EP0819137A1

EP0819137A1 - Peptides with growth promotion properties

Info

Publication number: EP0819137A1
Application number: EP96911304A
Authority: EP
Inventors: Brian Lee Buckwalter; Hong-Ming Shieh; Bosco Shang Wang
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 1995-03-31
Filing date: 1996-03-15
Publication date: 1998-01-21
Also published as: WO1996030405A1; MX9707433A; PL322585A1; KR19980703438A; BG101927A; HUP9801337A2; CA2216755A1; JPH11502844A; AU5422596A

Abstract

Peptides are described which have well-defined secondary structure, preferably a helical conformation, which mimic the corresponding region of porcine somatotropin (pST) and which enhance the activity of pST and promote growth of warm-blooded animals. These peptides compete with pST for binding to the PS-7.6 monoclonal antibody. These peptides contain therein the sequence of amino acids Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID No. 1), wherein the sequence differs from the native sequence of pST, as well as sequences in which the location of essential amino acids is shifted by three amino acids, representing almost one turn along the helix, which contain therein the sequence of amino acids Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID No. 18) and wherein the sequence differs from the native sequence of pST.

Description

PEPTIDES WITH GROWTH PROMOTION PROPERTIES

Field Of The Invention

This invention relates to peptides which elicit antibodies which enhance the activity of porcine somatotropin and promote growth of warm¬ blooded animals.

Background Of The Invention

Porcine somatotropin (pST) is a 191 amino acid long pituitary hormone which has diverse biological activities (Bibliography entry 1) . This hormone exists as a single chain polypeptide which is arranged in four anti-parallel α-helices (2) . Each α- helix contains 3.6 amino acids per turn. This periodicity places every third or fourth residue on the same face of the helix.

Administration of pST to farm animals increases muscle growth, feed efficiency and decreases fat accumulation (3,4). Endogenous amounts of pST are small; therefore, efforts have focused on the preparation of exogenous pST for use in large-scale agriculture. The widespread use of exogenous pST has been hampered by difficulties in formulation and administration. Long-term implants require the stabilization of the pST molecule and control of the release of pST over time. Alternatively, daily or frequent periodic injections of pST involve labor costs which may render the benefits of pST uneconomical.

Some efforts to reduce these difficulties have involved reducing the amount of pST needed through the addition of a component which potentiates the activity of pST.

Monoclonal and polyclonal antibodies complexed with somatotropin have been reported to further enhance the biological activity of somatotropin in vivo (5-9) . Although the mechanism of antibody-mediated growth enhancement is not totally clear, altered pharmacokinetics and/or bio- distribution of somatotropin has been proposed in part to explain the growth enhancement phenomenon (10) . It has also been suggested that the enhancement is due to the selective modulation of binding to subclasses of somatotropin receptors by the antibodies (5,6,11,12). Improvement of somatotropin binding to hepatic somatogenic receptors was also reported to be a possible explanation (13) . It is noteworthy that these antibodies by themselves do not potentiate growth in the absence of exogenous pST.

One attempt to increase animal growth immunologically has produced a monoclonal antibody designated PS-7.6, which consistently increases the biological activity of pST in a hypophyβectomized (hypox) rat assay (10) , even though in the absence of pST it does not by itself increase growth. However, passive immunization of farm animals with this growth- enhancing monoclonal antibody is not optimum economically because of the cost of monoclonal antibody and the labor required for the multiple dosing of antibodies.

Thus, there is a need for a new approach to growth potentiation which will reduce the difficulties of the current approaches. One such approach involves the design of peptides which mimic the region of pST which binds to PS-7.6 monoclonal antibody. The active immunization of animals with peptide fragments corresponding to the epitope recognized by PS-7.6 monoclonal antibody should generate antibodies functionally similar to PS-7.6 monoclonal antibody to enhance the endogenous pST activities. Thus, the identification of the epitope recognized by PS-7.6 monoclonal antibody is critical for the design of peptide vaccines that enhance animal growth performance. Most peptides possess the primary, but lack the secondary conformation of the native protein. Short peptides typically exist in solution as rapidly interconverting structures (random coils) . A conformation analogous to the desired region of pST will be present in only a small fraction of the peptides at a given time. Thus, there is a need to design peptides having a defined secondary conformation.

■qiimma-y of The Invention

Accordingly, it is an object of this invention to design peptides which have well-defined secondary structure, preferably a helical conformation, which mimic the corresponding region of pST.

It is a further object of this invention to design peptides which compete with pST for binding to PS-7.6 monoclonal antibody.

It is yet another object of this invention to develop compositions which utilize these peptides for the promotion of growth of warm-blooded animals. These objects of the invention are accomplished through a peptide which contains therein the sequence of amino acids Xaa-Xaa-Leu-Xaa-Xaa-Ile- Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID NO:IK wherein the sequence differs from the native sequence of pST. The invention also includes peptides wherein one or more of the first or second leucine or the valine of SEQ ID NO:l is replaced by normal (straight chain) leucine (Nle) . The invention is also directed to a peptide in which the location of the essential amino acids is shifted by three amino acids, representing almost one turn along the helix, which contains therein the sequence of amino acids Xaa-Xaa- Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa- Xaa-Val (SEQ ID NO:18) and wherein the sequence differs from the native sequence of pST. The peptides of SEQ ID NO:18 are further modified by limiting the third amino acid residue to isoleucine, generating the sequence of amino acids: Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa- Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19) and wherein the sequence differs from the native sequence of pST.

These peptides preferably contain serine as a promoter of helical conformation as the amino acid immediately amino-terminal to the first leucine of SEQ ID NO:l and as the amino acid immediately amino- terminal to the first isoleucine of SEQ ID NO:19.

The peptides of this invention are used with a pharmaceutically acceptable adjuvant, diluent or carrier to formulate compositions in methods for promoting the growth of a warm-blooded animal.

Brief Description Of The Figures

Figure 1 depicts the trypsin digests (lane 1), fractions #9 (lane 2) and #9-5 (lane 3) which are incubated with PS-7.6 monoclonal antibody, im unoprecipitated by magnetized goat anti-mouse IgG antibody, subjected to SDS-PAGE, electroblotted onto a PVDF membrane, probed with rabbit anti-pST polyclonal antibody followed by iodinated donkey anti-rabbit IgG antibody, and finally exposed to an x-ray film and autoradiography.

Figure 2 depicts the peptides pST(70-95), pST(64-95) and pST(54-95) (Figure 2A) , and pST(75-95) and pST(75-90) (Figure 2B) , which are synthesized and tested for their inhibitory effects on the interaction between PS-7.6 monoclonal antibody and ¹³⁵I-pST in a competitive RIA. Cold intact pST is also tested and serves as the positive control. Figure 3 depicts the results of a radioi munoassay which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST compared with: (1) the pGH(75-95) peptide, (2) a pGH(80-90) peptide, and (3) a pGH(81-90) peptide. Figure 4 depicts the amino acids of the pST(75-95) fragment, of which amino acid residues 75- 90 are sequentially substituted by alanine. These analogs are tested by competition RIA. The IC₅₀ of an effective competition for each residue is averaged from five separate experiments.

Figure 5 depicts a helical projection of pST(75-95) of pST. The shaded circles are the amino acids critical for the recognition by PS-7.6 monoclonal antibody. Figure 6 depicts the results of a radioimmunoassay which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST (referred to as "pGH" in Figure 5) compared with: (1) a peptide corresponding to the native sequence of amino acids 75-95 of pST (referred to as "pGH(75-95) " in Figure 5), (2) the peptide of SEQ ID NO:2 (referred to as "peptide-X") , and (3) a modified form of peptide-X where the amino-terminal amino group is modified by the addition of an acetyl group (referred to as "capped peptide-X") . Figure 7 depicts the results of a radioimmunoassay which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST compared with: (1) the pGH(75-95) peptide, and (2) a peptide of SEQ ID NO:21 not within the scope of this invention (referred to as "peptide-Y" in Figure 6).

Figure 8 depicts the results of a radioimmunoassay which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST compared with: (1) the pGH(75-95) peptide, (2) peptide-X, (3) peptide-Y, and (4) peptide-X wherein the first leucine is replaced by normal leucine (Nle) , generating SEQ ID NO:13 (referred to as "Nle80" in Figure 7) , (5) peptide-X wherein the second leucine is replaced by normal leucine (Nle) , generating SEQ ID NO:14 (referred to as "Nleβ7") , and (6) peptide-X wherein the valine is replaced by normal leucine (Nle), generating SEQ ID NO:15 (referred to as "Nle90").

Figure 9 depicts the effect on the growth of hypophysectomized rats receiving either no treatment (negative control, first bar) ; treatment with pST alone (positive control, second bar) ; treatment with pST plus swine antibodies before and after immunization with peptide-X (SEQ ID NO:2) from three different pigs (three groups of rats, third through eighth bars) .

Detailed Description Of The Invention

This invention comprises the design of peptides which mimic a helical region of pST. These peptides possess minimal sequence homology to pST in order that the peptides can retain a helical formation. These peptide mimics elicit antibodies which can recognize native pST.

Immunization of a target animal species with a peptide containing the epitope recognized by PS-7.6 monoclonal antibody (raised against native pST) produces antibodies functionally similar to PS-7.6 monoclonal antibody and promotes the growth of the animal.

Active immunization of animals with the epitope recognized by PS-7.6 monoclonal antibody is a practical alternative to the passive administration of PS-7.6 monoclonal antibody. This idea is supported by a recent report of Pell et al (14) who demonstrated that immunization of lambs with somatotropin fragment (residues 135-154) , a different region associated with the enhancement, increased the percentage of lean muscle growth.

In this invention, the epitope of pST which binds to PS-7.6 monoclonal antibody is mapped by a limited tryptic digestion of pST coupled with reverse- phase liquid chromatographic separation and immunoprecipitation. The critical amino acids of the epitope which interact with PS-7.6 monoclonal antibody are then identified by an immunologic analysis of a series of sequential alanine analogs of the epitope. The peptide which contains the epitope recognized by the monoclonal antibody designated PS-7.6, which is raised against recombinant pST, is shown to enhance the growth-promoting activity of pST in a hypophysectomized rat model and to promote growth in pigs.

A pST fragment corresponding to amino acid residues 70-95 (which contains the second helix of pST (2) ) is separated by reverse-phase high performance liquid chromatography and also immunoprecipitated by PS-7.6 monoclonal antibody. This fragment is found in a radioimmunoassay (RIA) to compete with radiolabelled pST for the binding to PS-7.6 monoclonal antibody in a dose-dependent fashion. However, sera from pigs immunized with pST(70-95) do not raise significant levels of anti-pST antibodies. This result suggests that the conformation of pST(70-95) does not match that of the true epitope or the peptide is not sufficiently immunogenic. Several peptides covering this potential epitope region of pST(70-95) are synthesized and assayed by competitive RIA. The results suggest that pST(75-95) is the optimal sequence recognized by PS- 7.6 monoclonal antibody. In contrast, pST(85-95) binds very weakly and non-reproducibly to PS-7.6 monoclonal antibody, while pST(81-95) is completely devoid of binding. The peptide pGH(80-90) binds almost as strongly as pGH(75-95), while pGH(81-90) binds very weakly to PS-7.6 monoclonal antibody. Furthermore, if the peptide is too small, such as pST(80-95), even though binding is good, aggregation is substantial due to the hydrophobic character of the individual amino acid residues. To reduce the aggregation problem and improve solubility, the peptide should be longer.

The design of peptides in the epitopic region pST(75-95) involves a number of factors. Most peptides possess the primary, but lack the secondary conformation of the native protein. Short peptides typically exist in solution as rapidly interconverting structures (random coils) . A conformation analogous to the desired region of pST will be present in only a small fraction of the peptides at a given time.

The goal of the design is to stabilize the helical conformation and thus to enhance the affinity of the antibody for the protein. This affinity requires structural complementarity between the binding site of the antibody and the epitope on the protein. Amino acid residues which contact the antibody binding site are relatively intolerant of substitution. Replacement of leucine by isoleucine is sufficient to abolish peptide binding to the antibody. Non-critical residues can be replaced by residues which promote or stabilize a desired structure, in this case an α-helix, which allows the peptide to better mimic the native protein conformation.

Among the strategies which can be employed to stabilize α-helices are: (1) interhelical hydrophobic association (15); (2) side-chain charge- charge interactions (16); (3) side-chain covalent linkages by a disulfide (17) or lactarn bond (18) ; (4) incorporating amino acid residues with high helical propensity (19) ; (5) charge-helix dipole stabilization (20) ; (6) end-capping of peptides (21) and metal-ion side chain stabilization (22) .

The criticality of the first leucine is demonstrated in Example 1 below, where the first leucine of a peptide (SEQ ID NO:2) is replaced by isoleucine to produce the peptide of SEQ ID NO:21. In contrast, non-contact residues may be replaced freely and still bind effectively. Thus, after identifying the essential antibody contact residues, the remaining residues of the peptide are replaced so as to achieve a stable helical conformation.

Sequential alanine substitution of each residue of pST(75-90) reveals that the residues of Leu", He" and Leu"⁷, plus either Leu" or Val", are critical for binding to PS-7.6 monoclonal antibody (as depicted in the helical projection of Figure 5) . Other residues are replaced by alanine without substantially altering the binding affinity. The region of amino acids 75-95 comprises the second helix of pST and the repeating pattern of i and i+3 (i+7) of the critical amino acids appears consistent with PS- 7.6 monoclonal antibody binding to a hydrophobic side of the helix. This enables peptides to be synthesized utilizing both the i and the i+3 (almost one helical turn) patterns. The sequence and the helical structure of the epitope recognized by PS-7.6 monoclonal antibody provide the basis for designing effective peptide vaccines to enhance the growth performance of animals.

The pST and peptides are generated as follows. Recombinant pST is produced by American Cyanamid Co., Pearl River, NY. Peptides are synthesized on a solid-phase automatic peptide synthesizer (9600, Milligen/Biosearch, Bedford, MA) . All reagents are purchased from Peninsula Laboratories, Inc., Belmont, CA. After being cleaved from the resin support, all peptides are purified by preparative reverse-phase high performance liquid chromatography (HPLC) on a C18 column (Rainin Instrument Corp., Woburn, MA). The purity of these peptides is always greater than 97% as determined by analytical HPLC, FAB mass spectrometry, and amino acid composition analysis. Alternatively, the peptides of this invention are also constructed by other techniques known in the art, such as solution-phase chemical synthesis or recombinant expression of a DNA nucleotide sequence in an appropriate host. Recombinant expression may also be in the form of a fusion of the peptide to a carrier, such as a maltose- binding protein or peptide. Mapping of the antibody-recognizing sites has been approached conventionally by the enzymatic digestion of the antigen (23,24) or the concurrent synthesis of multiple peptides on solid supports (25) . Initial attempts with multiple peptide synthesis were not successful and, therefore, the proteolytic degradation of pST is employed subsequently to complete the epitope mapping of PS-7.6 monoclonal antibody in this study. In the beginning, pST is treated at 37°C with various proteases, including chymotrypsin, trypsin and endoproteinase Glu-C. They all produce small pST fragments (m.w. < 4 kDa) . The rationale for selecting these small fragments is the convenience of solid phase synthesis of future peptide vaccine candidates. However, PS-7.6 monoclonal antibody fails to recognize any of these small fragments by RIA and Western Analysis. In addition, short peptides are generally conformationally flexible and produce a variety of inter-converting configurations in solution. As a result, resulting anti-peptide antibodies frequently interact with the corresponding native protein with a very low efficiency.

Results from the initial experiments of enzymatic degradation indicate that the epitope recognized by PS-7.6 monoclonal antibody is very sensitive to protease at 37°C. It is possible that the secondary/tertiary structure of pST might be slightly unfolded at this temperature, thus exposing the critical epitope region to the protease for a total degradation. In order to limit the degree of enzymatic digestion to protect the intact PS-7.6 epitope, especially the loops and turns (26) , the reaction temperature is restricted at 25°C for three days. Specifically, recombinant pST (100 mg) is subjected to limited tryptic digestion by incubation with 1 ml bovine pancreatic trypsin cross-linked to agarose (50 units enzymatic activity per ml of packed gel, Sigma Co., St. Louis, MO) in 200 ml phosphate buffer, pH 9.5, at 25°C for three days. The tryptic fragments are separated by preparative HPLC (C18 column, 21.4 mm x 25 cm, flow rate = 24 ml/min., UV at 235 nm., Dynamax-300A, Rainin) with a linear gradient of 0-100% of B buffer in 30 minutes (A buffer = 0.1% TFA in water; B buffer *- 0.1% TFA in acetonitrile) . All fractions are collected and analyzed by RIA to identify the active components. A predominant and stable tryptic fragment which is absent in the 37°C digestion is produced and designated as fraction #9. This fraction is eluted at a retention time of 18.89 minutes and comprises more than 60% yield of the initial pST digestion. It is later found to compete with ¹³⁵I-pST for the binding to PS-7.6 monoclonal antibody in RIA, suggesting that the fraction #9 contains the PS-7.6 epitope. The fraction #9 is further reduced by a 10 molar excess of dithiothreitol in 0.1 M ammonium bicarbonate solution at pH 9.5 and again fractionated by HPLC. One fraction, designated #9-5 and eluted at a retention time of 18.43 minutes, continues to exhibit an ability to compete with ¹²⁵I-pST for PS-7.6 monoclonal antibody binding.

A combination of immunoprecipitation and Western blotting experiments is carried out to analyze these samples. The trypsin digests, fractions #9 and #9-5, are incubated with PS-7.6 monoclonal antibody for 60 minutes at 37°C. Magnetic beads coated with goat anti-mouse IgG (Advanced Magnetics Inc., Cambridge, MA) are added to the mixture and incubated for 30 minutes at room temperature with gentle, constant shaking. The beads are washed several times and collected by a magnet. The proteins are separated from the beads by boiling in SDS-PAGE sample buffer and subjected to reduced Tris-tricine polyacrylamide gel electrophoresis on a 16% gel (PAGE, Novex, San Diego, CA) . The separated protein components are electroblotted from the gel onto a polyvinylidene difluoride microporouβ (PVDF) membrane (Applied Biosysterns, Foster City, CA) in a 10 mM 3- [cyclohexylamino] -1-propanesulfonic acid (CAPS) buffer at pH 11 containing 20% methanol. The membrane is treated with 5% nonfat milk followed by sequential exposures to rabbit anti-pST polyclonal antibody and iodinated donkey anti-rabbit IgG antibody (Amersham Corp., Arlington Heights, IL) . The PVDF membrane is finally exposed to an x-ray film for the development of autoradiography. To determine the amino acid sequence of the tryptic fragments, the membrane blots are stained briefly with a solution containing 0.1% Coomassie, 40% methanol and 10% acetic acid and the visible bands are excised. These bands are arranged in a single layer in the upper cartridge block of a gas-phase sequenator (Applied Biosysterns) and the phenylthiohydantoin-derived amino acids are determined by the procedure of Hankapiller & Hood (27) . The results are seen in Figure 1.

It is clear that the predominant component of pST after a limited digestion with trypsin is the 15 kDa material with minor contaminants of 10 kDa, 4 kDa and 2.5 kDa (lane 1). However, these minor components are enriched and become predominant in fraction #9 following HPLC fractionation (lane 2) . Finally, the 2.5 kDa fragment appears to be the only component present in the fraction #9-5, suggesting that the epitope recognized by PS-7.6 monoclonal antibody resides within this fraction (lane 3) . The 2.5 kDa fragment is then sequenced and found to correspond to amino acids 70-95 of pST. The chemical structure and immunologic properties of this fragment are confirmed by a solid phase peptide synthesis (28) , because the synthesized peptide is capable of competing with "⁵I-pST in a dose dependent fashion for the binding to PS-7.6 monoclonal antibody in RIA (Figure 2) . The competition by pST(70-95) is approximately 100-fold less active than that by cold pST.

In order to further characterize the precise epitope, three additional peptides are synthesized. Peptides pST(54-95) and pST(64-95) are produced by extending the amino acid residues of the N-terminus, while pST(75-90) is generated by truncating the residues at both ends. These peptides are tested in RIA and exhibit a similar competition effect as compared to the tryptic pST(70-95) fragment, suggesting that the residues between 75 and 90 contain the epitope (Figure 2) . The competition of these peptides is substantially lower than that of a control pST, probably due to the conformational flexibility inherent in the short peptides and the involvement of other discontinuous residues in the antibody recognition.

A separate competition RIA establishes the criticality of the Leu*⁰ residue. As depicted in Figure 3, the peptide pGH(80-90) binds almost as strongly as pGH(75-95), while pGH(81-90) binds very weakly to PS-7.6 monoclonal antibody.

The conformational flexibility of peptide antigens limits their practical utility as antigens. However, the limitation can be overcome by restricting the conformational flexibility of the peptides to mimic the secondary structure of the native protein antigen. It is also known that not every residue in an epitope is necessarily in contact with the antibody binding sites (29) . Because the secondary structure of the PS-7.6 monoclonal antibody epitope is considered important, the amino acid side chains of the pST(75-95) fragment that interact with PS-7.6 monoclonal antibody are determined by sequentially replacing each residue with alanine. The sequential substitution with alanine, which has a high propensity to form a helical conformation in peptides or proteins (30) , is less likely to change the accessible backbone conformation of pST(75-95) .

This approach, called alanine-scanning mutagenesis, has been very useful for identifying the critical amino acids of human somatotropin which interact with its receptor (31) . Alanine is assumed to leave the secondary structure unchanged, while eliminating one potential site of interaction with the antibody. If the peptide containing a substituted alanine still binds to the antibody, the replaced amino acid is considered to be a non-contact residue. Therefore, a series of alanine-substituted analogs of pST(75-90) is synthesized by the solid- phase method and their ability to compete for PS-7.6 monoclonal antibody binding with pST is evaluated by RIA. Side chains of residues are considered critical if the alanine replacement results in a significant increase in IC₅₀ to achieve an equivalent competition. Findings in Figure 4, which are summarized from five separate experiments, indicate that Leu⁸⁰ (see also Figure 3) , He⁸³ and Leu⁸⁷, which all are hydrophobic amino acids, are critical to PS-7.6 monoclonal antibody recognition, and that either Leu^7< or Val⁹⁰ is also critical. Although the bar value for position 89 is greater than that for position 90 in Figure 4, when p values are computed using Fisher's combined p-value test, the p value for position 90 is statistically significant (p=0.00162), while the p value for position 89 is not statistically significant (psO.16048) . Note that Figure 4 does not include the data from a sixth experiment, where the results are entirely inconsistent with the other five experiments.

Based on the tertiary structure of pST (2) and the projection of the second helix, these five critical residues presented by the shaded circles in Figure 5 are located on the same face of the helix, representing a hydrophobic ribbon to interact with PS- 7.6 monoclonal antibody.

In summary, the epitope of growth-enhancing antibody PS-7.6 monoclonal antibody is mapped by a limited tryptic digestion and the critical amino acids on the epitope are identified by the sequential alanine substitution. The α-helical projection of pST shows that the critical amino acids are a hydrophobic ribbon located on the same face of the second helix. The characterization of the sequence and secondary structure of the epitope provide the basis for designing effective peptide antigens that mimic the native conformation of the corresponding epitope of pST.

Based on the identification of critical amino acids in the epitope, a series of peptides is designed. In one embodiment of the invention, the peptides are selected from sequences containing the formula:

Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa- Xaa-Val-Xaa-Xaa (SEQ ID NO:l) wherein the sequence differs from the native sequence of pST. Examples of such peptides include: Tyr-Ser-Leu-Asp-Asp-He-He-Arg-Arg-Leu-Asp- Asp-Val-Ile-Arg-Arg-Ile (SEQ ID NO:2) , Tyr-Ser-Leu-Asp-Arg-Ile-Ile-Arg-Asp-Leu-Asp- Arg-Val-Ile-Arg-Asp-Ile (SEQ ID NO:3) , Tyr-Ser-Leu-Arg-Arg-Ile-Ile-Arg-Arg-Leu-Arg- Arg-Val-Ile-Arg-Arg-Ile (SEQ ID NO:4),

Tyr-Ser-Leu-Asp-Asp-Ile-Ile-Asp-Asp-Leu-Asp- Asp-Val-Ile-Asp-Asp-Ile (SEQ ID NO:5), Tyr-Ser-Leu-Asp-Asp-He-Ala-Arg-Arg-Leu-Asp- Asp-Val-Ala-Arg-Arg-Leu (SEQ ID NO:6), Tyr-Ser-Leu-Lys-Ala-He-Ala-Glu-Ala-Leu-Lys-

Ala-Val-Ala-Glu-λla (SEQ ID NO:7), Tyr-Ser-Leu-Lyβ-Glu-He-Glu-Lys-Leu-Leu-Lys- Glu-Val-Leu-Glu-Lys-Leu (SEQ ID NO:8), Tyr-Ser-Leu-Asp-Asp-He-Ala-Arg-Arg-Leu-Asp- Asp-Val-Ala-Arg-Arg-Ala (SEQ ID NO:9),

Tyr-Ser-Leu-Lys-He-Ile-Ile-Glu-Ile-Leu-Lys- Ile-Val-Ile-Glu-Ile (SEQ ID NO:10), Tyr-Ser-Leu-Lys-Glu-He-Glu-Lys-Leu-Leu-Lys- Glu-Val-Leu-Glu-Lys-Leu (SEQ ID NO:ll), and Tyr-Ser-Leu-Lys-Aib-He-Aib-Glu-Aib-Leu-Lys-

Aib-Val-Aib-Glu-Aib (SEQ ID NO:12), where Aib is 2-aminoisobutyric acid. In addition, one or more of the leucines or valine of SEQ ID NO:l can be replaced by normal leucine (Nle), as follows:

Tyr-Ser-Nle-Asp-Asp-Ile-He-Arg-Arg-Leu-Asp- Asp-Val-Ile-Arg-Arg-He (SEQ ID NO:13), Tyr-Ser-Leu-Asp-Asp-He-He-Arg-Arg-Nle-Asp- Asp-Val-Ile-Arg-Arg-He (SEQ ID N0:14), Tyr-Ser-Leu-Asp-Asp-lie-Ile-Arg-Arg-Leu-Asp-

Asp-Nle-Ile-Arg-Arg-He (SEQ ID NO:15), and Tyr-Ser-Nle-Asp-Asp-Ile-He-Arg-Arg-Nle-Asp- Asp-Val-Ile-Arg-Arg-He (SEQ ID NO:16) . Furthermore, a cysteine may be added to either or both ends of the peptides of SEQ ID NO:l. An example of such a peptide is:

Cys-Tyr-Ser-Leu-Asp-Asp-He-He-Arg-Arg-Leu-

Asp-Asp-Val-Ile-Arg-Arg-He (SEQ ID NO:17) .

In another embodiment of the invention, the location of the essential amino acid residues is shifted by three amino acids, representing almost one turn along the helix. These peptides are selected from sequences containing the formula:

Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-He-Xaa- Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:18) wherein the sequence differs from the native sequence of pST. In particular, these peptides include an isoleucine as the third residue and are selected from sequences containing the formula: Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-He-Xaa-

Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19) wherein the sequence differs from the native sequence of pST. An example of such a peptide is:

Tyr-Ser-Ile-Asp-Asp-Leu-Ile-Arg-Arg-He-Asp- Asp-Leu-He-Arg-Arg-Val (SEQ ID NO:20) .

These peptides preferably contain serine as a promoter of helical conformation as the amino acid immediately amino-terminal to the first leucine of SEQ ID NO:l and as the amino acid immediately amino- terminal the first isoleucine of SEQ ID NO:19.

The amino acid sequences of these peptides are described using the convention that the first essential residue is toward the amino-terminus and the last essential residue is toward the carboxy-terminus. The invention also encompasses identical sequences in opposite orientations where the first essential residue is toward the carboxy-terminus and the last essential residue is toward the amino-terminus. Both orientations have the same helical projection on the side chain (see Figure 5) and are functionally equivalent.

Other peptides within the scope of the invention are generated readily by using the methods and criteria described herein. The peptides of this invention are tested in various experiments, as described in detail in the Examples below.

First, radioi munoaβsayβ are performed to determine the extent to which various peptides (both within and outside the scope of this invention) compete with radiolabelled pST for binding to PS-7.6 monoclonal antibody. As detailed in Example 1 and shown in Figure 6, the peptide of SEQ ID NO:2 (referred to as "peptide-X") inhibits the interaction of PS-7.6 monoclonal antibody and radiolabelled pST in a dose dependent manner more strongly than a peptide corresponding to the native sequence of amino acids 75-95 of pST. A modified form of peptide-X, where the amino-terminal amino group is replaced by an acetyl group (referred to as "capped peptide-X") does not inhibit the interaction. In experiments not shown in Figure 6, the peptides of SEQ ID NOS:8-11 inhibit the interaction as well as peptide-X, while the peptides of SEQ ID NOS:3 and 12 inhibit the interaction, but at a lower rate than peptide-X.

As detailed in Example 1 and shown in Figure 7, a peptide not within the scope of the invention and having the sequence Tyr-Ser-Ile-Asp-Asp-Ile-He-Arg- Arg-Ile-Asp-Asp-He-Arg-Arg-Ile (SEQ ID NO:21; referred to as "peptide-Y") is much less effective in inhibiting the interaction than the pGH(75-95) peptide. Thus, the substitution of isoleucines for the first leucine and the valine in the peptide of SEQ ID NO:2 abolishes the binding to the antibody. As detailed in Example 1 and shown in Figure 8, peptide-X and three variants thereof (SEQ ID NOS:13-15) where the leucines or valine are replaced by normal leucine (Nle) are more effective than pGH(75-95) in inhibiting the interaction, whereas peptide-Y is again much less effective. In an experiment not shown in Figure 8, the peptide of SEQ ID NO:16 inhibits the interaction as well as peptide- X.

Next, the biological activity of these peptides is tested in hypophysectomized (hypox) rats. Hypox-rats are growth-deficient as a result of surgical removal of their pituitary glands. Hypox-rats serve as a useful model for studying the effect of somatotropin on growth (32) . As described in Example 2 and shown in

Figure 9, peptide-X (SEQ ID NO:2) is conjugated to ovalbumin and emulsified in Complete Freund's Adjuvant. Three pigs are immunized with peptide-X and receive two booster doses with Incomplete Freund's Adjuvant. Blood samples are taken from these animals and control sera are also harvested from the same pigs prior to the first injection. Antibodies are purified, mixed with pST and injected into hypox rats for five consecutive days. Rats receiving pST alone grow in a statistically signficant manner. Rats receiving pST plus antibodies from pigs #1 or #2 after immunization have significantly increased growth. Although the effect of the antibodies from pig #3 does not reach a statistically significant level (p*-0.08), there is visible growth enhancement. In contrast, pre-immunization antibodies are not effective. As a result of the hypox rat experiments, immunization trials are conducted with pigs.

Peptides may be administered alone or, more preferably, linked to a macromolecule which serves to enhance the production of antibodies In vivo. For example, the peptide may be conjugated to a protein such as keyhole limpet haemocyanin (KLH) . Other macromolecules within the scope of this invention include those known in the art such as human and bovine serum albumins, ovalbumin, myoglobins, β- galactosidase, penicillanase, heat shock protein and bacterial toxoids. Synthetic molecules such as multi- poly-DL-alanyl-poly-L-lysine and poly-L-lysine are also suitable.

The peptides may be administered by conventional routes such as subcutaneous injection, intramuscular injection and intravenous flow, as well as transdermal and oral administration. It is preferred to administer the peptides (or their conjugates) in association with a pharmaceutically acceptable adjuvant, diluent or carrier. It is particularly preferred to use a dosage regimen where an initial administration of the peptides is followed by one or more booster administration of the same peptides at regular time intervals.

As detailed in Examples 3-5, the immunization of pigs with the peptides of this invention results in the desirable results of an increase in feed efficiency, and an increased lean and decreased fat.

In Example 3, Table 1, finishing pigs receiving the peptide of SEQ ID NO:2 have a statistically significant increase in feed efficiency over the course of the experiment when compared to pigs receiving ovalbumin only. In Example 3, Table 2, the same pigs receiving the peptide display improved carcass characteristics, with a statistically significant increase in lean and decrease in fat when compared to pigs receiving ovalbumin only. It is interesting to note that the pigs receiving the peptide do not have a significant increase in most organ weights. In contrast, pST is known to increase organ weights. When the experiment of Example 3 is repeated in Example 5 with another group of finishing pigs receiving either the peptide of SEQ ID NO:2 or the peptide of SEQ ID NO:17 ("Cys-Peptide") , both groups of pigs have a statistically significant decrease in undesirable leaf fat when compared to pigs receiving ovalbumin only, while the pigs receiving the peptide of SEQ ID NO:2 have a statistically significant decrease in semitendinosiβ (see Tables 5 and 6) .

In Example 4, Tables 3 and 4, growing pigs receiving the peptide have a statistically significant increase in lean over the course of the experiment when compared to pigs receiving ovalbumin only. This is particularly significant during the treatment stages, as compared to the finishing period where treatment is stopped.

In order that this invention may be better understood, the following examples are set forth. The examples are for the purposes of illustration only and are not to be construed as limiting the scope of the invention.

Rιτ»τnp1 *^{*■ 1}

Competitive Radipimmungaagay

Radioimmunoassays are performed to determine the extent to which various peptides compete with radiolabelled pST for binding to PS-7.6 monoclonal antibody. For any given concentration of peptide, the lower the amount of pST which binds to the antibody in the assay, the greater the competition of the peptide to the antibody. Peptides which conformationally mimic native pST should displace pST from PS-7.6 monoclonal antibody more efficiently. The radioimmunoassays are performed as follows.

Removable microtiter wells are coated with PS-7.6 monoclonal antibody at 1 μg/well and the remaining microtiter well binding sites are blocked with 2% BSA. Iodinated pST (¹²⁵I-pST, specific activity = 180-250 μCi/μg, New England Nuclear/DuPont, Co., Boston, MA) is added together with competitive agents at various dilutions. After a 60 minute incubation, all wells are washed thoroughly to remove the unbound ^13SI-pST and the residual radioactivity, reflecting the amounts of ¹²⁵I-pST which remain associated with PS-7.6 monoclonal antibody in each well, is counted individually in a gamma counter.

In a first experiment depicted in Figure 6, a radioimmunoassay is performed which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST (referred to as "pGH" in Figure 6) compared with: (1) a peptide corresponding to the native sequence of amino acids 75-95 of pST (referred to as "pGH(75-95)^B in Figure 6), (2) the peptide of SEQ ID NO:2 (referred to as "peptide-X"), and (3) a modified form of peptide-X where the amino-terminal amino group is replaced by an acetyl group (referred to as "capped peptide-X") .

The results indicate that addition of pGH(75-95) to the wells inhibits the interaction of PS-7.6 monoclonal antibody and radioactive pST in a dose dependent manner. Peptide-X is also shown to inhibit the interaction in a similar, though somewhat stronger, fashion, whereas capped peptide-X fails to do so. Without being bound by theory, the failure of capped peptide-X in the competition assay may be due to the inter-peptide aggregation in the test solution. In a second experiment depicted in Figure 7, a radioimmunoassay is performed which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST compared with: (1) the pGH(75- 95) peptide, and (2) a peptide of SEQ ID NO:21 not within the scope of this invention (referred to as "peptide-Y" in Figure 7) .

The results indicate that addition of pGH(75-95) to the wells inhibits the interaction of PS-7.6 monoclonal antibody and radioactive pST in a dose dependent manner. In contrast, peptide-Y is shown to be much less effective.

In a third experiment depicted in Figure 8, a radioimmunoassay is performed which measures the competition for binding to PS-7.6 monoclonal antibody of radiolabelled pST compared with: (1) the pGH(75- 95) peptide, (2) peptide-X, (3) peptide-Y, (4) peptide-X wherein the first leucine is replaced by normal leucine (Nle), generating SEQ ID NO:13 (referred to as "NleβO" in Figure 8) , (5) peptide-X wherein the second leucine is replaced by normal leucine (Nle), generating SEQ ID NO:14 (referred to as "Nle87") , and (6) peptide-X wherein the valine is replaced by normal leucine (Nle) , generating SEQ ID NO:15 (referred to as "Nle90") .

The results indicate that addition of pGH(75-95) to the wells inhibits the interaction of PS-7.6 monoclonal antibody and radioactive pST in a dose dependent manner. Several peptides within the scope of this invention, including peptide-X, Nle80, Nle87 and Nle90, are found to be more effective than pGH(75-95) in this competition assay. However, peptide-Y is again shown to be much less effective. Example 2 Enhancement Of pST Activity In Hypox Rats With Swine Antibodies To A Peptide In this example, swine are injected with a peptide. Serum samples containing antibodies raised against the peptide are injected together with pST into hypox rats. The antibodies potentiate the ability of pST to promote the growth of the hypox rats. The experiment is conducted as follows. Peptide-X (SEQ ID NO:2) is conjugated to ovalbumin and emulsified in Complete Freund's Adjuvant. Three pigs are immunized with peptide-X and receive booster doses with Incomplete Freund's Adjuvant four and eight weeks thereafter. Blood samples are taken from these animals one week after the last boosting. Control sera are also harvested from the same pigs prior to the first injection. Antibodies are purified by ammonium sulfate precipitation, mixed with pST to provide a dose of 1 mg antibodies and 5 μg pST, and injected into hypox rats for five consecutive days. Three separate groups of rats each receives antibodies from one different pig; a fourth group of rats receives no injections, while a fifth group of rats receives only pST. The net weight gains of the rats of day 5 are depicted in Figure 9.

Treatment with pST alone stimulates growth in hypox rats in a statistically signficant manner (*, p<0.05). Rats receiving pST plus antibodies from pigs #1 or #2 after immunization have significantly increased growth. Although the effect of the antibodies from pig #3 does not reach a statistically significant level (p«0.08), there is visible growth enhancement. In contrast, pre-immunization antibodies are not effective. Example 3 TrminiTi-i ■*^■»«^■-ion Of Finishing Piσs With A Peptide This example sets forth the effects of immunization of finishing pigs (final growth phase from 70-80 kg and above) with a peptide of this invention on growth rate, feed efficiency and carcass composition.

The peptide (Tyr-Ser-Leu-Asp-Asp-Ile-He- Arg-Arg-Leu-Asp-Asp-Val-Ile-Arg-Arg-He; SEQ ID NO:2) is synthesized and conjugated to ovalbumin. For comparison, on ovalbumin antigen is generated by conjugating ovalbumin to itself.

Mixed-breed barrows (a new commercial DeKalb terminal cross genetic line) weighing 70-80 kg are purchased from Gold Kist Pork, Hillsborough, NC. Pigs are the target animal for the peptides and at least 25 pigs per treatment group are needed to obtain statistical significance between treatments. Animals are individually identified by numbered eartags and maintained in numbered pens. The pigs receive a diet which is medicated with chlortetracycline at 200 g/ton and are fed for about one week after the pigs are delivered. The pigs receive a Swine ST ration (20% crude protein) for the remainder of the experiment. Feed and water are available ad libitum.

Animals are assigned to each of the two treatments in a randomized complete block design. The animals are blocked by initial body weight. The following treatments are administered: &£____£ Antige n Number Pens

A Ovalbumi /ovalbumin 25 5

B Peptide/ovalbumin 25 5

The pigs are randomly assigned to treatment groups A or B within each of the blocks. Each block of 15 pigs start treatment when their average body weights are about 75 kg.

The pigs are allowed at least a 1-week pretreatment period in the pens before the first immunization injection. The flow of feed is adjusted to provide unlimited access with a minimum of wastage.

Pigs are immunized with 0.5 mg of peptide/ovalbumin conjugate totally emulsified with Complete Freund's Adjuvant using a subcutaneous (SC) injection in the neck area just behind the ears. All pigs receive one booster injection after a 4-week interval using the same quantity of conjugate, but with Incomplete Freund's Adjuvant. Control pigs receive the ovalbumin conjugate without the peptide. Pigs are observed daily. Any sick or injured animals receive appropriate veterinary care. Animals that do not recover within a few days or that are isolated to recover (e.g. lameness that is aggravated by penmates) are removed from the experiment. Body weights and feed consumption are determined weekly and more often if necessary as the pigs approach the slaughter weight.

Each pen of pigs is sacrificed when the mean live weight is 115±2 kg. Carcass measurements include chilled weight, backfat thickness, rib eye area, carcass length and weights of heart, liver, spleen, kidney, leaf fat and semitendinous muscle.

The data are analyzed using the analysis of variance procedures for a randomized complete block design. The experimental unit for the evaluation of weight gain and carcass measurements is the individual pig. Feed consumption and feed efficiency data use the pen as the experimental unit. Mean comparisons are made using Fisher's Protected LSD. The experiment proceeds on the following schedule. On Day 1, all pigs are weighed and treatment (including the initial immunization for Group B) starts for each block when the average body weight reaches 75 kg. Booster injections are administered 4 and 8 weeks after the initial immunization. Pigs are weighed and feed consumption is determined on a weekly basis. The pigs are then sacrificed at mean pen weights of 115±2 kg. The results of this experiment are shown in

Tables 1 and 2.

Table 1

The effects of active immunization of finishing pigs with a peptide on growth, feed consumption and feed efficiency

Control Immunized SEM

Item Ova/ova Peptide

No. pens/pigs 5/25 5/25

Body weight, kg Initial 76.2 76.3 0.3 4 Weeks 99.2 98.8 0.8

Final 116.2 117.1 1.0

Days on Experiment 51.2 51.0 1.3

Gain, g/day 1-4 Weeks 819 804 26.9 5 Weeks - End 743 805 27.5 1 Week - End 781 802 18.4

Feed intake, g/day 1-4 Weeks 3390 3250 59.7 5 Weeks - End 3316 3326 93.4 1 Week - End 3357 3285 54.5

Gain/Feed 1-4 Weeks .241 .247 .005 5 Weeks - End .222 .242 .009 1 Week - End .233 .244* .004

Significantly different than control, P<.05

Table 2

The effects of active immunization of finishing pigs with a peptide on carcass measurements and organ weights

Item Control Immunized SEM Ova/ova Peptide

No. pens/pigs 5/25 5/25

Carcass weight, kg 82.5 82.2 .8

Dressing percent 71.0 70.2 .3

Carcass length, cm 82.6 83.0 .4

Organ weight, g

Heart 370 373 8.6

Liver 1804 1829 37.8

Kidneys 375 384 8.7

Spleen 161 175 6.6

Leaf fat 1663 1389** 60.9

Semitendinoβus 394 434*** 8.2

Fat thickness, cm

1st rib 4.9 4.4* .14

Last rib 2.9 3.0 .10

Lumbar 2.6 2.3 .12

Backfat 3.5 3.2 .09

6th rib 3.6 3.1** .11

10th rib 3.7 3.2*** .11

Adjusted 10th rib 3.3 2.8*** .1 fat, cm

Loin eye area, cm² 44.2 44.6 .8

Adjusted loin eye 41.6 41.9 .8 area, cm²

Calculated lean (33)

Kg 38.1 39.7** .4

% 56.1 57.6* .5

Significantly different than control, P<.05

** Significantly different than control, P<.01 *** Significantly different than control, P<.001 Bxaiη l sa 4 Twmn -i na ion Of Growing Pigs With A Peptide

The immunization experiment of Example 3 is repeated using young growing pigs (approximately 15-20 kg) from the herd of American Cyanamid Company, Princeton, NJ. Two booster injections are given at four week intervals (during the growth phase to approximately 50-60 kg) . The animals are then maintained to sacrifice at a finishing weight of approximately 115 kg. The results of this experiment are shown in Tables 3 and 4.

Table 3

The effects of active immunization of growing pigs with a peptide on growth. feed consumption and feed efficiency

Item Control Immunized SEM

Ova/ova Peptide

No. pens/pigs 6/27 6/26

Body weight, kg

Initial 18.5 18.8 .3

4 Weeks 39.8 40.7 .6

8 Weeks 56.3 59.1* .8

12 Weeks 72.9 76.3* 1.1

16 Weeks 97.6 100.7 1.4

Final 113.0 114.1 1.5

Days on Experiment 135.0 131.2+ 1.4

Gain, g/day

1-4 Weeks 762.0 782.2 13.4

1-8 Weeks 675.4 718.9* 11.6

1-12 Weeks 647.8 683.7 12.3

1-16 Weeks 706.3 730.7 11.6

1 Week - End 704.0 728.5 11.6

8 Weeks - End 725.1 735.2 16.4

Lean tissue (34) 326.6 343.6* 5.8

Feed Intake, g/day

1-4 Weeks 1702 1632 37.2

1-8 Weeks 1670 1631 52.1

1-12 Weeks 1843 1857 70.5

1-16 Weeks 2131 2187 72.0

1 Week - End 2352 2401 70.6

8 Weeks - End 2872 3061 89.2

Gain/Feed

1-4 Weeks .448 .480* .011

1-8 Weeks .404 .444* .013

1-12 Weeks .351 .371 .010

1-16 Weeks .331 .337 .011

Week 1 - End .298 .305 .010

Week 8 - End .251 .242 .009

Lean tissue (34) .137 .144 .005

+ Significantly different than control, P<.10 * Significantly different than control, P<.05 Table 4

The effects of active immunization of growing pigs with a peptide on carcass measurements and organ weights

Item Control Immunized SEM

Ova/ova Peptide

No. pens/pigs 6/27 6/26

Carcass weight, kg 79.8 80.6 1.1

Dressing percent 71.1 71.0 .3

Carcass length, cm 83.2 83.5 0.5

Organ weight, g

Heart 400 439* 10.9

Liver 1796 1841 33.3

Kidneys 387 385 8.5

Spleen 150 158 5.0

Leaf fat 1441 998*** 54.7

Semitendinosis 425 461* 12.1

Fat thickness, cm

1st rib 4.3 4.2 .16

Last rib 2.2 2.2 .09

Lumbar 2.2 2.0 .09

Backfat 2.9 2.8 .09

6th rib 2.1 2.0+ .07

10th rib 2.7 2.5* .08

Adjusted 10th 2.5 2.2* .07 rib fat, cm

Loin eye area, cm² 38.2 42.1**

Adjusted loin eye 36.4 40.0*** .7 area, cm²

Calculated lean (33)

Kg 39.9 41.1** .3

% 56.2 58.5*** .4

+ Significantly different than control, P<.10

* Significantly different than control, P<.05

** Significantly different than control, P<.01

*** Significantly different than control, P<.001 Example 5 Immunization Of Finishing Pigs With A Peptide

The immunization experiment of Example 3 is repeated with the inclusion of a second peptide having a cystine linkage at the amino-terminus (SEQ ID NO:17) (Cys-peptide) , which is conjugated to ovalbumin as follows: Ovalbumin (10 mg) is dissolved in an aqueous solution (pH 8) and treated with iodoacetic anhydride (16 mg; 20-fold excess) and stirred for 2 hours at ambient temperature. The excess iodoacetic anhydride is separated by ultrafiltration and the modified ovalbumin is reacted with the Cys-peptide for four hours at ambient temperature and dialyzed to produce the final conjugate. The results of this experiment are shown in Tables 5 and 6. Note that two pigs receiving the conjugated Cys-peptide died during the course of the study.

Table 5

The effects of active immunization of finishing pigs with peptides on growth, feed consumption and feed efficiency

Control Immunized Immunized SEM

Item Ova/ova Peptide Cys- Peptide

No. pens/pigs 5/25 5/25 5/23

Body weight, kg

Initial 76.6 76.7 76.7 0.3

4 Weeks 100.8 102.2 102.2 0.9

Final 119.2 119.2 120.1 1.2

Days on Experiment 49.8 49.0 48.4 1.2

Gain, g/day

1-4 Weeks 866 909 903 30.2

4 Weeks - End 845 811 896 28.7

1 Week - End 856 870 902 23.8

Feed intake, g/day

1-4 Weeks

4 Weeks - End 3180 3333 3222 118.7

1 Week - End 3430 3440 3630 116.2

3285 3381 3389 100.8

Gain/Feed

1-4 Weeks .273 .273 .281 .008

4 Weeks - End .247 .236 .247 .009

1 Week - End .261 .257 .266 .008

Table 6

The effects of active immunization of finishing pigs with peptides on carcass measurements and organ weights

Control Immunized Immunized SEM

Ova/ova Peptide Cys- Peptide

No. pens/pigs 5/25 5/25 5/23

Carcass weight, kg 86.0 86.4 85.9 .9

Dressing percent 72.1 72.5 71.5 .3

Carcass length, cm 82.6 82.7 83.5 .5

Organ weight, g

Heart 397 400 415 8.4

Liver 1838 1810 1885 38.9

Kidneys 397 388 411 9.3

Spleen 166 167 183 6.9

Leaf fat 1569 1291** 1313* 73.1

Semitendinoβis 457 492* 467 10.0

Fat thickness, cm

1st rib 4.5 4.3 4.5 .13

Last rib 2.6 2.6 2.6 .10

Lumbar 2.0 2.0 2.3 .09

Backfat 3.0 3.0 3.1 .09

6th rib 2.4 2.3 2.3 .09

10th rib 3.0 2.9 2.9 .13

Adjusted 10th rib fat, 2.6 2.5 2.4 .1 cm

Loin eye area, cm² 46.8 46.7 46.8 1.1

Adjusted loin eye area, 43.6 43.6 43.4 1.1 cm²

Calculated lean (33)

Kg 40.6 40.7 40.9 .5

% 58.2 58.5 58.6 .7

* Significantly different than control, P<.05 ** Significantly different than control, P<.01 Bibliography

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34. National Pork Producers Council, Procedures to Evaluate Market Hog Performance (1983) . SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Buckwalter, Brian L. Shieh, Hong-Ming Wang, Boβco S.

(ii) TITLE OF INVENTION: Peptides With Growth Promotion Properties

(iii) NUMBER OF SEQUENCES: 21

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: American Cyanamid Company

(B) STREET: One Cyanamid Plaza

(C) CITY: Wayne

(D) STATE: New Jersey

(E) COUNTRY: U.S.A.

(F) ZIP: 07470-8426

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Gordon, Alan M.

(B) REGISTRATION NUMBER: 30,637

(C) REFERENCE/DOCKET NUMBER: 32,083-00

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-831-3244

(B) TELEFAX: 201-831-3305

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Xaa Xaa Leu Xaa Xaa lie Xaa Xaa Xaa Leu Xaa Xaa Val Xaa Xaa

1 5 10 15 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Tyr Ser Leu Asp Asp lie lie Arg Arg Leu Asp Asp Val lie Arg Arg

1 5 10 15

He

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Tyr Ser Leu Asp Arg He He Arg Asp Leu Asp Arg Val He Arg Asp 1 5 10 15

He

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Tyr Ser Leu Arg Arg He He Arg Arg Leu Arg Arg Val He Arg Arg 1 5 10 15

He

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE : protein (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :

Tyr Ser Leu Asp Asp He He Asp Asp Leu Asp Asp Val He Asp Asp 1 5 10 15

He

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANT -SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Tyr Ser Leu Asp Asp He Ala Arg Arg Leu Asp Asp Val Ala Arg Arg 1 5 10 15

Leu

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Tyr Ser Leu Lys Ala He Ala Glu Ala Leu Lys Ala Val Ala Glu Ala 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Tyr Ser Leu Lys Glu He Glu Lys Leu Leu Lys Glu Val Leu Glu Lys

1 5 10 15

Leu

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Tyr Ser Leu Asp Asp He Ala Arg rg Leu Asp Asp Val Ala Arg rg 1 5 10 15 Ala

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Tyr Ser Leu Lys He He He Glu He Leu Lys He Val He Glu He 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Tyr Ser Leu Lys Glu He Glu Lys Leu Leu Lys Glu Val Leu Glu Lys 1 5 10 15

Leu

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ( ix) FEATURE :

(A) NAME/KEY: Modified-βite

(B) LOCATION: 5

(D) OTHER INFORMATION: /label- Aib

/note* "Xaa at location 5 is Aib"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /label* λib

/notes "Xaa at location 7 is λib"

(ix) FEATURE:

(λ) NAME/KEY: Modified-βite

(B) LOCATION: 9

(D) OTHER INFORMλTION: /labels λib

/note** "Xaa at location 9 is λib"

(ix) FEλTURB:

(λ) NAME/KEY: Modified-βite

(B) LOCATION: 12

(D) OTHER INFORMλTION: /labels λib

/notes "Xaa at location 12 is λib"

(ix) FEATURE:

(λ) NAME/KEY: Modified-βite

(B) LOCATION: 14

(D) OTHER INFORMλTION: /labels λib

/notes "Xaa at location 14 is λib"

(ix) FEATURE:

(λ) NAME/KEY: Modified-βite

(B) LOCATION: 16

(D) OTHER INFORMλTION: /labels λib

/notes "Xaa at location 16 iβ λib"

(xi) SBQXTENCE DESCRIPTION: SEQ ID NO:12:

Tyr Ser Leu Lye Xaa He Xaa Glu Xaa Leu Lye Xaa Val Xaa Glu Xaa

1 5 10 15

(2) INFORMλTION FOR SEQ ID NO:13:

(i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRλNDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO

(ix) FEλTURB:

(λ) NAME/KEY: Modified-βite (B) LOCATION: 3 (D) OTHER INFORMλTION: /labels Nle

/notes "Xaa at location 3 is Nle"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 :

Tyr Ser Xaa λβp λβp He He λrg λrg Leu λβp λβp Val He λrg λrg 1 5 10 15

He

(2) INFORMλTION FOR SEQ ID NO:14:

(i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRλNDBDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO

(ix) FEλTURB:

(λ) NAME/KEY: Modified-βite

(B) LOCλTION: 10

(D) OTHER INFORMλTION: /labels Nle

/notes "Xaa at location 10 is Nle"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 14 :

Tyr Ser Leu λβp λβp He He λrg λrg Xaa λβp λβp Val He λrg λrg

1 5 10 15

He

(2) INFORMλTION FOR SEQ ID NO:15:

(i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEλTURB:

(λ) NAME/KEY: Modified-βite

(B) LOCATION: 13

(D) OTHER INFORMλTION: /labels Nle

/notes ^«χaa at location 13 is Nle" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 15 :

Tyr Ser Leu λβp λβp He He λrg λrg Leu λβp λβp Xaa He λrg λrg 1 5 10 15

He

(2) INFORMλTION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: Modified-βite

(B) LOCATION: 3

(D) OTHER INFORMλTION: /labels Nle

/notes "Xaa at location 3 iβ Nle"

(ix) FEATURE:

(λ) NAME/KEY: Modi iβd-site

(B) LOCλTION: 10

(D) OTHER INFORMλTION: /labels Nle

/notes "Xaa at location 10 is Nle"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Tyr Ser Xaa λβp λβp He He λrg λrg Xaa λβp λβp Val He λrg λrg

1 5 10 15

He

(2) INFORMλTION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(λ) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Cyβ Tyr Ser Leu λβp λβp He He λrg λrg Leu λβp λβp Val He λrg 1 5 10 15 λrg He

(2) INFORMλTION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa He Xaa Xaa Leu Xaa Xaa Xaa

1 5 10 15

Val

(2) INFORMλTION FOR SEQ ID NO:19:

(i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRλNDBDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Xaa Xaa He Xaa Xaa Leu Xaa Xaa Xaa He Xaa Xaa Leu Xaa Xaa Xaa

1 5 10 15

Val

(2) INFORMλTION FOR SEQ ID NO:20:

(i) SEQUENCE CHλRλCTERISTICS:

(λ) LENGTH: 17 amino acidβ

(B) TYPE: amino acid

(C) STRλNDBDNESS: βingle

(D) TOPOLOGY: linear (ii) MOLECULE TYPE : protein (iii ) HYPOTKETICλL : NO (iv) λNTI -SENSE : NO

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 20 :

Tyr Ser He λsp λβp Leu He λrg λrg He λβp λβp Leu He λrg λrg

1 5 10 15

Val

(2) INFORMλTION FOR SEQ ID NO:21:

(i) SEQUENCE CHARλCTBRISTICS:

(λ) LENGTH: 16 amino acidβ

(B) TYPE: amino acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Tyr Ser He λβp λβp He He λrg λrg He λβp λβp He λrg λrg He 1 5 10 15

Claims

What is claimed is:

1. A peptide comprising the sequence of amino acids Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu- Xaa-Xaa-Val-Xaa-Xaa (SEQ ID NO:l), wherein the sequence differs from the native sequence of porcine somatotropin (pST) .

2. The peptide of Claim 1 wherein the amino acid immediately amino-terminal to the first leucine is serine.

3. The peptide of Claim 2 wherein the peptide is selected from the group consisting essentially of the peptides having the sequence depicted in SEQ ID NOS:2 through 12.

4. The peptide of Claim 1 wherein a cysteine is added to either or both ends of the peptide.

5. The peptide of Claim 4 wherein the peptide has the sequence depicted in SEQ ID NO:17.

6. A peptide comprising the sequence of amino acids Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-He- Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:18) and wherein the sequence differs from the native sequence of pST.

7. The peptide of Claim 6 wherein the third residue is isoleucine, such that the peptide comprises the sequence of amino acids Xaa-Xaa-He-Xaa- Xaa-Leu-Xaa-Xaa-Xaa-He-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID NO:19) and wherein the sequence differs from the native sequence of pST.

8. The peptide of Claim 7 wherein the amino acid immediately amino-terminal to the first isoleucine is serine.

9. A composition for promoting the growth of a warm-blooded animal which comprises at least one peptide of Claims 1, 4, 5, 6, 7 or 8 together with a pharmaceutically acceptable adjuvant, diluent or carrier .

10. A method for promoting the growth of a warm-blooded animal which comprises administering to a warm-blooded animal the composition of Claim 9.