Monoclonal antibodies which preferentially bind to 22 K human growth hormone rather than its 20 K variant

We have established 13 hybridoma cell lines which secrete mouse IgG1 monoclonal antibodies (McAbs) to human growth hormone (hGH). Binding affinity and binding specificity of McAbs were analyzed by competitive radioimmunoassay. Among these McAbs, CL. B1 showed a high affinity of 9.8 x 10(8) l/mol, and all McAbs so far tested showed very weak cross-reactivity or none at all with human prolactin (hPRL) and human chorionic somatomammotropin (hCS; human placental lactogen). Analysis of binding sites of McAbs using hGH variant and fragments in both ELISA and RIA demonstrated that McAbs could be classified into two groups. All the McAbs obtained in this study bound to plasmin-digested fragment S2 (hGH 1-134 and 141-191) and fragment alpha 3 (hGH 1-134 and 147-191). However, five (such as 1D2) out of 13 McAbs bound to fragment F1 (hGH 1-134) and others (such as CL. B1) did not. The McAb CL. B1 in the latter group showed low affinity with 20 K hGH (residue 32-46 deleted in native 22 K hGH) in contrast to high affinity with hGH (22 K). This suggests that the former McAbs recognize an epitope located at the N-terminal two-third part of hGH. In contrast, the McAbs of the latter group are likely to recognize three-dimensional structure of native 22 K hGH.     

Isolation of dimeric forms of human pituitary growth hormone

A procedure is described which for the first time allows the isolation of noncovalently-linked dimeric human pituitary growth hormone. Isomers of this dimeric species were prepared as were also, for the first time, isomers of covalently-linked dimers. Chromatography on DEAE-Sepharose CL-6B revealed the existence of noncovalently-linked dimers composed of monomers of 22K hGH, 20K hGH and 20K1 hGH (the latter is a new form of 20K hGH with a scission in the peptide chain) and covalent dimers containing 22K hGH and 24K hGH (the latter a 22K hGH with a scission). The different dimers all occurred as charge isomers and subsequent HPLC on an anion exchanger followed by zone electrophoresis in agarose suspension made possible the isolation of four noncovalently-linked isomers: one form of (20K-20K)hGH, two forms of (20K-22K)hGH and one form of (22K-22K)hGH; and of three covalently-linked isomers: one form of (22K-22K)hGH and two forms of (22K-24K)hGH.     

Isolation of four isomers of the 20,000 dalton variant of human pituitary growth hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20,000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncovalently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-linked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Comparison of the acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects

The acute metabolic effects of 20,000-dalton human growth hormone (hGH20K) in man have not previously been tested. We compared changes in concentrations of free fatty acids (FFA), glucose, and insulin in nine growth hormone deficient children following injection of 22,000-dalton intact human growth hormone (hGH22K) and the smaller variant, hGH20K. There was a significant decline (37%) in the mean FFA concentration from baseline to 1/2 hour post-injection and from baseline to 1 hour post-injection (36%) in the children given hGH22K, but no such decline was seen after injection of hGH20K. No significant differences in mean insulin or glucose concentrations were noted between the two treatment groups, and glucose and insulin concentrations did not acutely change after injection of either hormone. The results of this study indicate that hGH20K has a diminished activity for suppression of FFA as compared to hGH22K. This suggests that GH residues 32-46, missing in hGH20K, constitute all or part of the region of hGH22K producing this response, or that the different primary structures of the two hormones result in tertiary structural differences and altered biological activity.     

The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities

The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action.     

The effect of growth hormone on the proliferation of human Th cell clones

The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and Th0 cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through prolactin receptor but through hGH receptors. The application of growth hormone binding protein (GHBP) inhibited the cell growth of Th1 clones. In Th2 and Th0 cells GHBP inhibited the hGH-stimulated cell proliferation. However, GHBP alone did not affect the proliferation of Th2 and Th0 cells. hGH was detected in the supernatant of Th1 clones in the presence of mite antigen but it was not detected in Th2 clones. hGH was detected in one out of 4 batches of Th0 clones. These data indicated that hGH was secreted from Thl clones, and that Th0 clones possessed characteristics of both Th2 and Th0 clones.     

Lipolytic and antilipolytic effects of human growth hormone, its 20-kilodalton variant, a reduced and carboxymethylated derivative, and human placental lactogen on chicken adipose tissue in vitro

The lipolytic and antilipolytic effects of human growth hormone (22K-hGH), its 20-kilodalton variant (20K-hGH), a reduced and S-carboxymethylated derivative (RCM-hGH), and human placental lactogen were examined using chicken adipose tissue explants in vitro. Lipolysis, as determined by glycerol release, was stimulated by 22K-hGH (biosynthetic and pituitary derived), 20K-hGH (pituitary derived), and RCM-hGH (modified biosynthetic). These growth hormone preparations also exhibited similar antilipolytic activity (i.e., transient inhibition of glucagon-induced lipolysis). However, unlike human growth hormone, human placental lactogen neither stimulated lipolysis nor inhibited glucagon-stimulated lipolysis. Some augmentation of glucagon-stimulated lipolysis was observed in the presence of human placental lactogen. These results indicate that the disulfide bridges (Cys53----Cys165; Cys182----Cys189) and amino acid residues 32-46 of hGH are not required for lipolytic or antilipolytic activities of human growth hormone on chicken adipose tissue.     

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Receptor binding properties and insulin-like effects of human growth hormone and its 20 kDa-variant in rat adipocytes

The natural 20 kDa-variant of human growth hormone (hGH) binds with high affinity to IM-9 human lymphocyte receptors, in agreement with its potency in biological assays for growth promoting and lactogenic activities. In contrast, 20 kDa-hGH has only 3% of the potency of 22 kDa-hGH in binding to the receptors of normal and hypophysectomized rat adipocytes. In agreement with the binding potency, 20 kDa-hGH is only 3% as potent as 22 kDa-hGH in stimulating lipogenesis in normal rat adipocytes preincubated for a few hours in hGH-free medium. The 20 kDa-hGH is also much weaker than 22 kDa-hGH in stimulating lipogenesis in adipocytes from hypophysectomized rats. These data strongly support the concept that the rat adipocyte receptor, which mediates the insulin-like effects of growth hormone, is different from the receptor found on human IM-9 lymphocytes. Preincubation of rat adipocytes with hGH induces a refractoriness to subsequent activation of lipogenesis by hGH but does not abolish the response to insulin, while preincubation with insulin slightly potentiates the hGH response and does not change the insulin response. Additivity studies and a detailed comparison of the lipogenic effects of insulin and hGH suggest that hGH shares only a subset of the metabolic pathways activated by insulin.     

The 20-kD human growth hormone reduces body fat by increasing lipolysis and decreasing lipoprotein lipase activity

AIM: The aim of this study was to estimate the lipolytic activity of the human growth hormone variant, 20-kD human growth hormone (20K-hGH). METHODS: Obese KV-A(y) mice were given daily subcutaneous injections of 20K-hGH (0.25, 0.5, 1.0 mg/kg), 22K-hGH (0.25 mg/kg) or saline as a control for 2 weeks. Body composition (fat, water and protein), lipolysis and lipoprotein lipase (LPL) activity were measured 24 h after the final injection. RESULTS: Both growth hormone isoforms significantly reduced relative fat pad and whole body lipids. In addition, 20K-hGH produced an inhibition of LPL activity in adipose tissue and stimulated lipolysis in adipocytes. CONCLUSION: These data strongly suggest that inhibition of LPL activity in adipose tissue and stimulation of lipolysis in adipocytes by 20K-hGH treatment reduce adipose tissue mass, resulting in body fat reduction.     

Immunoreactive growth hormone production by human lymphocyte cell lines

1. Two human lymphocyte cell lines, a T-cell line and a B-cell line, were shown to produce and secrete immunoreactive growth hormone (irGH). The irGH molecules secreted by the two cell lines appeared to be de novo synthesized and their molecular size was similar to that of pituitary GH as well as irGH secreted by peripheral blood lymphocytes. 2. Affinity-purified irGH molecules had human growth hormone (hGH)-like mitogenic activity on Nb2 cells. These findings indicate that the irGH molecules produced by H9 and IM9 were similar to hGH in structure. 3. However, the irGH messages could not be amplified by polymerase chain reaction (PCR) primers which had been demonstrated to be able to amplify reverse-transcribed hGH messenger RNA successfully, suggesting that the lymphocyte-derived irGH and pituitary hGH are not exactly identical molecules. 4. We conclude that the H9 and IM9 cells produce a growth hormone-related molecule whose structure is different from that in the anterior pituitary.     

Biosynthetic human growth hormone: effects on growth of Snell dwarf mice

Bacterially synthesized human growth hormone (bhGH) administered to Snell dwarf mice during 4 weeks, induced an increase in body length and weight to a comparable degree as obtained with pituitary-derived human growth hormone (hGH). At a dose of 150 mU/day both bhGH and hGH induced a significant stimulation over saline-treated controls, of the weight of the submandibular salivary glands, the m. quadriceps femoris and gastrocnemius, the heart, liver, kidneys, thymus and spleen. The weight of the brain and the thickness of the skinfold were not influenced by either of the preparations used. When organ weights were expressed as a function of body weight, the contribution of the kidneys to body weight was significantly higher with hGH than with bhGH. The other organs studied did not show differences. As a biochemical parameter of cartilage growth, the sulfate incorporation into costal and epiphyseal cartilage in vitro was measured, and it was found to be stimulated by both hormones after short-term treatment. Thus bacterially synthesized hGH behaves identically to pituitary-derived hGH with respect to body length, sulfate incorporation into costal and epiphyseal cartilage, body weight and organ growth of Snell dwarf mice, with one exception: increase of weight of the kidneys, as a function of body weight, was more pronounced after treatment with hGH than with bhGH.     

Binding specificity of monoclonal antibodies towards fragments of human growth hormone produced by plasmin digestion

To help define the immunological epitopes on human growth hormone (hGH), interaction of fragments of the hormone with 7 monoclonal antibodies (McAbs) was studied. Plasmin-digested hGH, containing two peptides (hGH1-134 and hGH141-191) joined by a disulphide bond, bound to each McAb with affinity similar to that of intact hGH. The purified C-terminal fragment, hGH141-191, showed low affinity for each McAb. The N-terminal fragment, hGH1-134, bound with quite high affinity to 2 McAbs (EB1 and EB3) but not to the other 5. We conclude that residues 1-134 of hGH contain the epitope to which McAbs EB1 and EB3 bind.     

Comparative study of biosynthetic human growth hormone immunogenicity in growth hormone deficient children

The immunogenicities of six recombinant human growth hormone (rhGH) preparations, from KABI (A rhGH191 and B rhGH192), Eli Lilly (C), Nordisk (D), Sanofi (E) and Serono (F), used to treat 260 GH-deficient children, have been compared using a common specific and sensitive procedure for antibody determination. For this purpose we developed two immunoassays: a competitive liquid radioimmunoassay using 125I-rhGH, and an immunometric solid enzymoimmunoassay in which the rhGHs were immobilized. Blood samples were collected from the GH-deficient children before treatment and after 3, 6, 9, 12, 18 and 24 months of therapy. Human GH antibodies were detected in children treated with 3 of the 6 rhGH preparations. Seven percent of the patients treated with hormone A, 14% with hormone B and 22% with hormone C formed antibodies against the respective rhGH. Differences in capacity and affinity of the hGH antibodies were observed between these anti-GH-positive groups. They could be divided into 2 groups according to their immunopotency. One group (7, 14 and 6% of the patients treated with hormones A, B and C, respectively) developed anti-hGH antibodies with very low binding capacities (30-100 fmol/ml). The other group (16% of the patients treated with hormone C) developed IgG-type antibodies to hGH with higher binding capacities (200-1,200 fmol/ml) and a measurable binding affinity (Ka = 10(8) M-1). These hGH antibodies partially inhibited the binding of labeled GH to its specific liver membrane receptor. However, because of their low titer, they did not inhibit growth in the treated children.(ABSTRACT TRUNCATED AT 250 WORDS)