NO174183B - Process for preparing a modified antigen - Google Patents

Description

The present invention relates to a method for producing a modified antigen for use in animal fertility control.

In GB-PS 1,567,764 and US-PS 4,302,386 it is described that hormones or other proteins found naturally in an animal can be chemically modified outside the animal's body (a term used here to include humans) so that, by injection into animals of the modified hormones or other proteins induces the formation of antibodies that react not only with the chemically modified hormone or other protein, but also with the unmodified hormone or protein found naturally in the animal, thereby reducing the level of natural hormone or other protein in the animal's body. Instead of using the whole hormone or another protein, the above-mentioned patents also describe the use of natural or synthetic fragments of such proteins in this so-called "isoimmunization" technique.

The above-mentioned patents mainly concern the use of their isoimmunization technique in fertility control so that the protein that is modified is a reproductive hormone such as follicle-stimulating hormone FSH, leutinizing hormone LH, human placental lactogen HPL, human prolactin HP and human chorionic gonadotropin HCG, or a peptide with an amino acid sequence which corresponds to some of these hormones. However, the above-mentioned patents also describe the applicability of the isoimmunization technique to other protein hormones, for example: 1. Gastrin for the treatment of Zollinger-Ellison syndrome; 2. Angiotension II for the treatment of hypertension; 3. Growth hormone and somatomedian for the treatment of diabetes and related micro- and macro-vascular diseases; 4. Parathyroid hormone for the treatment of kidney stones; 5. Insulin and glucagon for the treatment of hyper-insulinone; 6. Thyroid-stimulating hormone for the treatment of hyperthyroidism; 7. Secretin for treating irritable bowel syndrome.

The above-mentioned patents also describe the use of modified polypeptides derived from chordionic gonadotropin, LH or FSH, or a fragment thereof, for use in the treatment of certain carcinomas.

The main technique of the chemical modification described in the above-mentioned patents is the coupling of the hormone, another 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 above-mentioned patents also describe polymerization of the hormone, other protein or fragment thereof, by reaction with a bifunctional organic reagent (for example, a bifunctional imidoester such as dimethyl adipimidate, dimethyl superimidate or diethyl malonimidate) or dimerization by oxidation of a thiol group to form a disulfide bridge . All of these types of chemical modifications have deficiencies. Dimerization of hormone, other protein or fragment via a disulfide bridge does not introduce exogenous material into the animal to be treated but, because the chemically modified antigen introduced into the animal is only a dimer of a hormone, other protein or fragment thereof, which itself does not is immunogenic to the animal, such dimers are rarely successful in generating usable levels of antibodies. The modified polypeptides produced using carriers contain a very high proportion of non-endogenous material and will usually force the formation of significant amounts of antibodies against the carrier as well as against the hormone, other protein or fragments thereof. Although the generation of antibodies against the carrier may sometimes be useful, for example a vaccine based on an HCG fragment linked to diphtheria toxoid and intended for fertility control has the incidental advantage of also providing protection against diphtheria, there are many occasions where it is not desirable to force the formation of relatively large amounts of antibodies against the carrier, if, for example, one wishes to use a vaccine containing a modified polypeptide to treat a patient with a carcinoma or a serious viral infection, it may be desirable to avoid overloading the patient's immune system by to challenge it not only with the hormone, other protein or fragment thereof against which the antibodies are desired, but also with the carrier.

In theory, perhaps the most promising of the modification techniques described in the above-mentioned patents is polymerisation of the hormone, other protein or fragment thereof, by reaction with a bifunctional reagent. In theory, this technique has the advantage of introducing a relatively small proportion of non-endogenous material into the animal to be treated (and even this relatively small proportion of non-endogenous material can be chosen so that it is not highly immunogenic) while still providing a modified polypeptide large enough to be highly immunogenic. Unfortunately, experiments have shown that direct application of the bifunctional

organic reagent polymerization technique on most

hormones, other proteins or fragments thereof of practical interest, give very complicated mixtures of modified polypeptides correspondingly complicated immunogenic properties. Furthermore, the immunogenic properties of the polymerized polypeptides thus produced are not easily reproducible, but such reproducibility is essential in any material intended for pharmaceutical use, because the necessary safety and effectiveness tests cannot be carried out on a non-reproducible material.

It has now been found (although this knowledge is not embodied in the published literature) that the reason for the very complicated immunogenic properties and the lack of reproducibility present in polymers prepared by the bifunctional organic reagent polymerization technique is, regardless of the use of a bifunctional reagent, extensive cross-linking of the hormone, other protein or fragment thereof, since such cross-linking is probably due to the presence of free amino, thiol, carboxyl and possibly other groups (the exact groups involved depend of course on which groups the bifunctional organic reagent is intended to react with) at non-terminal positions in the hormone, protein or fragment thereof. Such cross-linking gives branching and three-dimensional structure in the resulting polymers. Not only does the relatively random cross-linking that thus occurs make the structure of the polymer itself unpredictable and not reproducible, but such cross-linking can also change the tertiary structure and shape of the hormones, other proteins or fragments thereof that are polymerized, which thus affects the immunogenic properties .

Accordingly, the applicant has concluded that in order to produce usefully modified polypeptides by bifunctional organic reagent polymerization techniques, it is essential to work in such a way that coupling of the polypeptide fragments to be polymerized occurs only at or near the ends of the fragments, thus giving a truly linear polymer substantially free of non-linear polymers of the fragments.

It has also been found that the isoimmunization technique described in the above-mentioned patent can be successfully extended to proteins which are neither endogenous nor significantly immunogenic to those 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.

According to this, the present invention relates to a method for producing a modified antigen for use in animal fertility control, comprising a zona pellucida or sperm antigen, or a peptide with a sequence corresponding to at least part of the sequence of such a zona pellucida or sperm antigen, which antigen or peptide is chemically modified outside the animal's body by either linking at least one of the antigens or peptides to a carrier, or linking a number of the peptides to each other to form a linear peptide, the modified antigen after administration to the animal's body having a greater capacity to induce the formation of antibodies than the zona pellucida or the sperm antigen before the chemical modification, and the method is characterized by what appears in the characterizing part of the claim.

As already mentioned, the linear polymeric modified polypeptides comprise inert polymers of polypeptide fragments essentially free of non-linear polymers. By saying that the polypeptide fragments used in the linear polymeric polypeptide have a molecular structure corresponding to a fragment of the protein against which antibodies are to be provoked, it is not necessarily meant that the entire amino acid sequence for each fragment must exactly correspond to a part of the protein against which antibodies are to be elicited, for example in certain cases certain substitutions of amino acids may be possible without affecting the immunogenic character of the fragment.

For example, the above-mentioned TJS-PS 4,302,386 describes a polypeptide called structure IV which is believed to be derived from the β-subunit of ECG but in which the cysteine residue in position 110 is replaced by oc-aminobutyric acid. In particular, when it is desired to provoke the formation of antibodies against an endogenous protein, the polypeptide fragments used to form the correct modified polypeptide must have a molecular structure corresponding to a fragment of the endogenous protein whose antibodies are to be formed, this does not, however, exclude the possibility that such fragments can really be derived from a protein of a different type because many proteins are either identical between species or differ so little in amino acid sequence that a significant cross-reactivity exists between antibodies to the corresponding proteins in the two species. For example, as noted below, porcine zona pellucid enzymes when injected into humans will produce antibodies that show significant activity against human zona antigens. If, for example, according to this one wishes to form a modified polypeptide to provoke the formation of antibodies in humans against zona pellucida antigens, suitable polypeptide fragments can be prepared from porcine zona pellucida antigens. Furthermore, the fragments used in the linear polymeric polypeptides can incorporate frequencies of amino acid without counterpart in the sequence of protein from which the fragment is derived. Furthermore, for example, the previously mentioned US-PS 4,302,386 describes certain polypeptide fragments designated structure (IV), (VIII), (IX), (X) and (XIV) which are all derived from the β-subunit of HCG but which incorporate spacer sequences comprising more proline residues.

It may at first seem surprising that a linear polymer of a polypeptide whose monomeric form is effectively non-immunogenic to an animal can be immunogenic to the same animal. Without wishing to be bound by theory, it is assumed that the increase in immunogenicity by polymerization is due to an increase in the physical size of the molecules, which enables them to be recognized more easily by the animal's immune system. It can be shown that at least some monomeric polypeptides are very weakly immunogenic and cause the animal's immune system to produce detectable amounts of antibodies, which amounts are however far too small to be effective. The immune system is not well adapted to recognize molecules such as the small polypeptides when the polypeptides are present in non-polymeric form.

It will be clear to the person skilled in the art that the polypeptide fragments used in the linear polymeric polypeptides can either be natural (ie derived from natural proteins) or can be produced synthetically. Obviously, a synthetic polypeptide will act in the same way as a naturally occurring one in that the immune system of the animal to which the modified polypeptide is administered will react in exactly the same way to both. Furthermore, the fragments in each linear polymer can be the same or different and need not necessarily be derived from the same protein.

The linear polymeric polypeptides produced according to the invention can be used to provoke the formation of antibodies to both endogenous and non-endogenous proteins by using suitable polypeptide fragments to form the polymers.

(The term "endogenous" is used herein to denote a protein native to the species being 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 considered to be endogenous to a sow, although obviously such a sperm antigen will usually be present in a sow's body. Further details of the use of non-endogenous protein fragments and which can be used in linear polymeric polypeptides are provided below in connection with the discussion of the antigens of the invention. With regard to endogenous proteins, fragments of which can be used to form the linear polymeric polypeptides, it can generally be stated that such endogenous proteins can include any of the proteins mentioned in the previously indicated US-PS 4,302,386. Furthermore, it should be pointed out that the use of the linear polymeric peptides based on growth hormone and/or somatomedian has not been confirmed in diabetic patients. Thus, the linear polymeric polypeptides based on these two hormones can be used to treat non-diabetic patients such as people suffering from acromegaly, who have elevated levels of growth hormone and/or somatomedian.

Particularly preferred linear polymeric polypeptides are those formed from fragments with a molecular structure corresponding to a fragment of human chorionic gonadotropin and especially corresponding to a fragment of the 3-subunit thereof. Two particularly preferred fragments for use in the linear polymeric polypeptides of the invention are:

Other HCG-derived fragments which can be used in the linear polymer polypeptides include those of the structure II-VIII, Villa and IX-XIV as indicated below, further details regarding the immunological nature of these fragments being given in the above-mentioned US-PS 4,302 .386:

Obviously, if it is desired to use one of the above-mentioned peptides which lacks a C-terminal cysteine as a second or later fragment in the invention's production of linear polymeric peptides, it will be necessary to add a C-terminal cysteine to the peptide; suitable methods for doing this are, of course, well known to those skilled in the art of polypeptide synthesis. Furthermore, some of the above-mentioned peptides will of course require blocking of non-terminal amino and/or thio groups before use.

The linear polymeric polypeptides based on HCG-derived fragments are usable in exactly the same way as the corresponding modified polypeptides where the same fragments are linked to carriers as described in the aforementioned US-PS 4,320,386. After the time of this patent, further discoveries have been made regarding the connections between HCG and immunologically similar substances and certain carcinomas. It appears (although the invention is not limited by this assumption) that certain carcinomas exudate chorionic gonadotropin or an immunologically equivalent material on their surface and therefore present a surface to the host immune system which superficially appears to be formed of material endogenous to the host and which is thus relatively non-immunogenic. Because of this known association between certain carcinomas and chorionic gonadotropin or chorionic gonaditropin-like substances, the HCG-derived linear polymeric polypeptides are useful for the treatment of HCG and chorionic gonadotropin-associated carcinomas. The same linear polymeric polypeptides are also of course usable for the fertility control as described in the previously mentioned US patent.

The fragments used in the linear polymeric polypeptides may also include fragments of zona pellucida and sperm antigens as discussed in detail below.

As already mentioned, the linear polymeric peptides are produced by polymerization of fragments of the protein to which the antibodies are to be provoked rather than the entire protein itself. This use of fragments instead of a whole protein has important advantages (and similar advantages are ensured by the use of fragments in the antigens and modified antigens produced according to the invention as discussed in detail below). It is well known by those skilled in the immunological field (see, for example, W.R.Jones, "Immunological Fertility Regulation", p. 11 et seq.) that one of the greatest potential risks of a vaccine, especially a contraceptive vaccine, is cross-reactivity with non-target antigens, enabling what is essentially an artificially induced autoimmune disease to cause immunopathological damage in, and/or loss of function of, the tissue bearing the cross-reactive substances.

Two possible mechanisms for such cross-reactivity are:

(a) the presence of shared antigenic determinants; a complex protein may contain components (amino acid sequences) identical to those present in the non-target antigen; (b) steric overlap between non-identical but structurally related parts of the protein and the non-target antigen.

Obviously, the changes given by both of these cross-reactivity models can be reduced by using in the linear polymeric polypeptides antigens and modified antigens according to the invention, a fragment of a complex protein instead of the whole protein. Because the fragment has a simpler sequence or the protein from which it is derived, there is less chance of shared antigenic determinants or steric

overlap with non-target proteins. In particular, cross-reactions can often be avoided by using fragments derived from part of the target protein (that is, the protein to which the antibodies are to be provoked) which are not equivalent in sequence to the non-target but cross-reactive protein. As described in the above-mentioned US-PS 4,320,386, one of the main problems when provoking antibodies to HCG is cross-reactivity of HCG antibodies with LH, this cross-reactivity being at least largely due to the identity of the amino acid sequence between LH and 1-110 the amino acid sequence in the β-subunit of HCG. Accordingly, when it is desired to form an HCG-derived linear polymeric polypeptide, the fragment used is preferably one with a molecular structure corresponding to part or all of the 111-145 sequence of the β-subunit HCG because it is only this 111- 145 sequence of p-HCG that differs from the corresponding LH sequence. However, research suggests that the fragments used in the linear polymeric polypeptides may contain sequences corresponding to the 101-110 sequence in p-HCG common to p-HCG and LH without inducing the formation of antibodies reactive to LH. Thus, fragments containing part or all of this common 101-110 sequence can be used in the linear polymeric polypeptides without causing cross-reactivity with LH. For example, structure II above 111-145 represents the amino acid sequence of p<->HCG. If desired, a fragment with the 101-145 amino acid sequence in p<->HCG can be used instead of the fragment with structure II in the linear polymeric polypeptides without significantly affecting the activity of the linear polymers and without causing cross-reactivity with LH.

Polymerization of the fragments to form the linear polymeric polypeptides can be carried out in any manner to link peptide fragments to form linear polymers thereof. The linear polymeric polypeptides can be divided into two distinct types. In the first type, individual peptide fragments are under head > tail by peptide bonds so that the entire polymer perceives only the fragments themselves without containing external material. Although such pure polymers have the advantage of not introducing external material into the body of the animal being treated, they are usually too expensive to be practical because the necessary fragments (whether produced by total synthesis or cleavage of a natural protein) in themselves are themselves very expensive and substantial losses occur during the polymerization. Furthermore, head > tail coupling of the fragments without intervening residues can give immunological determinants that have no counterpart in the unpolymerized fragment. If, for example, the fragment described above comprising the 105-145 sequence of HCG is polymerized by means of peptide bonds, a sequence: Pro-Ile-Leu-Pro-Gln-Asp-His-Pro-Leu-Thr will be produced at each joining between adjacent separated fragments, and this sequence can provoke the formation of antibodies that would not be produced by the fragment itself, and which may be undesirable. Because there is no "puncture" that can tell the immune system in the recipient animal where one fragment begins and another ends, in other words, the animal's immune system can inadvertently start reading the wrong residue and make unwanted antibodies by running the sequences off next to each other lying fragments together. For this reason, it is generally not recommended to use linear polymers where the fragments are linked together with peptide polymers, although such linear polymers may be useful in certain cases.

Various methods for linking polypeptide fragments via peptide bonds are known to the person skilled in the art. For example, a fragment to be linked can have its C-terminal carboxyl group blocked (for example by esterification) and be reacted with the other fragment that has its C-terminal amino group blocked, while its carboxyl group is activated by means of an activating agent. Obviously, blocking of non-terminal amino and -carboxyl groups may be necessary. Furthermore, as is well known to those skilled in the art, it can be advantageous to bind one end of the polymer thus produced to a carrier such as a polystyrene resin carrier whereby the polymer is only released from the carrier after the polymerization is complete.

In the second type of linear polymer polypeptides, the polypeptide fragments are connected to each other by means of residues derived from a bifunctional reagent which is used to effect polymerization of the fragments so that the final linear polymer is an alternating linear polymer of polypeptide fragments and coupling reagent residues. Although this type of polymer necessarily introduces certain external substances to the animal being treated, the proportion of external substances can be made considerably lower than it would be if the fragments were linked to a large carrier such as a diphtheria toxoid. The coupling agent, which is necessarily a bifunctional coupling agent, to give a truly linear polymer, can be chosen so that the residue it leaves in the polymer is not highly immunogenic (so that it does not impose a large burden on the immune system of the recipient animal, such as a large carrier molecule such as diphtheria toxoid would have done), and the presence of these residues in the polymer has the advantage of essentially eliminating spurious immunological determinants formed by joining the head of a fragment with the tail of the adjacent fragment as discussed above.

To discuss that a set linear polymer is produced during the polymerization process, a terminal of a first polypeptide fragment is reacted with the bifunctional coupling reagent such that the coupling reagent reacts with a group present at or near the terminal of the fragment; for example, the coupling reagent may react with an N-terminal amino group, a C-terminal carboxyl group, or a free thiol group present on a C-terminal cysteine. Obviously, the nature of the coupling agent used determines which group in the peptide reacts. To avoid any cross-linking and to ensure a reproducible result, it is important that only one site on the first fragment is available for reaction with the coupling agent so that the coupling agent can only bind to the first fragment on its one site. As the person skilled in the art will know, if it is desired to use a fragment containing more than one group which can react with the coupling agent, the excess sites can be blocked by binding to suitable protecting groups. The product formed by reaction of the first fragment with the coupling agent is then reacted with a second fragment (which may be the same or different from the first fragment) with a single site available to react with the second reactive group in the bifunctional bicoupling agent, thereby linking the first and second fragments with a residue derived from the linker. After any necessary purification of this dimeric product, this is then converted into a further proportion of a coupling agent which may be the same or different in relation to that which was used to effect the first coupling, thereby converting the free terminal of either the first or second fragment with the coupling agent. Naturally, it is important to ensure that only one site on the dimers is available for coupling with the coupling agent and, as will be apparent to the person skilled in the art, blocking or unblocking of potentially reactive groups on the dimeric polypeptide may be necessary. The reaction product of dimeric polypeptide with the coupling agent is then reacted with a third fragment having only a single site available for reaction with the remaining reactive group of the coupling agent to thereby yield a linear polymer containing three polypeptide fragments. Obviously, this process can be repeated until the desired size of the linear polymer is achieved.

It will be obvious to the person skilled in the art that the bifunctional coupling reagents used to prepare the linear polymeric polypeptides should be asymmetric, i.e. they should have two functional groups that react with different groups on the fragments that are polymerized because if, for example, attempts were made to reacting a bifunctional cross-coupling reagent with two functional groups that both reacted with amino groups of a first fragment with a single amino group would dimerize at least some of the first fragment via a residue derived from the bifunctional cross-coupling reagent. Such dimerization can in theory be avoided by using a very large excess of reagent, but in practice it is undesirable to run the risk of producing even a small proportion of dimers. In a similar way, at later stages in the polymerization process, it will be even less desirable to use symmetrical coupling reagents and thereby run the risk of dimerization of the partially formed polymers that have already been produced. In the preferred process for the production of linear polymer polypeptides as already described, the polymer chain is started with a first peptide without an unblocked thiol group and with an unblocked amino group only at the N-terminus (peptides containing thiol groups and/or amino groups other than the N-terminus can of course be used if all these thiol and amino groups are blocked with any conventional blocking agent). This first peptide is then reacted with an amino group activating agent, a preferred activating agent for this purpose is 6-maleimidocaproic acid a hydroxysuccinimide ester (MCS); (reaction of the peptide with a reagent is carried out optimally at pH 6.6). The activator reacts with the amino group at the N-terminus of the first peptide and forms an activated form of the first peptide; in the case of MCS, it is the ester portion of the reagent that reacts with the N-terminal group of the peptide. It is usually necessary to remove excess activator before continuing the manufacturing process. When excess activator is removed, the activated first peptide is converted into a second peptide with a C-terminal cysteine in a reduced state (that is, with an unblocked 3-thiol group), thereby causing coupling of the N-terminus of the activated first peptide to the C-terminus of the second peptide via an activator residue. Preferably, the resulting dimer is then purified as described in greater detail below. The dimer is then again reacted with an amino group activating agent and then with the second part of the second peptide or with a third peptide to thereby form a trimer. This procedure is repeated until the desired chain length is achieved.

In order to ensure reproducible responses from the immune systems of the treated animals, it is important that the linear polymeric polypeptides are used in the form of pure polymers where all the molecules contain the same number of fragments. In order to obtain such pure polymers, effective purification should be carried out after each polymerization step in the polymerization process. Due to the close chemical similarity between polymers containing different numbers of fragments, chemical purification is ineffective so that purification must be carried out by physical methods. Gel filtration can be used if desired, but the preferred purification method is reverse phase with high-pressure liquid chromatography, preferably using a molecular sieve as solid phase.

In this method for producing linear polymers, the first and second peptides can be identical in chemical configuration except that in the first peptide the C-terminal cysteine has a blocked thiol group. The first two preferred fragments for forming linear polymeric polypeptides for forming antibodies against HCG as mentioned above can be described as (111-145)-Cys and (105-145)-Cys, where the numbers indicate the amino acid sequence of the p<->subunit of HCG . It will be clear that when these fragments are to be used in the preparation of linear polymeric peptides by the method just described, the lysine residue in position 122 must have its amino group blocked, and in the case of the (105-145)-Cys fragment the non-terminal cysteine must in position 110 have their thiol group blocked, preferably with an acetamido-methyl group.

The linear polymeric peptides preferably contain from 4 to 14 fragments.

The modified antigens produced by chemical modification of non-endogenous proteins will now be described in greater detail. As the person skilled in the art will be aware, there are numerous pathogens and corresponding substances that are known and which are not endogenous in animals, which are capable of producing harmful effects in the animal's body, but which are not immunogenic to the animal in the sense that the introduction of pathogens or the other substance in the animal's body does not cause the animal's immune system to produce the quantities of correct antibodies that are necessary for the animal's immune system to be able to destroy the pathogen or the corresponding material. For example, herpes simplex type II virus is capable of producing a number of harmful effects in humans including the formation of painful lesions in the genital areas. Although this virus, like most viruses, has protein included in its structure, the viral protein is not strongly immunogenic in most people, so that only approx. 50$ of infected people produce antibodies against this virus. The lack of immune response to this virus in many people means that the virus can remain in the infected people for at least several years and this persistence of the virus in the infected individuals not only causes them to suffer from recurrent attacks of painful symptoms caused by this virus but also makes them long-term carriers of the virus. The persistence of the virus in infected animals is one of the factors that is largely responsible for the epidemic conditions herpes simplex infections have led to in several countries. By producing, according to the invention, an antigen, derived from a protein with 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 to enable it to produce larger amounts of antibodies against herpes simplex virus. Not only would this stimulation of immune systems reduce the appearance of symptoms associated with herpes simplex infection, but would also support control of the spread of the virus. In the same way, the immune response in humans and other animals against viruses such as colds, influenza or others can be increased by, according to the invention, producing modified antigens based on peptides with frequencies corresponding to the viral proteins in suitable viruses. If, as it appears, a virus is responsible for acquired immunodeficiency syndrome, AIDS, a modified antigen can also be used to provide immunity against this disease.

In general, the methods used according to the invention for the production of antigens based on non-endogenous substances such as viral proteins or peptides corresponding to parts thereof, are the same as those used to modify endogenous proteins or fragments thereof, as mentioned in the previously mentioned US-PS 4,302 .386. However, it should be understood that the preferred methods used to modify non-endogenous substances may differ in certain respects from those used to modify endogenous peptides. Because in general the non-endogenous peptide will provoke at least a limited immune response from the animal to which the antigen is administered, the requirements for modifying the non-endogenous protein or peptide to produce an antigen according to the invention will tend to be less stringent than those for the modification of an endogenous and entirely non-immunogenic protein or peptide. However, because the non-endogenous protein or peptide is modified to increase the immunogenic effect in the animal to which it is administered, it will generally be desirable that the carrier used to modify the non-endogenous protein or peptide (if chemical modification is to be carried out by coupling of the non-endogenous protein or peptide to a carrier rather than forming a linear polymeric polypeptide) to provide the antigen, a substance which itself provokes a strong response from the animal's immune system. For example, the carrier may be a bacterial toxoid such as diphtheria toxoid or tetanus toxoid.

The antigens are produced according to the invention as mentioned above by chemically modifying a protein that is not endogenous or immunogenic against the animal being treated, or a peptide with a sequence corresponding at least partially to the sequence of the protein, as this chemical modification is carried out outside the animal's body. The chemical modification can be accomplished by linking the protein or peptide to a carrier, by polymerizing the peptide to form a linear polymeric polypeptide, or by any other modification technique that renders the protein or peptide sufficiently immunogenic.

Preferred techniques for chemical modification of protein or peptide by coupling to a support include the following (further details for optimizing techniques for each of the following coupling reactions are described in the above-mentioned US-PS 4,302,386):

a. Treatment of a protein or peptide with a sulfhydryl group on it with an activator of the structure:

where X means a non-reacting group comprising substituted or unsubstituted phenyl or C 1-10 alkyl moieties, or a combination thereof, or an amino acid chain to achieve reaction between the maleimide group in the activator and the sulphide group in the protein or peptide; and treating the resulting activated protein or peptide at slightly alkaline pH with a carrier moiety that is biologically foreign to the animal and selected at a size sufficient to elicit an antibody response upon administration thereof to the animal's body.

b. Under neutral or acidic conditions, treating a support having no sulfydryl group but an amino group, with a

activator as indicated above, to force reaction between the activator and the amino group on the carrier, whereby the carrier is biologically foreign to the animal to be treated and is of a size sufficient to produce an antibody response after administration of the compound to the animal's body; and treating the resulting activated support with the protein or peptide, which must have a sulfhydryl group, thereby reacting the maleimide group in the activator at the sulfhydryl group on the protein or peptide. c. Treating a carrier which is biologically foreign to the animal to be treated and with an amino group with an activator present as an active ester of chloro, dichloro, bromo or iodoacetic acid to thereby cause a reaction between the activator and the amino group on the carrier and treating the resulting activated carrier with protein or peptide, which must have a sulfhydryl group, thereby reacting the activated carrier with the sulfhydryl group in the protein or peptide. d. Treating a protein or peptide that does not have a sulfhydryl group but does have an amino group, with an activator present as an active ester of chloro, dichloro, bromo, or iodoacetic acid, and treating the resulting halomethyl alkylation group-containing moiety with a sulfhydryl group-containing carrier which is biologically advanced for the animal to be treated to thereby react the sulfhydryl group in the carrier with the halomethyl alkylation group.

It will be clear 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 many natural proteins containing cysteine residues, these residues are not present in their thio form containing a free SH group; instead, pairs of cysteine residues are aligned by means of disulfide bridges to form cysteine. Such disulphide bridges are very important for determining the protein's conformation. In most cases, the disulfide bridges present in the native form of the protein are easily reduced to pairs of SH groups by mild reducing agents under conditions that leave the remaining parts of the protein molecule unchanged. When it is desirable to produce free SE groups in the proteins in order to carry out the coupling reaction described above, according to this, an appropriate way to obtain such free SH groups is to cleave disulfide bridges which are naturally present in the protein or another polypeptide which it is desirable to connect.

Formation of free SH by reduction of disulfide bridges in naturally occurring forms of proteins can also cause cross-reactivity for the antibodies formed when a modified polypeptide, prepared from the protein, is injected into an animal. Frequently, an antibody recognizes its corresponding antigen not only by the amino acid sequence in the antigen but also by the conformation (shape) of the antigen. According to this, an antibody that binds very strongly to this protein or other polypeptide in its natural conformation can bind much less strongly if at all to the same protein or polypeptide whose conformation is plastically changed by breaking up the disulphide bridges. According to this, the breaking up of disulphide bridges in proteins or other polypeptides can be a basis for reducing the cross-reactivity between antibodies to antigens with the same amino acid sequence along the end of the molecule.

As mentioned above, chemically modified antigens derived from zona pellucida or sperm antigens, or peptides with a sequence corresponding to at least part of the sequence for such a zona pellucida or sperm antigen, are produced according to the invention. These modified antigens are useful for fertility control. Antigens from the zona pellucida (the outer envelope of the egg) when injected into female primates are known to produce antibodies with antifertilization effects including preventing sperm attachment to a penetration of the zona pellucida of the unfertilized egg, and preventing dispersion of the zona pellucida in the fertilized egg before implantation (such dispersion of the zona is obviously an essential requirement for implantation). For this see W.R.Jones, "Immunological Fertilitiy Regulation" pages 160 et seq. Such anti-fertility effects are believed to be due to the formation of an antibody antigen precipitate on the zona as this renders the zona unable to undergo the normal sperm-binding reaction and also renders the zona insensitive to the action of the proteases that are normally responsible for dispersing the zona.

An antifertility vaccine based on zona antigens is particularly attractive because the zona antigen seems to be relatively free of side effects on other tissues and because methods have been developed to produce porcine zona antigens in large quantities; Pig and human zona antigens show very good cross-reactivity. As in the case of antifertility vaccines based on the P-HCG antigen discussed above and in the previously mentioned US-PS 4,302,386, better results will be obtained by modifying a zona antigen or a fragment thereof to provide a modified antigen according to the invention.

Several antigens, especially sperm enzymes, known to be present in sperm, can be used in the modified antigens according to the invention, see W.R. Jones, op.eit., page 133 et seq. The most promising of such antigens is the lactate hydrogenase known as LDH-C4 or LDH-X. Although of course lactate dehydrogenases are present in other tissues, LDH-C4 is distinct from other lactate dehydrogenase isoenzymes and appears to be sperm specific. Furthermore, the enzyme is not of a highly specific nature, and methods for its isolation and purification are known. Here, too, the best results will be achieved by modifying LDH-C4 or a fragment thereof to produce a modified polypeptide according to the invention. Several natural peptide fragments of LDE-C4 have been prepared, sequenced and shown to "bind to antibodies against the stem molecule. (see E.Goldburg, "LDGH-X as a sperm specific antigen" in T.Wegmann, and T.J.Gill (eds.) Reproductive Immunology , Oxford University Press, 1981).

Although theoretically an antifertility vaccine based on sperm antigens may be useful in males, the likelihood of testicular damage makes it more likely that such a vaccine will find its applicability in females. Circulating antibodies in the female's bloodstream are known to penetrate the genital fluids; for example, experiments on baboons with vaccines based on peptide of structure XII above conjugated with tetanus toxoid have shown the presence of HCG antibodies in the genital fluids. However, a potential problem with any vaccine based on sperm antigens is maintaining a sufficiently high level of antibody in the female genital fluids to complex with the large amounts of sperm involved.

The techniques used for the chemical modification of zona pellucida or sperm antigens, or the peptide derived therefrom, to form the modified antigens produced according to the invention, include all the techniques described in the previously mentioned US-PS 4,302,386 and also those described above techniques. Thus, the antigen or peptide may be linked to a carrier such as a tetanus toxoid or diphtheria toxoid, or may be polymerized to form a linear polymeric polypeptide. The preferred techniques for forming such linear polymeric polypeptides have already been discussed above while the preferred techniques for coupling antibodies or peptides to carriers are the same as those for coupling non-endogenous proteins or peptides, as already described above.

The obtained compounds can be used in connection with a vaccine containing a modified polypeptide, antigen or a modified antigen and a carrier comprising a mixture of mannide monooleate with squalane and/or squalene. This carrier has been found to have the effect of increasing the amount of antibodies elicited by the linear polymeric peptide, antigen, or modified antigen, when the vaccine is administered to an animal. In order to further increase the amount of antibodies that are formed when administering the vaccine, it is advantageous to incorporate an immunological adjuvant into this. The term "adjuvant" is used in its normal meaning for the expert in the immunological field, namely as a substance that increases the total immune response in the animal to which the vaccine is administered, that is to say that the adjuvant is a non-specific immunostimulator. Preferred excipients are muramyl dipeptides, in particular: NAe-nor Mur-L.Ala-D.isoGln;

NAc-Mur-(6-O-stearoyl)-L.Ala-D.isoGln; or

N Glycol-Mur-L. Abu-D.isoGln.

The vaccines can be administered parenterally to the animals to be protected; the usual routes of administration for the vaccine are intramuscular and subcutaneous injection. The amount of vaccine used will of course vary depending on various factors including the treatment conditions and the severity of the condition. However, generally unit doses of 0.1-50 mg in large mammals administered 1 to 5 times at intervals of 1 to 5 weeks give satisfactory results. Primary immunization can also be followed by "booster" immunization at 1 to 12 month intervals.

To prepare the vaccines, it is convenient to first mix the linear polymer peptide, antigen or modified antigen, with the muramyl dipeptide (or another excipient) and then emulsify the resulting mixture in mannide monooleate/squalene or squalane carrier. Squalane is preferred over squalane for use in the vaccines and is preferably used approx. 4 parts by volume of squalene and/or squalane per parts by volume mannide monooleate.

The animals treated with the linear polymeric peptide, antigen, or modified antigen include both humans and other animal species. The following example can be given as an illustration to show details of the production and use of a linear polymer peptide according to the invention.

The invention shall be illustrated in more detail with reference to the following example.

Example

Fragment A described above (a fragment with an amino acid sequence corresponding to the 105-145 sequence of beta HCG, with a cysteine residue attached to the C-terminus) was polymerized to obtain a hexamer. A first part of fragment A had both its thiol loops (on the non-terminal cysteine in position 110 corresponding to the position in p<->HCG, and on its C-terminal cysteine) and its non-terminal amino group (on the lysine residue in the position corresponding to position 122 in p<->HCG) blocked. This blocked form of fragment A was reacted with the bifunctional organic coupling reagent) or amino group activating agent) MCS in a buffered aqueous solution at pH 6.6 to thereby react the ester moiety MCVS with the N-terminal amino group in the first portion of fragment A. the resulting 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 cysteine was an unblocked thiol group, thereby reacting the remaining functional group of MCS with the free thiol group of the second part of fragment A and thus produce a dimer in which the N-terminus of the first part of fragment A was linked to the C-terminus of the second part of fragment A via an MCS residue. This dimer was then purified by gel filtration. The polymerization was then repeated in the same manner until a hexamer of fragment A had been prepared. Because the purification following each polymerization step was carried out by gel filtration rather than by reversed-phase high-pressure liquid chromatography, the hexamer was undoubtedly somewhat impure and contaminated by traces of pentamer, tetramer and so on, so that the results in the animal tests as described below could not be expected to be as good as when producing a pure hexamer.

To test the effectiveness of this hexameric polypeptide in provoking the formation of antibodies against HCG, the hexamer was converted into a vaccine using: Complete Freunds' Adjuvent" and injected into five rabbits. Each rabbit was given three injections of the vaccine intramuscularly in three weekly intervals with each injection containing 0.5 mg of hexamer. Three weeks after the first injection, each rabbit was bled once a week and the level of antibodies against HCG in the blood determined. The following mean values for the antibody level were found (figures in parentheses represent the confidence margins, i.e. mean + or - standard error):

The results stated above show that even the impure hexamer used in these experiments was much more strongly immunogenic than the very weakly immunogenic fragments from which they were obtained.

Claims (1)

Process for the production of a modified antigen for use in animal fertility control, comprising a zona pellucida or sperm antigen, 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 is chemically modified outside the animal's body by either linking at least one of the antigens or peptides to a carrier, or linking a number of the peptides to each other to form a linear peptide, the modified antigen after administration to the animal's body having a greater capacity to force the formation of antibodies than the zona pellucida or the sperm antigen before the chemical modification, characterized in that 1) a number of the peptides are bound to each other by a chemical modification technique which comprises: a) reacting a first of the peptides with a bifunctional coupling agent while the first peptide is present in a form with only a single seat able to react with coupling mid let as this site is at or near one of the termini of the first peptide, thereby bringing one of the functional groups in the coupling agent into reaction with a site on the peptide; b) reaction of the product from step a) with a second of the peptides while the second peptide exists in a form with only a single site capable of reacting with the second functional group of the coupling agent, this site being located at or near one of the second peptide's termini to thereby form a dimer peptide in which the first and second peptides are connected to each other via a residue of the coblin agent; c) reacting the resulting peptide with a bifunctional coupling agent while the peptide is in a form with only a single site ready for reaction with the coupling agent whereby this site is located at or near one of the termini of the peptide to thereby bring one of the functional groups in the linker to react with a site on the peptide; d) reacting the product from step c) with a further one of the peptides while the further peptide is in a form with only a single site capable of reacting with the second functional group in the coupling agent, whereby the site is at or near the termini of the further peptide thereby giving a polymeric peptide in which the first, second and further peptides are linked together via residues of the coupling reagent; and e) repeating steps c) and d) until the desired polymer length is achieved, or 2) a number of peptides are connected to each other via a chemical modification technique comprising: a) reacting a first of the peptides, this first peptide not has some unblocked thiol group and has an unblocked amino group at the N-terminus but no other unblocked amino group, with an amino group activating agent to thereby provide an activated amino group at the N-terminus of the first peptide; b) reacting the activated first peptide with a second of the peptides, the latter having a C-terminal cysteine bearing an unblocked thiol group but having no other unblocked thiol groups, thereby linking the N-terminus of the first peptide to the C-terminus of the second peptide; c) reacting the resulting compound in a form with an unblocked amino group at the N-terminus but no other unblocked amino groups, and no unblocked thiol groups, with an amino group-activating agent to thereby provide an activated amino group at the N-terminus of the resulting compound; d) reacting the activated compound prepared in step c) with a further one of the peptides having a C-terminal cysteine bearing an unblocked thiol group but having no other unblocked thiol groups to thereby link the activated N-terminal to the reactivated compound prepared in step c) to the C-terminus of the additional peptide; and e) repeating steps c) and d) until the desired polymer length is obtained, or 3) treating the antigen or peptide bearing a sulfhydryl group with an activator of the structure wherein X means a non-reacting group comprising substituted or unsubstituted phenyl or C^_^q alkyl moieties, or a combination thereof, or an amino acid chain, thereby forcing reaction of the maleimide group in the activator with the sulfhydryl group on the antigen or peptide; and treating the resulting antigen or peptide at slightly alkaline pH with a carrier moiety that is biologically foreign to the animal and selectively has a size sufficient to elicit an antibody response after administration to the animal's body, thereby providing the modified antigen, or 4) to providing a carrier which is biologically foreign to the animal, has a size sufficient to elicit an antibody response after administration and which does not have a sulfhydryl group but does have an amino group; treating the support under neutral or acidic conditions with an activator having the structure wherein X means a non-reactive group comprising optionally substituted phenyl or C^_^ q alkyl moieties, or a combination thereof, or an amine chain, to cause reaction between the activator and the amino group on the support; and treating the resulting activated carrier with the antigen or peptide, the antigen or peptide having a sulfhydryl group, thereby reacting the maleimide group in the activator with the sulfhydryl group on the antigen or peptide and thereby providing the modified antigen, or 5) providing a carrier that is biologically foreign for the animal, has a size sufficient to elicit an antibody response after administration to the animal's body and which does not have a sulfhydryl group but an amino group; providing a carrier which is biologically foreign to the animal, has a size sufficient to elicit an antibody response after administration to the animal's body and which does not have a sulfhydryl group but an amino group; treating the support with an activator present as an active ester of chloro, dichloro, bromo or iodoacetic acid to thereby react with the amino group on the support; treating the resulting activated carrier with the antigen or peptide having a sulfhydryl group, thereby reacting the activated carrier with the sulfhydryl group on the antigen or peptide to thereby give the modified antigen, or 6) treating the antigen or peptide not having a sulfhydryl group but having an amino group, with an activator present as an active ester of chloro, dichloro, bromo or iodoacetic acid, thereby reacting the active ester with the amino group on the antigen or peptide to thereby yield a moiety containing a halomethyl alkylating group ; and treating this portion with a carrier which is biologically foreign to the animal, has a size sufficient to elicit an antibody response after administration and contains a sulfhydryl group, thereby reacting the sulfhydryl group with a halomethylalkylating group to thereby provide the modified antigen.