.5
[0095] 30 volume hydrogen peroxide (Gifrer Barbezat, Decines,
France)
[0096] Ortho-phenylenediamine (OPD) (Sigma)
[0097] 2 N sulfuric acid (Prolabo, Paris, France)
[0098] b) Apparatus
[0099] Maxisorp plates (Nunc, Roskilde, Denmark)
[0100] Precision balance (Sartorius, Osi, Paris, France)
[0101] Multiskan II spectrophotometer, equipped with a 492 nm
filter (Flow laboratories, Helsinki, Finland)
[0102] Titersoft software (Flow Laboratories)
[0103] c) Method
[0104] The concentration of molecules to be adsorbed was determined
by checkerboard experiments for each antigen. It is about 1 .mu.g
per well.
[0105] The binding of the antigens to the solid phase is performed
in one night at +4.degree. C. in the amount of 100 .mu.l per well
of the solution of antigen in carbonate-bicarbonate buffer. The
plate is emptied. The sites of the solid phase still unoccupied are
saturated on incubation for one hour at 37.degree. C. with 200
.mu.l per well of 5% powdered milk reconstituted in PBS.
[0106] The samples to be tested are diluted in PBS to which 0.5%
BSA and 0.2% Tween 20 have been added: dilution to {fraction
(1/50)} for the search for anti-ovary and anti-corpus luteum
antibodies. The plate is emptied and washed 3 times in PBS. The
samples are distributed in the amount of 100 .mu.l per well. The
plate is incubated for 45 minutes at 37.degree. C.
[0107] The anti-isotypes are diluted in the same solution that was
used for the dilution of the samples:
[0108] {fraction (1/10,000)} for the anti-IgGs
[0109] {fraction (1/5000)} for the anti-IgAs
[0110] {fraction (1/5000)} for the anti-IgMs.
[0111] The plate is again emptied and washed 3 times in PBS. Then
the anti-isotypes are distributed in the amount of 100 .mu.l per
well. The plate is incubated for 45 minutes at 37.degree. C.
[0112] The developer solution is prepared on the spot, as follows
for one plate:
[0113] 12 ml of phosphate-citric acid buffer
[0114] 6 mg of OPD
[0115] 12 ml of H.sub.2O.sub.2.
[0116] After 3 further washings of the plate, 100 .mu.l of
developer solution are distributed into each well. After appearance
of a yellow colour of greater or lesser intensity, the reaction is
stopped with 50 .mu.l of H.sub.2SO.sub.4. The plate is read on the
spectrophotometer at 492 nm using the Multiskan II reader.
[0117] The results are firstly expressed as optical density (O.D.)
then calculated as ratios relative to a control sample at the limit
of positivity. Any ratio greater than 1 is considered as
positive.
EXAMPLE 2
Selection of Antigenic Peptides
[0118] The inventors firstly discovered that FSH, then the peptides
consisting of the sequence of amino acids 78-93 and 80-87 of the
.beta.-chain of FSH, identified respectively by SEQ ID No.1 and 5,
are simultaneously recognized by serums containing anti-ovary
antibodies and by 2 anti-.beta.-FSH monoclonal antibodies (property
of bioMrieux, 22A3G7; 5H3E8), which have the characteristic of
being immunoreactive with metrodine and ovarian extracts, while an
anti-.beta.-FSH monoclonal antibody (property of bioMrieux, 7G5A1)
only recognizing metrodine does not react with this sequence.
[0119] Given that the sequence of amino acids 78-93 of the
.beta.-chain of FSH contains 3 cysteines, 4 peptides of 16 amino
acids corresponding to the different disulfide bridge possibilities
were synthesized.
[0120] These four peptides are identified by the respective
references SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:
4, and are described at the end of the specification.
[0121] These four peptides obtained in lyophilized form were, after
reconstitution and dialysis, used as antigens in a series of ELISA
tests on 30 sera from patients.
[0122] Moreover, these same sera were tested against the crude
ovarian antigens.
[0123] The immunoenzymatic technique used is that described in
Example 1.
[0124] The results of the correlation obtained between the
reactivity of the sera toward the ovarian antigen and that of the
same sera toward each of the peptides tested, and expressed by an r
value, are shown in Table I below:
1 TABLE I SEQ ID No. 1 SEQ ID No. 2 SEQ ID No. 3 SEQ ID No. 4 IgG
AOA 0.74 0.44 0.79 0.59 IgA AOA 0.46 0.28 0.49 0.49 IgM AOA 0.81
0.75 0.71 0.69
[0125] It is found that the best correlation coefficients are
obtained for the IgMs on the one hand (which had already been noted
with the commercial FSH) and for the peptide SEQ ID NO: 1 on the
other hand.
[0126] The same results as those obtained for the peptide SEQ ID
No.1 were obtained with the peptide SEQ ID No.5, FSH and the
U-chain of FSH.
EXAMPLE 3
Antigenicity of the Peptide SEQ ID NO: 1
[0127] Two centre [sic] and ninety-five sera were tested in
duplicate, using the immunoenzymatic method described above, with,
firstly, the total ovarian extract, and, secondly, the peptide SEQ
ID NO: 1. For each antigen, the three immunoglobulin isotypes IgG,
IgA and IgM were studied.
[0128] a) Population Studied
[0129] The patients included in the study were all scheduled for
IVF. Their average age was 32.7.+-.4.2 years. The average duration
of the sterility was 4.8.+-.2.8 years.
[0130] The aetiology of the sterility which had led the couple to
IVF was of tubal origin in 24.9% of the cases, of masculine origin
in 18.2% of the cases, of idiopathic origin in 8.6%, due to an
ovarian dystrophy (PCO) in 3.8% of the cases, and due to an
endometriosis in 2.8% of the cases. In 41.7%, at least two of the
above aetiologies were present together, constituting mixed
aetiologies.
[0131] In 209 cases the serum was collected on the eighth day of
the stimulation, and in 84 cases the serum was collected 15 days
after the puncture.
[0132] b) Results
[0133] The results of [sic] the correlation obtained between the
reactivity of the sera toward the crude ovarian antigen and that of
the same sera toward the peptide SEQ ID NO:1 tested, and expressed
by an r value, are shown in Table II below:
2 TABLE II IgG SEQ ID IgA SEQ ID IgM SEQ ID No. 1 No. 1 No. 1 IgG
AOA 0.18 0.12 NS IgA AOA 0.12 0.11 0.01 IgM AOA 0.10 0.06 0.27
[0134] A correlation is observed between IgG AOA and IgG SEQ ID
NO:1 (r=0.18, p=0.002), and between IgM AOA and IgM SEQ ID NO:1 (r
0.27, p<0.00001). On the other hand, no correlation is observed
between IgA AOA and IgA SEQ ID NO:1 (r=0.10, p=0.06).
EXAMPLE 4
Applications of the Peptides of the Invention
[0135] Whatever the combination of number of IVF attempts made and
sterility aetiology, an increase in anti-peptide SEQ ID NO:1
antibodies was observed after puncture, with a statistically
significant difference for the IgM SEQ ID NO:1 (p=0.005) (see FIG.
1).
[0136] On the basis of the number of IVF attempts made, before and
after puncture, and as shown in FIG. 2, the following are
observed:
[0137] in first IVF attempt patients, an increase in anti-peptide
SEQ ID NO:1 antibodies, after the follicular puncture, whatever the
isotype considered,
[0138] in multiply-punctured patients, a decrease in the IgG and
IgA SEQ ID NO:1 post-puncture and a very clear and statistically
significant increase in the IgM SEQ ID NO:1 (p=0.01).
[0139] on the basis of the aetiology of the sterility and before
and after puncture
[0140] For the aetiology of tubal origin, included as it serves as
a reference for IVF (IgA p=0.04 and IgM p=0.027), an indisputable
increase is observed in the AOAs of isotypes A and M, after
puncture, but an increase in the anti-peptide SEQ ID NO:1, which is
clearer and statistically significant for these same isotypes (see
FIG. 3).
[0141] On the basis of the number of ovocytes and embryos
obtained
[0142] The presence of anti-peptide SEQ ID NO:1 antibodies appears
deleterious to the obtention of ovocytes and embryos.
[0143] An attempt was made to define a limit threshold depending on
the isotype concerned pre-puncture. From 151 measurements (a
certain number of attempts did not get as far as the follicular
puncture), it was possible to determine this threshold for the IgA
SEQ ID NO:1 at 0.75, for the ovocytes (9.4 vs. 11.8 ovocytes), and
at 0.60, for the embryos (3.1 vs. 4.9 embryos).
[0144] It should be noted that this threshold is lower than that
defined for the IgA AOAs (1.7 for the ovocytes and the
embryos).
[0145] On the basis of the outcome of the attempt
[0146] The mean levels of anti-peptide SEQ ID NO:1 antibodies were
compared in relation to whether the plasma HCG (chorionic
gonadotrophin) estimation was positive (biological pregnancy) or
negative (absence of implantation).
[0147] In accordance with FIG. 4, on average lower antibody levels
were found when the HCG was positive.
[0148] These results demonstrate that the peptide SEQ ID NO:1 makes
it possible sensitively and specifically to detect antibodies
displaying variations parallel to those of antibodies directed
against the total ovarian extract.
[0149] Moreover, follicular puncture stimulates the appearance of
anti-peptide SEQ ID NO:1 antibodies, and patients displaying such
antibodies in their serum have less chance of securing a pregnancy
after IVF.
BIBLIOGRAPHY
[0150] (1) C. A. SHIVERS and B. S. DUNBAR, (1977) Autoantibodies to
zona pellucida: a possible cause of infertility in women. Science,
197: 1087-1190
[0151] (2) P. BARBARINO-MONNIER et al., Fertility and Sterility,
Vol.56, No.5 (1991) 928-932
[0152] (3) P. BARBARINO-MONNIER et al., Human Reproduction, Vol.10,
No.8 (1995), 2006-2007
[0153] (4) H. E. MONCAYO-NAVEDA, R. MONCAYO, R. BENZ, A. WOLF and
Ch. LAURITZEN (1989) Organ specific antibodies against ovary in
patients with systemic lupus erythematosus. Am. J. Obstet.
Gynecol., 160: 1227-1229
[0154] (5) A. L. LEHNINGER, Translation of the second edition of:
Biochemistry, the molecular basis of cell structure and function,
Chapter 4: The Proteins: The constituent amino acids, p.69 to 74
(1979)
[0155] (6) R. MONCAYO and H. E. MONCAYO, Journal of Internal
Medicine (1993), 234: 371-378.
Sequence CWU 1
1
5 1 16 PRT human FSH 1 Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp
Ser Asp Ser Thr Asp 1 5 10 15 2 16 PRT human FSH DISULFID (5)..(7)
disulfide bond between the cysteine in position 5 and the cysteine
in position 7 2 Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp
Ser Thr Asp 1 5 10 15 3 16 PRT human FSH DISULFID (5)..(10)
disulfide bond between the cysteine in position 5 and the cysteine
in position 10 3 Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser
Asp Ser Thr Asp 1 5 10 15 4 16 PRT human FSH DISULFID (7)..(10)
disulfide bond between the cysteine in position 7 and the cysteine
in position 10 4 Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser
Asp Ser Thr Asp 1 5 10 15 5 8 PRT human FSH 5 Thr Gln Cys His Cys
Gly Lys Cys 1 5
* * * * *