Immuno-enhancement and -inhibition of GH-releasing factor by site-directed anti peptide antibodies in vivo and in vitro

Abstract

It is now well established that specific antibodies and binding proteins can potentiate rather than inhibit hormone activity. In order to investigate this phenomenon further, the current study was undertaken using a hormone with a characterised structure, in terms of receptor binding, and for which activity has already been manipulated in specific ways (prolongation of half-life, increased receptor affinity) using synthetic hormone analogues. GH-releasing factor (GRF) is a 40 or 44 residue peptide and is, together with somatostatin, responsible for the regulation of GH secretion. The effects of site-directed anti peptide antibodies were determined on the activity of GRF in vivo and in vitro as GH release. The peptide regions of GRF were: 1-14 (part of putative receptor-binding region) and 31-44 and 35-44 (sites thought to be distant from the receptor-binding region). Five sheep were administered GRF (1 microgram/kg), anti peptide immunoglobulin (Ig; a calculated tenfold excess binding to GRF dose), or GRF together with anti peptide Ig (preincubated for 1 h). GRF induced a significant increase in plasma GH concentration over the next 240 min, this was abolished when GRF was administered with anti 1-14 Ig (P < 0.05) and augmented (P < 0.05) when GRF was administered with anti 35-44 Ig; anti 31-44 had no effect on GRF activity. Anti 35-44 Ig alone induced an increase in GH secretion which was equivalent to that for GRF alone, implying that the antibody had interacted and potentiated with endogenous GRF. The Ig effects on exogenous GRF activity were confirmed for GH release in vitro using primary cultures of sheep pituitary cells, except that anti 31-44 Ig also augmented GH release (P < 0.05) when co-administered with GRF. (ABSTRACT TRUNCATED AT 250 WORDS)

Actions of an IGF-I-enhancing antibody on IGF-I pharmacokinetics and tissue distribution: increased IGF-I bioavailability

“…Indeed, there is some evidence that enhancing monoclonal antibodies might increase total hormone occupancy of receptor without changing receptor number (Massart et al 1993). This mechanism would require antibody to gain access to tissue compartments; indirect evidence from sheep passively immunised against GH-releasing factor suggests that this is possible in normal animals (Pell & James 1995). In addition, the latter study demonstrated that antibody-mediated enhancement of hormone activity can occur in vitro which has clear parallels with IGFBP action.…”

“…In addition to their classical role of protein neutralisation and facilitation of antigen clearance, specific antibodies can potentiate peptide hormone activity. This phenomenon was first demonstrated for insulin and epi¬ dermal growth factor but now extends to growth hormone (GH), thyroid-stimulating hormone, follicle-stimulating hormone, tumour necrosis factor and GH-releasing factor (reviewed in Pell & Aston 1995) and occurs in vivo and in vitro (Pell & James 1995). Antibodies have a similar molecular mass to that for the IGF-I-IGFBP-3-ALS ternary complex (150 kDa) and may therefore provide a model for further elucidation of the function of the IGF ternary complex and its importance for IGF-I stability and bioavailability.…”

Antibodies as molecular mimics of biomolecules: roles in understanding physiological functions and mechanisms

“…In terms of peptide hormone action, it is likely that the active site, i.e., the receptor-binding site on the peptide hormone, will be sterically hindered in the presence of specific antibodies, preventing receptor interaction and activation. Even though the sites of binding for all enhancing antisera have not been characterized, all findings to date suggest that enhancing antisera bind to peptide hormones at sites distant from the receptor-binding domain (13,49,86,87). This is shown for IGF-I in Fig.…”

“…It is known that the receptorbinding region of GRF resides in the NH 2 -terminal 29 residues (22). Specific site-directed anti-peptide antibodies were therefore generated against either the NH 2 -or COOH-terminal domains of GRF (87); their effects (with and without preincubation with GRF) were examined in vitro (GH release from cultured primary pituitary cells) and in vivo (circulating GH concentrations). Intriguingly, antibodies directed against the NH 2 -terminal region of GRF inhibited GRF activity, whereas antibodies directed against the COOH-terminal region enhanced GRF activity.…”

Immunological manipulation of growth

“…Subsequent studies have shown also that the activities of many peptide hormones can be potentiated: insulin (Gray et al 1985), human (h) growth hormone (GH;Holder et al 1985), thyroid-stimulating hormone (Holder et al 1987), bovine (b) GH (Pell et al 1989), follicle-stimulating hormone (Glencross et al 1993), tumour necrosis factor (Rathjen et al 1992), insulin-like growth factor-I (IGF-1; Stewart et al 1993) and GH-releasing factor (GRF; Pell & James, 1995). Further, immuno-enhancement has been demonstrated both in vivo and in vitro (Pell & James, 1995). It is theoretically possible, therefore, to design strategies to increase overall signalling of key pathways, either by neutralizing the activity of inhibitory factors or by potentiating the activity of stimulatory factors.…”