EP0819137A1 - Peptides avec des proprietes favorisant la croissance - Google Patents

Peptides avec des proprietes favorisant la croissance

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Publication number
EP0819137A1
EP0819137A1 EP96911304A EP96911304A EP0819137A1 EP 0819137 A1 EP0819137 A1 EP 0819137A1 EP 96911304 A EP96911304 A EP 96911304A EP 96911304 A EP96911304 A EP 96911304A EP 0819137 A1 EP0819137 A1 EP 0819137A1
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EP
European Patent Office
Prior art keywords
xaa
peptide
pst
leu
seq
Prior art date
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Application number
EP96911304A
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German (de)
English (en)
Inventor
Brian Lee Buckwalter
Hong-Ming Shieh
Bosco Shang Wang
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Wyeth Holdings LLC
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American Cyanamid Co
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Publication date
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Publication of EP0819137A1 publication Critical patent/EP0819137A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to peptides which elicit antibodies which enhance the activity of porcine somatotropin and promote growth of warm ⁇ blooded animals.
  • Porcine somatotropin 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 ⁇ -helices (2) . Each ⁇ - helix contains 3.6 amino acids per turn. This periodicity places every third or fourth residue on the same face of the helix.
  • 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.
  • 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.
  • compositions which utilize these peptides for the promotion of growth of warm-blooded animals 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- 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.
  • 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 135 I-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 IC 50 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 "Nle ⁇ 7") , 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) .
  • 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 produces antibodies functionally similar to PS-7.6 monoclonal antibody and promotes the growth of the animal.
  • 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.
  • RIA radioimmunoassay
  • 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.
  • pST(75-95) is the optimal sequence recognized by PS- 7.6 monoclonal antibody.
  • 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.
  • 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.
  • 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 ⁇ -helix, which allows the peptide to better mimic the native protein conformation.
  • Example 1 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.
  • non-contact residues may be replaced freely and still bind effectively.
  • the remaining residues of the peptide are replaced so as to achieve a stable helical conformation.
  • 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.
  • HPLC high performance liquid chromatography
  • 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.
  • 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) .
  • 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.
  • PS-7.6 monoclonal antibody fails to recognize any of these small fragments by RIA and Western Analysis.
  • 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.
  • 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.
  • 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.
  • PVDF polyvinylidene difluoride microporou ⁇
  • CA polyvinylidene difluoride microporou ⁇
  • 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.
  • 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.
  • 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 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) .
  • 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.
  • 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 ⁇ -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.
  • the peptides are selected from sequences containing the formula:
  • 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-
  • Aib-Val-Aib-Glu-Aib (SEQ ID NO:12), where Aib is 2-aminoisobutyric acid.
  • 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)
  • 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:
  • 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:
  • 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-Val (SEQ ID NO:18) wherein the sequence differs from the native sequence of pST.
  • 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-
  • 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.
  • 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.
  • radioi munoa ⁇ say ⁇ 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.
  • 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.
  • peptide-Y 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.
  • 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.
  • the peptide of SEQ ID NO:16 inhibits the interaction as well as peptide- X.
  • 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
  • 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.
  • the peptide may be conjugated to a protein such as keyhole limpet haemocyanin (KLH) .
  • KLH keyhole limpet haemocyanin
  • Other macromolecules within the scope of this invention include those known in the art such as human and bovine serum albumins, ovalbumin, myoglobins, ⁇ - 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.
  • 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.
  • 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.
  • 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.
  • Example 5 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 semitendinosi ⁇ (see Tables 5 and 6) .
  • 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.
  • 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 ⁇ g/well and the remaining microtiter well binding sites are blocked with 2% BSA.
  • competitive agents at various dilutions.
  • all wells are washed thoroughly to remove the unbound 13S I-pST and the residual radioactivity, reflecting the amounts of 125 I-pST which remain associated with PS-7.6 monoclonal antibody in each well, is counted individually in a gamma counter.
  • 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”) .
  • 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) .
  • 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 "Nle ⁇ O” 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 antibodies potentiate the ability of pST to promote the growth of the hypox rats.
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Example 3 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.
  • Example 3 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.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:4:
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANT -SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FEATURE FEATURE
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ⁇ NTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ⁇ NTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ⁇ NTI-SENSE NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention concerne des peptides qui ont une structure secondaire bien définie (de préférence avec une conformation hélicoïdale) et similaire à celle de la région correspondante de la somatotropine porcine (pST) et qui favorisent l'activité de la pST et la croissance des animaux à sang chaud. Ces peptides entrent en compétition avec la pST pour la fixation à l'anticorps monoclonal PS-7,6. Ces peptides contiennent la séquence des acides aminés Xaa-Xaa-Leu-Xaa-Xaa-Ile-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Val-Xaa-Xaa (SEQ ID No. 1) qui diffère de la séquence de la pST native, ainsi que des séquences dans lesquelles l'emplacement d'acides aminés essentiels est déplacé de trois acides aminés (ce qui représente presqu'un tour sur l'hélice) et qui contiennent la séquence des acides aminés Xaa-Xaa-Xaa-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Ile-Xaa-Xaa-Leu-Xaa-Xaa-Xaa-Val (SEQ ID No. 18), qui diffère de la séquence de la pST native.
EP96911304A 1995-03-31 1996-03-15 Peptides avec des proprietes favorisant la croissance Withdrawn EP0819137A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US41523995A 1995-03-31 1995-03-31
US415239 1995-03-31
PCT/US1996/003490 WO1996030405A1 (fr) 1995-03-31 1996-03-15 Peptides avec des proprietes favorisant la croissance

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EP (1) EP0819137A1 (fr)
JP (1) JPH11502844A (fr)
KR (1) KR19980703438A (fr)
AU (1) AU5422596A (fr)
BG (1) BG101927A (fr)
CA (1) CA2216755A1 (fr)
HU (1) HUP9801337A2 (fr)
MX (1) MX9707433A (fr)
PL (1) PL322585A1 (fr)
WO (1) WO1996030405A1 (fr)

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US6699466B1 (en) * 1999-08-05 2004-03-02 Research Corporation Technologies, Inc. IL-16 antagonist peptides and DNA encoding the peptides

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US5338836A (en) * 1992-07-29 1994-08-16 American Cyanamid Company Biologically active porcine somatotropin polypeptides and methods of using the same

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See references of WO9630405A1 *

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PL322585A1 (en) 1998-02-02
CA2216755A1 (fr) 1996-10-03
JPH11502844A (ja) 1999-03-09
AU5422596A (en) 1996-10-16
BG101927A (en) 1998-09-30
WO1996030405A1 (fr) 1996-10-03
MX9707433A (es) 1997-12-31
KR19980703438A (ko) 1998-11-05
HUP9801337A2 (hu) 1998-08-28

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