CA1223206A - Antigenic modification of polypeptides - Google Patents

Abstract

ANTIGENIC MODIFICATION OF POLYPEPTIDES
Abstract of the Disclosure Vaccines are described comprising antigens as described in U. S. Patent No.
4,201,770 or Canadian Patent No. 1,057,742 or 1,085,383 in Arlacel/Squalene or Squalane vehicles, optionally with the addition of immuno-stimulating adjuvants.
Antigens having similar activity may be produced by the linear polymerization of peptide fragments. Such vaccines may be used for contraception or treatment of hormone-related disease states and disorders, including hormone-associated car-cinomas.

Description

~L~Z32~6 ANTI~ENIC MODIFICATION OF POLYPEPTIDES
.

Background of the Invention -It is well known that antibodies are generated in humans and in other animals in response to the presence of foreign antigens. It is also known to confer immunity Oil an animal by administering an antibody formed elsewhere. For instance, patents 05 to Michelson (U.S. 3,553,317), Freedom (U.S. 2,388,260), Rouser (U.S. Lowe) and Peterson (U.S. 39376,198) relate to production of antibodies, which when injected into an animal of a different species or into a human being cause passive immunization. In patents to Fell (U.S. 2,301,532 and U.S. 2,372,066~, the patentee refers to active immunization using modified histamine in such animals as horses, 10 cows, etc. In a paper by R. G. Edwards in the British Medical Journal, Vol. 26, pages 72 to 78, published in 19~0 on "Irrlmunology of Conception and Pregnancy", he surveys the literature regarding the possibilities of utilizing immunological methods to influence or control fertility, surveying first production of antibodies against testes or spermatozoa. Much of the literature surveyed is directed to the 15 production of foreign antibodies which are injected into the subject (passive immunization Hormone antibodies have been studied for a long time and the effect of specific antisera have been recorded for many years. It is known that ad mini-striation of certain antibodies during pregnancy can suppress implantation or cause 20 fetal resorption. Several different approaches have been tried ranging from the induction of near permanent infertility in the case of agglutination of spermatozoa in the male to the disturbance of a single pregnancy by passive immunization with antibodies.
There are serious limitations to the use of passive immunization procedures 25 for human therapy. Since the antibodies are practically produced only in non-human animals, the repeated injection of animal proteins into humans is known to produce serious reaction in many individuals.
British patent Specification No. 1,058,82~ discloses that small molecules, referred to as "serological determinant peptizes", Jan be coupled to large protein 30 molecules, such as cattle albumin and the resultant conjugate then may be injected into animals for antibody production. The document lists proteins from which theserological determinant peptizes may be isolated prior to being used in the process taught, the collection including viruses and bacteria whose surface comepotent has the characteristics of a protein, toxins and hormones having protein I

23;~

structure and enzymes. My specific hormone is named in the document and no utility of anti hormone immunization is described. The patent specification references a publication entitled: "The Specificity of Serological Reactions", Dover Publications, Inc., New York, 1962, Chapter V, "Artificial Conjugated Antigens" by 05 I Land Steiner. This publication outlines various chemical methods and applies them passively to bind various toxic substances in the blood such as arsenic.
Thyroxine data provided in the publication suggests that such methods may be applied to hormones without indicating the therapeutic application, the publication teaching that specific antibodies may be formed to the small molecules and these10 antibodies are capable of neutralizing the biological action of a large protein from which the small peptize was a part.
Recently it has been discovered that doses of certain steroids consisting of synthetic non-protein hormones ("The Pill") when administered at stated intervals usually confer protection against pregnancy for a short time (possibly a month).15 This medication has sometimes been found to create undesirable side effects in creating undesirable metabolic changes and sometimes changes in the blood clotting mechanisms. Moreover, the effect of each dose is of such short duration that often it is of limited application, particularly in remote areas to persons not readily instructed Oil proper and contimling use.
There is need therefore of an effective safe method of creating a temporary but relatively long-time immunity against pregnancy which does not have serious side effects. There is also a need for an effective safe method of terminating apregnancy soon after conception which does not have serious harmful side effects.
Such need may be met by the neutralization of a reproductive protein which is 25 necessary for the normal events of conception and/or gestation There is also a need for a means for control of various disease states or maladies caused or influenced by unusual excesses of certain polypeptides such as gastric, angiotension II, or somatomedian.
In my United States Patent No. 4,201,770, and in Canadian Patents 1,~57,742 30 and 1,085,383, it is disclosed that antigens useful for controlling fertility or treating a disease in an animal (which term is used herein to include a human being may be formed by chemically modifying an endogenous protein of the animal or a fragmentthereof on a palpated which is immunologically equivalent to such a protein or fragment. The chemical modification requires coupling the protein, fragment or 35 palpated to a non-endogenous carrier, such as a bacterial toxoid. The alone-mentioned patents suggest that, apart from fertility control, such antigens may be - ~223~

useful in the treatment of diseases such as hypertension, diabetes and Zollinger-Allison syndrome. The aforementioned U.S. patent also suggests that the antigensmay be useful in treating certain breast and endocrine dependent breast tumors and cancers, and certain other cancers known to be associated with factors immulogical-05 lye similar to Human Chorionic Gonadotropin (HOG).
I have now discovered that the effectiveness of the aforementioned antigen scan be greatly increased by incorporating them into vaccines with certain selected vehicles and, optionally, immunostimulating adjutants. I have also discovered that similar antigens may be formed by linearly polymerizing palpated fragments 10 rather than coupling such fragments to carriers. Finally, there is also included herein experimental proof that such antigens are useful in treating at least one type of carcinoma.

us Accordingly, in one aspect this invention provides a vaccine comprising:
a modified palpated for isoimmuniogicaLly controlling biological action in a mammal by antibody formation, consisting of a protein hormone, a non-hormonal protein, or a fragment of either which has been chemically modified outside the body of said mammal, said protein hormone, non-hormonal protein or fragment having the properties of:
(a) in unmodified form, being non-immunogenic to said mammal and having a molecular structure similar to an endogenous protein hormone or a non-hormonalprotein, the biological function of which it is desired to inhibit, or fragment of either and (b) in modified form, causing antibodies to be formed in the body of the 25 mammal which inhibit the biological function of said endogenous protein hormone or non-hormonal protein following administration of the modified form into the body of said mammal; and a vehicle, said vehicle comprising a mixture of Arlacel A (Monday moonlit) with Sgualane and/or Skyline In a further aspect, this invention provides a modified palpated for isoimmunologically controlling biological action in a mammal by antibody formation, said modified palpated comprising a linear polymer of pupated fragments, each of said fragments, in its monomeric form, being non-immunogenic to said mammal and having a molecular structure similar to a fragment of an endogenous 35 protein hormone or a non-hormonal protein, the biological function of which it is ~223~

desired to inhibit, and said linear polymer causing antibodies to be formed in the body of the mammal, which antibodies inhibit the biological function of said endogenous protein hormone or non-hormonal protein following administration of said linear polymer into the body of said mammal.
OX Finally this invention provides a method for producing a modified palpated for isoimmunologically controlling biological action in a mammal by antibody formation, said method comprising:
(a) procuring a first peptize which is non-immunogenie to said mammal and has a molecular structure similar to a fragment of an endogenous protein or non-10 hormonal protein, the biological function of which it is desired to inhibit, said first peptize not having an unblocked C-terminal Sistine and having a free amino grouponly at its N-terminal;
(b) reacting said first peptize with an amino-group activating agent, thereby producing an activated amino group at the N-terminal of said first peptize, (c) reacting said activated first peptize with a second peptize having a C-terminal Sistine bearing a fresh group, thereby coupling the N-terminal of said first peptize to the C-terminal of the second peptize;
(d) reacting the resultant compound with an amino-group activating agent with an amino-group activating agent, thereby producing an activated amino-group20 at the N-terminal of said compound;
(e) reacting said activated compound produced in step (d) with a further peptize having a C-terminal Sistine bearing a fresh group, thereby coupling the activated N-terminal of said compound to the C-terminal of said further peptize;and (f) repeating steps (d) and (e) until a desired polymer length has bee achieved.
In the vaccines of my invention, the vehicle is preferably a mixture of Arlacel A and Skyline. Preferred adjuvan~s are muramyl dipeptides, especially:
Nunnery Mur-L.Ala-D.isoGln;
NAc-Mur-(6-0-stearoyl)-L.Ala-D.iso~ln; or NGlycol-Mur-L.o~Abu-D.isoGln Preferably, the unmodified form of the fragment used in my vaccines comprises a fragment of Human Chorionic Gonadotropin, preferred fragments being: Thr-Cys-As~As~Pro-Ar~Phe-~ln-Asp-Ser-Ser-Ser-Ser-LLucille-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Ar~Leu-Pro-Glyy-Pr~Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln;

32~i Cys-Pro-Pro-Pro-Pro-Pro-Pro-As~As~Pro-Ar~Phe-Gln-AAsp-Ser-Ser-Ser-Lys-Ala-Pr~Pro-Pro-Ser-Leu-Pro~Ser-Pro-Serr-Arg-Leu-Pro-Gly-Pro-Ser-~sp-Thr-Pro-Ile-Leu-Pro-Gln ~sp-~sp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Alla-Pro-Pro-05 Pro-Ser-Leu-Pro-Ser-Pro-Ser-~rg-Leu-Pro-Gly-Pro-Seer-As~Thr-Pro-Ile-Leu-Pro-Gln-Pr~Pro-Pro-Pro-Pro-Pro-Cys;
Asp-His-Pro-Leu-Thr-Cys-~sp-As~Pro-Ar~Phe-Gln-Asp--Son-Ser-Ser-S~r-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Prrouser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Glin; and lo Cys-Asp-HisPro-Leu-Thr-CysAsp-Asp-Pro-l~r~Phe-Gln--Asp-Ser-Sorcery Ser-Lys-Ala-Pro-Pro-Pr~Ser-l.eu-Pro-Ser-Pro-Ser-Ar~Leu-Pr~Gly-Pr~Ser-~sp-Thr-Pro-Ile-Leu-Pr~Gln Preferred fragments for forming the linear polymers of my invention are:
Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Alla-Pro-Pro-Pr~Ser-Leu-Pro-Ser-Pro-Ser-Ar~Leu-Pro-Gly-Pro-Ser--Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys and Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-~ln-Assp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Seer-Arg-Leu-Pr~Gyl-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Cys In my method for forming these linear polymers, desirably the first and second peptizes are of` the same configuration but the first peptize has the ASH group on its C-terminal Sistine blocked.
As already stated the vaccines of my invention may be used to treat Zollinger-Allison Syndrome. This syndrome or disease state is generally described as a 25 condition in which a hypersecretion of the palpated gastric, which is produced in the pancreas, brings about a state of hyperacidity in the stomach which results in a chronic digestive disorder. Heretofore, the only effective treatment for this disease state was the surgical removal of a part or total removal of the subject's stomach. Although survival of such patients is usually not threatened, the medical 30 state and life style of such individuals is severely affected by such treatment.
Treatment of such subjects with hasten coupled (produced according to the general method described herein) or otherwise chemically modified gastric can beused to enhance the production of antibodies against the hypersecretion of gastric and thereby alleviate or reduce the symptoms of this disease without surgical 35 intervention. Sufficient reduction by immunological means of this substance in the system of the body would be sufficient to avoid the complicated and serious ~Z3;~

consequences of the surgical treatment currently in use. In practice, an effective amount of modified gastric is simply injected into the patient as required to accomplish the control of the flow or presence of gastric.
another serious medical problem which is treatable by the application of the 05 vaccines of my invention is that of hypertension. In general terms, the state of hypertension is the abnormal level or fluctuation of one's blood pressure. The blood pressure in an individual is controlled by many physiological processes in the body.
However, one major substance affecting the regulation of such pressure is the hormonal palpated known as angiotension Il. In certain states of high blood 10 pressure (hypertension) it is difficult to medically control the secretion into, and therefore the level of angiotension II in, the circulatory system. By appropriate modification of this hormone and subsequent immunization with this altered modified pretenses hormone, it is possible to reduce the secretion of Anglo-tension If in patients with chronically elevated hormone levels. The predictable and 15 controlled reduction of this substance is beneficial to certain patients with chronic problems of hypertension. Modified angiotension If can be produced by the general protein modification technique described herein The resultant modified Anglo-tension II is simply injected into the patient in an amount sufficient to induceantibody response sufficient to control or regulate unmodified angiotension II to the 20 desired degree.
A further use for my vaccines is the treatment of diabetes and associated micro and macro vascular diseases. Currently, the treatment of diabetes is limited to dietary and/or drum treatment to regulate blood glucose levels. Recent scientific data support the concept that growth hormone and somatomedian (both polyp 25 peptizes) are intimately involved in the disease syndrome. These substances can be modified by the technique described herein and used in an effective amount to control the progress of this disease. In practice, modified growth hormone or modified somatomedian is injected into the body to develop antibodies for control of the normally secreted hormones.
Another health problem that can be treated by the use of the vaccines of this invention is that of certain endocrine or hormone dependent breast tuners or cancers. Certain of these cancers have been shown to be dependent upon the abundant secretion of the hormone prolactin for their continued survival. The inhibition of the secretion of prolactin has been shown to diminish the growth rate 35 and the actual survival of certain of these tumors. The immunization of such subjects with the hasten coupled or otherwise altered prolactin produced as ~23;~

described herein, would result in the systematic reduction of the level of this hormone circulating in the system and consequently, may result in the regression or remission of tumor growth. The consequence of this treatment would be far more favorable in terms of effective treatment of this disease since surgical removal of OX the breasts is a principal method of treatment currently available. It should be urlderstood that this treatment should be effective for only those tumors that are dependent upon the secretion of prolactin for survival.
Investigators also have determined, for example, that certain palpated entities are supportive factors to and secretions of neoplastic diseases in both man 10 and other animals. These entities have biochemically, biologically and immune-logically close resemblances Jo hormones, particularly to Chorionic Gonadotropin(COG), as well as to Luteinizing Hormone (LO). By applying the isoimmunization techniques of the invention, the function of such polypeptides or endogenous counterparts can be neutralized to carry out regulation of the malignancy. For 15 example, tumors in both male and female primates may be treated by isoimmuni-ration procedures developing antibodies to Chorionic Gonadotropin or Lightning Hormone or the noted entity analogous thereto. Further, neoplasms in primate Females may be regulated by isoimmunization procedures developing antibodies to endogenous Follicle Stimulating Hormone (FISH). This hormone, when associated 20 with a tumor state, tends to aggravate the timorous condition.
Example IV below shows that a beta-HCG/tetanus toxoid modified palpated confers upon rats substantially complete protection against an injection of tumor cells of the virulent rat mammary adenocarcinoma R 3230 AC, which is associated with CG-like material. The aforementioned modified palpated of the invention, 25 when given prior to injection of a dose of tumor cells which causes 100% mortality in unprotected rats, reduces the mortality to zero. Although this modified palpatedwas not administered in the form of a vaccine of the invention, the superior results obtained in Example III in fertility control using a vaccine of my invention show that the incorporation of such an antigen into a vaccine of my invention would also 30 improve the usefulness of such an antigen in treating ElCG associated carcinomas.
The immunochemical control asserted, as noted, neutralizes the naturally occurring hormone or the above-described entity biologically analogous thereto. As a consequence, the hormone or entity will not be available as would normally be the case, for example, the stimulation of some action of a target tissue. Conversely, 35 the neutralization of the biological activity of the hormone or analogous entity may serve to take away an inhibitory action which it otherwise might assert.

I

There are certain other disease states that may be treatable by the use of vaccines containing altered or modified hormonal or non-hormonal proteins as antigens. The disease states and the associated substances that may be used as modified antigens for immunological treatment of these diseases will be listed as 05 follows:
(1) modified parathyroid hormone for the treatment of kidney stones,

(2) modified insulin and/or glucagon for the treatment of hyperinsulinoma,

(3) modified thyroid stimulating hormone (TUSH) for the treatment of hype-thyroidism, and (a) modified secretion for the treatment of irritable bowel syndrome.
Another group of polypeptides which can be altered by the procedures described herein and used in the field of human fertility control are specific non-hormonal protein antigens isolated from placental tissue. There is direct evidence that inhibition of substances that are specific to the placental tissue and do not have 15 similar antigenic properties with other antigens from organs in other parts of the body, can result in the disruption of pregnancies by passive immunization. Such specific placental substances when modified to form modified polypeptides by theprocedures described herein can be injected into the body of an animal of the same species as an effective fertility control means with the mechanism being active 20 immunization similar to that described for the antigenic modification of hormones.
The particular advantages of these substances is that placental antigens are foreign to the non-pregnant female human subject and therefore are unlikely to cause anycross-reaction or disruption of normal body function in the non-pregnant female.While the invention is useful for the human species it will be appreciated that 25 it is also useful in connection with other animals. Similarly, while the reference herein with respect to fertility control is primarily directed to females such described techniques may be applicable to males, i.e. FS~I, its beta subunit andfragments thereof. Such immunization represents an effective fertility control procedure, providing no physiological consequences are encountered which may be 30 found to react adversely to the performance of other body constituents Whether the concerned hormone, non-hormonal protein or specific fragment thereof which is modified is naturally occurring or is a synthetic product is clearly immaterial. A synthetic protein molecule will perform the same function as the naturally occurring one, inasmuch as the body will react in an equivalent antigenic 35 manner.

I
g Thus, my vaccines render it possible to interfere with or treat various disease states or m medical problems which are caused or influenced by certain polypeptides by active immunization of a male or female animal by the production and use of antigens formed by administration of modified polypeptides. The modification of 05 the polypeptides forms antigens which are then administered into an animal in which immunization is to be developed. Said modification is accomplished by attaching to a palpated one or more foreign reactive (modifying) groups andtor by attaching two or more polypeptides to a foreign reactive group (i.e., a carrier) or both of the above, so that the body of the animal, recognizing the modified palpated as a 10 foreign object, produces antibodies which neutralize not only the modified protein but Also the natural protein which is responsible for the disease or medical problem being regulated. In order to produce an effective quantum of antibodies to the antigen or targeted functional palpated, it may be advantageous to administer the modified palpated together with an immunological adjutant. The term 15 "adjutant" is commonly referred to by those engaged in the field at hand as being a substance which will elevate the total immune response of an animal or person toany immunization thereof, i.e. the adjutant is a nonspecific immuno-stimulator.

iffy Descrie~ion of the Drawings Fig. 1 shows the levels of antibody to HUG produced in rabbits by various 20 modified peptizes in Example II below;
Fig. 2 shows the levels of antibody to HOG produced in rabbits by various te.anus-toxoi~coupled modified peptizes in Example II below;
Fig. 3 shows the levels of antibody to HOG produced in rabbits by various tetanus-toxoid-coupled modified peptizes having differing peptide:carrier ratios in 25 Example II below;
Fig. 4 shows the levels of antibody to HOG produced in rabbits by vaccines containing a tetanus-toxoid-coupled modified peptize and various adjutants in Example III ballooned Fig. 5 shows the levels of antibodies to HOG and peptize produced in mice of 30 various species by a vaccine comprising a tetanus-toxoid-coupled modified peptize coupled to various adjutants in Example III below.

G neural Description of the Invention In an effort to better define the modified polypeptides used in my vaccines, it is first considered appropriate to set out more precisely than hereinabove, examples ~23~

of the natural hormones and natural non-hormonal proteins modified according to this invention. They include Follicle Stimulating Hormone (FISH), Luteinizing Hormone (LO), Chorionic Gonadotropin (COG), e.g. immune Chorionic Gonadotropin, Placental Lactogen, e.g. Human Placental Lactogen (HAL), Prolactin, e.g. Human 05 Prolactin (all of which are pretenses reproductive hormones) gastric, Anglo-tension If, growth hormone, somatomedian, parathyroid hormone, insulin, glucagon, thyroid stimulating hormone (TUSH), secreting and other polypeptides which couldadversely affect body function.
The hormone, Chorionic Gonadotropin (COG) has been the subject of extensive 10 investigation, it being demonstrated in 1927 that the blood and urine of pregnant women contained a gonad-stimulating substance which, when i~uected into lab-oratory animals, produced marked gondola growth. Later, investigators demon-striated with certainty that the Placental Chorionic villa, as opposed to the pituitary, were the source of this hormone. Thus, the name Chorionic Gonadotropin or, in the 15 case of humans, Lyman Chorionic Gonadotropin (HOG) was given to this hormone of pregnancy. During the more recent past, a broadened variety of studies have beenconducted to describe levels of HOG in normal and abnormal physiological states,indicating its role in maintaining pregnancy. The studies have shown the hormone's ability to induce ovulation and to stimulate corpus luteum function, and evidence 20 has been evoked for showing its ability to suppress lymphocyte action. The immune-logical properties of the HOG molecule also have been studied widely. Cross-reaction of antibodies to HOG with human pituitary Lightning Hormone (LO), and vice-versa, has been extensively documented, see for example:
Paul, W. E. & Ross, FIT., Immunologic Cross Reaction Between HOG and Human Pituitary Gonadotropin. I, 75, 352-358 (1964) flux, D. X. & H C. H. Imm~mological Cross Reaction Among Gonad tropics. Act Endocrinolo~ic, 48, 61-72 (1965) Buicks K. D.; Off, A. H. & Golden J. Cross-Reaetion in Radio-Immunoassay between HOG and Plasma from Various Species. Journal of I, 42, 513-518 (196~) Franchimont, P. Study on the Cross-Reaction between HOG and Pituitary LO. 9 1, 65-68 (1970) Downer, M.; Brossmer, R.; Hilgenfeldt, U. & True, E. Immunological reactions of Antibodies to HOG with HOG and its chemical derivatives.
In Structure-Activitv Relationships of Proteins and Palpated irrupt Medic Foundation (1972) ~Z~3Z~

Further, these cross-reactions have been used to perform immunoassay for both COG and LO hormones. See:
Midgley, A. R. Jr. Radio immunoassay: a method for HOG and LO.
Endocrinology, 79,10-16 (1966) 05 Crosignani, P. G., Polvani, F. I Saracci R. Characteristics of a radio-immunoassay for HCE-LH. In Protein and Polvpeptide Hormones (Ed. M.
Margoulies) pp. 409, 411 Amsterdam: Éxcerpta l\~edica foundation (1969) Isojima, S; Naked O.; Kojama, K. & Adachi, H. Rapid radio immunoassay of human LO using polymerized antimony HOG as immunoadsorbent.
Journal of Clinical Endocrinology and Metabolism, 31, 693-699 (1970) In addition to providing for the modification of the entire hormone or selected palpated, the invention further provides for the utilization in the vaccines of modified subunits, for example the beta subunit of Chorionic ~onadotropin. Of particular interest, such subunits may be fragmented into smaller components herein 15 termed "fragments". The latter can be produced synthetically to exhibit an amino acid sequence sufficiently in analogous correspondence to a predetermined portion of the parent subunit. Such fragments generally are conjugated with a larger molecule or component foreign to the body, which may be termed a "carrier", in order to effectively evoke or raise a sufficient quantum of antibodies. The use of 20 the fragments, as thus conjugated, advantageously provides a high degree of specificity of antigenic reaction to the targeted hormone or its biochemical equivalent, i.e. the antibodies will not react with other body constituents. Of particular interest, the above-discussed cross reaction of HOG and LO can be avoided by utilization of fragments of the respective hormone due to the desirable 25 specificity of response thereto. Thus, when interested in obtaining an immune-logical reaction against the hormone, EICG, the undesirable immune reaction to the naturally occurring body constituent, LO, may be eliminated. Synthetic equivalents of the fragments offer enhanced practicality both from the standpoint of production costs and necessary maintenance of purity.
As indicated in the above discussion, when considered in isolation with respect to conception and pregnancy, COG only is present in female primates when they are in a pcst-conception state. However, as discussed above and later herein, an entity at least analogous thereto (having similar immunological properties to HOG) is seen to be present in conjunction with malignancies.
Subunits and fragments of the pretenses reproductive hormones include the beta subunit of natural Follicle Stimulating Hormone, the beta subunit of natural Human Chorionic Gonadotropin, fragments including, inter alias a 20-30 or 30-39 32~

amino acid peptize consisting of the C-terminal residues of natural Human Chore ionic Gonadotropin beta subunit, as well as specific unique fragments of naturalHuman Prolactin and natural Human Placental Lactogen, which may bear little resemblance to analogous portions of other protein hormones. Further with respect 05 to the type of novel chemical entities with which this invention is concerned, one may note for instance the chemical configuration of the beta subunit of HUG Thatstructure is as follows: 10 Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg-Cys-Arg-Pro-ne-Asnn-Ala-Thr-*
Leu~Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-lle-Thhr-Val-Asn-I
Thr-Thr-Ile-Cys~Ala-Gly-Try-Cys-Pro-Thr-Met-Thr-Arrg-Val-Leu-Gln-Gly-Val-Leu-Pro -Ala-Leu-Pr~Gln-Val-Val-Cys-Asn-Try-Arg -Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro -Gly-CysPr~Arg-Gly-Val-Asn-Pro-Val-Val -Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln-Cys -Allele -Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys -Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-5eer-Ser-Ser-* * 130 *
Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro Son -Pro-Ser-Arg-Leu-Pro-* 140 Gly-Pr~Ser -Asp-Thr ~Pro-Ile-Leu-Pro-Gln Structure (I) *indicates site location of carbohydrate moieties on natural palpated.
For specificity of antibody action it is necessary that distinctive peptizes be isolated or prepared that contain molecular structures completely or substantially completely dill event from the other hormones. The beta-subunit of HOG possesses a specific chain or chains of amino acid moieties which differ either completely or essentially from the palpated chain of 25 Human Luteinizing Hormone. These chains or fragments, when conjugated with a carrier, represent an additional aspect of this invention. Accordingly, the palpated Structures (II) and (III~ [ C-terminal portion of structure I] .
Asp-Asp-Pro-Arg-Phe-Gln-As~Ser-Ser-Ser-Ser -Lys-Ala-Pr~Pro-Prosier -Leu-Pro-Ser-Pro-Ser -Ar~Leu-Pro-Gly-Pro-Ser -Asp-Thr-Pro-lle-Leu-Pro-Gln ~L2Z3Z~

Structure (If) Gln-As~Ser-Ser-Ser-Ser - Lys-Ala-Pro-Pro-Pr~Ser - Leu-Pr~Ser-Prosier -Arg-Leu-Pro-Gly-Pro-Ser -As~Thr-Pro-Ile-Leu-Pro-Gln Structure (III) whether obtained by purely synthetic methods or by enzymatic degradation from the natural or parent palpated, [ Carson et at., J. Biological Chemistry, 284 (19), 6810, (1973)] when modified according to this invention, similarly provide materials with antigenic properties sufficient to provide the desired immunological response.
It will be understood, for example, thaw addition of a polytyrosine chain or a protein macromolecules (carrier) may assist in rendering Structure (If) antigenic so that the resulting administration of modified Structure (If) will provide the desired immune-logical action against natural HOG.
The beta subunit set forth at structure (I) is seen to represent a chemical sequence of 145 amino acid components. This structure has a high degree of structural homology with the corresponding subunit of Luteinizing Hormone (LO) to the extent of the initial 110 amino acid components. As indicated above, it may be found desirable, therefore, to evoke a high specificity to the Chorionic Gonadotropin hormone or an analogous entity through the use of fragments analogous to the C
terminal, 111-l45 amino acid sequence of the subunit. Structure (If) above may be observed to represent just that sequence. Structure ~III) is slightly shorter, representing the 116-145 amino acid positions within the subunit sequence.
Further palpated chains useful in promoting antibody buildup against natural HOG include the following structures labeled Structures (IV) through (XIV).
When modified according to this disclosure, such as by coupling to Focal 70 a synthetic copolymer of sucrose and epicholorohydrin having an average molecular weight of 70,000 + 10,000, good volubility in water, Stokes radius about 5.1, stable in alkaline and neutral media, available from Pharmacia Fine Chemicals, Pharmacia Laboratories, Inc., 800 Centennial Ave., Pussycat, NO 08854) or other modifier-carriers such as protein macromolecules described herein, these polypeptides provide immunogenic activity with which this invention is concerned All of thesepolypeptides are considered fragments of HOG by virtue of their substantial resemblance to the chemical configuration of the natural hormone and the immune logical response provided by them when modified as indicated herein.

* trademark ~22~2~i Cys-Pro-Pr~-Pro-Pro-Pro~Pro-Ser-Asp-Thr-Pro-~e-Leuu-Pro-Gin Structure (IV) Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Pro-Pro-Pro-Pro-Prreprocess 05 Structure Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Seer-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-IlLyle-Pro-Gln Structure (VI) Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Alla-Pro-Pro, Pro-Ser-Leu-ProrSer Structure(VII) Asp-Asp-Pro-Arg-Phe-GlnrAsp-Ser-ProrPro-Pro-Cys-Prro-Pro-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln Structure TV
Asp-Asp-Pro-Arg-Phe-Gln-AsprSer-Pro-Pro-Pro-Pro-Prreprocess-Pro-Pro-Pro-Pro-Pro-Pro-Ser-Asp-Thr-ProrIle-Leu-Prraglan Structure (Via) Asp-His-Pro-Leu-Thr-Aba-Asp,Asp-Pro-Arg-Phe-Gln-Assp,Ser-Ser-20 Ser-Ser-Lys-Ala-Pro~Pro-Pro-Ser-Leu-Pro,Ser-Pro-Seer-Arg~Leu -Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Pro-PPro-ProrPro-Preprocess Structure(IX) Asp-Asp,Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Alla-Pro-Pro-25 Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Seer-Asp-Thr-Pro-lle-Leu-Pro,Gln-Pro-Pro,Pro-Pro-Pro-Pro-Cys Z3;~

Structure (~) Asp-Asp-Pro-Arg-Phe-C~lr~Asp-Ser-Ser-Ser-Ser-Lys-AAla-Pr~Pr~
Pro-Ser-Leu-Pro-Ser-Pro-Ser-Ar~Leu-Pro-Gly-Pro-Serr-Asp-Th~
Pro-Ile-Leu-Pro-Gln-Cys 05 Structure (XI) Structure (IV) will be recognized as incorporating a Cyst component at the amino or N terminal which is associated with a Praline spacer sequence. These spacers serve to position the sequence which follows physically distant from the carrier-modifier.
The latter sequence may be observed to represent the Thea to Thea amino acid 10 component sequence of the subunit Structure (I). Structure (V) on the other hand, represents an initial sequence corresponding with the Thea to Thea components ofthe subunit Structure (I) followed by a sequence of six Praline spacer components and a carboxyl terminal, present as Sistine. The rationale in providing such a structure is to eliminate the provision of sites which may remain antigenically 15 neutral in performance. Structures (IV) and (V) represent relatively shorter amino acid sequences to the extent that each serves to develop one determinant site.
Consequently, as alluded to in more detail hereinafter, they are utilized in conjunction with a mixed immunization technique wherein a necessary two distinctdeterminants are provided by the simultaneous administration of two such frog-20 mints, each conjugated to a corresponding, separate carrier macromolecules Struck lure (VI) represents the Thea through Thea component sequence of structure (I).
Structure (VII) represents a portion of Structure (I), however, essentially, a sequence of the Thea to Thea components thereof is formed.
Structure (VIII) incorporates two sequences, one which may be recognized in 25 Structure (V) and the other in Structure (IV). These two sequences are separated by two spacer sequences of Praline components and one is joined with an inter mediately disposed Sistine component which serves a corljugation function as described later herein. With the arrangement, two distinct determinant sites aredeveloped in physically spaced relationship to avoid the development of an unwanted 30 artificial determinant possibly otherwise evolved in the vicinity of their mutual coupling. Structure (Vow) represents Structure (VIII) with additional Pro spacerresidues to provide a widened spacing of determinant sites.
Structure (IX) mimics sequences from Structure (I) with the addition of a Praline Spacer Sequence, a Sistine Component at the C-terminal, and an Abe 35 substituted for Cyst at the 110 position. The Abe designation is intended herein to ~3;2~Çii mean alpha-aminobutyric acid of Sistine. Structure (~) will be recognized as a combination of Structure (II) with a six residue Praline spacer sequence and a Cysteitle component at the C-terminal. Similarly, Structure (I) combines Structure ([I) with a Sistine component at the terminal without a Praline spacer sequence.06 Thr-Cys-~sp-Asp-Pro-Arg-Phe-Gln-Asp~er~er~eI ~er-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Glly-Pro-Ser-Asp-Thr-Pro-Ile-Leu -Pro-Gln Structure (XII) Asp-His-Pro-Leu -~hr-~ba-Asp-Asp-Pro-Arg-Phe-Gln-Asp~er~er~er-Ser-Lys-Ala-Pro-Pro-Pro~er-Leu-Pro~er-Pro~er-~rg-LLopper-~ly-Pro-Ser-~sp-Thr-Pro-lle-Leu-Pro-Gln-Cys Structure (XIII) Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro Arg-Phe-Gln-~sp~er-Ser~er~er-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro~er-Pro-SSer-Arg-Leu-Pro-Gly-Pro~Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln Structure (IVY) Structure IT will be recognized as having the sequence of Structure (II) with the addition of Theorizes components at its N terminal. Structure (~III) is similar to Structure (It) but does not contain the spacer conjugate. Structure (IVY will be20 recognized as being similar to Structure (II) with the addition of spacer components at the N terminal and a Cyst component for conjugation purposes.
When the vaccine of the invention is to be used for cancer control, it may be advantageous to use as the protein hormone or fragment a placental protein or fragment thereof, since there is some evidence that such placental proteins are 25 effective in stimulating the body's immune system so as to increase its resistance to cancer.
Particularly where the larger whole hormone or subunit type molecular structures are concerned, the number Ox foreign reactive groups which are to be attracted to the palpated and the number of polypeptides to be attached to a 30 foreign reaction group depends on the specific problem being treated. Basically, what is required is that the concerned palpated be modified to a degree sufficient to cause it to be antigenic when injected in the body of the host. If too littlemodification is effected, the body may not recognize the modified palpated as a foreign body and would not create antibodies against it. If the number of foreign 35 molecules added to the palpated is too great, the body will erect antibodies against the intruder antigen, but the antibodies will be specific to the injected ~;~23Z~6 antigen and will not neutralize the action of the concerned natural endogenous hormone or non-hormonal protein, i.e. they will be specific to the modifier.
N~odificfltion of the palpated is accomplished by attaching various kinds of modifying groups to pretenses hormone, non-hormonal proteins, subunits or 05 specific fragments thereof according to methods known in the art.
As is apparent, structures (IVY) are relatively small fragments, usually produced synthetically. To render them capable of eliciting antibody production, it becomes necessary to conjugate them with larger carrier-modifier molecules.
Generally about 5-30 peptize fragments will be coupled with one carrier molecule.
10 The body will, in effect, recognize these foreign carriers as well as the sequences represented by the fragments and form antibodies both to the carrier and to the sequences of the coupled fragments. Note that the carrier-modifiers are foreign to the body and thus antibodies to them will not be harmful to any normal body constituents. In the latter regard, it may be found preferable to utilize a carrier 15 which, through the development of antibodies specific to it, will be found beneficial to the recipient.
As indicated earlier herein, it also is preferred that the modification con-statute two or more immunological determinants represented on the native hormoneas palpated structures to which it is desired to evoke an antibody response. The20 effect is one of heterogeneity of antibody development. Thus, several fragment structures have been described above having at least two distinct amino acid sequences represented in the HOG beta subunit. These sequences may be so spaced as to derive the determinants in mutual isolation, while the spaced sequence fragment is conjugated with a larger, macro molecular carrier. Alternately, the 25 noted mixed immunization arrangement may be utilized wherein a first fragmentdeveloping one determinant is conjugated with a first carrier molecule and is administered in combination with a second, distinct fragment which is conjugatedwith a second carrier molecule, the latter of which may be the same as or different in structure from the first carrier. Thus, each macro molecular carrier must be 30 conjugated with hormone fragments such that each fragment represents two or more immunological determinants. These two necessary determinants can be evolved by mixing, for example, separate conjugate structures, for example Structures (IV) and (V) each of which, through forming antibodies separately to the distinct deter-Mennonites, will provide a population of antibodies reacting with two separate 35 determinants on the natural endogenous hormone.

~LZ23Z~

Inasmuch as the noted fragments are relatively small as compared, for instance, to if whole hormone or subunit thereof, a criterion of size is imposed upon the selection of a carrier. The carrier size must be adequate for the body immune system to recognize its foreign nature and raise antibodies to it. Additionally,I carrier selection preferably is predicated upon the noted antibody heterogeneity requirement, i.e. the carrier must itself evoke a heterogeneous immune response in addition to the fragments. For example, improved response may be recognized where the carrier is varied in structure, e.g. incorporating branching chains toenhance the recognition of both the carrier and the attached palpated as being of 10 a foreign nature.
As one example of whole hormone modification, modified dyes groups derived from sulfanilic as id may be attached to the subject polypeptides, see the Cinander et at and Phillips et at references cited subsequently for instruction on huge this "attachment" is accomplished, and to the extent necessary for an understanding of 15 this invention, such is incorporated herein by reference.
Additional modifying groups for modifying whole hormones or their subunits are those groups obtained by reaction of the polypeptides with dinitrophenol, trinitrophenol, and ~acetomercaptosuccinic android, while, suited for utilization as a carrier-modifier in conjunction with fragments, are polytyrosine in either 20 straight or branched chains, polyalanines in straight or branched chains, boo-degradable polydextran, e.g. polymerized sugars such as sucrose copolymerized with epichlorohydrin, e.g. Focal 70 and Focal 400 (a synthetic copolymer of sucrose and epichlorohydrin having an average molecular weight of 400,000 + Lowe intrinsic viscosity of 0.17 dug. specific rotation [Ox] 20 of +S6.5 available from Pharmacia 25 Fine Chemicals, Pharmacia Laboratories, Inc. 800 Centennial Ave., Pussycat, NO
08854) or a polyglucose such as Dextran* T 70 (a glucan containing alpha-l, 6-gluscosidic bonds and having an average molecular weight of approximately 70,000, synthesized microbiological by the action of Leuconostoc mesenteroides strain NRRL B-512 on sucrose), serum proteins such as homologous serum albumin, 30 hemocyanin from Keyhole limpet, a marine gastropod mollusk, viruses such as influenza virus (type A, B, or C) or polio myelitis virus, live or killed, Types 1, 2 and 3 of tetanus toxoid, diphtheria toxoid, cholera toxoid or somewhat less preferably, natural proteins such as thyroglobulin, and the like. Generally synthetic modifiers are preferred over the natural modifiers. However, carrier-modifiers found 35 particularly suitable for conjugation with the above-discussed fragment structures are Flagellin, tetanus toxoid, diphtheria toxoid and an influenza subunit, for '"trademark -:~Z3Z~

example, the preparation of which it described by Bach Meyer, Schmidt and Lowe, "Preparation and Properties of Q Novel Influenza Subunit Vaccine", Post-GraduateMedical Journal June, 1976) 52:360-367. This influenza subunit was developed as a vaccine which incorporates05 essentially only the two viral proteins Haemagglutinin and Neuraminidase. Con-twining substantially only these two essential immunogens, the subunit represents a preparation which does not contain other protein and lipid antigens which may befound to cause undesired side reactions A secondary benefit may be realized through the utilization for example, of the influenza subunit, polio myelitis virus, 10 tetanus toxoid, diphtheria toxoid, cholera toxoid or the like as a modifier-carrier, inasmuch as beneficial antibodies will be raised to that modifier-carrier as well as to the hormonal fragment conjugate thereto.
Flagellin is a protein described as forming the well of the main spiral filamentof the flagellum. Bacterial flagella, in turn, have been known as the active 15 organelles of cell locomotion, individual flagella (flagellum) occurring in suspension as individual spirals which, upon drying, collapse into filaments which describe a sine wave with a wave length of 2-3 microns and an amplitude of 0.~5-0.60 microns.
Generally, the flagellum consists of three morphologically distinct parts: a basal structure that is closely associated with the cytoplasmic membrane and cell wall, a 20 hook and the noted main spiral filament.
Purified flagellum is readily obtained by solubilization of flagella filaments below a pi value of about four, and subsequent removal of the insoluble material by centrifugation or filtration. As a group of related proteins, flagellins from different bacterial species have been predicted to have similar amin~acid compositions.
25 However, the amino acid composition of each flagellin species in unique. essentially all flagellins are described as containing no or only a few residues of Sistine,tryptophan, Tarzan, praline and histidine. Thus when conjugated with fragments in accordance with the invention, a thiolactonization procedure or the like is carried out as described later herein.
the molecular weights of various flagellin have been calculated, in all cases the values thereof of the monomeric subunits falling in the range of 30,000 to 50,000. From an immunological standpoint, a flageLlin molecule is highly immune-genie. For further and more detailed discourse describing bacterial flagella Endflagellin, reference is made to 'Advances in Microbial Physiology" 6, 219 (19713, 35 "B~c$erial Flagella" by R. W. Smith and Henry Coffer I

Tetanus toxoids have been the subject of study and production for many years.
Ire toxoid generally is evolved from a formalinization of tetanus Tyson, the latter beillg n protein synthesized by Clostridium Titan. Immunization currently is carried out utilizing soluble and absorbed tetanus toxoids and suggestions have been made 05 concerning the utilization of fluid tetamls toxoid in complex with antitoxin.Publications describing the toxin and toxoid are numerous, reference being made to the following:
1. Immunochemistry of Tetanus Toxin, Bison, et at, Journal of Biochemistry, 39, 171-181(1973).
10 2. Early and Enhanced Antitoxin Responses Elicited with Complexes of Tetanus Toxoid and Specific Mouse and Human Antibodies, Stoner et at, Journal of Infectious Diseases, 131, (3), 230-238 (1975).
3. Differences in Primary and Secondary Immunizability of Inbred Mice Strains, Ibsen, Journal of Immunology, 83, aye (1959).
15 4. Antigenic Thresholds of Antitoxin Responses Elicited in Irradiated Mice with Complexes of Tetanus loin and Specific Antibody, issue et at, Radiation Research, 25, 655-667 (1965).
5. Early and Enhanced Germinal Center formation and Antibody Responses in Mice After Primary Stimulation with ~ntigenisol-a ogous Antibody Complexes as Compared with Antigen Alone, Lucy et at, Journal of Immunology, 10~, 822-825 (1971).
6. Distinctive Muddler and Germinal Center Proliferative Patterns and Mouse Lymph Nodes after Regional Primary and Secondary Stimulation with Tetanus Toxoid, Burke et at, Journal of Imp munology, 112, (6),1961-1970 (1974).
Modification by removal of moieties is also contemplated by this invention.
Thus, for example, where certain of the natural proteins have carbohydrate moieties, these carbohydrate moieties may be removed according to methods known in the art by, for instance Nastily neuriminidase or Nastily glucosidase, 30 materials useful for removal of specific carbohydrate moieties.
These various means for modification are, as indicated above, known to persons skilled in the art. Certain of these means may be found in the following list of literature references, whereas various others of them may be found elsewhere in the literature by art-skilled persons:
1. Klutz et at., Arch. of Become. and Buffs., 96, 6Q5-612, (1966).
2. Kern, Chum. Rev. So 145 (1953).
3. Sofa et at., Become. J., 85, 223 (1962).
AL. Risen et at., J. Am. Chum. Sock 75, 4583 (1953).

~'~Z3Z~

5. Suntan et at., Fed Pro. (ABSTR) 25, 729 (1966).
6. Sokolowsky et at., J. Am. Chum. Sock 86,1212 (1964).
7. Tabachnick et at., J. Blot. Chum. 23D~ 1726, (1959).
8. Cramp ton et at., Pro. Sock Expert Blot. & Med. 80, 448 (1952).
05 oodfriend et at., Science 144,1344 (1964).
10. Sofa et at., J. Am. Chum. So., 78, 746 ~1955).
11. Cinder et at., Bruit. J. Exp. Patrol. 36, 515 (1955).
12. Phillips et at, J. of Blot. Chum. 240 I 699-704 (1965).
13. Earl J. of Blot. Chum., 244, 575 (1969).
I~/lethods for preparing the modified polypeptides of this invention also include the following.
In one preferred modification approach, the palpated fraction, for example, Structure IT is activated first following which it is conjugated with a carrier, for example the influenza subunit described above, tetanus toxoid or Flagellin. An 15 activating reagent may be utilized which exhibits differing functionality at its ends and by choice of reaction conditions, these end components can be made to react selectively. For example, the following activators A and B, having a maleiimido group and a substituted acid group, may be provided:

and "maleiimido" group where X is a non-reacting group made up of a substituted, or unsubstituted phenol or Cluck alkaline moiety, or a combination thereof. In this regard, the moiety substituted on the phenol should be non-interfering as is the remainder of the "X"
grouping.
X may, inter Ahab be selected from the following:
SHEA-, or , or I

The malei;mido grouping of the above reagents will react with sulfhydryl (SO) groups in the palpated fragments under conditions whereby the opposite end (active ester end) Ox the reagent does not react with the amino groups present in the fragment sequences. Thus, for example, palpated fragments such as Structure 05 (~11), containing a Cyst amino acid and hence, an SO group react as follows:

Structure (XII) + B Structure (XII) - S

Following the above, upon adjusting the pi to a slightly alkaline condition, e.g. 8, and adding the carrier protein accomplishes the fang conjugation:

Structure XII-Preferably, a carrier protein such as the above-noted Flagellin which does not contain SO groups, but does contain NO groups, may first be treated with activator A or B at pi 7 or lower at the active ester end giving:

Following the above, the activated carrier is reacted with a palpated fragment containing a SO group to derive a product similar to that discussed immediately 15 above.
Should the palpated fragment not contain an SO group, e.g. Structures (II), (III), (VI) and (VII), such structures can be modified first to introduce such a grouping by standard methods such as 'tthiolactonization", following which they are con-~ZZ3Z~

jugated utilizing the above-discussed selective bi-functional reagents. For a more detailed description of these reagents, reference is made to the following public-lions:
O. Weller and J. Rudinger, ~lelv. Chimp Act 58, 531-5~11(1975).
(US W. Trimmer, I Kolkenbrock I G. Pfleiderer, Hoppe~eyler's Z. Fishily.
Chum., 356,1455-1458 (1~75).
It is well known to those skilled in the art that, in many natural proteins containing Sistine residues, these residues are not present in their they'll form containing a fresh group; instead pairs of Sistine residues are linked by means of 10 disulfide bridges to form Sistine. Such disulfide bridges are very important in determining the conformation of the protein. In most cases, the disulfide bridges present in the natural form of the protein are easily reduced to pairs of OH groups by means of mild reducing agents under conditions which leave the remaining parts of the protein molecule unchanged. Accordingly, when it is desired to produce free-15 SEX groups in proteins in order to carry out the coupling reactions discussed above one convenient way of providing such frill groups may be to cleave disulfide bridges naturally present in the protein or other palpated which is desired to couple. In particular, the twelve Sistine residues present in the beta subunit of ICY (Structure (I) above) are, in the natural form of the subunit, coupled together 20 to form Sian disallowed bridges, so that the natural form of the protein has no fresh groups. To produce fresh groups for coupling reactions, any number of these bridges, from 1 to 6, may be broken using known techniques, as set out for employ in:
Bawl et at, Become. Buffs. yes. Comma 70, 525-532 (1976).
This particular article describes cleaving 3-5 of the six disulfide bridges in the beta 25 subunit of HCC~, but the same techniques may be used to break all Sue bridges if this is so desired.
The generation of fry by reduction of disulfide bridges in naturally occurring forms of proteins may also effect the cross-reactivity of the antibodies produced when a modified palpated produced from the protein is injected into an 30 animal. Frequently, an antibody recognizes its corresponding antigen not only by the amino acid sequence in the antigen by also by the conformation (shape) of the antigen. Accordingly, an antibody which binds very strongly to a protein or other palpated in its natural conformation may bind much less strongly if at all, to the same protein or palpated whose conformation has been drastically altered by 35 breaking dis~lfide bridges therein.

~Z232~6 Accordingly, the breaking of disulfide bridges in proteins or other polypeptidesmay provide a basis ire reducing the cross-reactivity between antibodies to antigens hiving the same amino acid sequence along parts of the molecule. For example, ithis been pointed out above that cross-reaction is frequently encountered between05 antibodies to beta-HCG and HUH because the first 110 residues in the beta-HCGsequence are the same as the corresponding part of the HUH molecule, and in the natural forms of both molecules, the conformations are also presumably very similar. It has been suggested above that one means of producing in an animal antibodies to beta-HCG which do not substantially cross-react with HUH is to supply 10 to the animal an antigen of the invention derived from a palpated which contains all or part of the residues 111-145 of beta-HCG but which lacks all or substantially all of the residues 1-110 of beta-HCG. In effect, this approach avoids antibody cross-reaction with HUH by chemically removing from the modified palpated of the invention the sequence of residues which is common to buttock and HUH. As an 15 alternative approach, by cleaving the appropriate number of disulfide bridges in the natural form of beta-HCG, it may be possible to so alter the conformation of residues 1-110 thereof that the antibodies formed when a modified palpated of the invention based upon this altered-conformation buttock is administered to an animal will no longer cross-react significantly with HUH. In other words, instead of 20 chemically severing the common sequence of residues from beta-~ICG in order to prevent cross-reaction, it may be possible to leave this common sequence of residues in the beta-HCG but to so alter the conformation of this common sequence that to an antibody the altered-conformation common sequence does not "look" like the natural form of the common sequence, so that an antibody which recognizes the 25 altered-conformation common sequence will not recognize the natural-conformation common sequence in HUH. Moreover, once the natural conformation of the sequence of residues 1-110 has been destroyed by breaking the disulfide bridges this common sequence will probably assume the helical conformation common in polypeptides lacking disulfide bridges, so that this part of the beta-HCG will not be 30 strongly immunogenic and most of the antibodies formed by a antigen of the invention based upon the altered-conformation buttock: will be antibodies to thesequence 111-145 which is not common with HUH.
As an alternative approach to the utilization Ox the maleiimido group reagents discussed above, an alkylation step may be used to cause conjugation. Conditions35 can be chosen such that in the presence of amino groups, essentially only SO groups will be alkylated. With this approach, a generalization of the reactions carried out may be expressed as follows:

~LZ;23;~

Structure (ZION Carrier-N~l.CO.CH2Br SO
Structure (ZION
CHICANO Carrier With this approach, the larger molecule carrier, e.g. Flagellin, tetanus toxoid 05 or the influenza subunit described herein is first modified by reaction of a fraction of its amino groups with an active ester of ehloro, dichloro, broom or idea acetic acid, such as:

Br.CH2CO.O-N

and this modified carrier is then reacted with the sulfhydryl group in a palpated 10 fraction, or a palpated fraction which has been modified to contain the SO group (e.g. thiolactonization) if it does not already have such a group. The present approach produces a trio ether linkage by alkylation of a free they'll (sulfhydryl group).
With the instant procedure, the roles of the fragment and carrier may be 15 reversed, the fragment being modified to contain the halo methyl alkylating group which would then react with sulfhydryl groups in the carrier, or a carrier suitably modified to exhibit a sulfhydryl group.
[t may be seen from an observation of the formulae of Structures (IV), (V), (IX), I (Al), (XII), (I 9 and (XIV) that a Cyst amino acid, which in a reduced state 20 provides an SO reactive group is located at either the C terminal or N terminal of the peptize structure. This location permits the peptize to be chemically linked to carrier molecules at either terminus. And some Structures (XIV), I I (X), (It have a six-Proline spacer chain (Pry between the ye residue and the remainder ofthe peptize sequence. This latter arrangement provides a chemical spacer between25 the coupled carrier and the sequences representing a fragment of the natural hormone. A six-Proline spacer can be added as a side chain spacer, for example at position 122 (Lye) in Structure (II), by initially adding an SO group (thiolactonization) to the free or unblocked epsilon amino group on this (Lye) residue. Then, utilizing the activator A, B above in which the component "X" is a chain of six Praline amino 30 acids, conjugation can be carried out. In the latter case, a spacer is provided between the carrier and peptize linked at an intermediate site, for example at ~2Z3;Z~6 position 122 in Structure (U). In the former case, only the space represented byconjugating reagent links the carrier and peptize.
l~lodifying groups, such as hemocyanin from Keyhole limpet, containing free amino groups, are prepared in buffer solution such as phosphate buffer, in sodium or chloride solution at a pal of 6-8. To this solution, tolylene diisocyanate TIC
reagent diluted from about lo to about 1-40 times with Dixon is added to the modifying group. The general procedure was disclosed by Singer and Schick, J.
Biophysical and Become. Cytology 9, 519 (1961). The amount of TIC added may range from .075 to 1,000 molar equivalents of the modifier used. The reaction may 10 be carried out at about -5 to about +10C., preferably 0 to 4C., for about 1/2 to 2 hours. any excess TIC may be removed by centrifugation. The precipitate may be washed with the above-mentioned phosphate loafer and the supernatants come brined.
This activated modifying group solution may then be combined with the I hormonal or non-hormonal palpated to be conjugated. Palpated is dissolved in the same phosphate buffer (5-30 mg/ml) and the volume of modifier and palpated combined according to the molar ratio of the two desired in the conjugate.
Combined solutions are reacted at 30 - 50C., preferably 35 - 40C., for 3-6 hours.
Separation of modified palpated and free unconjugated palpated may be 20 accomplished by conventional techniques, such as gel filtration.
Picogram amounts of 1l25 labeled palpated may be added as a tracer to the reaction mixture at the time of conjugation, and a quantity of palpated conjugated to modifying groups (molar ratio) may be determined by the amount of radioactivity recovered.
Included in the methods for modifying the hormones non-hormonal proteins and their fragments (unmodified polypeptides) are conjugation by use of water-soluble carbodiimide. The amino groups of the unmodified palpated are first preferably protected by acetylation. This (acetylated~ unmodified palpated is then con-jugated to modifier, such as natural protein modifier, e.g. hemocyanin from Keyhole 30 limpet, homologous serum albumin, and the like, or Dextrans, Focalize, or polyp Tarzan, preferably in the presence of guanidine such as guanidine Ill using lo ethyl-3-(3-dimethylamino propel) carbodiimide as activating agent. This method is generally disclosed by ire and Koshland, Jr., J. of Biological Chemistry 242, 2447 (1967). In the instant where Focal 70 is used, it is preferred that it be first treated 35 with ethylenediamine so as to render the final coupling more efficient. This treatment with ethylenediamine may be performed in solvent such as saline and Sue Dixon at about room temperature and a pi of about 9-12, preferably 10-11 for about I to about 2 hours. The conjugation itself between the unmodified palpated and the modifier may be performed in solvent such as Gleason methyl ester while maintaining the pi at about I preferably about ds.5-4.8. The temperature of 05 reaction is conveniently about room temperature and the reaction may be allowed to proceed for about 2-8 hours, preferably 5 hours. The resulting modified palpatedwith which this invention is concerned may be purified by conventional techniques, such as column chromatography.
The immunogenic substances for this invention may also be provided by 10 polymerization of unmodified palpated using bi-functional imidoester. The imidoester, such as dim ethyl adipimidate, dim ethyl suberimidate and deathly Malone-misdate, may be used to form the polymer in a nunnery similar to the generally described methods of Hart man and Would, Become. I 2439 (1967). The polymeric ration may take place conveniently at room temperature in aqueous solvent at a pi 15 of about 9-12, preferably about 10-11, over a period of 1/~-2 hours.
Said immunogenic substances may also be prepared by dimerization through a disulfide bond formed by oxidation of the trio group on a Cys-residue using iodosobenzoic acid and methods corresponding to known methods, such as room temperature reaction for about 10-40 minutes.
The modified polypeptides of the invention may also be formed by forming linear polymers of the polypeptides. In a preferred method of forming such linear polymers, the polymer chain is begun with a first peptize lacking a C-terminal Sistine and having an amino group only at its N-terminal (peptizes containing amino groups other than at the N-terminal may of course be used if all non-terminal 25 amino groups are blocked with any conventional blocking agent). This startingpeptize is reacted with 6-maleimido caproic azalea N-hydroxy succinimide ester (MCCOY) at a pi of 6.6, whereby the ester portion of the reagent reacts with the N-terminal amino group of the peptize. After removal of excess MCCOY, the thus-activated peptize is reacted under the same conditions with an equivalent amount of a second 30 peptize having a C-terminal Sistine in a reduced stated (i.e. having a free SKI
group. This reaction will cause the coupling of the N-terminal of the activated peptize to the C-terminal of the second peptize. The resultant diver is purified by gel filtration and then again reacted with MCCOY and a second equivalent of the second peptize, thereby producing a triter. This polymerization procedure can be35 repeated until the desired chain length has been achieved (polymers containing at least 12 peptizes have been prepared in this manner). It is critical that high-3Z~6 resolution chromatography be employed in the purification of the polymer at eachstep of the polymerization in order in ensure that all the final polymers have the seine chain length and are not missing one or more of the units.
In this method of forming linear polymers, the -first and second peptizes may 06 be identical in chemical configuration except that in the first peptize the C-terminal Sistine has a blocked they'll group. Where the modified palpated is to be used to Norm antibodies to lick, preferred fragments for this purpose are (111-145)-Cyst and (Swiss, where the figures refer to the bases in the beta subunit of ~CC~ (Structure I above). It will be appreciated that, when these fragments are to 10 be used in forming the linear polymers, the Lawson residue at position 122 must have its amino group blocked and, in the case of the (Swiss fragment, the non-terminal Sistine at position 110 must also have its they'll group blocked, preferably with an acetamidomethyl group.
The linear polymers preferably contain from about 4 to about 14 fragments.
lo Modified polypeptides may also be prepared using glutaric deluded as conjugating agent. According to a theory proposed by Richards and Knowles [J.
Mol. Blot. 37, 231(1968)], commercial glutaric deluded contains virtually no free glutaric deluded, but rather consists of a very complex mixture of polymers richin I, unstriated aldehydes. Upon reaction with natural protein modifiers such as20 homologous serum albumins, these polymers form a stable bond through the freeamino group, leaving alluded groups free. This intermediate product then reacts with unmodified palpated in the presence of alkali metal bordered, such as sodium bordered. This intermediate is formed at pi 7-10~ preferably 8-9, at about room temperature. The modified palpated is also conveniently obtained at 25 about room temperature after about 1/4 - 2 hours' reaction time. The resulting product is recovered in pure form by conventional techniques, such as gel filtration, dialysis and lyophilization.
Polymerized sugar modifiers such as Focal 70 or Dextran T 70 may also be prepared for conjugation by treatment with a cyan uric halide such as cyan uric 30 chloride to form a dihalotriazinyl adduce. The process may be performed in solvent such as dimethylformamide at about 0 - 20C., preferably 10 -kiwi for about 1/2 -

4 hours. The resulting intermediate product may then be dialyzed until essentially halogen ion free, and lyophilized and treated with unmodified palpated at pi 8-11, preferably about 9-10, for about 1/2 -12 hours at about 15 - 35C., conveniently at 35 room temperature. The resulting modified palpated may be recovered as indicated above.

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Said polymerized sugar modifiers may also be treated with alkali metal peridot, such as sodium peridot, at a pi of 3-6 at about 30-60C. for about I/2-4 hours, and the resulting intermediate conjugated with unmodified palpated nut a pi of about 7-11, preferably about 8-10, for about 1/4 to about 2 hours at a 05 temperatllre of about 15-80C.) preferably 20 - 60C. The resulting immunogenic substance according to this invention may be separated as indicated previously.
The modifying groups may vary in chemistry and number for any given palpated structure. However, they will attach to only certain amino acid moieties. In particular, when modifying with dyes groups they will chemically bond 10 to only the histidine, arginine, Tarzan and Lawson moieties or sites. Other modifying groups will bond to etude molecules at different sites and in different numbers. Consequently, depending upon the size and chemical make-up of a particular modified palpated desired, one skilled in the art will readily be able to calculate the maximum possible number of modifying groups as sociable with a 15 palpated. It is also recognized that several modifying groups may attach themselves to each other which in turn attach to a single amino acid moiety, but as used herein, reference to a number of modifying groups means the number of reaction sites to which a modifier has been attached.
As indicated above, a Tory leading to this invention was that the chemical 20 modification of an essential reproductive hormone would alter it such that it would exhibit antigenic properties so that when injected into an animal (including humans) it would cause the formation of antibodies which in turn would not only react to the injected modified hormone but also to the natural unmodified endogenous hormone as well. With this theory in mind, reproductive hormones of various species were25 modified and tested in baboons. The results illustrated that modified hormones of unrelated species do not produce the desired results, whereas modified hormones of the same or closely related species do produce the desired results. It will accordingly be clear that the palpated to be modified should be so related to the endogenous hormone or non-hormonal protein as to be either from the same animal 30 species or be the immunological equivalent thereof as modified.
additional experiments were conducted to test the validity of this concept in humans, i.e. modified human reproductive hormones injected into humans. Cot-electively, the results prove the conclusion drawn from the experiments with thebaboons, namely, isoantigenic immunization using modified human reproductive 35 hormones does produce contraception or interruption of gestation.

~23~(~6 It is known that fragments of endogenous hormones exhibit essentially no antigenic properties. However, should a large enough fragment of an endogenous hormone be slightly modified as indicated above, then antibodies will be formed which will react in the same way as if the modification is on a whole hormone, OX provided the large fragment is sufficiently distinctive in chemical and physical make-up as to be recognized as a specific part of the whole.
Whether the hormone or specific fragment thereof is naturally occurring or is a synthetic product is clearly immaterial. A synthetic hormone molecule will perform the same function as the naturally occurring one, being equivalent for the 10 purpose of this invention. In this connection, it will be noted that natural substances with which this invention is concerned possess carbohydrate moieties attached atcertain sites thereof whereas the contemplate corresponding synthetic polypeptides do not. Nevertheless, for the purpose of the instant specification and claims, the synthetic and natural polypeptides are treated as equivalents and both are intended 15 to be embraced by this invention. Examples of preferred antigens capable of being used in the vaccines of my invention are given in Example I-XX~ of aforementioned U. S. Patent D~,201,770.
Thus, where the word hormone or "hormone molecule" is used herein, the work "synthetic" may be added before "hormone" without changing the meaning of 20 the discussion. Similarly, the word, "fragment" may be inserted after "hormone" or "molecule" without changing the meaning, whether or not "synthetic" has been inserted before "hormone".
Throughout the above specification, the term "modified" has been utilized in referring to the chemical reaction by which the foreign molecules become chemical-25 lye attached to specific sites on the usually much larger palpated molecule Although specific mechanisms by which this is accomplished are described herein in detail, other appropriate mechanisms may be used if desired. It is clear that the modifier, i.e., the substance which modifies the concerned protein, can be a physically larger molecule or fragment thereof than the molecule or fragment which 30 it modifies. As noted above, such large molecules are deemed herein to be "carriers". Clearly, physical size of the fragment is not always critical; the criterion for effectiveness being that the body reaction generate antibodies in sufficient quanta and specific to the targeted hormone or endogenous substance The modified polypeptides of this invention may be administered parenterally 35 to the animals to be protected, preferably with a pharmaceutically acceptableinjectable vehicle. They may be administered in the form of injectable solutions or ~Z3~

suspensions. us indicated earlier, the adjutant serves as a substance which willelevate total immune response in the course of the immunization procedure.
L,ipasomes have been suggested as suitable adjutants. The insoluble salts of ~lurninum, that is, aluminum phosphate or aluminum hydroxide, have been utilized as 05 ~djuvants in routine cynical applications in man. Bacterial endotoxins or endow toxoids have been used as adjutants as well as polynucleotides and polyelectrolytes and water soluble adjuslants such as muramyl dipeptides. The adjutants developedby Freud have long been known by investigators; however, the use thereof is limited to non-human experimental procedures by virtue of a variety of side effects 10 evoked. The usual modes of administration of the entire vaccine are intramuscular and sub-cutaneous.
The amount of modified palpated to be administered will vary depending upon various factors, including the condition being treated and its severity.
However, in general, unit doses of 0.1- 50 my in large mammals administered one to 15 five times at intervals of one to five weeks provide satisfactory results. Primary immunization may also be followed by "booster" immunization at one to twelve month intervals.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE I
This example illustrates the use of a modified palpated capable of being used in my vaccines in repressing fertility in baboons.
A palpated of Structure (XII) above, identical to the residues 109-145 of I-HOG was prepared by total synthesis using the solid-phase synthesis method described in Regear et at, Become. 16, 2817 (1977). The purity of the peptize was 25 assessed using thih-layer chromatography, high-voltage electrophoresis and amino acid analysis. The peptize was conjugated to the amino groups of tetanus toxoid via the Sistine residue at position 110 by the method described in Lee et at, Mol.
Immunol. 17, 749 (1980). The resultant conjugated palpated contained 22 peptizesper 100,000 Dalton of the toxoid.
Male and female baboons (obtained from Primate Imports, Inc., Port Washing-ton, Jew York) were housed individually in metabolic cages measuring 89 x 73 x 114 cm (L x W x H) for for females and 168 x 94 x 165 cm for males, each cage being equipped with a "squeeze-bar" mechanism for restraining the baboon. Each male baboon was housed in a separate room in which were also housed six to ten females.
35 The room temperature was maintained at 21C with artificial light for 12 hours daily and the baboons were fed on Purina Monkey Meal (product of Ralston Purina, St.

~;232~)6 Louis, Missouri) mixed with corned beef, corn syrup and a vitamin mineral-supplement; phrasal fruit was given daily as a conditioning aid and water was provided Ed lobotomy. Daily observations were made of each female baboon to establish thepattern of sex stain turgescence/deturgescence and menstrual bleeding.
05 In order to ensure that the results of the test described below were statistically valid, the number of baboons required in each of the control and immunized groups was determined using arc sine transformation of the projected fertility rates and the resultant values applied to probability tables, as described in Suckle et alp Biometry, W H. Freeman and Co., San Francisco (1969), page 609. For an alpha level of p = 0.05 and a 90% confidence of detecting a significant reduction in fertility rate when the control rate is at least 70~6 and the immunized is not greater than 10%, a group size of 15 animals per group was determined to be required, and thus this was the group size used in the experiment.
accordingly, 30 female baboons whose cycle length varied by no more than 15 three days each side of its mean and who had exhibited progesterone levels of at least 3.0 ng/ml. for each of their last three menstrual cycles (thus indicating ovulation) were randomly assigned to each of two groups of 15 animals. Six male baboons, who had each proved their fertility by siring several offspring were selected for use in the experiments.
The control group of 15 baboons were immunized with pure tetanus toxoid while the other group received the aforementioned modified palpated conjugated with this toxoid. The antigens were dissolved in physiological saline mixed with an equal volume of Complete rounds Adjutant (supplied by Disco Laboratories, Detroit, Michigan and emulsified just prior to each immunization. The modified 25 polypeptide/toxoid conjugate was dissolved at a concentration of 4.0 mg/ml. and a dose of 2.0 my given to each baboon in the second group whereas the pure toxoid was dissolved in a concentration of 2.0 mg/ml. and a dose of 1.0 my. given to each baboon in the control group; since the conjugate comprises approximately 50% of the toxoid by weight, the dose of toxoid administered to each animal was 30 substantially the same. Each dose of the pure Todd or conjugate in the adjutant, approximately 1.0 ml. in volume, was injected intramuscularly into four separatesites into the animal, two in each thigh. The first (primary) immunization was given during the first five days of the menstrual cycle, with subsequent immunizationsgiven at 28 day intervals thereafter or until a pregnancy was confirmed. females35 that did not become pregnant received five or six immunizations during the course of the study depending upon the length of their individual menstrual cycles.

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food samples were collected from the female baboons without anesthesia via the cubical vein, five to six ml. of blood being drawn at weekly intervals beginning at 21 days after the primary immunization, and also immediately before and aftermating for antibody determinations. Blood samples for progesterone determinations 05 were drawn five and seven days after sex skin deturgescence and, in cycles in which mating occurs, samples for pregnancy testing were drawn daily commencing 12 daysafter deturgescel1ce and continuing until pregnancy was confirmed or menstruation begat After the blood samples were withdrawn, the serum was removed and samples not immediately tested were stored at -20C.
The serum samples thus obtained were tested for the presence of antibodies to libeled HOG, Structure IT baboon chorionic gonadotropin (bug) and tetanus toxoid by the methods described in Powell et at, J. Repro. Immunol. 2,1(1980). us described in this Powell et at paper, Structure (XIX) can be labeled with 1251 only after introduction of a Tarzan residue into the peptize. The G preparation, lo which was highly purified, had a biological potency of 10,800 lung while the bug preparation, which was only partially purified, had a biological potency of 850 IU/mg. Concentrations of labeled antigen capable of saturation of antibody combining sites at equilibrium were reacted with dilutions of serum for five days at C, followed by separation of the antigen-antibody complex from the unbound 20 labeled antigen using the double-antibody method. With the exception of tetanus toxoid, the concentrations of labeled antigens were adjusted so that equimolar quantities were reacted with the serum. The molecular weight of bug, which has not yet been established, was assumed to be the same as that of HOG, namely 38,000. The labeled antigen binding for llCG, bug and Structure (XII) was expressed 25 as moles/liter (M/L) x 10 I whereas for tetanus toxoid, due to its molecular heterogeneity, binding was expressed as micrograms/ml.
All 30 female baboons were mated during the course of their third menstrual cycle following the primary immunization and for the next two consecutive cycles if they did not become pregnant. Based upon their previously established menstrual 30 histories, each female baboon was transferred to a male's cage three days prior to expected ovulation. Cohabitation was continued until the day of sex skin detour-juiciness, 2-3 days after ovulation and the female was then transferred back to her individual cage. If the first mating did not make the female pregnant, subsequent matings were conducted with a differeIlt male baboon.
The serum levels of steroid hormones and bug were determined by the radio immunoassay methods described in Powell et at, Olin. Chum. 19, 210 (1973~ and ~ZZ,3~

Hodges et at, J. Olin. Endow Mutably. 39, 4s7 (1974). The assay for bug was conducted with an antiserum to Ovine-LE~ supplied by Dr. Chary Hodges, Bethesda,~arylarld; the supplier has previously shown, by means of unpublished data, that the billing of 125I-HCG to this antiserum can be displaced sensitively with bug, but not Us with baboon LO. The sensitivity of this assay was 5 mu of HCG/ml. Since antibodies produced in the female baboons immunized with the Structure (ZOO) conjugate were capable of binding the labeled HOG used in the HOG assay, this assay system could not be used to determine pregnancy in the baboons immunized with the conjugate. Accordingly, pregnancy testing in the conjugate-imrnunized 10 baboons was performed by measuring eastwardly and progesterone levels only. In the tetanus toxoid-immunized baboons, however, pregnancy was tested using the COG
assay as well as the steroid assays.
Data obtained on antibody levels, cycle levels and progesterone concentrations were evaluated by vflrious methods for randomized design experiments, as set out in 15 Jostle, B., Correlation Methods, in Statistics in Research, Ames, I. A., Iowa State College Press ~1954), page 17D~, while assessment of the mating data was accom-polished by the chi-squared procedure set forth in Mantel, Cancer Chemotherapy Imports 50,163 ~1966); this procedure compares the mating data in its entirety and not only in terms of individual matings.
20 Results The tetanus toxoid antibody level in both the baboons imm~mized with the pure tetanus toxoid and those immunized with the conjugate are shown in Table 1. Highantibody levels against tetanus toxoid were produced in both groups Ox baboons 60 days after primary immunization, with peak levels being reached in 90-120 days.
25 The differences between the tetanus toxoid antibody levels in the baboons immunized with the pure tetanus toxoid and with the conjugate were not significant at the p =
0.05 confidence level. Thus, it will be seen that, in addition to the anti-fertility effects observed below, the instant conjugate conferred a significant degree of protection against tetanus. Accordingly, by careful choice of the hasten to which 30 the palpated is conjugated in the instant modified palpated, the invention provides a method of protecting against a disease linked with the presence of the hasten as well as against pregnancy.

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The mean antibody levels produced Jo HOG, bug and Structure (XII) in the group of baboons immunized with the conjugate are shown in Table 2. Antibody levels to Structure (XII) reached a maximum during the little phase of the second menstrual cycle, approximately 60 days after the primary immunization, as did OX antibodies to HOG. While the mean antibody level to HOG and Structure (XII) were maintained by repeated booster imm~mizations, the responses of individual baboons varied considerably. There was a very close correlation between antibody levels to HOG and Structure (XII), r = 0.97. The mean levels of antibodies which were reacted with HOG were only 71% of those reacting with Structure (XII). However, 10 because of variation in levels between the individual animals, this difference in levels is not significant at the p = 0.05 level. Although the mean levels of antibody reacting with bug were only 4.5% of those reacting with Structure (XII) and 6.3% of those reacting with HOG, these bug antibody levels roached maximum levels by thefirst mating cycle and remained close to that level during the next two cycles lo There is a significant positive correlation (r = 0.78) between the bug antibody level and the Structure (XII) antibody level during these three cycles.

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Moreover, the antibody levels for HOG and bug shown in Table 3 reveal significant correlation between these two antibody levels during the three mating cycles. The correlation coefficients I are 0.55, 0.89, and 0.85 for the first, second, and third mating cycles respectively, the latter two correlation coefficients being OX sigllificant at the 1% level.
Table 4 below compares the cycle lengths and progesterone levels in the little phase of the menstrual cycles before and after the immunizations of the two groups of baboons. The pre-immunization portion of this table shows that the two randomly assigned groups of baboons showed no significant differences at the p = 0.05 level in 10 the cycle length or progesterone levels for the three cycles immediately proceeding immunization. Even though both groups of baboons were immunized using complete Fronds, no change significant at the p = 0.05 level in the cycle lengths or progesterone levels was apparent when the three pre-immunization cycles were compared with the post-imm~mization cycles.

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Table 5 shows the highly significant difference in the fertility rate between the two groups of baboons. On the first mating, 10 out of 15 of the tetanus toxoid-immunized baboons became pregnant, a of the 5 remaining baboons became pregnant after the second mating and the single remaining baboon became pregnant after the no third mating. Thus, of I matings, 15 resulted in pregnancy, giving a fertility rate of 7 1.~1%.
Of the 15 baboons immunized with the conjugate, one became pregnant after the first mating, one of the remaining 14 became pregnant after the second mating and 2 of the remaining 13 baboons became pregnant after the first mating. Thus, 42 10 matings resulted in four pregnancies for a fertility rate of 9.5%.
Chi-squared analysis of this data shows that this difference in fertility rate is highly significant (p < ~.0~05) even after adjustment for the small sample size.The antibody levels to tetanus toxoid from sofa obtained during the three mating cycles were assessed for correlation to the outcome of mating for all 15 animals. No correlation significant at the p = 0.05 level was found for either group of baboons. Similarly, although the post-mating antibody levels for menstrual cycles 3, 4, and 5 were quite variable, as shown by the rather large 95% confidence interval, no correlation significant at the p= 0.05 level was found between the antibody levels to Structure (ZOO, HOG or bug and the fertility of the conjugate-20 immunized group. Louvre, there was a significant difference (p< 0.025) between the mean bug antibody level in the four pregnant conjugate immunized baboons (1.4 M/L x 10 10) and the mean (9.8) and the 95% confidence interval (5.5.-14.1) levels of the same antibodies for awl matings, thus suggesting that these four baboons became pregnant because their bug antibody levels were insufficiently raised.
I have shown in the following published papers Excerpt Medic International Congress Series No. 402, pp. 379 (1976); and Physiological Effects of Immunity Against Reproductive Hormones, R. G.
Edwards and M. H. Johnson (Ed.), Cambridge University Press, pp. 249 (1975);
that immunizations with synthetic peptizes containing a C-terminal portion of B-30 HOG result in the production of antibodies capable of binding a neutralizing the biological activity of intact HOG and that these antibodies exhibit a low degree of reactivity with baboon COG. This is in accord with the results of this example, in which the conjugate based on Structure (ZOO, the 109-145 sequence of HUG
produced high levels of antibody to HOG but relatively low levels of antibody to35 bug. Nevertheless, despite the low levels of bug antibody, the conjugate was highly effective in preventing pregnancy. The pregnancy-preventing action of the ~Z3Z~

conjugate demonstrated in this example provides the statistically valid proof of the feasibility of this approach to fertility regulation in humans. It may reasonably be anticipated that the anti-fertility effects which the conjugate would produce inhumans would be considerably greater than that in baboons, given the much higherI level of antibodies to HOG produced in the baboons, as compared to the levels of antibody to bug.
The exact mechanism of action of the conjugate is not known, although presumably antibody neutralization of COG occurs in the peripheral blood soon after implantation and disrupts tropic hormone support to the corpus luteum of prig 10 Nancy and causes early abortion. However, since the duration of the menstrualcycle is not significantly effected, it appears that pregnancy must be disruptedalmost immediately after implantation.
EXAMPLE II
This Example further shows further preferred antigens usable in my vaccines.
The following peptizes, each hiving a sequence derived at least in part from that of HUG were prepared by the same method as in Example I (the numbers given refer to the sequence of bases in the full subunit of HOG, Structure (I) above:
a. 138-145, hereinafter referred to as Structure (XVI) Ser-Asp-Thr-Pr~n e-Leu-Pro-Gln b. 126-145, hereinafter referred to Structure (XVII) Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Seeras Thr-Pro-ne-Leu-Pro-Gln c 115-145, Structure (VI) above d. 111-145, Structure (II) above e. 109-145, Structure (XII) above f. 106-145, hereinafter Structure (XVIII) His-Pro-Leu-Thr-Cys-Asp-As~Pro-Arg-Phe-Gln-Asp-Serriser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pr~Serreargue-Leu-Pro-Gly-Pr~Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln g. 105-145, hereinafter Structure (ZOO) As~His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Aspposer-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pr~Ile-Leu~Pro-Glnn h Cys-(Pro)6-(111-145), Structure (XIV) above;
1. (prowesses, Structure (X) above; and ~'~23;~

Swiss), with the SO group of the Cyst residue at position 110 blocked with an acetamidomethyl (ACM) group, hereinafter referred to Structure (XX).
The peptizes of Structures (ZOO), (XIX) and (XX) contained a protected sulfhydryl group at the 110 Sistine position. The purity of all the peptizes wasdemonstrated using high-voltage electrophoresis, thin-layer chromatography and amino acid analysis.
The following carriers were used in the experiments: tetanus toxoid, polyp merited flagellin, poly-DL-alaninetlysine (polyalanine)~ (posy DALE poly(tyrosine, glutamic acid)/poly(alanine, Lawson) (TEAL) and polymerized sucrose (~icoll), de-scribed above. The carriers used were diphtheria toxoid obtained from Keynote Laboratories, Swift water, Pennsylvania) bovine gamma globulin (obtained from Swartz-Mann Laboratories, Orange burg, New York), Cornebacterium parvum (ox-twined from Burroughs-Wellcome, London, England), encapsulated meningococcal protein and pneumococcus polysaccharide.
A they'll group on each peptize was coupled to an amino group on the carrier by the same method as in Example I. The site at which the peptize was coupled to the carrier depended upon the point at which a they'll group existed or could be created on the peptize. Structure (XII) contains a Sistine at position 110 and accordingly, this peptize was conjugated by means of the they'll group produced at this position after cleavage of the synthetically produced disulfide diver of the peptize.
Structures (XVI), (XVII), (XVIII) end (XIX) had a they'll group introduced at the amino terminal group and were coupled to the carrier via this introduced they'll group.
Structures (11) and (Al) had they'll groups at the terminal amino group and also at the amino group at the Lawson residue at position 1~2, under the conditions used, approximately equal numbers of peptizes were attached to the carrier ail each of the two attachment sites. Structures (X) and (XIV) were coupled to carriers using the they'll group on the terminal Sistine residue.
After the coupling of the peptize to the carrier, the resulting conjugates were all purified by gel filtration or ultrafiltration and the ratio of the peptize to carrier was determined.
The animals used in these experiments were genetically heterogeneous rabbits of the New Zealand White variety, weighing 2-4kg. and inbred female mice of the Kiwi strain, 8-10 weeks old or retired breeders, weighing 20-30gm. each and obtained from Jackson Laboratories, Bar Harbor, Maine. For use in the rabbits, the conjugates were dissolved in saline and emulsified with an equal volume of Complete Fronds' adjutant, exactly as in Example 1. However, for immunization into mice, an adjutant was prepared by mixing 1.5 parts by volume of Arlacel A (obtained from Hilltop Research, Miamiville, Ohio) with 8.5 parts by volume of Clairol (Bate *trademark Chemical, Don Mills, Ontario, Canada) and autoclaving for 20 minutes at 15 lb.
pressure. Thereafter, heat-killed desiccated acetone-washed BOG bacteria (ox-twined from Keynote Laboratories, Wildly, Ontario, Canada) were added at the rate of 5mg. per 10 ml. of adjutant. Apart from the change of adjutant, the 05 solution used to immunize the mice was prepared in the same way as that used to immlmize the rabbits.
The mice were immunized three times, the primary immunization being given at day 0 with booster immunizations given at 21 and 38 days, the dose of conjugate injected on each occasion into mice was usually 100 ugh though multiples of this10 dose were given where stated. Elude samples were collected from the mice at weekly intervals starting at day 14.
The rabbits were immunized three times at 21 day intervals (the primary immunization at day 0 find booster immunizations at 21 and 42 days), 1 my. of the conjugate being given at each immunization. Blood samples were collected from the 15 rabbits weekly beginning at day 21. The sofa of the blood samples drawn from both the mice and the rabbits were separated from the cells and stored at -20C prior to analysis.
The levels of antibody to both the peptize used and HOG were measured by isotonically labeling the antigen with 1251 and reacting it with various dilutions of 20 the antisera. In most cases, 250 pg. of labeled antigen was incubated with 200 us of diluted serum for 120 hours at 4C. Antigen binding was determined at three or more serum dilutions using a double antibody technique, the results being expressed as nanogram (no) of antigen bound per milliliter of undiluted serum. The minimumsensitivity was 1 ng/ml. In some experiments, the level of antigen binding was 25 tested using two concentrations of antigen with two dilutions of serum and expressing the rests as M/L x 10 10 by the same methods as in Example XXXI.
The antigen binding levels found in the various groups of rabbits and mice were compared using the two-tailed Mann-Whitney U-test described by S. Siegel, "The Case of Two Independent Variables", in Non parametric Statistics, McGraw-Hill Book 30 Company, New York (1956), p. 116. Results where p < 0.05 were considered significant. In mice, in a few cases where the level of antigen binding was too low to be detected by the methods used, an arbitrary value of 0.1 ng/ml was assigned for statistical comparisons of values that were undetectable.
Results Mice were injected by the procedures set out above with doses of lug, I 8 and 1.6 fig. of conjugates of Structure (II) and ZOO with tetanus toxoid. Table 6 ~'~23;~

below shows the levels of antibodies to both the peptize used and HOG at 21 and 35 days after the primary immunization. Analysis of these results shows that the differences between peptize binding in the sofa between the mice injected with the two conjugates were significant at 21 days but not at 35 days. An increase in OX antibody levels to pept;des was observed at 35 days with increasing doses of antigen injected, but again there was no significant difference between the antibody levels of the mice injected with the two conjugates at the same dosage levels.

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To determine the effect of chain-length of the peptize on antibody response, groups of four rabbits were injected with conjugates of tetanus toxoid with the peptizes of Structures (XVI), VOW), (VI), (II), (XII), (XVIII), and (XIX). Sofa from animals immunized with these conjugates were reacted separately with equimolar 05 quantities of labeled peptize and labeled HOG, and the proportion of antibodies reactive to peptizes which were also reactive to HOG determined. The results areshown in Figure 1.
Contrary to what might be expected the maximum reactivity to HOG is not a simple function of the ehain-length of the peptize. Maximum reactivity to peptize 10 and HOG was obtained by rabbits receiving the conjugate of the peptize of Structure (XII), representing residues 109-145 of HUG The peptize of Structure (II), representing residues 111-145 of B-HCG, produced antibodies nearly as reactive as the peptize of Structure (XII), but the antibody levels produced by the longer peptizes of Structures (XVlII) and (XIX), representing respectively residues 106-145 and 105-145 lo of HUG were lower than those produced by the peptizes of Structures (XII) and(II). Not surprisingly, the shorter peptizes also resulted in a lower proportion of antibody reacting to HOG.
A further series of tests were effected to determine the effect of the hexaproline Sistine spacer sequences in the peptizes of Structures (XIV) and (X) on 20 antibody production. Conjugates of tetanus toxoid were prepared coupled to the peptizes of Structures (II) (the 111-145 sequence without any spacer), (XIV) (the 111-145 sequence with a N-terminal spacer) and (X) (the 111-145 sequence with a C-terminal spacer), all conjugates containing a peptide:carrier ratio of approximately 20-22 peptides/105 Dalton of toxoid.
Table 7 below shows the antibody levels to HOG and peptizes obtained in mice.
Twenty-one days after primary immunization the HOG antibodies were significantlyhigher in the mice immunized with the conjugates containing either of the spacerpeptizes than with the non-spacer peptize, Structure (II). Thirty-five days after primary immunization, the HOG antibody levels in mice immunized with the 30 conjugate containing the C-terminal spacer peptize of Structure (X) were signify scantly greater than those of mice immunized with the non-spacer peptize, but the difference in antibody levels between the latter and the mice receiving the conjugate of the N-terminal spacer peptize of Structure (XIV) was not significant.

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However, very different results were obtained when the serum antibodies were evaluated in terms of their ability to bind peptizes. More of the labeled peptize was bound by the antibodies produced by the conjugate of the non-spacer peptize of Structure (II) 35 days after the primary immunization than by the antibodies 05 produced by mice immunized with the conjugate of the N-terminal spacer peptize of Structure (XIV). There was no significant difference between the peptize bindingabilities of the sofa from mice immunized with the conjugates of the N-terminal and C-terminal peptizes of Structures (XIV) and (X) respectively at this time.
The tests in rabbits were carried out with the same three conjugates as in 10 mice, and also with the conjugate of tetanus toxoid and the peptize of Structure (XII) (the 109-145 sequence of HO used in the preceding series of tests. The antibody levels 10-13 weeks after the primary immunization are shown in Figure 2.
These results show that the mean antibody levels in rabbits immunized with the conjugate non-spacer peptize of Structure (II) were lower than those of rabbits 15 immunized with the spacer peptizes of Structures (XIV) and (X), but the rabbits injected with the conjugate of the peptize of Structure (XII) had mean antibody levels to HOG comparable to the rabbits immunized with the conjugates of the spacer peptizes of Structures (XIV) and (X).
further series of tests was carried out to determine the effects of different 20 carriers on antibody production. The peptize of Structure (XII), representing the 109-145 sequence of HUG was coupled to various carriers in a ratio of 15-28 peptizes per 105 Dalton of carrier and mice and rabbits were immunized with these conjugates. The antibody levels to the peptize and to HOG were tested in the sofa 21 and 35 days after the primary immunization. The results obtained in mice are 25 shown in Table 8 below. Although the large standard deviations makes the detection of significant differences difficult, the results do show that the tetanus toxoid conjugate elicited antibody levels to both the peptizes and HOG which were significantly higher than those produced by the conjugates of all the other carriers.
Mean antibody levels in the groups injected with the peptize conjugated with 30 ~lagellin and bovine gamma globulin were higher than those in which the peptize was linked to synthetic sugar (Focal) or palpated carriers. Table 9 shows the U-testanalysis of the date presented in Table 8.

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o ~L~2Z33z~6 The results obtained in rabbits are shown in Table 10 bullock. The highest antibody levels were obtained in the rabbits immunized with conjugates of bovinegamma globulin, tetanus toxoid and diphtheria toxoid, there being no significantdifference between the peak antigen titers of these three carriers. Significantly 05 lower antibody levels were found in rabbits immunized with bacterial carriers, while synthetic palpated and sugar carriers produced antibody levels which were significantly lower than those of bacterial carriers.

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o o ~LZ~5532~6 A final series of experiments were performed to determine the effect of the peptide:carrier ratio on antibody production. Conjugates of tetanus toxoid and the peptize of Structure (XII) with peptide:carrier ratios of from 5-33 peptizes per 105 doughtiness of carrier were prepared and groups of four rabbits were immunized with 05 these conjugates. The mean antibody levels to HOG produced 42, 63, and AL days after primary immunization are shown in Fig. 3. Statistical analysis of the data in Fig. 3 shows no significant difference with peptide:carrier ratio 42 days after primary immunization, but 63 days after primary immunization the antibody responses to conjugates containing 23 or more peptizes per 105 dQltons of toxoid are lo significantly greater than those of conjugates with lower peptide:carrier ratios. A
similar comparison 84 days after primary imm~mization shows that the antibody levels produced by conjugates containing 16 or more peptizes per 105 Dalton of carrier are significantly greater than those of conjugates Nit a lower peptide:ca nor ratio. Accordingly, it is believed that it is advantageous to use a conjugate containing between 20 and 30 peptizes per 105 Dalton of carrier.
When mice were immunized with the same conjugates, the responses were more variable and no linear dose-antibody response was observable, but the highest antibody levels were obtained in mice receiving conjugates containing 28-33 peptizes per 105 Dalton of carrier.
The above results show that antibodies formed to peptizes with 30 or more amino acids bind HOG better than those to peptizes with fewer residues. However,since the I and 41 residue peptizes (Structures (XVIII) and (XIX) above) were not as reactive to HOG as the 35 or 37 residue peptizes of Structures (II) and (ZOO above, it appears that no immunological determinant of ICY is present in the 105-1~9 region 25 of the beta subunit thereof. Based upon the foregoing results, the preferred peptizes for use in forming conjugates to produce antibodies to HOG are the peptizes of Structures (XII) (the 109-145 sequence without spacers), (XIV) and (X) (the 111~145 sequence with N-terminus and C-terminui spacers respectively). The addition of the seven-residue spacer sequence to either the N-terminus or the C-terminus of 30 the 111-145 peptize of Structure (II) produced higher antibody levels than the same peptize without spacer. It appears likely that a similar advantage can be produced by attaching similar spacer sequences to the 109-145 peptize of Structure (ZOO since this peptize without spacers elicited responses similar to the 111-145 peptize with spacer (cf. results for Structures (XIV) and (ZOO. On the other hand, it appears35 disadvantageous to attach peptizes to the carrier at both position 122 and the N-terminus (see results from peptizes of Structures (II) and (VI) above) since peptizes ~LZ23~

attached to the carrier at both positions did not elicit levels equivalent to those attached at either terminus alone. Probably coupling of the peptize at both its midpoint and its N-terminus affects its conformation and creates an immunological determinant dissimilar to that found on intact HOG.
05 Moreover the results presented above strongly suggest that the best carriers for use in humans or other primates are tetanus toxoid and diphtheria toxoid. While the antibody levels in rflbbits for bovine gamma globulin, tetanus toxoid and diphtheria toxoid are not significantly different, the antibody levels produces in mice with conjugates of the bovine gamma globulin are not as high as those 10 produced by conjugates of the two toxoids. Immunization of humans or other primates with tetanus and diphtheria toxoids is acceptable and even advantageous(since a single vaccination can then provide protection against tetanus or diphtheria as well as an isoimmunogenic action), whereas injections of non-primate gamma globulins may not prove safe. Conjugates of either tetanus toxoid or diphtheria 15 toxoid with a peptide:carrier ration of 20-30 peptizes per 105 Dalton of carrier evoked large titers of antibody reactive to HOG and would therefore appear to besuitable for an anti-HCG vaccine.
Example III
This example illustrates the variations in antibody levels produced by changes 20 in the adjutant and vehicle used in a vaccine of the invention.
Based upon the results in Example II above, the conjugates of tetanus toxoid with the peptizes of Structures (XIV) and (XII) were selected as most efficacious in generating antibodies to HOG and were thus used in these experiments to select the optimum adjutant and vehicle. The tetanus toxoid/peptide conjugates were pro-25 pared in exactly the same manner as in Example II and purified by gel filtration andlyophilization. The conjugate of the peptize of Structure IT contained 21-25 peptizes per 105 Dalton of carrier, while the conjugate of the peptize of Structure (IVY) contained 20-27 peptizes per 105 Dalton of carrier. A further conjugate was prepared by conjugating the same tetanus toxoid to both the peptize of Structure30 (XIV) and the synthetic muramyl deputed CUP 11637 (manufactured by Ciba-GeigyLimited Basic, Switzerland, - Formula (a) below). Using carbodiimide, the carboxyl group of the deputed was coupled to the amino groups of the tetanus toxoid, thereafter the peptize of Structure (IVY) was coupled to the tetanus toxoid via the remaining amino groups using the same procedure as in Example II. The resultant 35 conjugate contained five muramyl dipeptides and 31 peptizes of Structure ~XIV) per 105 Dalton of carrier respectively.

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total of eight different adjutants were tested. The first five of these adjutants were synthetic muramyl deputed hydrophilic analogies obtained from Ciba-Geigy Ltd., these five dipeptides being:
(a) CUP 11637, of the formula 05 NAc-nor Mural. Ala-D. is Gin;
(b) CUP 14767, of formula NAc-nor-Mur-L-Abu-D.isoGln;
(c) CUP 18177 of formula Newcomer (6-0-stearoyl)-L.Ala-DOisoGln;
(d) CUP 18741 of formula N~c-nor-Mur (6-0-stearoyl)-L.Ala-D.isoGln;
(e) CUP 19835 of formula NAc-Mur-L.Ala-D.isoGln-L.Ala-Cephalin.
10 The sixth adjutant was another muramyl deputed obtained from Syntax Corpora-lion, Palo Alto, California being:
(f) DT-l, of formula NGlycol-Mu~L.o~Abu-D.isoGln.
the last two adjutants were lipophilic adjutants manufactured by Ciba-Geigy Ltd., as follows:
(g) CUP 16940, of formula N-Palmitoyl-~-[2(R,S)-3-dipalmitoyloxy-propyl~ -L-Cys-L-Ser-L-Ser-L-Asn-L-Ala-L-Glu; and (h) CUP 12908, a highly purified lipoprotein from the cell membranes of E. Coil B.
The vehicles used in these experiments were:
(a) an aqueous solution of O.OlM sodium phosphate and 0.14M sodium chloride, of pi 7.0, hereinafter designated BUS;
(b) Incomplete Fronds' adjutant comprising 1.5 parts by volume of Arlaeel A (Muhammad monooleate)and 8.5 parts by volume of I~learol, both reagents being obtained from the same sources as in Example X~XII, the adjutant being referred to 25 hereinafter as IF;
(c) Squalene-Arlacel A, comprising four parts by volume Skyline (obtained from Sigma Chemicals, St. Louis, Missouri and one part by volume Arlacel A;
(d) Skyline - Arlacel A, comprising four parts by volume Skyline (ox-twined from Eastman Kodak, Rochester, New York) and one part by volume Arlacel 30 A;
(e) Peanut oil adjutant, comprising 10 parts by volume peanut oil (obtained from Merck, Munich, West Germany) and one part by volume egg lecithin;
(f) Liposomes adjutant, comprising 12 parts by weight egg lecithin and 1.6 parts by weight cholesterol; and (g) Alum adjutant, comprising 10 percent by weight potassium alum pro-cipitated with lo sodium hydroxide, as described in M. W. Chase and C. A. Williams ~223~6 (ens.), Methods in Immunology and Immunochemistry, Vol. I, Preparation of Antigens and Antibodies, academic Press, New York (1967), pp. 201-202.
The experimental animals used in these studies were the four inbred strains of mice Kiwi, C57BL/6, Dual and SOL, obtained from the same source as in OX Example Il. 'rho mice were retired breeders of more than 32 weeks of age and weighed 25-30 grams. Also used were genetically heterogeneous New Zealand White rabbits weighing 2-4kg. obtained from the same source. The mice were immunized subcutaneously with a primary immunization at day 0 and boosters at 21 and 38 days and, in some experiments, at day 55. Unless otherwise stated, each injection 10 comprised 200 ugh of conjugate and loo ugh of adjutant. Blood samples were collected from the mice 28, 35, 45, 52, 62, and 69 days after the primary it munization.
The rabbits were immunized intramuscularly three times at 21 day intervals, each injection comprising 500 ugh of conjugate and 500 go of adjutant unless 15 otherwise stated. Blood samples from the rabbits were collected weelcly on the sty day after the first immunization. In the case of the blood samples from both themice and the rabbits, the serum was separated from the remaining components of the blood and stored at -20C prior to analysis. Complete Fronds adjutant purchased or prepared as in Example II above was used as a reference adjuvan~. The 20 levels of antibody in the blood sofa reacting to the peptize in the conjugate or HOG
were measured using the same double-antibody technique as in Example II and the test results were evaluated using the same Mann-Whitney U-test as in that Exanlple.
In experiments in which antigen binding levels were pooled within each experimental group, Chi-squared analysis was used to determine significant differences.
25 results __ In a first series of tests, various adjutants were evaluated using IF as the vehicle. The Structure (XlI)/tetanus toxoid conjugate was combined separately with the adjutants CGP11637, CUP 1476?, CUP 12908 and CUP 1~940. The resultant conjugate/adjuvant mixtures were incorporated into IF emulsions and a parallel 30 series of emulsions were prepared using the conjugate (without any adjutant) in Complete Fronds' Adjutant (CFA). Each separate emulsions was administered to four groups each comprising five mice from one of the four inbred strains. Antibody levels to Structure (XII) and HOG were determined 52 days after the primary immunization and the results are shown in Table 11 below.

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pi I, u *o o o ~Z23Z~Çi The data in Table 11 reveal significant differences among the four strains of mice immunized with CFA. The differences between the levels of antibody to Structure (XII) between the Doyle and Kiwi groups on the one hand and the 05 C57BL/6 group on the other are significant at the p = 0.05 level. Moreover, the differences between the HOG antibody levels of the SOL group on the one hand andthe I)BA/l and the C57BL/6 group on the other ye also significant at the p = 0.05 level.
Table 12 below presents statistical comparisons of antibody levels produced in 10 mice receiving each of the four adjutants in comparison with the antibody levels levels in mice receiving the CFA.

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Of the four adjutants tested, only the lipophilic adjutant CUP 12908 induced responses in all four strains of mice that were not significantly different from those of the respective CFA groups, as regards antibody levels to either HOG or Structure (Ill). The other lipophilic adjutant CUP 16940, produced significantly lower levels I of antibody to Structure (XII) in the mouse strains DB~/l and Kiwi, and significantly lower levels of antibody to HOG in all four mouse strains. The hydrophilic adjutant CUP 14767 produced Structure (XII) antibody levels significant-lye lower than those produced by CFA in only one mouse group, the strain Dual while the Structure (XII) antibody levels produced by the other hydrophilic adjutant 10 CUP 11637 were significantly lower in the three mouse groups Dual Kiwi and SOL than in the corresponding CFA groups. The HOG antibody levels produced by both the hydrophilic adjutants COG 11637 and 14767 were not significantly different from those of the corresponding CFA groups, only the JO strain producing significantly different results in both cases.
lo Since the responses of the mice to immunization with the Structure (XIT)/tetanus toxoid conjugate were more dependent upon the adjutant used than the strain of mouse injected, genetic differences among the various strains were difficult to assess. However, the C57BL/6 strain mice did not show any significant differences in Structure ZOO antibody levels with any of the four adjutants tested, 20 as compared with the CFA immunized mice. On the other hand, mice of the Dual strain did show significantly lower Structure IT antibody levels with three of the four adjutants tested, as compared with the CFA immunized mice. Accordingly, in some of the later experiments only these two strains of mice were used for assessing genetic differences.
A parallel series of tests using the same four adjutants as well as the hydrophilic adjutant DT-l in conjunction with Structure (XII)/tetanus toxoid con-gigawatt and IF, produced somewhat different results, as shown in Figure 4, which shows the mean COG antibody levels averaged over the groups of four rabbits used, from 3-13 weeks after primary immunization. As with the mouse tests, a group of 30 four rabbits was immunized with the conjugate incorporated into CFA for come prison. Figure 4 shows that the HOG antibody levels in all groups were substantially constant prom the 9-13 weeks after primary immunization, and therefore statistical evaluation of the antibody levels was conducted after pooling data within each group during this five week period.
As compared with the CFA immunized rabbits only the rabbits receiving the adjutant CUP 16940 produced significantly lower HOG antibody levels (p<0.05). The 2~Z~

rabbits receiving adjutants CUP 11637 and DT-l produced HOG antibody levels significantly greater than the rabbits receiving adjutants CUP 16940 and 12908 (p<0.05), but not significantly higher than the rabbits receiving adjutant CUP 14767 or the rabbits receiving OF
05 Further tests were conducted in mice and rabbits to determine the effects of the various vehicles on antibody levels. In the mice tests, groups of five mice from each of the strains C57BL/6 and Dual were immunized with the conjugate of tetanus toxoid and Structure (XIV) (the 111-145 sequence of HUG with the N-terminal spacer sequence) in conjunction with one of the vehicles PBS, IF, 10 liposomes and Squalene/Arlacel. No adjutants were used in immunizing the micereceiving the vehicles, but a group of mice from each strain were immunized withthe conjugate incorporated into CFA for comparison purposes.
The mean Structure (XIV) and HOG antibody levels for each group of mice 35 and 52 days after the primary immunization are shown in Table 13, along with to lo standard deviations. Comparisons of the data in Table 13 as between the CFA
Control group with the groups receiving the various other vehicles by the alone-mentioned U-test are shown in Table I below. Table 14 shows that the Structure (XIV) antibody levels in the Dual mice were significantly higher than those in the C57BL/6 mice at either 35 or 52 days after the primary immunization. In terms of20 HOG antibody levels the Dual mice were significantly lower than those of the C57BL/6 mice 35 days after immunization, but not significantly lower 52 days after primary immunization.
The U-test factors in Table 14 show that, in the C57BL/6 mice groups 35 days after primary immunization, only the group receiving the IF did not haze 25 significantly lower levels of antibodies to both Structure (XIV) and HOG than those of the mice receiving CFA. However, the results 52 days after primary immunize-lion are strikingly different: no significant differences between Structure ~XlV) or HOG antibody levels existed at that time between the mice receiving CFA and those receiving BP~,IFA or liposomes vehicles. Indeed, the Squalene/Arlacel vehicle 30 immunized mice had levels of antibodies to both Structures (XIV) and lick which were significantly higher than those of any other vehicle including CFA. The responses of the Dual mice were not similar to those of the C57BL/6 mice.
Thirty-five days after primary immunization, the four groups of Dual mice immunized with the vehicles under test had Structure (XIV) antibody levels 35 significantly greater than those of the mice receiving CFA, but only the group of mice receiving liposomes vehicle had HOG antibody levels significantly lower than ~32C~6 the CFA group. In all the groups of DBA/i mice 52 days after primary immunize-lion, the antibody levels to both Structure (XIV) and HOG were not significantlydifferent from the CFA group.

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A similar series of tests were run in rabbits using the four vehicles Squalane/Arlacel A, Squalene/Arlacel B, peanut oil and alum. These tests were conducted using the same (Structure XIY)/tetanus toxoid conjugate, but the adjutant DT-l was used. Again, a control group of rabbits were it minced with the no conjugate emulsified in CFA without a vehicle. Antibody levels to HOG were measured throughout the 13 week immunization period and the data obtained from etch group of rabbits at the week of peak antibody level were pooled with the values obtained from thy same group one week earlier and one week later. The results are shown in Table 15 below. The HO antibody levels in rabbits receiving 10 Squalene/Arlacel were significantly higher than those of all other groups, including the rabbits receiving CFA. No significant differences in HOG antibody levels existed between the rabbits receiving Squalane/Arlacel and those receiving CFA, while the rabbits receiving peanut oil or alum vehicles had significantly lower HOG
antibody levels than those receiving CFA.
The efficacy of the Squalene/Arlacel A vehicle in increasing the antibody levels in the injected animals is surprising, especially since Squalene/Arlacel has not previously been used as a vehicle in a vaccine, although Skyline is used in topical preparations such as ointments and cosmetics. Although the Squalane/Arlacel A
vehicle was not as effective as the Squalene/Arlacel vehicle, it was as efficacious as 20 CFA. Both the Squalene/Arlacel and the Squalane/Arlacel vehicles should be clinically acceptable for use in human beings, since they appear to produce little or no irritation at the sight of injection, whereas CFA is known not to be clinically acceptable for use in human beings since it tends to produce intense irritation,assesses, etc. at the point of injection.

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To evaluate simultaneously combinations of various adjutants and various vehicles, and thus to detect any second order effects due to the interactions ofparticular adjutants with particular vehicles, groups of five or six mice from each of the strains C57BL/6 and Dual were immunized with preparations comprising the no same Structure (XIV)/tetanus tweaked conjugate used in the preceding tests incombination with one of the synthetic adjutants CUP 18177,187d~1 or 19834 and one of the three vehicles Squalene/Arlacel, liposomes and peanut oil. As in the preceding test described above with reference to Tables 13 and 14, HOG antibody levels were determined in sofa collected 35 and 52 days after primary immunization, and the 10 results are shown in Table 16 below. Lowe data in Table 16 reveal no significant differences between the levels of antibodies in the groups of mice injected with the same adjutant in different vehicles, nor between the groups receiving the same vehicle and different adjutants, at either 35 or 52 days after primary immunization (i.e. p > 0.05 in all cases). Furthermore, no differences significant at the p = 0.05 15 level were detected between the levels between corresponding groups of mice of difference strains.
Table 17 below shows data similar it? those in Table 16 but relating to Structure (ZOO) antibody levels instead of HOG antibody levels. Analysis of the date in Table 17 shows that 35 days after primary immunization in the C57BL/6 mice, 20 mice receiving the adjutant CUP 1~177 produced significantly higher levels ofantibodies than the group receiving adjutant CUP 19835 (p < 0.05). However, the similar differences at 52 days after primary immunization were not significant at the p = 0.05 level. Comparison of the three adjutants of the Dual mice 35 days after primary immunization shows no significant differences between the adjutants 25 in Squalene/Arlacel or peanut oil vehicles but the Structure (XIV) antibody levels produced by CUP 18177 in liposomes vehicle were significantly higher than those produced by CUP 19835 in the same vehicle (p < 0.05). The results 52 days after primary immunization show the same pattern of significant differences as the results 35 days after primary immunization.
While mean HOG antibody Levis were generally higher in mice immunized with Squalene/Arlacel vehicle and (Structure XIV) antibody levels were consistently higher using this vehicle? regardless of adjutant or mouse strain? these differences were not significant in view of the variability in the responses of the mice.
Although no significant differences were found between the two strains of mice as 35 regards to HOG antibody levels, the Dual mice produced higher mean Structure (XIV) antibody levels in all vehicle and adjutant groups than the C57BL/6 mice although the differences were not statistically different.

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A similar series of tests were then carried out in rabbits using the same Structure (~IV)/tetanus toxoid conjugate, the adjutants CUP 18177,18741, and 19835 tested in mice, plus the additional adjutants CUP 11637 and DT/l, using groups of four rabbits and Squalene/Arlaeel as the vehicle. Again, a control group of rabbits 05 were immunized with the conjugate incorporated into CFA without a vehicle. The mean HOG antibody levels in sofa collected from the various groups of rabbits weekly from 3-12 weeks after primary immunization are shown in Table 18 below, together with the corresponding standard deviations. The data in Table 18 show that the highest antibody levels were achieved in rabbits receiving the adjutants CUP10 11637 and DT/l 10 weeks after primary immunization. Comparison of the antibody levels produced by these adjutants with the control group receiving CFA showed that the increases in antibody levels with both adjutants we're significant at the p =
0.05 level. Adjutant CUP 18177 also produced higher antibody levels than CFA, the difference being significant at the p = 0.05 level, and the differences between the 15 antibody levels produced by the three adjutants CUP 11637, DT/I and CUP 18177 are not significant at the p = Owe level. The adjutants CUP 19835 and 18741 producedantibody levels which were lower than the CFA rabbits, the difference being significant at the p = 0.05 level.

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A final series of tests were run in mice to determine the effect of coupling one of the adjutants to the peptide/tetanus toxoid conjugate instead of merely administering the peptide/tetanus toxoid conjugate mixed with the adjutant. Groups of five mice from each of the strains C57 BLUE and Dual were immunized with the 05 Strut lure (XlV)/tetanus toxoid/CGP 11637 conjugate described above, separategroups of mice being immunized with the conjugate in association with each of the vehicles PBS, IF, liposomes and Squalene/Arlacel. As before a Control group fromeach strain was immunized with the conjugate incorporated into CFA for compare iron purposes.
lo Figure shows the mean HOG and Structure (XIV) antibody levels in the mice as a function of time after primary immunization (the coding for the various vehicles in Fig. 5 is the same as that in Fig. a) Figure pa shows the HOG antibody levels of the C57BL/6 mice. The elevated antibody levels produced by CFA and IF
35 days after primary immunization are not significant at the p = 0.05 level and15 were not sustained after the booster immunizations at 33 and 55 days. The mice receiving the conjugate in Squalene/Arlacel vehicles did not respond significantly until the third bleeding, 45 days a ton primary immunization, but there HOG
antibody levels increased progressively thereafter and were higher by an amount significant at the p = 0.05 level as compared with those of the other mouse groups.
20 The Structure (XV) antibody levels of the same mice shown in Figure 5b parallel those of the HOG antibody levels and statistical analysis shows that the same differences are significant.
The HOG antibody levels of the Dual mice shown in Fig.5c are considerably lower than those of the corresponding C57BL/6 mice. Fifty-two days after primary25 immunization, the HOG antibody levels in the Dual mice receiving the Squalene/Arlacel vehicle were not significantly different from those of the CFA
Control group (p owe), but were significantly higher than the levels achieved in the mice receiving the other vehicles up < 0.0). Sixty-nine days after primary immunization, there were no differences significant at the p = 0.05 level between 30 any of the groups of Dual mice. However, as shown in Fig. Ed, unlike the results obtained with the C57Bl/6 mice, in the Dual mice, the antibody levels to Structure (XIV) do not follow the antibody levels to HOG. The Structure (XIV) antibody levels in the Dual mice receiving CFA or IF were elevated 28 days after the primary immunization and thereafter declined until day 52. Later, the CFA immunized mice35 levels rose in response to the booster injection administered at day 55, while the antibody levels in the IF mice continued to fall. No differences significant at the ~'~Z3~

p = OWE level were found between the antibody levels in the mice receiving CFA
IF or Squalene/Arlacel 52 days after the primary immunization, but at this time the differences between the mice receiving CFA and those receiving PBS and liposomes vehicles were significant at the p = 0.05 level. Al ton 69 days from 05 primary immunization, while the difference between the CFA groups and the remaining vehicles were significant at the p = 0.05 level, the differences between the other groups were not significant at this level.
The experimental results described above clearly demonstrate the efficacy of some of the synthetic adjutants in increasing antibody production when administered 10 in conjunction with the modified palpated of the invention. Some of the adjutants, in particular CUP 11637 and 18177 and DT/l, when administered in certain vehicles, especially IF and SqualenetArlacel produced antibodies exceeding those produced by CFA. In general, the hydrophilic muramyl peptize adjutants were superior to the lipophilic adjutants in enhancing the antibody production caused by the hydrophilic 15 Structure (XIV)/tetanus toxoid conjugate. The experimental results also show that the delivery system used to administer the antigen and adjutant is of critical importance in producing an enhanced response to the conjugate. For example, the antibody responses of the C57BLt6 mice which received the conjugate in Squalene/Arlacel without adjutant were greater than those of the mice receiving 20 the conjugate in CFA. Adding an adjutant to this vehicle produced only a slight increase in the antibody levels. These observations are confirmed by those in rabbits receiving the conjugate and the synthetic adjutant in different vehicles, and different adjutants in the same vehicle. Squalane/Arlacel and peanut oil emulsions were efficacious, though less effective than Squalene/Arlacel.
To sum up, based upon all the foregoing data it appears that the optimum vehicle was Squalene/Arlacel, which was superior to the others in almost every formulation and that the best adjutants are CGB11637 and DT/l, which were more efficacious in various vehicles than other adjutants tested simultaneously. Squaw lane/Arlacel was also a highly acceptable vehicle although somewhat less of-30 ficacious than Squalene/Arlacel.
eye This example illustrates a use of a modified palpated in repressing a carcinoma which produces a chorionic gonadotropin-like material. This material by John A. Keen, Arnost Kaolin and Herman F. Acevedo is in press and will shortly 35 appear in ''Cancer".

siege The rat mammary adenocarcinoma R 3230 AC has been shown, using immune cytochemical, radio immunoassay (RIP) and bioassay methods in tissue sections and cell cultures to product a chorionic gonadotropin (CG)-like material; however this CG-like material cannot be detected in the sofa of the animals bearing the 05 carcinoma. The detection of the CG-like material is described in:
(a) Mullen et at, Immunohistochemical Detection of Ectopic Hormones in Experimental Rat Tumors, in FOG. Lehman (Ed.), Carcino-embryonic Proteins, Vol. 2, Elsevier-North Holland Biomedical Press, New York, Amsterdam (1979), pp. 751-758.
10 This adenocarcinoma can be propagated in cell culture and its cells retain their morphology and malignant characteristics. Intravenous injection of these cells into isologous Fischer 344 rats gives rise to numerous foci of this neoplasm in the lungs within 8-10 days, the animals then becoming very sick and then dying within 12-15 days.
The R 3230 adenocarcinoma (obtained from Dr. A. Ogden, Mason Research Institute, Worcester, Massachusetts) was cultured from explants of subcutaneous tumors grown for 20-21 days in Fischer 34~ female rats. The cultures were maintained in RPMI lG40 medium supplemented with 10% fetal cough serum. Upon reaching confluence, the cells were dispersed by means of trypsin-EDTA (1:250) and 20 passage five times. During the early stationary growth phase, the cultured cells were dispersed for in viva administration. The cell density was determined using a hemocytometer and viability was tested by the Try pan blue exclusion method. Onemillion cells were then injected into the tail vein of each of the experimental animals, which were 100 female Fischer 344 rats weighing 130-150g. The animals 25 were divided into two matched control groups of 15 animals each and a test groups of 70 animals. They were individually caged, fed Purina (Registered Trade Mark) chow and tap water ad lobotomy. All animals except six from the test group were sacrificed at intervals by cervical dislocation for necropsy studies. The lungs,livers, spleen and kidneys were examined and processed for paraffin sections and30 Ho staining in order to detect neoplastic foci, the diameter of the foci being measured with an eye piece micrometer. All animals were also bled before sacrifice and the serum tested for the presence of antibodies to COG.
The test group of 70 rats received, prior to the injection of carcinoma cells, a~-HCG/tetanus toxoid modified palpated of the invention. This HUG con-35 gigawatt, and its method of preparation, is described in U.S. patent 4,161,519 to Talwarand in the following papers written by the same author and others:

3Z~6 (b) Pro. Neil. Aged. eye. U.S.A. 73, 218-222 (1976);
(c) Contraception, 18,19-21(1978);
(d) Fertile Sterile 34, 328-335 (1980).
The conjugate was administered in saline once per week for a period of three weeks, 05 Approximately lo jug. being given at each injection and the last injection being given two weeks before administration of the carcinoma cells. In order to study the effect of the conjugate over an extended period a number of animals selected at random were sacrificed for autopsy, as follows (the day when the carcinoma cellswere injected being taken as day 0):
Number of Rats ~acri_ced :15 22 10 1~0 2 One of the two control groups was administered highly purified tetanus toxoid (obtained from Keynote Laboratories, Toronto, Ontario, Canada) at a rate of 20 0.7 ugh per injection, following the same injection schedule as the test group followed with the conjugate. Thus, this control group received a dose of tetanustoxoid substantially equal to that received in the conjugate by the test animals. The second control group received no treatment other than the tumor suspensions. Allthe Mammals from the test group were sacrificed for autopsy on dry 10.
Testing for the presence of HOG antibodies was performed using the HUG
RIP kit, Quantitative Method II (manufactured by Syrian Laboratories, Brain tree, Massachusetts. To the cold standard HOG, HOG free serum and 125I-HCG, an Alcott of the rat serum was added. After overnight incubation, the original protocol of the kit was followed in the subsequent procedure. If all the labeled HOG
30 was precipitated (thus indicating a high antibody titer the test was repeated with appropriately diluted rat serum.
All the animals in the two control groups, sacrificed 10 days after injection ofthe tumor cells, showed multiple lung foci of neoplastic cells in accordance with the normal progress of this carcinoma. None of the 30 animals in the control groups 35 showed neoplasms in any other organs. The qualitative histological appearance of the pulmonary tumor deposits was practically identical in every animal. The ~'~23~

neoplasm is a moderately differentiated adenocarcinoma rarely producing glandular alumina. The mitotic rates are high, but tumor necrosis and inflammatory reactions are absent. The tumor nodules range in size from a few cells to more than one millimeter in diameter and are uniformly distributed throughout the long parent 05 shim. Using a 2.5 x objective, from 5-10 tumor nodules were present in every low power camera field in the controlled animals. In contrast, the test animals that had received the B-HCG/tetanus toxoid conjugate rarely displayed more than 1 to 4 metastatic nodules not exceeding 0.3mm. in diameter in the entire lung sections 8-10 days after administration of the tumor cells, so that only a few camera fields could 10 be found containing a single metastatic focus. Most lung parenchyma were completely free of neoplasms. The histological appearance of the neoplasms in the test animals was the same as that in the controls.
In all the rats receiving the conjugate, a significant antibody titer was found,antibody levels capable of precipitating Moe of HOG standard per milliliter of 15 serum being consistently determined. This titer persisted for up to 120 days after receipt of the tumor cells and continued to protect at that time against formation of metastatic lung foci after a new intravenous seeding with the tumor cells. Incontrast, neither of the control groups showed any measurable HOG antibody levels, regardless of whether the rats had or had not received the tetanus toxoid.
Thus, the protective effects of immunization with the conjugate prior to injection with the tumor cells were demonstrated by the test animals being alive 20 days after injection of the tumor cells (at which time, 100% mortality would be expected in unprotected animals), by the absence of lung pathology in the animals sacrificed more than 20 days after injection of the tumor cells, by the long term 25 survival (more than six months) and lack of deterioration in the six test animals that were not sacrificed, and by the fertility of these six surviving test animals, several of which produced normal litters after termination of the 120 day observation period. This long term survival of all the six animals and absence of any deterioration therein is especially surprising in view of the surliness of the R 3230 carcinoma chosen for study, since previous work has indicated that no rats receiving the intravenous injection of tuner cells received by these test animals can be expected to survive for more than about 20 days.
Since there are no detectable levels of serum HO associated with this carcinoma in rats, the absence of pathological changes in the organs investigated 35 and the maintenance of the reproductive functions in the surviving test rats suggest that the antibodies act at the level of the cell membrane, probably in a cytotoxic ~2'~3~Z~6 I

manner, although the above experimental results are not sufficient to prove thishypothesis.
Example V
This example illustrates the use of a vaccine of the invention coupled to 05 diphtheria toxoid in repressing fertility in baboons.
modified palpated of the invention based upon Structure (XII) above was prepared in exactly the same manner in Example I except that the peptize was conjugated to diphtheria toxoid instead of tetanus toxoid. Also, the resultant conjugate, again containing about I peptizes per 100,000 Dalton of the toxoid, was 10 dissolved in a solution of the murarrlyl deputed CUP 11637 used in Example III, instead of the Complete Fronds' adjuvent used in Example I. The resultant conjugate/adjuvant mixture was emulsified with an equal volume of the 4:1 v/v Squalene/~rlacel A vehicle used in Example III. Again, the control animals received diphtheria toxoid in an amount equal to that received as part of the conjugate by the 15 test animals. All details regarding animal feeding, group size, animal housing, mode of administration of vaccine and blood testing were exactly as in Example I.
The length of the menstrual cycles and the little phase progesterone levels for both the test and control groups were measured for three menstrual cycles beforeimmunization and five menstrual cycles after immunization. The results are shown20 in Table 19 below (which is directly comparable with Table 4 of example I). The data in Table 19 shows that, as in the previous experiment, no significant differences existed between the lengths of menstrual cycle and little phase progesterone levels of the two groups of baboons either before or after immunize-lion.
tubule 20 below shows the antibody levels to Structure (XII), baboon chorionic gonadotropin and human chorionic gonadotPopin produced in the test animals injected with the conjugate during the five menstrual cycles following immunize-lion. As in example I, the antibody levels were determined from serum drawn fromthe early little phase of each menstrual cycle. Thus, the data in Table 20 may be 30 compared directly with those in Table 2 of Example I. Comparing Tables 2 and 20, it will be seen that the diphtheria-toxoi~containing conjugate produced slightlylower levels of antibodies to baboon chorionic gonadatropin than did the tetanus-toxoi~containing conjugate (though this difference does not appear to be sign nificant), but rather higher levels of antibodies to human chorionic gonadatropin 35 (and the differences between the two groups for menstrual cycles Nos. 4 and 5 would appear to be significant).

~LZ'~3~

As in sample It each of the baboons was mated during the course of the third menstrual cycle following immunization and also mated during the two subsequent menstrual cycles unless, of course, a previous mating produced a pregnancy. The results are shown in Table 21, which is directly comparable with Table 5 of Example 05 1. Of the control animals immunized only with diphtheria toxoid, 11 out of 15 became pregnant on the first mating, three of the remaining four became pregnant on thesecond mating and the remaining baboon was still not pregnant after the third mating Thus, a total of 20 matings produced 14 pregnancies and a fertility rate of 70%. On the other hand, in the test animals immunized with the conjugate, none of 10 the 15 baboons became pregnant during the first mating, only one of the remaining baboons became pregnant at the second mating, and at the third mating, another one of the 14 remaining baboons became pregnant. Thus, a total of 44 matings produced only two pregnancies for a fertility rate of 4.6%. Although statistical comparisons are difficult because of the very small numbers of pregnancies involved in the I conjugate-imm~mized baboons, it thus appears that the vaccine used in this Example containing Structure (XII)/diphtheria toxoid conjugate is more effective in prevent-in conception in baboons than the vaccine used in Example I.

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EXAMPLE VI
This example illustrates the effect of a vaccine of this invention in retarding the growth Ott Lewis Lung Carcinoma tumors in mice.
The mice used in these experiments were six-weelc old mice of the strain 05 C57B~/6, obtained from the same source as in Example II. The mice were divided into a test group of 20 animals and a control group of 17 animals. The test animals were immunized using the same vaccine as described in the second paragraph of Example V above, each injection comprising 200ug. of conjugate and lug. of adjutant. Following the first injection, booster immunizations were given 4, 12 and 10 23 weeks later. The control group of mice were given immunizations at the same time, but the vaccine used contained only the vehicle. Two days after the final immunization, a Lewis Lung Carcinoma containing approximately 1 million viable cells was implanted in each mouse. Measurements of the volume of the tumor were made 10,14 and 17 days after tumor implantation and then, on day 18, the mice were lo scarified and the tumor removed and weighed.

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The result in Table 22 above show that the conjugate immunized mice showed significantly smaller tumors than the control group, the average reduction in tumor volume and weight being about 40%.
EXAMPLE VII
This example illustrates the effect of a vaccine of the invention in increasing the survival rate of mice suffering from viral-induced leukemia.
The mice used in this experiment were six-week old female mice of the SJL/J
strain. test group and a control group each containing approximately 20 mice lo were selected at random and were immunized in the same manner as in Example VI
above, the test animals receiving the vaccine containing the peptide/Diphtheria Toxoid conjugated and the control group receiving the vaccine containing only the vehicle. However, in this experiment a different immunization schedule was followed: the original immunization was followed by booster immunizations a, 9, 20 15 and 29 weeks later. Six weeks after the first immunization (i.e. two weeks after the second immunization) each mouse was injected with approximately 1 million Friends' Leukemia Virus and the mice were observed daily for deaths. The results are shown in Table 23 below and from the results in this table it will be seen that the vaccine of the invention provided complete protection against mortality caused by the 20 leukemia virus, whereas more than 60% of the control group succumbed.

~;~Z3~

Comparison of survival rates in mice inoculated with Friends' Leukemia Virus following imm~mization with vehicle or conjugate.

__ ___ __ 05 Survival Rate - OWE
Days _ After Group Immunized With Inoculation Vehicle (Control) Conjugate 0 lQ0.0 100.0 22 87.5 100.0 29 75.0 100.0 98** 75.0 100.0 160 56.3 100.0 lo 163** 56.3 100.0 166 ~3.8 100.0 175 37.5 100.0 180 37.5 100.0 _ 20 **Day of booster immunization EXAMPLE VIII
This eacample illustrates the effect of a conjugate usable in the vaccines of the invention in retarding the growth of a sarcoma tumor in mice.
Sixty mice of the ARK strain were divided at random into a test group and a 25 control group each comprising 30 mice. The test group were then immunized in the same manner as in Example VI and VII with a vaccine comprising the same conjugate as in those Examples dissolved in Fronds Complete Adjutant, while the control group simply received the Fronds Adjutant. Booster immunizations were given to each group three and seven weeks after the first immunization. Ten days after the 30 third immunization, the mice were inoculated with approximately 2 mm . of Rouge Osteogenic Sarcoma cells, and the mice were observed to see if the tumor survived. In the mice in which the tumor survived, tumor volume measurements were made 22, 26, 29, 31, 33, 38, 40 and 43 days after inoculation with the ~;23~6 Sarcoma. The average tumor volume in the surviving mice of each group in which the tumor survive are shown in Table 24 below. The results in Table 24 show thatthe conjugate substantially reduced the rate of tumor growth. The later results should be interpreted with some caution because they are affected by the numbers05 of mice surviving; for example, at day 43, seven of the conjugate immunized mice were surviving but only five of the vehicle-immunized mice and naturally the mice having the largest tumors tended to succumb first so that the figure for averagetumor volume in the vehicle-immunized mice at day 43 is thus artificially reduced by the deaths of certain mice. Nevertheless, the results in Table 24 do show a lo significant reduction in the rate of tumor growth in the conjugate-immunized mice.
The mice were also bled ten days after the final immunization and serum tested for the presence of hug antibodies; all the mice immunized with the conjugate had produced such antibodies.

15 Comparison of tumor volumes in mice inoculated with ~idgeway Osteogenic Sarcoma following immunization with vehicle or conjugate Mean Tumor volume - cm3 Day of 20 Tumor Group Immunized With Growth Vehicle (Control) Conjugate _ 22 0.15 0.65 26 1.15 0.68 2529 2.30 1.20 31 3.45 2.24 33 2.33 3.~4 36 6.27 3.14 38 7.S5 4.40 3040 10.36 5.54 43 8.21 5.00 _ _ ~.Z;~326~

It will be appreciated that numerous changes and modifications can be made in the embodiments of the invention described above without departing from the scope of the invention. Accordingly, the foregoing description is to be construed in an illustrative and not in a limitative sense, the scope of the invention being defined 05 solely by the appended claims.

~2;23~6 SUPPLEMENTARY DISCLOSURE
AS already noted, the modified polypeptides used in the vaccines of the invention which are derived from endogenous protein hormones, non-hormonal proteins or fragments thereon, provoke, when administered into the bodies of OX appropriate mammals, antibodies to the endogenous proteins from which the modified polypeptides are derived. Cons-quaintly, not only can such modified polypeptides be used to influence the biological activity in a mammal to which they are administered by generating antibodies to an endogenous lo protein in the mammal, but the modified polypeptides used in the vaccines of the invention (whether prepared by coupling the endogenous protein or fragment thereof to a carrier, or by coupling a plurality of such fragments together) can also be used to generate antisera by introducing the modified polypeptides into the body of a mammal, thereby provoking the formation, in the mammal, of antibodies to the "end-genus protein"; note that in such a method, since the modified palpated need not be introduced into the same mammal, or even a mammal of the same species, as the animal from which it is derived or; in the case of a modified palpated based upon a synthetic rgment, the mammal whose protein it mimics, the so-called "endogenous protein" used in this method need not be endogenous to the mammal in which the antibodies are raised.
Following the raising of the antibodies in the mammal, some of the antibodies are recovered from the mammal, using conventional techniques which will be familiar to those skilled in the art of immunology. Techniques generating monoclonal antibodies may also be used to generate the desired antibodies. The antibodies thus generated can then be used for a variety of purposes. For example, such antibodies may be used for assaying the quantity of an endogenous protein in a mammal by bringing at least some of the recovered antibodies into contact with body tissue or 1 .) 9 232~

body fluid from the mammal and observing the formation or non-formation of the reaction process between the recovered antibody and the endogenous protein indicative of the presence or absence of the endogenous protein in the body 05 tissue or body fluid assayed. If, in this method, the endogenous protein assayed is one associated with pregnancy, this assay method can function as a pregnancy test. If, on the other hand, the endogenous protein assayed is one the presence or absence of which is associated with reduced fertility or infertility in the mammal from which the body tissue or body fluid is derived, the assay can function as a test for reduced fertility or infertility in such a mammal.

Brief Description of the Additional Drawings Figure 6 shows the close response curves generated in the radioimmunoassays described in Example IX below; and Figures 7 and 8 show the mean antibody levels to human chorionic gonadotropin in rabbits immunized with modified polypeptides, as described in Example IX below.
For reasons already noted, the need to avoid cross-reactivity with luteinizing hormone mainly restricts the chorionic gonadotropin-derived peptizes used to produce in the modified palpated used in the vaccines of the present invention to peptizes containing all or part of the 105-145 sequence of chorionic gonadotropin, since it is only this part of the chorionic gonadotropin sequence which differs significantly from luteinizing hormone. However, it has been found that there are antigenic determinants on the human chorionic gonadotropin molecule that will produce human chorionic gonadotropin-specific antibodies, which antigenic determinants are not located on the 105-145 sequence of human chorionic gonadotropin. Hitherto, it has been believed by most of those skilled in the art that these antigenic determinants which are not located on the 105-145 sequence (and which for this reason will hereinafter for convenience be referred to as the l'below-104" determinants) 32(:~

were wormed by the folding of the HOG molecule into a particular shape by the several dozily five bridges (six in all) in the beta-subunit of HOG, and that no linear amino acid sequence, other cyan portions of the 105-145 sequence, 05 would provoke the formation of antibodies which were specific to HUG These beliefs among skilled workers were based upon observations that monoclonal antibodies have been generated against HOG that react neither to human Litton-icing hormone nor to peptizes derived from the 105-145 sequence of HOG. No specific location of the relevant below-104 antigenic determinations has previously been disclosed so far as the present inventor is aware.
It has now been discovered that a peptize having a sequence corresponding to the sequence 40-52 of the beta-subunit of human chorionic gonadotropin reacts very well toe monoclonal antibody specific to the intact beta subunit but is not reactive to peptizes derived from the 105-145 sequence of the beta-subunit. However, attempts to product a modified palpated of the invention by coupling the 40-52 peptize to diphtheria toxoid, although successful, resulted in a modified palpated which gave very poor production of antibodies to human chorionic gonadotropin when the modified palpated was passed through rabbits.
Similar experiments using a peptize having a sequence corresponding the the sequence 38-54 of the beta subunit of human chorionic gonadotropin coupled to diphtheria toxoid produced slightly better production of antibodies to human chorionic gonadotropin when injected into rabbits, but these antibody levels were much lower than those produced by similar diphtheria toxoid coupled peptizes having sequences derived from the 105-145 region of the beta-subunit of HOG.
In view of the comparative failure of these experiments with peptizes derived from the 38-5~ region of the beta-subunit of HOG, the present inventor examined the accepted sequence for the beta subunit (set out in Structure I above) and noted that the 38-57 sequence of the beta subunit was ~Z~32~

bounded by two Sistine residues which, if coupled by a disulfide bridge, could result in the formation of a loop in the beta-subunit, which loop might be the relevant antigenic determinant. eased upon this hypothesis, peptizes having 05 sequences corresponding to the 38-57 sequence of the beta-subunit of human chorionic gonadotropin were Cynthia-sized, coupled to diphtheria toxoid, passed through rabbits and found to result in levels of antibodies to HOG compare able to those achieved using similar modified polypeptides derived from the 105-145 sequence of beta-HCG. Thus, peptizes comprising an amino acid sequence substantially similar to the 38-57 region of the beta-subunit of human chorionic gonadotropin can be used in the vaccines of the present invention.
Thea beta-HCG(38-57~ peptizes are, however used in a manner rather different from the buttock) peptizes previously discussed. Since the 38-57 region of the beta-subunit of human chorionic gonadotropin is sub Stan-tidally similar to the corresponding region of human Litton-icing hormone, follicle secreting hormone and thyroid stimulating hormone (and the same is true in other species), it is not advisable to use the beta-HCG(38-57) peptizes alone in the vaccines of the invention, since this involves a substantial risk of producing antibodies with an undesir-able degree of cross-reactivity with other hormone however, as noted above, it is advantageous for the modified polypep-tides used in the vaccines of the invention to comprise more than one antigenic determinant of the target protein, since this increases the antigenicity of the modified palpated. Accordingly, it is highly desirable that the beta-HCG(38-57) and analogous peptizes be used in the modified polypeptides in conjunction with a peptize which is more specific to human chorionic gonadotropin, in order that the resultant antibodies will possess the desired degree of specificity for this hormone. In particular, it is recomb ~'~23;2~i mended that the beta-HCG(38-57) peptize be used in conjunct lion with a peptize derived from, or similar to the 110-145 sequence of the same hormone subunit.
The joint use of the 38-57 and 110-145 peptizes may be 05 achieved in three separate ways. Firstly the beta-HCG(38-57) peptize may further comprise one or more amino acid sequences substantially similar to at least part of the 110-145 region of the same hormone subunit i.e. the two sequences may be chemically combined in the same peptize prior to modification of the peptize. Secondly, both peptizes may be chemically linked to the same carrier without first being chemically bonded to one another before being connected to the carrier Finally, the two peptizes may be bonded to serrate carriers and a mixture of the two resultant conjugates introduced into the animal to be treated.
Such polypeptides may comprise the 38-57 region of the beta-subunit of human chorionic gonadotropint or the analogous sequence of other mammalian chorionic gonadotro-pins, depending of course upon the mammal in which the modified palpated is to be used. This 38-57 sequence may be used alone, or the sequence may include adjacent regions substantially similar to the adjacent regions of the beta subunit of the appropriate horionic gonadotropin, even though the presence ox such adjacent regions isn't nieces-spry to produce proper antigenic properties in the modified palpated. For practical reason such~a~-the~ faulty of synthesizing very long peptizes, and~co~ it is dozier-able that the peptize having the amino acid eons cores- .
pounding to the 38-57 region of ~he~beta-subun~t-`not contain more than about 40 amino acid residue . - -Although sufficient for provoking sufficient antigenic activity, the simple amino acid sequence corresponding to the 38-S7 region of COG does have the disadvantage that it does not possess any convenient site at which coupling of the peptize to a carrier, or to other fragments used in the synthesis of the polymeric modified polypeptides can be effected. Accordingly, in order to provide the peptize with a convenient coupling site, it is preferred that the peptize have attached, to the portion of the amino acid sequence 05 corresponding to residue 38 of the beta-subunit of human chorionic gonadotropin, a spacer sequence of amino acid residues not substantially similar to the 30-37 region of the beta subunit of human chorionic gonadotropin, and further that the peptize have attached, to the N-terminal of this spacer sequence, a reactive residue suitable for coupling the peptize to a carrier, or to another fragment in the polymeric modified palpated of the invention.
Preferably, the spacer sequence comprises a plurality (conveniently 6) of praline residues and the reactive residue comprises an ala nine residue.
Alternatively, in order that the 38-57 peptize can be used in certain preferred coupling reactions (discussed above) which require the presence of a free sulfhydryl group on the peptize, one might add Jo one terminal (preferably the N-terminal) of the 38-57 peptize a Sistine residue.
However, if such an additional Sistine residue is added to the 38-57 peptize, care must be taken to ensure that, during the necessary cyclization of the peptize, the correct Sistine residues become linked by the disulfide bridge.
This is conveniently effected by placing a blocking group on the extra' Sistine residue before it is incorporated into the peptize and removing the blocking group only after the disulfide bridge has been formed. Appropriate blocking groups are well-known to those skilled in the art and some are discussed below.
As used in the vaccines of the invention, the peptize comprising an amino acid sequence corresponding to the 38-57 region of the beta subunit of HOG is used in a form in which the two Sistine residues corresponding to the Sistine residues at positions 38 and 57 of the beta-subunit of HOG
have their sulfur atoms linked in a disulfide bridge, since it appears to be only this form of the peptize, in which in effect the disulfide bridge closed the loop, which has strongly antigenic properties in viva. Nevertheless, since the amino acid sequence will normally be synthesized (e.g.
05 by the conventional solid state polymerization techniques discussed above) without the disulfide bridge, this invention extends to the peptize in both its bridged and unbridged forms In the present state of chemical Cynthia-skis, it is in practice necessary to Seychelles the 38-57 peptize before coupling it to a carrier (or to other peptize fragments) since the conditions necessary for cyclization (illustrated in Example IX below) cannot readily be produced after the peptize is coupled to a carrier (or to other peptize fragments As with other peptizes mimicking fragments of endogenous protein hormones, the peptize corresponding to the 38-57 range of the beta-subunit of HOG need not have an amino acid sequence identical to that occurring in the natural beta-subunit, provided that there is a sufficient degree of immunological similarity between the amino acid sequence of the peptize and that in the natural beta-subunit i.e.
provided the peptize, when modified according to the invention, provides sufficient antigenic activity to provoke antibodies having good reactivity with, and selectivity for, the natural HOG. Certain amino acid substation which can be made without substantially reducing the immunological similarity between the artificial peptize and the natural sequence of the beta-subunit will be well known to those skilled in the art, and the degree of immunological similar-fly of any proposed amino acid sequence can of course redetermined by routine empirical tests.
Not only do chorionic gonadotropins derived from other mammalian species have a region highly analogous to the 38-57 sequence of human chorionic gonadotropin, but a closely analogous region exists in other mammalian glycopro-loin hormones including luteininzing hormone, follicle _ 97 23~

secreting hormone and thyroid stimulating hormone. Cons-quaintly, peptizes derived from the regions of non-human chorionic gonadotropin and other mammalian glycoprotein hormones having an analogous region may also be used in 05 preparing the vaccines of the present invention. The regions of several specific mammalian glycopro loins analogous to the 38-57 region of HOG are given in detail below but those skilled in the art will have no difficulty in identifying an analogous region in other specific mammalian glycoproteins. As previously noted, peptizes having sequences similar, but not identical, to the natural sequence may also be used provided they are substantially immunologically equivalent to the natural sequence.
Examples of specific preferred peptizes having sequent cues analogous to the 38-57 region of HOG and useful in the modified polypeptides and processes ox the present invention are as follows:
Cys-Pro-Ser-Met-Lys-Arg-Val-Leu-Pro-Val-Ile-Leu-Pro-Pro-Met-Pro-Gln-Arg-Val-Cys;
(Structure XV) Cys-Pro-Thr-Met-Met-Arg-Val-Leu-Gln-Arg-Val-Leu-Pro-Pro-Leu-Pro-Gln-Val-Val-Cys;
(Structure XVI) Cys-Pro-Thr-Met-Thr-Arg-Val-Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys7 (Structure XVII) Cys-Tyr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asn-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-Cys;
(Structure XVIII) Cys-Tyr-Thr-Arg-Asp-Leu-Val-Tyr-Lys-Asn-Pro-Ala-Arg-Pro-Lys-Ile-Gln-Lys-Thr-Cys;
(Structure XIX) `

- 98 - ~223~

Cys-Pro-Ser-Met-Val-Arg-Val-Thr-Pro-Ala-Ala-Leu-Pro-Ala-Ile Pro-Gln Pro-Val-Cys;
(Structure XX) Cys-Met-Thr-Arg-Asp-Ile-Asp-Gly-Lys-Leu-Phe-Leu-Pro~(Lys-Tyr)-Ala-Leu-Ser-Gln-Asp-Val-Cys;
(Structure XXI) Structure XVII is the 38-57 region of human chorionic gonadotropin~ Structure XX is the corresponding sequence from equine chorionic gonadotropin. structure ZOO is the corresponding region ox human luteinizing hormone, and Structure XV is the corresponding region of ovine/bovine luteinizing hormone. Structure XVIII is the corresponding region of human follicle secreting hormone while Structure XIX is the corresponding region of equine follicle secreting hormone. The (Lester) portion of this follicle secreting hormone sequence is in parentheses because it represents an insert between positions 50 and 51 of the corresponding COG sequence, and thus has no direct equivalent in any of the other sequences given above.
It should be noted that there are some differences of opinion among those skilled in the field ox protein sequence determination as to certain minor details of the above sequences. See, for example:
Pierce and Parsons, Ann. Rev. Become.
50: ~6~-95 (1981).
In particular, some authorities dispute the existence of the aforementioned (Lester) insert in the human thyroid secreting hormone sequence, while other authorities dispute the existence of the Minoan at position 42 and the valise at position 55 of the human luteinizing hormone sequence. However, for reasons discussed above, even if the natural sequences do differ from those just given, the _ 99 _ ~23~

sequences just given are certainly sufficiently close to the natural sequences to produce a strong antigenic reaction when incorporated into vaccines of the invention.
As mentioned above, the main utility presently envies-no aged for vaccines of the invention incorporating modifiedpolypeptides derived from mammalian reproductive hormones or fragments thereof is as contraceptives and/or abortifactants by administration of the modified palpated to the female mammal. However, modified polypeptides derived from mama malign reproductive hormones or fragments thereof have variety of other uses. Since the modified polypeptides do provoke the production of antibodies to the endogenous reproductive hormone when injected into animals, they can be used, in ways which will be familiar to those skilled in the lo art, for the production of antibodies specific to the endogenous reproductive hormone from which the modified palpated is derived. The antibodies may be produced, for example, by injecting the modified palpated into a suitable mammal, extracting blood or other body fluid or I tissue from the mammal and harvesting the antibodies from the extracted blood, body fluid or tissue The antibodies thus produced may be used in a wide variety of tests and treatments. For example, since the antibodies thus produced are specific to an endogenous hormone, they may be used, in ways which will be familiar to those skilled in the art, to perform qualitative or quanta-native assays for the endogenous hormone in the tissues or body fluids of the mammal which produced the endogenous hormone to which the antibody is specific, the antibodies used in the vaccines of the present invention may be useful in diagnostic tests to determine whether hormone levels in a mammal are abnormal. For example, abnormal levels, usually lowered levels, of certain reproductive hormones are often associated with reduced fertility or infertility in man and other mammals, and consequently such antibodies may be used in tests for such conditions of reduced or absent fertility.

- 100- :1Z2BZ06 Such tests for reduced or absent fertility are not only useful in humans, but may also be desired by veterinarians charged with the care of valuable breeding animals such as stallions at stud or valuable pedigree bulls. For example, 05 a peptize having the 38-57 sequence of equine chorionic gonadotropin (Structure XX given above) can be used to prepare a modified palpated which can then be passed through a suitable mammal to generate antibodies to equine chorionic gonadotropin. Such antisera would be useful for infertility diagnosis in valuable thoroughbred horses.
The antibodies produced by the vaccines of the present invention may also be useful in pregnancy tests in man and other mammals. As previously noted, human chorionic gonadotropin was first discovered because it is present at relatively high levels in the urine of pregnant women, and detection of the elevated levels of human chorionic gonad-tropic in the urine ox pregnant women is the basis for most pregnancy tests. By virtue of their specificity to human chorionic gonadotropin IQr the corresponding gonadotropin in other mammalian species) antibodies produced by the vaccines of the present invention may be useful in such pregnancy tests. Such pregnancy tests are not only useful in humans;
for example, a pregnancy test may be highly desirable in a brood mare in order to ensure that she is in foal. In the absence of such a pregnancy test, an owner might incur an additional heavy stud fee unnecessarily.

EXAMPLE IX
This Example reports the results of experiments carried out to determine the immunogencity and cross-reactivity with LO, of various portions of the beta-subunit of HOG.
Synthetic polypeptides corresponding to 12-16 amino acid residue portions of the sequence of beta-HCG were prepared in the same manner as in Example I above These peptizes were then conjugated to diphtheria toxoid using the coupling techniques described in Example I above. In all cases, the - 1 o I lZ~3;~6 resultant conjugates contained approximately 30 molecules of the peptize per 100,000 Dalton of diphtheria toxoid. The result modified palpated conjugates were then mixed with Complete Fronds Adjutant and injected into rabbits using 05 the same techniques as in Example II above. Table 25 shows the results obtained by testing for the antibody levels to HOG and HUH in these experiments.

Mean Peak Antibody Levels in Sofa from Rabbits Immunized with Beta-hCG Peptize Conjugates.

Subunit Sykes Antibody levels M/Lx15-10 1-12 0092 0.05 10-22 0.62 0.05 20-32 1.90 0.05 30-42 15.70 ~20 40 52 1.80 0.05 50-62 0.55 S
60-72 1.78 0.90 70-82 4.66 0.05 80-92*
90-102 1.70 0.85 100-112 0.44 0.11 110-122 100.45 0.05 1~0~132 3.60 0.05 130-145 75.70 I

*Sequence not tested EXAMPLE X
This Example results the reports of experiments which identified the antigenic determinant in the 38-57 region of human chorionic gonadotropin. This example also describes ``~

- 102 - ~Z~2~

the preparation and use of modified polypeptides prepared from a peptize having this 38-57 sequence of human chorionic gonadotropin.
Synthetic peptizes each comprising 13 amino acids and 05 having a sequence corresponding to part of the sequence of the beta-subunit of human chorionic gonadotropin were prepared by the solid phase method described in Merrifield, 85, 2149 (1963). The peptize sequences were chosen so that altogether they covered the entire 145 amino acid sequence lo of the beta-subunit, and there was a two amino acid residue overlap between adjacent peptizes; thus, the peptizes covered the 1-12, 10-22, 20-32 etc. regions of the beta-subunit of human chorionic gonadotropin.
In the synthesis of the peptizes, (Boa Me Bzl) resins were used throughout and the completeness of amino group consumption checked after each amino acid addition. Peptizes were removed from the resin using hydrofluoric acid and the peptizes, which were synthesized on the resin and removed therefrom in the straight-chain, unbridged form, were I purified by a series of chromatographic steps employing reverse phase HPLC C-18 columns The peptizes were eluded with linear gradients of 0.1% trifluoroacetic acid, 0.05M
ammonium acetate or 0.05M phosphate buffers, these buffers containing 60% of acetonitrile.
The antigenic potency of each of the synthetic peptizes was tested by determining their reactivity with a mouse monoclonal antibody, designated H 18, raised against intact human chorionic gonadotropin. This H-18 antibody, provided by Dr. Christian Stahl of Basic, Switzerland was reactive with human chorionic gonadotropin but not with human luteinizing hormone or with a synthetic peptize having the 109-145 sequence of the beta-subunit of HOG. The reactivity of each peptize with the H-18 antibody was tested by determining the ability of the peptize to compete with 125I

- 103 ~232~

HOG in radioimmunoassays. The reactivity of the peptizes with the antibody was compared with that of unlabeled HOG
in the same assay.
One additional peptize was prepared, namely the peptize 05 Ala-tPro)6-(38-57), wherein (38-57) represents the 38-57 sequence of human chorionic gonadotropin, Structure XVII
above. The addition of the six praline residues provides a convenient spacer on this additional peptize, while the ala nine residue attached to the spacer sequence provides a convenient reactive site by means of which the peptize can be coupled to a carrier.
This Ala-(Pro)6-(38-57) peptize was prepared in cyclic form by oxidizing the sulfhydryl groups on the two Sistine residues. To effect this cyclization, the crude peptize, as removed from the resin, was diluted in distilled water to a concentration of glue at a pi of 7.5. To each liter of peptize solution was added 2.5ml. of 0.01M K3Fe(CN6) with stirring; this reagent serves to monitor the completion of formation of the disulfide bridge. Upon completion of the resultant oxidation reaction, the pi of the solution was adjusted to 4.5 and to each liter of the resultant solution was added 10g. of Borax 70 resin The resin and solution were mixed and filtered, and the resin was packed into a glass column and the peptize eluded therefrom with 70%
acetic acid. The subsequent purification of the peptize was effected by means of two chromatographic steps of reverse phase PLUCK as described above for the straight chain peptizes.
The purity of the peptizes was checked at various stages of the purification and in the final products using thin layer chromatography on silica gel and cellulose, paper electrophoresis and HPLC reverse phase chromatography. Amino acid analysis was also performed on all final produces. The existence of a "loop" in the cyclized Ala-(Pro)6-(38-57) peptize was confirmed by comparing the position of elusion at peak heights on reverse phase PLUCK and on Bejewel P-4 gel - 104 ~'~232~

filtration of the same quantity of intact loop peptize and the same peptize reduced by dithiothreitol, and having the resultant sulfhydryl groups blocked with N-ethyl malemide.
These tests for purity confirmed that all the purified 05 straight chain peptizes migrated as a single band on thin layer chromatography plates and on paper electrophoresis;
similarly, a single sharp peak was observed for each purified straight chain peptize during reverse phase HPLC
analyses. The amino acid analyses gave over 90% agreement in composition of expected and calculated amino acid values for all peptizes.
Recovery of the purified cyclic peptize from the crude fraction by the two reverse phase HPhC steps was only 3.5~
since only the center of the eluded peak on the first purification step was subjected to the second purification step. HPLC and Bejewel P-4 chromatography of the intact cyclic putted and of the corresponding peptize reduced and blocked on the sulfhydryl groups, revealed that both were of the same molecular weight and exhibited similar hydropho-Bassett. This suggests that the peptize was substantially pure, with only slight, if any, contamination of the cyclic peptize with oligomers of polymerized pep ides To determine the ability of the peptizes to bind with the H-18 antibody, a human chorionic gonadotropin prepare-lion obtained from Ayes, Geneva, Switzerland, and having specific activity of 11900IU/mg. was iodinated with Noel by the method described in Greenwood et at, Become. J. 89 123 (1963). The specific activities of the iodinated HOG
thus produced were from 35 to 60 microCi/microg. Doses of either unlabeled HOG or the synthetic peptizes were diluted in phosphate buffered saline (PBS) containing 5% bovine serum albumin in a volume of 100 micro. of iodinated HOG in I bovine serum albumin-PBS buffer was added to all tubes.
Thereafter, 100 micro. of H-18 monoclonal antibody diluted with PBS containing 20% normal calf serum was added. The resultant mixtures were incubated for two hours at 37C~

- 105 3Z~6 then for a further 16 hours at 4~C. Separation of the bound and free antigen was achieved by adding to each tube lml. of 20% polyethylene glycol, then centrifuging at 4C and 15009.
for 15 minutes. After recantation of the supernatant, the 05 radioactivity in the precipitate was determined and the calculation of antigen binding was performed by the prove-dune described in Feldman and Rod bard, Principles of Compete-live Protein winding Assays (O'Dell and Dow ens.), 158-203 (1971), Lippincott, Philadelphia. The results obtained are shown in Table 26 below, in which the binding constants are expressed as nanomols. of HOG binding inhibit ted by each nanomol ox peptize.

beta-hCG Peptize Sequence Nanomol hCG/nanomol peptize 12 0 r 00001 10 - 22 0.00001 20 - 32 .S
2030 - 42 0.00001 40 - 52 1.577 50 - 62 - 0.0008 60 - 72 ~0075 70 - 82 0.00001 80 - I 0.012 90 - 102 0.00001 100 - 112 0.00001 110 - 122 0.000~1 120 13~2 0 00001 130 145 0~00001 The data in Table 26 show that, although slight reactivity was found with a few other peptizes, only the peptize having the 40-52 sequence of the beta-subunit of HOG
competed significantly with HOG for binding the antibody. On - 106 - 2'2 3Z 6 a moles bound per liver of undiluted serum basis, the 40-52 peptize bound the molecular antibody approximately lo tires as efficiently as intact COG
In view of these observations, an extended peptize 05 containing the 38-54 sequence of the beta-subunit of HOG
were synthesized and tested in a similar manner. Figure 6 of the accompanying drawings shows that this extended peptize was no more efficient in binding to the monoclonal antibody than was the 40-52 peptize, thus suggesting that the entire epitome resides within the 40-5~ sequence To test the usefulness of the aforementioned synthetic peptizes in the preparation of modified polypeptides of the invention, the peptizes were coupled to diphtheria toxoid using the bifunctional reagent 6-maleimido caproic acyl-N-hydroxy succinimide ester (MCCOY), using the procedure and methods of analysis of the products described in Lee et at, Mol. Immunol. 17, 755 (1980). Briefly, MCCOY was coupled to the amino groups on the toxoid via the succinimide ester groups and the resulting MCS/toxoid product was purified and thereafter reacted with a they'll group on the peptizes via the maleimido grouping. The peptizes not containing Sistine were thiolated with Nastily homocysteine thiolac-tone at the amino terminus. The resultant toxoid/MCS/pep-tide conjugates were purified by gel filtration on a 1.6 x 60cm Sephacryl-200 resin column equilibrated with 0~2M
ammonium bicarbonate buffer. The conjugates thus prepared had a peptide:~oxoid ratio of 25-28 peptizes per 105 Dalton of toxoid. Such conjugates were prepared for each of the 13 amino acid peptizes, as well as the peptize with additional 30 residues of the 40-52 sequence The immunogenicity of the conjugates thus prepared was determined by immunizing rabbits and subsequently evaluating serum antibody level and antibody specificity; the prove-dunes used were in accordance with the detailed description in Powell et at, J. Repro. Immunol. 2, 13 (1980). Briefly, each conjugate was dissolved in saline together with an adjutant compound, namely N-acetyl-normuramyl-L-Ala-D-iso-glut amine, and the resultant solution emulsified with a wow. mixture of skyline and Monday moonlit, 2.3 parts of the saline solution being emulsified with one part US of the skyline/ Monday moonlit oil. Rabbits were immunized with doses of 0.5g. of conjugate and 0.2mg. of adjutant intramuscularly a three-week intervals. Blood samples were collected weekly beginning at the time of the second conjugate/adjuvant injection, and the serum levels of antibody were determined by reacting dilutions of the sofa with three concentrations of 125I-HCG. Antibody specificity was assessed by reacting the sofa with 125I-labelled pituitary hormones FISH, OH and T5H. The mean peak antibody levels determined are shown in Table 27 below.

Antibody HOG Beta Subunit Antigen Binding Nanomol (no) Sequence _ HOG HUH _ 20 1 - 12 0.107 0.007 10 - 22 0.072 0.007 20 - 32 0~234 0.0~7 30 - 42 ~.245 0.756 40 - 52 0.315 0~07 25 50 - 62 0.067 0.007 60 - 72 0.197 0.099 70 - 82 0.567 0.007 80 - go 1.~56 0.007 90 - 102 0.~24 0.095 100 - 112 0.062 0.018 110 - 122 12~670 0.007 120 - 13~ 0.410 0.007 130 - 145 8.022 0.007 From the date in Table 27, it will be seen that none of the conjugates of the synthetic peptizes elicited antibody levels as high as those achieved with intact HOG, the - 108 - ~Z~3Z~

beta-subunit of HOG or similar conjugates derived from the peptize having the 109-145 sequence of the beta-subunit of HOG. Although some of the antisera failed to react with 125I-labelled HUH, the degree of specificity of the antisera 05 to HOG was uncertain in view of the very low levels of binding to HOG.
In view of these disappointing results and especially the low levels of binding achieved with the 40-52 peptize, which was found in the experiment described above to contain an epitome competitive with one of the intact HOG molecule, a similar conjugate of diphtheria toxoid and the extended toxoid having the 38-54 sequence of the beta-subunit of HOG
was prepared and rabbits immunized with this extended conjugate in the same manner as previously described The results are shown in Fig. 7. From the data in Fig. 7, it will be seen that the conjugate prepared from the 38-54 peptize did produce antibody levels higher than those produced by the conjugate of the 40-52 peptize, even though the conjugate of the 38-54 peptize produced antibody levels much lower than those observed with the conjugate of the 109-145 peptize.
In view of this failure to achieve good antibody responses with the conjugate of the 38-54 peptize, the previously-mentioned Ala-(Pro)6-(38-57) peptize was synch-sized, purified, cyclized and conjugated by linking thiamine group of the terminal ala nine residue with MCCOY via the succinimide portion of the coupling reagent and thereafter linking the opposed end of the coupling reagent to diphtheria toxoid thiolated with Nastily homocysteine thiolactone. The resultant conjugate was used in immunization of rabbits conducted in the same manner as previously described.
The antibody levels raised to the conjugate of the cyclized peptize are shown in Fig. 8, where they are compared with antibody levels produced to the corresponding conjugate of the 109-145 peptize. The data in Fig. 8 indicate that the use of the cyclized 38-57 sequence stab-lived the epitome in the 4~-52 region by forming the disulfide bridge between adjacent Sistine residues to such an extent that antibody levels to the cyclized peptize were greater than those raised against the 37 amino acid peptize 05 having the 109-145 sequence. Furthermore, the conjugate of the cyclized peptize produced highly specific antibodies. No antisera from any of the four rabbits immunized with the conjugate of the cyclized peptize, in any of the five bleedings from each rabbit, showed detectable binding with 125I-labelled HUH, HFSH,or HUSH. The data indicated that reactivity of these hormones with the antisera was less than 2.2, 2.4 and 1.6 percent respectively of their reactivity with HUG
Although this work did not determine the boundaries of the epitome within the 40-52 sequence of HOG, examination of the difference in HOG and HUH in this region shows that three sequence positions have different amino acid nest-dues, and clearly this number of substations is adequate to render the immunological determinant(s) in this 13-residue sequence of HOG immunologically different from HUH

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A vaccine comprising:
a modified polypeptide for isoimmuniogically controlling biological action in a mammal by antibody formation, consisting of a protein hormone, a non-hormonal protein, or a fragment of either which has been chemically modified outside the body of said mammal, said protein hormone, non-hormonal protein or fragment having the properties of:
(a) in unmodified form, being non-immunogenic to said mammal and having a molecular structure similar to an endogenous protein hormone or a non-hormonal protein, the biological function of which it is desired to inhibit, or fragment of either and (b) in modified form, causing antibodies to be formed in the body of the mammal which inhibit the biological function of said endogenous protein hormone or non-hormonal protein following administration of the modified form into the body of said mammal; and a vehicle, said vehicle comprising a mixture of mannide monooleate with Squalane and/or Squalene.

2. A vaccine according to claim 1 wherein said vehicle comprises a mixture of mannide monooleate and Squalene.

3. A vaccine according to claim 1 or 2 wherein said vehicle comprises about four parts by volume of Squalene and/or Squalane and about one part by volume of mannide monooleate.

4. A vaccine according to claim 1 further comprising an immuno-stimulat-ing adjuvant.

5. A vaccine according to claim 4 wherein said adjuvant is a muramyl dipeptide.

6. A vaccine according to claim 5 wherein said muramyl dipeptide is of the formula:
NAc-nor Mur-L.Ala-D.isoGln;
NAc-Mur-(6-0-stearoyl)L.Ala-D.isoGln; or NGlycol-Mur-L.?Abu-D.isoGln.

7. A vaccine according to claim 1 wherein said unmodified form of said fragment comprises a fragment of Human Chorionic Gonadotropin.

8. A vaccine according to claim 7 wherein said fragment is of the formula:
Thr-Cys-Asp-Asp-Pro-Arg-Phe-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-lle-Leu-Pro-Gln.

9. A vaccine according to claim 7 wherein said fragment is of the formula:
Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Rro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln .

10. A vaccine according to claim 7 wherein said fragment is of the formula:
Asp-Asp-Pro-Arg-Phe-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-lle-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys.

11. A vaccine according to claim 7 wherein said fragment is of the formula:
Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-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-lle-Leu-Pro-Gln.

12. A vaccine according to claim 7 wherein said fragment is of the formula:
Cys-Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-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-lle-Leu-Pro-Gln.

13. A vaccine according to claim 8 wherein said modification of said fragment is effected by coupling said fragment to diphtheria toxoid.

14. A vaccine according to claim 13 wherein said fragment and said toxoid are present in said vaccine in an amount of about 20-30 fragments per 100,000 daltons of toxoid.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

15. A vaccine according to claim 1 wherein the modified polypeptide comprises an aminoacid sequence substantially similar to the region of a mammalian lutein-izing hormone, chorionic gonadotropin, follicle secreting hormone or thyroid stimulating hormone corresponding to the 38-57 region of the beta-subunit of human chorionic gonado-tropin

16. A vaccine according to claim 15 wherein said aminoacid sequence contains not more than about 40 aminoacid residues.

17. A vaccine according to claim 16 wherein, in said aminoacid sequence, the two cysteine residues corresponding to the cysteine residues at positions 38 and 57 of the beta-subunit of human chorionic gonadotropin have their sulfur atoms linked in a disulfide bridge.

18. vaccine according to claim 16 wherein the portion of said aminoacid sequence corresponding to residue 38 of the beta-subunit of human chorionic gonadotropin has attached thereto a spacer sequence of aminoacid residues not substantially similar to the 30-37 region of the beta subunit of human chorionic gonadotropin, and there is attached, to the N-terminal of said spacer sequence, a reactive residue suitable for coupling the aminoacid sequence to a carrier.

19. A vaccine according to claim 18 wherein the spacer sequence comprises a plurality of proline residues and the reactive residue comprises an alanine residue.

20. A vaccine according to claim 15 wherein said aminoacid sequence is one of the sequences (XV) to (XXI) described herein, or a sequence substantially immunologi-cally equivalent to one of these sequences.

21. A vaccine according to claim 15 wherein said aminoacid sequence further comprises an aminoacid sequence substantially similar to at least part of the 110-145 region of the beta-subunit of human chorionic gonadotropin.