NZ182272A

NZ182272A - Modified polypeptides for the immunological control of fertility,and pharmaceutical compositions

Info

Publication number: NZ182272A
Application number: NZ182272A
Authority: NZ
Inventors: V C Stevens
Original assignee: Univ Ohio State
Priority date: 1975-10-14
Filing date: 1976-10-08
Publication date: 1985-03-20
Also published as: JPS5251017A; GB1567764A; ZA766143B; SG63582G; BE847243R; AU513558B2; HK10883A; IE44541B1; IE44541L; NL7611212A; DE2646223C2; DE2646223A1; FR2327797B2; MY8500808A; AU1858476A; FR2327797A2

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 1 82272 18 22 7 2 n ' •• - - •• * IM-. 10 -IS Prsonvy ......... Jr.. Cooisieje C^sciScstion Ried; % *.'P CS««: mlM.V, fi-WMlb* ■ 20 MAti 1985 P.O. Jctsms!, No: ..... .. ah COW 15/12. NEW ZEALAND No.: Date: PATENTS ACT, 1953 COMPLETE SPECIFICATION "IMPROVEMENTS IN OR RELATING TO ORGANIC COMPOUNDS" }t/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 which Jx' we pray that a patent may be granted to gag^us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - (followed by page la) 18 22 7 2 iMrnovEMEMrPD- in on relating to organic compounds * Mew Ze^alcmei P«,1"e-tvt" c^choy\ Our pearlier application No. J 7 4-1^2 Of^ct&ajr* 5 describes /inter alia the chemical modification of natural endogenous polypeptides^which influence the reproductive process^to provide antigens capable of inducing formation of antibodies which neutralise not only the anti-genically modified polypeptide but also its unmodified endogenous counterpart, thus providing a means for immunological contraception. The modified polypeptides are produced from the natural, endogenous polypeptides in the 10 species involved or are immunologically equivalent to the modified polypeptides so produced. In practice, the polypeptides modified are derived from the species involved or from a closely related species. i'j *-»• ypec-i-Pi ce\t~ton More particularly, our earlier ^application -icx <Aoh4M describes the chemical modification of protein reproductive hormones and fragments thereof, to provide antigens for use in immunological contraception. The fragments of the protein hormones must be large enough and sufficiently distinctive in chemical and physical character to enable 20 them to be recognised as a specific part of the whole pro tein in question. They must also naturally possess the chemical nature, i.e. the requisite amino acid make-up, to enable them to be chemically modified in the manner desired. - lex- 1 8 22 72 118-^329/A The term "protein reproductive hormone" used herein includes those hormones essential to the normal events of the reproductive process. For the purposes of the present invention, the 5 term "fragment1^ with reference to a protein reproductive hormone| embraces both amino—acid chains forming part of the structure of the hormone and amino- acid chains which^ although not identical with chains forming part of the structure of the hormone^,are sufficiently similar for them 10 to be immunologically equivalent, i.e. to provoke essenti- ally the same antibody response upon administration. SpecTf ica.'t' ion Our earlier ^application specifically discloses the chemical modification of protein reproductive hormones, „«§> such as Follicle Stimulating Hormone (FSH) , Hbcutini-giTt^- 15 Hormone (LH) , Human Placental Lactogen (HPL) , Human Pro- ocP ^ lactin and Human Chorionic Gonadotropin (HCG), and fragments thereof. Specific fragments which may be modified in Speea-PiCAtW , accordance with our earlierytepplication include the 20 0-subunit of FSH and specific unique fragments of natural HPL or Human Prolactin, which fragments may bear little resemblance to analogous portions of other protein hormones, Preferred fragments include the S-subunit of HCG which, according to the two authoritative views, has either 25 structure I or II as follows Vindicates site locations a^ol Asx kip of carbohydrate moietiesl) - 2 - 7 2 10 Ser-Lys-Glu-Pro-Leu-Arg~Pro-Arg-Cys-Arg-Pro-Ile-Asn-Ala-Thr- 20 Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-Val-Asn- 40 Thr-Thr-Ile-Cys-Ala-Gly-Try-Cys-Pro-Thr-Met-Thr-Arg-Val-Leu- 50 g0 Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys-Asn-Try-Arg- 70 5 Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-Gly-Cys-Pro-Arg- 80 Gly-Val-Asn-Pro-Val-Val-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys- 90 100 Gln-Cys-Ala-Leu-Cys-Arg-Arg-Ser-Thr-Thr-Asp-Cys-Gly-Gly- 110 Pro-Lys-Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln- 12 0 * * 130 Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser- 140 10 Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro- Gln (Structure I) 10 Ser-Lys-Gln-Pro-Leu-Arg-Pro-Arg-Cys-Arg-Pro-Ile-Asn-Ala- 20 Thr-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys-Ile-Thr-29 * 40 15 Val-Asri-Thr-Thr-Ile-Cys-Ala-Gly-Tyr--Cys-Pro-Thr-Met-Thr- 50 Arg-Val-Leu-Gln-Gly-Val-Leu~Pro-Ala-Leu-Pro-Gln-Leu-Val- 60 70 Cys-Asn-Tyr-Arg-Asp-Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro- 80 Gly-Cys-Pro-Arg-Gly-Val--Asn-Pro-Val~Val-Ser-Tyr-Ala-Val- 90 Ala-Leu-Ser-Cys-Gln-Cys-Ala-Leu-Cys-Arg-(Arg)-Ser-Thr-100 110 20 Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr-Cys-Asp- 120 Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser- * 130 * 140 Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-jAsx-Thr-Pro-Ile- 147 ^ Leu-Pro-Gln-Ser-Leu-Pro W (Structure II) rpectFt'ccftion £2!§( As disclosed in our earlier ^application, for ffe specificity of antibody action, it is desirable that poly- - 3 - 1 8 227 peptides be modified which comprise molecular structures completely or substantially completely different to those of other protein hormones. In this connection, the p-subunit of HCG possesses a chain or chains of amino acids 5 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 10 the following formulae III and IV (C-terminal portions of structure I, above) and V and VI (C-terminal portions of Structure II, above): •k 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-* 15 Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln (Structure III) * * Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro- * ft Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln. 20 (Structure IV) * Tnr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys- * * Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro. (Structure V) -4 ~ 1 822 72 * Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-* * Pro-Ser-Pro-Ser-Arg-Leu-Pro-Glv-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro. (Structure VI) 5 Such structures may be obtained by purely synthetic methods or by enzymatic degradation of the parent polypeptide [Carlson et. al., J. Biological Chemistry, 284(19), 6810(1973)]. In one aspect, the present invention provides 10 further polypeptide chains which may be chemically modified to provide antigens for use in immunological contraception. In particular the present invention relates to the: chemical modification of the polypeptide chains of the following formulae VII, VIII, IX and X. Ey virtue 15 of their substantial resemblance to the chemical struc ture and configuration of HCG, and the immunological response provided by them when chemically modified, these fragments are considered to be fragments of HCG. Phe-Gln-Asp-Ser-Ser-Ser~Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-20 Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile- Leu-Pro-Gln (Structure VII) - 5 - '8227 118^129/A Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Gly (Structure VIII) 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-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro (Structure IX) 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-Pro~Asp~Thr-Pro-Ile-Leu~Pro--Gln-Ser-Leu-Pro (Structure X) Again, Such structures may suitably be produced by known synthetic methods. In one aspect, therefore, the present invention provides (i)a) an antigen for the immunological control of fertility which comprises a chemically modified polypeptide, the unmodified polypeptide being of structure VII, VIII, IX or X, stated above. Sf> eci -fCcatton As disclosed in our earlier ^application, the degree of chemical modification of the polypeptide is such that the resulting antigens will induce generation of antibodies which will neutralise not only the antigens ■ <j 1 8227 but also some, at least, of their natural counterpart, i.e. HCG, and this as well as the type of modification will depend on such factors as the nature of the polypeptide involved. If too little modification is 5 effected, the body may not recognise the modified poly peptide 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 10 not neutralise the natural endogenous protein counter part, i.e. HCG. In general, the chemical modification may involve attachment of foreign modifying groups to the polypeptide involved. The number of foreign modi-15 fying groups to be added will, of course, vary depending on the circumstances but, generally, it is preferred that 1 to 40, suitably 2 to 40, in particular- 5 to 30, for example 10 to 26 modifying groups per molecule of polypeptide be attached. Given modifying groups will 20 attach to particular amino acid sites in the peptide mol ecule, so that the maximum possible number of a given modifying group may be selected accordingly. It is also possible that several modifying groups may attach to each other and then attach to a single amino acid moiety but, 2 7 2 for the purposes of this invention, such a substitution .is regarded as attachment of a single modifying group. The chemical modification may alternatively comprise the attachment of 2 or more polypeptide molecules to a modi-5 fying group. For the purposes of the present invention, it is to be understood that where reference is made to attachment of modifying groups to the polypeptides (or vice versa) the attachment may be direct or via linking groups. The .10 polypeptide or modifier may, for example, suitably be activated with a modifying group for conjugation. The modifying groups may as indicated vary in chemistry depending on the circumstances. As disclosed SI, in our earlier, suitable modifying groups 15 include diazo groups. These may suitably be introduced by reaction with the appropriate number of moles of diazosulfanilic acid. Introduction of diazo groups into proteins is a well-known technique and may, for example, be effected as described, by 20 Cinader et al., J. Am. Chem. Soc. 7]}, 746 (1955); by Phillips et al., J. Biol. Chem. 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 25 Phillips et al. are preferred. - 8 - 1 8227 ll£^3-Z9/A Additional modifying groups include those derived from dinitrophenol, trinitro-phenol, S-acetomercaptosuccinic anhydride, polytyrosine or polyalanines (in either straight or 5 branched chains), biodegradable polydextran or, rather less preferably, natural proteins such as thyro-globulin. Generally/ synthetic modifiers are preferred to natural modifiers. The above reactions, as well as many other 10 suitable hapten coupling reactions are well known in protein chemistry. The following references may, for example, be cited in this connection:- 1. Klotz et al., Arch. Biochem. & Biophys. 96, 605-612(1966); 15 2. Khorana, Chem. Rev. 5_3, 145 (19 5 3); 3. Sela et al. , Biochem. J. 85_, 223 (1962); 4. Eisen et al. , J. Am. Chem. Soc. _75, 4583 (1953); 5 . Certano et-.al. , Fed. Proc. (ABSTR. ) 25, 20 729(1966); 6. Sokolowski et al., J. Am. Chem. Soc. 86, 1212(1964); 7. Goodfriend et al., Science 14 4, 1344 (1964) - 9 - 182272 8. Sela et-al. , J. Am. Chem. Soc. 7_8, 746 (1955) ; and 9. Bahl, J. Biol. Chem. 244, 575(1969). As disclosed in our earlier specification, the 5 chemical modification may alternatively or additionally comprise removal of moieties from the polypeptides. Thus, for example where certain of the natural proteins have carbohydrate moieties, these may be removed in conventional manner, using, for example, N-acetylneuraminidase or 10 N-acetyIglucosidase, materials useful for removing specific carbohydrate moieties. In a further aspect, the present invention relates to the use of additional modifying groups. More particularly, in this aspect, the invention provides (i)b) an 15 antigen for the immunological control of fertility, com prising a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or a fragment thereof, and the chemical modification comprising the attachment to the unmodified polypeptide of one or more 20 modifying groups derived from polymerised sugars, serum „ proteins, keyhole limpet hemocyanin, marine gastropod "• molluscs viruses, or bovine gamma globulin, or the attach- »•* ment of two or more unmodified polypeptide molecules to such a modifying group. 25 Suitable polymerised sugars for modifying the polypeptides include copolymers of sucrose with epichloro- - 10 - 182272 118^3329/A hydrin, such as Ficoll 70 or Ficoll 400 [Pharmacia Fine Chemicals, Pharmacia Laboratories Inc., 800 Centenial Avenue, Poscataway, N.J. 08854] or a polyglucose such as Dextran T70 [synthesised microbiologically by the action 5 of leuconostoc mesenteroides (a strain in NRRL B-512) on.sucrose. Glucan containing alpha-1,6-glucosidic bonds. Av. mol. wt. approx. 70,000]. Suitable serum proteins include homologous serum albumin or bovine serum albumin and suitable viruses include influenza virus (Type A, B or C) or polio-10 myelitis virus, live or killed (Type 1, 2 or 3). Any of the protein reproductive hormones or fragments thereof may be modified with these additional modifying groups in accordance with the present invention. It will be appreciated that such modification may comprise 15 attachment of the modifying groups to the polypeptide via linking groups and that this may be accomplished by reaction of the polypeptide, the modifier and a linker (activator) in any.order or simultaneously*with or without protection and subsequent disprotection of one or more reactive 20 groups in the polypeptide or modifier. Quite generally, such modification may be carried out in known manner, for example as described in the literature cited above. In a further aspect, however, the present invention provides processes for the production of antigens for - 11 - A. J. P: & s. 272 the immunological control of fertility comprising chemically modifying polypeptide which is a protein reproductive hormone or a fragment thereof. a) by attaching to the unmodified polypeptide 5 modifying groups derived from modifiers having free . amino groups, by activation of the modifier or polypeptide to be modified by reaction with tolylene di-isocyanate and reaction of the resulting activated product with the polypeptide to be modified or modifier, 10 respectively; or b) by attaching to the unmodified polypeptide < modifying groups derived from modifiers having free amino groups or being polymerised sugars, by conjugation 15 of the unmodified polypeptide with the modifier employing a water-soluble carbodiimide as activating agent; or c) By attaching to the unmodified polypeptide ■ Having fiwuvio <^rou.p5 modifvinq groups derived from jiatural protoino bv reac- :r modifying groups derived from ^natural protoino by reac-20j_^ tion of the Jhatural protein with glutaric dialdehyde and reaction of the resulting product with the polypeptide to be modified, in the presence of an alkali metal borohydride; or - 12 - 1 82272 im^l29/A d) by attaching to the unmodified polypeptide modifying groups derived from polymerised sugars by reaction of the polymerised sugar with a cyanuric halide, and reaction of the resulting dihalotriaziny1 adduct with the polypeptide to be modified; or e) by attaching to the unmodified polypeptide modifying groups derived from polymerised sugars by treatment of the sugar with an alkali metal periodate and reaction of the resulting product with the polypeptide to be modified. Process a), which, as indicated, may be employed for modification with modifiers having free amino groups, such as natural protein, e.g. keyhole limpet hemocyanin, serum proteins, e.g. homologous serum albumin or bovine serum albumin, polytyrosine or polyalanine, bovine gamma globulin or thyro-globulin, may suitably be effected as described by Singer and Schick, J. Biophys. Biochem. Cytology 9_, 519(1961). In particular the modifier is conveniently taken up in buffer solution such as phosphate buffer, in sodium chloride solution at a pH of, e.g. 6-8, and tolylene diisocyanate (TDIC) is then suitably added to this solution. The TDIC is suitably added diluted from 1:10 to 40^in dioxane and the amount of TDIC added may suitably range from 0.075 to 1000 molar equival- - 13 - 1 82272 118^^52 9 / A Vta^fd -!~h e^odl-Pi 'e-r' entsj[ The reaction may, for example, be effected at a temp- r~* eratui-e of from -5° to +10°C, preferably 0° to 4°C and the reaction time may, for example vary from about 1/2 to 2 hours. Any excess TDIC is then suitably removed by centri-5 fugation and the precipitate washed v/ith the above phosphate buffer and the supernatants combined. The activated modifier may then suitably be added to a solution of the polypeptide to be modified, in the same phosphate buffer (5 -30 mg/ml), the quantities of modifier and polypeptide being in 10 accordance with the molar ratio desired in the resulting modified polypeptide. The combined solution is suitably reacted at 30° to 50°C, conveniently 35-40°C, suitably for 3 to 5 hours. In an alternative embodiment, the first step may comprise addition of the TDIC solution to the 15 buffered polypeptide, followed by addition of buffered modifier to the resulting activated polypeptide, the conditions for the first and second steps being as described above. The modifiers for use in process b), which is 20 generally described by Moore and Koshland, J. Biol. Chem. 242, 2447 (1967), include natural proteins such as keyhole limpet hemocyanin, serum proteins such, as homologous or bovine serum albumin, polymerised sugars, such as Ficoll's (70 or IfOO 25 tyrosine. The polypeptide to be modified is suitably B 4#) or dextrans (Dextran T70), and polyalanine or poly - 14 - 1 8 22 7 protected initially by acetylation of its amino groups and the acetylated polypeptide is then conveniently conjugated to the modifier, preferably in the presence of a guanidine, e.g. guanidine hydrochloride, and in the presence, 5 preferably of l-ethyl-3- (3-dimethylaminopropyl)carbo-diimide as the water-soluble carbodiimide activator. VJhere a polymerised sugar, such as Ficoll, is the modifier it is preferred that this first be treated with ethylene diamine to render the final coupling more efficient and 10 this treatment is conveniently effected in a solvent, such as saline and dioxane, and suitably at about room temperature and a pH of about 9 to 12, preferably 10-11. The reaction time may, for example, vary from ^/4 to 2 hours. The subsequent conjugation of the protected poly-15 peptide and the modifier may conveniently be carried out in a solvent, such as glycine methyl ester, and at a pH of, for example, 4 to 5, preferably 4.5 to 4.8. The reaction temperature is suitably room temperature and the reaction time may, for example, vary from 2 to 8 hours, usually 20 about 5 hours. Process c), which may be used with natural protein modifiers, such as serum proteins, e.g. homologous serum albumin, is based on a theory of Richards and Knowles [J. Mol. Biol. 37, 231(1968)] that commercial glutaric 25 dialdehyde contains virtually no free glutaric dialdehyde, - 15 - 182272 but rather consists of a complex mixture of polymers rich in a,p--unsaturated aldehydes. Upon reaction v/ith natural protein modifiers, these polymers form a stable bond through the free amino group, leaving aldehyde groups 5 free. This intermediate product may then be reacted with the unmodified polypeptide in the presence of an alkali metal borohydride, such as sodium borohydride. The intermediate is suitably formed at pH 7-10, preferably 8-9 and at about room temperature. The subsequent conugation 10 is then conveniently effected at about room temperature and for a period of, for example, 1/4 to 2 hours. Process d), which may be employed with polymerised sugar modifiers such as sucrose copolymers and with epichlorohydrin, e.g. Ficolls, e.g. Ficoll 70 or 400, 15 or polyglucoses, e.g. Dextran T70, is effected by reaction of the modifier with a cyanuric halide, e.g. cyanuric chloride, e.g. at a temperature of 0° to 20°C, suitably in a solvent such as dimethyl formamide and conveniently for 1/2 to 4 hours. The resulting dihalotriaziny1 inter-20 mediate is conveniently dialysed until essentially halide ion free, and lyophilised and treated with the polypeptide, suitably at pH 8-11, preferably pH 9-10, and at room temperature, and conveniently for 1/2 to 12 hours. - 16 - 182272 Process e), which is also applicable for polymerised sugar modifiers, e.g. copolymers of sucrose with epichlorohydrin, e.g. Ficolls, e.g. Ficoll 70 or 400, or polyglucoses, e.g. Dextran T70, may conveniently be effected by treatment of the modifier v/ith an alkali metal periodate, e.g. sodium periodate, at a temperature of 30° to 60°C and a pH of 3 to G. The resulting intermediate is suitably reacted v/ith the polypeptide at a pH of about 7-11, preferably 0-10, and conveniently at a temperature of from 15° to 80°C, preferably 20° to 60°C. The reaction time may, for example, vary from V2 to 4 hours. The resulting product is suitably reduced with an alkali metal borohydride, e.g. sodium borohydride. The resulting modified polypeptides may be isolated and pu.rified using conventional techniques, such as gel .filtration r column chromatography f or dialysis and lyophilisation. Prior to conjugation (modification) 1 25 picogram amounts of I" labeled polypeptide may be added as a tracer to the reaction mixture. The quantity of polypeptide conjugated to modifying groups (molar ratio) may then be determined from the amount of radio-activity recovered. It will be appreciated that the methods a) to e), described above, may be employed for modification of any protein repreoductive hormones or fragments thereof, in - 17 - 1^72 particular those described above, to produce antigens for use in immunological contraception. As described above, the production of antigens for use in fertility control in accordance with our earlier 5 /Wplicarfejror» and the present invention can be accomplished by attaching one or more modifying groups to the selected polypeptide or attaching two or more polypeptide molecules to a modifying group. A further aspect of the present invention 10 provides antigens for the immunological control of fertil ity comprising a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or a fragment thereof, and the chemical modification comprising 15 (ii) polymerisation of the unmodified polypeptide via an appropriate bifunctional agent, or (iii) dimerisation of the unmodified polypeptide via a sulphur-sulphur bridge formed from the thiol group of a cysteine residue present in the 20 unmodified polypeptide or added to the unmodified polypeptide where this does not contain a cyst" eine residue. - 18 - 1 82272 118> The antigens of embodiment (ii) may be formed by polymerisation of the unmodified polypeptide with an appropriate bifunctional reagent, such as a bifunctional imidoester, e.g. dimethyl adipimidate, dimethyl suberimid-5 ate, or diethyl malonimidate, in conventional manner [e.g. Hartraann and Wold, Biochem. 6_, 2439 (1967)]. The polymerisation may thus be effected at room temperature in aqueous solvent and suitably at a pH of 9-12, preferably 10-11, and conveniently for a period of 1/4 to 2 hours. 10 The antigens of embodiment (iii) may be formed by oxidation of the thiol group in a cysteine i odos o -z o\c_ , residue, for example using /ie&ebcngoie acid, in known manner, e.g. room temperature reaction for about 10 to 40 minutes. Where the polypeptide to be modified possesses i 15 no cysteine residue, this may first be joined to the polypeptide in conventional manner. It will be appreciated that the chemical modification of embodiments (ii) and (iii) above may be applied to any protein reproductive hormone or fragment 20 thereof, in particular, however, those described above, for the purpose of producing antigens for use in immunological fertility control. The antigens of the present invention may suitably be admixed with a pharmaceutically acceptable - 19 - 182272 liquid carrier and administered parenterally. The dosage to be administered will of course, vary depending on various factors. However, in general, unit doses of 0.1 to 50 mg are indicated, suitably administered one to five 5 times at intervals of one to three weeks. The following Examples illustrate the invention. ~ 20 - 2 EXAMPLE 1: ' Structure X - Keyhole limpet hemocyanin [Process a) ] Hemocyanin from Keyhole limpet (KLH) solution (7 mg/ml) in 0.05 M sodium phosphate buffer in 0.2 M NaCl, pH 7.5, is prepared. Insoluble particles are removed by centrifugation. To 1 ml of this solution, tolylene di-isocyanate (TDIC) reagent is added (20 ,ul) diluted to 1/30 with dioxane, the amount being essentially the equivalent of the moles of lysyl residues in the KLH molecules. After 40 minutes at 0°C, the TDIC activated KLH solution is combined with 0.5 mg of synthetic 3-HCG peptide identified hereinabove as Structure X which is first dissolved in 25 jal of 0.05 M sodium phosphate buffer in 0.2 M NaCl, pH 7.5. The mixture is incubated at 37°C for four hours. The resulting product is purified by gel filtration. EXAMPLE 2: Structure III - Bovine gamma globulin [process a)] To an ice water bath cooled and vigorously stirred,0.23 ml of bovine gamma globulin (10 mg/ml) in 0.05 phosphate buffer with normal saline (PBS) pH 7.5, 50 ;ul 21 \1TI~12- of 1/10 TDIC in dioxane is added. After 40 minutes, the excess TDIC is removed by centrifugation (0°C, 10 minutes, 10,000 g) and the precipitate is washed twice with 0.1 ml of PBS. The combined supernatants are added to 7.7 g of the peptide of structure III dissolved in 0.8 ml of PBS, pH 7.5. The mixture is stirred at room temperature for 10 minutes, then .incubated at 37°C for 4 hours. The conjugate product is purified by dialysis. EXAMPLE 3: Structure VI - Bovine serum albumin [process a)] Bovine serum albumin (10 mg/ml) in PBS solution (0.25 ml) is treated v/ith 50 ;ul of 1/10 TDIC dioxane solution and conugaged to 7.5 mg of synthetic 0-HCG peptide of structure VI in 0.8 ml of PBS (pH 7.5) as in Example 2 to obtain the product. EXAMPLE 4: Structure VI - Poly (D , L-Lys-p. L-Ala) ["process a)] To an ice water bath cooled and vigorously stirred 0.6 ml of 3-HCG peptide of structure VI (10 mg/ml) in phosphate buffered saline, pH 7.5, is added 30 jil of 1/10 TDIC in dioxane. After 40 minutes, the excess TDIC is removed by centrifugation (10,000 g, 0°C, 10 minutes) and the precipitate is washed twice with 0.1 ml PBS. The combined supernatants are added to 3 mg. of ppl^j_ * jsmrofTOE . (D,L-Lys-D,L-Ala) dissolved in 0.3 ml. of PBS. * - 22 - 'J SEP 1978 1 8 22,7 2 118>3^9/A The mixture is incubated at 37°C for 4 hours. The product is then dialysed and lyophilised. EXAMPLE 5: Structure III - Keyhole "limpet hemocyanin [process b)] 5 Tv/o mg of the compound of Structure III con- 125 taining picogram amount of I labeled adduct and KLH (1.6 mg) is dissolved in 1 ml of 1.0M glycine methyl ester in 5 M guanidine hydrochloride. Ethyl dimethyl amino propylcarbodiimide (EDC) 19.1 mg is added to this LO solution. The acidity is adjusted to and maintained at pH 4.75 with 1 N HC1 at room temperature for 5 hours. The KLH-peptide conjugate is purified by passing it through a Bio-Gel p-60 2.2 x 28 cm column equilibrated with 0.2 M NaCl. 15 Ficoll 70 - Ethylenediamine [Starting material - process b)] One g of Ficoll 70 is dissolved in 1 ml each of normal saline and 2 M ethylene diamine (adjusted to pH 10 with hydrochloric acid) solution. The solution is kept at room temperature in a water bath and stirred 20 with a magnetic stirrer. Cyanogen bromide 4 g, dissolved in 8 ml of dioxane, is added to the Ficoll 70 solution. The acidity of the mixture is maintained at pH 10 - 10.5 - 23 - 10 15 for 8 minutes by adding drops of 2 N sodium hydroxide solution. An additional 2 ml of 2 M ethylene diamine, pH 10, solution is added, and stirring at room temperature is continued for 30 more minutes. The product is purified by passing it through a Bio—Gel p—60 column. This material can be used to couple Ficoll 70 to polypeptides in accordance with process b). EXAMPLE 6: Structure IX - Homologous Serum Albumin [process c)] To a 20 mg/ml solution of homologous serum albumin in 0.1 M borate buffer, pH 8.5, 1000% mole excess of 2 5% aqueous solution of glutaric dialdehyde is added at room temperature. The excess dialdehyde is removed by gel filtration in water using Bio-Gel p-2. The material collected at the void volume is lyophilised, and the dried product is redissolved in 0.1 M borate buffer, pH 8.5 (20 mg/ml), mixed with the required amount of polypeptide of Structure IX (20 mg/ml) in the same buffer at room temperature. Twenty minutes later, sodium borohydride in 250 percent molar excess of polypeptide-IX is added. The reaction is terminated after one hour. The conjugated product is purified by gel filtration on Bio-Gel p-60 column, dialysed free of salt and lyophilised - 24 - 18 227 2 HJ^l29/A EXAMPLE 7: Structure X - Ficoll' 70 [process d)] Ficoll 70 lg, NaHCO^ 500 mg, cyanuric chloride 3 g, H20 20 ml, and dimethylformamide 80 ml, are stirred at a temperature below 16°C for 2 hours. The product is dialysed against distilled water until Cl-free, then lyophilised. A polypeptide of Structure X (2 mg) contain- 125 ing a minute quantity of I -labeled analogue is incubated with 1 mg of this product in 0.2 5 ml of 0.2 M' sodium borate buffer, pH 9.5, for one hour at 20°C, and the product is recovered from a Bio-Gel p-60 2.2 x 28 cm column. When the above procedure is carried out and Dextran T70 is used in place of Ficoll 70, the corresponding modified polypeptide is obtained. EXAMPLE 8: Structure X - Ficoll 70 [process e)] Ficoll 70 lg, NalO^ 1.2 g, and KC1 0.42 g are dissolved in 1.5 ml of 1 M sodium acetate buffer, pH 4.5, and incubated at 37°C for 1 hour. Two mg (= 588 ;umoles) of polypeptide of - 125 Structure X above mixed with a minute quantity of I labeled analogue is incubated v/ith 2 mg of the product obtained above in 0.3 ml of 0.2 M borate buffer, pH 9.5 - 25 - 182272 118-3-3*29/A at 55°C for 1 hour. The reaction mixture is then chilled in an ice water bath and NaBH^ 1 mg is then added into this solution. The reduction reaction is terminated by passing the product through a Bio-Gel p-60 2.2 x 28 cm column equilibrated and eluted with 0.2 M NaCl. EXAMPLE 9: Structure III - Polymer Solid bifunctional imidoester dihydrochloride i (3 mole) is added in 2 mg portions at 5 minute intervals to a constantly stirred solution of 1 mole of polypeptide of structure III (1-20 mg/ml) in 0.1 M sodium phosphate, pH 10.5, at room temperature. Sodium hydroxide 0.1 N is added to maintain the acidity at pH 10.5. One hour after the addition of the diimidoester has been completed, a polymerised product according to this invention is obtained. EXAMPLE 10: Structure V or X dimer Iodosobenzoic acid dissolved in a slight excess of 1 N potassium hydroxide in 10% molar excess is added to the peptide structure V or X in phosphate buffer with normal saline at pH of 7.0. After thirty minutes at room temperature, the product polypeptide dimer is purified by gel filtration. - 26 - 1 8 22 72 118>3^29/A EXAMPLE 11; Test' Results Numerous rabbits are immunised with a variety of synthetic peptides conjugated to different modifying groups. Following two or three immunisations at 3 - 5 5 week intervals, sera from animals are assessed by determining their ability to bind in vitro to radiolabeled HCG. ,The specificity of this binding is studied by reacting the same sera against similarly labeled other protein hormones, particularly, pituitary LH. Sera are 10 further assessed by determining their ability to inhibit the biological action of exogenously administered HCG in bioassay animals. Thus, the increase in uterine weight of the immature female rat in response to a prescribed dose of HCG is noted. The dose of HCG is administered 15 subcutaneously in saline in five injections over a three- day period and the animal is sacrificed for removal of the uterus on the fourth day. The weight of the uterus increases in dose-response fashion to the hormone injections. When assessing the effects of antisera in 20 this response, varying quantities of test serum are admini stered intraperitoneally separately from the subcutaneous injection of hormone during the assay. This procedure permits the antiserum to be absorbed rapidly into the - 27 - 1 8 22 7 118->329/A rat's blood-stream and will permit interaction of it with hormone when the latter likewise enters this fluid. If the antiserum is capable of reacting with the hormone in a manner preventing stimulation of the uterus, the anti-5 serum is considered to be effective for biological inhib ition of hormone action. The frequency of animals showing a positive response to immunological binding and neutralisation of biological activity is presented in the following Table. The results set out in the following Table provide further evidence of the broad applicability of this invention as indicated previously in this specification. Using standard methods of testing in rabbits, 15 both immunological binding response and neutralisation of biological activity were established for the modified polypeptides indicated with the result as set out below: - 28 - </div>

Claims

<div class="application article clearfix printTableText" id="claims"> • I # Frequency of Positive Antibody Responses to Various HCG Peptide - Conjugates Number of Rabbits Smmunoloaicai Binding Responses Neutralization of Biological Attivity Peptide Carrier Immunized 35 amino acid 111-145 Bovine Gamma Globulin Morgan et al Keyhole Limpet Peptide 111 Hemocyanin io 10 10 • 5 6 & . 31 amino acid 115-145 Poly-D-L-Alanine Morgan et al Bovine Serum Albumin Peptide VI 10 1 2 9 12 5 6 44 amino acid 105-148 Keyhole Limpet ■ Hemocyanin Peptide X 10 8 & Natural 109-145 Keybole Limpet Keutman Hemocyanin Peptide V 10 10 & ^ additional tine needed for assessment 18227,'' L WHAT WE CLAIM IS: 1. An antigen,for the immunological control of fertility^which comprises a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or a fragment (as herein defined) thereof, and the 5 chemical modification comprising (i) the attachment of one or more modifying groups to the unmodified polypeptide, or the attachment of two or more unmodified polypeptide molecules to a modi-fying group, provided that the modifying groups are 10 derived from polymerised sugars, serum proteins, keyhole limpet hemocyanin, marihe gastropod mollusc herrocyanin,1 viruses or bovine gamma globulin, when the unmodified polypeptide is other than of structure VII, VIII, IX or X, 15 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 VII) Asp-Asp-Pro~Arg-Phe-Gln-/^.sp-Ser-Ser-Ser-Lys-Ala-20 pro-Pro-Pro-Ser-Leu-Pro-Ser-Gly (structure VIII) 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-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro ^ 1 5 SEP\978 * (structure IX) i ■ • - - 30 - 182272 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-Pro-Asp-Thr-Pro-lie-Leu-Pro-Gin-Ser-Leu-Pro (structure X) or (ii) or (iii and the chemical modification being such as to permit the chemically modified polypeptide to induce formation of antibodies which biologically neutralize the unmodified polypeptide as well as the chemically modified polypeptide. 2. An antigen for the immunological control of fertility which comprises a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or a fragment (as herein defined) thereof, and the chemical modification comprising the attachment of one or more modifying groups to the unmodified polypeptide, or the attachment of two or more unmodified polypeptide molecules to a modifying group, provided that the modifying groups are derived from polymerised sugars, serum proteins, keyhole limpet hemocyanin, marine gastropod mollusc hemocyanin, viruses or bovine gamma globulin, when the unmodified polypeptide has a structure other than of formula VII, VIII, IX or X, stated in Claim 1, and the chemical modification being such as to permit the chemically modified polypeptide to induce formation of antibodies which biologically neutralize the unmodified polypeptide as well as the chemically modified polypeptide. po)\$r*\e.V jplymoriGation of the unmodified polypeptide via an appropriate bifunctional reagent; dimerisation of the unmodified polypeptide via a sulphur-sulphur bridge formed from a cysteine residue present in the unmodified polypeptide or added to the unmodified polypeptide if it does not contain a cysteine residue, N-Z- PATENT OFFICE 15 SEP 1978 -182272 3. 7m antigen according to Claim 2, in which 1 to 40 modifying groups are attached per molecule~-of polypeptide. 4. /iii antigen according to Claim 2 or 3, 5 in which 2 to 40 modifying groups are attached per molecule of polypeptide. 5. An antigen according to any one of Claims 2 to 4, in which 5 to 30 modifying groups are attached per molecule of-polypeptide. 10 6. An antigen according to Claim 2, in which 2 or more polypeptide molecules are attached to the modifying group. 7. An antigen according to any one of Claims 2 to 6, in which the unmodified polypeptide is 15 of structure VII, VIII, IX or X, stated in Claim 1. 8. An antigen according to Claim 7, in which the modifying groups are diazo groups. 9. An antigen according to Claim 8, in which the diazo groups are introduced by reaction with diazo- 20 sulfanilic acid. 10. An antigen according to Claim 7, in which the modifying groups are derived from dinitrophenol, trinitrophenol, S-acetomercaptosuccinic anhydride, a polytyrosine, a polyalanine, biodegradable polyaextran 25 or thyroglobulin. jsl 2. patent OFFICE 15 SEP 1978 . 32 . 182272 11. An antigen according to any one of Claims 2 to 6, in which the modifying groups are derived from polymerised sugars, serum proteins, keyhole limpet hemocyanin, marine gastropod mollusc hemocyanin, viruses, or bovine gamma globulin. 12. An antigen according to Claim 11, in which the polymerised sugar is a copolymer of sucrose with epi-chlorohydrin, or a polyglucose, the serum protein is homologous or bovine serum albumin, and the virus is influenza virus Type A, B or C, or poliomyelitis virus, live or killed, Type,*; 1, 2 or 3. 13. An antigen according to Claim 12, in which the copolymer of sucrose with epichorohydrin is Ficoll 7 0 or 400 (as herein defined)., and the polyglucose is Dextran T70 (as herein defined). 14. An antigen according to Claim 11, in which the modifying groups are derived from Ficoll 70 (as herein defined), homologous or bovine serum albumin or keyhole limpet hemocyanin. 15. An antigen according to any one of Claims 11 to 14, in which the unmodified polypeptide is Follicle Stimulating Hormone, Luteinising Hormone, Human Placental Lactogen, Human Prolactin, Human Chorionic Gonadotropin or a fragment (as herein defined) of any of these. N.Z. PATENT OFFICE 15 SEP 1978 > ; 182272 16. An antigen according to Claim 15, in which the unmodified polypeptide is Human Chorionic Gonadotropin or a fragment (as herein defined) thereof. 17. An antigen according to Claim 16, in which the unmodified polypeptide is Human Chorionic Gonadotropin. 18. An antigen according to Claim 16, in which the unmodified polypeptide is the ft-subunit of Human Chorionic Gonadotropin. 19. An antigen according to Claim 16, in which the poly pe.pi~« de. unmodified Apoylpcptide is a 20 to 30 amino acid C-terminal fragment of the 3-subunit of Human Chorionic Gonadotropin. 20. An antigen according to Claim 16, in which the unmodified polypeptide is a 30 to 39 amino acid C-terminal fragment of the B-subunit of Human Chorionic Gonadotropin. 21. An antigen according to Claim 16, in which the unmodified polypeptide is of structure VII, VIII, IX or X, stated in Claim 1, or of structure III, IV, V or VI (* indicates site locations of carbohydrate moieties and "Asx" represents either Asp or Asn), * 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- •k Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln (Structure (III) - 34 - ISZ272 * * 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-Gin (Structure IV) * 5 Thr-Cvs-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Lys- * * Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro (Structure V) k Phe-Gln-Asp-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-* * 10 Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro- Ile-Leu-Pro-Gln-Ser-Leu-Pro (Structure VI). 22. An antigen according to Claim 21, in which the unmodified polypeptide is of structure IX or 15 X, stated in Claim 1, or III or VI, stated in Claim 21. 23. An antigen according to Claim 22, in which the unmodified polypeptide is of structure III, stated in Claim 21. 24. An antigen for the immunological control 20 of fertility which comprises a polypeptide of structure X, stated in Claim 1, chemically modified by the attachment of one or more modifying groups derived from keyhole limpet hemocyanin. - 35 - I E2Z72 25. An antigen for the immunological control of fertility which comprises a polypeptide of structure III, stated in Claim 21, conjugated to keyhole limpet hemocyanin. 5 26. An antigen for the immunological control of fertility which comprises a polypeptide of structure IX, stated in Claim 1, conjugated to homologous serum albumin. 27. An antigen for the immunological control 10 of fertility which comprises a polypeptide of structure X, stated in Claim 1, conjugated to Ficoll 70 (as herein defined) or Dextran T70 (as herein defined). 28. An antigen for the immunological control of fertility comprising a polypeptide of structure VI, 15 stated in Claim 21, conjugated to bovine serum albumin. 29. A process for the production of an antigen for the immunological control of fertility comprising chemically modifying a polypeptide which is a protein reproductive hormone or a fragment (as herein defined) w*-r 20 thereof/ j/j/K < _ 36 _ J?/ ■ mm a) by attaching to the unmodified polypeptide modifying groups derived from modifiers having free amino groups by activation of the modifier or polypeptide to be modified by reaction v/ith tolylene di-isocyanate and reaction of the resulting activated product v/ith the polypeptide to be modified , or modifier, respectively; or b) by attaching to the unmodified polypeptide modifying groups - derived from modifiers having free amino groups or being polymerised sugars by conjugation of the unmodified polypeptide with the modifier employing a water-soluble carbodiimide as activating agent; or c) by attaching to the unmodified polypeptide modifying groups derived frou modifiers having free amino groups by reaction of the modifier with glutaric-dialdehyde and reaction of the resulting product v/ith the polypeptide to be modified, in the presence of an alkali metal borohydride; or d) by attaching to the unmodified polypeptide modifying groups-. derived from polymerised sugars by \x. 182272 reaction of the polymerised sugar with a cyanuric halide, and reaction of the resulting dihalotriazinyl adduct with the polypeptide to be modified; or e) by attaching to the unmodified polypeptide modifying groups derived from polymerised sugars by treatment 6f the sugar with an alkali metal periodate and reaction of the resulting product with the polypeptide to be modified. 30. A process for the production of an antigen for the immunological control of fertility comprising chemically modifying a polypeptide which is a protein reproductive hormone or a fragment (as herein defined) thereof by attaching to the unmodified polypeptide modifying groups derived from modifiers having free amino groups .by activation of the modifier or polypeptide to be modified by reaction with tolylene di-isocyanate and reaction of the resulting activated product with the polypeptide to be modified or modifier, respectively. 31. A process according to Claim 30, in which a solution of tolylene di-isocyanate is added to a buffered solution of the modifier, the mixture allowed to react at -5° to +10°C, and the resulting activated modi- 1sSE?197S ' 182272 fier is added to a buffered solution of the polypeptide to be modified and the mixture allowed to react at 30° to 50 °C. 32. A process according to Claim 30 or 31, 5 in which the modifier is a serum protein, keyhole limpet hemocyanin or a polytyrosine, polvalanine, bovine gamma globulin or thyroglobulin. 33. A process according to any one of Claims 30 to 32, in which the polypeptide is as stated in any 10 one of Claims 15 to 23. 34. A process according to any one of Claims 30 to 33, in which the modifier is keyhole limpet hemocyanin and the polypeptide is of structure X, stated in Claim 1. 15 35. A process according to any one of Claims 30 to 33, in which the modifier is bcvine gamma globulin and the polypeptide is of structure III, stated in Claim 21. 36. A process according to any one of Claims 20 30 to 33, in which the modifier is bovine serum albumin and the polypeptide is of structure VI, stated in Claim 21. 37. A process according to Claim 30 or 31, in which the modifier is poly (D,L-Lvs-D,L-Ala) and the 25 polypeptide is of structure VI, stated in Claim 21. " 39 - 182 38. A process according to any one of Claims 30 to 37, substantially as herein described with reference to any one of Examples 1 to 4. 39. An antigen whenever produced by a process 5 according to any one of Claims 30 to 38. of fertility comprising a polypeptide of structure IIIr stated in Claim 21, conjugated to bovine gamma globulin. 10 of fertility comprising a polypeptide of structure VI, stated in Claim 21, conjugated to poly (D,L-Lys-D,L-Ala). for the immunological control of fertility comprising chemically modifying a polypeptide which is a protein 15 reproductive hormone or a fragment Xas herein defined) thereof by attaching 40. An antigen for the immunological control 41. An antigen for the immunological control 42. A process for the production of an antigen \ 5 1822 to the unmodified polypeptide modifying groups derived from modifiers having free amino groups or being polymerised sugars by conjugation of the unmodified polypeptide with the modifier emplpying a v/ater-soluble carbo diimide as activating agent. 43. A process according to Claim 42r in which the free amino groups of the polypeptide to be modified are protected prior to conjugation with the modifier and the protecting groups are removed subsequent to conjugation. 44. a process according to Claim 42 or 43 , in which, when the modifier is a polymerised sugar, this is treated v/ith ethylene diamine prior to conjugation with-the polypeptide to be modified. 45. A process according to any one of Claims 42 to 44, in which the v/ater-soluble carbodiimide is 1 — ethyl-3- (3--dimethylaminopropy 1) carbodiimide. 46-. A process according to any one of Claims 42. to 45, in which the conjugation is effected in the presence of guanidine and at a pH of from 4 to 5. 47. A process according to any one of Claims 42'. to 46, in v/hich the modifier is keyhole limpet hemocyanin, a serum protein, a polymerised sugar, or a poly-alanine or polytyrosine. I 62272 48. A process according to Claim 47, in which the serum protein is homologous serum albumin, and the polymerised sugar is Ficoll 70 or 400 (as herein defined) or Dextran T70 (as herein defined) . 49. A process according to any one of Claims 42 to 48,:.in which the polypeptide is as stated in any one of Claims 15 to 2 3. 50. A process according to any one of Claims 42 to 47, in which the modifier is keyhole limpet hemocyanin and the polypeptide is of structure III, stated in Claim 21. 51. A process according to Claim 42, substantially as herein described with reference to Example 5. 52. An antigen whenever produced by a process according to any one of Claims 4 2 to 51. 53. A process for the production of an antigen for the immunological control of fertility comprising chemically modifying a polypeptide which is a protein reproductive hormone or a fragment (as herein defined) thereof by attaching to the unmodified polypeptide modifying groups derived from modifiers having free amino groups by reaction of the modifier with glutaric dialdehyde and reaction of the resulting product with the polypeptide to be modified, in the presence of an alkali metal borohydride. - 42 - v/> 182272 54.. A process according to Claim 53,. in which the reaction of the modifier with glutaric . _ - -dialdehyde is effected at a pH of 7 to 10, and the conjugation of the resulting product with the polypeptide is effected in the presence of sodium borohydride. 55. A process according to Claim 53 or 54, in which the modifier is a serum protein. 56. A process according to any one of Claims 53 to 55, in which the polypeptide is as stated in any one of Claims 15 to 23. 57. A process according to any one of Claims 53 to 56, in which' the modifier -is_ homologous serum albumin and the polypeptide is of structure 1.&, stated in Claim 1. 58. A process according to Claim .53, substantially as herein described with reference to Example 6. 59. An antigen whenever produced by a process according to any one of Claims 53 to 58; 60. A process for the production of an antigen for the immunological control of fertility comprising chemically modifying a polypeptide which is a protein reproductive hormone or: a fragment (as herein defined) thereof by-attaching to the unmodified polypeptide modifying groups^ /T7 W*"-"?"'*'""'TIT*'." 1ftf. \mii derived from polymerised sugars by reaction of the polymerised sugar with a cyanuric halide, and reaction of the resulting dihalotriaziny1 adduct with the polypeptide to be modified. 5 61i- A process according to Claim 60,-. in which the reaction of the modifier with a cyanuric halide is effected at a temperature of 0° to 20°C in a solvent, and the resulting adduct is reacted with the polypeptide at pH 8-11. 10 62. A process according to Claim 60 or 61,.:,. in which the cyanuric halide is cyanuric chloride. 63,_ A process according to any one of Claims 60; to 62, in which the modifier is a copolymer of sucrose with repichlorohydrin or a polyglucose. 15 64. A process according to any one of Claims herein, 60 to 63, in which the modifier is Ficoll 70 or 400 (as/defined) or Dextran T70, (as herein defined). 65. A process according to any one of Claims 60 to 64', in which the polypeptide is as stated in any 20 one of claims 15 to 23. 66. A process according to any one of Claims 60..to 65, in which the modifier is Ficoll 70 (as herein defined)- and the polypeptide is of structure X, stated in Claim 1. - 44 - ux patent I 15 SEP 1978 rh<t;ved 182272 67. A process according, to any one of Claims .60 to 65, in which the modifier is Dextran T70 (as herein defined) and the polypeptide is of structure X, stated in Claim 1. 68., A process according to Claim 60,- substantially as herein described with reference to Example 7. 69,. An antigen whenever produced by a process according to any one of Claims 60 to 68 . 70. A process for the production of an antigen for the immunological control of fertility comprising chemically modifying a polypeptide which is a_ protein reproductive hormone or a fragment (as herein defined)' thereof by attaching to the unmodified polypeptide modifying groups derived from polymerised sugars by treatment of the sugar v/ith an alkali metal periodate and reaction of the resulting product with the polypeptide to be modified. 71. A process according to Claim 70^ in which the modifier is treated with the periodate at a temperature of. from 30° to 60°C and a pH of 3 to 6 and the resulting product is reacted with the polypeptide at a pH of 7 to 11 and a temperature of 15° to 80°C. 72., A process according to Claim 70 or 71 . in which the periodate is sodium periodate. 73. A process according to any one of Claims 70 to 72^ in v/hich the modifier is a copolymer of sucrose MZ- P&TEOT" OFRCE -45- 15S£P197M . RECfiVED '182272 with epichlorohydrin or a polyglucose. 74. A process according to any one of Claims 70 to 73, in which the modifier is Ficoll 70 or 400 (as herein defined) or Dextran T70.(as herein defined). 75.- A process according to any one of Claims 70_ to 74, in which the polypeptide is as stated in any one of Claims 15 to 23. 76.- A process according to any one of Claims 70- to 75, in which the modifier is Ficoll 70 (as herein defined) and the polypeptide is of structure X, stated in Claim 1. 77... A process according to Claim 70/.substantially as herein described with reference to Example 8. 78. An antigen whenever produced by a process according to any one of Claims 70. to 77. 79. An antigen for the immunological control of fertility which comprises a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or a "fragment (as herein"defined) thereof, and the -chemical modification comprising polymerisation of the unmodified polypeptide via an appropriate bifunctional reagent. 80. An antigen according to Claim 79^. in which the bifunctional reagent is a bifunctional imidoester. 81. An antigen according to Claim 79 or 80,-in which the bifunctional reagent is dimethyl adipimidate, - 46 - mx PAie<nr (QffTOS 15 SEP 1978 - RECtJVED dimethyl suberimidate or diethyl malonimidate. 82. An antigen according to any one of Claims 79 to 81/ in which the unmodified polypeptide is as stated in any one of Claims 15 to 23. 83- An antigen according to any one of Claims 79 to 82 , in which the unmodified polypeptide is of structure III, stated in Claim 21. 84. A process for the production of an antigen according to any one of Claims 79 to 83^ comprising reacting the unmodified polypeptide with the bifunctional reagent. 85. A process according to Claim 84, in which the reaction is effected at a pH of from 9 to 12. 86. A process according to Claim &4 , substantially as herein described v/ith reference to Example 9. 87. An antigen, whenever produced by a process according to any one of Claims 84 to 86. 88. An antigen for the immunological control of fertility which comprises a chemically modified polypeptide, the unmodified polypeptide being a protein reproductive hormone or"a fragment (as herein defined) thereof, and the chemical modification comprising dimerisation of the unmodified polypeptide via a sulphur-sulphur bridge formed n 7 PATENT OFFlCgj 15 SEP 1978 RECEIVED I 82272 from a cysteine residue present in the unmodified polypeptide or added to the unmodified polypeptide if it does not contain a cysteine residue. 89. An antigen according to Claim 88, in 5 which the unmodified polypeptide is as stated in any one of Claims 15 to 23. 90. An antigen according to Claim 83" or 89 , in which the unmodified polypeptide is of structure V, stated in Claim 21, or X, stated in Claim 1. 10 91. A process for the production of an anti gen according to any one of Claims 88 to 90 , comprising oxidising the thidl group in a cysteine residue present in or added to the unmodified polypeptide. 92. A process according to Claim 91, in which 15 the oxidation is effected v/ith iodosobenzoic acid. 93 _ A process according to Claim gir. substantially as herein described v/ith reference to Example 10 . 94. An antigen, whenever produced by a process 20 according to any one of Claims 91 to 93. 95. A pharmaceutical composition comprising an antigen according to any one of Claims 1 to 28, 39 to 4 1, 52, 59, 69, 78-to 8 3, 87, 90 and 94, in association with a liquid pharmaceutically acceptable carrier. DT;I!S/S« DAY OF A, j. PARK & SON AGENTS FOR THE APPLICANTS - 48 - </div>