Inhibition of binding of ovine placental lactogen to growth hormone and prolactin receptors by monoclonal antibodies

From a single cell fusion, five stable hybridomas secreting antiovine placental lactogen (oPL) antibodies were obtained. Three of these secrete immunoglobulin (Ig)G subclass, and the other two secrete IgM class antibodies. Ascites fluids were raised in mice for each clone and were used as the antibody component for the development of solid phase RIA. Three solid-phase RIAs were successfully established using individual IgG subclass monoclonal antibodies, but the IgM class antibodies were ineffective. In all three individual solid-phase RIAs, the binding of [125I]iodo-oPL to the immobilized antibody was inhibited by unlabeled oPL, but not by ovine pituitary PRL (oPRL), ovine GH (oGH), or ovine pituitary extract. Two of the IgG subclass antibodies were able to inhibit the binding of [125I] iodo-oPL to PRL receptors(s) and to GH receptor(s) in rabbit mammary gland and liver, respectively. One of these two IgG subclass antibodies was more effective at inhibiting the binding of oPL to PRL receptor(s) in rabbit mammary gland, whereas the other one is more effective in inhibiting the binding of oPL to GH receptor(s) in rabbit liver. These antibodies, however, could only weakly inhibit the binding of [125I]iodo-oPRL to rabbit mammary gland and were ineffective in inhibiting the binding of [125I]iodo-oGH to rabbit liver. The addition of monoclonal antibodies in both radioreceptor assay (RRA) for PRL (RRA-PRL) and for GH (RRA-GH) did not affect the parallelism of the displacement curve of oPL standard. Our results suggest that oPL might contain two distinct binding sequence(s): one responsible for the binding of oPL to PRL receptor(s) and the other responsible for the binding of oPL to GH receptor(s). These two binding sequences might overlap or be located adjacent to one another. The interaction of monoclonal antibodies with these binding sequences of oPL may block the binding of oPL with PRL and GH receptor(s). Alternatively, our studies suggest that the monoclonal antibodies do not bind to hormone receptor(s)-binding sequence(s) in oPL, but the interaction between oPL and monoclonal antibody might alter the conformational structure of the oPL which will consequently lead to a lower binding of oPL to PRL and GH receptor(s).     

The presence of lactogen but not growth hormone binding sites in the isolated rat hepatocyte.

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]alpha-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 degrees C, binding reached a steady-state after 2-5 h and had a half-life of dissociation of 2-3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26-74+/-3-73 fmol/10(6) cells and a binding affinity of 1-24 X 10(9)+/-0-17 X 10(9) (S.E.M.) 1/mol (n=10). There was a significant sex difference in binding (female greater than male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.     

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.     

Ovine placental lactogen and human growth hormone bind to different regions of the same receptors.

Anti-human growth hormone (hGH) polyclonal and monoclonal antibodies (MAb) failed to recognize ovine placental lactogen (oPL), indicating that the antigenic topographies of both hormones are different. Binding assays showed that oPL completely inhibited hGH binding to lactogenic receptors from Nb2-cells and to somatogenic receptors from rabbit or sheep liver; in contrast, oPL only bound to a subpopulation of rat liver receptors. Zinc ion increased hGH and oPL binding to Nb2-cell receptors and slightly inhibited both hormones' recognition by somatogenic receptors. However, ZnCl(2) increased hGH binding to rat liver microsomes but prevented that of oPL. Furthermore, MAb R7B4, recognizing lactogenic as well as somatogenic receptors, entirely blocked hGH binding to the various receptor systems but not affected oPL binding. Therefore, results presented in this paper suggest that oPL and hGH bind to different regions of the same receptors.     

Binding specificity of monoclonal antibodies towards the size variants of human growth hormone

The expression of antigenic determinants on size variants of human growth hormone (hGH) has been investigated using monoclonal antibodies of distinct combining-site specificity. Monomeric, dimeric, trimeric and polymeric (very high-molecular-weight) forms of hGH were separated by gel filtration on Sephadex G-100, and their antigenic potency was determined quantitatively by competition with 125I-labelled hGH for binding to each of four different monoclonal antibodies. With three of these antibodies the potencies of monomeric, dimeric and trimeric hGH were not significantly different, and the polymeric material was 11-13% as potent as the monomer. However, using one antibody (NA 71) the antigenic potencies of dimeric and trimeric hGH were lower (30-50%) than that of the monomer, and the polymeric material was only about 5% as potent as the monomer. These results suggest that the determinant with which antibody NA 71 interacts is close to the site of interaction between hGH monomers and apparently partially 'masked' in dimers, trimers and polymeric hGH.     

Pituitary regulation of human growth hormone binding sites in rat liver membranes.

We have studied the binding of 125I-human growth hormone (hGH) to crude 100,000 X g membrane preparations from rat liver, and have studied factors which might regulate the capacity and affinity of hGH binding sites. Membrane preparations have livers of pregnant rats bound between 8% and 18% of the 125I-hGH initially added, and 70%-80% of that bound was displaced by 1 mug of unlabeled hGH. Humans prolactin (hPrl) displaced 125 I-hGH in a manner parallel to hGH itself but with about one-third the potency. Ovine, porcine, and rat Prl, and rat and bovine GH were much less effective. Scatchard analysis of specific hGH binding by a variety of different rat liver membrane preparations revealed a single order of binding site in each case with a binding affinity of 0.93-1.62 X 10(-9) M-1. Membranes from pregnant rats had twice the binding capacity of membranes from nonpregnant female rats, and about six times the capacity of sites present in preparations from normal adult male rats and hypophysectomized (Hx) male or female rats. Female or male rats with extremely high circulating GH an Prl levels, due to the presence of transplantable GH/Prl secreting pituitary tumors showed a significantly greater binding capacity than did the pregnant rats. Estradiol (E2) treatment (25 mug/day for 10-12 days) of normal male rats led to an increase in specific hGH binding. Treatment of hypophysectomized male rats with bovine GH (100 or 500 mug/day) +/- E2 (25 mug/day) for 5-10 days stimulated both body weight gain and the incorporation of sulfate by cartilage from the treated rats, but no significant increase was observed in the characteristics of 125I-hGH binding. These results indicate that high levels of E2, GH, and/or Prl play an important role in the regulation of hGH binding sites in rat liver membranes. The restoration of binding sites in liver from hypophysectomized rats, however, apparently requires additional factors which are as yet unidentified. The role of the hGH binding sites in the physiologic actions of GH also remains to be determined.     

Placental lactogen and growth hormone receptors in human fetal tissues: relationship to fetal plasma human placental lactogen concentrations and fetal growth

The specific binding of human placental lactogen (hPL) and human GH (hGH) to particulate cell membranes from human fetal liver and skeletal muscle at 12-19 weeks gestation was examined. Fetal liver and muscle specifically bound [125I]hPL. This binding was inhibited by increasing concentrations of unlabeled hPL (half-maximal concentrations, 2.2 and 3.4 nmol/L, respectively). Scatchard analysis of the hepatic membrane binding revealed curvilinear plots with higher (Kd, 2.2 nmol/L) and lower (Kd, 24 nmol/L) affinity sites, while binding to muscle involved a single receptor class of Kd 5.6 nmol/L. The binding capacities for the two hepatic sites correlated positively with fetal body weight. [125I]hGH specifically bound to liver, but not muscle, with higher (Kd, 1.6 nmol/L) and lower (Kd, 8.6 nmol/L) affinity sites. [125I]PRL bound to hepatic membranes, but was preferentially displaced by hPL or hGH. Between 4 and 500 micrograms/L (mean, 82 micrograms/L, 3.8 nmol/L) hPL were present in fetal plasma. The findings identify distinct hPL receptors in human fetal liver and skeletal muscle and a hepatic hGH receptor in midgestation.     

Induction of differentiation of human myeloid leukemias: surface changes probed with monoclonal antibodies

The surface changes occurring in three acute myeloid leukemia cell lines (HL60, ML3, and KG1) induced to differentiate by a variety of agents (dimethylsulfoxide, retinoic acid, 12-O-tetradecanoylphorbol-13- acetate, and factors present in lymphocyte conditioned medium) were probed using monoclonal antibodies that are differentiation stage- and lineage-specific. In all cases, the differentiated phenotype was defective and varied with the inducing agent and the cell line used. HL60 proved to be the most sensitive to the effect of the inducers. Retinoic acid was better than DMSO, and TPA was better than the medium factors in the ability to induce granulocytic and monocytic differentiation, respectively, in HL60 cells. These findings indicate that the differentiation block in acute myeloid leukemias is heterogeneous and that each cell line has different phenotypic characteristics that are responsible for the extent of differentiation obtained with a given inducer. These results also suggest that the extent of the differentiation response in vitro may be improved by the use of more suitable inducers for each specific leukemic line.     

Effects of thyroid hormones on liver binding sites for human growth hormone, as studied in the rat.

Several weeks after thyroidectomy (T), female rats stopped growing, and their pituitary GH content had decreased to less than 2--3% of the values found for age-matched controls (C). The liver membranes of such animals were explored with human GH (hGH). It was found that in the severely hypothyroid T rat, the number, but not the affinity, of the lactogenic binding sites was markedly reduced. Treatment of these rats for 3 weeks with 1.75 micrograms or T4 or 0.5 micrograms T3/100 g body weight/day restored growth, increased pituitary GH content and restored the number of liver lactogenic binding sites were practically to normal. As regards the lactogenic binding sites, similar results were obtained when the severely hypothyroid rats were treated with a much lower T4 dose (0.2 microgram/100 g/day): this dose was clearly growth promoting, and restored to normal both the low circulating GH levels and the pituitary PRL content of the severely hypothyroid rat. The changes in plasma PRL were not clear. The lactogenic binding sites on liver membranes from rats which were both thyroidectomized and hypophysectomized were decreased in number. Treatment with 0.5 microgram T3/100 g/day for 30 days (but not for 12 days) resulted in an increase in the number of lactogenic binding sites, though it did not affect growth or the undetectable plasma GH levels. The effect on the lactogenic binding sites was less marked than in T rats with an intact pituitary. It would appear that minute amounts of thyroid hormones are needed for maintenance of liver lactogenic binding sites; it is possible that this not only occurs through mechanism(s) which involve the pituitary, but also through others which do not. The possible role of these receptors in growth processes is not yet clearly understood.     

The antigenic epitopes of human grown hormone as mapped by monoclonal antibodies

The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.     

Five-year follow-up of growth hormone antibodies in growth hormone deficient children treated with recombinant human growth hormone

OBJECTIVE The aim was to investigate the long-term evolution of circulating growth hormone antibodies (GH-AB) during and after treatment with methionyl-recombinant human growth hormone (met-rhGH). DESIGN AND PATIENTS The investigation was performed on serum samples of 46 growth hormone deficient children, treated for at least 12 months with met-rhGH. Twenty patients had never been treated with hGH (previously untreated patients, Group I). Twenty-six subjects were previously treated with pituitary extracted hGH (treated patients, Group II). MEASUREMENTS Serum levels of GH-AB were measured by radioimmunoassay using charcoal precipitation of free ligand. RESULTS Fifteen patients (75%) of Group I and three patients (12%) of Group II developed GH-AB. In 15 GH-AB positive patients the antibodies became detectable during the first year of treatment with met-rhGH. In three patients, however, the GH-AB appeared during the second year. Once present, the GH-AB remained detectable throughout the period of treatment with met-rhGH. In six patients in whom treatment with met-rhGH was stopped, GH-AB levels decreased rapidly. In nine patients in whom treatment with met-rhGH was changed to rhGH, the levels of GH-AB decreased and ultimately became undetectable. In two patients GH-AB remained present during administration of rhGH. No effect of GH-AB on the growth-promoting effect of met-rhGH could be documented, either during the first or during the second year of treatment. CONCLUSIONS This study confirms the high immunogenicity of met-rhGH, especially in patients not treated earlier with hGH. Once present, the GH-AB remain detectable throughout the period of treatment with met-rhGH. After stopping met-rhGH treatment or changing to rhGH the GH-AB disappear rapidly in most patients. No effect of GH-AB on the growth-promoting effect of rhGH could be documented.     

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.     

Localization of cofactor binding sites with monoclonal anti-idiotype antibodies: phenylalanine hydroxylase.

A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.     

The antigen binding sites of various hCG monoclonal antibodies show homology to different domains of LH receptor

The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Therefore, it can be hypothesized that the paratope of antibodies may exhibit homology with distinct domains of the receptor. By locating the hormone epitopes and determining the structure of the paratopes, it should be possible to identify the contact points between the ligand and the receptor. This hypothesis has been tested using hCG monoclonal antibodies (MAbs) recognizing different epitopes and having different effects on hormone binding and response. The β subunit and heterodimer specific antibodies inhibited both hormone binding and response, while the α subunit specific antibodies inhibited response without affecting binding. The single chain fragment variables (ScFvs) produced from these antibodies also retained the properties of the parent antibodies. The amino acid sequences of the ScFvs exhibited homology to different regions of the receptor; the β subunit specific antibody being homologous to the concave surface of the leucine rich repeats (LRR) of the receptor, particularly the concave surface of the LRRs, while the heterodimer specific antibody showed homology to the hinge region. The α subunit specific antibody showed homology to the transmembrane domain of the receptor. The exact locations of the epitopes of the monoclonal antibodies in the hormone molecule have also been identified. The data presented here also support the model of glycoprotein hormone–receptor interaction in which the hormone binds to the extracellular domain through the β subunit and then the α subunit is brought in contact with the transmembrane domain leading to signal transduction.     

Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue

The presence of specific receptors for synthetic growth hormone secretagogues (GHSs) has been investigated in non-tumoral and neoplastic human thyroid tissue using a radio-iodinated peptidyl GHS ((125)I-labelled Tyr-Ala-hexarelin) as ligand. Specific binding sites for Tyr-Ala-hexarelin were detected in membranes from non-tumoral and follicular-derived neoplastic thyroid tissue, but not in thyroid tumours (medullary carcinomas) of parafollicular (C cell) origin. The binding activity was greatest in well differentiated neoplasms (papillary and follicular carcinomas), followed by poorly differentiated carcinomas, non-tumoral thyroid parenchyma, follicular adenomas and anaplastic carcinomas. Both peptidyl (Tyr-Ala-hexarelin, hexarelin, growth hormone releasing peptide (GHRP6) and non-peptidyl (MK 0677) GHSs completely displaced the radioligand from binding sites of non-tumoral thyroid gland, but MK 0677 was significantly less potent. The IC(50) values were (1. 9+/-0.3)x10(-8) mol/l for Tyr-Ala-hexarelin, (2.1+/-0.2)x10(-8) mol/l for hexarelin, (2.4+/- 0.3)x10(-8) mol/l for GHRP6 and only (1. 5+/-0.4)x 10(-7) mol/l for MK 0677. Similar IC(50) values were found in neoplastic thyroid tissue. Scatchard analysis of the binding revealed a finite number of binding sites in non-tumoral (B(max): 1232+/-32 fmol/mg protein, n=3) and neoplastic (papillary carcinomas) thyroid tissue (B(max): 2483+/-380 fmol/mg protein, n=5), with dissociation constants (K(d)) of (3.8+/-0.3)x10(-9) and (4. 4+/-0.6)x 10(-9) mol/l, respectively. On the basis of this evidence, we investigated the effects of some GHS on the proliferation of three different human follicular thyroid carcinoma cell lines (NPA, WRO and ARO) in which the presence of specific GHS receptors was also demonstrated. Tyr-Ala-hexarelin, GHRP6 and MK 0677 were able to inhibit serum-stimulated [(3)H]thymidine incorporation in NPA cells at concentrations close to their binding affinity. These substances also caused a significant inhibition of cell proliferation, which was evident at the earliest time of treatment (24 h) in all the cell lines, and at the latest time (96 h) in NPA cells only. In conclusion, this paper confirms the existence of specific binding sites for GHS in normal thyroid tissue and demonstrates, for the first time, that these binding sites are present in papillary and follicular carcinomas, low in anaplastic carcinomas and absent in medullary carcinomas of the thyroid. This work also provides evidence of a growth-inhibitory effect of GHS on cell lines derived from follicular thyroid cancers.