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Endocrinology Vol. 141, No. 2 741-751
Copyright © 2000 by The Endocrine Society

ARTICLES

Enhancement of Insulin-Like Growth Factor I Activity by Novel Antisera: Potential Structure/Function Interactions1

Jennifer M. Pell, Rod A. Hill2, Claire E. H. Stewart3, Claire R. Weston and Helen C. Flick-Smith

The Babraham Institute, Cambridge, United Kingdom CB2 4AT

Address all correspondence and requests for reprints to: Dr. J. M. Pell, The Babraham Institute, Cambridge, United Kingdom CB2 4AT. E-mail: jenny.pell{at}bbsrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor I (IGF-I) is essential for normal growth and development, regulating cell proliferation, differentiation, and survival. Little IGF-I exists in the free form; rather, it is bound to one of a family of six specific IGF-binding proteins (IGFBPs). Usually, IGFBPs have a high affinity for IGF-I and inhibit its activity. Intriguingly, some IGFBPs also potentiate IGF-I action; the precise mechanism of this is unclear, but it is thought to include modification of the IGFBP to lower its affinity for IGF-I. We have previously generated a novel antihuman (h) IGF-I antiserum that, instead of inhibiting IGF-I activity, enhances it in vivo. As the enhancing anti-IGF-I antiserum and potentiating IGFBPs share several properties with regard to IGF action, the antibody may provide a model for examining the actions of enhancing IGFBPs. In this study we demonstrate that the antiserum can also enhance IGF-I activity in vitro, assessed as cell number of a bovine fibroblast cell line, suggesting that its actions might not merely be confined to changing the kinetics of IGF-I clearance or degradation. Epitope scanning using overlapping octamer and hexamer peptides spanning the entire sequence of IGF-I indicates that the enhancing antiserum recognizes a specific linear region spanning the C-terminal region of the C domain and the proximal A domain (residues Ser33 to Cys47), and that this recognition is not present in nonenhancing antisera. Further, this region is located on the opposite surface of IGF-I from putative type 1 receptor-binding residues, allowing the possibility that the antiserum might be able to modulate IGF-I receptor binding. Antibodies raised against a synthetic peptide corresponding to Ser33 to Cys47 of IGF-I also potentiated IGF-I activity in vivo. As IGF-I may be beneficial in various clinical conditions associated with catabolism or cell repair, we suggest that this potentiating anti-IGF-I antiserum has favorable properties that could form a basis for therapeutic strategy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) is a pluripotent polypeptide growth factor that is structurally related to insulin; it is essential for normal growth and development (1, 2, 3), regulating cell proliferation, differentiation, and survival (4). Peptide hormone activity is usually determined by two main mechanisms: the peptide concentration and its subsequent interaction with the cognate receptor. However, an additional tier of regulation exists for the IGFs via their association with a family of six high affinity and specific binding proteins (IGFBPs) (5, 6); over 97% of the IGFs are complexed to the IGFBPs (7). It has been suggested that IGFBPs are critical for the appropriate actions of IGF-I; they prevent pharmacological actions of free IGF, act as a short term reservoir of peptide, and target IGF activity to its receptor (5). Intriguingly, some of the IGFBPs do not merely inhibit, but also potentiate, IGF activity. The precise mechanism of this potentiation is unclear, but is thought to involve modification of the binding protein, reducing its affinity for IGF-I to be equivalent to or lower than that of the type 1 IGF receptor (5); it is speculated that this allows the receptor to compete more effectively for IGF-I peptide. The presence of at least six high affinity IGFBPs allows the coexistence of both common and individual regulation of IGF action as well as a mechanism for increased specificity of IGFBP expression at the cellular level. Thus, the IGFBPs provide a complex and sophisticated network to regulate IGF-I bioavailability, modulating and targeting its activity in a more rapid and efficient manner than could be achieved via transcriptional regulation of IGF-I synthesis.

The type 1 IGF and the insulin receptors consist of two {alpha}- and ß-subunits linked via disulfide bonds to form transmembrane-spanning, ligand-stimulated, autophosphorylated {alpha}2ß2-heterotetrameric tyrosine kinases. Conventional dogma suggests that the type 1 IGF receptor is associated primarily with mitogenic and the insulin receptor with metabolic activities. However, the signal transduction pathways activated by both ligands are interchangeable and demonstrate overlap and potential redundancy. Moreover, although IGF-I and insulin bind to their reciprocal receptors with only modest affinity, insulin can, in fact, signal mitogenic and IGF-I metabolic responses via their cognate receptors (8). Studies with insulin analogs (9) and cell lines derived from knockout mice lacking a functional type 1 IGF or insulin receptor (10) have demonstrated biased signaling away from the expected response. A key issue, therefore, must be the determination of signaling specificity or choice of downstream of signaling pathway.

We have generated an antiserum against human (h) IGF-I that, instead of inhibiting IGF-I activity as would be expected, potentiates its anabolic actions in vivo; it has a calculated affinity for IGF-I that is approximately equivalent to that of enhancing IGFBPs (11). The anti-IGF-I antiserum changes the kinetics of serum IGF-I in vivo, enabling a bioavailable reservoir of peptide to exist that escapes proteolytic degradation (12). Preliminary epitope scanning studies have demonstrated that one predominant linear region within IGF-I may represent a major epitope recognized by the antiserum (13); thus, despite being polyclonal, the antiserum appears to be of restricted specificity for IGF-I. In related studies, modulation of peptide hormone activity has been achieved previously using specific site-directed antibodies. For example, antipeptide antibodies directed against the C-terminal of GH-releasing factor will potentiate its ability to stimulate GH release (14), and a specific monoclonal antibody to a loop region of GH enhances its stimulation of weight gain in GH-deficient mice (15). In each case, the antibody is thought to associate with a nonreceptor binding epitope of the ligand, allowing the possibility that antibody binding does not prevent hormone-receptor interaction.

In this study we examine further the phenomenon of antibody potentiation of peptide hormone activity with respect to IGF-I action and potential structure/function relationships. The objectives were to 1) extend our observations on the potentiating anti-IGF-I antiserum from in vivo to in vitro models, 2) examine the epitope binding specificities of potentiating and nonpotentiating anti-IGF-I antisera, and 3) investigate further the predominant linear epitope initially identified in IGF-I-potentiating antisera. The data are discussed in the context of IGF-I and IGFBP interactions and potential regulatory mechanisms in peptide hormone action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Human recombinant IGF-I was a gift from Ciba-Geigy (St. Aubin, Switzerland). A 15-mer peptide region of IGF-I (termed peptide 15; Ser33 to Cys47; see Results for peptide selection) was synthesized by the Microchemical Facility at the Babraham Institute using the F-moc polyamide mode of peptide synthesis on a Milligen/Biosearch 9500 peptide synthesizer (Hertford, UK).

Antibody production and partial purification of immunoglobulins (Igs)
Polyclonal antibodies were raised against recombinant human IGF-I or to peptide 15 in adult sheep or rabbits; both peptides were conjugated to human {alpha}-globulin using glutaraldehyde, exactly as described previously (11). Anti-IGF-I and antipeptide 15 Igs were purified from the resultant sheep antiserum and also from sheep nonimmune serum (NI Ig) using ammonium sulfate precipitation (11). Protein concentrations of the Ig preparations were assayed by the method of Bradford (16), using BSA as the standard; the total protein concentration of the Ig preparations was adjusted to equivalent concentrations (8 mg/ml) using PBS (0.01 M sodium phosphate in 0.15 M saline, pH 7.4). Antibody titers for IGF-I were determined as described previously (11) using standard enzyme-linked immunosorbent assay (ELISA) techniques and liquid phase RIAs. In subsequent studies, the dilution or volume of Ig administered was calculated to complex the exogenous IGF-I administered.

Measurement of IGF-I activity in vitro: cell number
The bovine fibroblast cell line GM06034a (American Type Culture Collection, Manassas, VA) was used to investigate the action of the anti-IGF-I antibody in vitro. This cell line is well characterized in terms of IGF-I responsiveness and its IGFBP production, synthesizing only modest quantities of small mol wt IGFBPs in the basal state and IGFBP-3 in response to IGF-I (17). Cells were incubated with IGF-I in the presence of saline, and NI Ig or anti-IGF-I Ig was determined as described by Conover et al. (18). Cells were plated in 24-well plates at a density of 1.25 x 104 cells/ml in DMEM containing 10% FBS. The following day, designated day 0, the medium was changed to DMEM plus 0.1% BSA plus IGF-I (0, 0.1, 1.0, 10, or 100 ng/ml) plus additions of either anti-IGF-I Ig (1:500; calculated from liquid phase RIAs and ELISAs to be sufficient to complex the highest dose of IGF-I), NI Ig (1:500), or PBS; these had been incubated overnight at 4 C on day -1 to allow antibody and IGF-I complex formation. Medium was changed to fresh experimental medium on days 2 and 4. Cell number was measured using a hemocytometer on days 1, 3, and 5; each determination was performed in triplicate, and each "day" was an individual set of observations.

Epitope analysis of anti-IGF-I antiserum
Multiple pin peptide synthesis techniques (19) were used to construct two separate sets of overlapping and sequential octamers and hexamers onto plastic pins representing the entire sequence of hIGF-I, using an epitope scanning kit and following the manufacturer’s instructions exactly (Cambridge Research Biochemicals International, Northwich, UK). Briefly, for the octamers, the first plastic pin had a peptide corresponding to residues 1–8 of IGF-I, the second pin had residues 2–9 etc. through to the final octamer; hexamers were constructed in an analogous manner. Any antibody that binds to the pins was quantified using standard ELISA techniques, described below. The pins were assembled in plastic holders in an 8 x 12 configuration so that they could be placed in standard 96-well microtiter plates. The pin-bound peptides were incubated overnight at 4 C with the anti-IGF-I antiserum diluted 1:1000 in supercocktail [0.15 M NaCl and 0.01 M sodium phosphate buffer, pH 7.2, containing 0.05% (vol/vol) Tween-20] to which 0.1% (wt/vol) ovalbumin and 0.1% (wt/vol) BSA were added. The pins, retaining any peptide-bound antibody, were then rinsed in distilled water, washed in supercocktail (four times for 10 min each time with agitation at room temperature) and incubated in new microtiter plates for 1 h at 37 C containing horseradish peroxidase-conjugated antisheep second antibody at a dilution of 1:1000 (rabbit antisheep antibody, DAKO Corp., Glostrup, Denmark). The plates were then incubated at 37 C for 1 h and washed in supercocktail (three times for 10 min each time as before). Substrate color reagent was added (100 µl; 0.55 mg/ml 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid diammonium salt in 100 mM citrate buffered saline, pH 4.3, containing 1.2 µl 30% hydrogen peroxide/20 ml); absorbance at 405 nm was measured.

Estimation of the proportion of the total IGF-I-binding capacity of anti-IGF-I antisera by peptide 15
The proportion of the IGF-I-binding capacity of antisera that could be attributed to the linear peptide 15 region was estimated using an ELISA technique, in which the ability of peptide 15 to displace antibody binding to immobilized IGF-I was measured. ELISA plates were coated with 200 µl of a solution of IGF-I, ranging in concentration from 0.00098–2 µg/ml by doubling dilutions. The anti-IGF-I polyclonal Ig preparations (diluted 125-fold) were incubated overnight at 4 C with peptide 15 (512 ng/ml). One hundred microliters of this Ig/peptide 15 mixture were then added to ELISA wells coated with IGF-I and incubated at room temperature for 1 h. Ig that bound to the plates was measured by standard techniques using horseradish peroxidase-conjugated antisheep second antibody, as previously described.

Effects of antipeptide 15 Ig on IGF-I-stimulated weight gain in dwarf mice
Homozygous Snell dwarf mice (20) were bred at the Babraham Institute (Cambridge, UK) and fed good quality pelleted rodent feed (18% crude protein) and water ad libitum. Forty-eight 10-week-old Snell dwarf mice were allocated to one of eight treatment groups (n = 6/group); 0.4 ml anti-IGF-I Ig (as a positive control), antipeptide 15 Ig (derived from sheep F157), antipeptide 15 Ig (derived from sheep P260), or NI Ig plus 20 µg IGF-I or an equivalent volume of saline. The Ig and IGF-I or saline were preincubated at room temperature for 1 h before injection to allow complex formation. Previous studies had shown that the volume of Ig used (0.4 ml; the maximum amount that can be administered to dwarf mice of this size) was sufficient to complex with the exogenous IGF-I in vivo for the anti-IGF-I Ig (11). Thus, each Ig preparation was assessed in the presence and absence of IGF-I. Injections were performed for 7 days, when mice were killed by decapitation exactly 2 h after treatment. Trunk blood was collected, and liver and skeletal muscle (combined gastrocnemius, plantaris, and soleus) were dissected and weighed. Serum was prepared by centrifugation and stored at -20 C until analysis.

Measurement of serum IGF-I concentrations
Total plasma or serum IGF-I concentrations were determined by RIA after a modified acid-ethanol extraction (20) using a polyclonal rabbit anti-IGF-I antiserum generated at the Babraham Institute. Previous studies using 125I-labeled IGF-I and size exclusion chromatography demonstrated that the acid-ethanol treatment could dissociate any antibody-bound IGF-I in serum.

Statistical analysis
Differences between treatment groups in the dwarf mouse bioassay experiment were assessed using a 4 x 2 ANOVA with Ig (anti-IGF-I Ig and antipeptide 15 Ig, derived from two different animals, and NI Ig) and peptide (IGF-I or saline) as the main effects. Initial liveweight was used as a covariate for the analysis of whole body and tissue weights. Differences between treatment groups in cell number were assessed using a 3 x 4 ANOVA, with exogenous IGF-I and Ig preparation (or saline) as main effects.

Where significant differences were obtained for main effects (defined as P < 0.05), these were examined in more detail using t tests to compare individual treatment groups. Pooled SED values are presented for each analysis as well as individual SEM values where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of IGF-I antisera: potentiation of IGF-I activity in vivo
The ability of sheep or rabbit antisera to potentiate IGF-I activity was assessed as described previously in dwarf mice (11), which are panhypopituitary and lack the ability to synthesize GH. They, therefore, have very low endogenous IGF-I levels, are typically only 25–30% the size and weight of normal littermates, and provide an excellent in vivo bioassay for IGF-I activity, as assessed by whole body weight gain over several days (19). In support of our previous observations (11, 13), sheep anti-IGF-I antisera potentiated rather than inhibited IGF-I activity compared with sheep control nonimmune serum [weight gain (milligrams) of dwarf mice over a 7-day period; nonimmune serum alone, 79.7 ± 10.2; sheep anti-IGF-I antiserum alone, 82.2 ± 7.0; 20 µg IGF-I/day preincubated with nonimmune serum, 116.0 ± 21.0; 20 µg IGF-I/day preincubated with sheep anti-IGF-I antiserum, 190 ± 15.9; mean ± SEM; n = 6/group]. The titer (defined as 50% binding in a standard ELISA assay) and affinity of this anti-IGF-I antiserum were also in agreement with previous determinations, being 1:7000 and 2.05 x 10-8 liters/mol, respectively.

Additionally, antisera to hIGF-I were generated in three rabbits using similar methodologies, but these did not potentiate IGF-I activity when examined in the dwarf mouse bioassay. The weight gain of dwarf mice (investigated as part of the sheep anti-IGF-I antiserum bioassay described above) treated with 20 µg IGF-I/day preincubated with a representative rabbit anti-IGF-I antiserum was only 113.3 ± 15.5 mg, similar to the weight gain induced by IGF-I in the presence of nonimmune serum. However, the rabbit antisera typically had higher affinities and titers for IGF-I than did the sheep antisera, with values being 1:50,000 and 5.34 x 10-9 liters/mol, respectively.

Potentiation of IGF-I activity in vitro by anti-IGF-I Ig
To date, the effects of different anti-IGF-I Ig preparations on IGF-I activity have only been investigated in vivo. We have demonstrated that part of the mechanism of action of the potentiating anti-IGF-I Ig is to change the kinetics of IGF-I degradation and its transfer from blood to tissue compartments (12). Here we determined whether the sheep anti-IGF-I Ig could also potentiate IGF-I activity in vitro, as assessed by the stimulation of cell number in the bovine fibroblast cell line, GM06034a, which has been well characterized in terms of IGF-I responsiveness (17, 18). Figure 1Go shows fibroblast cell number incubated with serum-free medium containing BSA plus saline, nonimmune Ig, or sheep anti-IGF-I Ig together with increasing doses of IGF-I. After incubation for 1 day, no effect of IGF-I alone could be detected, but a clear dose-response relationship was observed on days 3 and 5 (P < 0.001 for both time points). When cells were incubated with NI Ig or anti-IGF-I Ig alone, cell numbers were similar to those observed for incubation with serum-free medium and saline. However, at all times examined, the anti-IGF-I Ig induced an increase in cell number when coadministered with IGF-I, although this was only significant at the highest IGF-I dose for the early time points. More importantly, with time there was a significant interaction between the anti-IGF-I Ig and IGF-I, such that on day 5 IGF-I (10 and 100 ng/ml) stimulated a further increase in cell number in the presence of the anti-IGF-I Ig compared with NI Ig or saline. Medium was changed on days 2 and 4 as well as at the start of the study; therefore, these effects of the anti-IGF-I Ig are unlikely to be due to IGF-I half-life alone.



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  		Figure 1. Anti-IGF-I Ig potentiation of IGF-I stimulation of cell number. Bovine fibroblasts were incubated with varying concentrations of IGF-I (0, 1, 10, and 100 ng/ml) that had been preincubated with saline (open bars), NI Ig (gray bars), or anti-IGF-I Ig (black bars) as described in Materials and Methods. Cell number was measured at 1, 3, and 5 days. Data were analyzed for each time point by ANOVA, with source of Ig (NI or anti-IGF-I) and dose of IGF-I as main effects. P values are presented for these main effects as well as any interaction between them. Bars with different letters (a and b) indicate significantly different responses (P < 0.05 or less), as analyzed by post-hoc t test, after significant effects had been observed by ANOVA. The pooled SED and individual group SEMs are presented.

 
Epitope scanning to demonstrate linear epitopes within different anti-IGF-I antisera: identification of a novel linear epitope in IGF-I-enhancing antisera
We previously used epitope scanning to define linear epitopes recognized by the potentiating anti-IGF-I antisera (13), and now use it as a means of assessing the specificity of the epitopes for IGF-I present in enhancing vs. nonenhancing antisera. Binding profiles of four different anti-IGF-I antisera to sequential octamer PIN peptides are presented in Fig. 2Go. A representative sheep IGF-I-enhancing antiserum is depicted in Fig. 2aGo, and a single major site of binding consisting of six octamer peptides (no. 35–40) was observed. The core region of this peak (peptides 36–38) was observed for all other enhancing sheep anti-hIGF-I antisera examined (six to date), although the peptides recognized by the antisera sometimes included peptide 34 and sometimes did not extend further than peptide 38. An additional minor binding peak, spanning peptides 51–53 was consistently observed. These binding sites were confirmed by epitope scanning using sequential hexamer peptides (data not shown). Thus, despite the fact that the sheep enhancing anti-hIGF-I antisera were polyclonal, they bound to IGF-I with a single predominant, functionally continuous epitope.



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  		Figure 2. Scanning epitope analysis of the binding of different anti-IGF-I antisera to sequential and overlapping octamer linear regions of IGF-I. A, Sheep enhancing antiserum; b, sheep inhibitory antiserum; c, NIDDK rabbit antiserum; d, Babraham rabbit antiserum. The PIN number on the abscissa indicates the N-terminal residue of each PIN-bound peptide.

 
Peptide binding of a sheep anti-hIGF-I inhibitory antiserum (a gift from Dr. Steve Hodgkinson) (21) is presented in Fig. 2bGo. No clear groups of sequential peptides were recognized, and a low signal to noise ratio was observed, indicating the presence of many epitopes in this preparation. The antiserum did not specifically bind peptides 36–40 above the general level. Like the anti-IGF-I inhibitory antiserum, binding of the NIDDK (a gift from the National Hormone and Pituitary Program) rabbit anti-hIGF-I antiserum (Fig. 2cGo) to the octamer peptides was characterized by multiple linear peaks of binding and a high background, indicative of the presence of many epitopes. Again, the octamer peptides recognized by the sheep enhancing antiserum did not appear to be identified specifically by the NIDDK antiserum. Lastly (Fig. 2dGo), the epitope-scanning profile of the rabbit anti-IGF-I antiserum generated at Babraham and having no IGF-I potentiating effects is shown. The pin peptides recognized included 35–37; however, binding was not specific to this region, and absorbance was, on the average, about 4-fold lower than that of the sheep enhancing antiserum. Significant additional binding to pin peptides, which included a number of residues derived from the B domain region of IGF-I, was also observed.

Structural homology of the major linear IGF-I-binding site recognized by the enhancing anti-IGF-I antiserum: identification of a putative linear structural determinant
Thus, from linear peptide analysis, the IGF-I-enhancing antiserum binds to IGF-I with restricted specificity, recognizing a region that covers a maximum of residues Ser33 to Cys47 (SerSerArgArgAlaProGlnThrGlyIleValAspGluCys) and a minimum, from the hexamer peptides, of Arg36 to Ile43. Figure 3Go represents a ribbon structure of IGF-I derived from the Brookhaven database coordinates of Cooke et al. (22) using Rasmol (R. Sayle, Glaxo, Middlesex, UK; shareware available via the internet). The region equivalent to residues 33–47 of IGF-I recognized by the anti-IGF-I Ig is highlighted in dark gray and spans the end of the C and the beginning of the A domains (C domain is equivalent to residues 30–41 and A domain to residues 42–62). For orientation, the N- and C-terminal amino acids are labeled, and the tyrosine residues thought to be important for type 1 receptor binding (23) are highlighted in light gray. To date, the region spanning residues 33–47 is not specifically attributed with any unique function of IGF-I.



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  		Figure 3. Ribbon-style depiction of the structure of IGF-I. Two images of IGF-I structure (180° to each other, around the y-axis) were derived from the nuclear magnetic resonance study of Cooke et al. (21 ) using Rasmol (R. Sayle, Glaxo; shareware via the internet). The linear region predominantly recognized by enhancing anti-IGF-I antisera (Ser33 to Cys47) is shaded dark gray. For reference, the three tyrosine residues important for type 1 receptor interaction and the N-terminal 16 residues that interact with IGFBPs are shaded in light gray; the domains are: B, residues 1–29; C, residues 30–41; A, residues 42–62; and D, residues 63–70. The figures demonstrate that the region Ser33 to Cys47 is on the opposite surface of IGF-I compared with the receptor-binding domains.

 
Mammalian IGF-I shows a very high degree of homology with human IGF-I, 100% in many mammals but with one (Pro66; sheep), three (Asp20, Ser35, Ala67; rat), or four (Asp20, Ser35, Ala67, Ser69; mouse) of the residues in the human sequence being different in selected mammals; IGF-I sequences for birds, frog, and fish display less homology with mammalian IGFs. The poor cross-reactivity of anti-hIGF-I antisera with rodent IGF-I is well documented (24), implying that these minor specific changes in amino acid sequence can have marked effects on structural determinants for antibody recognition and secondary structure. The linear peptide of IGF-I recognized by the enhancing antiserum (Ser33 to Arg46) was compared in IGF sequences derived from different species, hIGF-II and insulin (Fig. 4Go). The greatest deviations in homology in IGF-I derived from different species occur in residues equivalent to Ser33 to Arg46 and also in the carboxyl-terminal D peptide region of hIGF-I; additionally one of the three rat and one of the four mouse deviations are contained in the peptide region 33–47. Mature insulin lacks the C and D peptide regions of IGF-I, and IGF-II lacks two of the residues in the C peptide region, but does have a D peptide. Thus, the region recognized by the anti-IGF-I-enhancing antiserum is unique to mammalian IGF-I. We therefore decided to synthesize a 15-mer peptide (termed peptide 15) equivalent to residues Ser33 to Cys47 and to examine the effects of antibodies raised against this peptide on the activity of hIGF-I. Two sheep (F157 and P260) were immunized with peptide 15 as described in Materials and Methods, and Ig-enriched fractions were prepared by ammonium sulfate precipitation (11) of serum derived from the animals.



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  		Figure 4. Sequence homology of IGF-I, hIGF-II, and human insulin in the peptide 15' region (Ser33 to Cys47 of hIGF-I).

 
Predominance of antibody binding to the peptide region Ser33 to Cys47 (termed peptide 15) in anti-IGF-I-enhancing antisera
As the anti-IGF-I-enhancing antisera are effectively polyclonal and had only been analyzed for their ability to recognize linear epitopes within IGF-I, we sought to determine whether nonpeptide 15 (i.e. Ser33 to Cys47) binding, and presumably conformational, epitopes accounted for a high proportion of total IGF-I-binding capacity in the polyclonal anti-IGF-I antisera. This was estimated by comparing the ability of peptide 15 to inhibit the binding of IGF-I to the polyclonal anti-IGF-I antisera. Incubation of the sheep enhancing anti-IGF-I Ig to increasing concentrations of IGF-I resulted, as expected, in increased Ig binding (Fig. 5aGo). Incubation in the presence of peptide 15 inhibited this binding by about 50%, indicating that the epitope of IGF-I mimicked by peptide 15 accounted for a high proportion of the antiserum’s IGF-I-binding capacity. The binding of previous sheep enhancing anti-IGF-I antisera to IGF-I was also inhibited by peptide 15, and the degree of inhibition varied from about 34% to almost 100% (data not shown), depending on the host sheep from which the Ig preparation was derived. The polyclonal nature of each antiserum will, of course, be dependent on the nature of the host animal and the number of times it has been boosted. However, the presence of the peptide 15 epitope was a consistent observation in all IGF-I-enhancing preparations, determined by epitope scanning, direct binding to peptide 15, and displacement of IGF-I binding by peptide 15. As expected, peptide 15 completely blocked the binding of the sheep antipeptide 15 Ig preparations to IGF-I (Fig. 5bGo). However, in marked contrast to the sheep anti-IGF-I antisera, peptide 15 did not block rabbit anti-IGF-I Ig binding to IGF-I (Fig. 5cGo), supporting the observations derived from epitope scanning that this region of IGF-I was not dominant in these nonenhancing anti-IGF-I preparations.



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  		Figure 5. Predominance of peptide 15-binding determinants in IGF-I-potentiating anti-IGF-I antisera. Sheep anti-IGF-I (a), sheep antipeptide 15 (b), and rabbit anti-IGF-I (c) antiserum binding to increasing concentrations of IGF-I coated on to ELISA plates in the absence (closed circles) and presence of peptide 15 (open circles), as described in Materials and Methods.

 
Properties of anti-peptide 15 antisera
The titers of the sheep antipeptide 15 Ig preparations for IGF-I were 1:1000 (F157) and 1:3000 (P260) for 50% binding to IGF-I, which is lower than that usually found for antisera raised against intact IGF-I using our methodology (1:6000 to 1:7000); this decrease is often observed for the interaction of an antipeptide antiserum with the intact protein from which the peptide was derived. The affinities of the antipeptide 15 antisera for IGF-I were determined by Scatchard analysis using [125I]IGF-I in a liquid phase assay and were about 10-8 liters/mol (1.2 x 10-8 and 9.5 x 10-7 liters/mol for P260 and F157, respectively), equivalent to those we have reported for the polyclonal enhancing anti-IGF-I antiserum above and those reported by Stewart et al. (11).

Epitope scanning profiles for the interaction of antipeptide 15 antisera with octamer pin peptides are presented in Fig. 6Go; for reference, the original pin peptides recognized by the anti-IGF-I-enhancing antisera are indicated by the region between the dashed vertical lines. Both antipeptide 15 antisera had, as expected, a single major site of binding to IGF-I. For sheep F157, this peak of binding mimicked well that generated by the enhancing polyclonal anti-IGF-I antisera, with significant binding to the pin peptides also beginning at peptide 35; however, some minor binding was observed beyond peptide 40, extending to peptide 42. The antiserum derived from sheep P260 exhibited a more complex pattern of binding, which extended from peptides 32–43, but with a trough at peptide 35. These apparently different responses to immunization with peptide Ser33-Cys47 were confirmed using hexamer pin peptides (data not shown). Thus, it would appear that the antiserum derived from sheep F157 provided a better mimic of PIN peptide binding of the polyclonal anti-IGF-I antisera that did antiserum from sheep P260.



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  		Figure 6. Scanning epitope analysis of the binding of two different sheep antipeptide 15 antisera to sequential and overlapping IGF-I octamer peptides. a, Sheep F157; b, sheep P260. Epitope scanning was performed in an equivalent manner as with the antisera raised against intact IGF-I shown in Fig. 2Go.

 
Potentiation of IGF-I-stimulated weight gain in vivo by anti-peptide 15 Ig
The ability of Ig preparations derived from the peptide 15-immunized sheep F157 and P260 to modulate the ability of IGF-I to stimulate increased weight gain in dwarf mice was assessed and compared with that of a positive control polyclonal anti-IGF-I Ig and a NI Ig control preparation, as described in Materials and Methods. Figure 7aGo shows the average weight gain (for the 7 days of the study) of the dwarf mice treated with each of the four Ig preparations in the absence or presence of exogenous IGF-I. The weight gain of the dwarf mice in the absence of exogenous IGF-I varied from 17–27 mg over the 7-day period (average, 20 mg); the magnitude of this and its variability are normal for untreated or saline-treated dwarf mice. IGF-I stimulated an increase in weight gain in the presence of NI Ig, but this did not quite achieve statistical significance. However, when IGF-I was administered to mice that were also given the positive control anti-IGF-I polyclonal antiserum, weight gain was increased by 2.7-fold (P < 0.001) compared with that of the non-IGF-I-treated mice, and this was significantly greater (P < 0.01) than that of the mice given IGF-I plus NI Ig. Antipeptide 15 Ig preparations also potentiated increased weight gain in the presence of IGF-I by just over 2-fold compared with that in non-IGF-I-treated mice (P < 0.001 in each case); this increase was only achieved statistical significance (P < 0.05) compared with mice administered IGF-I plus NI Ig for the F157 Ig preparation. Serum IGF-I concentrations are presented in Fig. 7bGo and were about 13 ng/ml in non-IGF-I-treated mice and increased significantly (P < 0.05) to 23 ng/ml in mice in mice given IGF-I plus NI Ig. This increase was augmented further to 30 ng/ml with the polyclonal anti-IGF-I or antipeptide 15 antisera; however, these additional increases only achieved statistical significance (P < 0.05) for mice administered IGF-I plus the F157 antipeptide 15 and the polyclonal anti-IGF-I Ig preparations.



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  		Figure 7. Potentiation of IGF-I activity by anti-peptide 15 Ig in vivo. IGF-I activity was assessed as weight gain in dwarf mice (a); serum IGF-I concentrations are also presented (b). IGF-I was preincubated with NI Ig, antipeptide 15 Ig, or potentiating anti-IGF-I Ig as described in Materials and Methods; controls, to which no IGF-I was administered, were performed for each Ig. Significant effect of Ig preincubated in the presence vs. the absence of IGF-I: §, P < 0.05; §§, P < 0.01; §§§, P < 0.001. Significant effect of antipeptide 15 Ig plus IGF-I or anti-IGF-I Ig plus IGF-I vs. NI Ig plus IGF-I: *, P < 0.05; **, P < 0.01.

 
These data confirm our earlier observations on the action of the anti-IGF-I polyclonal antiserum (11) and also imply that antipeptide 15 antisera also have the potential to mimic the IGF-I-enhancing properties of the polyclonal anti-IGF-I Ig preparations. It is interesting that the preparation derived from sheep F157, which had a closer pin profile to the anti-IGF-I-enhancing antiserum as well as a 3-fold lower titer than that for P260, potentiated the anabolic actions of IGF-I better than did the preparation from P260.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here confirm and extend our recent observations on the activities of peptide hormone antisera. A key finding is the demonstration of enhancement of IGF-I activity in vitro; this is important because it indicates that even though we have found that the antiserum changes the IGF-I kinetics and degradation in vivo (12), it may also have additional, more direct effects on IGF-I action at the cellular level. Also, the antibody extended IGF-I activity beyond that which was achieved by IGF-I alone (at the doses examined), both as weight gain by mice in vivo (11) and as cell number in vitro (current study). An apparently unique dominant linear region is defined by the enhancing antiserum; antipeptide antisera directed against a synthetic peptide mimic of this antigenic determinant bind to native IGF-I and also potentiate IGF-I activity.

In comparable studies, animals were passively immunized with monoclonal or polyclonal anti-IGF-I antibodies whose affinity for IGF-I varied from 10-9–10-11 liters/mol (21, 25, 26); in each case, these preparations either inhibited or had no effect on IGF-I activity in vivo. Where reported, they inhibited IGF-I activity in vitro or inhibited IGF-I binding to type 1 receptor preparations (21, 26). Therefore, the antiserum presented here is behaving very differently. A panel of monoclonal antibodies raised against human recombinant IGF-I map to eight epitopic clusters (27), most being located in regions including part of the B domain or the N- and C-terminals of IGF-I (28). Epitopic cluster I (28) involves residues of the C domain and the first {alpha}-helix of the A domain; therefore, this could include part of the linear peptide defined by the potentiating antisera described here. All of these monoclonal antibodies inhibited IGF-I activity in vitro, and the degree of inhibition generally correlated with their affinity (10-6–10-10 liters/mol) (28). It has been calculated that as many as four different monoclonal antibodies could theoretically bind simultaneously to the IGF-I without structural hindrance, and simultaneous binding of antibodies directed to the D domain and IGF-I binding either to the type 1 receptor or to IGFBP-3 has been demonstrated (27).

The dominant linear region of IGF-I recognized by enhancing antisera includes the carboxyl-terminal of the C domain and the proximal amino-terminal of the A domain. Comparison of the sequences of IGF-I derived from different species, insulin, and IGF-II in the C domain and surrounding residues suggests that this region is one of the least homologous in mammalian vs. nonmammalian IGF-I, with mature insulin completely lacking and IGF-II having a shortened C peptide. Although we cannot unequivocally attribute the enhancing properties of our anti-IGF-I antiserum to this region, its absence in nonenhancing antisera and its presence in potentiating antisera provide us with compelling evidence. The receptor-binding region of IGF-I is defined mainly by residues at the carboxyl-terminal end of the B domain and proximal C domain, specifically Phe23, Tyr24, and Phe25, and Tyr31 and A domain Tyr60, which together form a hydrophobic patch on one face of the molecule (29). The C domain of IGF-I may also confer additional type 1 receptor binding specificity (23, 30). Loss of the C domain induces a 30-fold decrease in type 1 IGF receptor binding (23); however, insertion of the IGF-I C domain into insulin only results in about 25% native IGF-I binding (31), supporting the observation that non-C domain residues of IGF-I are also important for direct interaction with the receptor. The C peptide is thought to exist as an extended loop, with the pairing of Arg36 and Asp45 and also Arg37 with Glu46 resulting in the close association of Tyr31 with residues 23, 24, and 25 (22). Thus, it is likely that one function of the C peptide region is to confer structural stability, and it is possible that antibody binding to part of the C peptide also provides such stability.

The precise mechanism(s) of action of potentiating IGFBPs is poorly defined. It is clear that some modification of the binding protein is necessary, such as proteolytic cleavage, association with extracellular matrix or cell surface components, or dephosphorylation (reviewed in Ref. 5), reducing the native high affinity of IGFBPs for IGF-I (usually about 10-fold greater than that for the type 1 receptor) and presumably releasing IGF ligand and/or allowing more effective competition by the receptor for IGF peptide (32). However, it is difficult to reconcile this relatively straightforward hypothesis with some observations, for example the potentiation IGF-I-stimulated cell amino acid uptake by IGFBP-3 beyond the normal maximum achieved by IGF-I alone (33). This implies that in some circumstances additional mechanisms may occur. Similar to enhancing IGFBPs, potentiating anti-IGF-I antisera also have an affinity for IGF-I that is equivalent to or less than that of the type 1 IGF receptor (11). Also, antibody potentiation of hormone activity can occur when only a minor proportion of the hormone is complexed to antibody, i.e. when hormone is not limiting (34), implying a more positive mechanism than simple inhibition of hormone from degradation. Theoretically, IGF-I could bind simultaneously to the enhancing anti-IGF-I antibodies directed against residues Ser33 to Cys47 and the IGF type 1 receptor, as they are located on opposite faces of the molecule. Equivalent regions i.e. distant to receptor-binding domains, have been suggested for antisera that enhance GH-releasing factor (14) and GH (15). Further, the binding specific antibodies to an antigen can induce a conformational change in the antigen that increases its affinity for other proteins, such as another antibody (35) or a receptor (36), a putative mechanism of action that could account for the potentiation of peptide hormone activity in vitro (data presented here) (14). Simultaneous association of IGF-I with IGFBPs and the type 1 receptor has not been demonstrated. However, as the potentiating antibody and IGFBPs bind to different regions of IGF-I, IGFBPs associating predominantly with the N-terminal B domain, putative interaction with the type 1 receptor may be subject to different limitations.

A possible mechanism of action of the enhancing antibody might be to facilitate IGFBP potentiation of IGF-I activity; interactions between potentiating anti-IGF-I antisera and IGFBPs have not yet been examined. Whether the anti-IGF-I antibody can alter IGF-I distribution among IGFBPs will depend on their relative affinities and concentrations; further, changes in IGF-I activity induced by the antibody may change IGFBP synthesis or distribution.

A alternative mechanism for antibody (or IGFBP) enhancement of peptide hormone activity is that the binding of the antibody to the peptide in some manner changes the kinetics of hormone-receptor interaction, possibly slowing rates of ligand dissociation or receptor internalization (12, 14). The regulation of dissociation kinetics may be of physiological relevance in directing the specificity of insulin receptor signaling in terms of its mitogenic activity (37, 38). Insulin analogs with low dissociation rates are associated with increased mitogenic activity rather than the expected metabolic effects (9), and thus, insulin signaling specificity is governed by the kinetic properties of hormone-receptor interaction. A further intriguing property of the dissociation of both insulin and IGF-I from their cognate receptors is negative cooperativity; the consequence of this is that the kinetics of ligand-receptor interaction become dependent on ligand concentration. It has been proposed that this could limit overactivity at high ligand concentrations, controlling transforming potential and possibly explaining the weak mitogenic potential of insulin and IGF-I. It is tempting to speculate that potentiating IGFBPs or anti-IGF-I antibodies affect ligand-receptor kinetics, modulating either the magnitude or the specificity of the subsequent signaling response. This could be achieved in two ways: either direct interaction with ligand-receptor complexes or by changing IGF-I availability and local concentration in the perireceptor environment.

As a major circulating trophic factor, IGF-I has been investigated in several diseases and conditions that are associated with inhibited anabolism or decreased cell survival. Acute neurological disorders, such as severe stroke (39) or ALS (40), have been in part alleviated by administration of IGF-I. The hypercatabolic response to trauma, extensive surgery, sepsis, nutritional deprivation, and diabetes are all characterized by accelerated muscle wasting and negative nitrogen balance, which are difficult to reverse by traditional enteral or parenteral nutritional support. These conditions are often associated with disorders of the GH/IGF axis and with GH resistance, resulting in decreased IGF-I circulating concentrations and bioavailability (41, 42). Initial findings derived from animal models have been encouraging (43), but clinical trials may be subject to limitations that accompany exogenous IGF-I administration. IGF-I is usually given in a nonphysiological manner as non-IGFBP-bound peptide and, as a consequence, only restricted doses can be administered before severe side-effects occur, such as hypoglycemia, or negative feedback mechanisms manifest, for example decreased GH secretion. These problems have been addressed by coadministration of an IGF-I/IGFBP-3 complex (44) that enabled higher doses of IGF-I to be given safely and prolonged the serum half-life of IGF-I. However, little evidence for a potentiation of IGF-I activity was observed, analogous to that which can occur with enhancing IGFBPs or the potentiating anti-IGF-I antibody described here. One additional difficulty in catabolic conditions is increased IGFBP protease activity (45), which reduces the IGF-I reservoir and further limits total IGF-I bioavailability. We suggest that the potentiating anti-IGF-I antiserum described here has many favorable properties that could form the basis of future therapeutic strategy.


   Footnotes
 
1 This work was supported by a Babraham Institute Biotechnology and Biological Sciences Research Council Competitive Strategic Grant, a Medical Research Council Collaborative Studentship in association with Merck & Co. (Rahway, NJ; to C.R.W.), and a Rees Jones Fellowship (to R.A.H.). Back

2 Present address: Tropical Beef Center, P.O. Box 5545, Rockhampton Mail Center, Queensland 4702, Australia. Back

3 Present address: Division of Surgery, Level 7, Bristol Royal Infirmary, University of Bristol, Bristol, United Kingdom BS2 8HW. Back

Received July 23, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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