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1. (WO1989000166) GROWTH HORMONE RELATED PEPTIDE
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GROWTH HORMONE RELATED PEPTIDE

The present invention relates to biologically active molecules, particularly peptides, more particularly peptide fragments of growth hormone (including bovine growth hormone (bGH) , porcine growth hormone (pGH) , chicken growth hormone (cGH) , ovine growth hormone (oGH) and their mutant derivatives) , which can enhance or promote growth hormone activity. Human growth hormone (hGH) , rat growth hormone (rGH) , mouse growth hormone (mGH) , horse growth hormone (eGH) and salmon growth hormone (sGH) are other growth hormones of interest.

Polypeptide hormones are "important for both medical and veterinary application. One" such hormone, growth hormone, is found in vertebrates and is important for promoting somatic growth. Growth hormones from different species share both structural and functional characteristics. Growth hormones consist of amino acid sequences generally of about 191 residues in length. It is known that growth hormone can stimulate somatic growth, promote wool growth in sheep, affect body composition, improve food efficiency and promote lactation in appropriate species. Different aspects of the structural and functional characteristics of growth hormones have been described (Nicoll et al, 1986; Isaksson et al 1985; Wallis, 1978).

Antibodies to hormones have been shown to be capable of (i) enhancing hormone activity, (ii) have no effect on hormone activity or (iii) inhibit hormone activity (Thompson 1937; Rolands 1939; Goodfriend et al, 1970;

Shechter 1979a, 1979b ; Cole et al 1975; Aston' et al, 1986,1987 Ferguson 1954). More specifically, Aston et al (1986,1987) have shown that certain antibodies to growth hormones can enhance the biological activity of the hormone in vivo. It was concluded in these studies that enhancement of hormone by antibodies is characteristic of particular specificities, a property also described previously (Cole et al 1975; Goodfriend et al 1970) . However, in none of these studies is a regime available to indicate how consistently to produce an enhancing antiserum by using a small peptide. EP-A-0137234 discloses that the large 7 kiloDalton fragment of growth hormone can produce antibodies that enhance growth hormone activity; however, this fragment may not always be suited for this purpose due to its size and its limited ability to produce a consistent enhancing antiserum. Currently, manufacture of such a large polypeptide may be problematic by peptide synthetic routes.

Enhancement of hormonal activity by the administration of a peptide fragment of GH has been disclosed in WO-A-8404915) . In this particular disclosure it was shown that a short peptide derived from the amino terminal portion of the GH molecule potentiated hypoglycaemic activity; however this peptide was not administered in an immunogenic fashion. Both GH and insulin induce hypoglycaemia when administered to animals. The insulin-potentiating activity of peptide fragments of GH have also been described elsewhere (Pullin et al 1981; Ng et al 1980) .

This invention relates to various specific peptides and their derivatives which have the capability of inducing antibodies to enhance growth hormone activity. At least some of the peptides may have growth hormone activity themselves.

According to a first aspect of the invention, there is provided an antigenic molecule which causes antibodies to be raised against at least some of the 112 to 159 region of a natural growth hormone. This ,may be achieved by providing a molecule having antigenic equivalence to at least part of the 112 to 159 region.

According to a second aspect of the invention, there is provided a molecule (other than a natural growth hormone) at least part of which is antigenically equivalent to an oligopeptide selected from residues 112 to 159 of a natural growth hormone.

The 112 to 159 regions of various natural growth hormones have the following amino acid sequences:

112 120 130 140 150 159 bGH K ILKDLEEGIILALMRELEDGITPRAGQILKQITYDKFDTNMRISDDALLKNYI hGH LLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNY pGH KLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNY

OGH KLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRRDDALLKNY
eGH KLRDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNY mGH LLKDLEEGIQTLMGRLEDGSSRTGQIFKQTYSKFDTNSHNDDALLKNY

CGH KLKDLEEGIQALMRELEDGSPRGPQLLRPTYDKFDTHLRNEDALLKNY
rGH KLKDLEEGIQALMQELEDGSPRIGQILKQTYDKFDANMRSDDALLKNY

SGH VGINLLITGSQDGVLSLDDNDSQQLPPYGNYYQNLGGDGNVRRNYELL

Bovine, ovine, porcine and chicken growth hormones are preferred. Ovine growth hormone is very similar to bGH.

In the above, and throughout this specification, the a ino acid residues are designated by the usual IUPAC single letter nomenclature. The more recent single letter designat ions may be correlated with the classical three letter des ignations of amino acid residues as follows :

A = Ala G = Gly M = Met S = Ser
C = Cys H = His N = Asn T = Thr
D = Asp I = He P = Pro V = Val
E = Glu K = Lys Q = Gin W = Trp
F = Phe L = Leu R = Arg Y = Tyr

The oligopeptide will be of at least the minimum size necessary to confer antigenicity: usually it will be of at least six or seven res idues , but may be of any suitable length up to , for example , 20 amino acid residues . The best oligopeptides may be expected to correspond to topographical surface features of a natural growth hormone molecule, that is to say those f eatures having s ome three-dimens ional feature protruding from or extending into the ambient surface level o f the hormone . Pre ferred ol igopept ides correspond to regions 133-159 and 128-147.

Probably the most simple way of ensuring that at least part of the molecule is antigenically equivalent to the ol igopeptide is for that part of the molecule to comprise a sequence of amino acid residues which is identical to or conf ormationally similar to the ol igopeptide . Hovever , any other way of producing antigenic equivalence may be used: an example is to use an anti-idiotype antibody or other (even non-proteinaceous) analogue.

The invention therefore encompasses short peptides (preferably of less than 20 amino acid residues, but generally of at least six or seven amino acid residues, for example eight to twelve residues) sharing structural homology with growth hormone and which when administered to an animal can enhance hormone activity. Potentiation of hormone activity (in particular growth hormone activity) , may occur through direct or indirect effects on the hormone in question.

The invention therefore encompasses a peptide other than a natural growth hormone comprising the amino acid sequence:

GTPRAGQILKQTYDKFDTNMR

or an active fragment thereof and/or conservative mutant thereof. This sequence is taken from bGH, peptides 130 to 150.

The peptide will generally be antigenic and capable of stimulating the production of antibodies which, when in an appropriate formulation, potentiate the effect of growth hormone.

As stated above, an active subfragment of the specified sequence may be used. Active subfragments may consist of or include hexapeptides, including one or more of:

GTPRAG QILKQT YDKFDT
TPRAGQ ILKQTY DKFDTN PRAGQI LKQTYD KFDTNM RAGQIL KQTYDK FDTNMR AGQILK QTYDKF GQILKQ TYD FD

Active subfragments may also consist of or include heptapeptides, including one or more of:

GTPRAGQ ILKQTYD KFDTNMR TPRAGQI LKQTYDK PRAGQIL KQTYDKF RAGQILK QTYDKFD AGQILKQ TYDKFDT GQILKQT YDKFDTN QILKQTY DKFDTNM

The invention also encompasses a peptide other than a natural growth hormone comprising the amino acid sequence:

AGQILKQTYDKFDTNLRSDDA

or an active fragment thereof and/or conservative mutant thereof. The above sequence is taken from pGH, peptides 134 to 154.

Active subfragments may consist of or include hexa-peptides, including one or more of:

AGQILK QTYDKF DTNLRS GQILKQ TYDKFD TNLRSD QILKQT YDKFDT NLRSDD
ILKQTY DKFDTN LRSDDA LKQTYD KFDTNL KQTYDK FDTNLR

Active subfragments may also consist of or include heptapeptides, including one or more of:

AGQILKQ TYDKFDT NLRSDDA GQILKQT YDKFDTN QILKQTY DKFDTNL ILKQTYD KFDTNLR LKQTYDK FDTNLRS KQTYDKF DTNLRSD QTYDKFD TNLRSDD

The invention further encompasses a peptide other than a natural growth hormone comprising the amino acid sequence :

IQALMRELEDGSPRAGQILKQ

or an active fragment thereof and/or conservative mutant thereof or salts thereof . This sequence is taken from pGH, peptides 120 to 140.

Active sub f ragments may cons i st o f or i nc lude hexapeptides , including one or more of :

IQALMR ELEDGS PRAGQI QALMRE LEDGSP RAGQIL ALMREL EDGSPR AGQILK LMRELE DGSPRA GQILKQ MRELED GSPRAG
RELEDG SPRAGQ

Active subfragments may also consist of or include heptapeptides, including one or more of:

IQALMRE LEDGSPR . AGQILKQ
QALMREL EDGSPRA

ALMRELE DGSPRAG

LMRELED GSPRAGQ

MRELEDG SPRAGQI

RELEDGS PRAGQIL

ELEDGSP RAGQILK

Other preferred sequences include:

EDGSPRAGQIL

AGQILKQTYDK

RAGQILKQTYDKFDTNLRSDD

DTNLRSDDALL

KQTYDKFDTNLRSDDALLKNY

LRSDDALLKNY

and active fragments (including the hexa- and heptapeptides) thereof and/or conservative mutants thereof or salts thereof.

It should be noted that combinations of more than one of the above sequences may be used.

According to a further aspect of the invention, a method of promoting the activity of growth hormone (or a substance having growth hormone activity) comprises administering to a vertebrate an effective amount of a peptide or other molecule as described above. The invention thus encompasses the use of a peptide or other molecule as described above in the preparation of an agent for use in the promotion of the activity of growth hormone or a substance having growth hormone activity.

Peptides and other molecules in accordance with the invention may be presented in a variety of ways. For preference, an antigenic region (such as a peptide fragment or sub-fragment) in a molecule in accordance with the invention will contain the amino acid sequence of choice linked to a carrier peptide or protein. It is generally preferred to have a plurality, for example 5 to 10, copies of a peptide sequence (for example one or more of the above sequences) linked to the carrier. The carrier can for convenience be a generally large protein, which is inert in material respects, and which is derived from a different species or genus from that associated with the natural growth hormone. Examples of carriers include albumins such as human serum albumin, bovine serum albumin and ovalbumin (although not so many peptides will probably be able to be carried in this last case) . Alternatively, keyhole limpet haemocyanin can be used. The carrier will generally preferably come from a different species from that on which the fragment is based.

It is not essential that peptide sequences as described above be linked to albumins: they may be linked to other macro olecules, such as beta-galactosidase, especially of bacterial origin.

The invention encompasses molecules being peptides or having peptide regions which share substantial (eg greater than 30%, 50% or even 70%) sequence homology) with the above peptides. Similarly, conservative amino acid substitutions may not decrease the immunogenicity or antigenicity of peptides. Thus antigenically similar homologues will elicit antibody which binds to GHs in the same region as the above peptides define. It is well known that the use of homologues can be a means of circumventing 'self tolerance. Thus the use of the corresponding sequences from other species may be advantageous in this invention. Examples of homologous sequences to the preferred bovine, porcine and ovine growth hormones are those derived from the corresponding sequence regions of equine GH, mouse GH, chicken GH, trout or salmon GH or rat GH.

It is alternatively possible for molecules in accordance with the invention which are or which comprise peptides to be or to include polymers of sequences as described above. Appropriate sequences can be polymerised either by cross-linking of two cysteine residues to form disulphide bonds or by using external chemical coupling agents (such as carbodiimide, glutaraldehyde or other dialdehydes or di- (or poly-) functional carboxylic acids. As a further alternative, recombinant DNA techniques could be used to produce a peptide polymer.

It should be noted that the chemical coupling (which could for example take place through the agency of lysine residues) and disulphide bond formation are not limited to when the coupling residues are at the end of the sequence: internal residues could also be appropriate. Coupling residues, for example cysteine residues, may be added as desired.

It may be found that it is not necessary to couple any of the sequences described above with external peptides. They may be antigenic on their own. In such a case, it may be advisable to select particular adjuvants such as DEAE dextran and Merck 7426.

According to a further aspect of the present invention, there is provided a pharmaceutical or veterinary composition comprising a molecule as described above in conjunction with a pharmaceutically or veterinarily acceptable carrier. The composition may contain an adjuvant, for example, DEAE dextran, Merck 7426, saponin and aluminium hydrόgel. Alternatively or in addition Freund's complete adjuvant could be used. As noted above, certain adjuvants are more likely to be preferred in particular circumstances.

Compositions in accordance with the invention will normally be sterile, as they will be intended for implantation or injection. Intravenous injection is not preferred: subcutaneous injection is the route of choice, although possibly intramuscular and/or intraperitoneal injection could be used.

The preferred location of the subcutaneous injection would be at the back of the neck in the case of an animal, as that is a place where commercially useful meat is less likely to be damaged by tissue breakage or bruising.

The carrier will generally be an isotonic buffer plus saline, such as PBS or physiological saline.

Dosages will be generally as prescribed in accordance with the directions of the physician or veterinary surgeon, but 5 to 500 meg per dose, particularly 50 to 100 meg of peptide or other molecule may be found to be suitable for bovine or porcine applications.

A substance having or promoting growth hormone activity can be administered shortly after (or may be in some circumstances prior to or at the same time as) peptides or other molecules in accordance with the present invention. The activity of the growth hormone is then enhanced. This can lead to enhanced growth where growth is relevant; improved body compositions (for example, in pigs there may be less fat and more muscle on the back) ; better wool growth in sheep; improved growth efficiency (that is to say, better growth for a given unit amount fed) ; and enhancement of lactation in cows and sheep. This latter application is not only important for providing milk for human consumption, but it may also enable sheep to rear their young more effectively.

Examples of substances promoting growth hormone activity other than growth hormones themselves and other than molecules in accordance with this invention include antibodies to growth hormone inhibitors and' antibodies to other growth hormone antagonists, such as antibodies against somatostatin or leutinising hormone releasing hormone (LHRH) . The production of an antibody to somatostatin, for example, would increase circulating GH levels and may thus potentiate the effect of molecules in accordance with the present invention. Another substance which may be regarded as promoting growth hormone activity is growth hormone releasing hormone (GRF) , which may also be administered.

In another aspect, the invention provides antibodies raised against molecules of the first aspect. Such antibodies may be parenterally administered to animals, generally in an appropriate formulation to produce a growth hormone potentiating effect. Preferred formulation and administration details may be as described above, with changes as appropriate.

The invention will now be illustrated by the following examples. The examples refer to the accompanying drawings, in which:

Figure 1 show regions of high antigenicity in pGH and bGH;

Figures 2, 3 and 4 show that molecules in accordance with the invention enhance dwarf mice growth rates; and

Figures 5a to 5e show the effect of antipeptide antiserum on sheep growth and metabolism.

Example l Synthesis of bGH 130-150 peptide containing C-terminal Cys-Ala - Sequence 1

Synthesis of peptides was primarily performed by the Fmoc-methodology, essentially as described by Dryland and Sheppard (1986) . The peptide was assembled with the C-terminus covalently attached to a polymeric support consisting of polymerised polydimethylacrylamide (Atherton and Sheppard 1985 ) . This material is commercially available as Pepsyn KA resin for the purpose of peptide synthesis.

Attachment of the first amino acid to the resin via an ester bond is effected using dimethylaminopyridine as the catalyst . Throughout the synthesis dimethyl-formamide was used as the solvent. Temporary Na-amino group protection is the base-labile 9-fluorenyl-methoxycarbonyl (Fmoc) group, which is removed with 20% piperidine in DMF. Subsequent amino acids are " added sequentially by a repetitive protocol of amino deprotection, followed by coupling of an activated amino acid.

The hydroxy side-chains of serine , threonine and tyrosine are protected as t-butyl ethers, the carboxyl side chains of aspartic and glutamic acids as their t-butyl esters . The basic side chains of lysine and histidine are protected with the t-Boc group, but the guanidino side-chain functionality of arginine is protected as the 4 methoxy 2 , 3 , 6-trimethylphenyl-sulphonyl derivative (Atherton et al 1983) . Cysteine is protected as the S-acetamido methyl (ACM) or trityl derivative .

Amino acid derivatives were added as their pentafluorophenyl esters (PFP) with hydroxybenzo-triazole (HOBT) as the catalyst, but serine and threonine are added as the 3,4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine (DHBT) esters (Atherton et al 1986) . Occasionally, Fmoc amino acids were added as their pre-formed symmetrical anhydrides.

Acylation and deprotection steps were monitored spectrophotometrically as well as by the trinitro-benzene sulphonic acid (TNBSA) (Hancock and Battersby, 1976) test procedure and occasionally by amino acid analysis of the acid hydrolysate of resin bound peptide.

After the assembly of the peptide was complete, the peptide-resin bond was cleaved and most side chain protecting groups are removed simultaneously by the action of 95% trifluoroacetic acid (TFA) /scavenger mixture. Commonly used scavengers are water, phenol, ethanedithol , ethylmethylsulphide, anisol and thio-anisol.

The choice of scavengers and condition for cleavage varied with ;the composition of the peptide. TFA is removed in vacuo and scavengers extracted into ether. The crude peptide can be recovered from the aqueous layer by lyophilisation.

Peptides were synthesised by the t-Boc/benzyl strategy.

The support was either 4-(oxymethyl) -phenylacetamido-methyl (PAM) , or benzhydryla ine (MBH) resin. Temporary Na-protection is the acid labile t-butyloxycarbonyl (BOC) group, which is removed by trifluoroacetic acid (TFA) in dichloromethane. After neutralisation, the next Boc amino acid is added as the preformed symmetrical anhydride as , for example, programmed by Applied biosyste s 430A peptide synthesiser . The assembled peptide was cleaved from the resin with anhydrous hydrogen fluoride (HF) which also removes the usually benzyl-based side chain protecting groups .

Purif icat ion o f pept ides was ach ieved by gel filtration , ion exchange chromatography and high performance l iqu id chromatography (HPLC ) or a combination of these methods .

Homogeneity of peptides was assessed by analytical HPLC , thin layer chromatography (TLC) and electro-phoresis on thin layers of cellulose . Amino acid analysis of the acid hydrolysate and determination of the amino acid sequence of the synthetic peptide were also employed to indicate purity of the final product.

Peptides were optionally prepared with a Cys or Cys-Ala res idue ( s ) at the carboxy-terminal for ease o f synthesis or for alternative cross linking procedures .

Example 2 Synthesis of pGH 134-154 peptide containing C-terminal Cys-Ala - Sequence 2

Following the methodology of Example 1 , the above sequence was synthesised.

Example 3 Synthesis of bGH 120-140 peptide containing C-terminal Cys-Ala - Sequence 3

Following the methodology of Example 1, the above sequence was synthesised.

Examples 4 to 6 Preparation of carrier-linked i munogenic vaccines

Example 4

A synthetic peptide as prepared in Example 1 (Sequence 1) was cross-linked to 'carrier' (ovalbumin) by using glutaraldehyde. The immunogen consisted of 5 g peptide mixed with an equal weight of 'carrier' (ovalbumin, 5 mg) , dissolved in a total volume of 4ml phosphate-buffered saline (PBS) (25 mM sodium dihydrogen orthophosphate and 25mM disodium hydrogen orthophosphate containing 0.8% NaCl, pH 7.4). To this mixture glutaraldehyde (Sigma) solution was added. Generally the amount of cross-linking agent employed was 0.1 or 0.5 ml of a 0.5% solution (w/v) in water. The conjugation mixture was allowed to incubate at 20°C for between 30-60 minutes prior to dialysis and emulsification with adjuvant. It was found that dialysis was not a necessary step in the vaccine preparation and was sometimes omitted from the protocol .

Example 5

The procedure of Example 4 was followed, except that a synthetic peptide as prepared in Example 2 (Sequence 2) was used.

Example 6

The procedure of Example 4 was followed, except that a synthetic peptide as prepared in Example 3 (sequence 3) was used.

Examples 7 to 9 Preparation of carrier-free immunogenic vaccines

Preparation of 'carrier free' immunogen was achieved by repeating the general procedures of Examples 4 to 6 but omitting the ovalbumin from the conjugation mixture. The vaccine of Example 7 is based on Sequence 1, that of Example 8 is based on Sequence 2 and that of Example 9 on Sequence 3.

Examples 10 to 12 Vaccination protocols in sheep

Peptide vaccines as prepared in each of Examples 4 to 6 in a total volume of 4ml of PBS were emulsified with 7ml of Freund's complete adjuvant and injected subcutaneously (SC) at two distinct sites in sheep (Merino) . A second challenge (SC, two further sites) with the same vaccine was performed after 28 days in Freund's incomplete adjuvant and blood samples (jugular venepuncture) taken after 14 days and at weekly intervals thereafter. Further challenges with peptide vaccines were performed at 3-4 week intervals. Serum was prepared by allowing clotting of the blood whereas plasma was obtained by taking blood into heparinised tubes. Freund's adjuvants were purchased from Difco Laboratories.

Vaccination of cattle and pigs may be performed under similar conditions.

Examples 13 to 15 Preparation of serum or plasma samples and assay of anti-growth hormone antibodies in serum or plasma samples

Assays for antibody were performed with diluted serum or plasma whereas growth experiments were conducted with the immunoglobulin fraction of serum prepared by sodium sulphate precipitation. Sera were precipitated with two volumes of sodium sulphate (27% w/v in water) and collected by centrifugation. Subsequently, the globulin fraction was dialysed extensively against PBS (Johnstone and Thorpe, 1982) .

The immunogenicity and antigenicity of vaccines was determined by employing liquid-phase radioimmunoassay as described previously (Aston et al 1985). Essentially, serum or plasma was diluted in PBS (1/50-1/5000) and 0.05ml was assayed by the addition of 0.05ml of 125I labelled bGH or pGH. Binding of antibody in the serum to the tracer was determined by precipitation with a second antibody coupled to cellulose (SAC-CEL, Wellcome Diagnostics) . The word SAC-CEL is a trade mark.

Solid phase assays (ELISA) were performed as follows. bGH and pGH were coated onto solid phase microtitre plates as described previously (Aston et al 1985) 20

except that the plates were blocked with 1% bovine serum albumin BSA instead of haemoglobin. Sera were diluted across the plates (1/50, 1/500, 1/5000, 1/50000) in 1% BSA in PBS and incubated at 37°C for 2 hours or overnight at 4°C. Binding of anti growth hormone antibody in sera to the plates was determined by the addition of rabbit anti sheep immunoglobulin conjugate (peroxidase) (Dakko, 1/1000 in 1% BSA/PBS) . which was incubated at 0.1ml per well for 2 hours at 37°C. The substrate, 2 , 2-azino-di (3-ethyl benz-thiazolin-sulphonate(6) ) (ABTS) , (Boehringer Mannheim) was at a concentration of lmg/ml in citrate buffer pH 4.2 and contained Imcl of 30% hydrogen peroxide per 5ml. This was incubated at room temperature for five minutes before being read in a Dynatech MR600 microplate reader at 410nm.
*
Assay controls

Control sera or plasma samples were derived from sheep which had received 'carrier' protein cross-linked with glutaraldehyde in the absence of peptide.. The control vaccines were administered to groups of animals in parallel to the peptide vaccine treatments. Sheep group sizes were generally 5-10 animals but on occasions were as high as 50 animals per group.

The results are shown in Tables 1 and 2, which indicate the generation of growth hormone cross reactive antibodies by immunisation with peptides.

It is shown that immunisation of sheep with short peptides within the region 112-159 can elicit antibodies which bind to bGH and pGH (it is noteworthy that bGH and oGH differ in only one or two amino acids out of 191 and thus both hormones bind to the same antisera equally well) . The efficacy of antibody production varies with the sequence being used. It is also shown that certain sequences give a better response if 'carrier' is used whereas others this aspect is of lower importance. The extensive sequence homology between growth hormones from different species may enable use of a bGH sequence to elicit high affinity antibody against pGH and vice versa. It is clearly indicated in Table 1 that bGH sequences elicit antibody recognising pGH. The use of homologues rather than a 'self component to elicit auto antibody has been known for some time.

Table 1 shows the antibody response of sheep to peptide sequences from bovine and porcine GHs. Antibody was determined by liquid-phase radioimmunoassay (RIA) . (Positives retained counts of 200-2000cpm above the serum of control immunized sheep) .

22
Table 1
Sequence (1) bGH 130-150 )
) Contain C-terminal

Sequence (2) pGH 134-154 )
) Cys-Ala
Sequence (3) bGH 120-140 )



The efficacy of the response did not depend on whether a carrier was employed. The same results were obtained whether the peptides were cross-linked to an ovalbumin carrier or polymerised to themselves.

Table 2 shows another sheep experiment on the antigenicity of sequences pGH 134-154 and bGH 120-140. n=50/group. Serum assayed corresponded to that taken 2 weeks after the second injection with peptide vaccine. In this case, the peptides were polymerised to themselves with glutaraldehyde in accordance with Examples 8 and 9.

Table 2
% responding sheep
bGH pGH

Sequence (2) pGH 134-154 54 56 Sequence (3) bGH 120-140 60 74 (Binding to antisera ranged from between 400-2000 cpm above that of control serum)

Examples 16 to 18 Determination of growth enhancing antisera in dwarf mice

Example 16

Metabolic assay of growth in Snell dwarf mice was performed as described previously (Aston et al, 1986,1987; Holder et al, 1980). Essentially, male and female animals were randomised and allocated to cages so that no two mice of the same treatment group were in the same cage. Hormone or complex (prepared one hour prior to injection) with antipeptide antibodies were administered subcutaneously (0.1ml) on two consecutive days before injection of 35S labelled sodium sulphate (0.5 mcCi/g) (Amersham Radiochemicals) for a further 24 hours. Incorporation of radioactivity was measured in costal cartilage which articulated directly with the sternum after removal of the soft tissue. The mean growth rate of each treatment group was expressed as dpm/mg cartilage and represents the uptake of 35s-sulphate from six animals. Significance was determined by Student's T-test. The direct relationship between somatic growth and incorporation of radioactivity into cartilage has been described previously (Aston et al 1986, 1987; Holder et al, 1985). The results are shown in Figure 2.

Figure 2 clearly shows enhancement of dwarf mouse growth rate by anti GH antisera, as determined by 35S 24

sulphate incorporation into cartilage (Aston et al 1986,1987). Site directed antisera to bGH and pGH, raised by vaccinating sheep with peptide pGH 134-154 were examined for their effects in dwarf mice following complexing with pGH (50 eg) prior to injection. Control animals received antibodies from sheep which had received a control vaccine (FCA/ovalbumin only) . Each treatment group consisted of 6 animals. The three immune globulin preparations tested (A,B,C) were derived from different animals. Significance was determined by unpaired Student's T- test.

Example 17

In a second dwarf mouse experiment the growth enhancing activities of two anti-peptide antisera were examined. The results are shown in Figure 3. Mice were treated with bGH (50 meg) mixed with either control immune globulin (first column) or with immune globulin derived from sheep vaccinated with peptides pGH 134-154

(second column) , bGH 120-140 (third column) or a monoclonal antibody which enhances bGH activity (positive control ; fourth column) . Signif icant enhancement of the growth promoting activity of bGH was achieved with immunoglobulins raised with the above peptides .

Example 18

In a third dwarf mouse experiment further antisera raised to pGH 134-154 and bGH 120-140 were examined. The results are shown in Figure 4. Mice were treated with pGH (50 meg) either mixed with control globulin or immune globulin to pGH raised by vaccinating sheep with sequences pGH 134-154 or bGH 120-140. Growth rate was determined by incorporation of 35S-sulphate uptake into costal cartilage (Aston et al, 1986, 1987) . Levels correspond to means ± SEM. Weight gain was determined at the end of the experiment (hatched bars) and in all cases of treatment with pGH . (50 meg) + specific immune globulin (anti pGH 134-154 or bGH 120-140) significant increases in growth were observed (P<.001).

Example l9Effects of passive administration of anti-peptide i munoglobulin on sheep growth and metabolism

Sheep (pure Poll Dorset) were randomly allocated to one of four treatment groups (n=7) . The four groups were subjected to a pre-treatment period of 3 weeks and weighed twice per week. During the pre-treatment period blood was also taken at the times of weighing. On day 15, blood samples were taken hourly for 18 hours from a catheter. On day 18 an insulin tolerance test was performed on the animals. All animals were maintained on an ad lib diet of 16% crude protein farm mix (daily refusals were recorded) .

Treatments were bGH, bGH+anti-peptide immunoglobulin, anti-peptide immunoglobulin only, and control globulin.

During the treatment period (18 days) the animals were maintained on the same diet as above and weighed twice/week. On day 8 of treatment, hourly blood samples were taken (18) whereas on day 10, another insulin tolerance test was performed (ITT) . Eight animals were slaughtered on days 14, 15, 17 and the remaining four on day 18. At slaughter, liver was weighed and samples of subcutaneous fat were taken. The fat samples were analysed for lipolytic and lipogenic activity and also for lipid oxidation. Lipogenesis was determined by 1 C acetate incorporation into lipid, lipolysis by glycerol release (using BCL test kit 148270) and lipid oxidation by C02 production as described previously (Smith and Walsh, 1984,1988; Yang and Baldwin 1973)

The results are shown in Figures 5a to 5e. These show the effects of antipeptide antiserum (pGH 134-154) on sheep growth and metabolism. Immune globulin cross-reactive with oGH (also with bGH and pGH) , raised in sheep by vaccination with peptide pGH 134-154, was administered to growing lambs (Dorset) . The growth rate, liver weight, and metabolic activity of fat tissue was determined at slaughter. Treatments included (A) control immune globulin (from ovalbumin immunised sheep) , (B) anti ovine GH antiserum raised by immunising sheep with pGH 134-154, (C) bovine GH (lOmg) , and combination of (B) + (C) . Treatments were administered on alternate days. n=7 animals/group.

Treatment of lambs with anti-pGH 134-154 immune globulin increased growth rate (B) although to a lesser degree than did growth hormone (C) ; liver weights were similarly increased.

Growth hormone is known to be diabetogenic when administered to animals, thus agents that enhance endogeno s GH activity would be expected to manifest diabetogenic activity (resistance to insulin) . Treatment of lambs with bGH or bGH in complex with anti-pGH 134-154 antiserum results in significant diabetogenic activity. Administration of antiserum on its own also induced diabetogenic activity although to a lesser degree (Fig 5d) . The dose of insulin for the insulin tolerance test was 0.08 units per kg live weight. Units for the insulin tolerance test are millimoles glucose/litre as a change over a one hour period. The calculation of diabetogenic activity is in effect a measure of an animal's responsiveness to insulin: after treatment with growth hormone or antipeptide antiserum the animal is less responsive to insulin. The degree to which it is less responsive is calculated on each animal by conducting an insulin tolerance test during a control period and one after treatment; the difference is essentially the diabetogenic activity.

Another effect of growth hormone in vivo is to alter fat metabolism. Subcutaneous fat (perirenal) was removed and assayed for lipogenic activity and lipid oxidation by C acetate incorporation into lipid and glycerol release respectively. Treatment of lambs with anti peptide immunoglobulin (to pGH 134-154), resulted in significant suppression of lipid synthesis. Similarly, administration of bGH on its own or in combination with antipeptide antiserum significantly decreased lipogenesis (measured as the amount of acetate (in micromoles) incorporated into triglyceride per gram of lipid in two hours) . A similar profile of events was observed for lipid oxidation (Figure 5e) . (Lipid oxidation is measured as the amount of acetate (in micromoles) converted to carbon dioxide per gram of lipid in two hours.) Examples 20 to 41 Mapping of antigenic sites within the sequence region 112-159

Following the general procedure of Example 1, the following peptides, shown in Table 3, were prepared in order to map preferred antigenic sites within the sequence region 112-159 (n=5) . The peptides were injected into sheep either without a carrier or with an ovalbumin carrier, as in Example 4, and the proportion of sheep responding were noted.

Table 3
(NC= no carrier. OVA=ovalbumin. nd= not done)
SEQUENCE % RESPONDERS

NC OVA

ALMRELEDGS 0 20

KLKDLEEGIQALMRELEDGS 0 80

RELEDGSPRAG nd nd

DLEEGIQALMRELEDGSPRAG nd nd

EDGSPRAGQIL 80 100

EGIQALMRELEDGSPRAGQIL 40 40

SPRAGQILKQT 0 60

QALMRELEDGSPRAGQILKQT 80 20

AGQILKQTYDK 40 60

MRELEDGSPRAGQILKQTYDK 80 0

ILKQTYDKFDT 40 80

LEDGSPRAGQILKQTYDKFDT 40 40

QTYDKFDTNLR 40 20

GSPRAGQILKQTYDKFDTNLR 40 20 DKFDTNLRSDD 20
RAGQILKQTYDKFDTNLRSDD 80
DTNLRSDDALL 80
QILKQTYDKFDTNLRSDDALL 80
LRSDDALLKNY 40
KQT DKFDTNLRSDDALLKNY 40
DDALLKNY 40
YDKFDTNLRSDDALLKNY 60

The positives range in binding of 1 5-labelled bGH from 200-2000 cpm over background at two different dilutions. Titres of individual sera vary between 1/50-1/5000) (in solid-phase RIA the level of responders always between 80-100%) .

The results are shown graphically in Figure 1. The results indicate that the most antigenic regions lie between 133-159 and 128-147. The data also indicate that in cases the shorter peptides (about 10 amino acids) perform better than corresponding longer sequences of which the shorter sequence forms a part.

Examples 42 to 44

Peptide sequences pGH 134-154 and bGH 120-140 were prepared and injected into sheep or calves. Sheep or calf anti-growth hormone serum was thereby raised by the immunisation and titrated. Binding was determined in liquid phase RIA with either 125I-bGH or 125I-pGH. The results are shown in Table 4.

30

Table 4
Dilution of serum: 1/50 1/500 1/1000 1/5000

cpm/50 mcl of diluted
serum assayed.

125I-pGH 1750 1050 865 265

(sheep)
125I-bGH 2550 1890 995 375

(sheep)

125I-bGH 900 1097 925 505

(calf)

The efficacy of the response did not depend on whether a carrier was employed. The same results were obtained whether the peptides were cross-linked to an ovalbumin carrier or polymerised to themselves.
Example 45

Pigs were immunised with fragments bGH 120-140 and pGH 134-154, each conjugated with ovalbumin, in complete Freund's adjuvant (250 meg/pig) and challenged with the same dose in incomplete Freund's adjuvant after 28 days. Blood was taken after a further 10 days and assayed for antibody to bGH and pGH by solid phase ELISA. Essentially, plates were coated with bGH or pGH and binding of antiserum was determined with a peroxidase-conjugated rabbit immunoglobulin to swine Ig (Dako, Denmark) as described above (Examples 13 to 15) for the determination of sheep anti-hormone antibody in solid phase. Animals responded to both peptides, making antibodies which cross-reacted with the native hormones on the plates. Titres varied between 1/50 and 1/5000, although responses were better for the peptide bGH/pGH 120-140. The results are shown in Table 5.

Table 5
Peptide Response (%) - four animals per group
bGH pGH bGH 120-140 75 100 pGH 134-154 75 75

REFERENCES .

Aston, R. , Holder. A.T., Preece, M.A. and Ivanyi, J.

(1986) J. Endocrinol. 110, 381-388.

Aston, R., Holder, A.T., Ivanyi, J. and Bomford, R.

(1987) Molec. Immunol. 24, 143-150.

Holder, A.T. Aston, R. Preece, M.A. and Ivanyi, J. (1985) J. Endocrinol. 107, R9-R12.

Cole, H.H., Dewey, R. , Geschwind, I.I. and Chapman, M. (1975) Biol. Reprod 12, 516

Ferguson, K.A. (1954) Nature 174, 411

Ng, F., Henderson, T. , Bromley, J. and Bornstein, J (1983) 30, 63-71.

Pullin, C. O., Harcourt, J.A. , Ng, F.M. and Bornstein, J. (1981) Int. J. Peptide Protein Res. 18, 318-323.

Smith, R. W. and Walsh, A. (1984) Effects of lactation on the metabolism of sheep adipose tissue. Research in Veterinary Science Vol. 37.

Smith R.W. and Walsh, A. (1988) Effects of Pregnancy and lactation on the Metabolism of bovine adipose tissue. Research in Veterinary Science. Vol. 44.

Yang, Y. T.^and Baldwin , R.L. (1973) J. Dairy Science. 44

Isaksson, O.G.P., Edan, S. Jansson, J-O (1985) Ann. Rev. Physiol. 47, 483-499.

Nicoll, C.S., Mayer, G.L. , and Russell, S.M. (1986) Endocrine Rev. 7, 169-203.

Rolands, I.W. (1939) J. Endocrinol. 1, 177

Shechter, Y. , Chang, K. . , Jacobs, S. and Cuatrecasas, P. (1979) Proc. Natl. Acad. Sci. USA. 76, 2720

Shechter, Y. , Hernaez, L. , Schlessinger, J and Cuatrecasas, P. (1979) Nature 278, 835-838.

Thompson, K.W. Proc. Soc. Expl. Biol. NY 35, 640.

Goodfriend, T.L., Webster, M.E., McGuire, J.S. (1970) J. Clin. Endocrinol. Metab. 30, 565-572.

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



INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification ; (11) International Publication Number: WO 89/ 001 C 07 K 7/06, 7/10; // A 61 K 37/36 A3 (43) International Publication Date: 12 January 1989 (12.01.8

(21) International Application Number: PCT/GB88/00528 (74) Agents: SHEARD, Andrew, Gregory et al.; Kilburn
Strode, 30 John Street, London WC1N 2DD (GB).

(22) International Filing Date: 6 July 1988 (06.07.88)
(81) Designated States: AT (European patent), AU, BE (E

(31) Priority Application Numbers: 8715858 ropean patent), BR, CH (European patent), DE (E
8715859 ropean patent), DK, FI, FR (European patent),
8715866 (European patent), IT (European patent), JP, L
(European patent), NL (European patent), NO,

(32),Priority Dates: 6 July 1987 (06.07.87) (European patent), US.
6 July 1987 (06.07.87)
6 July 1987 (06.07.87)
Published
(33) Priority Country: GB With international search report
Before the expiration of the time limit for amending t
claims and to be republished in the event of the receipt

(71) Applicant (for all designated States except US): PEPamendments.
TIDE TECHNOLOGY LTD [AU/AU]; 4-10 Inman
Road, P.O. Box 444, Dee Why, NSW 2099 (AU). (88) Date of publication of the international search report:
26 January 1989 (26.01.89

(71X72) Applicant and Inventor: ASTON, Roger [GB/AU];
Lot 2340 Highs Road, West Pennant Hills, Sydney,
NSW 2120 (AU).

(54) Title: GROWTH HORMONE RELATED PEPTIDE

(57) Abstract
Antigenic molecules cause antibodies to be raised against at least some of the 112 to 159 region of a natural growt hormone when injected in vivo. The molecules can comprise portions of the 112 to 159 region. The antibodies are believe to bind to growth hormone on administration and enhance its effect.

FOR THE PURPOSES OF INFORMAHON ONLY

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