Monoclonal antibody enhanced stimulation of human growth hormone action in vitro using ovine costal cartilage growth plate chondrocytes.

Investigations into the mechanism underlying antibody-mediated enhancement of growth hormone action have been hampered by the lack of an in vitro assay system. In this work, we have used isolated ovine costal cartilage growth plate chondrocytes to demonstrate, for the first time, that monoclonal antibody EB1 can enhance the proliferative actions of human growth hormone on this cell type. Chondrocytes were cultured for 14 days prior to exposure to GH+/-monoclonal antibody EB1 for a 4-day treatment period. Human growth hormone alone promoted a significant dose-dependent increase in chondrocyte proliferation; maximal stimulation was achieved at about 3.3-10 ng/ml growth hormone, and at higher doses of growth hormone, the response declined. Monoclonal antibody EB1 was shown to enhance the proliferative activity of 10 ng/ml human growth hormone in a significant dose dependent fashion. In conclusion, our results demonstrate that an antibody capable of enhancing GH activity in vivo also has the capacity to potentiate GH activity in vitro. This system may provide an important tool for investigations into the mechanism of GH action, and how this is modified by GH enhancing MAbs.

Cross-linked growth hormone dimers have enhanced biological activity.

In this study we have investigated the effect on the bioactivity of pituitary-derived human growth hormone (hGH) and recombinant bovine (b) GH after the addition of various concentrations of the water soluble cross-linking agent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC; 6.25-100 mg/ml). The biological activity of resulting cross-linked reactions were determined by its ability to promote incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo. Administration of EDC-treated hGH solutions resulted in a significant enhancement of hormone activity in vivo compared with non-cross-linked samples. A similar significant enhancement of bGH activity in vivo was also observed when solutions containing recombinant bGH were cross-linked using EDC. For both hGH and bGH the degree of enhancement appears to be dose-dependent for the concentration of EDC (6.25-100 mg/ml for hGH; 6.25-50 mg/ml for bGH) present in the cross-linking reactions. SDS-PAGE analysis of EDC cross-linked solutions containing hGH and bGH spiked with 125I-hGH and 125I-bGH respectively revealed that dimeric GH was the primary cross-linked component. Increasing the concentration of EDC in cross-linking reactions resulted in increased formation of dimeric hGH and bGH. There was a significant correlation between the amount of GH dimer present and the increase in biological activity, suggesting that GH dimers were responsible for the enhanced biological activity. This was confirmed by the enhanced biological activity of a purified preparation of EDC cross-linked dimeric hGH. In conclusion, covalently cross-linked GH dimers reported here have enhanced bioactivity in vivo. However, since naturally occurring GH dimers are known to have reduced biological activity, this work suggests that the structure of EDC cross-linked GH dimers differs fundamentally from that of native dimeric hGH.

Recognition of porcine growth hormone by a panel of monoclonal antibodies.

A panel of murine monoclonal antibodies (MAbs) against porcine growth hormone (pGH) has been raised from BALB/c mice. MAbs were characterized for binding to growth hormones (GH), prolactins (PRL), and placental lactogen (PL) from different species and to the N-terminal peptides of GH. From their patterns of cross-reactivity MAbs were assigned into nine specificity groups. The sharing of pGH epitopes among hormones of different species was related to the sequence similarity to pGH, i.e., overlap was greatest for equine, ruminant, and rodent GHs and least for human GH, ovine, and porcine PRLs, and human PL. Partial epitope mapping was carried out by relating hormone cross-reactivity patterns with amino acid sequences. Two epitopes were localized to interhelical loops, around valine-73 and glycine-130, respectively. Direct mapping with synthetic peptides localized other epitopes (Groups 7, 8, and 9) to the N-terminal region of the GH molecule. Selected MAbs were studied for the enhancement of the somatogenic activity of pGH in the dwarf mouse bioassay, measuring weight gain and sulphate incorporation into costal cartilage. Only those antibodies with specificities for GHs and not PRL or PL showed significant enhancement in this assay.

Enhancement of FSH bioactivity in vivo using site-specific antisera.

The ability of site-specific antipeptide antisera to enhance the biological activity of ovine FSH (oFSH) in vivo was investigated using hypopituitary Snell dwarf mice. These animals were shown to respond to increasing doses of oFSH (3.3-90 micrograms/day), administered in two daily injections over a 5-day treatment period, in a highly significant dose-dependent fashion. The responses measured were increases in uterine weight, ovarian weight and the index of keratinisation in vaginal smears. The dose-dependent response to oFSH confirmed the suitability of this animal model for these investigations and suggested the suboptimal dose of oFSH (20 micrograms/day) for use in enhancement studies. Five peptides derived from the beta subunit of bovine FSH (bFSH) (A, residues 33-47; B, 40-51; C, 69-80; D, 83-94; E, 27-39) were used to generate polyclonal antipeptide antisera. Of these peptides, only A and B produced an antiserum (raised in sheep) capable of recognising 125I-bFSH in a liquid phase RIA. Antisera prepared against peptide A or peptide B were found to significantly enhance the biological activity of 20 micrograms oFSH/day over a 5-day treatment period. The response to antipeptide antisera alone did not differ significantly from that observed in PBS-injected control animals, neither did the response to FSH alone differ from that observed in animals treated with FSH plus preimmune serum. Thus the enhanced responses are dependent upon the presence of FSH plus antipeptide antiserum. Peptides A and B are located in a region thought to be involved in receptor recognition, this may have implications for the mechanism underlying this phenomenon and/or the structure/function relationships of FSH. That FSH-enhancing antisera can be generated by immunisation of animals with peptides A and B suggests that it may be possible to develop these peptides as vaccines capable of increasing reproductive performance, such as ovulation rate. The high degree of sequence homology between ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species.

Growth hormone stimulates the secretion of insulin-like growth factor binding protein-2 (IGFBP-2) by monolayer cultures of sheep costal growth plate chondrocytes.

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these--the 32 kDa BP--was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP-2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1-100 ng/ml). In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.

Location of an epitope defined by an enhancing monoclonal antibody to growth hormone: some structural details and biological implications.

We have previously shown that a murine monoclonal antibody (MAb OA15) prepared against ovine GH (oGH) can enhance the somatogenic activity of bovine GH (bGH), as determined by increased incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo We have now established that MAb OA15 can also enhance the biological activity of oGH and porcine GH (pGH) in vivo. Using multiple pin peptide synthesis techniques a set of overlapping immobilized octamers representing the entire bGH sequence were synthesized and tested for their ability to bind MAb OA15 using an enzyme-linked immunosorbent assay. The pattern of binding showed that OA15 defined a functionally continuous epitope comprising residues 91-102. This region includes the C-terminal end of helix 2 plus a portion of the adjacent loop linking helices 2 and 3. Polyclonal antisera to a synthetic peptide representing this epitope mimicked the ability of MAb OA15 to enhance oGH, bGH, and pGH. Window size analysis showed that the heptapeptide 94-100 (SRVFTNS) represents the minimum unit to retain full recognition of MAb OA15. The fact that pGH, bGH, and oGH have identical sequences in this region also explains the ability of OA15 to both cross-react with and enhance the biological activity of each of these GHs. Replacement net analysis (where each residue in the heptamer is substituted with each of the 19 naturally occurring L-amino acids) demonstrated that residues R95 and T98 are critical for antibody binding and also indicated that the substitution of V96 with I, as found in rat GH, would permit the observed binding of OA15 to this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody enhancement of FSH-induced uterine growth in snell dwarf mice.

The enhancing effect of bovine FSH monoclonal antibody (bFSH-MAb) on the gonadotrophic activity of FSH was investigated in dwarf mice using a uterine weight bioassay. Increasing doses of bovine FSH (NIH-FSH-B1; 3.3, 10 or 30 micrograms/day) were administered for 5 days to dwarf mice (groups of five) with or without administration of a bFSH-MAb preparation (USDA-bFSH-MC28; 100 micrograms protein/day) which at a dilution of 1:15,000 bound 50% of 125I-labelled bFSH (USDA-bFSH-BP3). The bFSH, at the doses given, gave no increases in uterine weight; when, however, these doses were given with bFSH-MAb, significant (three- to four fold) increases in uterine weight resulted.

Growth hormone control of tissue protein metabolism in dwarf mice: enhancement by a monoclonal antibody.

Monoclonal antibody (MAb) to GH has been shown to increase the anabolic response induced by the hormone in individual tissues of dwarf mice. Dwarf mice were treated with GH at a low and a high dose (2.5 and 50 mU/day respectively), with and without complexing to an MAb. Treatment was for 7 and 14 days, at which times protein synthesis rates in skeletal muscle, liver and heart were determined from incorporation of labelled phenylalanine following injection of a flooding dose. The MAb potentiated the actions of GH and produced increases in the rates of protein synthesis in each of the tissues to a significantly greater extent than did GH alone. The increase in protein synthesis rate induced by MAb appears to be mechanistically distinct from that observed by increasing the dose of GH. In skeletal muscle and liver there was a dose-response to the GH alone in terms of the RNA concentration, i.e. the capacity for protein synthesis, whereas in each tissue examined the MAb caused very little further response in the RNA concentration. The MAb-induced enhancement of protein synthesis rate was almost entirely due to an increase in the RNA activity, i.e. the efficiency of the synthesizing system. Complexing GH to a particular MAb, or to antisera of restricted epitope specificity, has previously been shown to enhance the in-vivo effects of GH on whole body protein content; the mechanism for this enhancement has not been adequately determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic structure of bovine growth hormone: location of a growth enhancing region.

Site-directed antisera generated by peptide immunization have been used to study the antigenicity of bovine growth hormone (bGH). Prediction of sequential antigenic sites has been performed using secondary structure information derived from the 'Protean' prediction routine. The structures predicted by this programme agree closely with the corresponding structure of GH recently derived from crystallographic studies. We have previously shown that the binding of monoclonal antibodies of particular epitope specificity to human or bovine GH results in significant enhancement of hormonal activity in vivo; however, the sites recognized by these antibodies were not known. Here we identify a sequence region, corresponding to a loop structure joining helices 3 and 4, which, is associated with the growth enhancement phenomenon. Antisera raised to either of two overlapping peptides (residues 120-140 and 134-154) significantly increase the biological activity of GH in vivo. Antisera directed to other regions on the GH molecule failed to demonstrate this property. Coincidentally, the sites recognized by the growth-enhancing anti-peptide antisera overlap with the site on GH which is highly susceptible to proteolytic cleavage; such cleavage has been shown in some cases to result in hormone enhancement.

A monoclonal antibody to human insulin-like growth factor-I: characterization, use in radioimmunoassay and effect on the biological activities of the growth factor.

A monoclonal antibody (BPL-M23) to insulin-like growth factor-I (IGF-I) was obtained following immunization of BALB/c mice with human IGF-I conjugated to ovalbumin. The affinity constant of BPL-M23 for IGF-I was 10.5 litres/nmol and the cross-reactivities of IGF-II, multiplication-stimulating activity III-2 and insulin were 0.8, 0.03 and less than 0.0001% respectively. Porcine, bovine, ovine and rabbit sera, but not rat or mouse sera, showed substantial reactivity with the antibody. Comparison of radioimmunoassay analyses of 54 human serum samples from normal subjects and acromegalic and GH-deficient patients using BPL-M23 and a polyclonal rabbit antiserum (R557A) to human IGF-I showed a high correlation, indicating the usefulness of the monoclonal antibody in radioimmunoassay. Monoclonal antibody BPL-M23 was capable of abolishing the sulphation, mitogenic and insulin-like activities of IGF-I in in-vitro bioassays, suggesting that these activities may rely upon the same receptor-binding site which is near to the antibody-binding site.

Potentiation of growth hormone activity in sheep using monoclonal antibodies.

Monoclonal antibody OA11 was raised against ovine GH; its effects on GH activity were examined in a target species relevant for animal production in vivo. The monoclonal antibody was found to enhance the galactopoietic response to exogenous GH in adult lactating ewes and also to potentiate the diabetogenic activity of both exogenous and endogenous GH in ewe lambs. Thus it was shown that GH activity may be manipulated above its usual dose-response range in normal, intact animals of commercial importance via immunological means.

The anabolic actions of growth hormone and thyroxine on protein metabolism in Snell dwarf and normal mice.

The individual effects of GH and thyroxine (T4) on protein metabolism were determined in dwarf and normal mice in vivo. The hormone deficiencies of dwarf mice (low serum concentrations of GH and T4) resulted in decreased protein synthesis rates in skeletal muscle and liver, but no difference in synthesis rates in heart. The efficiency of synthesis (g protein/g RNA per day; KRNA) was lower in all three tissues in dwarf compared with normal mice, but effects on RNA concentration were not consistent; there was no change in muscle, a decrease in liver and an increase in heart. Treatment of dwarf mice for 9 days with either human GH or T4 caused increases in body weight and length. Protein synthesis rates were increased in muscle, liver and heart by either hormone, though much more so with T4 than GH. In muscle and liver both GH and T4 treatment resulted in an increased RNA concentration, but T4 treatment also increased KRNA. In heart, both GH and T4 increased KRNA with no change in RNA concentration. GH caused no significant changes in protein degradation rates so that growth rates were increased. T4 increased degradation rates so that there was no increased net growth in muscle or liver; in heart, T4 did induce increased growth despite the large increase in degradation rate. Tibial length was increased by both hormones; GH treatment of dwarf mice also increased cartilage sulphate incorporation on day 9, but T4 treatment did not, suggesting that bone growth is transient with T4 treatment. Normal mice showed no changes in growth or tissue protein metabolism in response to GH, but following T4 treatment there was increased protein turnover due to higher tissue RNA concentrations, although only heart growth was increased. Thus normal mice showed almost no net response to GH or T4, but dwarf mice showed a large response to both hormones. The response was different, however, in that GH caused concomitant increases in growth rates whereas T4 altered body tissue proportions.

Monoclonal antibody enhancement of the effects of human growth hormone on growth and body composition in mice.

Dwarf mice were treated for 10 days with phosphate-buffered saline (PBS), human growth hormone (hGH) or hGH with monoclonal antibody EB1 (hGH/MAB-EB1); for each treatment there were three groups which received 50, 75 or 100% of the amount of food eaten when available ad libitum. The PBS control groups lost more or gained less weight than equivalent groups receiving hGH alone, and mice given hGH/MAB-EB1 showed a greater weight gain than those in comparable groups receiving hGH alone. When weight gain or loss was expressed as g/g food eaten, groups treated with hGH gained more or lost less than the PBS groups. Similarly, weight gain/g food was significantly greater in hGH/MAB-EB1 animals than in the comparable groups given hGH alone. A similar pattern of response was observed for increases in tail length and uptake of 35SO42- into costal cartilage in vivo. For mice given hGH alone, fat content was decreased compared with that in the equivalent group given PBS, and mice treated with hGH/MAB-EB1 had less fat than the equivalent group given hGH alone. Administration of hGH alone caused a concomitant increase in protein content and body weight such that, compared with mice given PBS, there was no significant increase in protein as a proportion of body weight. However, hGH/MAB-EB1 caused an increase in whole body protein which was significantly greater than that for the equivalent group given hGH alone, when expressed as per cent body weight. Monoclonal antibody EB1 has been shown to enhance the actions of hGH on growth and body composition in Snell dwarf mice and to increase food conversion efficiency.

Monoclonal antibodies can enhance the biological activity of thyrotropin.

In this work we demonstrate that monoclonal antibodies (MABs) to TSH can enhance the biological actions of TSH in vivo. Hypopituitary Snell dwarf mice were injected with TSH (25, 50, or 100 mU/day) alone or complexed with MAB-GC73 once per day for 5 days; control animals received PBS. Radioactive sulfate (35SO4(2-)) was also injected on the fifth day and animals were killed 20 h later. Thyroids were removed for histology, blood taken for T4 estimations by RIA, and 35SO4(2-) uptake into costal cartilage in vivo was measured. In control mice thyroid histology revealed small follicles comprised of small flattened epithelial cells with a high nuclear-cytoplasmic ratio; colloid was dark with little vacuolation. In animals treated with TSH alone there was moderate evidence of activation in most of these features. However, a marked response was noted in animals treated with TSH plus MAB-GC73; characteristically, there was little interfollicular tissue and the follicles, which were large and comprised of cuboidal cells, contained pale, finely vacuolated cytoplasm. Both TSH alone and TSH complexed with MAB-GC73 promoted a significant dose-dependent increase in serum T4 levels. The two higher doses of TSH plus MAB-GC73 promoted a significantly greater increase in serum levels of T4 than that in groups receiving the same dose of TSH alone. Uptake of 35SO4(2-) into costal cartilage showed a significant correlation with serum T4 levels. In similar experiments significant increases in salivary gland epidermal growth factor content of male dwarf mice were observed. This work demonstrated that MAB enhancement of hormone action is not restricted to human GH, suggesting a more general phenomenon.

Enhancement of bovine growth hormone activity in vivo by monoclonal antibodies.

Monoclonal antibodies (MAbs) prepared against ovine growth hormone (oGH) and recognizing several distinct or related specificities on bovine growth hormone (bGH), have been shown to strikingly enhance the growth promoting properties of the hormone in vivo as determined by 35SO2-4 incorporation in cartilage. The phenomenon is dependent on both hormone and antibody dose and is saturable in the presence of excess antibody. Competition binding analysis between pairs of antibodies has shown that they define distinct antigenic regions on bGH of which one is represented by four MAbs. The most potent growth enhancement was associated with a group of three MAbs (OA11, OA12 and OA13) binding to topographically closely related sites. Another MAb (OA14) with a specificity similar to OA11 and OA13 as defined by competition assay failed to enhance bGH activity. Two antibodies binding to a further two sites (OA15 and OA16) demonstrated modest growth enhancement activity when in complex with bGH, whereas the binding of another antibody (OA17) did not significantly affect hormonal activity. Univalent antibody fragments (Fab) derived from MAb OA11 were equi-potent to the bivalent form of the antibody in the enhancement of bGH activity. Determination of the effects of the different MAbs on the binding of 125I-bGH to liver microsome receptors revealed substantial increase in specific binding (3.5-fold) and is associated with some growth enhancing MAbs. However, not all growth enhancing MAbs increased receptor binding and in the case of one, OA16, clear cut inhibition of receptor binding was observed. Tentative conclusions have been drawn on the possible underlying mechanisms of the MAb mediated enhancement phenomenon.

Potentiation of the somatogenic and lactogenic activity of human growth hormone with monoclonal antibodies.

Monoclonal antibodies of certain epitope specificity have been shown to produce a marked dose-dependent enhancement of the somatogenic and lactogenic activity of human GH (hGH). Two antibodies (EB1 and EB2), binding to distinct antigenic determinants and expressed on both hGH and human chorionic somato-mammotrophin (hCS), significantly enhanced the hGH-stimulated uptake of 35S-labelled sulphate into cartilage. Similarly, these antibodies enhanced the lactogenic activity of both hGH and hCS in the pigeon crop sac test. Two hGH specific monoclonal antibodies (QA68 and NA71), defining a further two epitopes, exhibited only modest enhancing or inhibitory activity in these assays, whereas the binding of certain combinations of monoclonal antibodies resulted in either reversal of enhancement or inhibition of hormone activity. Univalent antibody fragments derived from EB1 were as enhancing as the intact antibody indicating that bivalency dependent mechanisms were not involved in the phenomenon. Enhancing monoclonal antibodies were relatively poor inhibitors of 125I-labelled hGH binding to liver microsomal receptors, which is in contrast with their previously described property of potent suppression of hGH interaction with lymphoid cell receptors. It is tentatively concluded that 'restriction' of hormone binding to particular hGH receptors, relevant to somatic growth or lactogenic activity, may play a role in the enhancement phenomenon of hGH in vivo.

Somatomedin C/insulin-like growth factor I: simplified purification procedure and biological activities of the purified growth factor.

Human somatomedin C has been purified from Cohn fraction IV paste by a simplified procedure using chromatofocusing, hydroxylapatite chromatography and reverse-phase high performance chromatography. The purified material has a specific activity by somatomedin C radioimmunoassay of 9160 units/mg (1 unit is defined as the amount of somatomedin present in 1 ml normal adult male human serum), representing a 650,000-fold purification, and possesses sulphation, mitogenic and insulin-like activities (specific activities of 3388 units/mg, 832 units insulin equivalents/mg and 1122 units/mg respectively). Somatomedin C is shown to be a potent stimulator of DNA synthesis (50% maximum stimulation at 150 fmol/ml) in isolated chondrocytes derived from costal cartilage, a major physiological target tissue.

Determination of the activity of clinical grade human growth hormone preparations by in vivo bioassay, in vitro cell proliferation assay and solid phase radioimmunoassay.

Preparations of clinical grade human growth hormone (hGH) have been examined for activity by an in vitro bioassay using NB2 lymphoma cells and by a monoclonal antibody (NA 71)-based radioimmunoassay. The activities observed have been compared with those obtained by the in vivo growth bioassay performed in mice of the Snell dwarf strain. The two in vitro assays correlated well with each other for specific preparations of hGH, although non-parallelism was observed between different preparations of the hormone. Some preparations of hGH were highly potent in the NA 71 immunoassay, but not in the NB2, cell-proliferation assay, suggesting the presence of antigenically active but biologically inactive hormone. Chromatographic studies on early hGH preparations revealed the presence of dimetric, trimetric and aggregated hormone as well as small quantities of prolactin. These were studied individually and shown to diverge in potency in the respective assays. It is concluded that the correlations between the in vivo bioassay in dwarf mice and the in vitro assays may be compromised by the varying potency of the constituent forms of the hormone and the non-linearity of the in vivo growth assay.

Monoclonal antibody-mediated enhancement of growth hormone activity in vivo.

This work demonstrates that complexing hGH with monoclonal antibody EBl (MAB-EBl) can produce a striking potentiation of the somatogenic actions of hGH in vivo in Snell dwarf mice. In short-term experiments significant increases in cartilage metabolism and body weight were noted; these responses were dose-dependent for both MAB-EBl and hGH concentration. Increased growth was also observed in long-term experiments. In marmosets where MAB-EBl cross-reacts with endogenous GH, MAB-EBl alone enhanced the actions of endogenous GH. A new perspective may be necessary to incorporate these results into the current concept of antibody action.