DK173981B1 - Small peptide(s) contg. bovine growth hormone partial sequence - used esp. for raising antibodies which will potentiate activity of hormone in vertebrate - Google Patents

Abstract

A peptide having primary structural homology to a sequence of amino acid residues of bovine growth hormone (bGH) in the region spanning positions 35 to 53 and antigenically equivalent peptides and salts are claimed. More specifically the following peptides are claimed: (a) NH2-Tyr-Ile-Pro-Lys-Glu-Gln-Lys-Tyr-Ser-Phe-Leu-Gln-Asn -Pro-Gln-Thr-Ser-Leu-Cys-COOH, (B) NH2-Thr-Tyr-Ule-Pro-Glu-Gly -Gln-Arg-Tyr-Ser-Ile-Gln-Asn-Thr-Gln-Val-Ala-Phe-Cys-COOH, (c) Ala-Tyr-Ile-Pro-Gla-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn -Ala-Gln-Ala-Ala-Phe-Cys, (d) Thr-Tyr-Ile-Pro-Glu-Asp-Gln-Arg -Tyr-Thr-Asn-Lys-Asn-Ser-Gln-Ala-Phe-Cys. (e) Thr-Leu-Leu-Pro-Asp-Glu -Arg-Arg-Gln-Leu-Asn-Lys-Ile-Phe-Leu-Leu-Asp-Phe-Cys or antigenically equivalent peptides or their salts, (f) bGH 26-43, (g) bGH 35-43, (h) bGH 37-48, (i) bGH 39-46, (j) bGH 43-54, (k) bGH 43-61 or human, avian, salmon or porcine analogues of peptides (f) to (k) or their salts. Also claimed are polynucleotide sequences encoding the peptides.

Description

in DK 173981 B1

The present invention relates to a pharmaceutical antigenic formulation comprising a peptide and agents for providing adjuvant and carrier functions.

Many polypeptide hormones, especially growth hormones, are important medical or animal agriculture, growth hormones are found in all vertebrates, the growth hormone (GH) of each species usually having an amino acid sequence slightly different from the amino acid sequence 10 of the GH nature. In general, the molecule comprises a single linear sequence of about, 191 amino acids. The amino acid sequence of human growth hormone (hGH) is described by Choh Hao Li in "Molecular and Cellular Biochemistry", 46, 31-41 (1982). Growth hormones are known to increase growth (somatogenesis), increase milk production (lactogenesis) and have an insulin-like hypoglycemic effect.

It is known from EP-A-137 234 (Wellcome) that certain antibodies against growth hormones may enhance the activity of the whole hormone, whereas previously it was generally believed that antibodies, at least in vivo, would antagonize the effect of the hormone. Such enhancing antibodies may be produced in situ by vaccinating the host animal with a suitable fragment of growth hormone, thus forming a class of limited specificity polyclonal antibodies which will enhance the activity of the endogenous hormone. A large (7K) fragment is described in this prior art as being suitable for such a purpose.

It is also known, e.g. from WO84 / 04915 (Amgen) that peptides corresponding to portions of the GH molecule may have useful biological activity, especially for the development of hypoglycemic effects when administered concomitantly with exogenous insulin. Administration of such peptides in an antigenic agent is not disclosed in WO84 / 04915, since the purpose of administration in this specification is not to induce antibody formation against the peptide.

It has now been found that certain partial sequences of the GK molecule are particularly suitable for inducing antibodies that will enhance the activity of the hormone in a vertebrate. These peptides are substantially smaller than the 10 7K fragment mentioned above and thus can be made easier and cheaper.

Thus, the present invention provides a pharmaceutical antigenic formulation comprising a peptide having 5 to 25 amino acid residues, wherein a 15 moiety comprising at least 45% of the peptide has an amino acid sequence corresponding to at least 35% of the region spanning from position 35 to 53 in bovine growth hormone or the corresponding regions 1 aviant or porcine growth hormone; or a variant of the peptide containing one or more deletions or conservative substitutions such that the tertiary structure of the peptide is unchanged; and means for providing adjuvant and carrier functions.

Said region from positions 35-53 in bovine 25 (and ovine) GH is: Nf ^ -Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-lle-GLn-Asn-Thr-Gln -Val-Ala-Phe-Cys-COOH.

The peptides used according to the invention preferably have less than 20 amino acid residues. The number of amino acid residues having structural homology to the hormone contained in the small peptide used according to the invention typically depends on the length of the small peptide and may vary from a sequence of a few amino acid residues to a sequence which is substantially comprises the entire small peptide. The sequence of amino acid residues with the structural homologue typically has a length of at least 5 amino acid residues, preferably at least approx. 8-10 amino acid residues.

As used herein, the term "amplify" means that the small peptide acts directly or indirectly to increase or enhance the activity of the hormone with which it has structural homology.

The term "conservative substitution" is defined functionally above. Examples of substitutions that may be conservative in this context include those substitutions which give essentially the same hydrophobic, size, charge and / or aromatic properties as the original amino acid sequence.

All such substitutions and modifications are well known to those skilled in the peptide chemistry. Potential substitutions include, for example: proline instead of glycine and vice versa, alanine or valine instead of glycine and vice versa, isoleucine instead of leucine and vice versa, tryptophan instead of tyrosine and vice versa, histidine instead of lycin and vice versa, serine instead of asparagine and vice versa, arginine instead of glutamate and vice versa, threonine instead of cysteine and vice versa, serine or alanine instead of threonine and vice versa, and glutamine instead of asparagine and vice versa.

The following are examples of regions of non-bovine GHs corresponding to the 35-53 region of the bovine growth hormone: 4 DK 173981 B1

Pigs and rat 35-53

Ala-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn-

Ala-Gln-Ala-Ala-Phe-Cys.

5 Poultry (35-53)

Thr-Tyr-Ile-Pro-Glu-Asp-Gln-Arg-Tyr-Thr-Asn-Lys-Asn-Ser-Gln-Ala-Ala-Phe-Cys.

The term "antigenic equivalent" means that the peptide can be used in a suitable formulation to induce antibodies in a vertebrate, which antibodies enhance the effect of the growth hormone in that vertebrate. In particular, peptides which are slightly shorter or longer than said regions or which substantially overlap these regions, e.g. 30-4Θ or 26-43, found to be antigenically equivalent to these. The term "slightly longer", "slightly shorter" and "substantially overlap" denotes peptides wherein at least 45% (preferably 50%, 60%, 70%, 80%, 90% 20 or 100%) of the equivalent peptide overlaps by at least 35% (preferably 40%, 50%, 60%, 70%, 80%, 90% or 100%) of said 35-53 regions. In particular, antigenically equivalent peptides shorter than said fragments, e.g. 35-43 or 35-48, are used.

With particular, though not exclusively, relation to bovine GH, the following sequences are useful in the practice of the invention: 26-43 r (Ala-Tyr), 35-43 (Thr-Tyr), 37-48 (Ile-Thr) , 39-46 (* Glu-Gln), 43-54 (Tyr-Phe), and 43-61 (Tyr-Pro).

It has been found that using a small pep time according to the invention from a species other than the animal to which the peptide is administered, e.g. administration of porcine 35-53 to sheep or cattle may be appropriate. Variations from the animal's own GH sequence may elicit a stronger immune response, while still providing antibodies that can recognize the animal's own Gtt.

In addition, peptides in which one or more of the 5 amino acid residues are chemically modified before or after preparation of the peptide can be used, provided that the effect of the peptide, namely the formation of specific antibodies in vivo, remains substantially unchanged.

Such modifications include formation of salts with 10 acids or bases, especially physiologically acceptable, organic or inorganic acids and bases, formation of an ester or amide of a terminal carboxyl group, and attachment of amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism. The peptides may be available as single copies or as multiple copies, e.g. tandem repeat units such as 35-53 + 35-53.

Such tandem repeat units or multiple repeat units may be sufficiently antigenic even to avoid the use of a carrier. It may be convenient for the peptide to be shaped as a loop with the N-terminal and C-terminal ends bonded together, or to add one or more Cys residues to one of the ends to increase the antigenicity and / or to enable disulfide bonds to form. . If the peptide is covalently linked to a carrier, preferably a polypeptide, the arrangement is preferably such that the peptide of the invention forms a loop.

According to current immunological theories, a carrier function must be present in any immunogenic formulation to stimulate or enhance the stimulation of the immune system. It is believed that carriers form (or together with the antigen form) a helper T cell cell top. The peptides may be bound together, e.g. by cross-linking, with a separate carrier, such as serum albumin, myoglobin, bacterial toxoids and dagger tail hemocyanin. Recently developed carriers that elicit T cell help in the immune response include the hepatitis B core antigen (also called nucleocapsid protein), putative helper T cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala. The Glu-Thr-Thr-Asn, β-galactosidase and the 163-171 peptide of interleukin-1. The last 10 compounds may alternately be regarded as a carrier or as an adjuvant or as both. Alternatively, multiple copies of the same peptide or of different peptides of the invention may be cross-linked. In this case, there is no separate carrier as such, but a carrier function can be provided by this crosslinking. Suitable crosslinking agents include those listed as such in the Sigma and Pierce catalogs, e.g. glutaraldehyde, carbodiimide and succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, the latter agent utilizing the -SH group on the C-terminal cysteine residue of the 35-53 region.

Suitable adjuvants are known to those skilled in the art of vaccine, e.g. Freund's complete or incomplete adjuvant, aluminum hydroxide, saponin, DEAE-dextran, muramyl dipeptide, mineral oils, neutral oils (such as miglyol), vegetable oils (such as arachis oil), "Iscbmer", liposomes, Pluronic polyols or Ribbon polyols e.g., GB-A-2 189 141).

30 "Pluronic" is a trademark.

The peptide of the invention can be bound to other antigens to produce a double effect. Eg. For example, the peptide can be bound to some or all of the somatostatin molecule so that, in addition to anti-GH antibodies, anti-somatostatin antibodies are formed which will promote growth, or it may bind to some or all of the sex hormone molecule to simultaneously provide of immune castration, or in part or all of the luteinizing hormone releasing hormone (LHRH).

The peptides and adjuvants and / or carriers may be formulated in any suitable manner as may be indicated by one of ordinary skill in the art using known or not yet discovered administration carriers and criteria. In particular, the formulations may be based on biodegradable polymers such as lactide-glycolide copolymers, such as those described in EP-A-58481 (ICI).

The antigenic formulations of the invention will usually be administered intravenously, subcutaneously or intramuscularly, although the intranasal, transdermal, oral and rectal route may be suitable for some formulations of the invention. The formulations will usually be sterile and (for parenteral use) 20 non-pyrogenic, and a unit dose will typically comprise 1 to 1000 µg of the subject small peptide, typically 10 to 500 µg, preferably 50 µg or less. One or more repetitions of immunizations may be appropriate, as is known in the field of immunological science. The formulations can generally be prepared and / or administered by a physician or veterinarian in accordance with his experience and expertise.

The peptides may be provided by known methods of peptide synthesis or by appropriate cleavage of the natural Gtt molecule. Peptide synthesis can be obtained following the general method of Stewart et al., Described in "Solid Phase Peptide Synthesis" (W Η Freeman, San Francisco, 1969) or by methods described by Marglin and Merrifield in Anual Reviews of Biochemistry, 39, 841-866 at 862 (1970), and by the following articles. Established methods for peptide synthesis by solid phase methods and similar methods are usually not suitable for large-scale production (although they may become so in the future), and thus commercial production of the peptides will normally occur by growing an appropriate organism transformed with a polynucleotide sequence which encodes the desired peptide.

Examples according to the invention are described below with reference to the drawings, in which:

Figure 1 shows the results of a preliminary dwarf mouse experiment. The columns represent 1 standard deviation with 6 animals per treatment.

Figure 2 shows the results of another dwarf mouse study, in which the activity of igG (globulin) from two sheep 772 (35-53 alone + FCA) and 775 (35-53 conjugated with KLH f FCA) was compared to OA11 at different 20 globulin concentrations.

772 pure - 10.5 mg protein / ml; 775 = 9.6 mg protein / ml, OA11 - 5 mg protein / ml. Columns = 1 standard deviation; n = 6th

Figure 3 shows the results of a third mouse 25 mouse test showing the activity of globulins from different sheep; all treated with 35-53 X-bundle + FCA (4600, 4609, .4602, 4612, 4610), or negative control immunization (4660, 4608). Columns = 1 standard deviation, n = 6.

Figure 4 shows how antibodies produced against b35-53 can in some cases, in various ways, bind to bovine growth hormone and the porcine molecule. The affinity for the latter is reduced by approx. 10 9 DK 173981 B1 times. The results of a dwarf mouse experiment are shown. Columns b 1 standard deviation, n = 6.

Figure 5 shows the results of an experiment with hypophysectomized rats performed as described below, which shows the weight gain of groups (n = 6) of rats over the 9 day period. Columns represent 1 standard deviation. All treated animals received hormone.

• anti-bovine 35-53 anti-serum collected from 4 immunized sheep positive for bovine growth hormone binding (RIA). x anti-serum as above from one of the four sheep. o negative control anti-serum.

(monoclonal antibody (treated as antiserum) OA15.

Π monoclonal antibody OA17 (see Aston et al., 1986).

Figure 6 shows the result of a further trial of hypophysectomized rats.

A: control sheep immunoglobulin.

B: anti-35-53 antiserum, gets 1064, 5 mg / ml.

C: anti-35-53 antiserum, gets 1064, 15 mg / ml pure.

Columns represent 1 standard deviation, n = 6.

Figure 7 shows the results of a further experiment with hypophysectomized rats, namely a comparison of antipept idantibody conjugated with either ovalbumin or somatostatin.

Columns represent 1 standard deviation, n = 6.

Each of these globulin preparations was complexed with bovine growth hormone prior to administration to the rats.

Figure 8 shows the results of a further test with hypophysectomized rats, which experiment illustrates how anti-bovine 35-53 can also potentiate porcine growth hormone. Rats received a dose of just 15 µg per day. day, but with the usual amount of anti-globulin. Columns represent 1 standard deviation, n = 6.

Figure 9 shows the results of another experiment with pituitary ectomized rats in which rats were treated with anti-peptide antibodies formed against a variety of peptides related to either bovine or porcine molecules. All were complexed with 10 porcine growth hormone prior to administration to rats.

Columns represent 1 standard deviation, n = 6.

APPROACHES

1 5 Preparation of Peptides

All peptides were prepared by the Fmoc polyamide solid phase peptide synthesis method.

Temporary amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly basic labile protecting group is induced using 20% piperidine in Ν, Ν-dimethylformamide.

Functional groups on side chains are protected as their butyl ethers (for serine, threonine and tyrosine), butyl esters (for glutamic and aspartic acid), butyloxycarbonyl derivatives (for lysine and histidine), tritylderivate (for cysteine) and 4-methoxy 2,3,6-trimethylbenzene sulfonyl derivative (for arginine). When glutamine or asparagine is the C-terminal residue, the 4,4'-dimethoxy-benzhydryl group is utilized to protect functional side chain amide groups.

The solid-phase support is based on a polydimethyl-acrylamide polymer consisting of the three monomeric dimethyl-lacrylamide (main chain monomer), bis-acryloylethylene diamine (crosslinking agent) and acryloylsarcosine methyl ester (functionalizing agent).

The cleavable peptide-to-resin-bound agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative.

All amino acid derivatives are added as their pre-made symmetrical anhydride derivatives except for asparagine and glutamine added using a reverse N, N-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole mediated coupling procedure.

All coupling reactions and reactions to remove protecting groups are followed using the ninhydrin, trinitrobenzenesulfonic acid or isotin test method.

Upon completion of synthesis, the peptides are cleaved from the resin support with the removal of side chain protecting groups by treatment with 95% trifluoroacetic acid containing a 5% scavenger mixture (scavenger mixture). Commonly used cleansers are ethanedithiol, phenol, anisole and water, the exact choice of which depends on the amino acids of the synthesized peptide. Trifluoroacetic acid is removed by evaporation in vacuo with subsequent trituration with diethyl ether to give the crude peptide. All the detergent present is removed by a simple extraction method which after lyophilization of the aqueous phase gives the crude peptide free of detergents.

Purification may be carried out by any or a combination of methods such as size-cut chromatography, ion-exchange chromatography and (first and foremost) reverse phase hplc.

Peptide analysis is performed using thin layer chromatography, reverse phase hplc, amino acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometry analysis as is well known to those skilled in the art.

5 I. Sheep experiments: preliminary

Methods: 1. Preparation of Immunogenic Agent 10 Cross-linking to dagger tail hemocyanin using glutaraldehyde: 10 mg of peptide (ovine / bovine sequence 35-53) was dissolved in 500 µl dimethylformamide and mixed with 10 mg dagga tail hemocyanin in 400 µl of 0.05M phosphate buffer, pH 7.8, 1 T5 ml of 0.02M glutaraldehyde solution in 0.05M phosphate buffer pH 7.8, was added dropwise over one hour with stirring at room temperature. The mixture was allowed to stir at room temperature for an additional 3 hours and then dialyzed against phosphate buffered saline, pH 7.2.

Half of the preparation was mixed with twice the volume of Freund's complete adjuvant and injected in two sheep subcutaneously in many places. 28 days later, the other half of the preparation was emulsified in the same manner and injected into Freund's incomplete adjuvant. Blood was drawn from the sheep weekly during immunization. The optimal antibody reaction was obtained approx. 14 days after the second immunization.

2. Sheep Serum Radioimmunoassay Antibody formation in sheep after immunization with GH peptide fragments 35-53 was determined by a direct liquid phase binding assay with 32 I-bGH substantially as described previously (Aston, et al., 1985).

13 DK 173981 B1 3. Growth Assay Growth-enhancing peptide antisera (anti-35-53) were assessed for activity in the dwarf mouse growth model as described previously (Aston, et al., 1986; 1987) following production of γ-globulins from sera. .

results

Sera from sheep immunized with peptides obtained from bovine / ovine GH 10 were assessed for their binding to 125 I-bGH and their effects on the bioactivity of bGH in vivo. Dwarf mice receiving bGH in a complex with anti-35-53 antiserum grew significantly better than mice treated with bGH and control globules.

The results are shown in Figure 1.

II. Sheep Experiment; investigative Methods; 20 1. Conjugation with ovalbumin or dagger tail hemocyanin (KLH) 1.4 mg of peptide (eg bovine 35-53) was dissolved in 140 μΐ dimethylformamide. 70 μΐ 10 mg / ml ovalbumin or 25 KLH in Dulbecco's phosphate buffered saline (PBS) was added and mixed thoroughly. 200 µl of freshly prepared 0.04M glutaraldehyde was added slowly with stirring over 10 minutes, after which the mixture was allowed to stand at room temperature for an additional 60 minutes. 0.7 ml of PBS was added and followed by an additional 100 μΐ 0.04M glutaraldehyde as above. This mixture was allowed to stand for 60 minutes at room temperature before dialyzing overnight at + 4DC against PBS.

2. Crosslinking 11.4 mg of peptide was dissolved in 140 µl dimethylformamide and 170 µl 0.04M glutaraldehyde was added as above. If no crosslinking or conjugation was necessary, the peptide was dissolved in dimethylformamide, dispersed in PBS but not dialyzed.

3. Negative controls

Negative control parallel samples for the above 10 were prepared without using peptide with ovalbumin (or KLH) or using polylysine (molecular weight 1000-2000 Da) and cross-linking.

4. Adjuvants and Administration - "Freunds" 15 After dialysis, the volumes of the above preparations were brought up to 4.5 ml with PBS and water-in-oil emulsions prepared using two volumes of Freund's complete adjuvant (FCA) (Difco or Sigma). This was achieved by sound treatment in the cold or using a Potter-Eleven homogenizer. Emulsions were tested for dispersion (or lack thereof) on a water surface. Injections were performed simultaneously at two sites (one on each side) in Cheviot sheep (9-12 months old, castrated males, 30-35 kg), 1 ml administered 25 each site. Another similar immunization was completed using freshly prepared peptide, conjugated in the same manner, but emulsified in Freund's incomplete adjuvant (FI + C) (Difco or Sigma). All subsequent immunizations were performed in the same manner but at 30 28 day intervals.

15 DK 173981 B1 5. Adjuvants and administration - other.

DEAE-dextran (fully hydrated overnight in double distilled water) (Pharmacia), Saponin (Sigma) and aluminum hydrogel were used alone and in combinations. After dialysis, 3.1 ml of PBS plus 7 ml of 5% DEAE-dextran (Dd) plus 2.8 ml of 5 mg / ml of saponin were added; or 5.9 ml of PBS plus 7 ml of 5% Dd; or 10.1 ml PBS plus 2.8 ml 5 mg / ml saponin. Aluminum ("AlOH") was used, if appropriate, at a final concentration of 1.0 mg / ml.

No emulsification was necessary, but care had to be taken to keep the ingredients in homogeneous suspension. 1 ml was administered in sheep as described above. Immunizations were performed at the same intervals.

6. Blood samples 10 ml blood samples were taken at neck vein puncture from the sheep tested just before each administration and at 3x1 week intervals thereafter. After allowing the blood to set at room temperature (ca.

The serum was removed after centrifugation for immediate antibody detection radioimmunoassay. larger samples of sera were similarly collected from ca. 150 ml of blood was frozen at -20 ° C for subsequent fractionation and use in growth assays.

7. Radioimmunoassay The detection of antibodies to peptides which would also bind to bovine growth hormone was determined by direct liquid phase binding as described previously (Aston et al., 1985; Chard, 1987).

16 DK 173981 B1

Results 1 Table 1 summarizes the results of the sheep experiments and these show the superiority of FCA in most conjugates (or peptide alone) using peptides 35-53. Small changes such as in porcine 35-53 introduced in get further improved reaction rate (and actual titer - not shown). Peptides around, with and within the 35-53 sequence gave at least moderate reactions.

10

Table 1

Radioimmunoassay for the detection of sheep sera antibodies which were capable of binding to inact, bovine liquid phase growth hormone.

Treatment% responsive Individuals after 56 days of

Conjugate adjuvant ters of 1/1000 or 20 more (n = 5).

Bovine 35-53 variations: KLH FCA 60 KLH AlOH none 25 KLH Saponin 20 KLH Dd 20 KLH Dd + AlOH. 20 Λ 30 17 DK 173981 B1

Ovalbumin XFCA (audio processed) 20 "XFCA 80" AlOH 20 "Saponin 60 5" Dd none · Dd + AlOH 80

Cross-linked FCA 80 "Saponin 20 10" Dd + Saponin 80 "Dd 20 alone

No FCA 40

AlOH 20 Saponin 20

Dd 20

Dd + AlOH 20

Other peptides - all above bovine (except indicated) 2o conjugated to ovalbumin and administered in FCA.

46-61 cys 60 43-53 20 35-43 Cys 20 25 Porcine 35-53 100 x Unless otherwise stated, emulsions were prepared by shear forces.

30 18 DK 173981 B1 III. Immunomodulatory Effects Procedure

An experiment was performed to demonstrate that the 5 35-53 sequence (or its analogs and related sequences) could be bound to another peptide or molecule of immunological interest and that the reaction against both peptides be improved.

In this context, and particularly relevant to the role of immuno-neutralization in animals, somatostatin was chosen for illustrative reasons and because of the difficulty in eliciting effective immuno-neutralizing antibodies (see Spencer, 1986).

The conjugation was completed using 15 (1-14) somatostatin (Sigma) as described above (II-I) except that somatostatin was only added to 4 mg / ml and no dialysis was performed in any of the treatments. All other conjugates were prepared as described above (II-I).

20o Radioimmunoassays for growth hormone binding antibodies were performed as above (IX-7) for somatostatin as described by Spencer et al., (1983) using 125 I-labeled Tyr somatostatin.

2 5 Results

The data summarized in Table 2 shows the usual. difficulty in inducing good antibody titers against somatostatin οή how the cross-linking to bovine 35-53 (not just internal cross-linking) overcomes this tolerance to the same.

DK 173981 B1 19

Table 2

Radioimmunoassays for antibodies against intact bovine growth hormone and / or against somatostatin following an immunization utilizing 35-53 somatostatin conjugation.

Treatment% antibody generators (n = 5) recognizing 10 bGH Somatostatin cross-linked 35-53: FCA 80 none PIA none none

Dd + Saponin 80 none 15 cross-linked somatostatin: PCA none 40 PIA none none

Dd + Saponin none none 20 35-53 sudden somatostatin: PCA 80 80 PIA 40 60

Dd + Saponin 80 40 25

Bound to somatostatin: 35-43 Cys 60 40 (cf. Table 1) Φ3-53 40 40 30 20 DK 173981 B1 IV - Experiments with pigs

Procedures: 1. general

Sequences 35-53 plus the T-cell epitope described previously were introduced into pigs after dissolution in dimethylformamide, dispersion in PBS (see II-2), and emulsification in FIA. No conjugation or crosslinking 10 was used in this method.

The peptide thus prepared was administered subcutaneously at 4 sites in the neck region of large white pigs (5 weeks old; body weight approximately 9 kg) to administer 500 µg of peptide per ml. pig. Another immunization using the same preparation was performed 28 days later. On this occasion everything was filed in the FIA. Blood samples were taken just prior to this immunization and weekly thereafter by means of vacuum at venous puncture (Corvac, Sarstedt, U.K.) of the pulmonary vein. The sera were tested for porcine growth hormone antibody recognition using an enzyme-linked immunosorbent assay (ELISA) based on Voller, 1979 in which they were subsequently cross-linked by competitive binding in a similar aqueous hormone assay.

25

2. ELISA

96-well plates treated for immunoassay consistency (Nunc, Immtmo grade, high binding capacity) were coated using 50 µg hormone / ml with 5 µg / well (100 µl) in sodium carbonate / hydrogen carbonate buffer, 0.05M, pH 9.5, and let stand overnight at + 4 ° C. The hormone solution was carefully removed and the wells washed once with PBS. A solution of 3% hemo-globin is added to block the wells by standing overnight at 4 ° C. This was removed and the wells were washed three times with PBS with 0.05% Tween. All plates were allowed to dry slowly at room temperature and stored at -20 ° C individually wrapped in adhesive film. The sera tested were added to each well for 1/50 and then log 10 dilutions (100 µl), which were left for 2 hours at room temperature. The sera were removed and the wells were washed three times in PBS, which was replaced with 100 µl of 10 rabbit anti-porcine IgG-alkaline phosphatase conjugate (Sigma) diluted to 10-3. This was removed and washed as above. 100 µl of p-nitrophenyl phosphate at 1.0 mg / ml was added and the absorbance of the wells was read using a Titertek Multiscan plus 2 with 405nm filters.

results

Table 3 shows that the presence of antibodies that recognize coated porcine growth hormone (and this will compete with aqueous hormone) could be detected in a number of the pigs.

25 30 DK 173981 B1 22

Table 3

Anti-pGH antibodies in peptide-immunized pigs after 42 days as measured by the ELISA method.

5

Peptide treatment% positive animals (n = 6) l / 50x l / 500x 35-53 T cell epitope 100 100 10 xanthisum dilution.

V - Biological Assays for GH Activity Methods: 1. Immuno-globulin preparations.

Sera from larger blood samples taken from particular animals (designated by the immunoassays) were fractionated by sodium sulfate precipitation (Johnstone & Thorpe, 1982) primarily to isolate the gamma globulins (IgG), which were dialyzed extensively against PBS before being re-frozen at -20 ° C. Prior to use in animal experiments, the purified (IgG) fractions were re-titrated to measure the possible effects of the precipitate.

2. The dwarf mouse method q q __

In this, the incorporation of JJSO4 into the rib cartilage of dwarf mice (pituitary deficiency) is used and is described elsewhere (Aston et al, 1986).

23 Method of hypophysectomized rats

These animals are rendered pituitary deficient by surgical removal of the pituitary gland. The test measures the overall effect of the hormone on the rat's body weight and the circulating 5 concentrations of somatomedin-C.

The surgical procedure on male Wister rats was performed by Charles River U.K. Limited (Margate, Kent, UK) and the rats were delivered 14 days later with a wall ranging from 125 to 145 g. They were weighed and observed 10 for an additional 7-10 days to ensure stable body weight and physiological characteristics (e.g. - appearance of testicles) consistent with good health and complete surgical intervention. Satisfactory animals are randomly assigned to obtain six animals per animal. treatment. These were injected daily with 0.5 ml of PBS containing approx. 1 mg of sheep IgG from the immunization treatment to be examined (including negative controls) to which 50 µg (exceptionally lo µg - see results) was added to bovine or porcine growth hormone as desired. Prior to administration, the hormone and IgG were mixed and the mixture was allowed to stand at room temperature for 60 minutes. Injections were performed subcutaneously and intrascapularly. The animals were weighed and injected daily for 8 days at the same time of day each time. On the ninth day, the 25 animals were weighed, terminal anesthetized, and a blood sample was taken from the aortic branch. EDTA plasma was frozen at -20 ° C for later evaluation of the relative total somatotnedin-C content using materials provided by the Nichols Institute (San Juan Capi-30 strano, CA 92675, USA).

24 DK 173981 B1

Results 1, Dwarf mouse model

From the data summarized in Figures 2, 3 and 45, it can be seen how dwarf mice receiving growth hormone complex with sheep anti-bovine (b) 35-53 antiserum incorporated substantially larger amounts of 3 µS (from

3 C

Na2 SO4) in their costal cartilage than the mice treated with bGH and control sheep globulin. This arises from a variety of immunization methods and is related to the antiserum dilution prior to complex binding (Figure 2). This phenomenon can also be seen when an antiserum formed against bovine 35-53, which also recognizes intact porcine growth hormone (though much weaker than it recognizes the homologous hormone), is complexed with that where an increased 35S incorporation into costal cartilage will is achieved.

2. Pituitary Ectomized Rats Model 20 Using a growth-related model, it can be seen (Figure 5-9) that a variety of anti-peptide sera will increase the activity of bovine and porcine growth hormones when administered to these surgically altered rats.

In this system, antisera against peptides with relation to the 35-53 region are active to enhance the response to. growth hormone (Figure 7). This phenomenon will cross species barriers as shown (Figure 7), provided the antisera will bind to the target hormone in question.

If the binding capacity or affinity is limited, in some cases it may be necessary to adjust globulin: hormone ratio to maximize the proportion of hormone-antibody complexes, as shown in Figure 8.

DK 173981 B1 VI - conjugate antibodies (Somatostatin)

Course of action

Using the in vitro method described previously (Hart et al., 1984), the activity of the anti-somatostatin antisera was measured following release of the growth hormone from ovine pituitary cells (in primary culture). Because the growth hormone binding properties of the antibodies would interfere with the assay, these were removed by passage through an affinity column to which bovine growth hormone was attached. An activated Sepharose CNBr column was prepared by the method described by the manufacturer (Pharmacia, Milton Keynes,

Bucks, U.K.).

Somatostatin-14 (Sigma) was added to wells in 10 μΐ immune (anti-somatostatin activity at RIA) or non-immune (anti-35-53 alone) sheep sera over the range 5-250x10-11 mol / L, and the inhibitory properties of this peptide. activity at the growth hormone release levels to the medium was determined.

results

Table 4 clearly shows how the inhibitory activity of somatostatin added in an antiserum containing somatostatin antibodies is remarkably reduced. Thus, it is important to note that the anti-serum induced against the bovine 35-53 + somatostatin conjugate appears to contain two populations of antibodies - one that binds to intact, bovine growth hormone 30 and enhances its activity, and another that binds to intact somatostatin-14 and neutralize it.

DK 173981 B1 26

Table 4

Effect of somatostatin on ovine pituitary cells grown in primary culture as described by Hart et al, 1984.

5

Somatostatin% by agent inhibition of GH release level anti-35-53 alone anti-somatostatin antisera antisera 5 5 5 10 25 10 5 125 70 20 250 90 70

References 15 1. Aston, R., Cooper, L., Holder, A.T., Ivanyi, J., and Preece, M.A. (1985). Molecular Immunol, 22 271-275.

2. Aston, R., Holder, A. T., Preece, M. A., and Ivanyi, J. (1986) J. Endocrinol. 110 381-388.

3. Aston, R., Holder, A. T., Ivanyi, J. and Bomford, R. (1987). Molec. Immunol. 2_4 143-150.

4. Chard, T. (1987). An Introduction to Radioimmu noassay and Related Techniques. 3rd edition

Elsevier, Amsterdam.

+ 5. Hart, I. C., James, S., Perry, B. N., and Simmonds, A. A., (1984), J. Endocrinol. 103 173-178.

6. Johnstone, A., and Thorpe, R. (1982), Immunochemistry in Practice, Blackwells, London.

35 7. Spencer, G.S.G. (1986). Control and Manipulation 27 DK 173981 B1 of Animal Growth. Pp 179-293, Butterworths,

London.

8. Spencer, G.S.G., Garssen, G.J., and Hart, I.C., 5 (1983). Livestock Production Science 1_0 25-37.

9. Voller, A., Bidwell, D.E. and Bartlett, A.

(1979). The Enzyme Linked Immunosorbent Assay,

Dynatech Europe, Guernsey.

10

Claims (6)

The formulation of claim 1, characterized in that the peptide has from 5 to 20 amino acid residues. A formulation according to claim 1, characterized in that the peptide is selected from the following 20 peptides: (a) NH2-Thr-Tyr-Ile-Pro-Glu-Gly-Gln-Arg-Tyr-Ser-Ile-Gln-Asn -Thr-Gln-Va1-Ala-Phe-Cys-COOH. (b) Ala-Tyr-11 e ~ P ro-G lu-G ly-G ln-Arg-Tyr-S er-I le-G ln-Asn-Ala-Gln-Ala-Ala-Phe-Cys ·. (C) Thr-Tyr-Ile-Pro-Glu-Asp-Gln-Arg-Tyr-Thr-Asn-Lys-Asn-Ser-Gln-Ala-Ala-Phe-Cysv Formulation according to claim 1, characterized in that the peptide is selected from the following peptides: DK 173981 B1 (d) bGh 26-43 (e) bGh 35-43 (f) bGh 37-48 (g) bGh 39 -46 5 (h) bGh 43-54 (i) bGh 43-61 Formulation according to any one of the preceding claims, characterized in that the peptide is bound to a carrier. Formulation according to claim 5, characterized in that the peptide is conjugated to (a) itself, (b) another peptide as defined in any one of claims 1 to 4, (c) a T cell epitope or (d) part or all of the somatostatin molecule. A formulation according to any one of the preceding claims, characterized in that the peptide, whether bound to a carrier or not, is admixed with an adjuvant.