RECEPTOR ANTAGONISTS Pegvisomant

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Pegvisomant: structure and function

S Pradhananga, I Wilkinson and R J M Ross Division of Clinical Sciences, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, UK

(Requests for offprints should be addressed to R Ross, Clinical Sciences, Northern General Hospital, Sheffield S5 7AU, UK; Email: [email protected])

Abstract

Pegvisomant is the pegylated form of mutant growth hormone (B2036). B2036 has increased affinity in one binding site and lowered affinity in its second binding site, it has been shown that this molecule still enables dimerisation of the growth hormone receptor at the cell surface but does not allow the necessary conformational changes for signalling. Pegylation decreases the antagonistic activity of B2036, however the rate of clearance of the pegylated B2036 is greatly reduced compared to the unpegylated form. Even though the antagonistic activity of pegvisomant is lower than B2036, the reduced rate of clearance makes it an effective clinical drug for the treatment of conditions such as acromegaly.

Journal of Molecular Endocrinology (2002) 29, 11–14

Introduction gated to this core B2036 molecule. Pegylation has little effect on the affinity of pegvisomant for GHBP The growth hormone (GH) antagonist, pegviso- but reduces affinity for the cell surface receptor. mant, is the first specific growth hormone receptor Therefore, there is a high dose requirement for antagonist to be developed for the treatment of pegvisomant in clinical practice; however pegy- acromegaly (Trainer et al. 2000, van der Lely et al. lation has a benefit in that it increases the half life 2001). In this review, the relationship between of clearance. pegvisomant structure and function is discussed. Growth hormone signals by binding two molecules of its receptor (GHR) at the cell surface. Background As a consequence there are two receptor binding Site 2 binding sites on the GH molecule (Fig. 1). The affinity of site 1, for the receptor, is greater than that of site 2. The discovery of the GH site 2 mutant antagonist The core molecule in pegvisomant is B2036, was serendipitous. The original observation came which is GH with a number of mutated amino whilst groups were looking for mutations that might acids. A G120K mutation at binding site 2 increase agonist activity. A mutation at G119A in greatly reduces the binding affinity of this site. the bovine GH molecule suppressed the growth of Additionally, B2036 also contains mutations in transgenic mice (Chen et al. 1990). Subsequent binding site 1, which increase the affinity of this research demonstrated that a similar mutation at binding site. These mutations enhance binding to G120R in the human molecule also acted as a GH growth hormone binding protein (GHBP, derived antagonist (Chen et al. 1994). With the characteris- from the extracellular domain of the GH receptor) ation of the crystal structure for GH binding to a but as our data suggests they do not change affinity homo-dimer of its receptor (de Vos et al. 1992), it for the cell surface receptor. was demonstrated that the mutation at G120A To generate pegvisomant, 4–6 moieties of prevented effective binding of the second GH polyethylene glycol (PEG) 5000 are then conju- receptor molecule. This single mutation at site 2

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Figure 1 The crystal structure of growth hormone complexed to the extracellular domains of its receptor. Growth hormone (red) can bind two molecules of its receptor (blue). Thus the growth hormone has two binding sites, binding site 1 (high affinity) and binding site 2 (low affinity). (Crystal structure determined by de Vos et al. 1992, Protein Database (PDB) code: 3 HHR.)

binding in the GH molecule is the basis of the GH affinity of site 1 for GHBP can be increased antagonistic activity of pegvisomant. (Lowman & Wells 1993). This potential increase in site 1 affinity could be an advantage in an antagonist, which had a mutated site 2 (Cunningham Site 1 binding et al. 1998). In B2036, there are 8 mutations at Using monovalent phage display, various residues binding site 1 (H18D, H21N, R167N, K168A, in the GH molecule have been demonstrated to be D171S, K172R, E174S and I179T). In practice, critical in binding at site 1. By mutating these these mutations at binding site 1 increase the residues, it has been demonstrated that the affinity of B2036 for recombinant GHBP by

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Figure 2 Hypothesis for interaction of GH, B2036 and B2036-PEG (pegvisomant) with the GHR. (A) GH binds through site 1 to GHBP; binding to the GHR through sites 1 and 2 triggers a conformational change, signalling and internalisation. (B) B2036 binds preferentially to GHBP, due to the site 1 mutations (1*), but this advantage is lost in the GHR dimer where other interactions are revealed within the trimeric complex. The site 2 mutation (X) blocks the conformational changes required for signalling, but internalisation still occurs. (C) B2036-PEG binds to GHBP through site 1, which is protected from pegylation by the mutation of the site 1 lysines, but the PEG moieties do conjugate in site 2 to the G120K mutation. Pegylation sterically reduces binding to the GHR dimer at the cell surface, although binding is sufficient at high concentration of B2036-PEG to block GHR signalling. (Reprinted with permission from Ross et al. 2001.) four- to five-fold compared with GH (Goffin et al. binding sites is doubled in the presence of a 1999, Ross et al. 2001). However, binding affinity to dimerisation blocking antibody (Ross et al. 2001). cell surface receptor in stable cell lines expressing This strongly suggests that the antagonist, B2036, the human GH receptor is equivalent between binds to a receptor dimer. It is clear that the B2036 and GH (Ross et al. 2001). The results on antagonist, B2036, prevents JAK2 phosphorylation binding studies suggest that although the site 1 and STAT5 signalling (Maamra et al. 1999). mutations create an increased affinity for GHBP, However, the antagonist receptor complex is still they do not change the affinity for the dimeric rapidly internalised into cells expressing the human complex at the cell surface. GH receptor (Maamra et al. 1999). This internalisation is associated with a down-regulation of GH binding and signalling with a refractory period of GH antagonists and receptor trafficking around 2–3 h (Ross et al. 2001). This is consistent There is now evidence that the B2036 antagonist with recycling and the presence of new receptor at binds to a receptor dimer but does not induce the cell surface. conformational changes required for signalling (Ross et al. 2001). This evidence comes from two sets of experiments. In Western blotting experi- Pegylation ments, there was evidence that the antagonist might associate with a receptor dimer in a 2:2 The half-life of native GH is short, as is that of molecular configuration (Harding et al. 1996). In B2036. In order to develop an effective treatment, recent experiments, we have demonstrated that it was recognised that an antagonist with delayed GH and B2036 have similar numbers of binding clearance would have to be generated. Pegylation sites at the cell surface and that the number of of GH reduces its affinity for the receptor but www.endocrinology.org Journal of Molecular Endocrinology (2002) 29, 11–14

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delays its clearance (Clark et al. 1996). If the pegvisomant compared with B2036 is that the molecular ratio of PEG to GH is optimised, a former has a greatly reduced rate of clearance, pegylated molecule with increased bioactivity can which facilitates clinical treatment using this be generated. PEG conjugates to lysines and the molecule. N-terminus, increasing the molecular size and hydrodynamic volume of a protein. Pegvisomant is B2036 with 4–6 PEG moieties covalently References conjugated. Chen WY, Wight DC, Wagner TE & Kopchick JJ 1990 Expression It was recognised early on that there was a lysine of a mutated bovine growth hormone gene suppresses growth of in binding site 1 of GH and, was therefore, mutated transgenic mice. PNAS 87 5061–5065. Chen WY, Chen N, Yun J, Wagner TE & Kopchick JJ 1994 In vitro to prevent pegylation in site 1. In addition, to and in vivo studies of the antagonistic effects of human growth increase the antagonist activity, the mutation hormone analogs. Journal of Biological Chemistry 269 15892–15897 generated at site 2 was a lysine, G120K, thus (Errata 269 20806). Clark R, Olson K, Fuh G, Marian M, Mortensen D, Teshima G, increasing the probability of pegylation at site 2. Chang S, Chu H, Mukku V, Canova-Davis E, Somers T, Cronin Comparing binding of site 2 only mutated GH M, Winkler M & Wells JA 1996 Long-acting growth hormones (pegylated GH.G120K) with pegylated B2036, produced by conjugation with polyethylene glycol. Journal of ffi Biological Chemistry 271 21969–21977. there is a four-fold greater binding a nity for Cunningham BC, Lowman HB, Wells JA, Clark RG, Olson K & pegylated B2036 (Ross et al. 2001). This is Fuh G 1998 Human growth hormone variants. US Patent #5 849 presumably explained by the lack of pegylation 535. Goffin V, Bernichtein S, Carriere O, Bennett WF, Kopchick JJ & in binding site 1 of B2036 compared with the Kelly PA 1999 The human growth hormone antagonist B2036 probability of pegylation of site 1 in the does not interact with the prolactin receptor. Endocrinology 140 GH.G120K. Thus, in the pegylated antagonist, 3853–3856. Harding PA, Wang X, Okada S, Chen WY, Wan W & Kopchick JJ there is an advantage to having the site 1 1996 Growth hormone (GH) and a GH antagonist promote GH mutations. However, pegylation does considerably receptor dimerization and internalization. Journal of Biological reduce the binding affinity for pegvisomant at the Chemistry 271 6708–6712. van der Lely AJ, Hutson RK, Trainer PJ, Besser GM, Barkan AL, cell surface. There is only about a four- to five-fold Katznelson L, Klibanski A, Herman-Bonert V, Melmed S, Vance reduction in binding of pegvisomant to GHBP ML, Freda PU, Stewart PM, Friend KE, Clemmons DR, but there is a much greater reduction of around Johannsson G, Stavrou S, Cook DM, Phillips LS, Strasburger CJ, Hackett S, Zib KA, Davis RJ, Scarlett JA & Thorner MO 2001 20-fold when binding is studied at the cell surface Long-term treatment of acromegaly with pegvisomant, a growth receptor. This is presumably explained by stearic hormone receptor antagonist. Lancet 358 1754–1759. hindrance. Lowman HB & Wells JA 1993 Affinity maturation of human growth hormone by monovalent phage display. Journal of Molecular Biology Even though the activity of pegvisomant is 234 564–578. reduced due to pegylation, its efficacy as a clinical Maamra M, Finidori J, Von Laue S, Simon S, Justice S, Webster J, drug is derived from its highly reduced rate of Dower & Ross R 1999 Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the clearance, which is a consequence of pegylation. full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling. Journal of Biological Chemistry 274 Conclusions 14791–14798. Ross RJM, Leung KC, Maamra M, Bennett W, Doyle N, Waters MJ & Ho KKY 2001 Binding and functional studies with the Figure 2 shows a hypothesis for the interaction of growth hormone receptor antagonist, B2036-PEG (pegvisomant), GH, B2036 and pegvisomant with the GHR. The reveal effects of pegylation. Journal of Clinical Endocrinology and site 2 mutation prevents a conformational change Metabolism 86 1716–1723. Trainer PJ, Drake WM, Katznelson L, Freda PU, Herman-Bonert required for signalling but does trigger internalisa- V, van der Lely AJ, Dimaraki EV, Stewart PM, Friend KE, tion of the antagonist receptor complex. The site 1 Vance ML, Besser M & Scarlett JAA 2000 Treatment of mutations enhance binding to GHBP but do not acromegaly with the growth hormone-receptor antagonist ffi pegvisomant. New England Journal of Medicine 342 1171–1177. change a nity at the cell surface. Pegylation de Vos AM, UltschM&Kossiakoff AA 1992 Human growth reduces the affinity of the antagonist for the cell hormone and extracellular domain of its receptor: crystal structure surface receptor because of stearic hindrance, but of the complex. Science 255 306–312. a high concentration is still effective in com- Received 10 January 2002 pletely blocking GH signalling. The benefits of Accepted 2 May 2002

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