CA1288341C - Physiologically active compositions - Google Patents

Abstract

ABSTRACT
A formulation which comprises an actual or functional fragment of a hormone and an immunological carrier or adjuvant, in combination with a pharmaceu-tically acceptable carrier may be used to increase a hormone-regulated response of a vertebrate beyond normal physiological levels; in the case of a growth hormone this may provide accelerated growth of economically important animals.

Description

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This invention relates to hormone activity in vertebrate species.
This application is a divLsional applica-tion of Canadian Appli-cation Serial No. 461,152, ~iled August 15, 1984.

The term "hormone" has been defined as encompassing any substance released from one part of the body and acting selectively on a; least one other, distant, part. Many such substances are secreted by one of the endocrine glands, an example being the secretion of growth horrnone from the pituitary gland. The basis for the selectivity for a given tarset tissue or tissues is the presence or absence in the tissue of receptors which bind the substance specifically. In thecase of the protein hormones, such as insulin, the receptors are on the cell surface. Interferon has been implicated in the defence of mammalian cells against viral attack. It is not secreted by an endocrine gland and does not havethe specificity of target tissue that, say. growth hormone has. Nevertheless, itis a large glycosylated protein molecule and binds to specific cell surface receptors. There are also cerSain similar substances which influen`ce the activity of cell by binding to specific rec~ptors on the cell sur~aca, for example tumour necrosis factor and lymphokines such as interleukins. In this specification, the term "hormone" is used to embrace all such protein or polypeptide entities (optionally glycosylated) and the like having a cell-surface receptor.

It has been shown that certain antibodies against insulin and against epidermal growth factor tEGF) potantiate or mimic the activity oF those hormones In vitro (Y.Schechter et al, Proc.Nat Acad.Scl.76(6),2720,(1979) and Y. Schechter et al, Nature 278, 835,(1979)). It is thought that this occurs because the antibodies, being bivalent, cause an aggregation of the hormone-receptor complexes on the cell-surface, such aggregation being involved in activating the second messenger within the cell; monovalent Fab fragments of antibodies do not cause such potentiation or mimicry. These studies have solely been concerned with characterising the insulin and EGF receptor~ and in identifying the mode of action of such hormones. Furthermore, not all such studies have demonstrated such potentiation (de Pirro et al, Diabetologia 19, 11B (1980) and Schechter et at, Proc. Nat.Acad.Sci, 75(12),5788 (1978)~. There has been no suggestlon that such potentiatlon ~Ivouk~ occur in vivo or that the phenomenon is more widely ~g ' ,: .
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applicable. Indeed, the generation of antibodies against insulin and other hormones ln VlVO was thought to be highly undesirable since the hormone-antibody comple:<es would be cleared by the body's irnrnune system and, far from being potentiated, the action of the hormone would be negated - see ~or example Schwartz ~., Endocrinology, 107(4),877; Fraisier, Endocrine Reviews 4(2),155 and Gause et al, Endocrinology 112(5),1559 on growth hormone, and 81ake ~ Kelch, Endocrinology 109(6),2175 on luteinising hormone releasing hormone.

It has now surprisingly been ~ound that the administration of certain specific antibodies to hormones can potentiate or mimic the activity of the hormone, provided that the epitope s~e~ificity of the antibody i~ chosen appropriately.

Accordingly, formulations are provided which comprise anti-bodies to a hormone, the epitope specificity of at least some of the antibodies being so chosen that the formulation will potentiate or mimicj when administered to a vertebrate, the administration of the hormone in that vertebrate.

Formulations are also provided which comprise complexes of (a) a hormone and (b) at least one type of antibody to that hormone, the epitope specificity of at least some of the antibodies being so chosen that the formulation will poten-tiate or mimic, when administered to a vertebrate, the administration of the hormone in that vertebrate.

A method is provided oE potentiating or mimicking hormone administration in a "normal" vertebrate (as herein defined) by administering to the vertebrate a formula-tion comprising antibodies to the hormone, the epitope specificity of at least some of the antibodies being so chosen that the formu-lation will potentiate or mimic, when administered to a verte-brate, the administration oE the hormone in that vertebrate.
The term "normal" is used herein to indicate an individual having suEficient endogenous amount oE the hormone in ques-tion for normal functioning o~ -the tissues regulated by that hormone.

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Further a method is provided of potentiating or mimicking hormone administration in a "normal" vertebrate (as herein defined) by administering to the vertebrate a formulation ccmprising complexes of (a) the hormone and (b) at least one type of antibody to that hormone, the epitope specificity of at least some of the antibodies being so chosen that the formu-lation will potentiate or minic, when administered to the verte-brate, the administration of the hormone in that vertebrate.
A method is also provided of treating a human or other verte-brate having abnormally low hormone-regulated tissue function by administering to the vertebrate a pharmaceutical formulation comprising antibodies to the hormone in question, the epitope specificity of at least some or the antibodies being so chosen that the formulation will potentiate or mimic, when administered to the vertebrate, the administration of the hormone in that vertebrate.
Still further a method is provided of treating a vertebrate having an abnormally low hormone-regulated tissue function by administering to the human or animal a pharmaceutical formu-lation comprising complexes of (a) the hormone in questionand (b) at least one type of antibody to that hormone, the epitope specificity of at least some of the antibodies being so chosen that the formulation will potentiate or mlmic, when administered to a vertebrate, the administration of the hormone in that vertebrate.
In accordance with the invention there is provided a formu-lation comprising an actual or functional fragment of a hormone and an immunological carrier or adjuvant, in c~mbination with a pharmaceutically acceptable carrier.

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In accordance with another aspect of the invention there is provided a me-thod of increasing a hormone-regulated response of a vertebrate having a sufficient endogenous amount of the said hormone for the normal functioning of the tissues regulated by the said hormone by administering to the vertebrate a preparation ccmprising at least one pre-selected actual or functional fragment of an appropriate hormone.
The potentiation or mimicry of the administration of growth hormone, insulin, thyroid stimulating hormone and interferon are particularly preferred aspects of the invention.
The clinical abnormalities which result fram a deficiency of a given hormone are in many cases well characterised and will not be listed here. However, examples include (from a deficiency of grown hormone) pituitary dwarfism, Turner's syndrome and cachexia (frcm a deficiency of insulin), diabetes and (from a deficiency of thyroid stimulating hormone) cretinism, simple goitre and myxedema.
By antibody "to" a particular hormone, we mean an antibody which will bind to that hormone. Thus, the antibody need not have been created in response to that specific hormone. For example several antibodies raised against growth hormone (GH) will cross-react with chorionic somatomammotropin (CS) because of the extensive sequence homology between the two hormones.
Furthermore, it may be possible to raise antibodies to a synthetic analogue or hormone of a portion of it.

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It is to be noted that the ant:ibodies need not necessarily be to the specific hormone of the species to which the formulation or method of the invention is being applled. Preferably, however, they are. It has been Found that not all antibodies to the hormone will potentiate or mimic the administration oF that hormone; instead, the ability of an antibody to act in accordance with the invention appears to depend on the specific determinant (ie. antigenic site) forthe antibody on the hormone. It will therefore readily be appreciated that polyclonal antibodies (that is tO say, a collection oF antibodies having a range of determinant specificities) are less suitable For use in formulations or methods in accordance with the invention, than are monoclonal antibodies. The man skilled in the art will readily, having read this specification, be able by means of routine experimentation to select a monGclonal antibody effective in carrying out the invention. Mixtures of suitable monoclonal antibodies may in some circumstances be used. However, it is nevertheless possible to use animal or human antisera raised by 'conventional' immunization provided that the epitope specificity of the antibodies is as described. Particularly preferred monoclonalantibodies for growth hormone (GH) and chorionic somatomammotropin (CS) are EB01 and EB02. Antibody QB01 is preferred for prolactin (PRL).

It has been found that the presence of the hormone in the animal is necessary for the antibody (when administered alone) to act in the manner described.
Thus, in the case of "normal" individuals, administration of the selected antibody alone will have the described effect but, for example, in individuals without endogenous GH, such as pituitary dwarf human children, GH must be administered as well as, but not necessarily sirnultaneously with, the antibody.

Instead of prepa. ing the antibody outside the animal, it is possible to raise antibodies of the appropriate specificity by injecting the animal with a pre-selected fragment of a suitable growth hormone molecule in combination with an adjuvant. The fragment will be so chosen as to comprise only the epitope or epitopes to which one or more of the hormone-potentiating antibodies are specific and may be derived by cleaving the hormone appropriately, or by synthesising a peptide fragment (or an analogue to such a fragment). 8y choosing portions of the hormone rich in hydrophilic residues, one is more likely I~SB/NDC/1st August, 198~

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to be creating or selecting a fragment (an "antigenic determinant") which is on the surface oP the complete hormone molecule and which will therefore cross-react with the complete molecule. Equally, the fragment should not contain the site of the hormone which binds to the cell-surFace receptor, nor any site which binds a third agent which causes a conformational change such that receptor binding is inhibited. Otherwise, the antibodies produced are likely to inhibit, rather than potentiate,the action of the hormone. Thus, in a successfulimmunisation of this type, a polyclonal collection of antibodies of narrow specificity is created within the animal in question, thus enabling less frequent injection of the animal than would be the case if exogenous antibodies were passively administered. It will be appreciated that, instead of an actual fragment, a functional fragment could be used, in which the undesirable epitopes of the molecule are present but are shielded from antibody access in some way. The term "fragment" is used in this specification to cover actual and functional fragments.

Accordingly, the present invention also provides a method of increasing a hormone-regulated response of a vertebrate by administering to the vartebrate a preparation comprising at least one pre-selected "fragment" tas herein defined) of an appropriate hormone, optionally in combination with an adjuvant.

The invention also encompasses such a preparation and methods of making such a preparation by conventional means. In such convential vaccines, immunological carriers are frequently used to enhance the immunogenicity of the antigen, for example keyhole limpet haemocyanin or tetanus toxoid.
Similarly, adjuvants are often included to stimulate the immune system, for example aluminium hydroxide, saponin or muramyl dipeptide. Generally, abou~
û.0û1 to 10~umoles of antigen should be present in a unit dose, preferably about 0.01 to 0.0~,~moles, although the selection oF a suitable amount of the antigen is well within the capabilities of one skilled in the art.

In the case of passive transfer of antibodies to a vertebrate, approximately 104-107, preferably 105-106 ABT50 units of antibodies should be administered in any suitable sterile medium, such as saline, to give a dose of 0.01 to 10ml, preferably about 0.5ml.

To take only three hormones as an example, namely GH, CS and PRL, 0 RSB/~lDC/1st August, 1984 3~
~~ - 7 - A690 formulatlons or methods in accordance ~,vith the invention are believed to oFferpotential in:

(a) accelerating the attainment of full growth o-F industrially important (ie. farmed) animals such as cattle, pigs and poultry or achieving such growth on reduced amounts of feed;
(b) increasing the growth of such animals beyond the normal maximum;

(c) increasing the duration or extent of lactation in mammals7 for example to obtain a greater milk yield frorn cattle or to enable a human mother to breast-feed an infant;
(d) increasing the proportion of lean meat to fat in farmed anima!s;

(e) increasing the growth of fleece, fur or other use~ul surface products of animals, for example sheep;

(f) treating a GH-deficient individual, for example a dwarf child, to enable normal growth to occur.

In all cases, it is believed that the use of formulations and/or methods in accordance with the invention may offer significant cost-saving and labour-saving advantages in comparison with the use of the hormone alone, not least because the potentiation of the hormone action is expected to result in fewer administrations being needed. Furthermore, a reduction of possibly harmful residues in the meat or milk of treated animals may be expected. Finally, because Farmed animals are frequently routinely injected with other vaccines, for example against foot and moutll disease, it would be extremely convenient to incorporate in such a vaccine a formulation in accordance with the present invention.

The invention will now be described by way of the following non-limiting Examples.

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Abbreviations _ hGH - human growth hormone hCS - human chorionic somatomammotropin hPRL - human prolactin bGH - bovine growth hormone MAB - monoclonal antibody PBS - phosphate-buff erred saline PMSf - phenyl methyl sulphonyl fluoride Ig immunoglobulin MHC - major histocompatibility complex SPRIA - solid phase radioimmunoassay wto weight at time 0 SDS-PAGE - SDS polyacrylamide gel electrophoresis EDTA - ethylene diamine tetracetic acid RIA - radio-immuno assay .
Statistical evaluation Arithmetic means and standard deviation values were calculated using conventional 25 methods. Differences between groups were assessed by unpaired Student's t-test.
Description of Figures Figure 1 relates to Example A and shows weight gain in dwarf mice with compositions in accordance with the invention;
Figure 2 relates to Exarnple C and shows corresponding weight gain in normal mics;
5 Figure 3 relates to Example D and shows the weight gain of the pigeon crop sac; and Figure 4 relates to Example E and shows the weight gain of marmosets.

RSB/NDC/1st August, 1984 3~
_ g A690 PREPARATNE EXAMPLES

-Example 1: Preparation of Monoclonal Antibody to Human Growth Hormone (hGH).

The antibodies employed in this study are available from Wellcome Diagno3tics, Temple Hill, Dartford, Kent. U.K. and have been characterized extensively (Ivanyi, 1982 a b, Aston and Ivanyi, 1983). BALB/c mouse spleen cells were fused with NSImyeloma cells and cloned by standard techniques (Ivanyi and Davis, 1980, 1981).
The antibodies derived were all of the IgG1 isotype and were non-precipitating when examined by double diffusion in agar. Four determinants have been defined on hGH by competition assays (QA68, NA71, EBO1 and EBO2) of which two are completely shared with hCS (EBO1 and EBO2). However, none of the antibodies cross-reacted with human prolactin. Antibody concentrations have been expressed as ABT50 values which correspond to the reciprocal antibody titre required to give 50 percent binding of 125I-hGH by RIA (Ivanyi, 1982a). Binding studies with proteolytically modified forms of hGH suggest that all four determinants are located in the first 1-139 redidues with the EBO1 determinant also represented in the sequence region 146 - 191 (Aston and Ivanyi, 1983).

Example 2:

PREPARATION OF Fab' FRAGMENT OF EBO1 Ascitic globulin (5mg/ml) of EBO1 was affinity purified on hGH (lOOmg) immobilized on CNBr-activated Sepharose. Retained material was eluted with glycine-HCl buffer pH 2.3 and tubes containing protein material were immediatelyadiusted to pH 7.5 with NaOH(lM). The purified antibody was concentrated to 20mg/ml (2ml) and dialysed against sodium phosphate buffer (0.5M9 pH 8.0) containing cysteine (0.OlM) and EDTA (.002M).

This material was digested with 0.4mg of papain (BDH) for 4 hours at 37C
followed by dialysis against PBS to remove the cysteine and EDTA.
Subsequently, the dialysate was applied to a column of DEAE cellulose (20cm x 1.2cm) and eluted with a linear gradient consisting of sodium phosphate buffer (0.005M - 0.3M, pH 8.0). The first peak to be eluted from the column contained no ~0 RSB/NDC/1st August, 1984 ~ 2~
- 10 - ~690 antibody heavy chain as determined by SDS-PAGE and retained an activity of 10 3 x ABT50.

Example 3: HWRMONES

Human growth hormone employed for injection was derlved from stocks of out-dated clinical grade material obtained by special agreement with the Institute of 10 Child Health, London, whereas hormone used in assays was of ~99~/~ purity. hGH is avaiable from RIA(UI~) Ltd, Washington, Co. Durham U.K. Radioiodination of hGH
was performed with lactoperoxidase resulting in a tracer of high specific activity (80 x 106 cpm/ g) (Linde et al, 1981). Monomeric 125I-h(:;H was separated from any aggregated material prior to assay by Ultrogel column chromatography.
15 Ultrogel is a trade mark of LKB Ltd, Cambridge, U.K. Antibody-hormone complexes for administration into animals were prepared by mixing the solutions for 1 hour prior to injection. In chronic experiments, where injections were given for several weeks, the complexes were prepared in batches enough for 1 week and stored at ~4C.

A soluble extract was prepared from the marmoset pituitary gland by homogenizingthe tissue in 0.05M sodium bicarbonate 2mM PMSF pH 8.S. The resulting homogenate was centrifuged at 10,000g for 20 minutes and the supernatant (20ml) was tested.
IOLOGICAL EXAMPLES

~e~ CUMULATIVE WEIGHT G~INS IN hGH-EE~O1 COMPLEX TREATED
DWARF MICE

Dwarf mice were bred from normal animals heterozygous for the dw gene or from a heterozygous femals mouse and a male homozygous dwarf mouse treated with thyroxine. The dwarf mice, weighing 9.1~ 0.L~g, were allocated at random to treatment groups of six animals and then distributed among several small cages each containing one representative of each treatment group. Hormones were ~0 RSB/NDC/1st August~ 198 34~

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injected subcutaneously in the back in 0.1rnl, For the periocis inciicated. Weights were measured at the onset, during and sometimes aFter treatment. Weight gains in short-term experiments or the cumulative welght gains over several days oF
study were e~pressed as relative values (%) related to initial whole body weights or as net weights (9). Tail lengths were measured by the method of Hughes and Tanner (1970).
Over a three week period, control mice, treated with phosphate-buffered saline (PBS), increased their relatiYe weight gains by about 15% (Figure 1). Mice treated with 10~9 hGH gained 22% over starting weight over the same period. However, hGH-EBO1 complexes raised the cumulative weight gain to 34%, which corresponds to an additional 12% increment over that achieved from treatment with hGH only.

Raising the hGH dose in the complex from 10~9 to 160,~9, increased the weight gains to 44% of the initial body weight. It is apparent (Figure 1) that the differences between treated groups and controls progressively increased over the21 day test period. Whilst hGH-MAB complexes produced a significant increment within 48h, the difference between the group treated with hGH only and the PBS-injected control group was not apparent until day 7.

Example B: GROWTH POTENTIATION BY EBO1 Fab' FRAGMENTS
In order to assess whether the bivalency of EBO1 antibody was a pre-requisite for growth potentiation, complexes of EBO1-Fab' -hGH were examined for their effects on 35So42 uptake in dwarf mice.

Dwarf mice within a relatively narrow weigh~ range (7 -109) were randomized by use of tables of random numbers (Fisher and Yates, 1957) and iniected with a dose of 35So42 related to body weight (0.5 Ci/g body weight) 24h after the final hormone injection (Herbai, 1g70). Mice were killed 20h later when rib cages wereremoved, placed in boiling water for 20 min, soaked overnight in saturated sodium sulphate and washed in tap water for 2 h and distilled water for 1 h. The bony RSB/NDC/1st August, 1984 portion of ribs together with about 1 mm o-f adjacent rib cartilage (costochondral junction) was then cut away leaving tne costal cartilages attached to the sternum.
All adhering soft tissue was removed and the five longest costal cartilages which articulated directly with the sternum were detached whole from each side of the rib cage and combined for each animal. Each pool of ten costal cartilages was then dried at room temperature overniyht, weighed and processed for the measurement of 55O 42 . The uptake of 35So42 by costal cartilage was expressed as 10 disintegrations per minute per mg of cartilage.

The levels of sulphate uptake by cartilage potentiated by EBO1 or EBOl-Fab' were not significantly different when comparing the antibody and fragments at the same ABT50 dose (Table 1). However, the growth observed in the presence of hGH
15 alone was signi ficantly less than that observed for Fab' -hGH.lncreasing the valency of the Fab' fragment by including a "second" either monoclonal anti-light chain antibody (TC187) or polyclonal anti-mouse Ig antibody did not significantly alter the 35so42~ uptake. Furthermore, Fab' fragments did not competitively inhibit the potentiating effect of EBOl antibody. Preparation of complexes of EBO1 with equimolar quantities of hGH and hCS also did not decrease the degree of potentiation. Such complexes would comprise mainly the species hGH-EBO1-hCS which would be expected to have decreased activity if bivalency was necessary, since hCS has only 10% somatotropic activity of hGH. Indeed, the EBO1-hCS complex resulted in significantly lower growth activity than the 25 corresponding complex with hGH.

~0 RSB/NDC/1st August, 1984 TABLE 1.
THE EFFECT OF ANTIBODY Fab FRAC,MENT-hGH COMPLEX ON_SULPHATE UPTAKE
A CTIVITY
_ 35so42~

uptake HORMONE ~9) ANTIBC ~u ANTI MOUSE Ig dpm/mg 8D
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hGH(160) . _ 1640 + 160 hGH(160) Ig(200) _ 4280 ~ 300 hGH(160) Ig(20) _ 2500 ~ 400 hGH(160) Fab(20) _ 2700 ~ 120*
hGH(160) Fab(20) TC1a7(12) 2300 1100 hGH(160) Fab(20) R-Poly(5) . 2000 ~ 100 hGH(160) Fab(20)+Ig(200) _ 4880 ~ 190 hCS(160) Ig(200) . 1200 170 hGH(80)~hCS(80) 19(200) ~ 3820 ~ 250 hGH(80hhCS(80) Ig(200) R-Poly(5) 4250 ~ 500 PBS _ _ 600 ~ 75 _ .

Dwarf mice (n = 6) were injected twiee with hormone plus antibody (0 and 24h) followed by 35So42 (48h) and harvested at 72h.
TC187 = Rat monoclonal anti-mouse L-chain; R-Poly = rabbit polyclonal anti-mause Ig.
*p<.D01 compared with hGH alone.

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Exampie C: POTENTIATION OF GROWTH IN JUVENILE BALB/c MICE

Since the stimulation of growth in dwarf mice is exercised over a background of very slow activity, it was of interest to ascertain if the antibody mediated potentiation effect could be demonstrated in normally, i.e. rapidly, growing juvenile mice. Three weel< old BALB/c mice weighing between 7 - 109 were randomized and injected as described above for dwarf mice. Weights were taken 3 1û times/week in chronic experiments. Group 1 received PBS twice weekly throughout; Group 2 had 160~ug hGH three times over 1 week and Group 3 the same thrice weekly for 4 weeks; Groups 4 and 5 had complexed 160~9 hGH/200~-g EB01 thrice weekly for 1 or 4 weeks resp0ctively; and Group 6 had daily injections ofthe said complex for 4 weeks~ Groups 5 and 6 grew by 31% and 37% more than PBS
injected controls (Fig. 2). A significant weight gain effect of hGH-EBO1 complexes was apparent as early as 48 hours after administration (Table 2)~
Animals receiving 10, 40 or 160~ug of hGH in the presence of EBOl demonstrated asignificant weight gain increment when compared with hGH only.

RSB/NDC/1st August, 1984 T A B L E 2.
SHORT-TERM WEIGHT GAINS IN JUVENILE BALB/c MICE INJECTED WITH

VARIOUS DOS~S OF hGH COMPLE)<ED WITH EBO1 ANTIBODY
_ hGH- Relative _ _ 10 ~9 ComplexWeight Gainp (significance) (160/200~9)~O + SD _ _ _ 13.0 + 11.0 ~ 16.0 + 12.0 <0.050 + 27.0 + 6.0 _ __.
_ 16.0+ 11.0 ~0 _ <0.050 + 29.0 + 8.0 _ _ _ _ 13.0 + 13.0 160 _ <0.025 ~ ~9.0_ 11.0 Three week old mice (Wto=B~0 + 1.0) were injected at time 0 and 24h.Weight gains, expressed as % of body weight, were determined at 48h.

Example D: POTENTIATION OF THE LACTOGENIC ACTIVITY OF hGH
Human growth hormone produces significant lactogenic activity as measured by its effects on pigeon crop-sac or mammary tissue in vivo and by its ability to displace 1251-hPRL from binding to mammary gland receptors. The pigeon crop-sac bioassay procedure measures the lactogenic activity of hormones and is analogous35 to other mammotropic assays involving either mammary gland or corpus luteum of rats or mice. Hormones, complexes or control solutions were administere~ (0.lml)from coded vials intradermally adjacent to each hemicrop, there being five birds in each group. The injection protocol was either one administration on day 1 only (2X104 ABT50) or three over 3fi hours (each of 2X103 A8T50) or combined with two RSB/NDC/1st August, 1984 '34~l further injections on day 2. Birds were killed on day 3 and the wet weight of the crop-sac mucosa of 2.5 cm diameter was determined (Nicoll, 1967). By administering the complex or free hormone intradermally, adjacent to each of thetwo individual hemicrops, the potentiation effect has been examined under conditions which excluded systemic hormone distribution. The weight of the crop-sac mucosa following the injection of 100 ~ug of hGH in three doses was about 100mg, whereas the control (PE35 or antibody only treated) mucosa weighed 10 -13mg (Figure 3).

Treatment with 10~ug of hormone alone produced a mucosa of 48mg, but in the presence nf EBO1 or EBO2 the mucosal weight increased to 108mg and 80mg respectively. As in dwarf mice, NA71 was without potentiating activity whereas QA68 significantly depressed the mucosal secretion. Furthermore, EBO1 potentiated the lactogenic effects proportionally by the same extent whether thehormone was administered in one or three doses. However, EBO1 failed to potentiate the lactogenic effect of saturating doses of hGH. Since EBO1 binds equally to hCS, we also examined the potentiation of the lactogenic activity of this hormone. The results show that antibody complexes with 10~ug hCS had doubled the weight of the crop-sac mucosa in comparison with controls receiving bhe hormone alone.

Example E: POTENTIATION OF GROWTH IN MARMOSETS

The potentiation effect in either murine growth or pigeon crop-sac responses wasdependent on the administration of exogenous hG5 I since none of the antibodies described here cross-reacted with the rodent growth hormone. However, we discovered that EaO1 antibody did bind to marrnoset growth hormone when tested 3D by immunoblot assay. This observation enabled the assessment of Ei301 potentiation of marmoset growth in the absence of any exogenously administered hormone (Figure 4). Sixteen animals, randomised on a weight basis, were divided into four groups receiving 0.4mg hGH only, hGH (0.4mg) ~ EBO1 (2mg), E801 (2mg) only or P8S three times per week. Two sister animals had to be removed from the experiment after 1 week due to continuous weight loss. Animals treated for 44 RSB/NDC/1st August, 1984 ~I Z~38~34~ ~69o days with PBS increasecl their weight by 689, whereas the group receiving EBQ1 only demonstrated a mean weight increass of about 1039. Groups receiving hormone only or complex had weight gains intermediate to those observed with EBO1 only and the control group. The relative weiyht gain (% over initial body weight) of animals receiving antibody was 89% whilst the control group increasedtheir body weights by only 61%. Despite continuous administration of heterologous antibody to the marmosets for 6 weeks, side effects, possibly of anaphylactic origin, have so far not been observed.

Example F- fURTHER PIGEON CROP SAC ASSAY
__ _ _ The pigeon crop sac assay of Example D was repeated with l~ug of highly purified (prep. L) or 3 ~9 of QBO1-MAB affinity purified (prep. R) hPRL alone or in complex with a constant dose, 2500 ABT50 of monoclonal antibodies. The preparation of QlBO1 antibodies was analogous to that described in Example 1 aoove ar-d has been published (Ivanyi & Davies, 1981).

The results, given in Table 3, show strong potentiation for antibody QBO1, for both preparations of hPRL. The figures represent mean values for six crops per group _ one standard deviation.

TABLE 3:
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_ibody Weiyht in Crop Sac (mg) Prepn. L Prepn. R
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QBO1 68.0 ~ 30.5 75.0 + 18.0 _ .. _ .
Control A (hPRL alone) 22.5 + 14.5 22.0 + 11.0 35 Contrrl B (uninjected) ~ 19.5 ~ 5.0 RSB/NDC/1st August, 1984 .. . . . . . . . . . . . .

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Example G: Dwarf Mouse Body Growth and Composition Dwarf (dw/dw) mice were oividecl into three groups of eighteen and fed on 100%, 75% or 50/~ of the usual ad iib consumption for 10 days. Within each diet, six mice were treated with saline, six with hGI I (40 milliunits) and six with hGH/EB01 (104ABT50 of antibody) complex. The mice were assessed for overall growth 504 uptake into intercostal cartilage, sulphate injected at day nine),, at content, weight change and length of tail. The results are presented in Tables 4 to 7.

It is apparent that the use of a hormone/antibody complex can compensate for a reduced diet and can also reduce the proportion o-f fat in an animal by preferentially causing growth of muscle, the latter advantage being even more marked with a reduced diet.

TABLE 4: Sulphate Uptake into Intercostal Cartilage ~ _ _ ~ DIET _ ¦
100% 75% 50%
_ -Treatment . _ Saline 2393 ~ 695~ 540+

hGH 5569 ~ 1 4492~ 2958 hGH+ 7956 l 5531~ 4840 30 The results are expressed as disintegrations per minute per milligram of tlssue;
mean ~ S.E.

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TA LE 5: Fat Content:

_ 100%_ DIF T 50%
Saline 2533 1502 108Z
~83 ~304 ~89 _ . -- _ _.
hGH 1857 1173 631 ~130 ~127 +152 _ _ _ --_ _ hGH+ 1164 733 347 EB01 ~99 +51 ~91 Figures are amounts of fat in milligrams TABLE 6: Weight Change as a % of Initial Weight (initial weight = 100%) _ _ DIET
-- 100% - 75% 50%
.. __ ~, Treatment Saline 104.67+ 94.5~ 87.83+
0.95 1.63 1.14 hGH 119.67+ 1 107.5~. 91.67+
1.94 1.61 0.8 hGH~ ~ 127.17~ 111.67+ 95.2+ .
. EB01 2.12 0.88 2.52 Figure expressed as mean + S.E.

RSE~/NDC/lst August, 1984 TABLE 7: Longitudinal Growth of Tail (Increase in mm).
_ ., -_ DIET _ _ 100% 75% 50%
_ Treatment _ Sa!ine 0.96 0.5+ 0.71+
+ 0.24 0.19 0.36 hGH 3.3}+ 3.29+ 2.04+
0.~ 0.47 0.28 _ _ .
hGH+ 5.21~ 4.96+ 4.05+
EB01 0.39 0.31 0.16 _ Figure expressed as mean + S.E.

Example H: Growth in Normal Mice The experiment of Example C was repeated, with the assay being for 35S04 uptake into intercostal cartilage rather than weight gain. Mice aged 4 weeks (weight 109~) 6 weeks (149) and 9 weeks (199) were used and injected sub-cutaneously Y/ith 0.1ml of either saline, hGH (100,ug) or hGH-EB01 complexes (100,~g/104A~T50) two days before administration of 504. Cartilage was removed 24h later. The results are given in Table 8 and shown significantly increased growth wlth the complexes (p<0.0005 for the oldest mice).

TABLE 8:
.
_ _ MICE AGE
Treatment 1 4 weeks 6 weeks 1 9 weeks Saline 1686+2B2 1176+85 505+32 hGH 1 1621+158 1330+83 605+54 hGH/EB01 2.431+307 173a+191 1071+85 _ _ (Jnits: counts/min/mg.cartilage RS8/NDC/1st August, 1984 < ~ 33~1 Example I: Use of Polyclonal Antibodies of Reduced Specificity A 7K (7000 Daltons) fragment was cleaved from the C-terminal of hGH with subtilisin followed by chromatography under denaturing conditions (Aston & Ivanyi 1983). Two mice were each injected with 5~,ug of the fragment emulsified with Freund's complete adjuvant but without a carrier and 21 days later received a further 50~ug without any adjuvant. Serum was taken 10 days after the second challenge, complexed with hGH (10~9) and injected into dwarf mice. The subsequent growth of mice was assayed by measuring 35504 uptake as above. The results (Table 9) show that the polyclonal antiserum potentiated gro~th.
15 TABLE 9:

Treatment 35504 uptake (c.p.m.~mg.cartllage) Saline 500+50 ZO _ _ hGH (10`~u~) 1365 i 146 hGH plus 4425~703 antiserum I
. __ hGH plus 3272+471 antiserum II _ REFERENCES

Aston, R and Ivanyi, J. The EMBO Journal 2 493-497 (1983).
3~ Fisher, R.A. and F. Yates (Ed.) "Statistical tables for biological, agricultural and medical research" Oliver Boyd (1957).
Herbai, G. Acta. Physiol. Scandinavica 8G (1970) 470-491.
Hughes, P.C.R. and Tanner, J.M. J.Anatomy 106 (1970~ 349-370.
Ivanyi J. In: Monoclonal Hybridoma Antibodies: Techniques and Applications 35 (Edited by Hussell, J.G.R.)CRC Press, Cleveland, Ohio (1982)pp 59-79.
Ivanyi J. Mol. Immunol. _ (1982) 1611-1618.
Ivanyi. J. and P. Davis. Mol.Immunnl. 17 (1980) 287-290 Ivanyi, J. and P. Davis. Protides of the Biolagical Fluids 29th Colloquium 1981.Ed. H. Peeters Pergamon Press, Oxforcl and New York, 1982. 855-860.

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Linde, S., B.Hausen, A. Lernmark. Analyt Biochem 107 (1980)165-176 Nicoll, C.S. ~:ndocrinology ~0 641-655 (1967).

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Example J: Potentiation of Ovine Growth Hormone Activity Monoclonal antiboclies to Dvine (ie. sheep) growth hormone toGH) were prepared in an analogous way to the method of Example 1, and are available from the Department of Experimental Immunobiology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, UK. Dwarf mice, which responcl to ` oGH, were divided into groups of six and treated with 50~ug of the hormone, either alone or complexed with one of four such antibodies, on two consecutive days prior to injection with 35504. Intercostal cartilage was removed 24 hours later, dissolved in formic acid and the radioactivity counted. The results are given in. 10 Table 1:

Table 10 Treatment MA3 titre Uptake of 35S04 (mean + S.E.; cpm/mg tissue) oGH alone - 1411 ~261 oGH ~ lDll H9 lX10-4 5871 ~1339 OGH + 4B62 D9 3.2X10 4 4323-~671 oGH ~ 2B11 4.2X10 3408 ~ 642 oGH~3B11 5.6X10 3434 +719 Saline - 557 ~79 j 20 Example K: Potentiation of Growth in Sheep , Groups of two sheep (mean weight 17kg) were treated with differing doses of anti-oGH antibody 2Bll (see Example J) or, as a control, mouse globulin, on two consecutive days before intraperitoneal injection of 35504 (1461uCi/kg).
Quadruplicate samples of intercostal cartilage were removed from each site 24 hours later and analysed as above. The results are given in Table 11:
' Table ll ~I , Treatment Incorporation of 35S04 (mean ~ S.D.; cpm/10mg cartilage) l ,.
106ABT50~B11 3094 ~ 630 0.2X106ABT502B11 5168 +24 ~04X106ABT5o2B11 2373 ~183 Control 2084 ~771 R~l~nr~l7~h Allnll~t~ l984 . .
.

-' 24 _ These results, which are highly significant by variance analyais, shuw that formulations in accordance with the invention can potentiate the action of endogenous GH in an economically important species.

Example L: otentiation of Growth in Sheep Example K was repeated additionally using a different monoclonal antibody, lD11 H9, and groups of five sheep, mean weight 24 kg. The results are given in Table 12:

Table 12 Treatment 504 Uptake (mean + S.E., cpm/lOmg) 8.~X105ABT502B11 3315 + 560 2X10 ABT501D11 H9 2818 + 343 control immunoglobulin 1908 + 299 Significantly ~p<0.05) increased growth is seen with the MAB-treated groups.

Example M: Potentiation of Thyroid Stimulating Hormone (TSH) Activity TSH is a glycoprotein produced by the pituitary gland and activating the thyroidgland in vertebrates. A deficiency of TSH causes involution of the thyroid glandand flattening of the epithelium. in humans, such a deficiency can be responsible ~ for cretinism, simple goitre and the panoply of abnormal conditions known; 20 collectively as myxedema. It may be treated with iodine compounds or thyroid gland extracts. Dwarf mice are hypopituitary and the thyroid gland is involuted.Treatment with TSH raises the serum T4 levels and causes some histological repair of the thyroid.

A monoclonal antibody (GC73) to TSH was prepared analogously ta those of Example l above and is available from the same address as in Example J. GC73 is specific for the ~-chain of TSH. Dwarf mice were randomly divided into gr~ups offive and treated accordingly to the regimes of Table 13 on five consecutive daysbeFore analysis oF the serum for T4 level by radioimmunoassay, and microscopic ¦ histological inspection of thyroid tissue, fixed in 10% formalin in saline solution i and then embedded in wax or plastic. The T4 data are gi~en in Table 13; the results were confirmed by microscopic examination.
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Table 13 Treatment T4 level (mean + S.E.) 0.1 international units TSH 62.8 ~ 3.7 0.05 units TSH 36.4 + 6.3 0.1 units TSH ~ 1 116.2 + 5.3 0.05 units TSH ~
72.2 + 11.75 ... Saline less than 5 10 ABT50GC73 only less than 5 .. ..

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Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A formulation comprising an actual or functional fragment of a hormone and an immunological carrier or adjuvant, in combination with a pharma-ceutically acceptable carrier.

2. A formulation according to claim 1, wherein said fragment is an actual fragment of a hormone.

3. A formulation according to claim 1, wherein said fragment is a functional fragment of a hormone.

4. A formulation according to claim 1, 2 or 3, wherein said fragment is a fragment of growth hor-mone.

5. A formulation according to claim 1, 2 or 3, wherein said fragment is a fragment of prolactin.

6. A formulation according to claim 1, 2 or 3, wherein said fragment is a fragment of chorionic somatomammotropin.

7. A formulation for increasing the growth or rate of growth of a mammal, the formulation compris-ing an actual or functional fragment of a growth hormone and an immunological carrier or adjuvant, in combination with a pharmaceutically acceptable carrier.

8. A formulation for increasing lactation in a mammal, the formulation comprising an actual or functional fragment of growth hormone, prolactin or chorionic somatomammotropin and an immunological carrier or adjuvant, in combination with a pharma-ceutically acceptable carrier.

9. Use of at least one pre-selected actual or functional fragment of an appropriate hormone for increasing a hormone-regulated response of a verte-brate having a sufficient endogenous amount of the hormone for normal functioning of the tissues regu-lated by the hormone.

10. The use according to claim 9, in which the response is regulated by growth hormone.

11. Use of at least one pre-selected actual or functional fragment of an appropriate hormone for the manufacture of a preparation for increasing a hor-mone-regulated response of a vertebrate having a sufficient endogenous amount of the hormone for normal functioning of the tissues regulated by the hormone.

12. A hormone-regulated response-increasing pharmaceutical formulation for increasing a hormone-regulated response of a vertebrate having a suf-ficient endogenous amount of the hormone for normal functioning of the tissues regulated by the hormone comprising at least one actual or functional frag-ment of an appropriate hormone, in association with a pharmaceutically acceptable carrier.

13. A formulation according to claim 12, wherein said fragment comprises an actual fragment of a hormone.

14. A formulation according to claim 12, wherein said fragment comprises a functional fragment of a hormone.

15. A formulation according to claim 12, wherein said fragment is a fragment of prolactin.

16. A formulation according to claim 12, wherein said fragment is a fragment of chorionic somato-mammotropin.