NZ174192A

NZ174192A - Antigens containing modified polypeptides

Info

Publication number: NZ174192A
Application number: NZ174192A
Authority: NZ
Inventors: V C Stevens
Original assignee: Univ Ohio State
Priority date: 1973-05-07
Filing date: 1974-05-07
Publication date: 1985-02-28
Also published as: DD113919A5; IL44779A0; DE2421943C2; JPS623128B2; JPS5069221A; IL44779A; AT356263B; NL7406098A; IE42002B1; CH622426A5; SE7406096L; AU6869574A; FR2228477B1; MY8100128A; ATA377074A; DE2421943A1; PL97318B1; GB1473601A; CA1057742A; IE42002L

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 1 74192 174192 i * Priority 7'.?.'7?.;. .W.'JP. .73.■ Complete Sj/spcJfjcaticn Filed: l.'f. .a •PubUoattort Dsto: , fat.7 >.0. Joun^l t^-o: ■■■■'■ in jiiiiiis NEW ZEALAND PATENTS ACT, 1953 No.: Date: * _ COMPLETE SPECIFICATION ANTIGENIC MODIFICATION POLYPEPTIDES X/We, THE OHIO STATE UNIVERSITY, of 190 North Oval Drive, Columbus, Ohio 43210 / United States of America, a non profit making organisation of Ohio, United States of America, hereby declare the invention for whicHK3P7 we pray that a patent may be granted to-'HSF/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - _ i _ followed by page 1a. — \Cv 1741S2 118 33-2-9 M0Dir-^eft?-i0N OF POL-YPBPTIDES- This invention relates to the chemical modification of polypeptides to provide antigens which may be used in active immunisation for the purposes of the control or treatment of various physiological processes, particularly reproduction. ■ It is well known that polypeptides, particularly proteins, are responsible for, contribute to or influence many physiological processes. For example, certain protein hormones and non-hormonal proteins are known to be essential for the normal events of the reproductive process. Unusual excesses of certain polypeptides, such as gastrin, angiotension II or somatomedian, are also known to cause or influence various disease states and maladies. The present invention is concerned with the control or treatment of such physiological processes by active immunisation (i.e. administration of antigens) causing formation of antibodies which neutralise (render biologically ineffectual) the endogenous polypeptides which are responsible for, contribute to or influence the"physiological process in question. - 2 - 1741 Immunology, as a means of controlling reproduction, has been the subject of much recent investigation. R.G. Edwards, in a paper entitled "Immunology of Conception and Pregnancy (British Medical Journal, 26, 72-78 [1970]), surveys the literature on this topic, for example that concerning the production and use of antibodies against testes or sperm. Hormone antibodies have also been studied for many years and the effects of specific antisera have been recorded. Most of the past approaches to contraception by immunological means have, however, used passive immunisation, that is injection of antibodies produced elsewhere. There are serious limitations to the use of passive immunisation for human therapy since the repeated injection of animal antibodies into humans is known to produce undesirable reaction in many individuals. It has now been found that by chemically modifying natural, endogenous polypeptides which play a role in the physiological process to be treated or controlled, antigens are obtained which are capable of inducing formation of antibodies which neutralise not only the an-ei"-gepieally modified polypeptide but also its unmodified endogenous counterpart. Thus, immunisation apparently takes place because of the inability of the antibodies _ 3 - 174192 produced to distinguish between the antjjgerrfcally modified polypeptides and their unmodified endogenous counterparts. The modified polypeptides are produced from the natural, endogenous polypeptides in the species involved or are immunologically equivalent to the modified polypeptides so produced. The polypeptides to be modified may be naturally occurring or synthetic. In practice, the polypeptides to be modified are derived from the species involved or from a closely related species. As used hereinafter in the specification and claims, the term "fragment" in relation to an endogenous polypeptide means a sequence of amino acids in the complete amino acid chain of the endogenous polypeptide. These fragments must be sufficiently large and distinctive in chemical and physical character to enable them to be recognised as a specific part of the whole. As used herein, the term "polypeptide which is immunologically equivalent to" in relation to an endogenous polypeptide or fragment thereof, means a polypeptide which, although not identical to the polypeptide or fragment in question, elicits, when modified in accordance with the invention, essentially the same antibody response as the endogenous polypeptide or fragment concerned. The invention accordingly provides an antigen for use in active immunisation for the control or treatment of a physiological process in a particular species, comprising a chemically modified polypeptide, the unmodified polypeptide being a) an endogenous polypeptide which influences such physiological process in that species and selected - 4 - 1r- A -> r\c% from protein reproductive hormones, non-hormonal protein antigens isolated from placental tissue, gastrin, Angiotension II, growth hormone, somato-median, parathyroid hormone, glucagon, thyroid stimulating hormone or secretin; b) a fragment (as herein defined) of such endogenous polypeptide; or c) a polypeptide which is immunologically equivalent (as herein defined) to such endogenous polypeptide or fragment thereof, and the chemical modification (i) comprising (a) the attachment of one or more modifying groups to the unmodified polypeptide and/or (b) the removal of one or more moieties from the unmodified polypeptide, and (ii) being such that as to permit the chemically modified polypeptide to induce in that species, formation of antibodies which biologically neutralise the endogenous polypeptide as well as the chemically modified polypeptide. The invention also provides a pharmaceutical composition comprising an antigen of the invention, in association with a pharmaceutically acceptable liquid carrier, and in a form suitable for parenteral administration. !.. wmvmk - 5 - 1741 rlj^-3329 H Polypeptides which may be chemically modified to provide the antigens of the invention include protein reproductive hormones, such as Follicle Stimu-lating Hormone (FSH) , Lcutinicing Hormone (LH), Human Placental Lactogen (HPL), Human Prolactin and Human Chorionic Gonadotropin, and specific fragments thereof. Specific fragments of protein reproductive hormones which may be modified in accordance with the invention include the p-subunit of FSH and specific 10 unique fragments of natural HPL or Human Prolactin, which fragments may bear little resemblance to analogous portions of other protein hormones. Preferred fragments include the p-subunit of HCG which, according to the two authoritative views, has either structure I or II as 15 follows (''•'indicates site locations of carbohydrate moieties) 10 * Ser-Lys»Glu-Pro-Leiv-Are-Pro-/ire-Cys-Ars-Pro-Ile-Asn-Ala-Thr«Leu~Ala 20 ■ * Val-Glu--Lys~Glu-Gly-Cys-Pro-Val-Cyr;--ilc-,rhr-Val"Asn-Thr-Thr~Ilc-Cy s !i0 ' 50 Ala-Gly-Ti'y-Cj's-Pro-Thr-l-ie't-Wir-ArE-Val-Leu-Gln-Gly-Val-Lou-Fro-Ala 60 20 IfiU"PrQ-Gln~V&l»Val-Cys-Asn--Try-Arg"-Asp--Ve.l-.Ar£i~Phe«Gl'u~Ser-Iie-Arr; 70 80 Leu-Pro~Gly-Cys-Pro«Arg-Gly-V&l-Asn--Pro-Val"Val-Scr-iyr-Al&--Val»Ala 90 100 I/eu-Ser-Cys-Gln-Cys~Ala-Lcu~Cys~Arg~Arg-Ser-TJ'n-«Thr~Asp-Cyi;-Gly»Gly 110 Pro-Iiys~Asp-}!is-Pro-I,eu-Tlir-Cys-Asp-Asp-Pro-Arg~p}ie~Gln--A5p-Ser~Ser ISO « s 130 Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly * l';0 2 5 Fro-Sei'-Asp-Tlir-Pro-Ile-Lau- Pro-Gin (Structure I) - 6 - 17 41 .118-33-39 10 i( ■ Ser~Lys-Gln~Pro-LEu-Arg-Pro-./U'g"Cys~Arg-Pro~Ile~Asn--Ala-.Thr-Leu~ _20 29 ft Ala-Val-Glu-iiys-Glu-Gljr-Cys-Pro-VRl'-Cys-Ile-Thr-Val-Asn-Tljr-T/ir- 1^0 He-Cys-Ala~Gly-Tyr-Cys-l'ro-!riir-Met-Thr-ArB-Yal~L"eu-Gln»Gly-Val~ 50 60 Leu-Pro-Alfi-Leu-Pro-Glx-Lou-Val-CyB-Asn-TiT-Ai-g-Asp-Yal-Ara-Phe- 70 80 Glu-.£5er-Ilo-Arg~Leu-Pro~Gly"Cys~PrO"Arg-Gly-Val~Asn-Ero-Val-Val~ 90 Sei'-j?„vr-/Lla~Val-AI.a-Leu-Ser-Cys-Gln-Cys-Ala-Lcu-CyG~Arg" (Arg)» 100 110 Ser-Thr~Tlir~Asp-Cys«-Gly-Gly~Rro~Lys-Asp-Hi5-Bro-Leu-Thr-Cj's-A8p- « 120 Asp-Px,o-Arg~Phs~Gln-Asp-Ser~Ser-Sc-r-LyS"A.lG-Fi,'o-IVo-Pro--Ser-Leu.~ 130 » iko Pro-Ser-.Ero-Ser-Arg-Leu-Pro-Gly-.P'j.'o-Pro-Asx-Tiir-Pro-Ile-Leu-'PrO"-lit? Gln-Ser~Leiv~Pro (Structure II) For specificity of antibody action, it is desirable that polypeptides be modified which comprise molecular structures completely or substantially completely different to those of other protein hormones. In this connection, the p-subunit of IICG possesses a chain or chains of amino acids which differ greatly from those of LH and such chains may also be modified in accordance with the invention. Such chains include 20-30 or 30-39 amino acid peptides consisting of the C-terminal residues of the p-subunit of HCG. More particularly, suitable such chains include those of the following formulae III and IV (C-terminal portions of structure I, above) and V and VI (C-terminal portions of Structure II, above): 174192 ^-M-8-332.9 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-Ile-Leu-Pro-Gln (Structure III) •k "k 5 Gln-Asp-Ser-Ser-Ser-Ser-I.ys-Ala-Pro-Pro-Pro-Ser-Leu-Pro- "ft "ic Ser-Pro-Ser--Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln. (Structure IV) •k Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys- * * 10 Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly- Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro. (Structure V) je Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu- "k * Pro-Sex—Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-15 Ile-Leu-Pro-Gln-Ser-Leu-Pro. (Structure VI) Such structures ir.ay be obtained by purely synthetic methods or by enzymatic degradation of the parent polypeptide [Carlson et. al., J. Biological Chemistry, 20 284(19), 6810(1973)]. A polytyrosine chain may also be added to the structures III to VI and the resulting polypeptide modified in accordance v?ith the invention. nif - 8 - Another group of polypeptides which may be modified according to the invention are specific non-hormonal protein antigens isolated from placental tissue and fragments thereof. Other proteins which may be modified according to the invention and used for active immunisation in the treatment of various disease states and maladies, include gastrin the hormonal polypeptide known as Angiotension II, growth hormone, Somatomedian, para-thyroid hormone, glucagon, thyroid stimulating hormone (TSH) and secretin. (Somatomedian is a substance isolated from human blood which has insulin-like activity and which regulates the production of Growth Hormone (Rompp's Chemie Lexicon).) The degree of chemical modification of the polypeptides, .in accordance with the invention, is, as indicated, such that the resulting antigens wi11.induce generation of antibodies which will neutralise not only, the antigens but also some, at least, of their natural endogenous counterparts and this as well as the type of modification will depend on the specific problem being treated as well as the nature of the polypeptide involved If too little modification is effected, the body may not recognise the modified polypeptide as a foreign intruder and may therefore not generate antibodies against it. If, on the other hand, too much modification is effected, the body will generate antibodies specific to the injected antigen which will not neutral- 17 *192 ~ 9 - <4i8~-33^ ilse the natural endogenous protein involved. In general, the chemical modification involves owe gy movt addition of ^foreign modifying groups to the polypeptide involved (hapten-coupling). The number of foreign modifying groups to be added will, of course, vary depending on the circumstances but, generally, it is preferred that 1 to 40, preferably 2 to 40, more preferably 5 to 30, and most suitably 10 to 26 modifying groups per molecule of polypeptide be attached. Given modifying groups will attach to particular amino acid sites in the peptide molecule, so that the maximum possible nurrber of a given modifying group which will attach to a given polypeptide can readily be calculated. The nature of the modifying group may be selected accordingly. It is also possible that several raodifying groups may attach to each other and then attach to a single amino acid moiety but, for the purposes of this invention, such a substitution is regarded as attachment of a single modifying group. The modifying groups may as indicated vary in chemistry depending on the circumstances. Suitable modifying groups include diazo groups. These may suitably be introduced by reaction with the appropriate number of - 10 - MrS-rm- raoles of diazosulfanilic acid. Introduction of diazo groups into proteins is a well-known technique and, may, for example be effected as described, by Cinader et al., J. Am. Chera. Soc. 7_8, 746 (1955); by 5 Phillips et al., J. Biol. Chera. 244, 575 (1969) ; by Tabachnick et al., J. Biol. Chem. 234(7), 1726(1959) or by Crampton et al., Proc. Soc. Ex. Biol. & Med. 80, 448(1952). In general, the methods of Cinader et al. and Phillips et al. are preferred. 10 Additional modifying groups include those introduced by reaction of the polypeptides with dinit.ro-phenol, trinitrophenol, S-acetomereaptosuccinic anhydride, polytyrosine or polyalanines (in either straight or branched chains), biodegradable polyaextran or, 15 rather less preferably, natural proteins such as thyro- globulin. Generally, synthetic modifiers are preferred to natural ir.odifiers. The above reactions, as well as many other suitable hapten coupling reactions, are well known in 20 protein chemistry. The following references may, for example, be cited in this connection 1. Klotz et al., Arch. Biochem. & Biophys. 96, 605-612(1966); 2. Khorana, Chem. Rev. 53, 145(1953); 17 11 - -3.1 8 1 09 3. Sela et al-, Biochem. J. 85_, 223 (1962) ; 4. Eisen.et al., J. Am, Chem. Soc. 7_5, 4583 (1953); 5. Certano et al., Fed, Proc. (ABSTR-;) 2_5, 729 (1966) ; 5 6. Sokolowski et al. , J. Am, Chem. Soc. _86_, 1212 (1964); 7. Goodfriend et al., Science 144, 1344(1964); 8. Sela et al. , J. Am. Chem. Soc. 7_8, 746 (1955), and 9. Bahl, J. Biol, Chem. 244, 575 (1969) . 10 The chemical modification may alternatively or ^ DfC CW<L t*vCV-^ UhVHvd* U Cq yjy? additionally comprise removal of^moieties from the^poly- peptides. Thus, for example where certain of the natural proteins have carbohydrate moieties, these may be removed in conventional manner, using, for example, 15 N~ac.etylneursminidase or N-acetylglucosidase, materials useful for removing specific carbohydrate moieties. As indicated, the modified .polypeptides of the invention are indicated for use as antigens in active immunisation for the purposes of control or treatment of 20 various physiological processes. More particularly, the modified protein reproductive hormones or fragments thereof, of the invention are indicated for use as antigens in active immunisation - 12 - 192 for the purposes of controlling reproduction. Thus, these materials induce formation of antibodies which neutralise not only the antigens but also the natural endogenous protein reproductive hormones which are 5 essential for the normal events of the reproductive process, thus disrupting the natural processes of conception and/or gestation. These antigens are therefore indicated for use in contraception and for terminating pregnancies soon after conception. The specific 10 non-hormonal protein antigens isolated from the placental tissue and modified according to this invention are also indicated for such use. There is direct evidence that inhibition of substances which are specific to the placental tissue and do not have similar antigenic 15 properties to antigens from other organs, can result in disruption of pregnancies by passive immunisation. Such specific placental substances when modified in accordance with the invention can be injected into the body of the same species as a fertility control means by 20 active immunisation. The advantage of these substances is that placental antigens are foreign to the nonpregnant female humans and are therefore unlikely to cause any cross-reaction or disruption of normal body functions in the non-pregnant female. - 13 - It will be understood while the methods of controlling reproduction described are mainly applicable to females, certain antigens, in particular FSHf its p-subunit and fragments thereof, when modified in accordance with the invention, may be applicable to males. Gastrin, when modified in accordance with the invention is indicated for use in the treatment by active immunisation of the digestive disorder known as the Zollinger-Ellison Syndrome. This state is generally described as a condition in which there is hyper-secretion of the polypeptide gastrin, which is produced in the pancreas and brings about a state of hyperacidity in the stomach resulting in a chronic digestive disorder. Injections of modified gastrin will enhance formation of antibodies against the hypersecretion of gastrin and thereby alleviate the disease without the need for the surgical treatment which is the only effective treatment presently known. Anyiotension II, when modified in accordance with the invention, is an antigen for use in the treatment of hypertension. In general terms, the state of hypertension is the abnormal level or fluctuation of ones blood pressure. The blood pressure in an individual is controlled by many physiological processes in the body. However, one major substance affecting the regulation of such pressure is the hormonal polypeptide known as Angiotension II. In certain states of high blood pressure (hypertension) it is difficult to medically control the secretion and therefore the level of Angiotension II in the circulatory system. By the appropriate modification of this hormone and subsequent immunisation with the resulting antigen, it is possible to reduce the secretion of angiotension II in patients with chronically elevated hormone levels. The predictable and controlled reduction of this substance is beneficial to certain patients with chronic problems of hypertension. Growth Hormone and Somatomedian, when modified according to the invention are indicated for use as antigens in immunological treatment of diabetes and associated micro and macro-vascular disorders. Currently, the treatment of diabetes is limited to dietary and/or drug treatment to regulate blood glucose levels. Recent scientific data support the concept that Growth Hormone and Somatomedian (both polypeptides) are intimately involved in the disease syndrome. Antigens of the invention are suitable admixed - 15 rl with a pharmaceutically acceptable liquid carrier and administered parenterally. The dosage to be administered will, of course, vary depending on various factors, including the condition being treated and its severity. However, in general, unit doses of 0.1 to 50 mg are indicated, suitably administered one to five times at intervals of one to three weeks. As indicated, the theory leading to the present invention was that the chemical moolification of a protein essential for reproduction or influencing a particular disease state would render it antigenic such that it would cause formation cf antibodies which would, at least partially, neutralise the endogenous protein in addition to the antigen. With this in mind, reproductive hormones of various species were modified and tested in baboons as described in Example 1, hereinafter. The results demonstrated that modified hormones of unrelated species do not produce the desired result whereas modified hormones of the same species or closely related species do. The remaining Examples 2 to 9 also illustrate the invention. 17419 Example Adult female baboons were studied for at least one menstrual cycle for patterns of urinary estrogens, plasma, progestin, and in some cases urinary LH. Only those animals displaying normal patterns of these horiaones vere inmunized. The criteria for normality and the procedures for housing aniiaals are veil known and vill not be described. Gonadotropin Preparations Human Luteinizing Hormone (HLK) partially purified preparation from human pituitaries with a biological potency of 2„5 units per jag. (nih-lh-si). Human Follicle Stimulating Hormone (KFSii) - a partially purified preparation frora human pituitaries with a biological potency of 86 units per rag. (NIH-FSH-SI). Human Chorionic Gonadotropin (HCG) - a highly purified preparation from human pregnancy urine with biological potency of .13,200 lU/nig, (2nd IRP-IICG). Monkey Luteinizing Hormone (MLH) - a crude preparation from rhesus monkey .pituitaries with a biological potency of 0„75 units per nig. (K.T.H-IjH-S'1). Ovine Luteinizing Hormone (OLH) (NIH-LH-S5). Baboon Luteinizing Hormone (BLH) partially purified baboon pituitary preparation vith & biological potency of lel units per mg. (HIH-LH-S1). All preparationss excepting the OLH, vere prepared in the inventor's laboratory. LH and HCG biological activity was determined by the ovarian ascorbic acid depletion test and the FSH preparation assayed by the ovarian augmentation assay. Hormones vere altered as antigens by coupling with a hapten in varying ratios of hapten to hormone as described by Cinader et al., supra. For convenience,, the Cinader process is discussed herein although Phillips, ^4 jras 4/ t X. supra, may provide a more stable bond under certain circumstances. In this procedure, the protein hormone serves as a carrier and the hapten is coupled to it by diazo bonds,, Although a variety of hapten groups were coupled to different hormones, the same basic procedure was used for any combination. Fifteen to thirty-five haptenic groups per hormone molecule were found most useful for preparing immunizing antigens. The basic reaction consisted of diazotizing the hapten (sulfanilic acid) by adding it to a solution of 0.11 W HC1 and then slowly adding this solution drop-wise to a 1 percent solution of NaNOg with constant stirring at U°C. Diazotisation was considered complete when free HNOp was detected in the reaction mixture. Although the above reaction was accomplished at 2»°C,, optimum temperatures for the reaction normally are about 0-6°C.5 although lt°C. is preferred. The hapten-protein coupling was performed by dissolving the protein hormone in an alkaline buffer, pH 8.0. The diazotized hapten was added slowly to the hormone solution with continuous stirring at )|°C, The pll of the reaction was constantly monitored and kept near 8.0. After all the hapten vs,s added, the pH was finally adjusted to 8.0, stirred for 1-2 hours and allowed to stand at h° overnight. 'The mixture was thoroughly dialyzed for 6-8 days against distilled water to remove unreacted hapten. Although the number of diazo groups per hormone molecule could be regulated by the number of moles of hapten and hormone reacted, a- 35 parallel control experiment with S~ label3.ed Bulfc.nj.lic acid to evaluate the precise composition of the hapten-protein samples was performed with each diazotization. The same hormone preparation to be used for immunization was used in the control experiment. After the reaction was completed, an aliquot was taken from the reaction mixture and the remainder thoroughly dialyzed. Equal volumes of the dialyzed and undialyzed solutions were counted by liquid scintillation. By comparing the counts of the dialyzed 17419 -vr— . and. iindialyz.ed samples, the moles of hapten coupled to each mole of hormone was calculated since the unreacted he.pten was removed by dialysis. For this calculation, a molecular weight of 30,000 was assumed for all gonadotropin preparations. Following dialysis, hapten-hormones vere lyophiliz,ed and stored at 1»°C. Diazo-HCG X35 groups/molecule) and IiLH (26 groups/molecule) were bioassayed by the ovarian ascorbic acid depletion method and found to retain 62 and 85 percent respectively of the activity of the unaltered hormones from which they were derived. Hong of the other hormones were assayed for biological activity. Immunisation Procedures Female baboons received their initial immunisation on days 3-5 of the menstrual cycle and the second and third injections one week apart. The fourth injection was given 2-3 weeks after the third, A few animals received a fifth injection at 70-80-days after the first injections. All antigens were administered subcutaneously in a suspension of mannide man-oleate or peanut oil. Doses of antigens for each injection varied between 3 and 5 mg. Injection sites were inspected daily for 5 days s.fter each immunization for local reactions. Monitoring Kffects of Icimunination. Daily 2k hour urine specimens and frequent serum samples vere collected during at least one menstrual cycle prior to immunizations and following immunizations until the effects of treatment vere assessed. Urinary LH, urinary estrogens and plasma progestins were measured. Anti- bodies vere detected in post-immunisation serum, samples by reacting 0.2 ml. of a 1:1000 dilution of serum in phosphate-buffered saline (pH 7.'0 131 0,5 perccnt normal baboon serum with 250 pg of 1 labelled hormone. Sera were reacted with both the unaltered immunizing hormone and unaltered baboon LH for antibody detection. A purified baboon LH preparation (1.9 x HIH-LH-Sl) was used a3 a tracer antigen. Antigen-antibody complexes 1741 . . were precipitated with ovine anti-baboon gamma globulin after a 2b hr. incubation at 4°C. Antibody levels vere expressed as pg of labelled hormone bound. Significant antibody levels ware considered to be those 131 that would bind 5.0 pg or more of the 1 labelled antigen, Aritisera vere fractionated by gel filtration of Sephadex G-200 according to the procedure of Fahey and Terry (at p. 36, Experimental Tevy^ j Immunology,^F.A. Davis Co., Philadelphia, Pa,, 19^7, incorporated by reference to the extent necessary to understand the invention) to determine the proportion of IgM and'JgG antibodies in the baboon sera. Since the IgG fraction in this procedure contained a portion of IgA and IgD antibodies 5 only IgM and total titers vere determined. The IgM fraction froia 131 the column vas reacted vith 1 hormones and the binding capacity determined. The vo3.um.es of the fractionated sera vere adjusted so that antibody levels would be comparable to thoss of whole seruic. Antibody Production Ho significant reactions were observed at the site of injection following any iraaunization. On U occasionss a slight induration (2-3 cm in diameter) was seen whem mannide manoleate vas used as a vehicle but the redness and swelling disappeared within 'i-5 days. Antibodies were detected against the immunizing antigen id.thin 3-5 weeks in all animals. The extent, duration and cross reactivity of these antibodies is recorded. Generally speaking, higher levels were observed to heterologous gonadotropin immunization than to homologous ones. The cross-reactivity of induced antibodies with baboon LH was studied on. each animal. Cross-reaativity of aritisera at peak levels was recorded. Although relatively high antibody activity against human Lll and HCG were seen, relatively little reaction with baboon L1I occurred. An intermediate cross-reaction was noted with anti-ovine LH and a high degree of cross-reactivity wa3 seen with anti-monkey LH. Diaao-huiaan FSH waa -ao — weakly antigenic in the baboon. The duration of antibody production was generally longer with the human and sheep gonadotropin immunisation than with those of monkey or baboon origin. Peak antibody levels usually occurred at the time when the antibodies had shifted to principally the IgG type. Early antibodies had a larger proportion of IgM type and vere generally more cross-reactive with baboon LH„ The change in the proportion of the total antobody propulatlon that was IgM vac recorded fros the time antibodies were first detected. Significant cross-reactivity to baboon LH was observed in anti-human gonadotropins when IgM was abundant but dropped sharply as the antisera shifted to nearly all IgG. This drop in cross-reactivity did not occur with monkey and baboon immunizations. Again, the ovine LH immunizations produced an intermediate change in reactivity with the shift from IgM to 1'gG. Effects on the Menstrual Cycle The effects of immunization upon the events of the menstrual cycle were determined by observing changes in sex skin turgescence and levels of pituitary and/or ovarian hormones. Based on these parameters, the delay or retardation of ovulation, from the expected tine,, as Judged by the control cycle was calculated*, One animal immunized with KCG had no interruption in ovulation arid another immunized with IfFSII was delayed for only one cycle. Two animals injected with JJLH and two injected with HCG had ovulation delays equivalent to two menstrual cyclesc A third animal immunized with HLH was delayed a calculated 86 days, Ovine LH immunizations produced an 88 day delay in ovulation. Immunizations with diaso-nonkey or baboon LH resulted in longer disruption of the menstrual cycle. Calculated delays in ovulation for tha two animals receiving monkey LH was 1^6 and 122 days whereas the animals receiving altered baboon LH were retarded from ovulation 22h and 210 days. Effects on specific hormone patterns following -immunization with HLH in one animal were recorded. The interval between menses was considered, to represent a "cycle". Urinary estrogens and plasma progestin patterns indicated that no ovulation occurred during the cycle of immunization which was 85 days in duration. Urinary estrogens vere elevated during treatment but did not reflect a typical pattern. Plasma progestins were not elevated until about day 19 of the first post-treatment cycle. Patterns of both estrogens and progestins vera within normal limits during the second post-treatment cycle. Antibody levels vere elevated from about day 35 of the treatment cycle until 289 days from the first detection of antibodies. An LH assay was not available when this animal vas studied and no data on plasma or urinary levels of this hormone vas obtained. Hormonal patterns following an immunisation vith diazo-baboon LH vere recorded. In this animal, antibody levels were lower and per-sisteds in general, for a shorter period than did immunizations with huraa.t; gonadotropins. During the treatment cycles levels of urinary estrogens and plasma progestins followed a normal pattern but were qualitatively lower than normal. Urinary LH patterns fluctuated markedly due to the injections of disac-LH during this period. Ko conclusive evidence of ovulation was obtained for the treatment cycle. The first post-treatment cycle lasted 2h6 days. During this cycle urinary LJI and estrogens were elevated on days 35-^1 but there was no subsequent elevation in plasma progestins-that would indicate ovulation had occurred. Following day h2 of this cycle5 there vas no significant elevation in any of the three hormone levels until day 231 when significant elevations of urinary estrogens and LH occurred. These rises were followed 3 days later by an elevation in plasma progestins indicating the presence of a functioning corpus luteuiiw A second post-treatment menstrual cycle was of normal duration and the endocrine patterns were normal. Antibodies to unaltered baboon EH attained maximum levels by about day 70 of the post-treatment cycle and reraained relatively constant until day 190 when, a steady decline was observed. By day 215 of this cycle, antibody levels were barely detectable. Approximately 16 days after this —1,"U - 174192 time, a peak of LH commensurate -with a normal midcycle elevation vas observed. From this point the animal appeared to have the normal function of the pituitary-ovarian axis. Hormonal patterns in animals vith other heterologous gonadotropin immunizations vere similar to animal receiving HLH and other animals receiving monkey or baboon LH were similar in response to animal receiving baboon LH. These results in baboons indicated that the modification of a reproductive hormonef by the procedures outlined^ did render it antigenic and the antibodies thus formed did neutralize natural endogenous hormones if the natural hormone vas obtained from the species receiving the immunisations with modified hormone, EXAMPLE- 2 HCG is a hormone naturally present only in pregnant vomea. HCG is also commercially available. LH hormone is immunologically and biologically identical to HCG hormone, even though there are chemical dif** ferences. Since they are biologically identical and HCG is readily available from commercial sources it was presumed that the effectiveness of this immunological procedure could be evaluated by injecting modified HCG into non-pregnant vromen and monitoring the blood levels of LH. Antibodies formed will neutralize both the LK and the modified HCG. Women have a pattern of LH levels; the level is substantially constant until the middle period between menstrual cycles, immediately prior to ovulation;"at that point the LH level rises greatly and helps induce the ovulation. Monitoring the LH level and the antibody level will show that the procedure used did or did not cause the production of antibodies capable of neutralizing the endogenous reproductive hormonef namely LH. A vojnan aged 27 years was selected for study. Hormone vas obtained, purified and modified. The "modified human hormone (HCG) vas •*7419 injected, into the subject. It is veil known that antibodies to HCG react identically to LH as veil as HCG. The .effect of the immunization vas elevated, principally by monitoring blood levels of LH hormone. Finally the results were evaluated. Preparation of Hormone Clinical grade HCG derived from pregnancy urine vas obtained from the Vj.tsur.erican Corp., Little Falls, Hew Jersey, This material has an immunological potency of 2600 lU/sng. Contaminants vere detected in. this preparation. Purification consisted of chromatography and elution. Fractions vere dialyzed and lyophyliz.ed. The most potent fraction contained approximately ?600 lU/mg., however, it vas heterogenous on oloyacrylsrd.de gel electrophoresis. The fraction was further purified by gel filtration. The elution profile revealed two major protein peaks. The most potent HCG vas found in the first peak and had an immunological potency of 13,670 I'J per mg. This fraction vas subjected, to polyacrylami&e gel electrophoresis. Further purification by gel filtration showed no evidence of heterogenity of the HCG at this stage. Consequently, materials for study xrere processed according to the above procedure. 131 The contamination of this purified HCG was tested with I used for identification and a sample vas reacted with antinera against several proteins offering potential contamination. Those proteins were follicle stimulating hormone, human groirth hormone, whole human serum, human albumin, transferin, alpha one globulin, alpha tiro globulin and oroBDEiucoid. Ho detectable binding of the purified HCG vas observed with .any antisera at a dilution of 1:50 of each. These negative results, calculated against potential binding of the respective proteins, indicated that contamination with any vas less than 0.005 percent. 1741 - - -Alteration of Hormone Hormone vas altered, by coupling with a hapten (sulffanilazo)» This method couples the hapten molecules to the protein via the amino group of the aliphatic or aromatic portion of the hapten. The number of hapten molecules coupled to each KCG molecule (Ha-HCG) can. be regulated and for this study, forty haptenic groups per HCG molecule vere used for preparing the immunising antigen* Following the hapten-eoupling process, the H&-IICG "as sterilized end tested., Subject The subject vas multiparous and had terminated her reproductive capabilities by prior elective bilateral salpingectomy. She vas in good health and had regular cyclic menstruation. She underwent complete histroy, physical examination and laboratory evaluation including blood count, urinalysis, latex fixation and Papanicolau smear. She had no history of allergy. To demonstrate normal functioning of the pituitary-ovarian axis prior to itemization, blood sempl.es were obtained every other day from the first day of menses for 10 days, then daily for 10 days and finally, every other day until the next menses. Serum determinations of FSH, LH, estrones estradiol and progesterone vere performsd. These studies indicated an ovulatory pattern. Immunization Procedures Ten rag. of the Ha-HCG antigen vas dissolved in 1.0 ml, of saline and emulsified with an equal volume of oil. Prior to injection, scratch tests to antigen and vehicle vere performed. Immunizations vere begun in the luteal phase of the treatment cycle to prevent superovulation from the administered HCG. Four injections at two veek intervals vere given to the subject. The first two of these vere administered in oil subcut-aneously (1.0 ml. in each upper arm); the final two injections vere given —-Z.-ST- — ■274192 in saline only via the intradermal route, Following each injection, blood pressure readings vera taken and the subject observed for allergic reactions. Monitoring Effects of Inraunizatioils Blood samples were collected at weekly intervals beginning two weeks after the initial injection to test for the presence of humoral and cellular antibodies. Following completion of the immunisation schedule;, blood samples were collected in the same manner as in the control cycle to assess effects of immunization on hormonal patterns of the menstrual cycle. Since antibodies to HCG react identically to LH as with KCG, LH was monitored e.s an index of effectiveness of the procedure. A third cycle was similarly studied six months after Initial immunization. Upon completion . of the study, physical and pelvic examinations and laboratory evaluations were repeated. Serum samples frora the control and post™treatment cycles were assayed for FSH, LH, estrone, estradiol and progesterone. The subject vas tested for delayed hypertensivity before immunisation and at two week intervals until the injection schedule was completed. k}r in vitro lymphocyte transformation test„ Results Temporal relationships of serum pituitary and gonadal horaonss in the control cycles of the subject were recorded. Antibody titers to h'CG were detected in the subject after tvo injections. Menses occurred at regular intervals during the insauniRations. Following the initial injection in aannide raanoleate, soma itching and swelling at the injection site occurred. Subsequent intradermal injections in saline produced no reactions and it -was concluded that the local reactions were induced by the mannide znanoleate, Lymphocyte trancforaation tests on plasma samples were negative. In the post-treatment cyclet baseline follicular and. luteal phase LH'levels were not noticeably changed in the subject. Very small midcycle eleeations in LH levels vere observed as compared to the normal large increases* FSH patterns in the post-treatment cycle vere normal. This indicated that the antibodies were neutralizing the action of endogenous LH. The subject showed an ovulatory progesterone pattern but attain relatively high antibody titers to LH and HCG after only two injections of Ha~HCG» The subject vas studied during another cycle approximately six Eionths from the first immunization. Significant antibody titers were found. LH patterns indicated a small midcycle elevation. FSH patterns were essentially noroal. Thus, the specificity of anti~HCG antibodies to Lli vas shown but not to FSH. EXAMPLE 3 Another woman aged 29 years was selected for further study. Hormone was obtained, purified, and modified as in Example 2.. This modified horaone was injected into this subject in the same way as in Example II, The subject was monitored and tested as in Example 2, The results were similar to the results found in Example 2 except that (l) the levels of estrone and estradiol vas substantially normals (2) the subject acquired significant antibody titers late in the post-immunization cycle, and (3) in the cycle studied after six months this subject shoved no significant midcycle elevation in LH patterns. EXAMPLE Jj_ Another woman aged 29 years was selected for further study. Hormone was obtained and purified and modified as in Example 2. This modified hormone was injected into this subject in the same way as iu Exaiaple .2, The subject wan monitored and tested as in Example 2. The results vere similar to the results found in Example 2 cxo.ept that (l) baseline follicular and luteal phase LH levels were noticeably depressed in the post-treatment cycle, (2) no midcycle elevations irere observed in LH, (3) estrone levels were elevated during the follicular phase of the post-imraunizstion cycle, and (it) during the six months study there vas no significant .midcycle elevation in LH patterns. EXAMPLE } . Another woman aged 35 years was selected for further study. Hormone was obtainedj purifieds and modified as in Example 2. This modified hormone vas injected into this subject in the same way as in Example 2. The subject was monitored and tested as in Example 2. The results were similar to the results found in Example 2 except that (l) baseline follicular said luteal phase LH levels were noticeably depressed in the post-treatment cycle, (2) a very small mid« cycle elevations of LH were observed, (3) levels of FSH patterns in the post-treataent cycle were depressed, and (U) levels of both estrone and estradiol were reduced, during the follicular phase of the post-immunisation. ■ EXAMPLE Jl Another woman aged 28 years was selected for further study. Hormone was obtained, purified, and modified as in Example -2. This •modified hormone was injected into this subject in the same way as in Exenple 2. »fhe- subject vas monitored and tested as in Example 2, The results were similar to results found in Example 2 except that (1) baseline follicular and luteal phase LH levels were depressed in the post-treatment cycle, (2) no peaks vere observed in midcycle levels of LH, (3) estrone levels appeared elevated in the follicular phase of the post immunization cycle, and (U) LH patterns indicated no significant jaificycle elevation in the six month post-immunization cycle. -is - 1741 EXAMPLE 7_ Another woman aged 28 vas selected for further study. Hormone vas obtained, purified, and modified as in Example 2. This modified hormone, was injected into this subject in the sane vay as in Example 2. The subject waa monitored and tested as in Example 2. The results were siirdlar to results found in Example 2 except-that (l) antibody titers to HCG vere not detected until after three injections s (2) baseline follicular and luteal phase LH levels were depressed in the post-treatment cycle, (3) no peaks nor midcycle elevation in the LH were observed, (if) estrone levels were elevated during the follicular phasef and (5) no significant antibody titers vere found in the six month cycle. All the above examples show the practicality of injecting modified hormones for the purpose of neutralizing an endogenous reproductive hormone and thereby offering a procedure for the prevention of conception or the disruption of gestation. EXAMPLE. 8 Data obtained in earlier experiments and discussed in Examples 1 to 7showed that 8. modified natural reproductive hormone, when injected into an animal of species from, which it was derived, would produce antibodies.that would neutralise the action of the unmodified endogenous natural hormone in the body of the animal. Hormones used in experiments 1 to 7 were FSH, LH and HCG. Hew experiments were performed, based on this 3mowledgev to identify another reproductive hormone (placental' lactogen) that could be used in a similar fashion. Preparation of Horraona A purified preparation of placental lactogen was prepared from placentae of baboons since it was intcndc-d to use t/lodified placental -■QJ\ - 27419 lactogen to immunize baboons. Placentae vere extracted and purified on column chroEatograph according to previously published procedures. The purity waa tested by polyscrylaaide gel, electrophoresis and by radioimmunoassay. The material obtained showed a high degree of purity on electroi>horesis and-radioimmunoassay showed no contamination with other placental hormones. Honnone Modification and Iranimizo.tions The baboon placental lactogen (BPL) vas altered by coupling with the diasoniim salt of sulfanilic acid as outlined for other hormones in Example X, The number of diazo molecules per BPL molecule in this instance was 15. Itaunization procedures vere also similar to those described in Example I for other hormones. Results^ Within !i~6 veelis after the first injection of diasio-BPL, antibody levels to natural unmodified BPL in_ vitro were detected, in 6 female baboons. Levels rose to a plateau vithin 8-10 weeks and remained there for several months. Hormonal measurements indicated that there were no effects on the normal events of the menstrual cycle due to the immuniza^ tions. Since BPL is normally secreted only in pregnancy, this was not a surprising observation. •All six females vere mated vrith a male of proven fertility three times (once each in three different cycles during the fertile period). Pregnancy diagnosis by hormonal measurement vas performed after each mating. From the 18 ratings, there vere 13 conceptions as judged by pregnancy tests, The animals that were pregnant had menstrual bleeding 7-12 days later than vas expected for their normal menstrual cycles. Subsequent hormonal measurencntcconfirmed that these 13 pregnancies vers terminated by abortions approximately one veek after the time of expected menses, -3o - These findings suggest that the antibodies formed in the animals body after immunization had no effect on the non-pregnant menstruaJ. cycle but when pregnancy was established, they neutralized the baboon placental lactogen in the baboon placenta and the result vas abortion very early after conception. When in Examples 1 to 8 above, structures 1 to 7 are modified by use of diazosulfanilic acid, dinitrophenolP or S-aceto mercaptosiiccinic anhy&ri&e or structures III -VI '-modified by addition of polytyrosine or polyalanine„ are used, sireilar results may be obtained. Similarly, vhen FSH, soinatciaedian, grovth hormone or angiotension II modified by vise of diazosulfanilic acid or trinitrophenolj, the results obtainable upon administration of the' purified modified po.Iy--» peptide into a male or female human or animal indicate the stimulation of an bodies which neutralise all or some of. the modified polypeptide as well as corresponding endogenous polypeptide, 'Example 9 The subjects used in the studies reported in the example are femal baboons. All baboons vere adults of reproductive age. A description of subjects and the conditions of experimentation have been described in Example 1. The animals were studied using highly purified beta «■ subunits of HCG using a preparation with a biological activity of leas than 1,0 IU/mg. Animals vere immunized with lh-26 moles/mole of polypeptide,of diaxasulfanilic acid coupled subuaits in mannide manoleate. Antibody levels vere assessed by determining the binding of 3 25 serum dilutions with I ' labelled antigens. Cross-reactivity of antisera vas measured by direct binding of labelled antigens and by displacement radioimaunoaesoys. Antifertility effects in actively immunized animals vere tested by mating females with males of proven fertility. Effects in pregnant baboons pajs'sively immunized with either sheep or baboon </div>

Claims

<div class="application article clearfix printTableText" id="claims"> --*>> - onti-i>-HCG vere determined by monitoring serum levels of gonadotropins and sex Bteroid hormones before and after immunizations. Eight female baboons ware immunized with the modified beta subunit of HCG. Significant'.antibody levels vere attained in all animals. Baboon immunizations with modified beta subunit of HCG- resulted in high antibody levels reacting to HCG, human LH and baboon CG but not to baboon LH. All animals remained Ovulatory, however» no pregnancies resulted from numerous Eatings with males of proven fertility. Passive immunization of non-immunized pregnant baboons with sheep anti-S-HCG serum produced abortions within 36-^; hours. I74ir2 - 32 - - WHAT WE CLAIM IS:

1. An antigen for use in active immunisation for the control or treatment of a physiological process in a particular species, comprising a chemically modified polypeptide, the unmodified polypeptide being 5 a) an endogenous polypeptide which influences such physiological process in that species and selected from protein reproductive hormones, non-hormonal protein antigens isolated from placental tissue, gastrin, Angiotension II, growth hormone, soma-10 tomedian, parathyroid hormone, glucagon, thyroid—stimulating hormone or secretin} b) a fragment (as herein defined) of such endogenous polypeptide; or c) a polypeptide which is immunologically equivalent 15 (as herein defined) to such endogenous polypeptide or fragment thereof, and the chemical modification (i) comprising (a) the attachment of one or more modifying groups to the unmodified, polypeptide and/or (b) the removal of one or more moieties from the unmodified polypeptide, and (ii) being such as to permit the chemically modified polypeptide to induce in that species, formation of antibodies which biologically neutralise the endogenous polypeptide as well as the chemically modified polypeptide. 174192 - 33 -

2. An antigen according to Claim 1, for use in active immunisation for the control of fertility, in which the unmodified polypeptide is a) a protein reproductive hormone; b) a fragment (as herein defined) thereof; or c) a polypeptide which is immunologically equivalent (as herein defined) to such protein reproductive hormone or fragment thereof.

3. An antigen according to Claim 2, in which the protein reproductive hormone is Follicle-Stimulating Hormone, Luteinising Hormone, Human Placental Lactogen, Human Prolactin or Human Chorionic Gonadotropin.

4. An antigen according to Claim 3, in which the protein reproductive hormone is Follicle-Stimulating Hormone.

5. An antigen according to Claim 4, in which the unmodified polypeptide is Follicle—Stimulating Hormone or the B-subunit thereof.

6 An antigen according to Claim 3, in which the protein reproductive hormone is Human Placental Lactogen or Human Prolactin.

7. An antigen according to Claim 3, in which the protein reproductive hormone is Human Chorionic Gonadotropin. g.

An antigen according to Claim 7, in which the unmodified polypeptide is Human Chorionic Gonadotropin . nifiqi t - 34

9. An antigen according to Claim 7, in which the unmodified polypeptide is the R-subunit of Human Chorionic Gonadotropin.

I 0. An antigen according to Claim 7, in which the unmodified polypeptide is a 20 to 30-amino acid C-terminal fragment of the B-subunit of Human Chorionic Gonadotropin.

II . An antigen according to Claim 7f in which the unmodified polypeptide is a 30 to 39-amino acid C-terminal fragment of the B-subunit of Human Chorionic Gonadotropin.

12. An antigen according to Claim 7, in which the unmodified polypeptide is of structure III (* indicates site locations of carbohydrate moieties), * Asp-Asp-Pro-Ary-Phe-Gln-Asp-Ser-Scr-Ser-Ser-Lys-Ala-* ★ Pro-Pro-Pro-Ser-Leu-Fro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-* Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln,

13. An antigen ncdording to Claim 7 f which the unmodified polypeptide is of structure IV (* indicates site locations of carbohydrate moieties), * it Gln-Asp-Ser-Ser-Ser-Scr-Lys-A-l a-Pro-Pro-Pro-Ser--Leu-Prc-* * 5er-Pro~Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln.

14. An antigen according to Claim 7 , in which the unmodified polypeptide is of structure V (* indicates site locations of carbohydrate moieties), * Thr-Cys- Asp-Zisp-Pro-Arg-Phe-Gln -h a p - Si c-r • - r> c-r^Sifirr^l.y r,-ALt * * M/± ' Pro-Pro-Pro-Ser-Leu-Pro-Ser-Tro-Ser-Z^g/fflzfu-Pro- II { Pro-Pro-Asx-Thr-P rc-1 le"Leu--Pro--Gln-5eJ(-IJfcae55T937g Lcei Ilt+I - 35 -

15. An antigen according' to Claim 7> , in which the unmodified polypeptide is of structure VI (* indicates site locations of carbohydrate moieties)', * rhe-Gln-Asp-Scr-Scr-Ser-Lys-Ala-Fro-Pro-Pro-Ser-Leu-* * Pr o-Ccr-Pr o-Ser -7\r y-Leu--Pro~Gly--Pro-Pro~A;;>:-Thr~Pro-T lo-I,c!U--Pro-Gln--Ser-Leu~Pro.

16. An antigen according to Claim 1, in which the unmodified polypeptide is a) a non-hormonal protein antigen isolated from placental tissue? b) a fragment (as herein described) thereof; or c) a polypeptide which is immunologically equivalent (as herein defined) to such antigen or fragment thereof.

17. An antigen according to Claim 1, for use in active immunisation for the treatment of the Zollinger-Ellison syndrome, in which the unmodified polypeptide is a) gastrin; b) a fragment (as herein defined) thereof; or c) a polypeptide which is immunologically equivalent (as herein defined) to such gastrin or fragment thereof.

18. An antigen according to Claim 1} for use in active immunisation for the treatment of hypertension, in which the unmodified polypeptide is a) Angiotension II; b) a fragment (as herein defined) thereof^^qpiTj^s^ ox or c) a polypeptide which is immunologically equival'^^t (as herein defined) to such Angiotens IxyiS III fragment thereof. 174192 - 36 -

19. An antigen according to Claim 1, for use in active immunisation for the treatment of diabetes and associated micro- and macro- vascular disorders, in which the unmodified polypeptide is 5 a) Growth Hormone or Somatomedian; b) a fragment (as herein defined) of such Growth Hormone or Somatomedian; or c) a polypeptide which is immunologically equiv alent (as herein defined) to such Growth 10 Hormone or Somatomedian or fragment thereof. 2q.

An antigen according to any one of the preceding Claims, in which the chemical modification comprises the attachment of one or more modifying groups to the unmodified polypeptide. ^

21. An antigen according to Claim 20, in which 1 to 40 modifying groups are attached per molecule of the unmodified polypeptide.

2 2. An antigen according to Claim 21, in which 2 to 40 modifying groups are attached per molecule of the 2 0 unmodified polypeptide.

2 3. An antigen according to Claim 2 2, in which 5 to 30 modifying groups are attached per molecule of the unmodified polypeptide. < y 19 NOV 1984 if j, r-! A ■* HO 1 i £±iJ*> -37 -

24. An antigen according to Claim 23, in which 10 to 26 modifying groups are attached per molecule the unmodified polypeptide.

25. An antigen according to any one of Claims 1 to 24, in which the modifying groups are diazo groups.

26. An antigen according to Claim 25, in which the diazo groups are introduced by reaction of the polypeptide with diazo sulfanilic acid.

27. An antigen according to any one of Claims 1 to 24. in which the modifying groups are derived from dinitrophenol,trinitrophenol, S-aceto-mercaptosuccinic anhydride, a polytyrosine, a polyalanine, biodegradable polydextran, or thyroglobulin.

28. Human Chorionic Gonadotropin modified by introduction of 35 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

29. Human Leutinising Hormone modified by introduction of 26 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

30. Human Chorionic Gonadotropin modified by introduction of 40 diazo groups per molecule, the diazo groups being derived from diazotised sulfanilic acid.

31. Baboon Placental Lactogen modified by introduction of 15 diazo groups per molecule, the diazo Ilk)41 - 38 - groups being derived from diazotised sulfanilic acid.

3 2. The p-subunit of Human Chorionic Gonadotropin modified by introduction of 14 to 26 diazo groups per molecule, the diazo groups being derived from diazo-5 tised sulfanilic acid.

3 3. An antigen according to Claim 1, substantially as herein described with reference to any one of the Examples.

3 4. A pharmaceutical composition comprising 10 an antigen according to any one of Claims 1 to 3 2, in association with a pharmaceutically acceptable liquid carrier, and in a form suitable for parenteral administration .

3 5. A pharmaceutical composition according 15 to Claim 34, in which the liquid carrier is mannide mono- oleate, saline or an oil.

36. A pharmaceutical composition according to Claim 34 or 35 in unit dosage form and containing 0.1 to 50 mg of the antigen. 20

37. A pharmaceutical composition according to Claim 34, substantially as herein described with reference to any one of the Examples. THE OHIO STATE UNIVERSITY By ^"their authorised Agents., A.J. PARK & SON. Per *^4^- >~J 113$ </div>