Article pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding Henrik S. Ramírez-Andersen,*,† Carsten Behrens,† Jens Buchardt,† Johannes J. Fels,† Charlotta G. Folkesson,† Chen Jianhe,§ Leif Nørskov-Lauritsen,† Per F. Nielsen,† Mats Reslow,† Christian Rischel,† Jing Su,§ Peter Thygesen,† Charlotte Wiberg,† Xin Zhao,§ Xia Wenjuan,§ and Nils L. Johansen† Bioconjugate Chem. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 08/31/18. For personal use only.
†
Novo Nordisk A/S Global Research, DK-2760 Maaloev, Denmark Novo Nordisk Research Center China, 20 Life Science Park Road, Changping District, Beijing 102206, China
§
S Supporting Information *
ABSTRACT: The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.
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INTRODUCTION
frequency. Another sustained release formulation of hGH, based on hyaluronate microparticle delivery technology, LB03002 from LG Life Science, had obtained marketing authorization in the EU, but it was revoked in 2017 by the European Medicines Agency as they did not launch.6−8 Altus Pharmaceuticals have developed ALTU-238, a formulation of crystalline hGH, coated with poly(arginine) allowed for in vivo PK release profiles of over several days in animal models, but it was discontinued in phase II.9 Products with prolonged elimination phase include the polyethylene glycol (PEG)ylated hGH analogues PHA794428, ARX201, and NNC126-0083 (described as compound 3 below), were all terminated during clinical development.10−12 However, a PEG-hGH product developed by GeneScience Pharmaceuticals, Jintrolong, has been marketed in China since 2014 and is currently undergoing further clinical trials.13 Furthermore, a long-acting growth hormone antagonist Pegvisomant (Somavert) is a growth hormone molecule marked for treatment of acromegaly with between four and five PEG chains covalently bond per hGH, which affords a half-life of approximately 15 h in rats.14 Finally, a GH albumin fusion protein, Albutropin (TV-1106) has also been terminated in clinical development.15,16
Recombinant human growth hormone (hGH) has been in clinical use since the mid-1980s for treatment of both pediatric and adult patients with growth hormone (GH) disorders. Numerous patients have benefited from a positive clinical outcome of hGH replacement therapy. However, an extensive renal clearance, rapid proteolysis, and ligand−receptor internalization result in the short half-life, and as a result, hGH must be administered as a daily subcutaneous (sc) injection. The frequent administration has turned out to be a drawback of current treatment, as it represents a burden for the patients and for parents of pediatric patients undergoing daily hGH treatment and can affect the overall treatment adherence.1−4 A long-acting hGH treatment with prolonged effect and sufficient stability for a once-weekly administration will be an attractive alternative, especially for young and adolescent patients. Such a treatment can, in principle, be achieved by either prolonging the absorption phase of the hormone, prolonging its elimination phase, or by using a combination of both. Several attempts have been made to generate a long-acting hGH. Nutropin Depot from Genentech was the first approved depot formulation of hGH. This product was withdrawn from the market because of manufacturing difficulties, injection site reactions, and initial burst release.5 However, Nutropin Depot showed that a clinically relevant effect of hGH could be obtained with a reduced injection © XXXX American Chemical Society
Received: June 29, 2018 Revised: August 14, 2018
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DOI: 10.1021/acs.bioconjchem.8b00463 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry Table 1. Assessment of hGH Half-Life Prolongation Potentiala Analogue (#)
GH protein
Attachment point
Protractor/albumin binder
Attachment chemistry
Connector
− −
− −
− −
− −
2
hGH hGH (K41A, R64A, K172A, G120R)b hGH
N-Phe1
mPEG(40k)-10 kDa PEG-
propyl
3
hGH
Gln141
aminoethyl
4
hGH
N-Phe1
mPEG(40k)-glycerylpropyl-benzylIc
Reductive alkylation mTGase
5
hGH(K41A, R64A, K172A, G120R)b
N-Phe1
Ic
aminoethyl
hGH 1
aminoethyl
Reductive alkylation Reductive alkylation
BAF (#/hGH)
Half-life (rat, iv) (h)
MRT (rat, iv) (h)
1 >1000 (n = 2) 4.71 (n = 6) 10 (n = 4)
0.21 1.2
0.15 0.19
5.6
12
5.0
15.5
2.5
3.7
12.1
7.7
5.83 (n = 6) 152 (n = 2)
a
Abbreviations: BAF, BAF-3 cells (a murine pro-B lymphoid cell line derived from the bone marrow) was originally IL-3 dependent for growth and survival. IL-3 activates JAK-2 and STAT, which are the same mediators that GH is activating upon stimulation. After transfection of the GHR the cell line was turn into an hGH-dependent cell line. This clone can be used to evaluate the effect of different hGH samples on the survival of the BAF-3GHR, #/hGH, EC50(#)/EC50(hGH), GH, growth hormone; Gln141, glutamine position 141; hGH, human growth hormone; IL-3 interleukin-3; iv, intravenous; mPEG, methoxy terminated PEG; N-Phe1, N-terminal phenylalanine; PEG, polyethylene glycol; MRT, mean residence time; mTGase, microbial trans-glutaminase. bInactive GH analogue (refs 33,67). cSee Figure 1 and Table 2 for details.
approximately 70% of hGH is cleared by non-capacity-limited glomerular filtration.29 A major component of the remaining clearance is believed to be receptor-mediated internalization followed by lysosomal degradation.29 In long-acting hGH molecules, the method of clearance can be addressed by increasing their molecular size, as seen in PEGylated hGH molecules. Although hGH has been reported to escape lysosomal degradation to some degree, the amount of receptor-mediated degradation can be lessened further by reducing receptor affinity, which might result in loss of efficacy or by conjugation to molecules that are capable of rescuing internalized molecules from lysosomal degradation by binding to the neonatal Fc receptor.30 This rescue concept has been utilized in the hGH-albumin conjugate albutropin.31,32 In the attempt to identify a long-acting hGH molecule, hGHalbumin fusions were deselected because of the risk of immunogenicity from the connecting regions of such molecules and chose to pursue half-life prolongation of hGH molecules to which noncovalent albumin binding moieties were conjugated. Initially a series of reference analogues (Table 1) were prepared for benchmarking and to reveal the maximal protraction achievable by using noncovalent albumin binding technology. First the compounds were screened for GH receptor binding/activation in a BAF-3 cell-based assay (a murine pro-B lymphoid cell line derived from the bone marrow), which was originally interleukin-3 (IL-3) dependent for growth and survival. IL-3 activates JAK-2 and STAT, which are the same mediators that GH is activating upon stimulation. After transfection of the GH receptor (GHR), the cell line was turned into a hGH-dependent cell line. This clone was used to evaluate the effect of different hGH samples on the survival of the BAF-3 GHR.33 The protraction potential was estimated from their pharmacokinetics (PK) properties in Sprague− Dawley rats (Table 1). The analogues were based on wild-type hGH and on an analogue 1, which was specifically designed to have minimal interaction with the GHR and thus not to undergo receptor-mediated clearance. Analogue 1 was based on published Ala-scan data on hGH and mutations selected to abolish binding to site 1 and 2 in the GH receptor.34 The protractors used for half-life extension comprised a 40-kDa branched PEG or an albumin binder (Table 1 and Figure 1) similar to the one performing very well in protracting the GLP-1 compound Semaglutide.35 The PEG or the albumin binder was
Projects that combine a prolonged absorption and elimination phase are VRS-317, TransCon GH (ACP-001), and MOD4023, which either are in clinical development or have been discontinued.17−20 Recently, advances in protein fusion technology have led to the development of a new class of long-acting hGH compounds, GH-Fc fusion proteins. These new compounds are generated by fusing hGH with the Fc-domain of immunoglobulin G, prolonging the half-life of the resulting fusion molecule considerably via Fc binding to the neonatal Fc receptor (FcRn) in the endosome, which reduces its lysosomal degradation.21,22 Utilizing this Fc-fusion technology, Hanmi Pharma is currently in clinical development with a long-acting GH-Fc molecule HM10560A23 and Genexine Inc. together with Handok Inc. are conducting a phase II trial with a similar molecule, GX-H9.24 Long-acting hGH formulations have recently been reviewed by Cawley et al.,25 Høybye et al.,5 and Yuen et al.26 In spite of the pursuit of many different approaches aiming at a long-acting hGH molecule, only Jintrolong has so far reached the market, in China. In this study, we describe how prolongation of hGH half-life via noncovalent albumin binding led to the identification of a frontrunner candidate with extended half-life and in vivo efficacy.
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RESULTS AND DISCUSSION Like most other protein hormones, hGH acts by interacting with a specific receptor on the surface of cells. Human GH’s effect on height appears to be stimulated by at least two biological mechanisms. First, hGH exerts some of its effects by binding to receptors on target cells, where it activates the MAPK/ERK pathway.27 Through this mechanism, hGH directly stimulates proliferation of chondrocytes of cartilage. Second, hGH also stimulates the production of insulin-like growth factor 1 (IGF-1)28 through the JAK-STAT signaling pathway.27 The liver is a major target organ of hGH for this process and is the principal site of IGF-1 production. IGF-1 has growth-stimulating effects on a wide variety of tissues including osteoblast and chondrocyte activity to promote bone growth. Clearance of hGH. Clearance is the major determinant for the duration of action of an hGH molecule. It is estimated that B
DOI: 10.1021/acs.bioconjchem.8b00463 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 1. Abbreviations of subunits constituting the albumin binder linkers used to derivatize hGH analogues as shown in Tables 1−5. Protractor, the fatty acid/tetrazole part that binds to albumin; Linkage, the linker part that provides spacing between albumin and hGH; and Connector, the part of the linker that connects the albumin binder to hGH.
weekly hGH therapy. This assumption was made even though PK scaling to humans of PEGylated hGH and albumin binder hGH analogues can be different.38 Introduction of albumin binder I (Figure 2 and Table 2) via reductive alkylation (Scheme 1) at the N-terminus of hGH afforded analogue 4, with a half-life of 2.5 h being in-between the half-lives of analogues 1 and 2. However, introduction of the same albumin binder I to the inactive hGH analogue 1 resulted in analogue 5, with a very long half-life of 12.1 hbelieved to be close to maximum half-life extension for hGH in rats using the albumin binder technology. From the initial data it was concluded that an albumin binder at the hGH N-terminus would not provide sufficient protraction for a once-weekly hGH analogue. PK data from the inactive analogues 1 and 5 indicated that receptor-mediated clearance has a major impact on the PK. Albumin Binder/hGH Analogues Based on Wild-Type hGH. In a first attempt to improve PK of the albumin binder
selectively conjugated to the N-terminus of hGH by either reductive alkylation or to the side chain of Gln141 by a microbial transglutaminase (mTGase)-catalyzed exchange of the sidechain amide into a β-amino alcohol, followed by periodate oxidation to the corresponding aldehyde and then coupled to the protractor by reductive alkylation.36,37 Analogue 1, with a greater than a 1000-fold reduction in affinity for the GHR, clearly showed increased half-life of 1.2 h compared with 0.21 h for hGH, demonstrating the impact of the receptor-mediated clearance. The 40 kDa PEGylation of hGH either at the N-terminus or at the Gln141 position afforded analogues 2 and 3, which showed a significant increase in halflife with 5.6 and 5.0 h, respectively, with only a slight reduction in GHR affinity. With the knowledge that a very similar PEGylated GH38 analogue already had demonstrated the potential for once-weekly hGH treatment, it was assumed that an albumin binder-conjugated hGH analogue possessing a halflife approaching 5 or 6 h could potentially be used for onceC
DOI: 10.1021/acs.bioconjchem.8b00463 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 2. Structure of selected albumin binder reagents I, IX, and X used for analogues 4, 5, 15, and 19. Syntheses of the albumin binders was done on solid phase using standard Fmoc strategy DIC/HOBt peptide synthesis coupling protocols, followed by purification on prep-HPLC.
Table 2. Linker Composition for Albumin Binders I−X Used to Derivatize hGH and hGH Analoguesa (#)
Protractorb
Linkageb
Connectorb
MS found
MS calculated
I II III IV V VI VII VIII IX X
C20 diacid C20 diacid Tetrazolyl C16 acid C20 diacid C18 diacid C18 diacid Tetrazolyl C16 acid Tetrazolyl C16 acid Tetrazolyl C16 acid C20 diacid
Trx-γGlu-Glu-2xADO-Gly Trx-γGlu-Glu-PEG24-εLysamide-benzyl SulfonamideC4-ADO-2xγGlu-ADO-εLysamide-benzyl Trx-γGlu-Glu-2xADO-εLysamide-benzyl 2xADO-εLysamide-benzyl Cyssulfone-γGlu-εLysamide-benzyl SulfonamideC4-C12−2xγGlu-ADO-εLysamide-benzyl SulfonamideC4-ADO-2xγGlu-ADO-εLys-benzyl SulfonamideC4−2xγGlu-ADO-εLysamide-benzyl Trx-γGlu-Glu-PEG3-ethyl
aminoethyl aminoethyl aminoethyl aminoethyl aminoethyl aminoethyl aminoethyl aminoethyl aminoethyl maleimide
1128.38 2114.64 1268.71 1276.76 851.10 841.04 1320.67 1269.49 1123.35 1094.39c
1128.35 2114.58 1268.48 1276.56 851.08 841.02 1320.64 1269.46 1123.32 1093.35
a
Abbreviations: ID, identity; MS, mass spectrometry. Identity (ID) (MS found) determined by LCMS is shown and compared with calculated values. bFor details see Figure 1. cM+1.
Scheme 1. Reductive Alkylation to the N-Terminal Phe1 with Aldehyde I
Scheme 2. mTGase-Mediated Conjugation of Anilides to Position Gln40 and/or Gln141 in hGH
D
DOI: 10.1021/acs.bioconjchem.8b00463 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
increase. However, its half-life (2.5 h) was lower than that of analogue 7. This may be as a result of insufficient space between the two albumin binders to allow for simultaneous binding of two albumin molecules. A longer PEG24 linker was introduced in analogue 9, but this addition did not impact on its half-life. However, a shorter linker in combination with a protractor moiety with a C2 chain length reduction, as in analogues 10 and 11, had a dramatic negative impact on half-life (
