EP1087784A1 - Synthetic somatostatin immunogen for growth promotion in farm animals - Google Patents

Abstract

The invention provides peptides comprising somatostatin, or a sequence homologous to somatostatin, which is covalently linked to a helper T cell epitope and optionally to other immunostimulatory sequences. The present invention provides for the use of such peptides as immunogens to elicit the production in mammals of high titer polyclonal antibodies, which are specific to somatostatin. The peptides are expected to be useful in lowering somatostatin levels in mammals, thereby increasing growth rate and food utilization efficiency.

Description

SYNTHETIC SOMATOSTATIN IMMUNOGEN FOR GROWTH PROMOTION IN FARM ANIMALS

FIELD OF THE INVENTION This invention relates to a peptide composition that is useful as an immunogen for growth promotion in farm animals. The immunogenic peptides of the subject composition contain helper T cell epitopes (Th) which comprise multiple class II MHC binding motifs and have somatostatin at either the C- or N- terminus. The peptides, optionally, contain an invasin domain which acts as a general immune stimulator. The helper T cell epitopes and the invasin domain enable the immune response against the somatostatin self-peptide .

BACKGROUND OF THE INVENTION Recent understanding of neuro-endocrine and hormonal factors involved in growth, together with rapid advances in biotechnology, have provided potential avenues for the improvement of growth rate in a variety of farm animal species. A method for animal growth promotion that modifies the regulatory effects of growth hormones through immunoneutralization is a valuable alternative to the present methods which use antibiotics, anabolic steroids, and growth hormones as growth promoters. This application of antibiotics to animal husbandry is believed to be a driving force for the selection of antibiotic resistance in certain pathogenic bacterial species and has resulted in adverse consequences in the control of human infectious disease (Witte, Science, 1998; 279:996). The application of anabolic steroids promotes growth in farm animals, but is unpopular among consumers. Steroid use is constrained in Europe both by legislation and by public opinion (Buttery and Dawson, Proc Nutr Soc, 1990; 49:459). Recombinant DNA technology has enabled the production of metabolic hormones in large quantity for direct administration to animals. Such application can promote growth. It is, however, just one of the steps that need to be taken if the somatotrophic hormones are to be used as a means

SUBSTITUTE SHEET (RULE 25) of improving animal performance .

The principal somatotrophic hormones are: somatotropin (growth hormone, GH) , somatomedin-C (msulin-like growth factor 1, IGF-1), somatocrinm (GH releasing factor, GRF) and somatostatin (GH release inhibiting factor) . The major potential applications for the somatotrophic hormones are m growth and lactation, however the hormonal control of these phenomena relies upon a complex interaction between many different hormones (Spencer, Livest Prod Sci, 1985, 12:31). Thus, simple application of a single hormone may not be sufficient to enhance productivity.

It appears that GH plays two distinct roles. It has a positive effect in stimulating an increase in muscle protein synthesis (most, if not all, of which is mediated by IGF-1) and a potent catabolic effect by its ability to breakdown fats. The overall effects are an increase in the lean content of the carcass and a decrease in carcass fat, often an increase in growth, and universally an improvement in food conversion efficiency (Buttery and Dawson, Proc Nutr Soc, 1990; 49:459; and, Spencer, Reprod Nutr Develop, 1987; 27 (2B) :581) .

The effects on growth of GH administration are not reliable for its use in practice; probably because administration of exogenous hormones ignores the sensitive interrelationship between various hormones and ignores the possible effect of elevation of plasma levels on receptor populations . Effective application of GH requires frequent in_jections so as to provide a continuous physiologically effective serum level of hormone. This results in increased risk of upsetting these balanced relationships and causing adverse effects.

Thus, although pure preparations of growth hormones can be made m large quantities by recombinant DNA technology, this technology does not provide the modulated mechanism for delivery that is needed for the desired effect on growth. Moreover, the use of recombinant growth hormone m food animals, most recently m dairy cattle, has resulted m widespread consumer opposition and regulatory obstacles. The use of growth hormone to promote lean deposition in ruminants and other farm animals has not received regulatory approval within the European Economic Community. The regulatory and political situation is similar for the application of IGF-1 or GRF to promote growth in farm animal species.

As an alternative to increasing the levels of growth stimulating hormones it may prove equally, or even more, effective to remove endogenous growth inhibitors. The major inhibitor of total somatic growth is somatostatin. Somatostatin is a cyclic peptide of fourteen ammo acids and its structure is conserved across species. It is synthesized as a ninety-two ammo aciα prosomatostatm molecule from which six peptides including somatostatin itself and a twenty-eight ammo acid form of somatostatm are known to be derived (Reichlm, J Lab Clin Meα, -987; 109:320). Somatostatin inhibits the release of many gastro-mtestmal hormones as well as inhibits release of GH, insulin, thyroid hormones, thereby affecting both tne ability of the animal to absorb nutrients and its subsequent ability to direct these nutrients into tissue growth.

The use of a somatostatin antagonist has been found to stimulate growth n rats (Spencer et al . , Life Sc , 1985, 37:27), but this kind of treatment also suffers from the drawbacks of GH, IGF-1 and GRF n that it requires daily in_jections. A practical alternative is the use of the immune response to induce lmmunoneutralization of somatostatin. The use of the immune system to promote growth may be more acceptable to consumers and regulatory agencies than direct administration of hormones or synthetic steroids . The lmmunoneutralization of somatostatm by vaccine was first explored in sheep by Spencer et al . (Livest prod Sci, 1983, 10:469) . In a preliminary study using twin St. Kilda lambs, active immunization against somatostatin resulted m the treated lambs growing at 176% of the rate of the control lambs (Spencer et al . , Anim Prod, 1981, 32:376). Subsequent studies have been unable to reproduce this figure, but an improvement of 15-20% in growth rate is more usual. The somatostatin molecule is the same in all farm animal species, and it has now been shown that active immunization against somatostatin can stimulate growth commercially important breeds of sheep (Spencer et al . , Livest Prod Sci, 1983, 10:25; Laarveld et al., Can J Anim Sc , 1986, 66:77), cattle (Lawrence et al . , J Anim Sci, 1986, 63: (Suppl) 215), pigs and chickens (Spencer et al . , Pom Anim Endocr, 1986, 3:55) . A summary of successful examples demonstrating effective immunization against somatostatin for farm animals is shown in Table 1.

In addition to stimulating growth rate and leading to a 20% reduction m rearing time (Spencer, Reprod Nutr Develop, 1987; 27(2B):581), active immunization against somatostatin also has a beneficial effect on food conversion efficiency. In addition to the saving on food by virtue of more rapid growth, the animals actually utilize their food more efficiently during the growing period (Spencer et al . , Livest Prod Sci, 1983, 10:469), at least partly as a result of changes in gut motility (Fadlalla et al., J Anim Sci, 1985, 61:234; Faichney et al . , Can J Anim Sci, 1985, 64 (Suppl) 93).

The treatment does not have any marked effect on carcass composition (Spencer et al . , 1983, ibid.) but there are indications that, when killed at equal weights, treated animals may be leaner. Taken all experimental data together, active immunization appears to be a powerful, safe, and effective tool to enhance growth (Spencer, Pom Anim Endocr, 1986, 3:55) . Several lmmunogenic forms of somatostatin have been designed and tested as reported in the literature. For example, somatostatin has been conjugated with protein carriers to enhance lmmunopotency . However, protein carriers are too expensive for economical use in farm animals. Further, effective immunization with somatostatin depends on the conjugation site between somatostatin and the carrier. Most if not all of the somatostatin protein carrier conjugates were prepared by glutaraldehyde coupling, employing cross linkage between the lysine residues present on somatostatin and the carrier protein. The two lysines on somatostatin available for coupling reside within a 12-mer functional loop thus may result in significant loss of the native somatostatin structure and reduction in crossreactivity to somatostatin when such conjugates are used as vaccines.

Moreover, protein linkage to somatostatin is problematic because the majority of immune responses are directed to the carrier rather than to somatostatin (the mass of the carrier molecule (s) is much greater than that of somatostatin) and immunization with hapten carrier conjugates frequently leads to carrier-induced immune suppression (Schutz et al . , J Immunol, 1985, 135:2319). Accordingly, an immune enhancer that is suitable for live stock use, inexpensive and capable of stimulating an early and strong immune response to somatostatin has been sough . This immune enhancer should avoid carrier-induced suppression.

An important factor affecting immunogenicity of a synthetic peptide for an somatostatin immunogen is its presentation to the immune system by T helper cell epitopes. Formerly, those were provided by a carrier protein with the concomitant disadvantages discussed above. These may also be supplied as hybrid polypeptides by recombinant DNA expression systems (Riggs, US 4,812,554; US 4,563,424; and Xu et al . ,

Science in China (Series B) , 1994; 37:1234). These may also be more simply and less expensively supplied by a synthetic peptide comprising the target hapten B cell site and T-helper epitopes (Th) appropriate for the host. Such peptides react with helper T-cell receptors and the class II MHC molecules, in addition to antibody binding sites (Babbitt et al., Nature, 1985, 317:359) and thus stimulate a tightly site-specific antibody response to the target antibody binding site (target site) . A wholly synthetic peptide immunogen for somatostatin would enjoy the following advantages over carrier conjugates and recombinant polypeptides : the product is chemically defined for easy quality control, it is stable, no elaborate downstream processing is needed, no elaborate production plant is required, and the engendered immune response is site- specific so that undesirable responses such as epitopic suppression are avoided. Immunogenicity of synthetic somatostatin immunogens can be optimized by (1) combining somatostat with selected promiscuous Th sites to which the majority of a population are responsive; (2) combining somatostatin with an enlarged repertoire of Th through combinatorial chemistry and thereby accommodate to the variable immune responsiveness of a population, and (3) the stabilization of a desirable conformational feature of somatostatm by cyclic constraint.

Epitopes termed promiscuous Th evoke efficient T cell help and can be combined with B cell epitopes that by themselves are poorly lmmunogenic to provide potent immunogens. Well-designed promiscuous Th/B cell epitope chimeric peptides are capable of eliciting Th responses and resultant antibody responses m most members of a genetically diverse population expressing diverse MHC naplotypes . Promiscuous Th can be provided by specific sequences derived from potent immunogens including measles virus F protein and hepatitis B virus surface antigen. Many known promiscuous Th have been shown to be effective n potentiating a poorly lmmunogenic peptide corresponding to the decapeptide hormone LHRH (US 5,759,551) .

Potent Th epitopes range in size from approximately 15-30 ammo acid residues in length, often share common structural features, and may contain specific landmark sequences. For example, a common feature is amphipathic helices, which are alpha-helical structures with hydrophobic ammo acid residues dominating one face of the helix and with charged and polar residues dominating the surrounding faces (Cease et al . , Proc Natl Acad Sci USA, 1987; 84: 4249-4253). Th epitopes frequently contain additional primary ammo acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Also, Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single Th epitope (Partidos et al . , J Gen Virol, 1991; 72:1293). Most, if not all, of the promiscuous T cell epitopes fit at least one of the periodicities described above. These features may be incorporated into the designs of idealized artificial Th sites, including combinatorial Th epitopes. In regard to the design of combinatorial Th sites, lists of variable positions and preferred amino acids are available for MHC-binding motifs (Meister et al . , Vaccine, 1 1995; 13:581-591); and, a method for producing combinatorial Th has been disclosed for library peptides termed structured synthetic antigen library or SSAL (Wang et al . , WO 95/11998). Thus, the 1,4,5,8 rule can be applied together with combinatorial MHC-b ding motifs in the assignment of positions for the invariant and degenerate sites of an SSAL and for the selection of residues for these sites, so as to vastly enlarge the range of immune responsiveness to an artificial Th (WO 95/11998) .

Peptide immunogens are generally more flexible than proteins and tend not to retain any preferred structure. Therefore it is useful to stabilize a peptide immunogen by the introduction of cyclic constraints. A correctly cyclized peptide immunogen can mimic and preserve the conformation of the targeted epitope and thereby evoke antibodies with cross- reactivities on that site on the authentic molecule (Moore, Chapter 2 in Synthetic Peptides A User' s guide, ed Grant, WH Freeman and Company: New York, 1992, pp 63-67) .

Peptide immunogens that have been designed with the peptide technologies and peptide design elements discussed above, i.e., design of promiscuous potent Th epitopes, Th SSAL combinatorial peptides, and cyclic constraint, are the basis for effective synthetic somatostatin immunogens. Such peptides are preferred for their presentation of the somatostat by optimized positioning and cyclization, and for broadly reactive Th responsiveness. Hence, it has been found that peptides containing particular structural arrangements of a Th epitope alone or linked to a general immune enhancer, e.g., an invasin domain (US 5,759,551) and somatostatin its intact form where the functional site withm the 12 mer loop structure is not disturbed (as target antigen) , are effective m stimulating the production of antibodies against somatostatin.

SUMMARY OF THE INVENTION

The present mvention relates to an lmmunogenic peptide composition comprising synthetic peptides, which are capable of inducing antibodies against somatostat that lead to the suppression of somatostatin levels, promote growth and improve food conversion efficiency m farm animals. In particular, peptides of this mvention have a Th epitope linked to a carboxyl- or ammo- terminal somatostatin (SEQ IP NO:l) or a peptide analog of somatostatin. Optionally, the peptides have an invasin domain (SEQ ID NO : 2 ) as a general immune stimulator. These peptides are effective as immunogens, capable of increasing serum growth hormone level in immunized hosts to promote daily weight gam m farm animals .

Another aspect of this invention provides an antigenic composition comprising an immunologically effective amount of a peptide composition m accordance with this invention and one or more pharmaceutically acceptable vaccine formulations and instructions for dosage such that lmmunotherapeutic antibodies directed against the targeted somatostatin site are generated. Such peptide compositions are useful for growth promotion farm animals.

A further aspect of the invention relates to a method for increasing circulating somatotropic hormone levels in a mammal by administering one or more of the subject peptides to the mammal for a time and under conditions sufficient to induce functional antibodies directed against said somatostatm.

Yet another aspect of the invention relates to an lmmunogenic synthetic peptide of about 30 to about 90 ammo acids which contains a helper T cell (Th) epitope, somatostatin (SEQ IP NO:l) or a peptide analog of somatostatin, spacers to separate the lmmunogenic domains and optionally general immunostimulatory sites, for example, an invasin domain (SEQ ID NO:2). These three lmmunogenic domain elements of the peptide and spacer can be covalently joined in any order provided that either the immunoreactivity of the peptide hapten is substantially preserved or that immunoreactivity to the somatostatin self-peptide can be generated.

DETAILED DESCRIPTION OF THE INVENTION This invention is directed to a novel peptide composition for the generation of high titer polyclonal antibodies with specificity for somatostatin. The high site- specificity of the peptide composition minimizes the generation of antibodies that are directed to irrelevant sites on carrier proteins. Therefore, the invention is further directed to an effective method for the growth promotion farm animals .

Somatostatm (SEQ ID NO:l) is a short cyclized peptide hormone which, by itself is non-immunogenic, more so for being a self-antigen. This short peptide can be immunopotentiated by chemical coupling to a carrier protein, for example, keyhole limpet hemocyanm (KLH) or by fusion to a carrier polypeptide through recombinant DNA expression, for example, hepatitis B surface antigen. Major deficiencies of such "somatostatm-carπer" vaccines is that the largest portion of antibodies generated by the combinations are the non-functional antibodies directed against the carrier protein or polypeptide and the potential for epitopic suppression. The immunogens of the present mvention are wholly synthetic peptides which minimize the generation of irrelevant antibodies to elicit an immune response more focused to somatostatin . However, because somatostatin is a non- lmmunogenic T cell-dependent antigen, it is completely dependent on extrinsic Th epitopes for lmmunogenicity . These are provided for the peptides of the invention as covalently linked promiscuous Th epitopes. The immunogens of the invention are all of site-specific immunoreactivity to provide for effective growth promotion m livestock.

Specific examples are provided in the present invention as embodiments of the peptides of the invention. These examples provide for the linkage of synthetic immunostimulatory elements to the somatostatin peptide such that potent somatostatm-reactive antibodies are generated, in a genetically diverse host population. These antibodies, turn, lead to inhibition of the function of somatostatin, thus resulting in an effective growth promotion for livestock.

For active immunization, the term "immunogen" referred to herein relates to a peptide composition which is capable of inducing antibodies against somatostatin, leading to inhibition or suppression of somatostatm levels in a mammal. The peptide composition of tne present invention includes peptides which contain promiscuous helper T cell epitopes (Th epitopes) . The peptides are covalently attached to the somatostatm peptide, with a spacer (e.g. Gly-Gly) , so as to be adjacent to either the N- or C-termmus of the target somatostatm peptide, in order to evoke efficient antibody responses. The immunogen may also comprise a generalized immunostimulatory element, for example, a domain of an invasin protein from the bacteria Yersinia spp (Brett et al . , Eur J Immunol, 1993, 23: 1608-1614) (SEQ ID NO:2). The invasin domain is attached through a spacer to a Th peptide.

The peptides of this invention can be represented by the formulas:

H₂N- (A) _n- (somatostatm peptide) - (B) ₀- (Th)_m-X or H₂N- (A)n- (Th)_m- (B) o- (somatostatin peptιde)-X wherein

H₂N is the N-terminal -NH₂ of the peptide conjugate, each A is independently an ammo acid or a general immunostimulatory sequence; each B is chosen from the group consisting of ammo acids, -NHCH(X)CH₂SCH₂CO-, -NHCH (X) CH₂SCH₂CO (ε-N) Lys-, -NHCH(X)CH₂S-succιnιmιdyl (ε-N)Lys-, and -NHCH (X) CH₂S- (succmimidyl) -; each Th is independently a sequence of ammo acids that comprises a helper T cell epitope, or an immune enhancing analog or segment thereof; somatostatm peptide is somatostatin or a crossreactive and immunologically functional analog thereof;

X is an ammo acid α-COOH or α-CONH₂; n is from 1 to about 10; m is from 1 to about 4; and o is from 0 to about 10.

The peptide immunogen of the present invention comprises from about 20 to about 100 ammo acid residues, preferably from about 25 to about 80 ammo acid residues and more preferably from about 25 to about 65 ammo acid residues.

When A is an ammo acid or a general immunostimulatory element, e.g., Inv, it can be covalently linked to either the N-terminal of the peptide immunogen as shown by the formulas, or to the C-termmal (not shown) .

When A is an ammo acid, it can be any non-naturally occurring or any naturally occurring ammo acid. Non- naturally occurring ammo acids include, but are not limited to, β-alanine, ornithme, norleucme, norvalme, hydroxyprolme, thyroxme, γ-ammo butyric acid, homoserme, citrullme and the like. Naturally-occurring amino acids include alanme, argmme, asparagme, aspartic acid, cysteine, glutamic acid, glutamme, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Moreover, when m is greater than one, and two or more of the A groups are amino acids, then each amino acid may be independently the same or different.

When A is an invasin domain, it is an immune stimulatory epitope from the invasin protein of a Yersinia species. This immune stimulatory property results from the capability of this invasin domain to interact with the βl integrin molecules present on T cells, particularly activated immune or memory T cells. The specific sequence for an invasin domain found to interact with the βl integrins has been described by Brett et al { Eur J Immunol , 1993) . A preferred embodiment of the invasin domain (Inv) for linkage to a promiscuous Th epitope has been previously described in US 5,759,551 and is incorporated herein by reference. The said Inv domain has the sequence:

Thr-Ala-Lys-Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-Thr-Tyr-Gln-Phe

(SEQ ID NO:2) or is an immune stimulatory homologue thereof from the corresponding region in another Yersinia species invasin protein. Such homologues thus may contain substitutions, deletions or insertions of amino acid residues to accommodate strain to strain variation, provided that the homologues retain immune stimulatory properties .

In one embodiment, n is 1 and A is -NH₂. In another embodiment, n is 4 and A is -NH₂, an invasin domain (Inv), glycine and glycine, in that order.

B is a spacer and is an amino acid which can be naturally occurring or the non-naturally occurring amino acids as described above. Each B is independently the same or different.. The amino acids of B can also provide a spacer, e.g., Gly-Gly, between the promiscuous Th epitope and the somatostatin peptide (e.g., SEQ ID NO:l) and crossreactive and functional immunological analogs thereof. In addition to physically separating the Th epitope from the B cell epitope, i.e., the somatostatin peptide and immunological analogs thereof, the Gly-Gly spacer can disrupt any artifactual secondary structures created by the joining of the Th epitope with the somatostatin peptide and crossreactive and functional immunological analogs thereof and thereby eliminate interference between the Th and/or B cell responses . The amino acids of B can also form a spacer which acts as a flexible hinge that enhances separation of the Th and IgE domains . Examples of sequences encoding flexible hinges are found in the immunoglobulin heavy chain hinge region. Flexible hinge sequences are often proline rich. One particularly useful flexible hinge is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO:3), where Xaa is any amino acid, and preferably aspartic acid. The conformational separation provided by the amino acids of B permits more efficient interactions between the presented peptide immunogen and the appropriate Th cells and B cells and thus enhances the immune responses to the Th epitope and the antibody-eliciting epitope and their crossreactive and functional immunological analogs thereof.

Th is a sequence of amino acids (natural or non- natural amino acids) that comprises a Th epitope. A Th epitope can consist of a continuous or discontinuous epitope. Hence not every amino acid of Th is necessarily part of the epitope. Accordingly, Th epitopes, including analogs and segments of Th epitopes, are capable of enhancing or stimulating an immune response to the somatostatin peptide and immunological analogs thereof. Th epitopes that are immunodominant and promiscuous are highly and broadly reactive in animal and human populations with widely divergent MHC types (Partidos et al . , 1991; US 5,759,551). The Th domain of the subject peptides has from about 10 to about 50 amino acids and preferably from about 10 to about 30 amino acids. When multiple Th epitopes are present (i.e., m ≥ 2), then each Th epitope is independently the same or different. Th segments are contiguous portions of a Th epitope that are sufficient to enhance or stimulate an immune response to the somatostatin peptide (SEQ ID NO:l) and immunological analogs thereof.

Th epitopes of the present invention include those derived from foreign pathogens including but not limited to, as examples, hepatitis B surface and core antigen helper T cell epitopes (HB_S Th and HB_C Th) , pertussis toxin helper T cell epitopes (PT Th) , tetanus toxin helper T cell epitopes (TT Th) , measles virus F protein helper T cell epitopes (MV_F Th) , Chlamydia trachoma tis major outer membrane protein helper T cell epitopes (CT Th) , diphtheria toxin helper T cell epitopes (DT Th) , Plasmodium falciparum circumsporozoite helper T cell epitopes (PF Th) , Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th) , and Escherichia coli TraT helper T cell epitopes (TraT Th) . The pathogen-derived Th selected here as representative examples of promiscuous Th were listed as SEQ ID NOS:2-9 and 42-52 in US 5,759,551; as CT Th Pll in Stagg et al., Immunology, 1993; 79; 1-9; and as HBc peptide 50-69 in Ferrari et al., J Clin Invest, 1991; 88: 214- 222; and incorporated herein by reference. Further, Th epitopes include idealized artificial Th (e.g., SEQ ID NOS:14) and artificial SSAL Th (e.g., SEQ ID NOS : 7 , 30, 31) . Peptides comprising SSAL Th are produced simultaneously in a single solid-phase peptide synthesis in tandem with somatostatin and other sequences. Th sites also include functional immunological analogs. Functional Th analogs include immune- enhancing analogs, crossreactive analogs and segments of any of these Th epitopes. Functional Th analogs further include conservative substitutions, additions, deletions and insertions of from one to about 10 amino acid residues in the Th epitope which do not essentially modify the Th-stimulating function of the Th epitope.

The synthetic peptides of this invention, as described by the formulas

(A)_n- (Th)m- (B) ₀- (somatostatin peptide) or (A) _n- (somatostatin peptide) - (B) ₀- (Th) _m, have the Th epitope covalently attached through spacer B to either the N terminus or C terminus of the somatostatin peptide and crossreactive and functional immunological analogs thereof . Crossreactive and functional immunological analogs of the somatostatin peptide (e.g., SEQ ID NO:l) accordmg to the invention, may further comprise conservative substitutions, additions, deletions, or insertions of from one to about four amino acid residues provided that the peptide analogs are capable of eliciting immune responses crossreactive with the somatostatin peptides. The conservative substitutions, additions, and insertions can be accomplished with natural or non-natural ammo acids as defined herein. Preferred peptide immunogens of this invention are the peptides containing the somatostatm peptides or crossreactive and functional immunological analogs thereof; a spacer (e.g., Gly-Gly) ; a Th epitope that s an HB_S Th (SEQ ID NO:15), HB_c Th (SEQ ID NO:4), MV_F Th (SEQ ID NOS:21,29), PT Th (SEQ ID NO:6), TT Th (SEQ ID NO:5); CT Th (SEQ ID NO:27), DT Th (SEQ ID NO:28), an artificial Th (e.g., SEQ ID NOS : 7 , 14 , 30, 31) or an analogue thereof; and, optionally, an Inv domain (SEQ ID NO : 2 ) or analog thereof.

Peptide compositions which contain cocktails ofthe subject peptide immunogens with two or more ofthe Th epitopes may enhance immunoefficacy in a broader population and thus provide an improved immune response to the somatostatin peptide

The peptide immunogens of this invention can be made by chemical synthesis methods which are well known to the ordinarily skilled artisan. See, for example, Fields et al . , Chapter 3 in Synthetic Peptides : A User' s Guide, ed. Grant, W. H. Freeman & Co., New York, NY, 1992, p. 77. Hence, peptides can be synthesized using tne automated Merπfield techniques of solid phase synthesis with the α-NH₂ protected by either t- Boc or F-moc chemistry using side chain protected am o acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431. Preparation of peptide constructs comprising SSALs for Th epitopes can be accomplished by providing a mixture of alternative amino acids for coupling at a given variable position. After complete assembly of the desired peptide immunogen, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups on the amino acid side chains. The free peptide is purified by HPLC and characterized biochemically, for example, by amino acid analysis or by sequencing.

Purification and characterization methods for peptides are well known to one of ordinary skill in the art.

The subject immunogen may also be polymerized. Polymerization can be accomplished for example by reaction between glutaraldehyde and the -NH₂ groups of the lysine residues using routine methodology. By another method, the synthetic

"A-Th-spacer- (somatostatin peptide) " or "(somatostatin peptide) -spacer- (Th) _m-A" immunogen can be polymerized or co-polymerized by utilization of an additional cysteine added to the N-terminus of the synthetic "A-Th-spacer- ( somatostatin peptide) or "(somatostatin peptide) -spacer- (Th) _m- (A) _n" immunogen. The subject immunogen may also be prepared as a branched polymer through synthesis of the desired peptide construct directly onto a branched poly-lysyl core resin (Wang, et al . , Science, 1991; 254:285-288) .

Alternatively, the longer synthetic peptide immunogens can be synthesized by well known recombinant DNA techniques . Any standard manual on DNA technology provides detailed protocols to produce the peptides of the invention. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated into a nucleic acid sequence, and preferably using optimized codon usage for the organism in which the gene will be expressed. Next, a synthetic gene is made, typically by synthesizing overlapping oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and recombinants are obtained and characterized. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods .

The efficacy of the peptide composition of the present invention can be established by injecting an animal, for example, rats, with an immunogenic composition comprising peptides of the invention, e.g., SEQ ID NOS: 8-13, 16-20, 22 followed by monitoring the humoral immune response to the somatostatin and crossreactive and functional immunological homologues thereof, as detailed in the Examples. Another aspect of this invention provides a peptide composition comprising an immunologically effective amount of one or more of the peptide immunogens of this invention in a pharmaceutically acceptable delivery system. Accordingly, the subject peptides can be formulated as a peptide composition using adjuvants, pharmaceutically-acceptable carriers or other ingredients routinely provided in peptide compositions . Among the ingredients that can be used in this invention are adjuvants or emulsifiers including alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL) , QS21, ISA51, ISA35, ISA206 and ISA 720 as well as the other efficacious adjuvants and emulsifiers. The formulations include formulations for immediate release and/or for sustained release, and induction of systemic immunity, which may be accomplished by, for example, immunogen entrapment by or coadministration with microparticles . Such formulations are readily determined by one of ordinary skill in the art. The present immunogens can be administered by any convenient route including subcutaneous, oral, intramuscular, or other parenteral or enteral route. Similarly the immunogens can be administered as a single dose or multiple doses. Immunization schedules are readily determined by the ordinarily skilled artisan. The peptide composition of the instant invention contain an effective amount of one or more of the peptide immunogens of the present invention and a pharmaceutically acceptable carrier. Such a composition in a suitable dosage unit form generally contains about 0.5 μg to about 1 mg of the immunogen per kg body weight. When delivered in multiple doses, it may be conveniently divided into an appropriate amount per dosage unit form. For example, an initial dose, e.g. 0.2-2.5 mg; preferably 1 mg, of immunogen represented as a peptide composition of the present invention, is to be administered by injection, preferably intramuscularly, followed by repeat (booster) doses. Dosage will depend on the age, weight and general health of the animal as is well known in the vaccine and therapeutic arts. The immune response to synthetic somatostatin peptide immunogens can be improved by delivery through entrapment in or on biodegradable microparticles of the type described by O'Hagan et al . ( Vaccine, 1991; 9: 768-771). The immunogens can be encapsulated with or without an adjuvant in biodegradable microparticles, to potentiate immune responses, and to provide time-controlled release for sustained or periodic responses, and for oral administration, (O'Hagan et al, 1991; and, Eldridge et al . , 1991; 28: 287-294).

Specific peptide immunogens and compositions are provided in the following examples to illustrate the invention. These examples are for purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLE 1 TYPICAL METHODS TO SYNTHESIZE

SOMATOSTATIN PEPTIDE CONSTRUCTS

Peptides listed in Tables 2 and 3 were synthesized individually by the Merrifield solid-phase synthesis technique on Applied Biosystems automated peptide synthesizers (Models 430, 431 and 433A) using Fmoc chemistry. Preparation of peptide constructs comprising structured synthetic antigen libraries (SSALs), e.g., artificial Th site termed "1,4,9 PALINDROMIC" (SEQ ID NO:7), can be accomplished by providing a mixture of the desired amino acids for chemical coupling at a given position as specified in the design. After complete assembly of the desired peptide, the resin was treated according to standard procedure using trifluoroacetic acid to cleave the peptide from the resin and deblock the protecting groups on the amino acid side chains. For cyclic peptide, the cleaved peptide was dissolved in 15% DMSO in water for 48 hrs to facilitate intradisulfide bond formation between cysteines.

The cleaved, extracted and washed peptides were purified by HPLC and characterized by mass spectrometry and reverse phase HPLC.

Peptides marked by "b" in the peptide code column were synthesized as target antigenic peptides in tandem with Th sites as shown. Th sites used include, for example, the HBs Th taken from hepatitis B virus (SEQ ID NO:15), and the novel artificial Th site termed "1,4,9 PALINDROMIC" (SEQ ID NO:7). Peptides marked by "c" are variants of the "b" constructs synthesized in tandem with the Inv domain immunostimulatory peptide (SEQ ID NO:2). Peptides marked by "d" are the reversal of the "b" constructs (e.g., somatostatin-Th) and peptides marked by "e" are the reversal of the "c" constructs (e.g., somatostatin-Th-Inv) . The "b", "c", "d" and "e" constructs were synthesized with gly-gly spacers for separation of the target antigenic site from the Th site, and separation of the Th from the Inv immunostimulatory site.

EXAMPLE 2

TYPICAL METHODS TO EVALUATE IMMUNOGENICITY OF SOMATOSTATIN PEPTIDES

Somatostatin Peptide immunogens (e.g., SEQ ID NOS: 8-

13,16-20,22 and 24 as shown in Tables 2 and 3) were evaluated on groups of 4 or 5 rats as specified by the experimental immunization protocol outlined below and by serological assays for determination of immunogenicity .

Standard Experimental Design:

Immunogens: (1) individual peptide immunogen; or

(2) mixtures comprising equal molar peptide immunogens as specified in each example.

Dose: 100 μg in 0.5 mL per immunization unless otherwise specified

Route: intramuscular unless otherwise specified

Adjuvants: (1) Freund's Complete Adjuvant (CFA) / Incomplete Adjuvant (IFA); or

(2) 0.4% Alum (Aluminum hydroxide); CFA/IFA groups received CFA week 0, IFA in subsequent weeks. Alum groups received same formulations for all doses Dose Schedule: 0, 2 and 4 weeks; 0, 3, and 6 weeks or otherwise specified.

Bleed Schedule: weeks 0, 3, 6 and 8 or otherwise specified

Species: Sprague-Dawley rats

Group Size: 4 or 5 rats/group Assay: specific ELISAs for each immune serum's anti-peptide activity, solid-phase substrate was the cyclized somatostatin peptide (SEQ ID NO:l) .

Blood was collected and processed into serum, and stored prior to titering by ELISA with the target antigenic peptides.

Anti-somatostatin antibody activities were determined by ELISAs (enzyme-linked immunosorbent assays) using 96-well flat bottom microtiter plates which were coated with the cyclized somatostatin peptide (SEQ ID NO:l) as immunosorbent. Aliquots (100 μL) of the peptide immunogen solution at a concentration of 5 μg/mL were incubated for 1 hour at 37°C. The plates were blocked by another incubation at 37°C for 1 hour with a 3% gelatin/PBS solution. The blocked plates were then dried and used for the assay. Aliquots (100 μL) of the test immune sera, starting with a 1:100 dilution in a sample dilution buffer and ten-fold serial dilutions thereafter, were added to the peptide coated plates. The plates were incubated for 1 hour at 37°C.

The plates were washed six times with 0.05% PBS/Tween® buffer. 100 μL of horseradish peroxidase labeled goat-anti-species specific antibody was added at appropriate dilutions in conjugate dilution buffer (Phosphate buffer containing 0.5M NaCl, and normal goat serum) . The plates were incubated for 1 hour at 37°C before being washed as above. Aliquots (100 μL) of o-phenylenediamine substrate solution were then added. The color was allowed to develop for 5-15 minutes before the enzymatic color reaction was stopped by the addition of 50 μL 2N H₂SU4. The A9_2nm of the contents of each well was read in a plate reader. ELISA titers were calculated based on linear regression analysis of the absorbances, with cutoff A49_2nm set at 0.5. This cutoff value was rigorous as the values for diluted normal control samples run with each assay were less than 0.15.

Th peptide-based ELISAs. The Th peptide based ELISAs were performed essentially the same as the somatostatin ELISA described herein above except for the antigen coating steps, where microtiter wells were coating for 1 hr at 37° with the designated individual Th peptide derived from its corresponding somatostatin vaccine construct (e.g., peptides with SEQ ID NOS: 14 and 15) at 5 μg/mL. EXAMPLE 3

IMMUNOGENICITY STUDIES OF SOMATOSTATIN

ANTIGENIC PEPTIDES INCORPORATING

VARIOUS IMMUNOSTIMULATORY ELEMENTS

The somatostatin peptide immunogens shown in Table 2 illustrate variations of the peptides of this invention represented by the formulas:

(A) _n- (Th)_m- (B) o- (Somatostatin peptide) or (A) _n( Somatostatin peptide) - (B)₀~ (Th)_m wherein: A is an amino acid, NH₂, or Inv (SEQ ID NO:2); when A is an amino acid or Inv it can be linked to either the N-terminal or the C-terminal;

B is glycine; Th is a helper T cell epitope derived from foreign pathogens, e.g., HBcso-egTh (SEQ ID NO: 4) , TT₆i5-63iTh (SEQ ID NO:5), PTi49-i76Th (SEQ IP NO: 6), or and artificial Th e.g., 1,4,9 PALIPROMIC Th (SEQ IP NO:7); n is 1, m is 1 and o is 2 These peptides were synthesized among others and immune sera were generated for immunogenicity evaluation. Most of the peptides shown in Table 2, incorporating various forms and orientations of Th epitopes, elicited high titer somatostatin-specific antibodies in the immunized hosts. In contrast, the somatostatin peptide (pl348a, SEQ IP NO:l) lacking Th was devoid of immunogenicity. However, certain Th are to be preferred over others. For example, in Table 2, p2134b (SEQ IP NO:8) having HBc Th (SEQ ID NO:4), p2384b (SEQ ID NO:20) having SynTh(l,2,3) (SEQ ID N0:14), an artificial Th site, and p2138b (SEQ ID NO: 10) having PT149-176 Th (SEQ ID NO: 6) are more immunogenic than p2135b (SEQ ID NO: 9) having TT Th (SEQ ID NO:5) and all of these are preferable to p2136b (SEQ ID NO:24) having CTA8 Th (SEQ ID NO:23) which is scarcely immunogenic at all. Also, for optimum immunogenicity, the orientation of the Th site to the somatostatin target site must be specified. Compare the kinetics of the antibody response for p2253b (SEQ ID NO:ll) to p2255d (SEQ ID NO:12). It is preferable to place 1,4,9 PALINDROMIC Th (SEQ ID NO: 7) on the C-terminus of somatostatin. From the comparison of pl344b (SEQ ID NO:16) to pl349b (SEQ ID NO:22), the better placement for MV_F258-₂77 (SEQ ID NO: 21) is on the N-terminus of somatostatin. It is clear that the selection and arrangement of each Th site must be specified for the preferred peptides of the invention. For the somatostatin peptides shown in Table 3, where somatostatin constructs comprising a promiscuous Th epitope were already found to be immunogenic, attachment of Inv to the "Th" constructs, can improve the immunogenicity of the somatostatin peptide. The comparisons of immunogenicities for pl344b (SEQ ID NO:16) with pl343c (SEQ ID NO:17) and pl346b (SEQ ID NO:18) with pl345c (SEQ ID NO:19) shows that the addition of the Inv domain (SEQ ID NO: 2) to the N-terminus of the Th constructs improved immunogenicity in terms of percentage of responding animals, in the intensity of the somatostatin-specific antibody titer, and in the longevity of that antibody response (>35 weeks) . However, the comparison of immunogenicities of p2134b (SEQ ID NO : 8 ) with p2134c (SEQ ID NO:25) and with p2134d (SEQ ID NO:26) illustrates that in combination with particular Th sites Inv may not be immunostimulatory, and that a particular orientation on the N- terminus is preferred over the C-terminus orientation for the combination of Inv with the HBc Th site (SEQ ID NO:4). For the example of p2135e (SEQ ID NO: 13), the combination of Inv with TT₆i₅-6₃i Th (SEQ ID NO: 5) on the C-terminus did result in an effective immunogen. Thus, the addition of Inv and the orientation of Th and Inv must be specified for the preferred peptides of the invention.

For constructs having potent site-directed immunogenicity for somatostatin, the antibody titers directed at the immunostimulatory elements, e.g., Inv-MVF₂58-277 Th of pl343c (SEQ ID NO: 17) and KKK-HBS19-32 Th-GG of pl346b (SEQ ID NO: 18) from Table 3, were <1 Logio in comparison to those of >3 Logio for somatostatin. Thus, the immune response generated by the synthetic peptides of the present invention were directed almost exclusively to the somatostatin target site.

EXAMPLE 4

ADPITIONAL ANTIGENIC PEPTIDES OF THE INVENTION Somatostatin peptide antigens illustrating variations of the peptides of this invention represented by the formulas:

(A) n- (Th)_m- (B) ₀- (somatostatin peptide) or (A)n- (Somatostatin peptide) - (B) _D- (Th)_m

wherein : Th is a helper T cell epitope derived from any of the foreign pathogens as shown in Table 4; or, an helper T cell epitope from any of the artificial Th epitopes as shown in Table 5;

A is an amino acid, ocNH₂, or an invasin domain (SEQ ID NO: 2) ; when A is an amino acid or Inv it can be linked to either the N-terminus or the C-terminus;

B is glycine; n is 1, m is 1 and o is 2 are synthesized and immune sera generated.

Table 1 Immunization against Somatostatin for Growth Promotion

Carrier protein-somatostatin conjugates were used as the vaccines for immunization.

Table 2 Immunogenicity of Somatostatin Antigenic Peptides Incorporating Various Th

bTest results for pooled sera from ELISA-reactive animals.

Table 3. Improvement on Immunogeniciity of Somatostatin Antigenic Peptides with the Attachment of an Invasin Domain

(0 c

CD C/> c m en x m m H c in

M

"Sequence of Somatostatin: AGCKNFFWKTFTSC (Seq. ID No.l) bSSeeqquueennccee ooff IInnvv ((ii..ee.. iinnvvaassiinn aaddjjuuvvaanntt ddoommaaiinn)):: TTAAKKSSKKKKFFiPSYTATYQF (Seq. ID No.2) Test results for pooled sera from ELISA-reactive animals.

Table 4 Amino Acid Sequences of Pathogen-derived Th Epitopes

Table 5 Amino Acid Sequences of Artificial Th Epitopes

Claims

CLAIMSWe claim:

1. A peptide conjugate comprising a helper T cell epitope sequence (Th) covalently attached to somatostatin or a crossreactive and immunologically functional analog thereof .

2. A peptide conjugate of Claim 1 wherein said peptide conjugate is represented by the formula

H₂N-(A)_n-(somatostatinpeptide)-(B)₀-(Th)_m-X or

H₂N-(A)_n-(Th)_m-(B)₀-(somatostatinpeptide)-X wherein

H₂N is the N-terminal -NH₂ of the peptide conjugate, each A is independently an amino acid or a general immunostimulatory sequence; each B is chosen from the group consisting of amino acids, -NHCH (X) CH₂SCH₂CO-, -NHCH (X) CH₂SCH₂CO (╬╡-N) Lys-, -NHCH (X) CH₂S-succinimidyl (╬╡-N) Lys-, and -NHCH (X) CH₂S- (succinimidyl) -; each Th is independently a sequence of amino acids that comprises a helper T cell epitope, or an immune enhancing analog or segment thereof; somatostatin peptide is somatostatin or a crossreactive and immunologically functional analog thereof;

X is an amino acid ╬▒-COOH or -C0NH₂; n is from 1 to about 10; m is from 1 to about 4; and o is from 0 to about 10.

3. A peptide conjugate of Claim 1 wherein said peptide conjugate is represented by the formula

H₂N- (somatostatin peptide) - (B) ₀- (Th)_m- (A)_n-X or H₂N- (Th)_m- (B) ₀- (somatostatin peptide) - (A) _n-X wherein

H₂N is the N-terminal -NH₂ of the peptide conjugate, each A is independently an amino acid or a general immunostimulatory sequencer- each B is chosen from the group consisting of amino acids, -NHCH (X) CH₂SCH₂CO-,

-NHCH (X) CH₂SCH₂CO (╬╡-N) Lys-,

-NHCH (X) CH₂S-succinimidyl (╬╡-N) Lys-, and

-NHCH (X) CH₂S- (succinimidyl) -; each Th is independently a sequence of amino acids that comprises a helper T cell epitope, or an immune enhancing analog or segment thereof; somatostatin peptide is somatostatin or a crossreactive and immunologically functional analog thereof;

X is an amino acid ╬▒-COOH or ╬▒-C0NH₂; n is from 1 to about 10; m is from 1 to about 4; and o is from 0 to about 10.

4. A peptide conjugate of claim 2 or claim 3, wherein each B is chosen from the group consisting of natural and unnatural amino acids .

5. A peptide conjugate of any one of claims 1-4 wherein said somatostatin peptide is somatostatin.

6. A peptide conjugate of any one of claims 1-4 wherein said Th is an SSAL epitope.

7. A peptide conjugate of any one of claims 1-4 wherein said Th has an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 SEQ ID NO:30, and SEQ ID NO:31.

8. A peptide conjugate of claim 2 wherein said peptide conjugate has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

9. A peptide conjugate of claim 2 or claim 3 wherein at least one A is an invasin domain.

10. A peptide conjugate of claim 2 wherein n is 3, and (A) ₃ is (invasin domain) -Gly-Gly.

11. A peptide conjugate of claim 9 or claim 10 wherein said invasin domain has the amino acid sequence of SEQ ID NO: 2.

12. A synthetic peptide of about 25 to about 90 amino acids, which comprises the amino acid sequences of

(a) an invasin domain,

(b) a helper T cell (Th) epitope, and (c) somatostatin or a crossreactive and immunologically functional analog thereof.

13. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO:25.

14. A peptide or peptide conjugate of any one of claims 1 to 13 wherein said peptide stimulates an immune response to somatostatin in a mammal.

15. A peptide or peptide conjugate of claim 14 wherein immunization of a mammal with said peptide conjugate causes a reduction in somatostatin levels in said mammal.

16. A pharmaceutical composition comprising an immunologically effective amount of a peptide or peptide conjugate of any one of claims 1-15, and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition of claim 16, wherein said immunologically effective amount of said peptide or peptide conjugate is about 0.5 ╬╝g to about 1 mg per kilogram body weight per dose.

18. A method for inducing anti-somatostatin antibody production in a mammal which comprises administering to said mammal a pharmaceutical composition of claim 16 or claim 17.

19. A method for increasing growth rate in a mammal which comprises administering a pharmaceutical composition of claim 16 or claim 17 to said mammal.

20. A method of increasing growth rate in a mammal which comprises administering to a mammal an amount of a pharmaceutical composition of claim 16 or claim 17 sufficient to reduce somatostatin levels .

21. A composition comprising a mixture of two or more peptides or peptide conjugates of any one of claims 1-15.

22. A pharmaceutical composition comprising an immunologically effective amount of a composition of claim 21 and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition of claim 22, wherein said immunologically effective amount of said composition is about 0.5 ╬╝g to about 1 mg per kilogram body weight per dose .

24. A method for inducing anti-somatostatin antibody production in a mammal which comprises administering to said mammal a pharmaceutical composition of claim 22 or claim 23.

25. A method for increasing growth rate in a mammal which comprises administering a pharmaceutical composition of claim 22 or claim 23 to said mammal.

26. A method of increasing growth rate which comprises administering to a mammal an amount of a pharmaceutical composition of claim 22 or claim 23 sufficient to reduce somatostatin levels.

27. A branched polymer comprising a lysine, trilysine, or heptalysine core, covalently attached to two, four, or eight peptide conjugates, respectively, of any one of claims 1-15.

28. A polymer comprising one or more peptide conjugates of any one of claims 1-3 and claims 5-15, cross-linked by a bifunctinal crosslinking agent.

29. A Th epitope peptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:14, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 SEQ ID NO:30, and SEQ ID NO:31.