Note: Descriptions are shown in the official language in which they were submitted.
105774~
ANTIGENIC MODIFICATION OF POLYPEPTIDES
This inven~ion relates to the chemical modification
of polypeptides to provide antigens which may be used in
active immunisation for the purposes of the control or treat-
ment of various physiological processes, particularly re-
production.
It is well known that polypeptides, particularly
proteins, are responsible for, contribute to o~ influence
many physiological processes. For example, certain pro-
tein hormones and non-hormonal proteins are known to be
essential for the normal events of the reproductive process.
Unusual excesses of certain polypeptides, such as gastr~n,
angiotension II or somatomedian, are also known to cause
or influence various disease states and maladies.
The present invention is concerned with the con-
trol or treatment of such physiological processes by active
immunisation (i.e. administration of anitgens) causing
formation of antibodies which neutralise (render biologically
ineffectual) the endogenous polypeptides which are responsible
for, contribute to or influence the physiological pr~cess
in question.
-- 1 --
~057 74~
Immunology, as a means of controlling reproduction,
has been the subject of much recent investigation. R.G.
Edwards, in a paper entitled "Immunology of Conception and
Pregnancy (British Medi~al Journal, 26, 72-78 ~1970]),
surveys the literature on this topic, for example that con-
cerning the production and use of antibodies against testes
or sperm. Hormone antibodies have also been studied for
many years and the effects of specific antisera hav~ been
recorded. Most of the past approaches to contraception by
immunological means have, however, used passive immunisation,
that is injection of antibodies produced elsewhere. There
are serious limitations to the use of passive immunisation
for human therapy since the repeated injection of animal
antibodies into humans is known t~ produce undesirable
reaction in many individuals.
It has now been found that by chemically modify-
ing natural, endogenous polypeptides which play a role in
the physiological process to be treated or controlled,
antigens are obtained which are capable of inducing form-
ation of antibodies which neutralise not only the anti-
genically modified polypeptide but also its unmodified
endogenous counterpart. Thus, immunisation apparently
takes place because of the inability of the anîbodies
105774'~
produced to distinguish between the antigenically modified
polypeptides and their unmodified endogenous counterparts.
m e modified polypeptides are produced from the natural,
endogenous polypeptides in the species involved or are immuno-
logically equivalent to the modified polypeptides so produced.
In practice, the polypeptides to be modified are derived from
the species involved or from a closely related species.
As used hereinafter in the specification and claims,
the term "modified polypeptides" refers to polypeptides which
are
a) protein hormones
b) non-hormonal proteins
or c) fragments of either a) or b).
Such polypeptides may be naturally occurring or synthetic. As
used herein, the term "fragment" in relation to endogenous poly-
peptide means a sequence of amino acids in the complete amino
acid chain of the endogenous polypeptide. m ese fragments must
be sufficiently large and distinctive in chemical and physical
character to enable them to be recognised as a specific part of
the whole. m ey must naturally also possess the chemical nature,
i.e. the requisite amino acid make-up, to enable them to be
chemically modified in the manner desired. As used herein, the
term "polypeptide which is immunologically equivalent to" in
relation to an endogenous polypeptide or fragment thereof, means
a polypeptide which, although not identical to the polypeptide
or ~ragment in question, elicits, when modified in accordance
with the invention, essentially the same antibody response as
the endogenous polypeptide or fragment concerned.
~he present invention accordingly provides an antigen
for use in active immunisation for the control or treat-
, ,i~f
~OS'774'~
ment of a physiological process in a particular species,
comprising a chemically modified polypeptide~ which chemically
modified polypeptide is derived from an endogenous polypeptide,
which influences such physiological process in that species, or
is immunologically equivalent to a chemically modified polypeptide
so derived, the chemical modification being such as to permit
the chemically modified polypeptide to induce, in that species,
formation of antibodies which biologically neutralise the endo-
genous polypeptide as well as the chemically modified polypeptide.
The present invention also provides a process for the
production of an antigen for use in active immunisation for the
control of fertility, in a particular species, comprising
chemically modifying a polypeptide which is a) an endogenous
protein reproductive hormone in that species; b) a fragment
thereof; or c) a polypeptide which is immunologically equivalent
to such protein reproductive hormone or fragment thereof, the
chemical modification comprising a) the attachment of one or more
foreign modifying groups to the unmodified polypeptide;, or b)
the removal of one or more moieties from the unmodified polypep-
tide, and being such as to permit the chemically modified poly-
peptide to induce upon administration the formation of antibodies
which biologically neutralise the protein reproductive hormone
as well as the chemically modified polypeptide.
The invention also provides the antibodies generated in
the species in response to the action of the antigens of the
invention, and antisera containing such antibodies.
m e invention als~ provides a method of controlling or
influencing a physiological process in a species comprising
injecting into that species an immunologically effective amount
of the antigens or antibodies of the invention. The invention
particularly provides such a method of contraception.
-
774~
Polypeptides which may be chemically modified
to provide the antigens of the invention include protein
reproductive hormones, such as Follicle Stimulating
Hormone (FSH), Leutinising Hormone (LH), Human Placental
Lactogen (HPL), Human Prolactin and Human Chorionic
Gonadotropin, and specific fragments thereof.
Specific fragments of protein reproductive
hormones which may be modified in accordance with the
inv~ntion include the ~-subunit of FSH and specific
unique fragments of natural HPL or Human Prolactin,
which fragments may bear little resemblance to analogous
portions of other protein- hormones. Preferred fragments
include the ~-subunit of HCG which, according to the two
authoritative views, has either structure I or II as
follows (*indicates site locations of carbohydrate
moieties):-
*
Ser-Lys-Glu-Pro-Leu-A~g-Pro-Arg-Cys-Arg-Pro-Ile-Asn-Ala-Thr-
Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn-
Thr-Thr-Ile-Cys-Ala-Gly-Try-Cys-Pro-Thr-Met-Thr-Arg-Val-Leu-
~ ~0
Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys-~-sn-Try-Arg-Asp-
Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-Cys-Pro-Arg-Gly-Val-
Asn-Pro-Val-Val-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln-Cys-Ala-
100
Leu-Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-His-
110 120 *
Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Cln-Asp-Ser-Ser-Ser-Ser-
Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-
* 140
Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Cln
(Structure I)
lOS7'~4;~
10 *
Ser-Lys-Gln-Pro-Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile-Asn-Ala-Thr-
20 29
Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn-
Thr-Thr-Ile-Cys-Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr-Arg~Val-Leu~
50 60
Gln-Gly-Val~Leu-Pro-Ala-Leu-Pro-Glx-Leu-Val~Cys-Asn-Tyr-Arg-
Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-Cys-Pro-Arg-Gly-
Val-Asn-Pro-Val-Asn-Pro-Val-Val-Ser-Thy-Ala-Val-Ala-Leu-Ser-
go 100
Cys-Gln-Cys-Ala-Leu-Cys-Arg~Arg)-Ser-Thr-Thr~Asp-Cys-Gly-
110
Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys-ksp-Asp-Pro-Arg-Phe-Gln-
* 120 * 130 *
Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-
140
Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Glu-Ser-Leu-
147
Pro
(Structure II~
For specificity of antibody action, it is des-
irable that polypeptides be modified which comprise
molecular structures completly or substantially completely
different to those of other protein hormones. In ~:
this connection, the ~-subunit of HCG possesses a chain
or chains of amino acids which differ greatly from those
of LH and such chains may also be modified in accordance
with the invention. Such chains include 20-30 or 30-39
amino acid peptides consisting of the C-terminal residues
of the ~-subunit of HCG. More particularly, suitable such
chains include those of the following formulae III and IV
(C-terminal portions of structure I, above) and V and VI
(C-terminal portions of Structure II, above):
iO5774;~
Asp-Asp-Pro-Arg-Phe-GlnrAsp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-
* *
Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-
Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln
(Structure III)
Gln-Asp-Ser-Ser*Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-
Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-
Leu-Pro-Gln
(Structure IV)
Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser~5er-Ser-Lys-
Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-
Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro.
(Structure V)
Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-
* *
Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-
Ile-Leu-Pro-Gln-Ser-Leu-Pro.
(Structure VI)
Such structures may be obtained by purely syn-
thetic methods or by enzymatic degradation of the parent
polypeptide [Carlson et. al., J. Biological Chemistry,
284(19), 6810(1973)]. A polytyrosine chain may also be
added to the structures III to VI and the;resulti'n'g''pdly-
peptide modified in accordance with the invention.
- 7 -
i~)5774~
Another group of polypeptides which may be mod-
ified according to the invention are specific non~hormonal
protein antigens isolated from placental tissue.
Other proteins which may be modified according
to the invention and used for active immunisation in
the treatement of various disease states and `maladies,
include gastrin, the hormonal polypeptide known as
Angiotension II, growthl hormone, Somatomedian, para-
thyroid hormone, insulin, glucagon, thyroid stimulating
hormone (T8H) and secretin.
The degree of chemical modification of the
polypeptides, in accordance with the invention, is, as
indicated, such that the resulting antigens will induce
generation of antibodies which will neutralis~ not only
the antigens but also some, at least, of their natural
endogenous counterparts and this as well as the type of
modification will depend on the specific problem being
treated as well as the nature of the polypeptide involved.
If too little modification is effected, the body may
not recognis~ the modified polypeptide as a foreign
intruder and may therefore not generate antibodies
against it. If, on the other hand, too much modific-
ation is effected, the body will generate antibodies
specific to the injected antigen which will not neutral-
lOS774~
ise the natural endogenous protein involved.
In general, the chem~cal modification involves
addition of foreign modifying groups to the polypeptide
involved (hapten-coupling). The number of foreign
modifying groups to be àdded will, of course, vary
depending on the circumstances but, generally, it is
preferred that 1 to 40, preferably 2 to 40, more prefer-
ably 5 to 30, and most suitabley 10 to 26 modifying
groups per molecule of polypeptide be attached.
Given modifying groups will attach to particular amino
acid sites in the peptide molecule, so that the max-
imum possible number of a given modifying group which
will attach to a given polypeptide can readily be cal-
culated. The nature of the modifying group may be
selected accordingly. It is also poss~ble that se~eral
modifying groups may attach to each other and then attach
to a single amino acid moiety but, for the pu~poses of
this invention, such a substitution is regarded as
attachment of a single modifying group.
The modifying groups may as indicated vary in
chemistry depending on the cirrcumstances. Suitable mod-
ifying groups include diazo groups. These may suitably
be introduced by reaction with the appropriate number of
_ g _
1~)5774~
moles of diazosulfanilic acid. Introduction of diazo
groups into proteins is a well-known technique and may,
for example be effected as described, by Cinader et al.,
J. Am. Chem. Soc. 78, 746(1955); by Phillips et al.,
J. Biol. Chem. 244, 575(1969); by Tabachnick et al.,
J. Biol. Chem. 234~), 1726(1959) or by Crampton
et al., Proc. Soc. Ex. Biol. & Med. 80, 448(1952).
In general, the methods of Cinader et al. and Phillips
at al. are preferred.
Additional modifying groups include those
introduced by reaction of the polypeptides with dinitro-
phenol, trinitrophenol, S-acetomercaptosuccinic anhydride,
polytyrosine or polya~anines (in either straight of
branched chains), biodegradable polydextran or,
rather less preferably, natural proteins suGh as thyro-
globulin. Generally, synthetic modifiers are preferred
to natural modifiers.
The above reactions, as well as many other
suitable hapten coupling reactions, are well known in
protein chemistry. The follo~ing references ma~, for
example, be cited in this connection:-
1. R~otz et al., Arch. Biochem. & Biophys. 96,
605-612(1966);
2. Khorana, Chem. Rev. 53, 145(1953);
-- 10 --
105774'~
3. Sela et al., Ba~ochem. J. 85, 223tl962);
4. Eisen et al., J. Am. Chem. Soc. 75, 4583(1953);
5. Certano et al., Fed. Proc. (ABSTR,)25, 729
(1966);
6. Sokolowski et al., J. Am. Chem. Soc. 86, 1212
(1964)'
7. Goodfriend et al., Science 144, 1344(1964);
8. 8ela et al., J. Am. Chem. Soc. 78, 746(1955);
and
9. Bahl, J. Biol. Chem. 244, 575(1969).
The chemiGal modification may alternatively or
additionally comprise removal of moieties from tne poly-
peptides. Thus, for example where certain of the nat-
uaal proteins have carbohydrate moieties, these may be
removed in conventional manner, using, for example,
N-acetylneuraminidase or N-acetylglucosidase, materials
useful for removing specific carbohydrate moieties.
As indicated, the modified polypeptides of the
invention are indicated for use as antigens in active
immunisation for the purposes of control or treatment of
various physiological processes.
More particularly, the modified protein repro-
ductive hormones or fragments thereof, of the invention
are indicated for use as an~igens in active immunisatton
l~)S774~
for the purposes of controlling reproduction. Thus,
these materials induce formation of antibodies which
neutralise not only the antigens but also the natural
endogenous protein reproductive hormones which are
essential for the normal events of the reproductive
process, thus disrupting the natural processes of
conception and/or gestation. These antigens are there~
fore indicated for use in contraception and for termin-
ating pregaancies soon after conception. The specific
non-hormonal protein antigens isolated from the placental
tissue and modified according to this invention are
also indicated for such use. There is direct evidence
that inhibitaion of substances which are specific to
the-placental tissue and do not have similar antigenic
properties to antigens from ~her organs, can result in
disruption of pregnancies by passive immunisation. Such
specific placental substances when modified in accord-
ance with the invenfion can be injected into the body
of the same species as a fertility control means by
active immunisation. The advantage of these substances
is that placental antigens are foreign to the non-
pregnant female humans and are therefore unlikely to
cause any cross-reaction or disruption of normal body
functions in the non-pregnant female.
1~)5774Z
It will be understood while the methods of
controlling reproduction described are mainly applic-
able to females, certain antigens, in particular FSH,
its ~-subunit and fragments thereof, when modified in
accordance with ~e invention, may be applicable to
males.
Gastrin, when modified in accordance with the
invention is indicated for use in the treatment by
active immunisation of the digestive disorder known as
the Zollinger-Ellison~ Syndrome. This state is generally
described as a condition in which there is h,~per~secretion
of the polypeptide gastrin, which is produced in the
pancreas and brings about a state of hyperacidity in the
stomach resulting in a chronic d~gestive disorder.
Injections of modified gastrin will enhance formation
of antibodies against the hypersecretion of gastrin and
thereby alleviate the disease without the need for
the surgical treatment which is the only effective
treatment presently known.
Angiotension II, when modified in accordance
with the invent~on, is an antigen for use in the treat-
ment of hypertension. In general terms, the state of
hypertension is the abnormal level or fluctuation of
ones blood pressure. The blood pressure in an individual
- 13 -
1057'~4;~
is controlled by many physiological processes in the body.
However, one major substance affecting the regulation
of such pressure is the hormonal polypeptide known as
Angiotension II. In certain states of high blood
pressure (hypertension) it is difficult to medically
control the secretion and therefore the level of Angio-
tension II in the circulatory system. By the appropri-
ate modification of this hormone and subsequent immuni-
sation with the resulting antigen, it is possib~le to
reduce the secretion of angiotension II in patients
with chronically elevated hormone levels. The predict-
able and controlled reduction of this substance is
beneficial to certain patients with chronic problems of
hypertension.
Growth Hormone and Somatomedian, when modified
according to the invention are indicated for use as
antigens in immunological treatment of diabetes and
associated micro and macro-vascular disorders. Currently,
the treatment of diabetes is limited to dietary and/or
drug treatment to regulate blood glucose levels.
Recent scientific data support the concept that Growth
Hormone and Somatomedian (both polypeptides) are intim-
ately involved in the disease syndrome.
Antigens of the invention are suitable admixed
- 14 -
lOS7742
with a pharmeceutically acceptable liquid carrier and
administered parenterally. The dosage to be administered
will, of course, vary depending on various factors,
including the condition being treated and its severity.
However, in general, unit doses of 0.1 to 50 mg are
indicated, suitably administered one to five times at
intervals of one to three weeks.
As indicated, the theory leading to the present
invention was that the chemical modification of a pro-
tein essential for reproduction or influencing a partic-
ular disease state would render it antigenic such that
it would cause formation of antibodies which would, at
least partially, neutralise the endogenous protein in
addition to the antigen. Wi~h this in mind, reproductive
hormones of various species were modified and tested in
baboons as described in Example 1, hereinafter. The
results demonstEated that modified hormones of unrelated
species do not produce the desired result whereas
modified hormones of the same species or closely related
species do. The remaining Examples 2 to 9 also illus-
trate the invention.
Example I
Adult female baboons were studied for at least one
menstrual cycle for patterns of urinary estrogens, plasma,
progestin, and in some cases urinary LH. Only those animals -
displaying normal patterns of these hormones were immunized.
The criteria for normality and the procedures for housing
animals are well known and will not be described.
3~ - 15 -
1~)5774Z
Gonadotropin Preparations
Human Luteinizing Hormone (HLH) - partia~ly purified
preparation from human pituitaries with a biological potency
of 2.5 units per mg. (NIH-LH-SI).
Human Follicle Stimulating Hormone (HFSH) a par-
tially purified preparation from human pituitaries with a
biological potency of 86 units per mg. (NIH~FSH~SI~.
Human Chorionic Gonadotropin (HCG) - a highly
purified preparation from human pregnancy urine with biolog-
ical potency of 13,200 IU/mg. (2nd IRP-HCG).
Monkey Luteinizing Hormone (MLH) - a crude pre-
paration from rhesus monkey pituitar~es w~th a b~olog~cal
potency of 0.75 units per mg. (NIH-LH-SI).
Ovine Luteinizing Hormone (OLH) (NIH-LH-S5).
Baboon Luteinizing Hormone (BLH) - partially
pu_ified baboon putuitary prearation with a biological potency
of 1.1 units per mg. (NIH-LH-Sl).
All preparations, excepting the OLH, were prepared
in the inventor'ls laboratory. LH and HCG biological activity
was determined by the ovarian ascorbic acid depletion test
and the FSH preparation assayed by t~e ovarian augmentation
assay.
Hormones were altered as antigens by coupling with
a hapten in varying ratios of hapten to hormone as des-
cribed by Cinader et al., supra. For conven~ence, the
Cinader process is discussed herein although Phillips, supra,
may provide a more stable bond under certain circumstances.
In this procedure, the protein hormone serves as a carrier
and the hapten is coupled to it by diazo bonds. Although
a variet~ of hapten groups were coupled to different hormones,
the same basic procedure was used for any combination.
~ 16 -
105774'~
Fifteen to thirty-five haptenic groups per hormone molecule
were found most useful for preparing immunizing antigens.
The basic reaction consisted of diazotizing the hapten
(sulfanilic acid) by adding it to a solution of 0.11 N HCl
and then slowly adding this solution dropwise to a 1 percent
solùtion of NaN02 with constant stirring at 4C. Diazotiza-
tion was considered complete when free HNO2 was detected ~n
the reaction mixture. Although the above reaction was
accomplished at 4C., optimum temperatures for the reaction
normally are about 0-6C., although 4C is preferred.
The hapten-protein coupling was performed by dis-
solving the protein hormone in an alkaline buffer, pH 8.0,
The diazotized hapten was added slowly to the hormone sol~-
tion with continuous stirring at 4C. The pH of the reaction
was constantly monitored and kept near 8Ø After all the
hapten was added, the pH was finally adjusted to 8.0, stirred
for 1-2 hours and allowed to stand at 4 overnight. The
mixture was thoroughly dialyzed for 6-8 days against distilled
water to remover unreacted hapten.
Although the number of diazo groups per hormone
molecule could be regulated by the number of moles of hapten
and hormone reacted, a parallel control experiment with S35
labelled sulfanilic acid to evaluate the precise composition
of the hapten-protein samples was performed with each
diazotization. The same hormone preparation to be used for
immunization was used in the control experiment. After the
reaction was completed, an aliquot was taken from the
reaction mixture and the remainder thoroughly dialyzed.
Equal volumes of the dialyzed and undialyzed solut~ons were
counted by liquid scintillation. By comparing the counts of
the dialyzed and undialyzed samples, the moles of hapten
coupled to each mole of hormone was calculated since the un-
- 17 -
10S774'~
reacted hapten was removed by dialysis. For this calculation,
a molecular weight of 30,000 was assumed for all gonadotropin
preparation.
Following dialysis, hapten-hormones were lyophilized
5% and stored at 4C. Diazo-HCG (35 groups/molecule) and HLH
(26 groups/molecule) were bioassayed by the ovarian ascorbic
acid depletion method and found to retain 62 and 85 percent
respectively of the activity of the unaltered hormones from
which they were derived. None of the other hormones were
assayed for biological activity.
Immunization Procedures
Female baboons received their initial immunization
on days 3-5 of the menstrual cycle and the second and third
injections on~ week apart. The fourth injection was g~ven 2~3
weeks after the third. A few animals received a fifth injec-
tion at 70-80-days after the first injections. All antigens
were administered subcutaneously in a suspension of mannide
manoleate or peanut oil. Doses of antigens for each in~ection
varied between 3 and 5 mg. Injection sites were inspected
daily for 5 days after each immunization for local reactions.
Monitoring Effects of Immunization
Daily 24 hour urine specimens and frequent ser~n
samples were collected during at least one menstrual cycle
prior to imrnunizations a~d following immunizations until the
effects of treatment were assessed. Urinary LH, urinary
estrogens and plasma progestins were measured. Antibodies were
detected in post-immunization serum samples by reacting 0.2 ml.
of a 1:1000 dilution of serum in phosphate-buffered saline (pH
7.4) 0.5 percent normal baboon serum with 250 pg of 1131 label-
led ho~none. Sera ~lere reacted with both the unaltered
ir~nunizing hormone and unaltered baboon LH for antibody detec-
tion. A purified babGon LH preparation (1.9 x NIH-L~-Sl) was
- 18 -
105774'~
used as a tracer antigen. Antigen-antibody complexes were
precipitated with ovine anti-baboon gamma globulin after a
24 hr. incubation at 4C. Antibody levels were expressed as
pg of ~abelled hormone bound. Significant antibody levels were
considered to be those that would bind 5.0 pg or more of the
1131 labelled antigen.
Antisera were fractionated by gel filtration of
"Sephadex G-200"* according to the procedure of Fahey and Terry
(at p. 36, Experimental Immunology, F.A. Davis Co., Philadelphia,
Pa., 1967) to determine the proportion of IgM and IgG antibodies
in the baboon sera. Since the IgG fraction in this procedure
contained a portion of IgA and IgD antibodies, only IgM and
total titers were determined. The Ig~ fraction from the column
was reacted with 1131 hormones and the binding capacity deter-
mined. The volumes of the fractionated sera were adjusted sothat an~ibody levels would be comparable to those of whole serum.
Antibody Production
No significant reactions were observed at the site of
injection following any immunization. On 4 occas~ons, a slight
induration (2-3 cm in diameter) was seen when mannide manoleate
was used as a vehicle but the redness and swelling disappeared
within 4-5 days. Antibodies were detected against the immu-
nizing antigen within 3-5 weeks in all animals. The exten~t,
duration and cross reactivity of these antibodies is recorded.
Generally speaking, higher levels were observed to heterologous
gonadotropin immunization than k~ homologous ones.
The cross-reactivity of induced antibodies with baboon
LH was studied on each animal. Cross-reactivity of antisera at
peak levels was recorded. Although relatively high antibody
activity against human LH and HCG were seen, relatively little
reaction with baboon LH occurred. An intermed~ate cross-reaction
was noted with anti-ovine L~l and a high degree of cross-reacti-
*Trademark for a synthetic organic compound derivedfrom cros~-lin~ed dextran, in the ~orm of macroscopic beads.
-- 19 --
lOS774;~
vity was seen with anti-monkey LH. Diazo-human FSH was weekly
antigenic in the baboon,~ The duration of antibody production
was generally longer with the human and sheep gonadotropin
immunization than with those of monkey or baboon origin.
Peak antibody levels usually occurred at the time when
the antibodies had shifted to principally the IgG type. Early
antibodies had a larger proportion of IgM type and were generally
more cross-reactive with baboon LH. The change,in the propor-
tion of the total antibody population that was IgM was recorded
from the time antibodies were first detected. Significant cross~
reactivity to baboon LH was observed in anti-human gonadotropins
when IgM was abundant but dropped sharply as the antisera shifted
to nearly all IgG. This drop in cross-reactivity did not occur
with monkey and baboon immunizations. Again, the ovine LH im-
munizations produced an intermediate change in reactivity with
the shift from IgM to IgG.
Ef~cts on the Menstrual Cycle
The effects of immunization upon the events of the
menstrual cycle were determined by observing changes in sex skin
turgescence and levels of pituitary and/or ovarian hormones.
Based on these parameters, the delay or retardation of ovalution
from the expected time, as judged by the control cycle was calcu-
lated. One animal immunized with HCG had no interruption in
ovulation and another immunized with HFSH was delayed for only
one cycle. Two anima~s injected with HLH and two injected with
HCG had ovulation delays equivalent to two menstrual cycles. A
third animal immunized with HLH was delayed a calculated 86 days.
Ovine LH immunizations produced an 88 day delay in ovulation.
Immunizations with diazo-monkey or baboon LH resulted
in longer disruption of the menstrual cycle. Calculated delays
in ovulation for the two animals receiving monkey LH was 146 and
122 days ~hereas the animalc receiving altered baboon L~ were
~ - 20 -
lOS7'~4;~
retarded from ovulation 224 and 210 days.
Effects on specific hormone patterns following
immunization with ~LH. in one animal were recorded. The
interval between menses was
_ 20a -
iO5774;~
considered to represent a "cycle. Urinary estrogens and plæma progestin
pattems indicated that no ovulation occurred during the cycle of immunization
which was 85 days in duration. Urinary estrogens were elevated during treat-
ment but did not reflect a typical pattem. Plasma progestins were not
elevated until about day 19 of the first post-treatm~ent cycle. Pattems of
both estrogens and progestins were within no~mal limits during the second
post-treatment cycle. Antibody levels were elevated from about day 35 of the
treatment cycle until 289 days frGm the first detection of antibodies. An
IH assay was not available when this animal was st~ied and no data on plasma
10 or urinary levels of this hormane was obtained.
Hormonal patterns following an immunizatic,n with diazo-baboon IE~
were reoorded. In this animal, antibody levels were lower and persisted, in
general, for a shorter period than did imnn~izations with human gonadotrapins.
~ring the treatment cycle, levels of urinary estrogens and plasma progestins
follcwed a normal pattem but: were quantitatively lGwer than normal. Urinary
IH pattems fluctuated ma~cedly due to the injections of diæo-~ during
this period. No conclusive evidenoe of ovulation was obtained for the treatment
cycle. Ihe first post-treatment cycle lasted 246 days. During this cycle
urinary IA and estrogens were elevated on days 35-41 but there was no subsequent
20 elevation in plasma prcgestins that would indicate ovulation had occurred.
~bllc~ing day 42 of this cycle, there was no significant elevation in any of
the three hormone levels until day 231 when significant elevations of urinary
estrogens and I~l occurred. These rises were followed 3 days later by an
elevation in plasma progestins indicating the presenoe of a functioning corpus
luteum. A sec~nd post-treatment m~enstrual cycle was of normal duration and
the endccrine pattems were no~nal.
Antibodies to unaltered baboon I~ attained maxin~m levels by about
day 70 of the post-treatment cycle and remained relatively c~nstant until
day 190 when a steady decline was observed. By day 215 of this cycle,
30 altibody levels were barely detectable. Al~proximately 16 days after this
C:
1~3S774'~
time, a peak of LH commensurate with a normal midcycle ele-
vation was observed. From this point the animal appeared to
have the normal function of the pituitary-ovarian axis.
Hormonal patterns in animals with other heter~ogousgonadotropin
immunizations were similar to animal receiving HLH and other
animals receiving monkey or baboon LH were similar in response
to animal receiving baboon LH.
These results in baboons indicated that the mod-
ification of a reproductive hormone, by the procedures outlined,
did render it antigenic and the antibodies thus formed did
neutralize natural endogenous hormones if the natural hormone
was obtained from the species receiving the immunizations
with modified hormone.
EXAMPLE 2
HCG is a hormone naturally present only in pregnant
women. HCG is also commercially available. LH hormone is
immunologically and biologically identical to HCG hormone,
even though there are chemical differences. Since they are
biologically identical and HCG is readily available from
commercial sources it was presumed that the effectiveness of
thls immunological procedure could be evaluated by injecting
modified HCG into non-pregnant women and monitoring the baood
levels of LH. Antibodies formed will neutralize both the LH
and the modified HCG.
Women have a pattern of LH levels; the level is
substantially constant until the middle period between
menstrual cycles, immediately prior to ovulation; at this
point the LH level rises greatly and helps induce the
ovulation. Monitoring the LH level and the antl'body level
will show that the procedure used did or did not cause the
production of antibodie3 capable of neutralizing the endoger.ous
reproductive hormone, namely LH.
1~)S774;~
A women aged 27 years was selected for study.
Hormone was obtained, purified and modified. The modified
human hormone (HCG) was injected into the subject. It is
well known that antibodies to HCG react identically to LH
as well as H~G. The effect of the immunization was elevated,
principally by monitoring blood levels of LH horTnone. Finally
the results were evalutated.
Preparation of Hormone
Clinical grade HCG derived from pregnancy urine
was obtained from the Vitamerican Corp., Little Falls, New
Jersey. This material has an immunological potency of 2600
IU/mg. Contaminants were detected in this preparation.
Purification consisted of chromatography and elution.
Fractions were dialyzed and lyophilized. The most potent
fraction contained approximately 7600 IU/mg., however, ~t
was heterogenous on polyacrylamide gel electrophoresis.
The fraction was further purified by gel filtration.
The elution profile revealed two major protein peaks. The
most potent HCG was found in the first peak and had an
immunological potency of 13,670 IU per mg. This fraction
was subjected to polyacrylamide gel electrophoresis. Further
purification by gel filtration showed no evidence of hetero-
genity of the HCG at this stage. Consequently, materials for
study were processed according to the above procedure.
The contanimation of this purified HCG was tested
with 1131 used for identification and a sample was reacted with
an~isera against several proteins offering potential contam-
ination. Those proteins were follicle stimulating hormone,
human growth hormone, whole human serum, human albumin,
transferi~, alpha one globulin, alpha two globulin and
oro~ornucoid. No detectable binding of the purified HCG was
ob~erved with any antisera at a dilution of 1:50 of each.
- 23 -
l~S774~
These negative results, calculated against potential binding
of the respective proteins, indicated that contamination with
any was less than 0.005 percent.
Alteration _ Hormone
Hormone was altered by coupling with a hapten
(sulfanilazo). This method couples the hapten molecules to
the protein via the amino group of the aliphatic or aromatic
portion of the hapten. The num~er of hapten molecules coupled
to each HCG molecule (Ha-HCG) can be regulated and for this
study, forty haptenic groups per HCG molecule were used for
preparing the immunizing antigen.
Following the hapten-coupling process, the Ha-HCG
was sterilized and tested.
Subject
The subject was multiparous and had terminated her
reproductive capabilities by prior elective bilateral
salpingectomy. She was in good health and had regular cyclic
menstruation. She underwent complete history, physical
examination and laboratory evaluation including blood count,
urinalysis, latex fixation and Papenicolau smear. She had
no history of allergy.
To demonstrate normal functioning of the pituitary-
ovarian axis prior to immunization, blood samples were ob-
tained every other day from the first day of menses for lO
days, then daily for lO days and finally, every other day
until the next menses. Serum determinations of FSH, LH,
extrone, estradiol and progesterone were performed. These
studies indicated an ovulatory pattern.
Immunization Procedures
Ten mg, of ~he Ha-HCG antigen was dissolved in 1.0
ml. of ~aline and emulsified with an equal volume of oil.
Prior to injection, scratch tests to antigen and vehicle were
- 24 -
l~S774;~
performed. Immunizations were bequn in the luteal phase of
the treatment cycle to prevent superovulation from the
administered HCG. Four injections at two week intervals were
given to the subject. The first two of these were administered
in oil subcutaneously (1.0 ml. in each upper arm); the final
two injections were given in saline only via the intradermal
route. Following each injection, blood pressure readings
were taken and the subject observed for allergic reactions.
Monitoring Effects of Immunizations
-
Blood samples were collected at weekly intervals
beginning two weeks after the initial injection to test for
the presence of humoral and cellular antibodies. Following
completion of the immunization schedule, blood samples were
collected in the same manner as in the control cycle to
assess effects of immunizaiton on hormonal patterns of the
menstrual cycle. Since antibodies to HCG react identically to
LH as with HCG, LH was monitored as an index of effectiveness
of the procedure. A third cycle was similarly studied six
months after initial immuni2ation. Upon completion of the
study, physical and pelvic examinations and laboratory
evaluations were repeated.
Serum samples from the control and post-treatment
cycles were assayed for FSH, LH, estrone, estradiol and
progesterone.
The subject was tested for delayed hypertensivity
before immunization and at two week intervals until the
injection schedule was completed by an in vitro lymphocyte
transformation test.
Results
Temporal relationships of serum pituitary and
gonadel hormones in the control cycles of the subject were
recorded. Antibody titers to HCG were detected in the subject
- 25 -
lOS774~
after two injections. Menses occurred at regular intervals
during the immunizations.
Following the initial injection in mannide manoleate,
some itching and swelling at the injection site occurred.
Subsequent intradermal injections in saline produced no
reactions and it was concluded that the local reactions were
induced by the mannide manoleate. Lymphocyte transformation
tests on plasma samples were negative.
In the post-treatment cycle, baseline follicular
and luteal phase LH levels were not noticeably changed in the
subject. Very small midcycle elecations in LH levels were
observed as compared to the normal large increases. FSH
patterns in the post-treatment cycle were normal. This
indicated that the antibodies were neutralizing the action
of endogenous LH.
The subject showed an ovulatory progesterone pat-
tern but attained relatively high antibody titers to LH and
HCG after only two injections of Ha-HCG.
The subject was studied during another cycle
approximately six months from the first immunization.
Significant antibody titers were found. LH patterns in-
dicated a small midcycle elevation. FSH patterns were
essentially normal. Thus, the specificity of anti-HCG anti-
bodies to LH was shown but not to FSH.
EXAMPLE 3
Another woman aged 29 years was selected for further
study. Hormone was obtained, purified, and modified as in
Example 2. This modified hormone was injected into this
subject in the same way as in Example II. The subject was
monitored and tested as in Example 2.
The re~ults were similar to the results found in
Example 2 except that (1) the levels of estrone and estradiol
- 26 -
it)S774~
was substantially normal, (2) the subject acquired significant
antibody titers late in the post-immunization cycle, and (3)
in the cycle studied after six months this subject showed
no significant midcycle elevation in LH patterns.
EXAMPLE 4
Another woman aged 29 years was selected for further
study. Hormone was obtained and purified and modified as in
Example 2. This modified hormone was injected into this sub-
ject in the same way as in Example 2. The subject was moni-
tored and tested as in Example 2.
The results were similar to the results found in
Example 2 except that (l) baseline follicular and luteal p~ase
LH levels were noticeably depressed in the post-treatment cycle,
(2) no midcycle elevations were observed in LH, (3) estrone
levels were elevated during the follicular phase of the post-
immunization cycle, and (4) during the six months study there
was no significant midcycle elevation in LH patterns.
EXAMPLE 5
Another woman aged 35 years was selected for further
study. Hormone was obtained, purified, and modified as in
Example 2. This modified hormone was injected into this sub-
ject in the same way as in Example 2. The su~ect was
monitored and tested as in Example 2.
The results were similar to the results found in
Example 2 except that (l) baseline follicular and luteal
phase L~ levels were noticeably depressed in the post-treat-
ment cycle, (2) a very small midcycle elevations of LH were
observed, (3) levels of FSH patterns in the post-treatment
cycle were depressed, and (4) levels of both estrone and
estradiol were reduced, during the follicular phase of the
post-irmtluni~ation .
- 27 -
lOS774'~
EXAMPLE 6
_ _
Another woman aged 28 years was selected for further
study. Hormone was obtained, purified, and modified as in
Example 2. This modified hormone was injected into this sub-
ject in the same way as in Example 2. The subject was moni-
tored and tested as in Example 2.
The results were similar to results found in
Example 2 except that (1) baseline follicular and luteal phase
LH levels were depressed in the post-treatment cycle, (2) no
peaks were observed in midcycle levels of LH, (3~ estrone
levels appeared elevated in the follicular phase of t~e post
immunization cycle, and (4) LH patterns indicated no signif-
icant midcycle elevation in the six month post-immunization
cycle.
EXAMPLE 7
Another woman aged 28 was selected for furthér
study. Hormone was obtained, purified, and modified as in
Example 2. This modified hormone was injected into this
subject in the same way as in Example 2. The sub~ect was
monitored and tested as in Example 2.
The results were similar to results found in
Example 2 except that (1) antibody titers to HCG were not
detected until after three injections, (2) baseline
follicular and luteal phase LH levels were depressed in the
post-treatment cycle, (3) no peaks nor midcycle elevation
in the LH were observed, (4) estrone levels were elevated
during the follicular phase, and (5) no significant antibody
titers were found in the six month cycle.
All the above examples show the practicality of
injecting modified hormones for the purpose of neutralizing
an endogenou3 reproductive hormone and thereby offering a
procedure for the prevention of conception or the disruption
28
105774'~
of gestation.
EXAMPLE 8
Data obtained in earlier experiments and discussed
in Examples 1 to 7 showed that a modified natural reproductive
hormone, when injected into an aminal of species from which
it was derived, would produce antibodies that would neutralize
the action of the unmodified endogenous natural hormone in
the body of the animal. Hormones used in experiments 1 to 7
were FSH, LH and HCG. New experiments were performed, based
on this knowledge, to identify another reproductive hormone
(placental lactogen) that could be used in a similar fashion.
Preparation of Hormone
_
A purified preparation of placental lactogen was
prepared from placentae of baboons since it was intended to
use modified placental lactogen to immunize baboons.
Placentae were extracted and purified on column chromatograph
according to previously published procedures. The purity
was tested by polyacrylamide gel, electrophoresis and by
radioimmunoassay; The material obtained showed a high degree
of purity on electrophoresis and radioimmunoassay showed no
contamination with other placental hormones.
Hormone Modification and Immunizations
The baboon placental lactogen (BPL) was altered by
coupling with the diazonium salt of sulfanilic acid-~,as out~ined
for other hormones in Example I. The number of diazo molecules
per BPL molecule in this instance was 15. Immunization pro-
cedures were also similar to those described in Example I for
other hormones.
Results
Within 4-6 weeks after the first injection of diazo-
BPL, antibody levels to natural unmodified BPL in vitro were
detected in ~ female baboons. Levels rose to a plateau ~ithin
lOS7'74i~
8-10 weeks and remained there for several months. Hormonal
measurements indicated that there were no effects on the
normal events of the menstrual cycle due to the immuniza-
tions. Since BPL is normally secreted only in pregnancy,
this was not a surprising observation.
All six females were mated with a male of proven
fertility three times (once each in three different cycles
during the fertile period). Pregnancy diagnosis by hormonal
measurement was performed after each mating. From the 18
matings, there were 13 conceptions as judged by pregnancy
tests. The animals that were pregnant had menstrual bleeding
7-12 days later than was expected for their normal menstrual
cycles. Subsequent hormonàl measurements confirmed that
these 13 pregnancies were terminated by abortions app~ x-
imately one week after the time of expected menses.
These findings suggest that the antibodies formed
in the animals body after immunization had no effect on the
non-pregnant menstrual cycle but when pregnancy was estab-
lished, they neutralized the baboon placental lactogen in
the baboon placenta and the result was abortion very early
after conception.
When in Examples 1 to 8 above, structures 1 to 7
~re modified by use of diazosulfanilic acid, dinitrophenol,
or S-aceto mercaptosuccinic anhydride or structures III-VI,
modified by addition of polytyrosine or polyalanine, are
used, similar results may be obtained.
Similarly, when FSH, somatomedian, growth hormone
or angiotension II modified by use of diazosulfanilic acid or
trinitrophenol, the results obtainable upon administration of
the purified modified polypeptide into a male or female human
or animal indicate the stimulation of antibodies which
neutralise all or some of the modified polypept~de as well as
~ 30 -
774~
corresponding endogenous polypeptide.
Example 9
The subjects used in the studies reported in the
example are female baboons. All baboons were adult~ of re~
productive age. A description of subjects and the conditions
of experimentation have been described in Example 1. The
animals were studied using highly purified beta subunits of
HCG using a preparation with a biological activity of less
than 1.0 IU/mg. Animals were immunized with 14-26 moles/mole
of polypeptide, of diazasulfanilic acid coupled subunits in
mannide manoleate.
Antibody levels were assessed by determining the
binding of serum dilutions with I125 labelled antigens.
Cross-reactivity of antisera was measured by direct binding
of labelled antigens and by displacement radioimmunoassays.
Antifertility effects in actively immunized animals were
tested by mating females with males of proven fertility.
Effects in pregnant baboons passively immunized w~th e~t~er
sheep or baboon anti-~-HCG were determined by monitoring
serum levels of gonadotr~pins and sex steroid hormones before
and after immunizations.
Eight female baboons were immunized with the modified
beta subunit of HCG. Significant antibody levels were atta~ned
in all animals.
Baboon immunizations with modified beta subunit of
HCG resulted in high antibody levels reacting to HCG, human
LH and baboon CG but not to baboon LH. All animals remained
ovulatory, however, no pregnancies resulted from numerous
mating~ with males of proven fertility. Passive immunization
of non-immunized pregnant baboons with sheep anti-~-HCG serum
produced a~ortions within 36-44 hours.
- 31 ~
