JPS623128B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides chemical modifications of polypeptides to provide antigens for use in active immunization for the purpose of regulating or treating various physiological processes, particularly reproduction. Polypeptides, particularly proteins, are known to contribute to, influence, or be involved in many physiological processes. For example, certain classes of protein hormones and non-hormonal proteins are known to be necessary for the normal occurrence of phenomena during the reproductive process. It is also known that excess of certain polypeptides, such as gastrin, angiotension or somatomedian, usually causes and influences various pathological conditions and diseases. The present invention describes active immunization methods (i.e. , administration of antigens) to regulate and manage this physiological process. Immunology as a means of regulating reproduction has recently been the subject of various studies. R. G. Edwards, British Medical Journal, Vol. 26, pp. 72-78;
In 1970, in a paper entitled ``Immunization of Conception and Pregnancy,'' he reviewed the literature on this subject, including the production and use of antigens for testicles and spermatozoa. Hormonal antibodies have also been studied for many years, and the effects of specialized antisera have also been recorded. Most of the work to date to achieve immunological methods of contraception has been through passive immunization, ie, injection of antibodies produced elsewhere. Passive immunization of humans is highly limited because repeated injections of animal antibodies into humans are known to cause undesirable reactions in many individuals. Chemical modification of naturally occurring endogenous polypeptides that act in the physiological processes to be processed and regulated results in antibodies that neutralize not only the antigenically modified polypeptides but also their unmodified endogenous counterparts. It has now been found that antigens can be obtained which can also be produced. That is, immunization apparently occurs because the antibodies generated are unable to discriminate between antigenically modified polypeptides and their unmodified endogenous counterparts. The modified polypeptides are produced from naturally occurring endogenous polypeptides in related species, or are immunologically equivalent to the modified polypeptides thus produced. In practice, the polypeptides to be modified are derived from related or closely related species. As used herein and in the claims, the term "modified polypeptides" refers to the polypeptides described below. (a) Protein hormones; (b) non-hormonal proteins; or (c) fragments of (a) or (b) above. These polypeptides are naturally occurring or synthetically produced. The fragment (c) must be of sufficient size to exhibit distinct physical and chemical properties to be recognized as a special part of the total protein in question. These must, of course, have the necessary amino acid composition that can be chemically modified in the desired manner. Accordingly, the present invention provides antigens for use in active immunization methods to treat or modulate physiological processes in specific species, including chemically modified polypeptides derived from endogenous polypeptides (such as An endogenous polypeptide is one that is immunologically equivalent to a chemically modified polypeptide that affects such a physiological process in a species) or derived therefrom;
In addition, the chemical modification is characterized in that it can generate antibodies that biologically neutralize the endogenous polypeptide as well as the chemically modified polypeptide. The present invention also provides a method for producing such an antigen, which is characterized by chemically modifying a suitable polypeptide to the necessary degree. The present invention also provides antibodies produced in response to the action of the antigens of the present invention and antisera containing the antibodies. The invention also provides a method for modulating and influencing physiological processes in a species, which comprises injecting the species with an immunologically effective amount of the antigens or antibodies of the invention. It is something. In particular, the invention provides a method of contraception. Chemically modifiable polypeptides that provide the antigens of the present invention include proteinaceous reproductive hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH),
Refers to such as human placental lactogen (HPL), human prolactin and human hair gonadotropin and specific fragments thereof. Specific fragments of proteinaceous reproductive hormones that can be modified according to the present invention include β of FSH;
- Subunits and specific fragments of natural HPL or human prolactin, which fragments have little similarity to similar parts of other proteinaceous hormones. Preferred fragments include the β-subunit of HCG according to the following two authoritative formulas:

【table】

【table】

[Table] Regarding the specificity of antibody action, it is desirable that the polypeptides to be modified have a molecular structure that is completely or substantially completely different from other proteinaceous hormones. The β-subunit of HCG then has a chain that is very different from the single or multiple chains of amino acids of LH, and such chains are those that are modified according to the present invention. Such chains constitute the 20-30 or 30-39 C-terminal residues of the β-subunit of HCG.
It contains amino acid peptides. More specifically, suitable such chains are: and (C-terminal portion of the above structure) and and (C-terminal portion of the above structure):

【table】

【table】

【table】

[Table] Those with these structural formulas can be obtained by purely synthetic methods or by enzymatic decomposition of the parent polypeptide [Carlson et.al., Journal of
Biological Chemistry, Volume 284 (19),
No. 6810 (1973)]. Polytyrosine chains can be added to the structural formula and the resulting polypeptide modified according to the invention. Another group of polypeptides modified by the present invention are specific non-hormonal protein antigens isolated from placental tissue. Other proteins modified according to the present invention and used in active immunization in the treatment of various pathological conditions and diseases include the hormonal polypeptide known as angiotension, gastrin, growth hormone, somatomedian, and parathyroid. These include the hormones insulin, glucagon, thyroid stimulating hormone (TSH) and secretin. The degree of chemical modification of polypeptides according to the present invention is such that the antigens produced as described above produce antibodies that neutralize not only the antigen but also at least some of its natural endogenous counterpart. , and the type of modification will depend on the nature of the polypeptide involved as well as the particular problem being addressed. If the degree of modification is too small, it will be unclear whether the modified polypeptide is a foreign contaminant, and whether antibodies will be produced against it.On the other hand, if the degree of modification occurs excessively, This will result in the production of antibodies specific to the injected agent that do not neutralize the relevant natural endogenous proteins. Generally, this chemical modification involves the addition of exogenous modifying groups to the polypeptide of interest (hapten conjugation).
It also includes. The number of exogenous modifying groups added depends of course on the surrounding environment, but in general, it is 1 to 40 modifying groups per polypeptide molecule.
Preferably 2 to 40, more preferably 5 to 30, particularly preferably 10 to 26 modifying groups are suitable. Since a given modifying group is added to a particular amino acid position in a peptide molecule, the maximum possible number of a given modifying group added to a given polypeptide can be easily calculated. The nature of this modifying group can be freely selected. Although several modifying groups can be attached to each other and to one amino acid molecule moiety, according to the purpose of the present invention,
Such substitution is considered to be the addition of one modifying group. As mentioned above, this modifying group will vary depending on the surrounding chemical context. Suitable modifying groups include diazo groups, which are preferably introduced by reaction with an appropriate number of moles of diazosulfanilic acid. Methods for introducing diazo groups into proteins are carried out using known techniques, such as those described by Cinader et.al., Journal of the American Chemical Society, Vol. 78, No. 746.
(1955); Phillips et.
al) Journal of Biological Chemistry, Vol. 244, p. 575 (1969); Tabachnick et.al, Journal of Biological Chemistry, Vol. 234 (7),
1726 (1959) or Crampton et.al., Proceedings of the Society for Experimental Biology and Medicine, Vol. 80,
No. 448 (1952). In general, the methods of Chinada et al. and Phillips et al. are superior. Other modifying groups include these polypeptides and dinitrophenol, trinitrophenol, S-acetomercaptosuccinic anhydride, polytyrosine or polyalanine (both linear and branched), biodegradable Polydextran, which can be produced, or, to a lesser extent, those produced by reaction with natural proteins such as thyroglobulin. In general, synthetic modifiers are superior to natural ones. As with many other suitable hapten conjugation reactions,
The above-mentioned reactions are also well known in the field of protein chemistry. The following documents may be cited in this connection: 1. Klotz et.al., Artypus of Biochemistry and Biophysics, Vol. 96, pp. 605-612 (1966); 2 Khorana, Chemical Review, Vol. 53, p. 145 (1953); 3 Sela et.al., The Biochemical Journal, Vol. 85, p. 223 (1962)
4. Eisen et.al., Journal of the American Chemical Society, Vol. 75, p. 4583 (1953); 5. Certano et.al., Federación・Proceedings (Abstract), Volume 25,
No. 729 (1966); 6 Sokolowski et.al., Journal of the American Chemical
Society, Volume 86, Page 1212 (1964
7 Goodfriend et.al., Science, Vol. 144, p. 1344 (1964); 8. Sela et.al., Journal of
The American Chemical Society, No.
78, p. 746 (1955); and 9 Bahl, The Journal of Biological Chemistry, vol. 244, p. 575.
Page (1966). This chemical modification consists of selectively or additionally removing substructures from the polypeptides. For example, if certain natural proteins have carbohydrate groups, they can be processed according to conventional methods, such as with N-acetylneuramidase or N-acetylglucosidase, which are used to remove specific carbohydrate groups. It is done. As mentioned above, the modified polypeptides of the present invention can be used as antigens in active immunization for the regulation and treatment of various physiological processes. Furthermore, the modified protein reproductive hormones or fragments thereof of the present invention are used as antigens in active immunization methods for the purpose of regulating reproduction.
These substances generate antibodies that neutralize not only the antigen but also the natural endogenous proteinaceous reproductive hormones necessary for the phenomena that normally occur in the reproductive process, interrupting the natural process of conception and/or pregnancy. These antigens are therefore used for contraception and for terminating pregnancy soon after conception. Certain non-hormonal protein antigens isolated from placental tissue and materials modified according to the invention are used for this purpose. Direct evidence of suppression of substances that are unique to placental tissue and have no similar antigenic properties in antigens obtained from other organs is the fact that pregnancies can be terminated by passive immunization. This specific placental material, when modified according to the invention, can be injected into individuals of the same species as a fertility control means by active immunization. The advantage of these substances is that placental antigens are not relevant to non-pregnant women and do not pose any risk of cross-reactivity or abortion to normal physical activity in non-pregnant women. It is noteworthy that although the reproductive control methods described above apply primarily to females, some antigens, particularly FSH, its β-subunits and fragments, can also be applied to males when modified according to the present invention. . The gastrin modified according to the invention is used to treat the gastrointestinal disease known as Zollinger-Ellison syndrome by active immunization. This condition is generally described as hypersecretion of the polypeptide gastrin in the pancreas, leading to hyperacidity in the stomach and resulting in chronic gastrointestinal disease. Injection of modified gastrin stimulates the production of antibodies against gastrin hypersecretion and alleviates the disease without surgical intervention, which is currently the only effective treatment. Angiotension modified according to the present invention is an antigen used in the treatment of hypertension. Generally, a hypertensive condition refers to an abnormally high or fluctuating blood pressure. Although an individual's blood pressure is regulated by many physiological processes in the body, one major substance regulating blood pressure is a hormonal polypeptide known as angiotension. In conditions of high blood pressure (hypertension), it is difficult to control the secretion and therefore the concentration of angiotension in the circulatory system by drugs. By appropriately modifying this hormone and then immunizing it with the antigen produced, it is possible to reduce angiotension secretion in patients with chronically high levels of the hormone. Preliminary and controlled reduction of this substance is advantageous for certain patients suffering from chronic hypertension. Growth hormone and somatomedia modified according to the present invention can be used as antigens in the immunological treatment of diabetes and related micro- and macro-vascular diseases. Currently, treatments for diabetes are limited to dietary and/or drug treatments that regulate blood glucose levels. Recent scientific data supports the idea that growth hormone and somatomedia (both polypeptides) are closely related to this syndrome. The antigens of the invention are preferably administered intravascularly in a mixture with a pharmaceutically acceptable liquid medium. The dosage will of course depend on a variety of factors, including the condition and severity being treated, but will generally be administered 1 to 5 times at intervals of 1 to 3 weeks.
A dose of 0.1 to 50 mg is administered. As mentioned above, the present invention theoretically provides that antigenicity can be obtained by chemically modifying a protein that is necessary for reproduction or that affects a specific pathological condition, and that in addition to the antigen, at least a portion of the endogenous protein can be modified. generate antibodies that neutralize the target. In this regard, reproductive hormones of various species were modified and tested on baboons as described in Example 1 below. The results showed that modified hormones from unrelated species did not give the desired results, whereas modified hormones from the same or closely related species showed the desired results. Other Examples 2 to 9 also illustrate the invention. Example 1 Urinary estrogen, blood cell, progestin and urinary LH types during one menstrual cycle of adult female baboons were studied. Only animals that showed a normal pattern of these hormones were immunized. The normal standards and methods of animal husbandry are well known and will not be described here. Gonadotropin Preparations Human Luteinizing Hormone (HLH) - A partially purified preparation obtained from the human pituitary gland with a biological potency of 2.5 units per mg (NIH-LH-SI). Human Follicle Stimulating Hormone (HFSH) - A partially purified preparation obtained from the human pituitary gland with a biological potency of 86 units per mg (NIH-FSH-
SI). Human hair gonadotropin (HCG) - highly purified preparation obtained from human pregnant urine, 13,200 IU/mg
(2nd IRP-
HCG). Monkey Luteinizing Hormone (MLH) - A crude preparation obtained from the pituitary gland of Rhesus monkeys with a biological potency of 0.75 units per mg (NIH-LH-SI). Ovine luteinizing hormone (OLH) (NIH-LH-
S5). Baboon Luteinizing Hormone (BLH) - A partially purified preparation obtained from the baboon pituitary gland with a biological potency of 1.1 units per mg (NIH-LH-SI). All the above formulations except OLH were manufactured in the inventor's laboratory. LH and HCG biological activities were determined with an ovarian ascorbic acid reduction test, and FSH preparations were analyzed with an ovarian enlargement assay. Hormones have been conjugated with haptens in various proportions to provide antigens (see Chinader et al., supra). For convenience, we will discuss the Chinada method here, although Phillips (cited above) has announced a more stable bond under certain conditions. In this method, protein hormones act as carriers to which haptens are conjugated with diazo bonds. Different groups of haptens have been conjugated to different hormones, but the same basic method was used for all combinations. It has been found that the attachment of 15 to 35 haptenic groups per hormone molecule is most convenient for producing immunizing antigens. The basic method was to diazotize the hapten (sulfanilic acid) by adding it to a 0.11N hydrochloric acid solution, and then slowly diazotizing this solution at 4° C. with constant stirring and adding it dropwise into a 1% solution of NaNO ₂ . Completion of diazotization was determined by the detection of free HNO ₂ in the reaction mixture. Although the above reaction was carried out at 4°C, the appropriate temperature for this reaction is usually around 0°C.
to 6°C, preferably 4°C. Hapten-protein conjugation is performed by dissolving the protein hormone in an alkaline buffer solution with a pH of 8.0.The diazotized hapten solution is slowly stirred at 4°C and added to the hormone solution (arbitrary concentration) until the pH is around 8.0. Constantly inspected to maintain the same. After adding all the haptens, the pH is finally 8.0
The mixture was stirred for 1 to 2 hours and left at 4°C overnight. The mixture was completely dialyzed against distilled water for 6 to 8 days to remove unreacted hapten. The number of moles of hapten per hormone molecule is determined by adjusting the number of moles of both reactants, and at the same time, the exact composition of the hapten-protein sample is measured using S ^35- labeled sulfanilic acid to diazotize each molecule. The reaction was tested. The same hormonal preparation used for immunization was used in this control study. After the reaction was completed, a certain amount was taken out from the reaction mixture, and the remainder was completely dialyzed.
Radioactivity was measured by solution scintillation in the same volume of dialyzed and non-dialyzed samples, and the counts of both solutions were compared to calculate the number of moles of conjugated hapten per mole of each hormone. It is calculated as unreacted haptens are removed by dialysis. This calculation estimated a molecular weight of 30,000 for all gonadotropin preparations. After dialysis, the hapten-hormone was freeze-dried at 4°C.
It was stored in Diazo-HCG (35 groups/molecule) and HLH (26 groups/molecule) were bioanalyzed in the ovarian ascorbic acid reduction test and the activity of the unchanged hormone in the original hormone was found to be 62 and 85%, respectively. . Immunization method The first immunization is carried out during the 3rd to 5th day of the female baboon's menstrual period, then the second and third injections are given weekly, and the fourth injection is 2-3 days after the third. Injected a week later. Some animals received a fifth injection 70-80 days after the first injection. All antigens were suspended in mannide manoleate or peanut oil and administered subcutaneously; each injection was administered at a dose of 3.
The dose was varied between 5mg and 5mg. The injection site is 5 after each injection.
Local reactions were examined daily for several days. Examination of the efficacy of immunization Prior to immunization, 24-hour urine and often serum samples were collected daily for at least one menstrual period, and after immunization until the efficacy of the treatment was examined. Urinary LH, urinary estrogen, and blood cell progestin were measured. Antibodies in post-immunization serum samples were determined by reacting 0.2 ml of a 1:1000 dilution of 0.5% normal baboon serum in phosphate-buffered saline (PH 7.4) with 250 pg of ^I131 -labeled hormone. was detected. Serum and unchanged immunizing hormone and intact baboon LH for antibody detection.
were reacted with both. Purified baboon LH preparation (1.9×
NIH-LH-SI) was used for antigen tracking. The antigen-antibody complex was precipitated with sheep anti-Higanma globulin after 24 hours at 4°C. Antibody content is expressed as Pg of labeled hormone conjugate. Significant antibody content was considered to be 5.0 Pg or more of I ¹³¹ labeled antigen bound. Antisera were from Fahey and Terry (Fahey &
Terry) [Experimental Immunology,
Published by FADavis Co., Philadelphia, Pennsylvania, 1967.
The proportions of 1 gM and 1 gG antibodies in baboon serum were determined by fractionation by gel filtration using Sephadex G-200 (trade name) according to the method described in [See page 36]. Since the IgG fraction contains IgA and IgD antibody portions, only IgM and total titers were determined. The IgM fraction obtained from the column was reacted with I ¹³¹ hormone to determine binding ability. The volume of fractionated serum was adjusted to allow comparison with antibody content in whole serum. Antibody Generation No appreciable reactions were found at the injection site after either immunization. In 4 cases, slight induration (2-3 cm in diameter) was observed when mannide manoleate was used as the vehicle, and the redness and swelling disappeared within 4-5 days. 3- for all animals
Antibodies to the immunizing antigen were detected within 5 weeks and the extent, duration and cross-reactivity of these antibodies were recorded. In general, there were higher concentrations of heterogeneous compared to homogeneous gonadotropin immunizations. The cross-reactivity caused by baboon LH antibodies was studied for each animal and the cross-reactivity of the antiserum at the highest concentration was recorded. Relatively high antibody activity against human LH and HCG was observed, but
In the case of LH, relatively little activity was observed. In the case of anti-sheep LH, an intermediate cross-reaction was observed;
A high degree of cross-reactivity was observed in the case of anti-monkey LH.
Diazo-human FSH was weakly antigenic in baboons. Human and sheep gonadotropin immunizations produced longer periods of antibody production than monkeys or baboons. The peak in antibody content usually occurred when antibodies were mainly transferred to the IgG type. Early antibodies were mostly of the IgM type, but there were generally many cross-reactions with baboon LH. Changes in total antibody propagation or IgM proportions were recorded from the time antibodies were first detected. A clear cross-reactivity to baboon LH was observed in the anti-human gonadotropins when IgM was high, but this decreased sharply as the antiserum was almost entirely converted to IgG. This reduction in cross-reactivity did not occur with monkey and baboon immunizations. Furthermore, sheep
After LH immunization, there was a moderate change in reactivity as the transition from IgM to IgG occurred. These results are shown in detail in Tables 1 and 2 below.

【table】

【table】

[Table] Effect on the menstrual period The effect of immunization on the menstrual period was measured by observing changes in the swelling of the genital skin and the content of the pituitary gland and/or the content of ovarian hormones. Based on these indicators, the delay in ovulation from the expected period was determined and calculated based on the regulation cycle. In some animals immunized with HCG, ovulation was not prevented, whereas in other animals immunized with HFSH, it was delayed by one cycle. Two animals injected with HLH and two animals injected with HCG delayed ovulation by two menstrual cycles.
The third animal with HLH injection was delayed by 86 days. Ovine LH-immunized animals had an 88-day ovulation delay. Immunization with diazosar or baboon LH resulted in disruption of the long menstrual cycle. The ovulation delays in the two animals injected with monkey LH were 146 and 122 days, compared to 224 and 210 days in the animals receiving the modified baboon LH. Effects on specific hormone types following HLH immunization were recorded in one animal. The interval between menstrual periods is considered to represent a "cycle." Urinary estrogen and blood cell progestin levels persisted, indicating that ovulation did not occur during the 85-day immunization cycle. Urinary estrogen increased during the treatment period but did not follow a typical pattern. Blood cell progestin did not rise until approximately day 19 of the first post-treatment cycle. Both estrogen and progestin patterns remained within normal ranges throughout the cycle after the second treatment. The content of antibodies increased from about day 35 of the treatment cycle to day 289 of the first antibody detection. LH analysis was not applied when this animal was examined and no data were available on blood cell and urine concentrations of this hormone. Hormone type was recorded after immunization with diazo baboon LH. In this animal, antibody concentrations were lower than when immunized with human gonadotropins;
In general, the duration was also short. During the treatment cycle, urinary estrogen and blood cell progestin concentrations were normal but in low amounts. During this period, the urinary LH type was clearly changed by injection of diazo LH, but no reliable evidence of ovulation was obtained during this period. The first post-treatment cycle lasted 246 days. During this period, urinary LH and estrogen rose for 35 to 41 days, but there was no subsequent rise in blood cell progestin indicating that ovulation had occurred. After day 42 of the cycle, no increase in any of the three hormones was observed until day 231, when a clear increase in urinary estrogen and LH occurred. Three days after these increases, an increase in blood cell progestin was observed, indicating that the corpus luteum was at work. After the second treatment, the menstrual cycle was of normal duration and the endocrine pattern was normal. Antibodies against unchanged baboon LH were detected at approximately
The highest concentration was reached on day 70 and remained relatively constant until day 190, with a steady decline. 215 of this cycle
Antibody content is evident until day 1, and from this time on about 16 days LH increases commensurate with the normal mid-cycle rise.
peak was observed. From this time on, the animals were observed to exhibit normal activity of the pituitary-ovarian axis. Hormone type of other heterogeneous gonadotropin-immunized animals was similar to that of HLH-treated animals, and other animals treated with monkey or baboon LH showed response types similar to those of baboon LH-treated animals. . Examples of these results are 1A, 1B, 2A,
Shown in Figures 2B and 2C. These results in baboons suggest that the aforementioned modifications of the reproductive hormones render them antigenic and that the antibodies thus generated are linked to naturally occurring endogenous sex hormones, unless the natural hormones are modified by a species individual immunized with a modified hormone. As it is obtained from
This shows that it has neutralized the Example 2 HCG is a hormone that occurs naturally only in pregnant women and is also available commercially. The LH hormone is immunologically and biologically identical to the HCG hormone, although there are chemical differences. Both are biologically identical and HCG
Since LH is available on the market, it was expected that the effectiveness of this immunological method could be assessed by injecting modified HCG into non-pregnant women and testing the blood levels of LH. The antibodies generated will neutralize both LH and modified HCG. Women have a stereotype of LH concentrations, ie, concentrations are essentially constant until midway through the menstrual cycle and just before ovulation, when LH concentrations rise significantly to stimulate ovulation. Testing LH and antibody levels will tell you whether you have produced or not produced antibodies that can neutralize endogenous reproductive hormone (LH). A 27-year-old woman was selected as the subject. Purified and modified hormones. Modified human hormones (HCG) were administered to women. It is well known that antibodies against HCG have the same effect as LH. Testing mainly on the blood concentration of LH hormone showed that the immune effect increased. Finally, the results were evaluated. Hormone Production Vitamerican Corp., Little Falls, New Jersey
Made from pregnant women's urine obtained from
Clinical grade HCG with an immunological titer of 2600 IU/mg was used. Since this preparation contained impurities, it was purified by chromatography and elution, and fractions were dialyzed and freeze-dried. The potency of the most potent fraction is approx.
7600 IU/mg, but was heterogeneous according to polyacrylamide gel electrophoresis. This fraction was further purified by gel filtration, and the eluate contained two main protein peaks. The highest titer HCG was present in the first peak, with an immunological titer of 13670 IU/mg. This was subjected to polyacrylamide gel electrophoresis and further gel filtration to eliminate HCG heterogeneity. This was used for research using the above method. The impurity of this purified HCG was assayed using I ¹³¹ ,
Antisera against some proteins showing strong impurity were reacted. These proteins were follicle stimulating hormone, human growth hormone, whole human serum, human albumin, transferrin, alpha 1 globulin, alpha 2 globulin and orosomucoid. There was no sign of purified HCG binding to any of the antisera at a dilution of 1:50.
This negative result is calculated based on the binding strength of each protein, and the impurity of each is 0.005%.
It was shown that: Modification of hormones Hormones were modified by coupling them with hapten (sulfanylazo). That is, the protein and hapten molecule are coupled together via the amino group of the aliphatic or aromatic portion of the hapten. each
The number of hapten molecules that couple with HCG molecules (Ha
-HCG) can be prepared and in this experiment 40 hapten groups per HCG molecule were used to produce the immunizing antigen. Following the hapten-capture step, Ha-HCG was sterilized and subjected to testing. Target woman The target woman was a multiparous woman who had already lost her fertility due to electrical bilateral salpingectomy, and was in good health.
Her menstrual cycles were normal. The woman's past history was examined, as well as physiological examinations (blood tests, urine analysis, latex adsorption, Papanicolaou smear, etc.). The woman had no history of allergies. Prior to immunization, blood was collected every other day for 10 days from the first day of menstruation, then every day for 10 days, and finally every other day until the next menstruation, to demonstrate the normal functioning of the pituitary-ovarian axis. Serum FSH, LH, estrone, estradiol, and progesterone were assayed, and these results were typical for ovulation. Immunization method: Dissolve 10 mg of Ha-HCG antigen in 1.0 ml of saline,
Suspensions were made in equal volumes of oil and a drag test for antigen and solvent was performed prior to injection. Immunization was started during the ovarian luteal phase of the treatment cycle to prevent overeating with the administered HCG. Four injections were given at two week intervals. Two of these were injected subcutaneously in oil (1.0 ml in each upper arm); the other two were injected intradermally in saline. Blood pressure was checked after each injection to check for allergic reactions. Assay of Immunization Efficacy Blood was drawn weekly starting 2 weeks after the first injection to test for the presence of antibodies in body fluids and cells. Once the immunization schedule was completed, blood was collected as in the control cycle to determine the effect of immunization on the hormonal pattern of the menstrual cycle. LH was tested as an indicator of the effectiveness of this method since antibodies to HCG act similarly to LH to HCG. The third cycle was examined 6 months after the first cycle. After this was completed, the physiological and pelvic examination and laboratory evaluation were repeated. FSH, LH, and blood from control and post-treatment cycles
Analysis of estrone, estradiol and progesterone was performed. An experimental lymphocyte transfer assay was used to examine the delay in hypertensive levels between women's immunization and completion of the injection schedule every two weeks. Results Temporal correlations of serum pituitary and gonadal hormones during control cycles were recorded. Antibody titers against HCG were assayed after two injections. Menstrual cycles remained normal during immunization. Itching and swelling were observed at the injection site after the first injection of mannide manoleate. No reaction occurred after the next intradermal injection in saline, and this local reaction was determined to be due to mannide manoleate. The results of blood cell lymphocyte transfer test were negative. Basal follicular and ovarian luteal phase LH during post-treatment cycle
There was no noticeable change in content. The increase in LH content was small in the middle of the cycle compared to the normal increase. FSH type during the post-treatment cycle was normal. This indicates that the antibodies neutralize the action of endogenous LH. The female subject showed ovulatory progesterone, but only two injections of Ha-HCG reduced LH and
It showed that the titer of antibodies against HCG became relatively high. The women continued to be tested during cycles for approximately 6 months after the initial immunization and had satisfactory antibody titers. LH type showed an increase in the middle of the cycle. FSH type was essentially normal. Thus, anti-LH
The specificity was shown to be that it acts like an HCG antibody but not against FSH. Example 3 Another 29 year old woman was selected. Hormones were prepared, purified, and modified as in Example 2. Example 2
This modified hormone was injected into women, as in
Inspected and tested. The results were similar to those of Example 2, but
(1) the estrone and estradiol contents were essentially normal; (2) the woman obtained a clear antibody titer at the end of the cycle after immunization; and (3) the LH type during the 6-month later cycle. The difference was that no obvious mid-cycle increase was observed. Example 4 Another 29 year old woman was selected. Hormones were produced as in Example 2, purified, modified, administered as in Example 2, and subjects were examined and tested. The results were similar to those of Example 2, except that (1) the LH content in the basal follicle and ovarian luteal phase clearly decreased during the post-treatment cycle, and (2)
No mid-cycle increase in LH was observed, (3) estrone content increased during the follicular phase of the post-treatment cycle, and (4) no mid-cycle increase in LH type was evident during the 6-month study. . Example 5 Another 35 year old woman was selected for the study. Hormones were produced as in Example 2, purified, modified, injected intravaginally as in Example 2, and tested. The results were similar to those of Example 2, except for the following points. (1) The LH content of basal follicles and ovarian luteal phase decreased obviously during the post-treatment cycle. (2) There was a very slight increase in LH in the middle of the cycle. (3) The type of FSH content during the treatment cycle decreased. (4) The contents of both estrone and estradiol decreased during the follicular phase after immunization. Example 6 Another 28 year old woman was selected for the study. Hormones were manufactured as in Example 2, purified, modified, injected into women as in Example 2, tested,
Tested. The results were similar to those of Example 2, except for the following points. (1) The LH content of basal follicles and ovarian luteal phase during the post-treatment cycle decreased. (2)
No peak in the middle of the LH cycle was observed. (3) Estrone content in the follicular phase of the post-immunization cycle appeared to be increased. (4) No obvious mid-cycle increase in LH type was observed during the 6-month post-immunization cycle. Example 7 Another 28 year old woman was selected for the study. Example 2
Hormones were produced, purified, modified, injected into women as in Example 2, tested,
Tested. The results were similar to those of Example 2, except for the following points. (1) Until after 3 injections
No antibody titer against HCG was observed.
(2) basal follicular and ovarian luteal phase during the post-treatment cycle;
LH content decreased. (3) No LH peak was observed in the middle of the cycle. (4)Estrone content increased during the follicular phase. (5) No obvious antibody titers were found during the 6-month cycle. All of the previous examples demonstrated the practicality of injecting modified hormones to neutralize endogenous reproductive hormones. So there is practicality to methods of preventing conception and destroying pregnancies. EXAMPLE 8 Natural reproductive hormones modified according to the foregoing experiments and materials discussed in Examples 1 to 7, when injected into an animal of the species from which it was derived, remain unmodified endogenously in the body of that animal. It has been shown to produce antibodies that neutralize the effects of natural hormones.
The hormones used in the experiments of Examples 1 to 7 were FSH, LH and HCG. Based on this finding, new experiments were conducted to identify other reproductive hormones (placental lactogens) that could be used as well. Hormone Production A purified preparation of placental lactogen was produced from baboon placenta for the purpose of using the modified placental lactogen to immunize baboons. Briefly, placenta was extracted and purified on a column chromatograph by previously published methods, and purity was tested by polyacrylamide gel electrophoresis and radioimmunoassay. The obtained material was found to be highly pure and free from contamination with other placental hormones by electrophoresis and radioimmunoassay. Hormone Modification and Immunization Baboon placental lactogen (BPL) was modified by conjugation with diazonium salts of sulfanilic acid as described for other hormones in Example 1. The number of diazo molecules per BPL molecule was 15 in this example. The method of immunization is similar to that described in Example 1 for other hormones. Results The content of antibodies against native unmodified BPL was detected in vitro in 6 female baboons within 4-6 weeks after the first injection of diazo-BPL. Within 8-10 weeks the content increased and remained constant for several months. Hormone measurements showed that the immunization had no effect on the normal state of the menstrual cycle. Since BPL is secreted only during pregnancy, the above findings were not particularly new. All six females were mated with one fertile male and given three opportunities for fertilization (one for each of three separate cycles during the fertilization period). After each copulation, hormones were measured to diagnose pregnant women. Pregnancy tests confirmed 13 conceptions from 18 matings. Pregnant animals experienced menstrual bleeding 7-12 days later than scheduled for their normal menstrual cycle.
Subsequent hormone measurements revealed that these 13 pregnant animals aborted approximately 1 week after their scheduled menstrual period. These findings suggest that antibodies produced after immunization do not affect the menstrual cycle of non-pregnant animals, but in the case of pregnancy, they neutralize the baboon placental lactogen in the baboon placenta, causing miscarriage immediately after conception. has been proven. In the case of Examples 1-8 above, 1 to 7 are modified with diazosulfanilic acid, dinitrophenol or S-acetomercaptosuccinic anhydride, and the structural formula is modified by adding polytyrosine or polyalanine, Both methods yield similar results. Similarly, FSH, somatomedium, growth hormone or angiotension is modified with diazosulfanilic acid or trinitrophenol and this purified modified polypeptide is administered to a male or female human or animal. The result is to stimulate the production of antibodies that neutralize all or some of the modified polypeptide as well as the corresponding endogenous polypeptide. Example 9 The experimental animals in this example were baboons, all of which were adults in the reproductive period. The animals and experimental conditions are the same as in Example 1. A highly purified β-subunit preparation of HCG of less than 1.0 IU/mg was used in the experiment. Animals were immunized with 14 to 26 mol/mol of polypeptide subunits conjugated with diazosulfanilic acid in mannide manoleic acid. Antibody content was determined by binding of diluted serum with I ¹²⁵ labeled antigen. Antiserum cross-reactivity was measured by direct binding of labeled antigen and radioimmunoassay. Antifertility efficacy in effectively immunized animals was tested by mating females to fertile males. Sheep or baboon anti-β-HCG
The efficacy was determined by measuring the content of serum gonadotropins and sex steroid hormones before and after immunization in pregnant baboons passively immunized with . Eight female baboons were immunized with the β-subunit of modified HCG and clear antibody content was observed in all baboons. High antibody content acting against HCG, human LH and baboon CG was observed in baboons immunized with the β-subunit of modified HCG, but no antibody content against baboon LH was observed. All animals retained the ability to ovulate, but pregnancy did not occur in many females mated to fertile males. Nonimmunized pregnant baboons passively immunized with sheep anti-β-HCG serum aborted within 36 to 44 hours. The configuration and embodiments of the present invention are summarized as follows. (1)(a) Endogenous polypeptides that affect the physiological processes of specific animal species (including humans); (b) fragments of the above endogenous polypeptides; (c) modified endogenous polypeptides when modified; An unmodified polypeptide selected from polypeptides that are immunologically equivalent to its fragments is chemically modified, and the degree of modification is determined such that the modified polypeptide is isolated from the endogenous polypeptide in the animal species. The chemical modification is capable of inducing the production of antibodies that biologically neutralize the modified polypeptide, and the chemical modification comprises: (i) attaching one or more exogenous modifying groups to the unmodified polypeptide; or ( ii) A method for producing an antigen for use in an active immunization method for regulating or treating a biological process in the above animal species, characterized in that one or more substructures are removed from the unmodified polypeptide. (2) The method according to Structure/Embodiment Item 1, which chemically modifies a fragment of an endogenous polypeptide. (3) The method according to Structure/Embodiment 1, wherein the chemical modification comprises removing one or more partial structures from the unmodified polypeptide. (4) The antigen is used in an active immunization method for the regulation of fertility, and the unmodified polypeptide is a proteinaceous reproductive hormone, (b) a fragment thereof, or (c) a proteinaceous reproductive hormone or its fragment. The method according to configuration/embodiment item 1, wherein the polypeptide is immunologically equivalent to the fragment. (5) The unmodified polypeptide contains follicle-stimulating hormone,
5. The method of embodiment 4, wherein the luteinizing hormone is selected from human placental lactogen, human prolactin, or human hair gonadotropin. (6) The method according to Structure/Embodiment Item 5, wherein the unmodified polypeptide is follicle stimulating hormone. (7) The method according to Item 6, wherein the unmodified polypeptide is selected from follicle stimulating hormone or its beta subunit. (8) The method according to Structure/Embodiment Item 5, wherein the unmodified polypeptide is selected from human placental lactogen or human prolactin. (9) The method according to Structure/Embodiment Item 5, wherein the unmodified polypeptide is selected from human dichohair gonadotropin or a fragment thereof. (10) The method according to Structure and Embodiment Item 9, wherein the unmodified polypeptide is human dichorionic gonadotropin. (11) The method according to Structure/Embodiment Item 9, wherein the unmodified polypeptide is a fragment of human dichorionic gonadotropin. (12) The method according to Structure/Embodiment item 9, wherein the unmodified polypeptide is the beta subunit of human hair gonadotropin. (13) The method according to Item 9, wherein the unmodified polypeptide is selected from a C-terminal fragment of 20-30 amino acids in the beta subunit of human dichohair gonadotropin. (14) The method according to Item 9, wherein the unmodified polypeptide is selected from a C-terminal fragment of 30 to 39 amino acids in the beta subunit of human dichohair gonadotropin. (15) Unmodified polypeptide has the structural formula 9. The method according to configuration/embodiment item 9, which has the following. (16) Unmodified polypeptide has the structural formula The method according to Structure/Embodiment Item 9, which has the following. (17) Unmodified polypeptide has the structural formula 9. The method according to configuration/embodiment item 9, which has the following. (18) Unmodified polypeptide has the structural formula The method according to Structure/Embodiment Item 9, which has the following. (19) The method according to any one of Arrangements/Embodiments 1 to 18, wherein the chemical modification comprises bonding one or more exogenous modifying groups to the unmodified polypeptide. (20) The method according to Structure/Embodiment Item 19, wherein 1 to 40 modifying groups are bound per mole of unmodified polypeptide. (21) The method according to Structure/Embodiment Item 20, wherein 2 to 40 modifying groups are bound per mole of unmodified polypeptide. (22) The method according to Structure/Embodiment No. 20, wherein 5 to 30 modifying groups are bonded per mole of unmodified polypeptide. (23) The method according to Structure/Embodiment Item 21, wherein 10-26 modifying groups are bound per mole of unmodified polypeptide. (24) The exogenous modifying group is a diazo group, or dinitrophenol, trinitrophenol, S-acetomercaptosuccinic anhydride, polytyrosine,
The method according to Structure/Embodiment No. 4-23, wherein the group is derived from polyalanine, biodegradable polydextran, or thyroglobulin.

[Brief explanation of the drawing]

Figures 1A, 1B, 2A, 2B and 2C are all diagrams showing the effect of immunization on the menstrual period in Example 1. In addition, Figures 1A and 1B show the immunization effect of female baboons (11-66) using diazo-human LH based on the hormonal pattern of the menstrual cycle, and Figures 2A, 2B, and 2C show the immunization effect based on the hormonal pattern of the menstrual cycle. Figure 2 shows the immunization effect of female baboons (1-68) using diazo-baboon LH based on .

Claims (1)

[Scope of Claims] 1. (a) An endogenous polypeptide that affects the physiological process of a specific animal species (including humans); (b) a fragment of the endogenous polypeptide; (c) a fragment of the endogenous polypeptide when modified; An unmodified polypeptide selected from polypeptides that are immunologically equivalent to the native polypeptide or its fragments is chemically modified, and the degree of modification is such that the modified polypeptide is within the above-mentioned range in the above-mentioned animal species. The chemical modification is capable of inducing the production of antibodies that biologically neutralize the raw polypeptide and the modified polypeptide, and the chemical modification comprises: (i) attaching one or more exogenous modifying groups to the unmodified polypeptide; or (ii) the production of an antigen for use in active immunization for the regulation or treatment of biological processes in the above animal species, characterized in that one or more substructures are removed from the unmodified polypeptide. Method.