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1Darbepoetin alfa (Aranesp®) molecular characteristics and basic research
2Presentation overview The evolution of protein therapeuticsStructure and function of recombinant human erythropoietinImportance of sialic acid contentDiscovery and development of darbepoetin alfamolecular characteristicsimplications for clinical use
3The evolution of protein therapeutics Recognition that proteins can be useful ‘hormone-like’ therapeutics eg, insulin (1920s)First purified from animal and human tissues eg, insulin, growth hormone, factor VIIIRecombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983Key pointsThe development of recombinant protein technology to produce drugs for human therapy has been one of the most significant achievements of biotechnology in recent decades.Therapeutic proteins are often recombinant versions or functional equivalents of naturally occurring molecules in the body. Research in this area has made substantial progress since the 1950s when the structure of DNA was determined.Many recombinant human proteins have since been generated and have proved invaluable as targeted treatments for a range of diseases, eg, recombinant human erythropoietin for anaemia, insulin for diabetes, interferon alpha-2b for the management of hairy cell leukaemia and recombinant methionyl human granulocyte colony-stimulating factor for the treatment of chemotherapy-induced neutropenia.Research is now focusing on the relationship between pharmacokinetic and pharmacodynamic properties of molecules, with the aim of engineering proteins that possess enhanced therapeutic characteristics.
4The evolution of protein therapeutics Recognition that proteins can be useful ‘hormone-like’ therapeutics eg, insulin (1920s)First purified from animal and human tissues eg, insulin, growth hormone, factor VIIIRecombinant protein therapeutics – Amgen scientists were among the leaders cloning erythropoietin in 1983Now a new era where protein therapeutics are modified to enhance their properties as therapeutics eg, darbepoetin alfa (Aranesp®)Key pointsMolecular modifications, such as the creation of sialic acid-containing carbohydrate molecules or the derivitisation of molecules, such as the attachment of polyethylene glycol molecules, can increase the half-life of drugs and allow extended dosing intervals, thus relieving the burden of frequent dosing schedules.Such advances have already been made in the design and development of haematopoietic growth factors such as darbepoetin alfa (Aranesp®).
5Darbepoetin alfaDarbepoetin alfa is a biochemically distinct recombinant erythropoietic protein that stimulates the production of red blood cellsThe discovery of darbepoetin alfa resulted from basic research into the structure and function of rHuEPO and its attached carbohydrateThe longer serum residence time and greater biological activity of darbepoetin alfa results from the addition of two extra sialic acid-containing carbohydrate side chainsKey pointsAmgen hypothesised that if it could create a novel super-sialated erythropoiesis stimulating protein, then that novel protein might last longer in the circulation, and exhibit increased potency. Such an enhanced erythropoietic agent might offer patients the benefit of less frequent dosing.Darbepoetin alfa is an erythropoietic protein that was, therefore, rationally designed to contain an increased number of carbohydrate chains and sialic acid molecules compared with recombinant human erythropoietin (rHuEPO).The increased sialic acid content of darbepoetin alfa results in a greater serum residence time and increased in vivo biological activity compared with rHuEPO.1Reference1. Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.
6Structure of EPO bound to an EPO receptor Carbohydrate side chainsrHuEPO has four carbohydrate side chainsrHuEPO has a theoretical maximum of 14 sialic acidsKey pointRecombinant human erythropoietin (rHuEPO) has four carbohydrate side chains, with a maximum number of 14 sialic acid residues, which are distal to the receptor-binding sites.BackgroundStructural determination of rHuEPO has been possible through techniques such as nuclear magnetic resonance spectroscopy and X-ray crystallography. This slide illustrates the overall topography of rHuEPO when bound to the erythropoietin (EPO) receptor.The carbohydrate addition sites are clustered at one end of the molecule, distal from the receptor-binding site. The four carbohydrate side chains contribute approximately 40% of the mass of the hormone.1Reference1. Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.
7Typical tetra-antennary carbohydrate Sialic acidN-linked carbohydrateKey pointsThe three N-linked carbohydrate chains present on recombinant human erythropoietin (rHuEPO) can each contain up to four sialic acid residues.An additional O-linked carbohydrate chain can contain up to two sialic acid residues.Therefore, the maximum number of sialic acid residues that can be found on one molecule of rHuEPO is 14.BackgroundAmgen’s research showed that the number of sialic acid-containing branches attached to carbohydrate side chains varies between rHuEPO isoforms. Some rHuEPO isoforms have two, three or four carbohydrate side chains. These are known respectively as biantennary, triantennary and tetraantennary.On the more highly sialated isoforms, the greater number of sialic residues is responsible for the observed increase in erythropoietic potency. For example, isoforms with tetraantennary side chains are more potent than those with triantennary side chains. This is because the pharmacokinetic profile of the rHuEPO changes as the sialic acid content increases. Specifically, it remains in the circulation longer.The number of branches in carbohydrates and therefore the number of sialic acids is variable
8In vivo activity in mice increases with greater sialic acid content Longer serum half-lifeHigher receptor bindingGreater in vivo activity30252015Increase in Hct at day 30 (points)10Key pointsExperiments on isolated recombinant human erythropoietin (rHuEPO) isoforms have demonstrated that the carbohydrate moieties of erythropoietin have significant effects on the in vivo biological activity of the hormone; increased sialic acid-containing carbohydrate is associated with greater in vivo activity in mice.1,2Early on, Amgen researchers realised that the rHuEPO expression system produced not just one form of the molecule, but a mixture of many different isoforms. Each of the isoforms identified in the laboratory was assigned an individual number, and subsequent studies showed that rHuEPO released as product post-purification consisted of a mixture of isoforms 9–14.Isoforms with a greater sialic acid content have a longer serum half-life but a lower receptor binding affinity. Thus, serum clearance (serum half-life) is the primary determinant of in vivo activity, rather than receptor binding affinity.2BackgroundA series of experiments were carried out to explore the relationship between carbohydrate content and in vivo biological activity in rHuEPO.1–3 rHuEPO isoforms 8 through 14 were individually purified, and the efficacy of each isoform in stimulating erythropoiesis in mice was measured. Mice were injected with a vehicle control or an equimolar dose (2.5 µg/kg of each peptide) of each of the individual isoforms, three times per week, for 1 month.Isoforms having a greater sialic acid-containing carbohydrate content were the most potent in inducing an increase in haematocrit (Hct).3 By day 30, the group mean Hct of isoform 14-treated animals increased by 26.2 ± 2.7 points (to 76.2%), compared with an increase of only 6.3 ± 3.5 points for the isoform 8-treated group.2ReferencesEgrie J, et al. Glycoconj J. 1993;10:263.Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.Egrie J, et al. Blood. 1997;90:56a.5891011121314Adapted from Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.Isoform
9Darbepoetin alfa development strategy Introduce N-linked glycosylation consensus sequences (Asn-Xxx-Thr/Ser) into rHuEPOIdentify individual variants that have the desired propertiesTest optimal combinations of variantsKey pointsThe first step in developing the new erythropoietic molecule was to introduce additional carbohydrate. In the recombinant human erythropoietin (rHuEPO) expression system, one particular amino acid sequence triggers glycosylation of the polypeptide backbone.At one end of this sequence is asparagine (Asn); at the other end is either serine (Ser) or threonine (Thr). Scientists refer to this amino acid sequence as an N-linked consensus glycosylation sequence. To add extra carbohydrate chains to rHuEPO, it was therefore necessary to add extra N-linked glycosylation sequences.Numerous candidate molecules were generated. With each molecule, it was necessary to determine whether carbohydrate was added and the effect on folding and erythropoietic potency.Finally, combining certain amino acid alterations might result in a molecule that shows greater activity than any single alteration on its own.
10Amino acid sequence of rHuEPO ALA1PRO32ARG4LEU5CYS7ASP8SER910ILE6VAL1112GLU1314TYR1516171819LYS20212223ASN2425THR26GLY282930273132333435363738394041424344454647PHE484950TRP515253MET54555657GLN58596061626465666768697071727374757677787980818283848586878889909192939596979899100101102103104105106107108109110111112GlY113114115116117118119120121122123124125126127128129130131132133134135136137138161160159158157156155154153152151150139140141142143144145146147148149162163164165NH2COOHHIS94N-glycosylation sitesDisulphide linkagesO-glycosylation siteKey pointsIn order to create a new erythropoietic molecule, Amgen had to start at a very basic level, namely the primary structure of recombinant human eyrthropoietin (rHuEPO).Amgen scientists recognised that adding new carbohydrate chains to rHuEPO would require alterations to the 165-amino acid sequence of the polypeptide backbone.In order to alter the amino acid sequence, one needs to know, for example, which amino acid residues are involved in stimulating the erythropoietin receptor, and what critical folding and structural elements must be preserved in order to retain the full bioactivity of the molecule.Altering specific amino acids involves a technique known as site-directed mutagenesis; an individual mutation is targeted at a particular site on the backbone.Early observations had already shown that certain amino acids had to remain intact for rHuEPO to retain its full bioactivityresidues 7 and 161, as well as residues 29 and 33, are cross-linked by disulphide bridges. Disrupting these inactivates rHuEPO.four amino acid residues in rHuEPO are glycosylation sites. Three of the glycosylation sites are N-linked (orange in diagram), and one is O-linked (green in diagram). The carbohydrate is important for activity.
11rHuEPO has two receptor binding sites Key pointThe active sites indicated in red are crucial for the binding of recombinant human erythropoietin (rHuEPO) to the erythropoietin (EPO) receptor. Alteration of the amino acid residues in this area leads to a loss of activity.BackgroundThe spatial relationships between the more and the less crucial residues become clearer when we look at crystal structures of rHuEPO.The crystal structure depicted on the right-hand side of the slide shows how the amino acid side chains are arranged around the four polypeptide helices and associated connecting polypeptide loops of rHuEPO.The space-filling model of rHuEPO depicted on the left-hand side of the slide shows that the crucial residues, shown in red, are clustered together into two distinct groups. Changing these residues destroys activity.Amino acid residues critical for activity are grouped together in two separate regions, providing important evidence that rHuEPO binds to not just one, but two EPO receptors. The one rHuEPO molecule: two EPO receptors theory supports evidence suggesting that rHuEPO itself has two functionally indispensable domains – presumably one to bind with each receptor.The effect of mutations on in vitro activity is indicated:red <2% active, orange <20% active, yellow <70% active
12The following needed to be addressed in order to make darbepoetin alfa Would the glycan addition be efficient?Would the molecule be properly folded/stable?Would the ability to stimulate erythropoiesis be retained?Would in vivo activity be increased?Key pointIn the development of a novel erythropoietic protein certain challenges needed to be addressed. Extensive research was directed at ensuring that the resultant protein would retain a similar conformation to that of endogenous erythropoietin (EPO) and thus retain (and increase) the erythropoietic activity of the native hormone.BackgroundTo ensure that large enough quantities of the protein could be manufactured, it was important to ensure that the process of glycan addition was efficient.It was also important to ensure that the protein would fold in a manner comparable to the native hormone, and hence exhibit comparable stability. The three-dimensional conformation of a protein may be exquisitely sensitive to changes in the amino acid sequence, and there were concerns that certain amino acid mutations would produce a protein that would not fold correctly.Finally, it was important to ensure that not only would the resultant protein retain the erythropoietic activity of endogenous EPO, but that the in vivo activity of the new molecule would be increased.
13Discovery of new glycosylation sites in rHuEPO Good bioactivityPoor bioactivityGlycosylatedPartial glycosylationUnglycosylatedNH2a1B1a2a3B2a4COOHS126N24T26N38T40N83S85N-linked carbohydrate consensus sequences were introduced into rHuEPO at positions indicated by vertical linesEach molecule was tested to see if it had the desired propertiesTwo positions, Ala30 and Trp88, were selected for further development workKey pointsSince in vivo biological activity increases with increasing carbohydrate, it was hypothesised that increasing the sialic acid-containing carbohydrate of erythropoietin (EPO) (beyond the maximum of 14 sialic acids in recombinant human erythropoietin [rHuEPO]) would create a molecule with enhanced in vivo activity.To test this hypothesis, recombinant DNA technology was used to introduce consensus glycosylation sequences into the rHuEPO molecule, thereby allowing the addition of N-linked carbohydrate chains.1Numerous candidate molecules were generated, with one or more new carbohydrate addition sites. With each molecule, it was necessary to determine whether carbohydrate was added and whether the molecule had the correct tertiary structure and retained biological activity.1Darbepoetin alfa differs to endogenous human EPO at five amino acid positions, with two positions (alanine [Ala30] and tryptophan [Trp88]) allowing for additional carbohydrate attachment.1BackgroundN-linked consensus glycosylation sequence triggers glycosylation of the polypeptide backbone. At one end of this sequence is asparagine; at the other end is either serine or threonine.Reference1. Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.
14Optimisation of glycosylation sites Key pointsThe amino acid sequence of darbepoetin alfa differs from that of endogenous erythropoietin (EPO) at five amino acid positions, allowing for the addition of two N-linked carbohydrate chains.Darbepoetin alfa exhibited comparable immunoreactivity to recombinant human erythropoietin (rHuEPO), suggesting that the folding and stability of darbepoetin alfa would probably be similar to that of rHuEPO.BackgroundThese tests of protein folding relied upon the fact that a certain monoclonal antibody (9G8A) could accurately distinguish the folding patterns of native from non-native proteins. Thus a rHuEPO variant with a folding pattern markedly different from that of the native hormone would bind more tightly to the antibody. The amount of binding of 9G8A to an altered protein can therefore be used as an index of the extent to which its folding pattern is distorted.An immunoassay with antibody 9G8A was used to assess the immunoreactivity, and hence the likely degree of folding distortion, of the EPO variants generated. The reactivity of the variants was expressed as a percentage of reactivity of rHuEPO.The challenge now was to find a combination of mutations that would preserve, or even enhance, the erythropoietic activity of rHuEPO.Changing the peptide backbone to asparagine (Asn88) and threonine (Thr90) produced a molecule with only three N-linked side chains (the same number as rHuEPO); yet the immunoreactivity was about four times that of rHuEPO.When residue 87 was changed to serine (Ser), a fourth N-linked carbohydrate chain was added to molecule NM177, but the immunoreactivity was nearly eight times that of rHuEPO. However, when residue 87 was changed to valine (Val), an additional carbohydrate side chain was added and the immunoreactivity was normalised.After testing combinations of different molecules, it was determined that the molecule now known as darbepoetin alfa, with its five N-linked carbohydrate chains and comparable immunoreactivity to that of rHuEPO, would probably have a suitable folding and stability profile.A two-fold increase in 9G8A immunoreactivity is suggestive of an altered conformation. Val87 substitutions allow carbohydrate addition at position 88 and normalisation of the conformationAla = alanine; Asn = asparagine; His = histidine; Leu = leucine; Pro = proline; Ser = serine; Thr = threonine; Trp = tryptophan; Val = valine
15Comparison of rHuEPO and darbepoetin alfa Receptor 1rHuEPOReceptor 2Carbohydrate side chainsNew carbohydrate side chainsReceptor 1Darbepoetin alfaReceptor 2Three N-linked carbohydrate chainsMaximum 14 sialic acidsMW ~ 30,400 daltons40% carbohydrateFive N-linked carbohydrate chainsMaximum 22 sialic acidsMW ~ 37,100 daltons51% carbohydrateKey pointsDarbepoetin alfa was rationally designed to contain an increased number of carbohydrate chains and sialic acid molecules through the creation of two additional glycosylation sites.Darbepoetin alfa contains up to eight sialic acid residues more than recombinant human erythropoietin (rHuEPO) resulting in a three-fold longer serum half-life and greater in vivo biological activity.1Darbepoetin alfa binds to and activates the same erythropoietin (EPO) receptor as endogenous EPO and rHuEPO.2BackgroundDarbepoetin alfa is a biochemically distinct molecule, which has an increased sialic acid content and thus an increased molecular weight (MW) when compared with rHuEPO. Each additional N-linked carbohydrate chain increases the MW of the protein by approximately 3,300 daltons, correlating to four additional sialic acid residues.1Thus, in comparison with rHuEPO, the two extra carbohydrate chains on darbepoetin alfa increase the MW by 22% and the maximum number of sialic acid residues from 14–22.1The result of this modification is a three-fold longer serum half-life and increased in vivo biological activity.2References1. Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.2. Macdougall IC, et al. J Am Soc Nephrol. 1999;10:
16Darbepoetin alfa has a longer half-life than rHuEPO: single-dose PK of IV administration 1001010.10.01Darbepoetin alfa (oncology; 0.5 µg/kg, n = 20)*1Darbepoetin alfa (dialysis; 0.5 µg/kg, n = 11)2rHuEPO (dialysis; 100 IU/kg, n = 10)2Mean (SD) baseline-correctedserum concentration (ng/mL)t1/2 = 38.8 hourst1/2 = 25.3 hoursKey pointsDarbepoetin alfa has a longer serum half-life than recombinant human erythropoietin (rHuEPO) in dialysis patients with anaemia (25.3 hours vs 8.5 hours).In a separate study, darbepoetin alfa was administered to patients with nonmyeloid malignancies receiving chemotherapy at a dose of 2.25 µg/kg once weekly. The normalised serum half-life for a dose of 0.5 µg/kg was 38.8 hours.1Data from a study in patients with cancer who were not receiving chemotherapy reported a serum half-life for darbepoetin alfa of 50 hours following subcutaneous administration.2BackgroundMacdougall and colleagues conducted a randomised study to compare the pharmacokinetics (PK) of a single dose of darbepoetin alfa (0.5 µg/kg) and an equimolar dose of rHuEPO (100 IU/kg) after intravenous administration to dialysis patients with anaemia.3The results of immunoassays showed that darbepoetin alfa has an approximately three-fold greater terminal half-life and decreased clearance compared with rHuEPO.Increased sialic acid-containing carbohydrate content of darbepoetin alfa resulted in a longer serum half-life.ReferenceHeatherington A, et al. Proc Am Soc Clin Oncol. 2002;21:256b. Abstract 2844.Smith R, et al. Proc Am Soc Clin Oncol. 2002;21:367a. Abstract 1465.Macdougall I, et al. J Am Soc Nephrol. 1999;10:2392–2395.t1/2 = 8.5 hoursTime post-IV injection (hours)*Oncology patients received 2.25 µg/kgData shown are normalised for 0.5 µg/kgSD = standard deviation; IV = intravenous1Heatherington A, et al. Proc Am Soc Clin Oncol. 2002;21:256b. Abstract and poster; 2Macdougall I, et al. J Am Soc Nephrol. 1999;10:
17In vivo activity in mice increases with increasing number of glycans 8059Fe incorporated(% of maximum)6040rHuEPO (three chains)20NM279 (four chains)Key pointsA bioassay of erythropoietic activity in mice was used to compare the potency of three moleculesrecombinant human erythropoietin (rHuEPO), with three N-linked carbohydrate side chainsNM279, with four side chainsdarbepoetin alfa, with five side chains.In this assay, the incorporation of radioactive iron (59Fe) into animal red blood cells was measured at various concentrations of test molecule, and the results plotted on a logarithmic scale.The assay showed that as the number of carbohydrate side chains increased from three to four to five, the erythropoietic response shifted progressively to the left.As these results were plotted on a logarithmic scale, it became clear that the addition of sialic acid residues to darbepoetin alfa is associated with a dramatically increased erythropoietic activity in vivo.Darbepoetin alfa (five chains)101001,00010,000100,0001,000,000ng/mL sample
18The anti-EPO monoclonal Ab F12 does not neutralise EPO bioactivity Effect of antibodies on EPO in vitro bioactivity120100Non-neutralising anti-EPOmonoclonal Ab (F12)Neutralising anti-EPOmonoclonal Ab (D11)polyclonal Ab (862)8060In vitro activity (%)*40Key pointsAlthough intuition suggested that an antibody that may bind to the altered peptide sequence of darbepoetin alfa would not be neutralising, this concept required testing. An attempt was therefore made to develop experimental assays that would provide quantitative evidence.For this experiment it was necessary to use recombinant human erythropoietin (rHuEPO) as F12 does not bind to darbepoetin alfa. rHuEPO was incubated with two known neutralising antibodies as well as F12, the antibody directed against the peptide region that was changed to produce darbepoetin alfa. With increasing concentrations of either of the known neutralising antibodies, the bioactivity of rHuEPO decreased sharply. By contrast, even when the concentration of F12 in the incubation medium was increased a thousand-fold, the rHuEPO bioactivity remained intact.The experiment with F12 shows that, whether shielded by glycans or not, the peptide region at which rHuEPO has been changed to produce darbepoetin alfa does not provoke a neutralising antibody effect. These findings suggest that, even if it were possible to generate an antibody to the polypeptide that was changed to make darbepoetin alfa, the bioactivity of darbepoetin alfa may not be neutralised.20*In vitro activity assay measures formation of erythroid colonies from human bone marrow in soft agar0.0010.010.1110100Amount of Ab added (µg/mL)EPO = erythropoietin; Ab - antibody
19Development of darbepoetin alfa A new erythropoietic protein, biochemically distinct from rHuEPOIncreased sialic acid content, resulting ina longer circulating half-life (2–3-fold greater than rHuEPO)less frequent dosing requirementsincreased biological activityPharmacokinetics offer potential for higher response rates and faster onset of actionKey pointsThe discovery of darbepoetin alfa resulted from basic research on the structure and function of recombinant human erythropoietin (rHuEPO) and its attached carbohydrate.Darbepoetin alfa has similar immunoreactivity, folding and stability properties to rHuEPO, although it has been altered at five amino acid positions, and has five N-linked carbohydrate side chains.The increased serum residence time and in vivo bioactivity of darbepoetin alfa derive from its increased sialic acid content, resulting from two additional carbohydrate chains. These properties have been confirmed in both preclinical and clinical studies.The greater potency of darbepoetin alfa and its longer duration of action allow it to be dosed less frequently than rHuEPO. The convenience of administering darbepoetin alfa at extended intervals benefits both patients and healthcare professionals.The above characteristics also have clinical implications in terms of potentially enabling darbepoetin alfa to produce higher and faster responses (increases in haemoglobin) than rHuEPO.
20Darbepoetin alfa: conclusions Darbepoetin alfa has a similar conformation to rHuEPODarbepoetin alfa binds to and activates the same receptor as rHuEPODarbepoetin alfa has increased sialic acid-containing carbohydrate resulting in increased in vivo activity and a prolonged half-life1Provides the opportunity to dose less frequently: QW, Q2W, Q3W or Q4W2–4Clinical benefits have been demonstrated (high and rapid haematological responses at convenient dosing schedules)5Key pointsDarbepoetin alfa was rationally designed to have a similar conformation to recombinant human erythropoietin (rHuEPO) and to bind to and activate the same erythropoietin receptor.Darbepoetin alfa has increased sialic acid-containing carbohydrate content resulting in increased in vivo activity and allowing for less frequent administration.1BackgroundThe greater potency of darbepoetin alfa and its longer duration of action allow it to be dosed less frequently than rHuEPO.2–5 The convenience of administering darbepoetin alfa at extended intervals benefits both patients and healthcare professionals.1Egrie JC, et al. Br J Cancer. 2001;84(suppl 1):3-10.2Glaspy JA, et al. Br J Cancer. 2002;87:3Kotasek D, et al. Eur J Cancer In press. 4Kotasek D, et al. Proc Am Soc Clin Oncol. 2002;21:356a. Abstract 1421.5Glaspy JA, et al. Cancer. 2003;97:QW = once every week; Q2W = once every 2 weeks; Q3W = once every 3 weeks; Q4W = once every 4 weeks