Note: Descriptions are shown in the official language in which they were submitted.
~335~:7
BACXGROUN~ AND PRIOR ART
Mammalian spermatozoa have been kn~n to be
antigenic or many years. Moxe recently, it has been
demonstrate~ that mammalian sperm co~tain an antigenic
enz~meJ which is known as the C4 isozyme of lactate
dehydrogenase (LD~-X, LDH C4 ) . LDH-C4 h~s been isolated
. .
- in pure crystalline f~rm xom mouse testes. Goldberg .(1972)
J. Biol. ChemO 247:2~44-2048. The enzyme has a molecular
weight of 140,000 and is composed of four id~ntical C
subunits. The amino acid se~ue~ce and ~hree-dimensional
structure of LDH-C4 has been studied and partia~ly detexmined
by a n~mber of investigators. See Musick et al ~1976) ~ Mol.
Biol. 104:659-668; and Wheat et al (1977~ Bîochem. ~ Bioph~s.
Res. Comm., 74, No. 3:1066-1077. h~eat et al determined the
sequence of the e;sential thiol peptide ~rom amino acid 159
to 171, and found this to be nearly identical to essential
thiol peptides from other vertebrate LDE i~oz~mes.
- I~ 1974 r Dr. ~rwin Goldberg reviewed ~he ef~ects o~
immunization with LD~-X ~LDH-C4) on fertili~y, and advanced
the possibility that l'by using a defined macromolecular
constituent of sperm it becomes possible to elucidate its
primary structure in terms of amino acid se~uence r to map
specifically the antigenic determinan.t (s) responsible for
induci~g infertility, and then to construct sy~the~ic
pep~ides containing these determinants. Possessing ~he
capability for synthesi~ing a molecule with such properties,
makes the immunological approach to fertility control
feasible. ~r Karolinska Symposia on Research Methods in
Reproductive ~ndocrinology, 7th Symposia: Immunological
Approaches to Fertility Control, Geneva, 1974 202L222.
-- 2
~83~5Z7
However r such synthetic antigenic peptides remained a goal
and not an achievement although their theoretical desir-
ability had been recognized. In 1979, Dr. Erwin Goldberg
summarized the state of the art as follows:
"In conclusion, and on a practical basis
immunotherapy for birth control requires more
than effectiveness, specificity, reversibility,
and absence of systemic side reaction. Rather
large amounts of the antigen must be available
in unequivocally pure form. This condition
probably cannot be met by a natural product
enzyme antigen from sperm or testes. Rather,
contraceptive technology requires a synthe-
sizable peptide fragment retaining anti-
genicity and provoking a response which
impairs fertility. Completion of the struct-
ural analysis of LDH-C4 should allow mapping
of antigenic determinants and synthesis of
such peptides for use in a new contraceptive
technology," "Recent Advances in ReProduction
and Regulation of Fertilityli' G.P. Talwar,
editor, Elsevier/North Holland ~iomedical
Press (1979).
SUMMARY OF INVENTION
It has now been discovered that an antigenic peptide
can be prepared by synthesizing a linear sequence of seven
amino acids comprising: methionine-glutamine-lysine-aspartic
acid-leucine-glutamic acid-leucine. All of the amino acids
used to prepare the foregoing peptide are in their L-form.
30 The N-terminal is methionine and the C-terminal is leucine.
Although not known with certainty, it is believed that the
foregoing sequence of seven amino acids corresponds to amino
acids 325 to 332 of LDH-C4. This is contrary to a recently
published tentative sequence. See Musick et al (1979)
J. Biol. Chem., 254, No. 16:7621-7623~
DESCRIPTION OF INVENTION
The present invention relates to a novel antigenic
linear peptide having a chain length of seven amino acids.
35;~
The formula of this peptide can be represented as follows:
N-Met~Gln-Lys-Asp-Leu-~lu-Leu-C
In the foregoing formula, the letter "N" designates
the N-terminal amin~ acid, while the letter "C" designates
the C-terminal amino acid. Met, Gln, Lys, Asp, Glu, and
1eu represent the L-amlno acid forms, respectively, of
methionine, glutamine, lysine, aspartic acid, glutamic acid,
:: and leucine.
The peptide compound of the present inve~tion can be
10 synthesized from its constituent amino acids. For example,
the synthesis can be carried out by the Merrifield solid phase
method, as described in J.A.C.S. 85:2149-2154 ~1963). This
solid phase method for synthesizing se~uences of amin~ acids
is also described in Stewart and Young, ~
(W. H. Freeman and Co~, San Francisco, 1969), pages
1-4. In this procedure, the C-terminal amino acid, such as
leuci~e for the compound of this in-~ention, is attache~ to
chloromethylated polystyrene divinylbenzene copolymer beads.
Each subsequent amino acid, with suitab-le protecting group,
is then added sequentially to the srowing chain. For exam~le,
as described in the Merrifield article, the protective group
may be a carbobenzoxy group. By the procedure of coupling,
deprotection, and coupling of the next amino acid, the desired
amino acid sequence and chain length can be produced. ~s a
final step, the protective ~roup is removed from the ~terminal
amino acid (YiZ. methionine), and the C-terminal amino acid
is cleaved from the resin, using a suitable reagent, such as
trifluoroacetic acid and hydroyen bromide. Sinca this syn-
thesis procedure is well known, it is not believed that it
will be necessary to further describe it herein~ The peptide
of this invention can be prepared by this sy~thesis procedure
for use in reducing the fertility of mammals.
-- 4 --
~33~2~
To utilize the antigenic peptide of this invention
in ~he form o~ a ~ertility reducing vaccine, the peptide is
coniugated to a carrier molecule, which is preferably a
protein which itself elicits an antigenic response and which
can be safely administered. For example, the peptide can
be coupled to tetanus toxoid for administr~tion by intra-
muscular injection. ~or example, a mixture of l~Mole tetanus
.. toxoid, 60yNoles antigenic ~eptide, and lB millimoles
l-ethyl-3-(3 dimethyl aminopropyl~ carbodiimide hydrochloride
reacted in water (pH6) for 12 houxs at room temperature an~
24 hours at 4 gives a product containing 3~5 moles o~
~eptide/mole tetanus toxoid. Excess reactants can be
removed by dialysis or gel ~iltration. See Pique et al,
Immunoche _ st~, 15:55-60 (1978~. Alterna~ively, the peptide
may be coupled using bisdiazotized benzidine ~B~ssiri et al,
ndocrinology, 90:722 (1972)] or glutaraldehyde.
~ or intramuscular injection, the coupled peptide may
be suspended in a sterile isotonic saline solution, or other
conventional vehicle, and, if desired, an adjuvant may be
included. A preferred use of such a vaccine is for administra-
tion to human ~emales. Anti~odies will be formed, which will
appear in the oviduct ~luids and thereby achieve a significant
reduction in fertility. For this purpose, the amount to be
administered will range from about 1 to 10 milligrams ~mg)
of the antigenic peptide.
.... .
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