CA1264000A - Antigenic modification of polypeptides - Google Patents

Abstract

ANTIGENIC MODIFICATION OF POLYPEPTIDES
Abstract Modified polypeptides capable of provoking the formation of antibodies in an animal may be produced by forming a linear polymer of polypeptide fragments, each having a molecular structure similar to a fragment of the protein to which antibodies are to be provoked. Such linear polymers can be made more immunogenicthan the proteins to which they are related without introducing undesirable extraneous materials into the animal being treated, but have reproducable immunogenic properties. Proteins which are not endogenous or immunogenic to an animal can be chemically modified so as to make them more immunogenic. Also, modified antigens useful for fertility control can be produced by chemical modi-fication of zona pellucida or sperm antigens. These modified polypeptides, antigens and modified antigens are desirably administered in the form of a vaccine having a vehicle comprising a mixture of mannide monooleate with Squalene and/or Squalene.

Description

~2~ V{) A~TIGENIC MODIFICATION OF POLYPEPTIDES

This invention relates to antigenic modification of polypeptides. More particularly, this invention relates to specific techniques for modification of polypeptides, to antigens for provoking the formation, by the immune system of an animal, of antibodies to exogenous proteins e.g. proteins found in viruses and other 05 pathogenic organisms, and to processes for fertility control using chemically-modified antigens.
In our British patent No. 1,567,764 and in our U.S. patent No. 4,302,386 it is disclosed that hormones or other proteins found naturally in an animal can be chemically modified 10 outside the body of the animal (a term which is used herein to include human beings) so that, when injected into the animal, the modified hormones or other proteins provoke the formation of antibodies which react not only with the chemically-modi~led hormone or other protein but also with the unmodified hormone or other protein found naturally in the animal, thereby reducing the level of the natural15 hormone or other protein in the body of the animal. Instead of using the entire hormone or other protein, the aforementioned patents also disclose the use of natural or synthetic fragments of such proteins in this so-called "isoimmlmization~' technique.
The aforementioned patents are mainly concerned with the application of their 20 isoimmunoization technique in controlling fertility, so that the protein which is modified is a reproducti~re hormone, such as Follicle Stimulating Hormone (FSH),Leutinising Hormone (LH), Human Placental Lactogen (llPL), ~luman Prolactin and Human Chorionic Gonadotropin (HCG), or a peptide having an amino acid sequence corresponding to a part of one of these hormones. However, the aforementioned 25 patents also disclose the applicability of the isoimmunization technique to other protein hormones, for example:
1. Gastrin, for the treatment of Zollinger-Ellison Syndrome;

2. Angiotension 11, for the treatment of hypertension;
~ 3. Growth hormone and somatomedian, for the treatment of diabetes and associated micro and macro vascular diseases;
4. Parathyroid hormone for the treatment of kidney stones;
5. Insulin and glucagon, for the treatment of hyperinsulinoma;
6. Thyroid stimulating hormone, for the treatment of hyperthyroidism;
7. Secretin, for the trea~ment Or irritable bowel syndrome.

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The ~forementioned patents also disclose the use of rnodified polypeptides derived from chorionic gonadotropin, LH or FSH, or a frugment thereof, for use in treating certain carcinomas.
The main technique of chemical modification disclosed in the aforementioned ûS patents is coupling of the hormone, other than protein or fragment thereof to a large ~'carrier" molecule such as diphtheria toxoid, tetanus toxoid or a synthetic polymer; these carrier molecules are not endogenous to the animal to be treated.The uforementioned patents also describe polymerization of the hormone, other protein, or frsgment thereof by reaction with a bifunctional organic reagent (for 10 example a bi-functional imidoester such as dimethyl adipimidate, dimethyl sube~
imidate or diethyl malonimidate) or dimerization by oxidation of a thiol group to form a disulfide bridge. All these modes of chemical modification have dis-advantages. Dimerization of the hormone, other protein or fragment ViQ a disulfide bridge does not introduce exogenous material into the animal being treated but, 15 since the chemically-modified antigen udministered to the ~nimal is only a dimer of a hormone, other protein or fragment which is not itself immunogenic to the animal, such dimers are rarely successful in provoking useful levels of antibodies. The modified polypeptides produced using carriers contain a very high proportion of non-endogenous materiul, and will usually provoke the formation of substantial quan-20 tities of an~ibodies to the carrier as well as to the hormone, other protein orfra~nt ther~of. Altho~h the forrnation of antibodies to the carrier may sometimes be useful (for example, a vaccine based upon 8 HCG frugment coupled todiphtheria toxoid and intended for fertility control has the incidental advantage ot also conferring protection against diphtheria), there are mnny occasions upon which 25 it is not desirable to provoke the formation of relatively large quantities of antibodies to the carrier; for example, if one wishes to use a vaccine containing e modified polypeptide to tre~t B patient with a c~rcinoma or a serious viral infection, it may be desirable to avoid overstraining t~le patient's immune system by ch011enging it not only with the hormone, other protein or fragment thereof to which 30 antibodies ure desired, ~ut slso with the carrier.
In theory, perhups the most promising of the modification techniques disclosed in the aforementioned patents is polymerization of the hormone, other protein orfragment thereof by reaction with a bifunctional reagent. This technique has, intheory, the advuntages of introducing a relutively small proportion Or non-35 cndogenous ma~erial into the animal bcing treuted (and even this relatively small ~s proportion of non-endogenous materinl can bc choscn so that it is not strongly o~

immunogenic), while still providing a modified polypeptide large enough to be stron~ly immunogenic. Unfortunately, experiments have proved that straight-forward application of the bifunctional org~nic reagent polymerization technique to most hormones, other proteins or fragments thereof of practical interest produces 05 very complicated mixtures of modified polypeptides having correspondingly com-plicated immunogenic properties. Furthermore, the immunogenic properties of the polymerized polypeptides thus produced are not readily reproduceable, but such reproduceability is essential in any material intended for pharmaceutical use, since the necessary tests of safety and efficiency cannot be performed on non-repro-10 duceable material.
We have now found (though this knowledge is not in the published literature)that the reason for the very complicated immunogenic properties and the lack of reproduceability present in polymers produced by the bifunctional organic reagent polymerization technique is that, notwithstanding the use of a bifunctional reagent, 15 extensive cross-linking of the hormone, other protein fragment thereof occurs, such crosslinking presumably being due to the presence of free amino, thiol, carboxyland perhaps other groups (the exact groups involved depending of course upon which groups the bifunctional organic reagent is designed to react with) at non-terminal positions on the hormone, protein or fragment thereof. Such cross-linking produces 20 branching and three-dimensional structure in the resultant polymers. Not only does the relatively random cross-linking thus produced render the structure of the polymers themselves unpredictable and non-reproduceable, but such cross-linking may well alter the tertiary structure and shape of the hormone, other protein orfragment thereof being polymerized, thus affecting its immunogenic properties.
Accordingly, we have concluded that to produs~e useful modified polypeptides by the bifunctional organic reagent polymerization technique, it is essential tooperate in such a way that coupling of the polypeptide fragments being polymerized occurs only at or near the terminals of the fragments, thus producing a true linear polymer substantially free of non-linear polymers of the fragments.
It has also been found that the isoimmunization technique described in the af orem entioned patent csn usefully be extended to proteins which are not endo-genous nor substantially immunogenic to the animal to be treated. Finally, it has been found that modified antigens for use in fertility control can be produced by chemical modification of zona pellucida or sperm antigens.
A~cordingly, in one aspect this invention defines a modified polypeptide for provoking the formation, in the body of an animal, of antibodies to a protein, the .` f:
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modified polypeptide being char~cterized in that it comprises a linear polymer ofpolypeptide fragments, each of the fragments, in its monomeric form, being substanti~lly non-immunogenic to the anim~l and having a molecular structure similar to Q frQgment of the protein to w~iich ~ntibodies are to be provoked, the 05 linear polymer, after administration into the body of the animal, having a gre~ter capacity to provoke the formation of the antibodies than the protein, the linetlr polymer being substantially free of non-linear polymers of the fragments.
In another aspect, this invention provides Q method for producing a line~r polymeric polypeptide for provoking the formation, in the body of an animQl, of 10 antibodies to one or more proteins which is substantially non-immunogenic to the animal, the method being characterized by:
(a) procuring a first peptide h~ving a molecular structure similar to Q
fragment of the or one of the proteins;
(b) reacting this first peptide with a bifunction~l coupling reagent while 15 the first peptide is in ~ form having only a single site capable of reacting with the coupling reagent, this site being at or adjacent one of the termini of the firstpeptide, thereby c~using one of the functional groups of the coupling reagent toreact with the one site on the peptide;
(c) reacting the product of step (b) with a second peptide having fl 20 molecular structure similar to a frsgment of the or one of the proteins while the second peptide is in a form having only n single site capable of reacting with the other functional group of the coupling reagent, this site being at or adjacent one of the termini of the second peptide, thereby forming a dimeric peptide wherein thefirst and second peptide ~re interconnencted vi~ a residue of the coupling reagent;
(d) reacting the resultant peptide with a bifunctional coupling reagent while the peptide is in a form h~ving only e single site capable of reacting with the coupling reagent, this site being at or adjacent one of the termini of the peptide, thereby causing one of the function~l groups of the coupling reagent to re~ct with the one site on the peptide;
(e) rencting the product of step (d) with ~ further peptide having a molecular structure simil~r to Q fragment Df the or one of the proteins while this further pepUde is in a form having only Q single site capable of rencting with the other functionfll group of the coupling reagent, this site being at or adjecent one of the termini of the further peptide, thereby torming a polymeric peptide wherein thc 35 first, second and further peptides are interconnected via residues of the coupling raagent;
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(f) repeating steps (d) and (e) until the desired polymer length has been achieved.
In another aspect, this invention provides a method for producing a modified polypeptide for provoking the formation, in the body of an animal, of antibodies to a 05 protein which is substantially non-immunogenic to the animal, the method being characterized by:
a. procuring a first peptide having a molecular structure similar to a fragment of the protein, the first peptide not having an unblocked thiol group and having an unblocked amino group only at its N-terminal but no other unblocked amino group;
b. reacting the first peptide with an amino group activating agent, thereby producing an activated amino group at the N-terminal of the first peptide;
c. reacting the activated first peptide with a second peptide having a molecular structure similar to Q fragment of the protein, the second peptide having a C-terminal cysteine bearing an unblocked thiol group but not having any other unblocked thiol groups, thereby coupling the N-terminal of the first peptide to the C-terminal of the second peptide;
d. reacting the resultant compound in a form having an unblocked amino group at its N-terminal but no other unblocked amino groups, and no unblocked thiol group, with an amino-group activating agent, thereby producing an activated amino-group at the N-terminal of the resultant compound;
e. reacting the activated compound produced in step (d) with a further peptide having a molecular structure similar to a fragment of the protein, this further peptide having a C-terminal cys$eine bearing an unblocked thiol group, but not having any other unblocked thiol groups, thereby coupling the activated N-terminal of the reactivated compound produced in step (d) to the C-terminal of the further peptide; and f. repeating steps (d) and (e) until the desired polymer length has been achieved.
In another aspect, this invention provides an anti~en for provoking the formation, in the body of an animal, of antibodies to a protein which is not endogenous nor substantially immunogenic to the animal, characteri~ed in that the 35 antigen comprises the protein, or a peptide having a sequence corresponding to at least part of the sequence of the protein, which protein or peptide has been ~.~

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chemically modified outside the body of the animal, the antigen having a greatercapacity to provoke the form~tion of the antibodies than the protein in its unmodified form.
In another aspect, this invention provides a process for preparing an antigen ofns the invention, which process is characterized by:
procuring the protein which is not endogenous or immunogenic to the animal, or the peptide having a sequence corresponding to at least part of the sequence of the protein; and chemically modifying the protein or peptide outside the body of the animal, 10 thereby producing the antigen according to claim 3.
In another aspect, this invention provides a modified antigen for use in fertility control in an animal characterized in that it comprises an antigen derived from the zona pellucid~ or from sperm, or a peptide having a sequence corres-ponding to at leQst part of the sequence of such a zona pellucida or sperm antigen, 15 which antigen or peptide has been chemically modified outside the body of theanimal, the modified antigen, after administration into the body of the animal, having a greater capacity to provoke the formation of antibodies than the unmodified antigen from which it is derived.
~inally, this inv~ntion provides a vaccine for provoking the formation, in the 20 body of an animal, of antibodies to a protein, this vaccine being characterized in that it comprises a modified polypeptide, antigen or modified antigen of the invention and a vehicle comprising a mixture of mannide monooleate with squalaneand/cr squalene.
As already mentioned, the linear polymeric modified polypeptides of the 25 invention comprise inert polymers of polypeptide fragments substantially free of non-linear polymers. In saying that the polypeptide fragments used in the linearpolymeric polypeptide of the invention have a molecular structure similar to a fragment of the protein to which antibodies are to be provoked, we do not necessarily imply that the entire amino acid sequence of each fragment must 30 correspond exactly to part of the protein to which antibodies are to be provoked; for example, in certain cases certain substitutions of amino acids may be possible without affecting the immunogenic character of the fragment. For example, our aforementioned U.S. p~tent No. 4,302,386 describes a polypeptide, designated Structure (IX), which is notionally derived from the beta subunit of HCG but in 35 which the cysteine residue at the 110 position is replaced by alpha-aminoblltyric acid.
In particular, when it is desired to provoke the formation of antibodies to an , .. ~............................................................. .

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endogenous protein, the polypeptide fragments used to form the appropriate modified polypeptide of the invention must have a molecular structure similar to a fragment of the endogenous protein which antibodies are lo be provoked, this does not exclude the possibility thnt such fragments might actually be derived from a05 protein in a different species, since mnny proteins are either identical between species or differ from one another so little in amino ncid sequence that considerable cross-reactivity exists between antibodies to the corresponding proteins in the two species. For example, as men~ioned below, zon~ pellucida enzymes from a pig will, when injected into humans, produce antibodies which display considernble activity 10 against human zona antigens. Accordingly, for example, if one wishes to form a modified polypeptide for provoking the formation of antibodies, in humans, to zona pellucida untigens, appropriate polypeptide fragments may be prepared from zona pellucida antigens of pigs. Also, the fragments used in the linear polymeric polypeptides of the invention may incorporate sequences of amino acids having no15 counterpart in the sequence of the protein from which the Eragment is notionally derived. Again, for example, our aforementioned U.S. patent No. 4,302,386 describes certain polypeptide fragments, designated Structure (IY), (VIII), (IX), (X) and (XIV) which are notionally derived from the beta subunit of HCG but which incorpornte spncer sequences comprising multiple proline residues.
It may at first appear surprising thAt 8 linear polymer of a polypeptide, the monomeric form of which is effectively non-immunogenic to an animal9 can be immunogenic to the same animal. It is believed (though the invention is in no way limited by this belief) that the increase in immunogenicity upon polymerization is due to the incrense in physical size of the molecule, which enables the molecule to 25 be recognized much more easily by the animal's immune system. It can be shownthat at least some monomeric polypeptides ars very weakly immunogenic and cause the animal's immune system to produce detectable quantities of antibodies, whichquantities, however are much too small to be effective. Immune systems are not well-adapted to recognize molecules as small ns the small polypeptides when the 30 polypeptides are present In polymeric form.
It will be Qpparent to those skilled in the art that the polypeptide fragments used in the linear polymeric polypeptides of the invention may be either nfltural (i.e.
derived from natural proteins) or mny be produced synthetically. Obviously, a synthetic polypeptide will perîorm in the same manner ns A nnturally occuring one in 35 as much as the immune system of the animal to which the modified polypeptide is administered will react in exactly the same way to both. Also the frngments within , .:

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q OOl() each linear polymer may be the same or different and need not necessarily be derived from the same protein.
The linear polymeric polypeptides of the invention may be use~ to provoke the formation of antibodies to both endogenous and non-endogenous proteins by using 05 appropriate polypeptide fragments to form the polymers. (The term "endogenous" is useà herein to denote a protein which is native to the species to be treated, regardless of whether the antigen is endogenous to the particular individual animal being treated. Thus, for example, for present purposes a porcine sperm antigen is regarded as being endogenous to a sow even though obviously such a sperm antigen10 will not normally be present in the body of a sow.) Further details OI the use of non-endogenous proteins fragments of which may be utilized in the linear polymeric polypeptides of the invention is given below in connection with the discussion of the antigens of the invention. As regards endogenous proteins, fragments of which may be used to form the linear polymeric polypeptides of the invention, in general it may 15 be stated that such endogenous proteins may include any of the protein.s mentioned in our aforernentioned U.S. patent No. ~,302,386, as described above. Incidentally, it should be noted that the use of the linear polymeric polypeptides of the invention based upon growth hormone and/or somatomedian is not confined to diabetic patients. Thus, the linear polymeric polypeptides of the invention based upon these 20 two hormones may be used to treat non-diabetic patients, such as persons suffering from acromegaly, who have excessive levels of growth hormone and/or somato-median.
Particularly preferred linear polymeric polypeptides of the invention are those formed from fragments having a molecular structure similar to a fragment of 25 Human Chorionic Gonadotropin, and in particular similar to a fragment of the beta-subunit thereof. Two particularly preferred fragments for use in the linear polymeric polypeptides of the ihvention are:
As~As~Pr~Ar~Phe-Gln-As~Ser-5er-Se~Ser-Lys-Ala-Pro-Pr~
Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pr~Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Cys (hereinafter designated fragment A); and As~E~is-Pro-Le~Th~Cys-Asp-As~Pr~Arg-Phe-Gln-Asp-Ser-Se~
Ser-Ser-Lys-Ala-Pr~Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gyl-Pro-Ser-As~Th~Pr~Ile-Leu-Pro-Gl~Cys.
Other HC~derived fragments usable in the linear polymeric polypeptides of the 35 invention include those of Structure (Il~(VIII), ~VIIIa) and (IX~(XIV) as set forth below, further details of the immunological nature of these fragments being given in the aforementioned U.S. patent No. 4,30~,386:
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As~As~Pr~Ar~Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pr~Pr~
Pro-Ser-Leu-Pro-Ser-Pro--Ar~Leu-Pr~Gly-Pro-Ser -As~Thr-Pro-Ile-Leu-Pro-Gln Structure (II) 05 Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Ar~Leu-Pro-Gly~Pro-Ser-As~Thr-Pro-Ile-Leu-Pro-Gln Structure (III) Cys-Pro-Pro-Pro-Pro-Pro-Pro-Ser-As~Thr-Pro-Ile-Leu-Pro-Gln Structure (IV) As~As~Pro-Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Pro-Pro-Pr~Cys Structure (V) Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser Ar~Leu-Pro-Gly-Pro-Ser-As~Thr-Pro-Ile-Leu-Pro-Gln Structure (VI) As~As~Pro-Ar~Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser -Leu-Pro-Ser Structure (VII) ~ .
As~As~Pro-Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Cys-Pro-Pro-Pr~
Ser-As~Thr-Pro-ne-Leu-Pro-Gln ~Structure (VIII) As~As~Pr~Ar~Phe-Gln-As~Ser-Pro-Pro-Pro-Pro-Pro-Pro-Cys-; ~ Pro-Pro-Pro-Pro~Pro-Pro-Se~As~Thr~Pro-Ile-Leu-Pr~Gln ~ ~ ~ 25 Structure (VI[Ia) :

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As~His-Pro-Leu-Thr-Aba-As~As~Pro-Ar~Phe-Gln-As~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-Pro-Pro-Pro-Pr~
Pro-Pro-Cys 05 Structure (IX) Asp-AsE~Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pr~Ser-Pro-Ser-Arg~Leu-Pr~Gly-Pro-Ser-Asp-Th~
Pro-Ile-Leu-Pro-Gln-Pro-Pro-Pro-Pro-Pro-Pro-Cys Structure (X) As~Asp-Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pr~
Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pr~Gly-Pro-Ser-Asp-Thr-Pro-Tle-Leu-Pro-Gln-Cys Structure (XI) 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-Pr~Ser-Asp-Thr-Pro-Il~Leu-Pro-Gln Structure (XII) Asp-His-Pro-Leu-Thr-Aba-As~As~Pro-Ar~Phe-Gln-Asp-Ser-Ser-Ser-Se~Lys-Ala-Pr~Pr~Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys Structure (XIII) Cys-Pro-Pro-Pro-Pro-Pro-Pro-Asp-As~Pro-Ar~Phe-Gln-As~Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Ar~
Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln Structure (XIY) Obviously, if it is desired to use one of the above peptides which lacks a C-: terminal cysteine as a second or later fragment in preparing the linear polymeric peptides of the invention, it will be necessary to add a C-terminal cysteine to the peptide; appropriate methods for doing so are of course well known to those skilled 30 in the field of polypeptide synthesis. Also, some of the above peptides will of course reguire blocking of non terminal amino and/or thio groups before use.

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gL2~ 0()~) The linesr polymeric polypeptides of the invention based upon HCG-derived fragmenes are usable in exactly the same manner as the corresponding modified polypeptides in which the same fragments are coupled to carriers, as described in our aforementioned U.S. patent No. 4,302,386. Moreoever, since this patent was ns written, further dis~overies have been made con-~erning the aSsociQtion between HCG and immunologically similar materials and certain carCinOmQS. It appears (though the invention is in no way limited by this belief) that certain carcinomas exude chorionic gonadotropin or ~n immunologically-similar material on their surfaces, thereby presenting to the immune system of the host animal a surface, 10 which, superficially, appears to be formed of material endogenous to the hostanimal, and which is thus relatively non-immunogenic. Because of this known association between certain carcinomas and chorionic gonadotropin or chorionic gonadotropin-like materials, the HCG-derived linear polymeric polypeptides of the invention flre useful for the treatment of HCG and chorionic gonadotropin-15 associated carcinomas. The same linear polymeric polypeptides of the inventionare also of course useful for fertility control, as described in the ~forementioned U.S. patent.
The fr~gments used in the linear polymeric polypeptides of the invention may also include fr~gments of the zona pellucids and sperm antigens discussed in detail 20 below.
As alre~dy mentioned, the line~r polymeric peptides of the invention are produced by polymerizing fragments of the protein to which antibodies are to be provoked rather than the entire protein itself. This use of fragments r~ther than an entire proteins hes important advantages (and similar advnntages are sec~red by 25 the use of rragments in the ~ntigens and modified antigens of the invention discuæed in det~il below). It is well recogni~ed by those skilled in immunology (see e.g. W.R. Jones, "Immunological Fertility Regulation", Blackwell Scientific Pub-lications, Vic~oria, Austrnlia (1982) (the entire disclosure of this work is herein incorporated by reference), pages 11 et seq.) that one of the greatest potential30 hazards Or a vaccine, especially a contraceptive vaccine, is cross-reactivity with no~tnrget ~ntigens, producing what is essentially an artificiall~induced aut~
immune disease capable of causing immunopathological lesions in, and/o; loss of function of, the tisues carrying the crossreactive substances. Two possible mechanisms for such cross-re~ctivity are:
(a) Presence of shared antigenic determinallts; a complex protein may contain components (amin~acid sequences) identical to those present in fl non-target antigens;

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(b) steric overlap between non-identical but structurally relnted parts of the protein and no~target antigens.
Obviously, the chnnges pvsed by both these modes of cross-reactivity may be lessened by using, in the linear polymeric polypeptides, nntigens end modified 05 antigens of the invention, a fregment of Q complex protein rnther thnn the whole protein. Since the fragment has a simpler sequence thnn the protein from which it is derived, there is less chance of shared nntigenic dete~mi~r~s or sterie overlnp with non-tnrget proteins. In particular, eross-reactions c~n often be avoided byusing frngments derived from n portion of the target protein (i.e. the protein to 10 which nntibodies ~re to be provoked) which is not similar in sequence to the non-target but cross-renctive protein. ln particular, as described in our nforementioned U.S. patent No. 4,302386, one of the major problems in provoking nntibodies to HCG
is cross-reactivity of HCG antibodies with LH, this cross-reactivity being at least largely due to identity of amino acid sequence between LH and the 1-110 amino acid 15 sequence of the bet~ subunit of HCG. Accordingly, when it is desired to form an HC~derived lline~r polymeric polypeptide of the invention, the fragment used is prefernbly one having n molecular structure similar to pnrt or all of the 111-145 sequence of the bet~ subunit of HCG, since it is only this 111-145 sequence of bet~-HCG which difers from the corresponding sequence of LH. However, rese~rch 20 indieates th~t the fr~ments used in the linear polymeric polypeptides of the invention mny contain sequences corresponding to the 101-110 sequence of beta-HCG which is comsnon ~o bet~-HCG ~nd LH without inducing the formation of ~ntibodies re~ctive to LH. Thus, one can use in the linear polymeric polypeptides of the invention frQgments conteining part of or all of this common 101-110 sequence 25 without c~using cross-re~ctivity with LH. For ex~mple, Structure (U) ~bove represents the 111-145 ~mino acid sequence of betn-HCG. If desired, A fragment having the 101-145 ~mino ~cid sequence of betn-HCG could be substituted for the ragment oî Stuucture (Il) in the linenr polymeric polypeptides of the inventionwithout subst~nti~lly eftecting the activity of the linenr polymers ~nd without 30 c~using er~s-reactivity with LH.
Polymerizstion of the fragments to form the linear polymeric poly~eptides of the invention may be effected in any manner for coupling peptide frngments to form linear polymers thereof kslown ~o those skilled in the art. Thc linenr polymcriepolypeptides oi the invention may ~e divided into two distinct types. In the firs~
35 type, the individunl peptide fr~gments ore linkcd head-to-t~U by pcptide linkngcs, so that the wholc polymer compriscs solely the frngmcnts thcmsclvcs nnd docs not -ov contain Any extraneous m~terial. Although such pure polymers do have the advant~ge of not introducing any extraneous material into the body of the animalbeing treated, they are usually too expensive to be practical, since the necesary fragments (whether produced by total synthesis or cleavage of Q n~tural protein) are 05 themselves very expensive Elnd substantW losses occur during the polymerization process. Furthermore, the head-to-tail coupling of the fragments~ without any intervening residues, may produce immunological determinants which have no counterpart in the unpolymerized fragment. For example, if the fragment described above, comprising the 105-145 sequence of HCG, is polymerized by means of peptide 10 linkages, a sequence:
Pro-Ile-Leu-Pro-Gln-As~His-Pro-Leu-Thr will be produced at each junction between adjacent fragments, and this sequence may provoke the formation of antibodies which would not be produced by the fragment itself, and which may be undesirable. In colloquial terms, since there is no 15 "punctuationl' to tell the immune system of the recipient animal where one fr~gment begins and another ends, the animal's immune system may inadvertently start reading at the wrong residue and produce unwanted antibodies by running the sequences of adjacent fragments together. For this reason, in general, we do notrecommend the use of linear polymers in which the fragments are connected by 20 peptide polymers, though of course such linear polymers may be useful in certain instances.
Various methods of coupling polypeptide fragments via peptide bonds are known to those skille~ in the art. For example, one fragment to be coupled may have its C-termin~l carboxyl group blocked (e.g. by astearification) and be re~cted 25 with the other fragment, which has its N-terminal amino group blocked, but its carboxyl group activnted by means of an activating agent. Obviously, blocking ofnon-terminal amino and carboxyl groups may be necessary- Also, aswell knowrl to those skilled in this field, it may be advantageous to attach one end of the polymer being produced to a support, such as polystyrine resin support, the polymer 30 only being detached from the support after polymerization is completed.
In the second type of linear polymer polypeptide of theinvention~the peptide fragments are connected to one another by means of residues derived from a bifunctional reagent used to effect polymerization of the fragments, so thAt thefinal linear polymer is an alternating linear polymer of polypeptide fr~gments and 35 coupling reagent residues. Although this type of polymer necessarily introduces some extr~neous m~teri~l into the nnimal being treated, the proportion of extrA

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neous material can be made consi~erably lower than it would be if the fragments were coupled to a large carrier, such as diph~heria toxoid. The coupling reagent, - which is necessarily a bifunctional coupling reagent to produce a true linear polymer, can be chosen so that the resides it leaves in the polymer are not strongly 05 immunogenic (so that they do not plas!e the strain on the immune system of the recipient animal that, for example, a large carrier molecule such as diphtheria toxoid would) and the presence of these residues in the polymer has the advantage of substantially eliminQting false immunological determinants produced by conjunction of the head of one fragment with the tail of an adjacent fragment, as discussed 1 0 above.
To ensure that a true linear polymer is produced during the polymerization process, one terminal of a first polypeptide fragment is reacted with the bi-functional coupling reagent so that the coupling reagent reacts with a group present at or adjacent one terminal of the fragment; for example, the coupling reagent may 15 react with a N-terminal amino group, a C-terminal carboxyl grou~ or a free thiol group present on a C-terminal cysteine. Obviously, the nature of the coupling reagent used determines what group on the peptide reacts. In order to avoid any cross-linking and to ensure a reproduceable product, it is important that only one site on the first fragment be available for reaction with the coupling reagent so that 20 the coupling reagent can only attach to the first fragment at this one site. As those skilled in this field are aware, if it is desired to use a fragment containing more than one group which could react with the coupling reagent, the excess sites may be blocked by attaching suitable protective groups thereto. The product formed by reaction of the first fragment with the coupling reagent is then reacted with a 25 second fragment (which may be the same or different from the first fragment) having a single site available to react with the second reactive group of the bifunctional bicoupling reagent, thereby coupling the first and second fragments by a residue derived from the coupling reagent. Following any necessary purification of this dimeric product, it is then reacted with a further portion of a coupling agent 30 which may be the same or ~ifferent reagent from that used to effect the firstcoupling), thereby reacting the free terminal of either the first or second fragment with the coupling reagent. Naturally, it is important to ensure that only one site on the dimer is available for coupling to the coupling reagent, and as will be apparent to those skilled in the art, blocking or unblocking of potential reactive groups on the 35 dimeric polypeptide may be necessary. The product of the reaction of the dimeric polypeptide with the coupling reagent is then reacted with a third fragment hanng .
. . .

.~.
:
- ., , o~

only a single site av~ilable for reaction with the remaining reactive group of the coupling reagent, thereby producing a linear polymer containing three polypeptide fragments. Obviously, this process can be repeated until the desired size of lineur polymer has been produced.
05 It will be apparent to those skilled in this field that the bifunctional coupling reagents used to prepare the linear polymeric polypeptides of the invention should be asymmetric i.e. they should have two functional groups which react with different groups on the fragments being polymerized, since, for example, if one attempted to react a bifunctional bicoupling reagent having two functional groups 10 which both reacted with amino groups with a first fragment having a single amino group, st least some of the first fragment would be dimerized via a residue derived from the bifunctional bicoupling reagent. Such dimerization may in theory be avoided by using a very large excess of the coupling reagent, but in practice it is undesirable to run the risk of producing even a small proportion of dimer. Similarly, 15 in later stages of the polymerization process, it will be even more undesirable to use symmetric coupling reagents, thereby running the risk of dimerizing the partially formed polymers already produced. In the preferred process for producing the linear polymeric polypeptides in the invention already described, the polymer chain is begun with a first peptide having no unblocked thiol group and having an unblocked 20 amino group only at its N-terminal (peptides containing thiol groups and/or amino groups other than at the N-terminal may of course be used if all these thiol andamino groups are blocked with any conventional blocking agent). This first peptide is then reacted with an amino group nctivating agent, a preferred activating agent for this purpose being 6-maleimido caproic acyl N-hydroxy succinimide ester (MCS);
25 reaction of the peptide with this reagent is optimally effected ~t a pH of 6.6). The activating agent reacts with the amino group at the N-terminal of the first peptide to form an activ2ted form of the first peptide; in the case of MCS, it is the ester portion of the reagent which reacts with the N-terminal group of the peptide. It is normally ~hen necessary to remove excess activating agent before continuing the 30 preparative process. Once the excess activating agent is removed, the activated first peptide is reacted with a second peptide having a C-terminal cysteine in areduced state (i.e. having an unblocked 3-thiol group), thereby causing coupling of the N-terminal of the activated first peptide to the C-terminal Or the second peptide viaan activating sgent residue. Desirably, the resultant dimer is purified as 35 described in more detail below. Next, the dimer is again reacted with an amin~
group activating agent and then with a second portion of the second pcptide or with ~ . .
. , : ,, :

01~3~) a third peptide, thereby producing a trimer. This procedure is repeated until the desired chain length has been achieved.
In order to secure reproduceable responses from the immune systems o~
treated animals, it is important that the linear polymeric polypeptides of the 05 invention be used in the form pure polymers in which all the molecules contain t}ie same number of fragments. To achieve such pure polymers, effective purification should be used after each polymerization step of the polymerization process.
Because of the close chemical similarity between polymers containing difIerent numbers of fragments, chemical purification is ineffective, so purification must be 10 effected by physical methods. Gel filtration may be used if desired, but our preferred purification method is reverse-phase, high-pressure liquid chroma-tography, preferably using a molecular sieve as the solid phase.
In this method of forming linear polymers, the first and second peptides may be identical in chemical configuration except that in the first peptide the C-15 terminal cysteine hus a blocked thiol group. The first two preferred fragments forforming linear polymeric polypeptides of the invention to form antibodies to HCG
mentioned above may be described as (l~l-145)-Cys and (105-145~Cys, where the figures refer to the amino acid sequence in the beta subunit of HCG. It will be appreciated that, when these fragments are to be used in forming linear polymeric 20 polypeptides of the invention by the method just described, the lysine residue at position 122 must have its amino group blocked and, in the case of the (105-145~Cys fragment, the non-terminal cysteine at position 110 must have its thiol group blocked, preferably with an acetamidomethyl group.
The linear polymeric peptides of the invention preferably contain from about 4 25 to about 14 fragments.
The modified antigens of the invention produced by chemically modifying non-endogenous proteins wi~l now be described in more detail. As those skilled in the art are aware, there are numerous pathogens and similar materials known which are not endogenous to animals, which are capable of produ~ing harmful effects in the 30 animal's body but which are not immunogenic to the animal, in the sense that introduction of the pathogen or other material into the body of the animal fails to elicit from the animal's irnmune system production of the quantity of appropriate antibodies necessary for the animal's irnmune system to destroy the pathogen or similar material. ~or example, the Herpes simplex Type Il virus is capable of 35 producing a number of harmful effects in man, including the production of painful lesions in the genit~ areas. ~lthough this virus has, like most viruses, protein :
-, ': ~ ' .' ~:.
.. ~
~ - ~ - . :.

~4(~3~3 included in its structure, the viral protein is not strongly immunogenic in rnost human beings, so that only about 5096 of infected human beings produce antibodies to the virus. This lack of immune response to the virus by many human beings means that the virus can remain in the infected human beings for at least several 05 years, and this persistence of the virus in the infected individuals not only causes those individuals to suffer recurrent attacks of the painful symptoms caused by the virus, but also renders them long-term carriers of the virus. This persistence of the virus in infected individuals is one of the factors largely responsible for the epidemic proportions which ~ _ simplex infections have reached in several 10 countries. By prep~ring an antigen OI this invention derived from a protein having a sequence similar to that of at least part of the sequence of a Herpes simplex viral protein, it is possible to stimulate the human immune system so as to render it capable of producing large quantities of antibodies to the Herpes simplex virus. Not only should this stimulation of the immune system reduce the occurrence of 15 symptoms associated with Herpes simplex infection, but it should help to control the spread of the virus. Similarly, the immune response of humans and other animals to viruses such as colds, influenza and other viruses can be increased by preparingmodified antigens of the invention based upon peptides having sequences corres-ponding to viral proteins of the appropriate virus. If, as appears likely, a virus is 20 responsible for acquired immune deficiency syndrome (AIDS) a modified antigen of this invention could also be used to produce immunity to this disease.
In general, the methods used for preparing the antigens of the invention based upon non-endogenous materials, such as viral proteins or peptides corresponding to parts thereof, are the same as those used for modifying endogenous proteins or 25 fragments thereof, as described in our aforementioned U.S. patent No. 4,302~386.
However, it will be appreciated that the preferred methods used for modifying non-endogenous materials may differ in certain respects from those used for modifying endogenous peptides. Since, in general, the non-endogenous peptide will provoke at least a limited immune response from the animal to which the antigen is to be 30 administered, the requirements for modification of the non-endogenous protein or peptide to produce an antigen of the invention will tend to be less stringent than those for modification of an endogenous and completely non-immunogenic protein or peptide. However~ since the non-endogenous protein or peptide is being modified to increase its immlmogenic effect in the animal into which it is to be administered, in 35 gener~l it will still be desirable that the carrier used to modify the non-endogenous protein or peptide (if chemical modification is to be effected by coupling the non-. .: .

- -:.. ,;. :: ..
-`
.
., .
.. . . . .
~,~, ' '~

~ ~ .

~6~0~3 endogenous protein or peptide to n carrier, rather forming a linear polymericpolypeptide of the invention) to produce the antigen of the invention be a materialwhich itself provokes ~ strong response from the animal's immune system. For example, the carrier may be a bacterial toxoid such as diphtheria toxoid or tetanus oS toxoid.
The antigens of the invention are prepared by chemically modifying Q protein which is not endogenous or immunogenic to the animal to be treated, or a peptidehaving a sequence corresponding to at least part of the sequence of the protein, this chemical modification being effected outside the body of the animal. The chemical 10 modification m~y be effected by coupling the protein or peptide to a carrier, by polymerizing the peptide to form a linear polymeric polypeptide of the invention or by any other chemical modification technique which will make the protein or peptide sufficiently immunogenic. Preferred techniques for chemical modificationof the protein or peptide by coupling to a carrier include the following (further 15 details of optimum techniques for each of the following coupling reactions are given in our aforementioned ll.S. patent No. 4,302,386):
a. Treating a protein or peptide having a sulfhydryl group thereon with an activator of the structure:

~--X~ --X--CON~

wherein X represents a non-reacting group comprising substitutedor unsubstituted phenyl or Cl-10 alkylene moieties, or a combinAtion there-of, or an amino acid chain, so as to cause reaction of the maleiimide group of the activator with the sulfhydryl group on the protein or peptide; and treating the resulting activated protein or peptide at slightly Mlkaline pH with a carrier moiety biologically foreign to the animal and selected haYing a size sufficient to elicit antibody response following the administration thereof into the body of the animal.
b. Treating, under neutral or acid conditions, a carrier not hnving M
sulfhy~yl group but having an amino group with an activ~tor as defined in the preceding parsgraph, so as to cause reaction of the aCtivAtor with .
~, ~ .
, ~: :. .. : : - :
. ~ . .

::
:
: . ..
: . ~.. . . .

~L2 ~ Lq ~

the amino group on the c~rrier, the carrier being biologically foreign to the animal to be treated and having a size sufficient to elicit antibody response following administration thereof into the body of the animal;
and treating the resulting activated carrier with the protein or peptide, 05 which must have a sulfhydryl group thereon, thereby reacting the maleiimide group of the activator with the sulfhydryl group on the protein or peptide.
c. Treating a carrier biologica~ly foreign to the animPl to be treated and having Rn amino group with an activator present as Rn active ester of chloro-, dichlor~, brom~ or iodo- acetic ~ci~ so as to cause reaction of the activator with the amino group of the carrier and treating the resulting activated c~rrier with the protein or peptide, which must hàve a sulfhydryl group thereon, thereby reacting the activated carrier with the sulfhydryl group of the protein or peptide.
d. Treating ~ protein or peptide that does not have a sulfhydryl group but has an amino group with an activator present RS an active ester of chlor-, dichlor~, bromo- or iodo- acetic acid, and treating the resulting halomethyl Qlkylating group containing moiety with a sulfhydryl group-containing carrier biologically foreign to the animal to be treated so as to react the sulfhydryl group of the carrier with the halomethyl alkylating group.
It will be noted that several of the preferred coupling techniques described above require the presence of a sulfhydryl group in the protein or peptide.
It is well known to those skilled in the art that, in mQny natural proteins 25 containing cysteine residues, these residues are not presene in their thio form containing a free-SH group; instead pairs of cysteine residues are lined by means of disulfide ~ridges to form cystine. Such disulfide bridges are very important in determining the conformation of the protein. In most cases, the disulfide bridges present in the natural form of the protein are eRsily reduced to pairs of -SH groups 30 by means of mild reducing agents under conditions which leave the remaining parts of the protein molecule unchanged. Accordingly, when it is desired to produce free -SH groups in proteins in order to carry out the ~oupling reactions discussed ~bove, one conver~ient way of providing such fre~SH groups may be to cleave disulfide bridges naturally present in the protein or other polypeptide which is desired to ;; 35 couple.
: ~r~
~3 - ~ : : ,. : : :
,. : ~ ~ - - ~ . . .
- ~

~2~

The generation of free-SH by reduction of disulfide bridges in naturally occurring forms a~proteins may also affect the cross-reuctivity of the antibodies produced when a modified polypeptide produced from the protein is injected into an animal. Frequently, an antibody recognizes its corresponding antigen not only by05 the amino acid sequence in the antigen but also by the conormation (shape) of the antigen. Accordingly, an antibody which ~jinds very strongly to a protein or other polypeptide in its natural conformntion may bind much less strongly, if at all, to the same protein or polypeptide whose conformation has been drastically altered by breaking disulfide bridges therein. Accordingly, the breaking of disulfide bridges in 10 proteins or other polypeptides may provide a basis for reducing the cross-reactivity between antibodies to antigens having the same amino acid sequence along parts of the molecule.
As mentioned above, this invention extends to chemically modified antigens derived from zona pellucida or sperm antigens, or peptides having a sequence 15 corresponding to at least part of the sequence of such a zona pellucida or sperm antigen. These modified antigens of the invention are useful for fertility control. It is known that antigens from the zona pellucida (the outer covering of the ovum) when injected into female primates produce antibodies having anti-fertilization effects, including prevention of sperm attachment to, and penetration of, the zona 20 pellucida of the unfertilized ovum, and prevention of dispersal of the zona pellucida of the fertilized ovum prior to implantation (such dispersal of the zona apparenUy being an essentisl prerequisite for implantation). See e.g. W.R. Jones, "Immuno-logical Fertility Regulation", Blackwell Scientific Publications, Victoria, Australia (1982), pages 160 et seq. Such anti-fertility effects are believed to be due to 25 formation of an antibody-antigen precipitate on the zona, this precipitate rendering the zons unable to undergo its normal sperm-binding reaction and also rendering the zona insensitive to the action of the protreases normally responsible for dispersal of the zona.
An anti-fertility vaccine based upon zona antigens is especially attractive 30 because zona antigens appear to be relatively free of side-effects on other tissues and because methods have been developed for producing swine zona antigens in large quantities; swine ~nd human zona antigens show very good cross-reactivity. As inthe case of anti-fertility vaccines based upon the beta-HCG antigens discussed ~bove and in our aforementioned U.S. patent No. 4,302,386, better results will be 35 obtained by modifying n :~ona antigen or a fragment thereof to produce a modified antigen of this invention.

~,, , ~: : ' ,:
.

3L"~ Lq~O ~3(3 Several antigens, especiaLly sperm enzymes, known to exist in sperm, may be usèd in the modified antigens of this invention; see W.R. Jones, op. cit., pages 133 et seq. The most promising such antigen is the lactate hydrogenase known as LDH-C4 or LDH-~. Although of course lactate dehydrogenases are present in other tissues, 05 LDH-C4 is distinct from other lactate dehydrogenase isoenzymes and appears to be sperm-specific. Moreover, the enzyme is not strongly species specific, and methods for its isolation and purification are known. Again, the best results will be obtained by modifying LDH-C4 or a fragment thereof to produce R modified polypeptide of this invention. Several natural peptide fragments of LDH-C~ have been prepared, 10 sequenced and shown to bind to antibodies against the parent molecule. (See E.
Goldburg, "LDH-X as a sperm-specific antigen", in T. Wegmann and T.J. Gill (eds.~, Reproductive Immunology, Oxford University Press, 1981).

Although theoretically an anti-fertility vaccine based on sperm antigens might 15 be useful in males, the likelihood of testicular damage renders it more likely that such a vaccine will find its utility in females. It is known that circulating antibodies in the female bloodstream do penetrate the genital fluids; for example experiments in baboons with vaccines based upon the peptide of Structure (XII) above conjugated with tetanus toxoid have shown the presence of HCG antibodies in the genital fluids.
20 However, one possible problem with any vaccine based on sperm antigens is maintaining a sufficiently high antibody level in female genital fluids to complex ,with the large amounts of sperm involved.
The techniques used for chemical modification of zona pellucida or sperm antigens, or peptides derived therefrom, to form the modified antigens of the 25 invention include all the techniques discussed in our aforementioned ~.~. patent No 4,302,386 and also those techniques discussed above. Thus, the antigen or peptide may be coupled to a carrier, such as tetanus toxoid or diphtheria toxoid, or may be polymerized to form a linear polymeric polypeptide of the invention. The preferred te¢hniques for forming such linear polymeric polypeptides have already been 30 discussed above, while the preferred techniques for coupling the antigens or peptides to carriers are the same as those for coupling non-endogenous proteins or peptides, as already discussed above.
~ inally, as mentioned above, this invention extends to a vaccine containing a modified p~lypeptide, antigen or modified antigen of the invention and a vehicle35 comprising a mixture of mannide monooleate with squalane and/or squalene. It has been found that this ~ehicle has the effect of increasing the quantity of antibodies , : : :

oov provoked by the linear polymeric polypeptide, antigen or modified antigen of theinvention when the vaccine is administered to an animal. To further increase thequantity of antibodies provoked by administration of the vaccine, it is advantageous to include in the vaccine an immunological adjuvant. The term 'ladjuvant" is used in 05 its normal meaning to one skilled in the art of immunology, namely as meaning a substance which will elevate the total immune response of the animal to which the vaccine is administered i.e. the adjuvant is a non-specific immuno-stimul~tor.
Preferred adjuvants are muramyl dipeptides, especially:
NAc~nor Mur-L.Ala-D.isoGln;
NAc-Mur-(6-0-stearoyl~L.Ala-D.isoGln; or N Glycol-M ur-L.~Abu-D.isoGln The vaccines of this invention may be administered parenterally to the animals to be protected; the usual modes of administration of the vaccine being intramuscular and sub-cutaneous injections. The quantity of vaccine to be employed will of course 15 vary depending upon various factors, including the condition being treated and its severity. However, in general, unit doses of 0.1-50 mg. in large mammals admin-istered with one to five times the intervals of 1 to 5 weeks provide satisfactory results. Primary immunization may also be fo~lowed by llbooster'l immunization at 1 to 12 month intervals.
To prepare the vaccines of the invention, it is convenient to first mix the linear polymeric polypeptide, antigen or modified antigen of the invention with the muramyl dipeptide (or other adjuvant) and then to emulsify the resultant mixture in the mannide monooleate/squalene or squalane vehicle. Squalene is preferred to squalane for use in the vaccines of the invention, and preferably about ~ parts by 25 volllme of squalene and/or squalane are used per part by volume of mannide m onooleate.
The animals to be treated with the linear polymeric polypeptides, antigens, modified antigens and vaccines of this invention include both humans and other animal species.
The following example is now given, though by way of illustration only, to show details of the preparation and use of a linear polymeric polypeptide of theinvention.
EXAMPLE
Fragment A described above (a fragment having an amino acid sequence 35 corresponding to the 105-I45 sequence of beta-llCG, with a cysteine residue added to the C-terminal ther~of) was polymerized to form a hexamer. A first portion of

3~

fragment A had both its thiol groups (on the non-terminal cysteine at the position corresponding to position 110 of beta-HCG, and on its C--terminal cysteine) and its non-terminal amino group (on the lysine residue at the position corresponding toposition 122 in beta-HCG) blocked. This blocked form of Fragment A was reacted 05 with the bifunctional organic coupling reagent (or amino group activating agent) MCS in a buffered aqueous solution at pH 6.6, thereby reacting the ester portion of the MCS with the N-terminal amino group of the first portion of Fragment A. The resultant product was then reacted with a second portion of Fragment A, which was used in the same form as the first portion of Fragment A except that the C-terminal 10 cysteine bore an unblocked thiol group, thereby reacting the remaining functional group of the MCS with the free thiol group on the second portion of Fragment A and producing a dimer in which the N-terminal of the first portion of Fragment A wascoupled to the C-terminal of the second portion of Fragment A via an MCS residue.
This dimer was then purified by gel filtration. The polymerization was then 15 repeated in the same manner until a hex~mer of Fragment A had been produced.
Because the purification following each polymerization step was effected by gel filtration rather than by reverse-phase, high-pressure liquid chromatography, the hexamer was undoubtedly somewhat impure and contaminated by traces of penta-mer9 tetramer etc., so that the results in the animal tests results described below 20 could not be expected to be as good as would be produced using a pure hexamer.
To test the effectiveness of this hexameric polypeptide in provoking the formation of antibodies to HCG, the hexamer was formed into a vaccine using Complete Freunds' Adjuvant and injected into five rabbits. Each rabbit was giventhree injections of the vaccine intramuscularly at 3 week intervals, each injection 25 containing 0.5 mg. of the hexamer. Starting three weeks after the first injection, each rabbit was bled weekly and the level of antibodies to HCG in the blood determined. The following average values of antibody level were found (the figures in parenthesis represent the confidence limits i.e. average + or - standard error):

:-- .

' v TABLE
Weeks After First Injection Antibody concentration (moles/liters x 10 10) 3 5 (2-7) 18 (13-22) 30 (21-39) 6 45 (31)-60) 7 58 (32-83) 8 61 (34-86) 9 77 (~0-108) 110 (53->125) 11 100 (50->125) 12 79 (30-1l9) 13 53 (22-83) The, the above results show that even the crude hexamer preparation used in 15 these experiments was much more strongly immunogenic than the very we~kly immunogenic fragment from which it was derived.

.

: .

.

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Claims (26)

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

1. A polymeric polypeptide for provoking the formation, in the body of an animal, of antibodies to a protein, the polymeric polypeptide being characterized in that it comprises a linear polymer of polypeptide fragments, each of the fragments, in its monomeric form, being substantially non-immunogenic to the animal and having a molecular structure similar to a fragment of the protein to which antibodies are to be provoked, each adjacent pair of fragments in the linear polymer being connected via a residue of a bifunctional coupling agent, the linear polymer, after administration into the body of the animal, having a greater capacity to provoke the formation of the antibodies than the protein, the linear polymer being substantially free of non-linear polymers of the fragments.

2. A modified polypeptide according to claim 1 wherein each of said fragments, in its monomeric form, has a molecular structure similar to a fragment of Human Chorionic Gonadotropin.

3. A modified polypeptide according to claim 2 wherein each of said fragments is of the chemical configuration:

4. A modified polypeptide according to claim 2 wherein each of said fragments is of the chemical configuration:

5 . A method for producing a linear polymeric polypeptide for provoking the formation, in the body of an animal, of antibodies to one or more proteins which is substantially non-immunogenic to the animal, the method comprising:

(a) procuring a first peptide having a molecular structure similar to a fragment of the or one of the proteins;
(b) reacting this first peptide with a bifunctional coupling reagent while the first peptide is in a form having only a single site capable of reacting with the coupling reagent, this site being at or adjacent one of the termini of the firstpeptide, thereby causing one of the functional groups of the coupling reagent toreact with the one site on the peptide;
(c) reacting the product of step (b) with a second peptide having a molecular structure similar to a fragment of the or one of the proteins while the second peptide is in a form having only a single site capable of reacting with the other functional group of the coupling reagent, this site being at or adjacent one of the termini of the second peptide, thereby forming a dimeric peptide wherein thefirst and second peptide nre interconnencted via a residue of the coupling reagent;
(d) reacting the resultant peptide with a bifunctional coupling reagent while the preptide is in a form having only a single site capable of reacting with the coupling reagent, this site being at or adjacent one of the termini of the peptide, thereby causing one of the functional groups of the coupling reagent to react with the one site on the peptide;
(e) reacting the product of step (d) with a further peptide having a molecular structure similar to a fragment of the or one of the proteins while this further peptide is in a form having only a single site capable of reacting with the other functional group of the coupling reagent, this site being at or adjacent one of the termini of the further peptide, thereby forming a polymeric peptide wherein the first, second and further peptides are interconnected via residues of the coupling reagent;
(f) repeating steps (d) and (e) until the desired polymer length has been achieved.

6 . A method according to claim S wherein each of said first, second and further peptides has a molecular structure similar to a fragment of Human Chorionic Gonndotropin.

7. A method according to claim 5 wherein said bi-functional coupling reagent is 6-maleimido caproic acyl N-hydroxy succinimide ester.

27 .

8. A method according to claim 5 wherein said first, second and further peptides are of the same configuration but said first peptide has the -SH group on its C-terminal cysteine block.

9 . A method according to claim 5 wherein said first, second and further peptides have the chemical configuration:
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-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys said peptides having the amino groups on their lysine residues blocked.

10. A method according to claim 5 wherein said first, second asld further peptides have the chemical configuration:
Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys said peptides having the amino groups on their lysine residues and the thiol groups on their non-terminal cysteine residues blocked.

11. A method according to claim 1O wherein said blocking of said thiol group is effected with a acetamidomethyl group.

12. A method for producing a linear polymeric polypeptide for provoking the formation, in the body of an animal, of antibodies to a protein which is substantially non-immunogenic to the animal, the method comprising (a) procuring a first peptide having a molecular structure similar to a fragment of the protein, the first peptide not having an unblocked thiol group and haYing an unblocked amino group at its N-terminal but no other unblocked amino group;
(b) reacting the first peptide with an amino group activating agent, thereby producing an activated amino group at the N-terminal of the first peptide;
(c) reacting the activated first peptide with a second peptide having a molecular structure similar to a fragment of the protein, the second peptide having a C-terminal cysteine bearing an unblocked thiol group but not having any other unblocked thiol groups, thereby coupling the N-terminal of the first peptide to the C-terminal of the second peptide;

(d) reacting the resultant compound, in a form having an unblocked amino group at its N-terrninal but no other unblocked amino groups, and no unblocked thiol groups with an amino-group activating agent, thereby producing an activated amino-group at the N-terminal of the resultant compound;
(e) reacting the activated compound produced in step (d) with a further peptide having a molecular structure similar to a fragment of the protein, this further peptide having a G-terminal cysteine bearing an unblocked thiol group but not having any other unblocked thiol groups, thereby coupling the activated N-terminal of the reactivated compound produced in step (d) to the C-terminal of the further peptide; and (f) repeating steps (d) and (e) until the desired polymer length has been achieved.

13. A method according to claim 12 wherein each of said first, second and further peptides has a molecular structure similar to a fragment of Human Chorionic Gonadotropin.

14. A method according to claim 12 wherein said first, second and further peptides have the chemical configuration:
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-Cys said peptides having the amino groups on their lysine residues blocked.

15.A method according to claim 12 wherein said first, second and further peptides have the chemical configuration:
Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser-Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln-Cys said peptides having the amino groups on their lysine residues and the thiol groups on their nor-terminal cysteine residues blocked.

16. A process for preparing an antigen for provoking the formation, in the body of an animal, of antibodies to a protein which is not endogenous or substantially immunogenic to the animal, the process being characterized by:
procuring the protein, or a peptide having a sequence corresponding to at least part of the sequence of the protein, the protein or peptide having a sulfhydryl group thereon;
treating the protein or peptide with an activator of the structure wherein X represents a non-reacting group comprising substituted or unsubstituted phenyl or C1-10 alkylene moieties, or a combination thereof, or an amino acid chain, so as to cause reaction of the maleiimide group of the activator with the sulfhydryl group on the protein or peptide; and treating the resulting activated protein or peptide at slightly alkaline pH with a carrier moiety biologically foreign to the animal and selected having a size sufficient to elicit antibody response following the administration thereof into the body of the animal, thereby producing the antigen.

17. A process for preparing an antigen for provoking the formation, in the body of an animal, of antibodies to a protein which is not endogenous or substantially immunogenic to the animal, the process being characterized by:
procuring a carrier which is biologically foreign to the animal, has a size sufficient to elicit antibody response following the administration thereof into the body of the animal and does not have a sulfhydryl group but has an amino group;

treating the carrier under neutral or acid conditions with an activator of the structure wherein X represents a non-reacting group comprising substituted or unsubstituted phenyl or C1-10 alkylene moieties, or a combination thereof, or an amino acid chain, so as to cause reaction of the activator with the amino group on the carrier; and treating the resultant activated carrier with the protein, or a peptide having a sequence corresponding to at least part of the sequence of the protein, the protein or peptide having a sulfhydryl group thereon, thereby reacting the maleiimide group of the activator with the sulfhydryl group on the protein or peptide and producing the antigen.

18. A process for preparing an antigen for provoking the formation, in the body of an animal, of antibodies to a protein which is not endogenous or substantially immunogenic to the animal, the process being characterized by:
procuring a carrier which is biologically foreign to the animal, has a size sufficient to elicit antibody response following the administration thereof into the body of the animal and does not have a sulfhydryl group but has an amino group;

treating the carrier with an activator present as an active ester of chloro-, dichloro-, bromo- or iodo-acetic acid so as to cause reaction with the amino group on the carrier; and treating the resultant activated carrier with the protein, or a peptide having a sequence corresponding to at least part of the sequence of the protein, the protein or peptide having a sulfhydryl group thereon, thereby reacting the activated carrier with the sulfhydryl group on the protein or peptide and producing the antigen.
19 A process for preparing an antigen for provoking the formation, in the body of an animal, of antibodies to a protein which is not endogenous or substantially immunogenic to the animal, the process being characterized by:
procuring the protein, or a peptide having a sequence corresponding to at least part of the sequence of the protein, the protein or peptide not having a sulfhydryl group but having an amino group thereon;
treating the protein or peptide with an activator present as an active ester of chloro-, dichloro-, bromo- or iodo-acetic acid, thereby reacting the active ester with the amino group on the protein or peptide to produce a halomethyl alkylating group containing moiety: and treating the halomethyl alkylating group containing moiety with a carrier which is biologically

Claim 19 cont'd (1) foreign to the animal, has a size sufficient to elicit antibody response following the administration thereof into the body of the animal and contains a sulfhydryl group, thereby reacting the sulfhydryl group with the halomethyl alkylating group to form the antigen.

20 . A modified antigen for use in fertility control in an animal comprising an antigen derived from the zona pellucida or from sperm, or a peptide having a sequence corresponding to at least part of the sequence of such a zona pellucida or sperm antigen, which antigen or peptide has been chemically modified outside thebody of the animal, the modified antigen, after administration into the body of the animal having a greater capacity to provide the formation of antibodies than theunmodified antigen from which it is derived.

21 . A modified antigen according to claim 20 wherein said chemical modi-fication comprises polymerization of said antigen or peptide by connection of said antigens or peptides to one another via residues of a bi-functional reagent used to couple the antigens or peptides together.

22 . A modified antigen according to claim 20 wherein said chemical modi-fication has been effected in a manner as defined in claim 5 or 12

23 . A modified antigen according to claim 20 wherein said chemical modi-fication has been effected in a manner as defined in claim 16 or 17.

24 . A modified antigen according to claim 20 wherein said chemical modi-fication has been effected in a manner as defined in claim 19.

25. A vaccine for provoking the formation, in the body of an animal, of antibodies to a protein, the vaccine comprising a modified polypeptide according to claim 1 or 2 and a vehicle comprising a mixture of mannide monooleate with Squalane and/or Squalene.

26. A vaccine for provoking the formation, in the body of an animal, of antibodies to a protein, said vaccine comprising an antigen according to claim 20 and a vehicle comprising a mixture of mannide monooleate with Squalane and/orSqualene.