JP2002518033A - Synthetic somatostatin immunogen for promoting growth of farm animals - Google Patents

Abstract

  (57) [Summary] The present invention provides a peptide comprising somatostatin or a sequence homologous to somatostatin covalently linked to a helper T cell epitope and optionally other immunostimulatory sequences. The present invention provides the use of such peptides as immunogens to induce the production of high titer polyclonal antibodies specific for somatostatin in mammals. These peptides are expected to be useful in lowering somatostatin levels in mammals and therefore increasing growth rate and feed utilization efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to peptide compositions useful as immunogens for promoting the growth of farm animals. The immunogenic peptide of the composition of the invention comprises a multi-class II MHC binding motif and a helper T having somatostatin at either the C or N terminus.
Contains cellular epitopes (Th). The peptide optionally contains an invasin domain that acts as a general immunostimulator. This helper T cell epitope and invasin domain enable an immune response to the somatostatin self peptide.

BACKGROUND OF THE INVENTION Recent understanding of neuro-endocrine and hormonal factors involved in growth, combined with the rapid advancement of biotechnology, has potential for increasing the growth rate of various farm animal species. Provides a path. Methods for promoting the growth of animals by altering the regulation of growth hormone through immune neutralization include antibiotics as growth promoters,
It is a valuable alternative to current methods utilizing anabolic steroids and growth hormone. The application of this antibiotic to farm animals has been the driving force for the selection of antibiotic resistant bacteria in certain pathogenic bacterial species and has had deleterious consequences in controlling human infectious diseases (Witte, Science , 1998; 279: 996
), Application of anabolic steroids promotes the growth of farm animals, but is not common among consumers. Steroid use is regulated in Europe both by law and by public interest (Buttery and Dawson, Proc Nutr Soc, 1990; 49: 4).
59). Recombinant DNA technology has enabled large-scale production of metabolic hormones for direct administration to animals. Such an application can promote growth. However, if somatostatin is used as a means to improve animal function, it is only one step to take.

[0003] The main somatotropin hormones are somatotropin (growth hormone GH), somatomedin-C (insulin-like growth factor 1, IGI-1), somatocrinin (GH)
Release factor, GRF) and somatostatin (GH release inhibitor). Although the major potential uses for somatotropin hormones are in growth and lactation, hormonal regulation of these events relies on complex interactions between a wide variety of hormones (
Spencer, Livest Prod Sci , 1985, 12:31 ). Thus, simple utilization of a single hormone may not be enough to increase productivity.

[0004] GH appears to play a dual role. It has a positive effect stimulating increased muscle protein synthesis and a potential catabolic effect due to its ability to degrade fat (most if not all are mediated by IGF-1).
The overall effect is increased carcass redness and reduced carcass fat, often increased growth, and, as always, increased food conversion efficiency (Buttery and Dawson, Proc.
utr Soc, 1990; 49: 459; and Spencer, Reprod Nutr Develop, 1987; 27 (2B)
: 581).

[0005] The effect of GH administration on growth is unreliable in practical use. Presumably, administration of exogenous hormones ignores the sensitive interrelationships between the various hormones and ignores the possible effects of elevated plasma levels on the receptor population. Effective application of GH requires frequent injections to provide continuous and physiologically effective serum levels of hormones. This increases the risk of breaking such a balanced relationship and has harmful effects.

[0006] Thus, despite the fact that pure preparations of growth hormone can be made in large quantities by recombinant DNA technology, this technology does not allow for the regulation of transport required for the desired effect on growth. It does not provide the mechanism performed. In addition, the use of recombinant growth hormone in food animals (most recently dairy cows) has created a wide range of consumer opposition and regulatory barriers. The use of growth hormone to promote redness accumulation in ruminants and other farm animals has not received regulatory approval within the European Economic Association. This regulatory and political issue has led to the IG to promote the growth of farm animal species.
Similar to the application of F-1 or GRF.

[0007] As an alternative to increasing the levels of growth stimulating hormone, removing endogenous growth inhibitors may prove to be as effective or even more effective. The main inhibitor of systemic growth is somatostatin. Somatostatin is a cyclic peptide of 14 amino acids, and its structure is conserved between species. It is synthesized as a 92 amino acid prosomatostatin molecule and then contains 6 somatostatin itself.
It is known that species of peptides and somatostatin in the form of 28 amino acids are induced (Reichlin, J Lab Clin Med, 1987; 109: 320). Somatostatin inhibits the release of a number of gastrointestinal hormones and also inhibits the release of GH, insulin, thyroid hormone, and thus the ability of animals to absorb nutrients, and subsequently induces such nutrients into tissue growth Inhibits ability.

Although the use of somatostatin antagonists has been shown to stimulate rat growth (Spencer et al., Life Sci , 1985, 37:27 ), this type of treatment has
It also suffers from the disadvantage that daily injections of IGF-1 and GRF are required.
A practical alternative is the use of an immune response to induce somatostatin immune neutralization. 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. Vaccine-mediated somatostatin immunoneutralization was first pioneered in sheep by Spencer et al. ( Livest Prod Sci , 1
983, 10: 469).

[0009] Twin St. In previous studies using Kida lamb,
Active immunity also affects treated lambs that grow at 176% of the control lamb growth rate.
(Spencer et al.,Anim Prod., 1981, 32: 376). Subsequent research
Cannot be reproduced, and it is more common to improve the growth rate by 15 to 20%. Somato
The statin molecule is the same in all farm animal species, and somatostatin
Sheep whose active immunity is of commercial importance (Spencer et al.,Livest Prod Sci, 1983,
 10:25; Laarveld et al.Can J Anim Sci, 1986, 66:77), cow (Lawrence)
J Anim Sci, 1986, 63: (Suppl) 215), pigs and chickens (Spencer et al. Dom Anim Endocr , 1986, 3:55) have been shown to stimulate the growth of varieties.
You. Effective Fruit Demonstrating Effective Immunity to Somatostatin in Farm Animals
Table 1 shows an outline of the embodiment.

[0010] In addition to stimulating the growth rate and reducing the breeding period by 20% (Spen
cer, Reprod Nutr Develop, 1987, 27 (2B): 581), active immunity to somatostatin also has a beneficial effect on food conversion efficiency. In addition to food savings due to more rapid growth, these animals are effectively fed during growth, at least to some degree as a result of changes in gut motility (Fadalla et al., J Anim Sci , 1985, 61: 234; Fai
chney et al., Can J Anim Sci , 1985, 64 (Suppl) 93)
encer et al., Livest Prod Sci , 1983, 10: 469). This treatment has no significant effect on the slaughter composition (Spencer et al., 1983, supra), but when sacrificed at the same weight, the treated animals have been shown to be more reddish. Taken together, all experiments seem to indicate that active immunity is a powerful, safe and effective means for promoting growth (Spencer, Dom Anim Endocr , 1986, 3:55).

Several immune forms of somatostatin have been designed and tested as reported in the paper. For example, somatostatin has been conjugated to a protein carrier to enhance immunity. However, protein carriers are too expensive for economical use in farm animals. Furthermore, effective immunization with somatostatin depends on the junction between somatostatin and the carrier. Most, if not all, somatostatin protein carrier conjugates are prepared with glutaraldehyde and employ cross-linking between somatostatin and lysine residues present on the carrier protein. The two lysines on somatostatin, which are useful for coupling residues within the dodecameric functional loop, result in a significant loss of native somatostatin structure and reduced cross-reactivity to somatostatin when such conjugates are used as vaccines sell.

In addition, protein ligation to somatostatin is problematic because most immune responses are more specific for carriers than somatostatin (the mass of the carrier molecule is much larger than that of somatostatin) and the hapten carrier conjugate Immunization often results in carrier-induced immunosuppression (Schutz et al., J. Immunol. 19
85, 135: 2319). Therefore, an immunopotentiator which is suitable for livestock use, is inexpensive and can stimulate an early and strong immune response to somatostatin is eagerly desired.
This immunopotentiator should avoid carrier-induced suppression.

An important factor affecting the immunogenicity of a synthetic peptide with respect to the somatostatin immunogen lies in its presentation to the immune system via a T helper cell epitope. Heretofore, they have been provided by carrier proteins, with the disadvantages mentioned above. These may also be provided as hybrid polypeptides via 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). They can also be provided simply and inexpensively by synthetic peptides comprising a target hapten B cell site and a T-helper epitope (Th) appropriate for the host. Such peptides react with helper T-cell receptors and class II MHC in addition to the antibody binding site (Babbitt et al., Nature
, 1985, 317: 359), thus stimulating a stringent site-specific antibody response to the target antibody binding site (target site). Fully synthetic peptide immunogens for somatostatin have the following advantages over carrier conjugates and recombinant polypeptides: The product is chemically defined for ease of quality control, it is stable,
It does not require cumbersome down stream processing, does not require messy production blunts, and the resulting immune response is site-specific, thus avoiding unwanted responses such as epitope suppression.

The immunogenicity of the synthetic somatostatin immunogens includes (1) combining somatostatin with selected promiscuous (cluttered) Th sites where the majority of the population is responsive; and (2) combining somatostatin with Th-through combinatorial chemistry. In combination with the repertoire, thereby adapting to the different immunoreactivity of the population; and (3) cyclization constraint (cycl)
The desired conformational characteristics of somatostatin can be optimized by ic constraints.

[0015] The epitope called promiscuous Th induces efficient T cell help and can itself be combined with weakly immunogenic B cell epitopes to provide a strong immunogen. A well-designed promiscuous Th / B cell epitope chimeric peptide is capable of inducing a Th response, and the resulting antibody responds in most members of a genetically diverse population expressing various MHC haplotypes Sex. Promiscus Th can be provided by specific sequences from potential immunogens such as the measles F protein and hepatitis B virus surface antigen. Many known promiscuous Ths have been shown to be useful in enhancing weak peptides of the immunogen corresponding to the decapeptide hormone LHRH (US 5,759,551).

[0016] Strong Th epitopes range in size from about 15 to 30 amino acid residues in length, often share common structural features, and can include specific characteristic sequences. For example, a common feature is the amphipathic helix, which is an alpha-helical structure, with one surface of the helix occupied by hydrophobic amino acid residues and the charged and polar residues occupying the outer surface (Cease et al., Proc. Natl Acad Sci USA, 19
87; 84: 4249-4253). Th epitopes often include other primary amino acid patterns, such as Gly, or charged residues, followed by a few hydrophobic residues, followed by charged or polar residues. This pattern defines a so-called Rothbard arrangement. Also, Th epitopes often follow the 1,4,5,8 rule, where hydrophobic residues follow at positions 4,5 and 8 of the positively charged residue. Since all such 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 fall into at least one of the above cycles. These features can be incorporated into the design of an ideal artificial Th site containing a combinatorial Th epitope. For the design of combinatorial Th sites, various positions and a list of suitable amino acids are available for the MHC-binding motif (Meiste
r et al., Vaccine, 1 1995; 13: 581-591); and a method for making combinatorial Th has been disclosed for an assembled synthetic antigen library or a library peptide called SSAL (Wang et al., WO95 / 11998). Thus, 1,4,
The 5,8 rule, together with the combinatorial MHC binding motif, is applied to the location of invariant and degenerate sites of SSAL and to the selection of residues at such sites, greatly expanding the range of immune responsiveness to artificial Th. (WO95 / 11998).

[0017] Peptide immunogens are generally more flexible than proteins and tend not to include any suitable structures. Therefore, it is useful for stabilizing a peptide immunogen by introducing a cyclization constraint. Properly cyclized peptide immunogens can mock and retain the conformation of the targeted epitope, thus inducing antibodies with cross-reactivity to sites on the intrinsic molecule (Moore, Synthetic Peptides A User's Gui
de Chapter 2, Grant, WH Freeman and Company: New York, 1992, pp.63-67
).

The above peptide engineering and peptide design elements, ie, promiscuous strong T
The design of h epitopes, Th SSAL combinatorial peptides, and peptide immunogens designed by cyclization constraints are the basis for effective synthetic somatostatin immunogens. Such peptides are preferred for presentation of somatostatin due to its optimized position and cyclization, and for a broadly reactive Th response. Thus, a Th epitope linked to a single or general immunopotentiating factor, such as invasin (US 5,759,551), and somatostatin (as the target antigen) in its intact form with uninterrupted functional sites within the 12-mer loop structure. Peptides comprising a particular structural arrangement of are useful in stimulating the production of antibodies to somatostatin.

SUMMARY OF THE INVENTION [0019] The present invention relates to an immunogenic peptide composition comprising a synthetic peptide capable of inducing antibodies to somatostatin that is associated with suppressing somatostatin levels, promoting growth and improving feed conversion efficiency in farm animals. . Specifically, the peptides of the present invention are carboxy or amino terminal somatostatin (SEQ ID N
O: 1) or with a Th epitope linked to a peptide analog of somatostatin. Optionally, the peptide has an invasin domain (SEQ ID NO: 2) as a common immunostimulator. These peptides are effective as immunogens that can increase the serum growth hormone of the immunized host and promote daily weight gain in farm animals.

Another aspect of the present invention is the use of an immunologically effective amount of a peptide composition of the present invention and one or more pharmaceutically acceptable vaccine preparations to specifically immunize against targeted somatostatin. An antigen composition is provided which comprises together with instructions for the dose at which the therapeutic antibody is made. Such peptide compositions are useful for promoting growth in farm animals.

[0021] A further aspect of the present invention relates to a method for increasing circulating somatotropin hormone levels in a mammal, wherein the mammal is provided with one or more peptides of the invention specific for the somatostatin. By administering for a time and under conditions sufficient to induce a functional antibody.

Still another aspect of the invention is the use of helper T cell (Th) epitopes, somatostatin (SEQ ID NO: 1) or peptide analogs of somatostatin, immunogenic domains and any general immunostimulatory sites, For example, it relates to an immunogenic synthetic peptide of about 30 to about 90 amino acids including a spacer separating the invasin domain (SEQ ID NO: 2). The three immunogenic domain elements of the peptide and spacer can be in any order provided that the immunoreactivity of the peptide hapten is substantially preserved or that immunoreactivity to the somatostatin self-peptide can be generated. May be covalently bonded.

[0023] DETAILED DESCRIPTION OF THE INVENTION The present invention relates to 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 specific for unrelated sites on the carrier protein. Accordingly, the present invention further relates to effective methods for promoting the growth of farm animals.

Somatostatin (SEQ ID NO: 1) is a short cyclized peptide hormone, which is itself non-immunogenic but a self-antigen. This short peptide can be immunopotentiated by chemical coupling to a carrier protein, such as keyhole limpet hemocyanin (KLH), or fusion to a carrier polypeptide, such as hepatitis B surface antigen, via recombinant DNA expression. Such "somatostatin-carrier"
The major disadvantage of vaccines is that most of the antibodies produced by the combination are specific for a carrier protein or polypeptide and are non-functional in their ability to suppress epitopes. The immunogens of the invention are totally synthetic peptides, which minimize the generation of irrelevant antibodies and induce a more focused immune response to somatostatin. However, since somatostatin is a non-immunogenic T-cell dependent antigen, it is completely dependent on an exogenous Th epitope for immunogenicity. These covalent promiscuous Th epitopes are provided for the peptides of the invention. The immunogens of the present invention are all site-specific immunoreactive to provide effective livestock growth promotion.

Specific examples of embodiments of the peptides of the present invention are provided in the present invention. These examples provide for the linking of a synthetic immunostimulant to a somatostatin peptide such that potent somatostatin-reactive antibodies are produced in a genetically diverse host population. These antibodies lead to an inhibition of the function of somatostatin and consequently to an efficient growth of livestock.

With respect to active immunity, the term “immunogen” as referred to herein means a peptide composition capable of inducing antibodies to somatostatin, resulting in the inhibition or suppression of somatostatin levels in a mammal. The peptide composition of the present invention includes a peptide containing a promiscuous helper T cell epitope (Th epitope). These peptides are covalently linked to the somatostatin peptide by a spacer (eg, Gly-Gly) and thus the N or C of the target somatostatin peptide.
Adjacent to either end to induce an effective antibody response. This immunogen is derived from common immunostimulants such as the domain of the invasin protein from the bacterial species Yersinia (Brett et al., Eur J Immunol. 1993, 23: 1608-1614 (SEQ.
ID No. 2) may be included. The invasin domain is added to the Th peptide via a spacer.

The peptide of the present invention can be represented by the following formula: H ₂ N- (A) _n- (somatostatin peptide)-(B) _o- (Th) _m -X
Or H ₂ N- (A) _n- (Th) _m- (B) _o- (somatostatin peptide) -X
Wherein H ₂ N is the N-terminal α-NH ₂ of the peptide conjugate, each A is independently an amino acid or a general immunostimulatory sequence, and each B is an amino acid, —NHCH (X) CH ₂ SCH ₂ CO-, -NHCH (X)
_{CH 2 SCH 2 CO (ε-} N) Lys -, - NHCH (X) CH 2 S- succinimidyl (ε-N) Lys-, and -NHCH (X) CH ₂ S- (succinimidyl) - selected from the group consisting of Wherein each Th is an amino acid sequence that independently constitutes a helper T cell epitope, or an immune enhancing analog or segment thereof, and the somatostatin peptide is somatostatin, or a cross-reactive and immunologically functional analog thereof. X is the amino acid α-COOH or α-CONH ₂ , n is 1 to about 10, m is 1 to about 4, and o is 0 to about 10).

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

When A is an amino acid or a general immunostimulatory factor such as Inv, it is covalently attached to either the N-terminus of the peptide immunogen as shown in the formula or to the C-terminus (not shown). Good.

When A is an amino acid, it can be any non-naturally occurring amino acid or any naturally occurring amino acid. Non-naturally occurring amino acids include, but are not limited to, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline and others. Naturally occurring amino acids include: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, Tyrosine and valine. Moreover, m is greater than 1 and A is greater than or equal to 2
When is an amino acid, each amino acid can be independently the same or different.

When A is the invasin domain, it is Yersinia
Immunostimulatory epitopes from species invasin proteins. This immunostimulatory results from the ability of this invasin domain to interact with the β1 integrin molecule present on T cells, especially activated immune or memory T cells. The specific sequence of the invasin domain found to interact with β1 integrin is Bret
t, et al. (Eur. J. Immunol., 1993). A preferred embodiment of the invasin domain (Inv) for binding to a promiscuous Th epitope is described previously in US Pat. No. 5,759,551, which is incorporated herein by reference. Was. The Inv domain has the sequence Thr-Ala-Lys-
Ser-Lys-Lys-Phe-Pro-Ser-Tyr-Thr-Ala-
It has Thr-Tyr-Gln-Phe (SEQ ID NO: 2) or its immunostimulatory homologue from the corresponding region in the invasin protein of other Yersinia species. Thus, such homologs can contain amino acid residue substitutions, deletions or insertions, as long as they retain immunostimulatory properties, to accommodate bacterial strain varieties.

In one embodiment, n is 1 and A is α-NH ₂ . In another embodiment, n is 4 and A is in the following order in the invasin domain (In
v), glycine and glycine.

B is a spacer, which can be an amino acid which can be a naturally occurring or non-naturally occurring amino acid as described above. Each B is independently the same or different. The amino acids of B can also provide a spacer between the promiscuous Th epitope and the somatostatin peptide (eg, SEQ ID NO: 1) and its cross-reactive and functional immunological analogs, eg, Gly-Gly. . Gl
The y-Gly spacer, in addition to separating Th epitopes from B cell epitopes, ie, somatostatin peptides and immunological analogs thereof,
Any artificial secondary structure created by binding the epitope to the somatostatin peptide and its cross-reactive and functional immunological analogs, thereby eliminating interference between Th and / or B cell responses Can collapse. The amino acid sequence of B can also form a spacer that acts as a flexible hinge that enhances the separation of Th and IgE domains. Examples of flexible hinge forming sequences are found in the hinge region of immunoglobulin heavy chains. Flexible hinge sequences are often rich in proline. One particularly effective flexible hinge is the sequence Pro-P
Provided by ro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 3), wherein Xaa is any amino acid, preferably aspartic acid. The conformational separation provided by the B amino acids allows for a more efficient interaction between the displayed peptide immunogen and the appropriate Th and B cells,
Thus, it enhances the immune response to Th and antibody-derived epitopes and their cross-reactive and functional immunological analogs.

Th is an amino acid sequence (natural or unnatural amino acid) comprising a Th epitope. Th epitopes can consist of continuous or discontinuous epitopes. Therefore, not all amino acids of Th need be part of the epitope. Thus, Th including analogs and segments of Th epitopes
Epitopes can enhance or stimulate an immune response to somatostatin peptides and their immunological analogs. Epitopes that are immunodominant and promiscuous are highly and widely reactive in populations of animals and humans with a wide variety of MHC types (Partidos et al., 1991; US Pat. No. 5,759,551). The domain of the subject peptide has about 10 to about 50 amino acids, preferably about 10 to about 30 amino acids. When multiple Th epitopes are present (ie, m ≧ 2), each T
The h epitopes are independently the same or different. A Th segment is a contiguous portion of a Th epitope that is sufficient to enhance or stimulate an immune response to a somatostatin peptide (eg, SEQ ID NO: 1) and its immunological analogs.

Epitopes of the invention include, but are not limited to, for example: Hepatitis B surface and core antigen helper T cell epitopes (HBs Th
And HBc Th), pertussis toxin helper T cell epitope (Pt Th)
, Tetanus toxin helper T cell epitope (TT Th), measles virus F protein helper T cell epitope (MV _F Th), trachoma chlamydia (Ch)
lamydia trachomatis) major outer membrane protein helper T cell epitope (CT Th), diphtheria toxin helper T cell epitope (D
T Th), Plasmodium falciparum
m) circumsporozoite helper T-cell epitope (PF Th), Schistosoma mansoni
mansoni) triosephosphate isomerase helper T cell epitope (
SM Th) and Escherichia coli Trat helper T cell epitope (TraT Th). T derived from the selected pathogen
h are listed in U.S. Patent No. 5,759,551 as SEQ ID NOs: 2-9 and 42-52; as CT Th P11, Stagg et al., Immunology, 1993; 79
1-9; and as HBc peptides 50-69, Ferrari et al., J. Am.
Clin. Invest., 1991; 88: 214-222 (all of which are incorporated herein by reference). In addition, Th epitopes include ideal artificial Th (eg, SEQ ID NO: 14) and artificial SSAL Th (eg, SEQ ID NO: 7, 30, 31). Peptides comprising SSAL Th are produced in tandem with somatostatin and other sequences simultaneously in a single solid phase peptide synthesis. These sites contain functional immunological analogs. Functional Th analogs include any immune enhancing, cross-reactive analogs and segments of these Th epitopes. Functional Th analogs further include, among Th epitopes, from 1 to about 1 that do not substantially alter the Th stimulatory function of the Th epitope.
Includes conservative substitutions, additions, deletions and insertions of zero amino acid residues.

The present invention represented by the following formula (A) _n- (Th) _m- (B) _o- (somatostatin peptide) or (A) _n- (somatostatin peptide)-(B) _o- (Th) _m Is covalently linked via a spacer B to either the N- or C-terminus of the somatostatin peptide and its cross-reactive and functional immunological analogs.

A cross-reactive, immunologically functional analog of a somatostatin peptide (eg, SEQ ID NO: 1) according to the present invention is that the peptide analog elicits a cross-reactive immune response with the somatostatin peptide. Where possible, it may further comprise conservative substitutions, additions, deletions or insertions of 1 to about 4 amino acid residues. Conservative substitutions, additions, and insertions can be achieved using natural or unnatural amino acids as defined herein.

A preferred peptide immunogen of the present invention is a somatostatin peptide or a cross-reactive and functional immunological analog thereof; a spacer (eg, Gly-Gly); a Th epitope, ie, HB _S Th (SEQ ID NO: 15). , HB _C Th (SEQ I
D NO: 4), MV _F Th (SEQ ID NOS: 21, 29), PT T
h (SEQ ID NO: 6), TT Th (SEQ ID NO: 5), CT
Th (SEQ ID NO: 27), DT Th (SEQ ID NO: 28)
), Artificial Th (eg, SEQ ID NOS: 7, 14, 30, 31) or analogs thereof; and optionally Inv domain (SEQ ID NO: 2) or analogs thereof.

[0039] Peptide compositions containing a mixture of a subject peptide immunogen and two or more Th epitopes can enhance immune efficiency in a broader population, thus improving the immune response to the somatostatin peptide Can be provided.

The peptide immunogen of the present invention can be produced by a chemical synthesis method well known to those skilled in the art. See, for example, the following references: Fields et al., Chapter 3,
Synthetic Peptides: A User's Guide, Grant, WH, Freeman & Co., New
York, NY, 1992, p.77. Therefore, automated Merrifield for solid-phase synthesis using α-NH ₂ protected by t-Boc or Fmoc chemistry.
d) Following techniques, for example, Applied Biosystems (Applied B)
Iosystems) Peptides can be synthesized using amino acids side-chain protected on a peptide synthesizer 430A or 431. Production of peptide constructs comprising the SSAL for a Th epitope can be achieved by providing a mixture of alternative amino acids for coupling at a given variable position.

After assembly of the desired peptide immunogen is complete, the resin is treated according to standard procedures to cleave the peptide from the resin and deblock functional groups on amino acid side chains. The free peptide is purified, for example, by HPLC and biochemically characterized, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those skilled in the art.

The subject immunogen can also be polymerized. Polymerization in accordance with routine methods, for example, glutaraldehyde can be accomplished by reaction with the -NH ₂ group of lysine residues. According to another method, a synthetic immunogen, for example, polymerizing an “A-Th _m -spacer- (somatostatin peptide)” or “(somatostatin peptide) -spacer- (Th) _m -A”, or a synthetic immunogen “ Co-polymerization with other immunogens by utilizing an additional cysteine added to the N-terminus of "A-Th-spacer (somatostatin peptide)" or "(somatostatin peptide) -spacer (Th) _m- (A) _n " Can be done. Also, by synthesizing the desired peptide construct directly on a branched poly-lysyl core resin,
The subject immunogen can be prepared as a branched polymer (Wang et al., Scie
nce, 1991; 254: 285-288).

Alternatively, longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Many standard manuals on DNA engineering are
Detailed protocols for producing the peptides of the invention are provided. To construct a gene encoding a peptide of the invention, the amino acid sequence is back-translated into a nucleic acid sequence, preferably using codons optimized for the organism in which the gene is expressed. Next, a synthetic gene is made, typically by synthesizing overlapping oligonucleotides encoding the peptide and the necessary regulatory elements. The synthetic gene is inserted into a suitable vector to form a recombinant and characterized. The peptide is then expressed under conditions appropriate for the chosen expression system and host. The peptide is purified and characterized by standard methods.

The efficacy of the peptide composition of the present invention is described in detail in Examples. An immunogenic composition comprising a peptide of the present invention in an animal such as a rat, for example, SEQ ID NO: 8-13, 16-20, 22 Injections and then follow up the humoral immune response to somatostatin and its cross-reactive and functional immunological homologs.

Another aspect of the present invention provides a peptide composition comprising an immunologically effective amount of one or more peptide immunogens of the present invention in a pharmaceutically acceptable delivery system. Thus, the subject peptides can be formulated as a peptide composition using an adjuvant, a pharmaceutically acceptable carrier, or other ingredients routinely provided in the peptide composition. Examples of ingredients that can be used in the present invention include adjuvants or emulsifiers such as alum, incomplete Freund's adjuvant, liposin, saponin, squalene, L121, emulsigen, monophosphoryl lipid A (MAL), QS21, ISA51, ISA35, ISA206, and ISA720, and other known effective adjuvants and emulsifiers. The formulations can be formulated for immediate and / or sustained release, and for inducing systemic immunity, which can be achieved, for example, by capture of the immunogen or co-administration with microparticles. Such formulations are readily determined by those skilled in the art, and methods of making, storing, and sterilizing such formulations are known in the art. The immunogens of the present invention can be administered by any convenient route, e.g., subcutaneously, orally, intramuscularly,
Or it can be administered by other parenteral or enteral routes. Similarly, immunogens
It can be administered as a single dose or as multiple doses. One of skill in the art will readily determine the immunization schedule.

The peptide compositions of the invention comprise an effective amount of one or more peptide immunogens of the invention and a pharmaceutically acceptable carrier. Such compositions will generally contain from about 0.5 μg to about 1 mg of immunogen / kg body weight in a suitable dosage unit form. When introduced in multiple doses, the compositions may be conveniently divided into appropriate doses / dosage unit forms. For example, an initial dose may be provided as a peptide composition of the invention, eg, 0.2-2.5 mg, preferably 1 mg, of the immunogen administered by injection, preferably as an intramuscular injection, and then repeated. (Booster) Administer. Dosages will depend on the age, weight and general health of the animal, as is well known in the vaccine and therapy arts.

Improving the immune response to synthetic somatostatin peptide immunogens by delivery trapped in or on biodegradable microparticles described by O'Hagan et al. (Vaccine, 1991; 9: 768-771). Can be. The immunogen is encapsulated in biodegradable microparticles in the presence or absence of an adjuvant to enhance the immune response and provide a sustained or periodic response and time-controlled release for oral administration (O'Hagan et al., 1991; Eldridge et al., Molec. Immunol., 1991
28: 287-294). Immunogens for particular peptides and peptide conjugates are provided in the Examples below to illustrate the invention. These examples are for illustrative purposes only and should not be construed as limiting the invention in any manner.

Example 1 Exemplary Methods for the Synthesis of Somatostatin Peptide Constructs The peptides listed in Tables 2 and 3 were individually applied to the applied biosystems automated peptide synthesizer (Models 430) using Fmoc chemistry and the melifilled solid phase synthesis technique. 431 and 433A). The constructed synthetic antigen library (SSAL), for example, “1,4,9 PALINDROM”
The preparation of a peptide construct comprising an artificial Th, referred to as "IC" (SEQ ID NO: 7), can be achieved by subjecting the mixture of desired amino acids for chemical coupling to the predetermined positions specified in this design. After complete assembly of the desired peptide, the resin is treated to cleave the peptide from the resin and deblock the protecting groups on the amino acid side chains according to standard procedures utilizing trifluoroacetic acid. In the case of a cyclic peptide, the cleaved peptide is dissolved in a 15% aqueous solution of DMSO for 48 hours to promote inter-disulfide bond formation between cysteines.

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

The peptide labeled “b” in the peptide code column was synthesized in tandem with the indicated Th site as the target antigenic peptide. The Th sites used include, for example, HBr Th (SEQ ID NO: 15) from Hepatitis B virus and "1,
4,9 PALINDROMIC ”(SEQ ID
NO: 7). The peptide labeled "c" is a variant of the "b" construct synthesized in tandem with the Inv domain immunostimulatory peptide (SEQ ID NO: 2). The peptide labeled "d" is an inverted version of the "b" construct (e.g., somatostatin-Th), and the peptide labeled "e" is an inverted version of the "c" construct (e.g., somatostatin-Th). -Inv). "B", "c", "d" and "
The "e" construct was synthesized with a Gly-Gly spacer to separate the target antigen site from the Th site and to separate Th from the Inv immunostimulatory site.

Example 2 Typical Method for Evaluation of Somatostatin Peptide Immunogenicity A somatostatin peptide immunogen (eg SEQ ID NO shown in Tables 2 and 3)
: 8-13, 16-20, 22 and 24) were evaluated based on groups of 4 or 5 rats identified by the experimental immunization protocol outlined below and serological assays for immunogenicity determination.

Standard Experimental Design: Immunogen: (1) Individual peptide immunogens; or (2) Mixtures containing equimolar peptide immunogens identified in each example. Dose: 100 μg in 0.5 ml per immunization unless otherwise specified Root: Intramuscular adjuvant unless otherwise specified: (1) Freund's complete adjuvant (CFA) / incomplete adjuvant (IFA); or ( 2) 0.4% alum (aluminum hydroxide); CFA / IFA group received CFA at week 0 and IFA at subsequent weeks. The alum group received the same formulation for all doses. Dosing schedule: 0, 2, and 4 weeks; 0, 3, and 6 weeks, or as otherwise indicated. Blood collection schedule: 0, 3, 6 and 8 weeks unless otherwise specified Species: Sprague-Dawley rats Group size: 4 or 5 rats / group Assay: Specific ELISA for anti-peptide activity of each immune serum. The solid phase substrate was a cyclized somatostatin peptide (SEQ ID NO: 1).

[0053] Blood is collected and processed into serum and ELISA with the target antigen peptide
Stored until titer test by A.

Anti-somatostatin antibody activity was determined by ELISA (enzyme-linked immunosorbent assay) using a 96-well flat-bottom microtiter plate coated with cyclized somatostatin peptide (SEQ ID NO: 1) as immunosorbent. . 5
Aliquots (100 μl) of the peptide immunogen solution at a concentration of μg / ml were
Incubated for hours. These plates were blocked by a further incubation with a 3% gelatin / PBS solution at 37 ° C. for 1 hour. The blocked plate was dried and used for the assay. Aliquots (100 μl) of test immune sera, starting at a 1: 100 dilution in sample dilution buffer, then serially diluted 10-fold, were added to peptide-coated plates.
These plates were incubated at 37 ° C. for 1 hour.

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 an appropriate dilution in conjugate dilution buffer (phosphate buffer containing 0.5 M NaCl and standard goat serum). The plates were incubated at 37 ° C. for 1 hour and washed as described above. O
-An aliquot (100 μl) of the phenylenediamine substrate solution was added. 5-15
The color reaction of the enzyme was stopped by the addition of 50 μl of 2N H ₂ SO ₄ for 5 min. A _{492 nm} of the contents of each well was read on a plater reader. EL
ISA titers were calculated based on linear regression analysis of absorbance, with a cut-off A of _{492 nm} of 0.5
Was set to. This cutoff value is strict because the value of the diluted standard control sample run in each assay is less than 0.15.

Th Peptide-Based ELISA The Th peptide-based ELISA is performed essentially in the same way as the somatostatin ELISA described herein except for the antigen coating step, wherein microtiter wells are loaded with a corresponding somatostatin vaccine construct (eg, , SEQ ID NOs: 14 and 15) at 5 [mu] g / ml for 1 h at 37 [deg.] C.

Example 3 Study of the Immunogenicity of Somatostatin Antigen Peptides Containing Various Immunostimulating Factors The somatostatin peptide immunogens shown in Table 2 represent variants of the peptides of the invention represented by the following formula: _{n - (Th) m - (} B) o - ( somatostatin peptide) or (A) _n - (somatostatin _{peptide) - (B) o - (} Th) m ( wherein, A is amino acid, ArufaNH ₂ or Inv (SEQ ID NO: 2); when A is an amino acid or Inv, it may be linked to the N-terminus or C-terminus; B is glycine; Th is a helper T cell epitope from a foreign pathogen, such as HB _{C50- 69} Th (
SEQ ID NO: 4), TT _615-631 Th (SEQ ID NO: 5), P
T _149-176 Th (SEQ ID NO: 6) and / or artificial Th, eg 1,4
, 9 PALINDROMIC Th (SEQ ID NO: 7); n is 1, m is 1, and o is 2.).

In particular, these peptides were synthesized and immune sera were generated for immunogenicity assessment. Most of the peptides shown in Table 2 containing Th epitopes in various forms and orientations induced high titers of somatostatin-specific antibodies in immunized hosts. In contrast, the somatostatin peptide lacking Th (p1348a, SEQ ID N
O: 1) lacked immunogenicity. However, a given Th was more favorable than others. For example, in Table 2, HB _C Th (SEQ ID NO: 4)
P2134b (SEQ ID NO: 8) with SYN Th (1, 2, 3
) (SEQ ID NO: 14), p2384b with artificial Th site (SEQ
P2138b (SEQ ID NO: 10) having ID NO: 20) and PT _149-176 Th (SEQ ID NO: 6) is TT Th (SEQ ID NO: 10).
NO: 5) is more immunogenic than p2135b (SEQ ID NO: 9), and they are all less immunogenic CTA8 Th (SEQ ID NO: 9).
More preferred is p2136b (SEQ ID NO: 24) having D NO: 23). Also, for optimal immunogenicity, the orientation of the Th site relative to the somatostatin target site must be specified. The kinetics of the antibody response of p2253b (SEQ ID NO: 11) is compared to that of p2255d (SEQ ID NO: 12). 1,4,9 PALINDROMIC T on the C-terminus of somatostatin
h (SEQ ID NO: 7) is preferably arranged. p1344b (SEQ
The best configuration for MV _F258-277 (SEQ ID NO: 21) is on the N-terminus of somatostatin, based on a comparison of pD349b (SEQ ID NO: 22) with p1349b (SEQ ID NO: 22). It is clear that the choice and arrangement of each Th site must be specified for a preferred peptide of the invention.

For the somatostatin peptides shown in Table 3, if the somatostatin construct comprising the Prosmiths Th epitope has already been found to be immunogenic,
Addition of Inv to the "Th" construct may improve the immunogenicity of the somatostatin peptide. p1344b (SEQ ID NO: 16) and p1343c (SEQ
ID NO: 17) and p1346b (SEQ ID NO: 18) and p13
The immunogenicity contrast of 45c (SEQ ID NO: 19) is similar to that of the Inv domain (SE
Addition of Q ID NO: 2) to the N-terminus of the Th construct indicates immunogenicity in terms of the proportion of responding animals, the strength of the somatostatin-specific antibody titer, and the life span of the antibody response (> 35 weeks). It was shown to improve. However, p2134b (SEQ ID
NO: 8), p2134c (SEQ ID NO: 25) and p2134d (
Comparison of immunogenicity with SEQ ID NO: 26) shows that Inv may not be immunostimulatory in combination with certain Th sites, and that Inv and HB _C Th sites (
It shows that certain orientations on the N-terminus are preferred over C-terminal orientations in combination with SEQ ID NO: 4). Regarding the example of p2135e (SEQ ID N
O: 13), Inv on the C-terminus and TT _615-631 Th (SEQ ID NO: 5).
) Resulted in an effective immunogen. Therefore, the addition of Inv and Th and I
The orientation with nv must be specified for the preferred peptides of the invention.

[0060] For constructs with strong site-specific immunogenic against somatostatin, immune stimulators, eg, Table 3 p1343c derived Inv-MVF _258-277 Th (SE
QID NO: 17) and KKK-HB _S19-32 Th-GG of _p1346b (S
The antibody titer specific to EQ ID NO: 18) was <1 Log ₁₀ , whereas for somatostatin was> 3 Log ₁₀ . Thus, the immune response generated by the synthetic peptides of the present invention was almost exclusively specific for the somatostatin target site.

Example 4 Additional Antigen Peptides of the Invention Peptides Somatostatin peptide antigens representing variants of the peptides of the invention represented by the following formula were synthesized and immune sera were prepared: (A) _n- (Th) _m- (B) _o- (somatostatin peptide) or (A) _n- (somatostatin peptide)-(B) _o- (Th) _m (where Th is a helper T cell derived from any foreign pathogen shown in Table 4) Is an epitope or a helper T cell epitope from any of the artificial Th epitopes shown in Table 5; A is an amino acid, αNH ₂ or invasin domain (SEQ ID NO:
: 2); when A is an amino acid or Inv, it may be linked at either the N-terminus or the C-terminus; B is glycine; n is 1, m is 1, and o is 2).

[0062]

[Table 1]

[0063]

[Table 2]

[0064]

[Table 3]

[0065]

[Table 4]

[0066]

[Table 5]

[0067]

[Table 6]

[Sequence list]

[Procedure amendment]

[Submission date] August 2, 2001 (2001.8.2)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 171 C07K 19/00 C07K 14/655 C12N 15/00 ZNAA 16/28 A61K 37/02 19 / (37) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, B , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (29)

[Claims] 1. A peptide conjugate comprising a helper T cell epitope sequence (Th) covalently linked to somatostatin or a cross-reactive and immunologically functional analog thereof. 2. The peptide conjugate according to claim 1, wherein the peptide conjugate is represented by the following formula: H ₂ N- (A) _n- (somatostatin peptide)-(B) _o- (Th) _m -X
Or H ₂ N- (A) _n- (Th) _m- (B) _o- (somatostatin peptide) -X
Wherein H ₂ N is the N-terminal α-NH ₂ of the peptide conjugate, each A is independently an amino acid or a general immunostimulatory sequence, and each B is an amino acid, —NHCH (X) CH _{2 SCH 2 CO-, -NHCH (X} ) CH 2 SCH 2 CO (ε-N) Lys-, -NHCH (X) CH 2 S- succinimidyl (ε-N) Lys-, and -NHCH (X) CH ₂ S Each Th is independently an amino acid sequence comprising a helper T cell epitope, or an immunopotentiating analog or segment thereof; the somatostatin peptide is somatostatin or a crossover thereof. A reactive and immunologically functional analog; X is the amino acid α-COOH or α-CONH ₂ ; n is 1 to about 10; m is 1 to about 4 And o is 0 to about 10). 3. The peptide conjugate according to claim 1, wherein the peptide conjugate is represented by the following formula: H ₂ N- (somatostatin peptide)-(B) _o- (Th) _m- (A) _n -X
Or H ₂ N- (Th) _m- (B) _o- (somatostatin peptide)-(A) _n -X
Wherein H ₂ N- is the N-terminal α-NH ₂ of the peptide conjugate, each A is independently an amino acid or a general immunostimulatory sequence, and each B is an amino acid, —NHCH (X) _{CH 2 SCH 2 CO-, -NHCH (} X) CH 2 SCH 2 CO (ε-N) Lys-, -NHCH (X) CH 2 S- succinimidyl (ε-N) Lys-, and -NHCH (X) CH ₂ Selected from the group consisting of S- (succinimidyl)-; each Th is independently an amino acid sequence comprising a helper T-cell epitope, or an immune enhancing analog or segment thereof; X is an amino acid α-COOH or α-CONH ₂ ; n is 1 to about 10; m is 1 to about 4 And o is 0 to about 10). 4. The peptide conjugate according to claim 2, wherein each B is selected from the group consisting of natural and unnatural amino acids. 5. The peptide conjugate according to claim 1, wherein said somatostatin peptide is somatostatin. 6. The peptide conjugate according to claim 1, wherein said Th is an SSAL epitope. 7. The Th is 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, SEQ
The peptide conjugate according to any one of claims 1 to 4, having an amino acid sequence selected from the group consisting of ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. 8. The peptide conjugate according to SEQ ID NO: 8, S
EQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11
, 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
The peptide conjugate according to claim 2, which has an amino acid sequence selected from the group consisting of: 9. The peptide conjugate according to claim 2, wherein at least one A is an invasin domain. 10. n is 3 and (A) ₃ is (invasin domain)
3. The peptide conjugate according to claim 2, which is -Gly-Gly. 11. The peptide conjugate according to claim 9, 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, comprising: (a) an invasin domain; (b) a helper T cell (Th) epitope; and (c) somatostatin or its cross-reactivity and immunology. A synthetic peptide comprising the amino acid sequence of: 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. The peptide or peptide conjugate of any one of claims 1 to 13, wherein said peptide stimulates a mammalian immune response to somatostatin. 15. The peptide or peptide conjugate of claim 14, wherein immunization of the mammal with the peptide conjugate reduces somatostatin levels in the mammal. 16. A pharmaceutical composition comprising an immunologically effective amount of the peptide or peptide conjugate of any one of claims 1 to 15 and a pharmaceutically acceptable carrier. 17. The pharmaceutical composition of claim 16, wherein said immunologically effective amount is from about 0.5 mg to about 1 mg of said peptide or peptide conjugate per kg body weight per administration. 18. A method for inducing the production of anti-somatostatin antibodies in a mammal, the method comprising administering to the mammal a pharmaceutical composition according to claim 16 or 17. 19. A method for increasing the growth rate of a mammal, comprising administering to the mammal a pharmaceutical composition according to claim 16 or 17. 20. A method for increasing the growth rate of a mammal, wherein the mammal has an amount sufficient to reduce somatostatin levels.
A method comprising administering a pharmaceutical composition as described. 21. A composition comprising a mixture of two or more of the peptides or peptide conjugates of any one of claims 1 to 15. 22. A pharmaceutical composition comprising an immunologically effective amount of the composition of claim 21 and a pharmaceutically acceptable carrier. 23. The pharmaceutical composition of claim 22, wherein said immunologically effective amount of said composition is from about 0.5 μg to about 1 mg / kg body weight per administration. 24. A method for inducing the production of an anti-somatostatin antibody in a mammal, comprising administering to the mammal the pharmaceutical composition according to claim 22 or 23. 25. A method for increasing the growth rate of a mammal, comprising administering to the mammal the pharmaceutical composition of claim 22 or 23. 26. A method of increasing the growth rate, comprising administering to a mammal a pharmaceutical composition according to claim 22 or 23 in an amount sufficient to reduce somatostatin levels. 27. A branched polymer comprising a lysine, trilysine or peptalidine core covalently linked to each of 2, 4 or 8 peptide conjugates according to any one of claims 1 to 15. 28. The method according to claim 1, which is crosslinked by a divalent crosslinking agent.
A polymer comprising one or more peptide conjugates according to any one of claims 15 to 15. 29. SEQ ID NO: 4, SEQ ID NO: 5, SE
Q ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14, S
EQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 2
3, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID N
A Th epitope peptide selected from the group consisting of O: 29, SEQ ID NO: 30, and SEQ ID NO: 31.