- Email: [email protected]
Recombinant proteins in rheumatology – recent advances
New Biotechnology Volume 28, Number 5 September 2011
REVIEW
Review
Recombinant proteins in rheumatology – recent advances Achim Rothe and Andrea Rubbert Department of Internal Medicine I, University Hospital Cologne, Joseph Stelzmann Street 9, 50931 Cologne, Germany
New targeted anti-inflammatory drugs have revolutionized the therapeutic strategies in rheumatology. Recombinant DNA selection technologies have enabled the isolation and humanization of specific antibody fragments of any specificity that can be ‘armed’ to deliver effective anti-inflammatory ‘payloads’. Antibodies and other targeted provide the opportunity to block key soluble mediators of inflammation in their milieu, or alternatively to block intracellular inflammation-triggering pathways by binding to an upstream cell-surface receptor. Designed proteins can be improved with respect to desired pharmacokinetic and pharmacodynamic effects. They facilitate the delivery of the required immunosuppressive effect. However, the individual extent of desired and undesired effects of a particular biologic therapy can be broader than initially predicted and requires careful evaluation during clinical trials. This review highlights advances in the application of recombinant antibody technology for novel biologic therapies in rheumatology. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The engineering of biologic modulators. . . . . . . . . . . . . . . . . . . . . . . . . . . Intact antibodies and antibody fragments . . . . . . . . . . . . . . . . . . . . . . . Fusion proteins with antibody Fc fragments (immunoadhesins). . . . . . . The generation of biologic immune modulators. . . . . . . . . . . . . . . . . . . . . Humanization and transgenic mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . Library and display technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Affinity maturation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical and clinical applications of targeted biologics in rheumatology . Inhibition of cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-cell ablation or modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benefits and risks of biologics in rheumatology . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
503 503 503 504 504 504 504 504 504 505 505 507 508 508 508 508 509
Corresponding author: Rothe, A. ([email protected])
502
www.elsevier.com/locate/nbt
1871-6784/$ - see front matter. Crown Copyright ß 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.nbt.2011.03.019
REVIEW
Introduction Rheumatic diseases are characterized by aberrant cellular and humoral immune responses. Accordingly, the primary goal for protein-based therapeutics in rheumatology is to target and remediate the disease-causing upregulated cytokines, to block cell surface receptors that are crucially involved in initiating and maintaining the inflammatory cascade and to counteract autoimmune T and B cells. This review summarizes the molecular structures and design of antibody-based immune-modulators and discusses their production and properties as therapeutic agents. In addition, therapeutic formulations, focusing on the clinically most relevant targets and current in-trial status (TNF, IL-6, Table 1) are described.
The engineering of biologic modulators Intact antibodies and antibody fragments Intact antibodies, such as the Y-shaped IgG, are highly specific targeting reagents and provide congenital defense against patho-
genic organisms and toxins. So far, almost all engineered therapeutic antibodies exclusively use the bivalent format of IgG. The antigen-binding domains are located on the two antigen binding (Fab) tips, which enable the antibody to bind to two antigens simultaneously. This feature increases their functional activity and influences their pharmacokinetic profile, as described later. Recruitment of effector functions, such as complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent phagocytosis, is mediated by the fragment crystallizable (Fc) domain through interactions with Fc receptors (Fc receptors for globulins). The Fc domain also provides the antibody with an extended circulation time through recycling within vascular endothelial cells via the neonatal Fc receptor (FcRn) [1]. Because of its two binding surfaces and Fc domain, intact IgG antibodies can result in cross-linking of soluble or cellsurface target antigens. The inclusion of post-translational modification, such as glycosylation, can also be used to profoundly alter the circulation time of recombinant proteins [2].
TABLE 1
Antibody- and receptor-based biologics that have been approved or are preclinical or in-trial for rheumatologic diseases Format
(Generic) name
Target
Maximum serum half-life
Indication in rheumatology
Stage
Company/ developer
Chimeric IgG Humanized IgG ScFv-Fc (SMIP) Human IgG Ligand-Fc Chimeric IgG
Rituximab Epratuzumab TRU-015 Denosumab OPG-Fc Infliximab
CD20 CD22 CD20 RANKL RANKL TNF
16 14 12 32 N/A 10
Approved Phase II/III Phase II Phase III Preclinical Approved
Genentech UCB Trubion/Wyeth Amgen Amgen Centocor
Human IgG
Adalimumab
TNF
13 days
Approved
CAT/Abbott
Human IgG
Golimumab
TNF
13 days
Approved
Centocor
Receptor-Fc
Etanercept
TNF
3 days
Approved
Amgen/Wyeth
Fab-PEG
Certolizumab
TNF
14 days
dAB
TNF
Ligand-Fc
Atacicept (TACI-Fc) Belimumab Tocilizumab ALD518 (BMS-945429) Anakinra
BLyS
Tailore days through protein engineering 63 days
Approved (Phase II/III) Preclinical
UCB Pharma
Human variable domain
RA SLE RA Bone erosion in RA Bone erosion in RA RA, Ankylosing spondylitis, Psoriasis arthritis RA, Ankylosing spondylitis, Psoriasis arthritis RA, psoriatic arthritis, ankylosing spondylitis RA, (ankylosing spondylitis, Psoriasis arthritis) RA (ankylosing spondylitis, Crohn) Autoimmune diseases
RA, SLE
Phase II/III
BLyS IL-6R IL-6
16 days 10 days 30 days
RA, SLE RA RA
Phase III Phase III Phase II/III
Merck Serono/ ZymoGenetics GSK/HGSI Roche/Chugai BMS
IL1-receptor
4–6 h
RA
Approved
Amgen
Canakinumab
IL-1ß
14 days
CAPS, (Gout)
Novartis
Human IgG
Secukinumab
IL-17
14 days
Receptor-Fc
Rilonacept
IL-1
8.6 days
RA, psoriasis, ankylosing spondylitis CAPS (Gout)
Approved (Phase II/III) Phase II
Receptor-Fc Ligand-Fc Receptor-Fc
BR-3-Fc Abatacept Aflibercept (VEGF-Trap) Bevacizumab
BAFF CD80/86 VEGF
15 days 23 days 14 days
VEGF
20 days
Humanized IgG Humanized IgG Humanized IgG Modified receptor antagonist Human IgG
Humanized IgG
days days days days days
Domantis/Peptech
Novartis
RA, SLE RA –
Approved (Phase II) Phase I Approved –
Regeneron Biogen IDEC/Genentech Orencia/Bristol-Myers Squibb Regeneron/Sanofi Aventis
–
–
Genentech/Roche
This table is based on information in the public domain but is not comprehensive, due to the confidential embargo of company commercial information. Abbreviations: BAFF, B-cell activating factor; BLyS, B-lymphocyte stimulating factor; Fc, fragment crystallizable region; IL, interleukin; RA, Rheumatoid arthritis; RANKL, receptor activator of nuclear factor kB ligand; ScFv, single chain variable fragment; SLE: Systemic lupus erythematosus; TACI, transmembrane activator and calcium-modulating and cyclophilin interactor; TNF, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.
www.elsevier.com/locate/nbt
503
Review
New Biotechnology Volume 28, Number 5 September 2011
REVIEW
Review
Antibody fragments (e.g. monovalent Fab fragments) and engineered antibody designs (e.g. single-chain variable fragments [scFv], diabodies, triabodies, minibodies and single-domain antibodies) have proven to be valid alternatives to full-size IgG antibodies [3]. Fab fragments may be coupled to polyethylene glycol (PEG) that extends the half-life of the drug but may avoid potentially Fc-mediated side effects. PEGylation may facilitate accumulation of the compound in inflamed tissues [4]. In contrast to fullsize IgG antibodies, PEGylated Fab fragments will most probably not cross the placenta. Receptor antagonists (nonimmunoglobulin protein scaffolds) protein engineering has enabled the production of a wide range of different nonimmunoglobulin scaffolds with diverse origins, biodistribution and pharmacokinetic properties, such as the recombinant IL-1 receptor antagonist anakinra, which is approved for the treatment of patients with active rheumatoid arthritis (RA) [5]. Anakinra is a small recombinant, nonglycosylated 153-residue (17 kDa) domain of the human IL-1 receptor antagonist. The majority of these nonimmunoglobulin scaffolds, such as ankyrins and anticalins are also small, single protein domains [6] and are less than half the size of the 30 kDa scFv antibody fragment, which comprises two 14 kDa V-domains. Many of these small novel scaffolds have stability and production advantages over antibody domains because they do not require disulfide bonds and posttranslational modifications. Their immunogenicity in therapeutic formulations, however, is still under early evaluation [6].
Fusion proteins with antibody Fc fragments (immunoadhesins) As an alternative to antibodies or scaffolds, the extracellular soluble domain of a cell-surface receptor can be fused with the Fc domain of an antibody, thus combining the unique binding specificity of the receptor domain with the capacity to mediate antibody effector functions or to increase serum half-life. These constructs commonly represent more stable and easier to produce compounds than their original physiological structures. Etanercept is a TNF receptor-Fc fusion protein and the most successful engineered immunoadhesin for RA therapy as yet [7]. Abatacept represents a fusion protein of soluble CTLA-4 (cytotoxic lymphocyte antigen-4) and the Fc fragment of IgG1 and inhibits T cell costimulation. Abatacept is currently approved for patients with active RA who failed to methotrexate and/or TNF inhibitors. Another example is the transmembrane activator and calciummodulating and cyclophilin interactor (TACI)-Fc fusion protein atacicept, which similarly comprises the extracellular binding domain from the TNF receptor family and has been studied for the treatment of arthritis, SLE and multiple sclerosis [8]. Fv-like fragments of T-cell receptors (single-chain T-cell receptor) can also be fused to IgG1 heavy chains to create properties similar to monoclonal antibodies, but that are targeted to antigens derived from intracellular targets [9].
The generation of biologic immune modulators Humanization and transgenic mice Murine monoclonal antibodies provide very effective therapeutics but their deleterious immune response can prevent multiple administrations. A preferred method of reformulation is ‘humanization’, which involves grafting the murine Fab/Fv (targeting regions) onto a human Fc backbone. To generate a fully human 504
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
antibody, transgenic mice have been developed that lack the native mouse immune repertoire and instead harbor most of the human antibody repertoire in their germline. A humoral immune response in these mice, following the injection of an antigen, leads to the development of human antibodies that can be recovered by classic hybridoma technology. The transgenic mice also have a fully operational affinity maturation system of somatic hypermutation and selection, so the resultant human antibodies have high affinity [10].
Library and display technologies As an alternative to using live animals, antibody library selection (entirely in vitro) has become a fast process for the discovery of human antibodies, using either natural or synthetic immune repertoires [11,12]. Specific high-affinity antibodies can be selected by linking phenotype to genotype in vitro, which results in gene isolation after a stepwise enrichment process based on antibody affinity. Adalimumab, a fully human antibody to TNF, which has FDA approval for the treatment of RA was selected from human phage display libraries using a guided selection strategy [11]. In phage display, the recombinant library is expressed as fusion constructs on the surface of phage particles before entering the selection process. Antibodies are generally displayed as monovalent fragments, and in vitro display technology can effectively supersede natural in-cell selection. Several technologies are now available for display and selection of high-affinity antibody fragments, including ribosome, messengerRNA and DNA linkages that lead to the isolation of a vast range of engineered protein-based reagents [12].
Affinity maturation The improvement of the binding characteristics of a low-affinity binding molecule is achieved by an affinity maturation process involving mutation followed by selection of the higher-affinity mutants. Random mutagenesis, as well as engineered interface mutations, have been proven effective for affinity maturation [13– 15]. In vitro display systems provide ideal tools for inherent affinity maturation. The fully human antibody belimumab, which is directed against BLyS, was isolated from a naive human phage display library [12,16]. Affinity optimization was performed by randomization of the last six amino acids of the heavy-chain CDR3 region. Belimumab is currently being tested in clinical trials for the treatment of rheumatic diseases and approval for use in SLE is expected [17].
Production The production of stable, high-affinity antibody fragments in high yield for preclinical and clinical trials can be elaborate and expensive. Expression systems, including bacteria, yeasts, plants, insect and mammalian cell lines and even cloned transgenic animals, have been evaluated and compared [18]. Presently, the preferred media for the bulk production of recombinant proteins are devoid of proteins or components that can elicit deleterious immunogenicity in the final recombinant product. Bacteria are favored for the expression of small, nonglycosylated antibody fragments, but yields can vary for each construct. Mammalian or plant cells are favored hosts for high-yield expression of larger intact antibodies and minibodies due to the efficient protein folding processes
New Biotechnology Volume 28, Number 5 September 2011
provided by the eukaryotic endoplasmic reticulum. Staphylococcal protein A affinity columns facilitate the purification of Fcfusion proteins. Several strategies have been developed to simplify recombinant antibody expression and purification, such as terminal polypeptide tags for affinity purification, although this adds a complication of potential, unwanted immune responses. The production of smaller non-immunoglobulin scaffolds, however, can be cheap and efficient, because they can be efficiently expressed in bacteria or produced by chemical synthesis.
REVIEW
of actions of a new biologic might be well-characterized, acute or long-term adverse events are unpredictable. Initial toxicity and immunogenicity experiments are commonly performed in rodents; however, the rodent immune system is distinct and different to humans. The establishment of settings closer to a human system such as transgenic, knock-in models or human cord-blood transplanted mice [26] would be of great benefit. In addition, computer-based prediction of immunogenicity should be utilized at an early stage in the process of engineering human biopharmaceuticals [27].
Pharmacokinetics
Preclinical and clinical applications of targeted biologics in rheumatology Targeting cell-surface receptors B cells play a crucial role in autoimmune diseases such as rheumatoid arthritis, SLE or ANCA associated vasculitis. They may not only be regarded as the source of autoantibodies, but also function as antigen presenting cells and producers of proinflammatory cytokines [4]. The therapeutic chimeric anti-CD20 antibody rituximab has been approved for the treatment of patients with active RA who have failed to TNF inhibitors. Rituximab does not only improve signs and symptoms of disease, but also inhibits radiographic progression and improves physical function. It has been recently demonstrated that rituximab induces a significant decrease in RANK+ osteoclasts within the synovial tissue [28]. Not surprisingly, the likelihood of achieving a clinically relevant response is significantly greater in patients who are rheumatoid factor and/or CCP positive [29,30]. Rituximab treatment may well be judged as the most efficient blockade against recirculating and potentially pathologic B cells eventually enabling ‘re-booting’ of the immune system. Rituximab can generally be administered repetitively without major side effects; although a noteworthy first-dose reaction has been observed so commonly that the infusion rate is now increased slowly to minimize possible allergic reactions [29]. Antichimeric antibodies to rituximab have been occasionally reported but do not generally seem to cause treatment abrogation. Different mechanisms of action have been proposed, ranging from B-cell depletion to the generation of decoy cellular immune complexes [31]. The most crucial issue for successful CD20 immunotherapy is to dictate the most effective engagement strategy for the surface CD20 epitope as modulated by the choice of antibody and choice of effector function [32]. Therefore, targeting the same cell surface protein will not necessarily result in comparable clinical efficacy and safety. Ocrelizumab as a humanized anti-CD20 directed monoclonal antibody has been evaluated in randomized controlled trials in RA but its development for RA and SLE was recently stopped because of an unfavorable balance between efficacy and safety [33]. However, Ocrelizumab is still being explored for patients with multiple sclerosis. Ofatumumab (HuMax-CD20R) represents a fully human antiCD20 directed monoclonal antibody that is currently in clinical development. SLE represents a prototypic autoimmune disorder that is characterized by potential involvement of multiple organs and the presence of autoantibodies. Retrospective cohort analyses and www.elsevier.com/locate/nbt
505
Review
The evaluation of pharmacokinetics is most easily performed with radiolabeled antibodies, which not only are important clinical therapeutic reagents, but also represent effective tools for in vivo imaging [19]. Intact IgG antibodies have the ability to bind to two antigens, which greatly increases their functional affinity and confers high-retention times (avidity) on many cell-surface receptors and polyvalent antigens. The Fc component of intact antibodies also provides long serum half-lives (>10 days) through interaction with the FcRn, as discussed previously. This FcRn binds and transports IgGs both within and across cells and rescues them from a default degradative pathway [20,21]. For example, serum rituximab levels can still be detected 3 months after the last infusion [22]. By contrast, smaller proteins, such as antibody fragments, exhibit a short serum half-life, which can be extended by the addition of polyethylene glycol (PEG; pegylation). Pegylation of a protein increases the apparent size through hydration and is a simple process that can be completed in 1 day and the pegylated protein can be purified in hours. This approach has been successfully applied to antibody fragments and other proteins and peptides, without destroying their tertiary structure or interfering with their biologic activity [23,24]. Certolizumab pegol consists of a Fab fragment of a humanized anti-TNF coupled to PEG with a serum half life of around 14 days and represents a new TNF inhibitor that is currently approved for rheumatoid arthritis and Crohn’s disease. Pharmacokinetics relies on computational algorithms to assess the appropriate characteristics for cell targeting by inclusion of rates for diffusion, binding, internalization and systemic clearance [25]. In tumor targeted therapy the size of an antibody-based therapeutic is crucial for penetration into the solid tumor mass. This feature is eventually less important in rheumatology, where target antigens are often circulating and may be generally more accessible. However, treatment of arthritis may require that therapeutic antibodies or compounds get access into the synovium where proinflammatory cytokines and activated lymphocytes are abundant. Anti-TNF antibody therapy, for example, is associated with a spectrum of anti-inflammatory effects and the concentration of adalimumab in the synovial fluid after intravenous administration reaches 31–96% of the serum concentration. The chronic nature of rheumatologic diseases necessitates longterm biopharmaceutical intervention; accordingly, any associated adverse events should be carefully considered and addressed. Removing the Fc domain in intact antibodies or immunoadhesins certainly abrogates many of the associated side effects, but might also impair the efficacy of the therapy. This leaves a small therapeutic window between loss of efficacy and the associated toxicity mediated by the Fc-domain. Thus, although the mechanisms
REVIEW
Review
open-label Phase I/II trials suggested efficacy of using rituximab as monotherapy or in combination with cyclophosphamide in patients with active SLE who failed to standard therapy. However, the placebo-controlled Phase II/III trials LUNAR and EXPLORER in patients with SLE failed to meet their primary and secondary endpoints (Genentech/Biogen IDEC press release 2008/2009). Possible reasons may have been the relatively short duration of the trials, the suitability of the outcome parameters selected and the confounding effects of oral steroids that the patients had to take initially. Apart from case reports and case series, a recent randomized controlled trial (RAVE) has demonstrated that rituximab has comparable efficacy to cyclophosphamide in patients with ANCA-associated vasculitis [34]. Based on these results, rituximab will hopefully be approved for use in this indication. The HERMES trial represents a Phase II study in patients with relapsing-remitting multiple sclerosis and has demonstrated significant reductions in gadolinium-enhancing cerebral lesions and prevention of clinical relapses following a single course of rituximab [35]. Of note, a placebo-controlled Phase II/III trial of rituximab in primary progressive multiple sclerosis (OLYMPUS) did not prove efficacious (Press release Genentech/Biogen IDEC 2008).
[()TD$FIG]
New Biotechnology Volume 28, Number 5 September 2011
Numerous case reports and cohort studies have reported on the benefit of rituximab in various other autoimmune disorders such ¨ gren’s syndrome, myositis, mixed cryoglobulinemia, as Sjo myasthenia gravis and pemphigus. Of note, levels of autoantibodies do not necessarily predict response to rituximab or correlate with the clinical response to rituximab. A different approach targeting the CD20 receptor and designed to balance effector functions, such as antibody-dependent cellmediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), uses the fusion protein of an anti-CD20 scFv antibody fragment to the human IgG Fc-domains CH2 and CH3. This so-called small modular immunopharmaceutical TRU-O15 had been evaluated in clinical trials for the treatment of RA. The IL-1 receptor antagonist anakinra represents an example of a modified, nonglycosylated form of a natural ligand (IL-1), in this case for competitive blockade of the IL-1 receptor. Because of a superior efficacy of other biologics in RA, the use of anakinra in RA has decreased. Interestingly, anakinra has been reported to be highly efficacious in Still’s disease and hereditary fever syndromes [36]. Receptor blockade of RANKL activity is a key intervention strategy relevant for both osteoporosis and bone destruction in arthritis [37]. An Fc-fusion protein linked to the RANKL physiological decoy
FIGURE 1
Structural comparison of engineered, targeted monoclonal antibodies, fusion protein and PEGylated Fab fragment directed against TNFa (adapted from Weir et al. Therapy 2006;3:535–545). 506
www.elsevier.com/locate/nbt
receptor osteoprotegerin (OPG-Fc) blockades RANKL activity, and a comparison of this format to the fully human anti-RANKL antibody denosumab is reviewed by Tarner and colleagues [38,39]. Denosumab has recently been approved for the treatment of osteoporosis in men and postmenopausal women. Interestingly, a randomized controlled clinical trial of denosumab in RA revealed that denosumab significantly inhibited erosive joint destruction, but had no benefit regarding disease activity [40].
Inhibition of cytokines TNF is an inflammatory cytokine produced by cell compartments of the innate and adaptive immune system, which contributes to the pathogenesis of various rheumatic diseases. High TNF levels may be detected in the synovial fluid and synovial tissue of patients with RA, where TNF triggers cell proliferation and the production of other proinflammatory cytokines. TNF inhibitors are well-established in the therapy of various rheumatic diseases by neutralizing soluble and membrane-bound TNF and directing cell death either through cytokine deprivation or through Fc-mediated mechanisms [41]. Anti-TNF complexes have been postulated to also have an agonistic effect on cytokine production via binding to cell-surface Fcg receptors, for example on natural killer cells [42]. For clinical use, currently five different TNF inhibitors are available (Fig. 1). The chimeric monoclonal antibody infliximab and the human antibodies adalimumab and golimumab are IgG anti-TNF agents (i.e. intact, full-size antibodies). By contrast, etanercept represents a fusion protein of the soluble TNF-receptor and the Fc domain of human IgG and certolizumab is a pegylated, humanized anti-TNF Fab fragment [42,43]. The latter Fab construct (certolizumab) lacks the Fc-portion and thus does not induce apoptosis through complement activation or ADCC. Differences among the available TNF inhibitors relate to the molecule structure, application (sc versus iv) or half-life. TNF inhibitors have successfully been used in RA, psoriatic arthritis, ankylosing spondylitis, juvenile arthritis, Crohn’s colitis or psoriasis. At present, not all of them are approved for all indications. Of note, if patients show intolerance to one TNF blocker (injection site reaction to the sc compounds, infusion reaction to infliximab) or if they develop antibodies to one compound that results in secondary inefficacy, switching within the class of TNF inhibitors may represent a therapeutic option. An increased risk for serious infections and tuberculosis as well as opportunistic infections, the development of malignant lymphoma and inflammatory demyelinating disease has been reported [17]. Screening for latent tuberculosis before starting a TNF inhibitor is mandatory. In patients with a history of demyelinating disorders or in patients with cardiac insufficiency, TNF inhibitors are contraindicated. Several national registries (Germany, Great Britain and Sweden) have been established and have followed patients on TNF inhibitors for more than 10 years. These registries have not revealed a higher incidence of malignancies associated with the use of TNF blockers. However, potentially other therapeutic options may be preferred in patients with a history of a recent malignancy. The smallest available anti-TNF biologics are single-domain proteins that potentially offer a greater penetration (and hence affinity) to TNF. One human single-domain antibody (14 kDa) originally developed by Domantis, is now undergoing a Phase II trail as ART621 (Arana Therapeutics Ltd.) but very limited data are
REVIEW
currently available [3,44]. Domain antibodies are also engineered for long-term serum half-life either through pegylation, fusion with a carrier protein or through expression as multivalent formats. In one example a domain antibody targeting human serum albumin was recently fused to an IL-1-receptor antagonist and considerably extended the half-life of this receptor blocker resulting in a potentially more efficient arthritis therapy [45]. The blockage of cytokines that increase inflammation represents an attractive strategy for the development of targeted protein-based therapy. IL-1 represents a key player in inflammation; however, the term IL-1 refers to two cytokines, namely IL-1a and IL-1b that are encoded by different genes. IL-1 effects are potentially counteracted by endogenous inhibitors such as the IL-1 receptor antagonist (IL1Ra) and the IL-1 receptor type II (Il-1RII). Inhibition of IL-1 by anakinra or canakinumab, a human antibody directed against IL-1b, has been studied mainly in RA with limited success. Interestingly, the blockade of IL-1 has proven significant efficacy in other disorders, mainly cryopyrinopathies, Still syndrome and IL-1Ra deficiency. Canakinumab and rilonacept (IL-1 Trap) have recently been approved for patients with cryopyrin-associated periodic syndromes (CAPS). More recently, IL-1 appears as an attractive target in patients with refractory gout based on the findings that urate crystals induce inflammation through the NALP3 inflammasome that leads to IL-1 production. Currently, in addition to anakinra, clinical trials have been set up to assess rilonacept and canakinumab on gout flare treatment and prevention. IL-6 has recently been characterized as a pleiotropic driver in acute and chronic inflammation. IL-6 mediates its effects by binding to a soluble or membrane bound IL-6 receptor (IL-6R), that may then assemble with gp130, that is necessary for signal transduction. IL-6 may therefore be targeted by monoclonal antibodies directed to either the receptor, an example of this is the chimeric antibody tocilizumab, or directed against IL6 itself. IL-6 has been shown to be crucial for joint inflammation in arthritis, but is also crucial for systemic features of inflammation such as fever, anemia, fatigue and the raise of acute-phase proteins. Tocilizumab has recently been approved in patients with active rheumatoid arthritis who failed to conventional DMARDs and/or TNF inhibitors and has proven highly efficacious in patients with juvenile arthritis. Tocilizumab represents also a promising therapeutic option for other autoimmune diseases such as adult-onset Still’s disease, Crohn’ disease and Takayasu arteritis. Recent studies have demonstrated the importance of IL-12 and IL-23 in the immunopathogenesis of psoriasis. The p40 subunit common to IL-12 and IL-23 is an attractive target for treatment of psoriasis and psoriatic arthritis. Two compounds, ustekinumab (approved in 2009 for treatment of plaque psoriasis) and ABT-874 have been studied and resulted in significant improvement of cutaneous lesions. Of the cytokines relevant to autoimmunity, IL-17 and its family have recently received attention, as in murine models of autoimmune diseases, IL-17 producing Th17 cells play a pivotal role in pathogenesis. The role for IL-17 in driving human autoimmune diseases is less clear, but IL-17A may be detected in synovial fluid of RA patients and in T cell rich areas of RA synovial tissue. Thus far, monoclonal antibodies have been developed and are currently being assessed in rheumatoid arthritis, psoriasis and ankylosing www.elsevier.com/locate/nbt
507
Review
New Biotechnology Volume 28, Number 5 September 2011
REVIEW
Review
spondylitis. Other IL-17 directed approaches may include subunits of the IL-17 receptor complex (IL-17RA and IL-17RC) [46]. Cytokines such as BLyS and a proliferation-inducing ligand (APRIL) are required for B-cell maturation and may contribute to autoantibody production and disease severity. A fusion protein consisting of the BLyS receptor TACI coupled to a Fc-domain has been shown to decrease serum levels of rheumatoid factors, but significant amelioration of patients’ symptoms was not observed. The humanized anti-BLyS antibody belimumab [47] was isolated from a naive human phage display library [12] and its affinity optimized by randomization and selection of the best six amino acids of the heavy-chain CDR-3 region. Even though initial trials in RA were disappointing, belimumab has achieved excellent Phase III trial for the treatment of systemic lupus erythematosus [48]. A B-cell modulation approach with a construct similar to etanercept uses a fusion protein of the B-cell activating receptor BR3 and a Fc domain of a human IgG1 to target the B-cell activating factor BAFF (BR3-Fc). By trapping soluble BAFF, this construct inhibits B-cell activation and leads to apoptosis [49]. Data from clinical trials have not yet been reported.
T-cell ablation or modulation Monoclonal antibodies targeting T-cell surface antigens, such as CD4, CD5 and CD52 have not shown clinical benefit for rheumatoid diseases [41]. However, abatacept, an Fc-fusion protein targeting CD28 has been approved for the treatment of patients with RA refractory to MTX and/or TNF inhibitors [50]. Abatacept combines the extracellular domains of the cytotoxic T-lymphocyte-associated antigen 4 with the Fc-portion of an IgG1 without the complement activating properties. By binding to the co-stimulatory molecules CD80/CD86 on antigen-presenting cells with greater avidity than CD28, abatacept blocks CD28 binding and increases the threshold for T-cell activation [50]. In 2006, TGN1412, manufactured by TeGenero Immuno Therapeutics, an activating anti-CD28 antibody, came into public prominence when clinical trials led to multi-organ failure and cytokine storm after first administration in human [51]. A massive T-cell activation through FcgR crosslinking by the Fc-domain of the agonistic TGN1412 antibody might have contributed to these drastic effects. By contrast, abatacept offers a different modulation response and only causes serious adverse effects when combined with other disease-modifying biologic agents [50].
Angiogenesis inhibition Angiogenesis is involved in the pathogenesis of inflammatory arthritis. Consequently, the levels of its mediator, VEGF, are elevated in the synovial fluid of RA patients, [52] whereas levels are reduced in patients treated with TNF-antagonists [53]. VEGFantagonists, such as the VEGF-Trap molecule consisting of a bait receptor based on VEGFR1 and VEGFR2 fused to an IgG1 Fcfragment [54] and the anti-VEGF antibody bevacicumab [55] may represent promising biologics for future evaluation in rheumatic diseases [52]. Indeed, single domains are also being exploited to bind and block VEGF activity [56].
Benefits and risks of biologics in rheumatology Engineered proteins, most commonly based on antibodies or antibody-like scaffolds, are now providing a new wave of thera508
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
peutic products. Indeed, the biotechnology industry is embracing this opportunity because designed biologics now outnumber and surpass the number of new small-molecule drugs approved annually by FDA. Antibodies and immunoadhesins that directly target cytokines for their systemic removal (ligand ablation) have become an effective therapeutic strategy (e.g. adalinmumab and infliximab), and in some indications the selective targeting of cytokine receptors (e.g. anakinra) can deliver a highly effective clinical outcome. Powerful display technologies have enabled the rapid isolation of fully human proteins, often with novel and unique targeting specificities. Some display systems, such as ribosome or messenger RNA display, even enable a simultaneous affinity maturation process and have derived new biologics with exceptionally high binding affinities. Antibody fragments can be further engineered as multivalent constructs with increased functional affinity. Fc-fusion proteins mediate effector functions through their constant domain and these properties can now be fine-tuned by molecular design. In addition, Fc-fusions offer an increased size advantage and an extended serum half-life [1]. The high affinity and avidity of engineered protein-based biologics, however, might not always be associated with maximum efficacy and the best safety profile. We postulate that a construct of lower affinity might equally translate its effect and presumably be even more versatile through consecutive binding-release events. When administering biologics, and in respect of the past experiences with intact antibodies and Fc-fusion constructs, one has to consider that in vivo effects are unpredictable. Even when animal trials demonstrate a safe profile, there remains uncertainty when administering a highly active targeted protein into a human, as demonstrated by the extreme reaction to the CD28 activating antibody TGN1412 [57]. Independent of the structure and the origin of a biologic agent, immunogenicity is often unexpected and unpredictable. For example, even the fully human antibody adalimumab invokes an anti-human-antibody response in 12% of patients when administered as monotherapy [41]. Antibodies to therapeutic antibodies may be associated with a secondary loss of efficacy as reflected by lowering of serum concentrations. Antiidiotype-responses against the antigen-binding surface, commonly of neutralizing character, and anti-isotype responses against the constant domains may be detected. Nevertheless, a serious systemic inflammatory response syndrome in humans, such as that found to TGN1412, is rare. Furthermore, engineered protein-based biologics have widened the therapeutic spectrum for rheumatic diseases and facilitate effective immunotherapeutic strategies as a potent addition to the classic disease-modifying antirheumatic drugs. If we learn from previous mistakes made and incorporate the knowledge of protein structure using recent technological advances in protein engineering the impact of engineered biologics in rheumatology in the future will be immense.
Conclusion Biologic reagents have significantly broadened the therapeutic options for patients with rheumatic diseases. New engineered antibody/based drugs aimed at different targets in the inflammation cascade. They are able to block signaling pathways at definite, predicted stages with different efficacies. Safety issues and adverse events such as a possible increased risk of infection or secondary malignancies (resulting from the reduction of natural immuno-
New Biotechnology Volume 28, Number 5 September 2011
surveillance) have occurred and need to be addressed. The limiting effect of downstream alternative signaling, biasing the therapeutic effect, has to be considered when developing biologics blocking pathways at the intercellular or cellular level. Nevertheless, specific
REVIEW
biologic reagents represent novel and powerful mediators of anti/ inflammatorial effects in rheumatological therapy and engineered antibodies are widely used in the clinical routine as essential therapies against a range of rheumatoid diseases.
1 Olafsen, T. et al. (2006) Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment. Nat. Protoc. 1, 2048–2060 2 Walsh, G. and Jefferis, R. (2006) Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 24, 1241–1252 3 Holliger, P. and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136 4 Dorner, T. et al. (2009) B-cell-directed therapies for autoimmune disease. Nat. Rev. Rheumatol. 5, 433–441 5 Cohen, S.B. et al. (2004) A multicentre, double blind, randomised, placebo controlled trial of anakinra (Kineret), a recombinant interleukin 1 receptor antagonist, in patients with rheumatoid arthritis treated with background methotrexate. Ann. Rheum. Dis. 63, 1062–1068 6 Hosse, R.J. et al. (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci. 15, 14–27 7 Moreland, L.W. et al. (1997) Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N. Engl. J. Med. 337, 141–147 8 Wang, H. et al. (2001) TACI–ligand interactions are required for T cell activation and collagen-induced arthritis in mice. Nat. Immunol. 2, 632–637 9 Mosquera, L.A. et al. (2005) In vitro and in vivo characterization of a novel antibodylike single-chain TCR human IgG1 fusion protein. J. Immunol. 174, 4381–4388 10 Lonberg, N. (2005) Human antibodies from transgenic animals. Nat. Biotechnol. 23, 1117–1125 11 Hoogenboom, H.R. (2005) Selecting screening recombinant antibody libraries.. Nat. Biotechnol. 23, 1105–1116 12 Rothe, A. et al. (2006) In vitro display technologies reveal novel biopharmaceutics. Faseb J. 20, 1599–1610 13 Lipovsek, D. and Pluckthun, A. (2004) In vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51–67 14 Marks, J.D. (2004) Antibody affinity maturation by chain shuffling. Methods Mol. Biol. 248, 327–343 15 Valjakka, J. et al. (2002) Crystal structure of an in vitro affinity- and specificitymatured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains. J. Biol. Chem. 277, 44021–44027 16 Baker, K.P. et al. (2003) Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum. 48, 3253–3265 17 Bayry, J. et al. (2007) Monoclonal antibody and intravenous immunoglobulin therapy for rheumatic diseases: rationale and mechanisms of action. Nat. Clin. Pract. Rheumatol. 3, 262–272 18 Chambers, R.S. (2005) High-throughput antibody production. Curr. Opin. Chem. Biol. 9, 46–50 19 Kenanova, V. et al. (2007) Radioiodinated versus radiometal-labeled anticarcinoembryonic antigen single-chain Fv-Fc antibody fragments: optimal pharmacokinetics for therapy. Cancer Res. 67, 718–726 20 Ward, E.S. et al. (2005) From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling. Mol. Biol. Cell 16, 2028–2038 21 Woof, J.M. and Burton, D.R. (2004) Human antibody-Fc receptor interactions illuminated by crystal structures. Nat. Rev. Immunol. 4, 89–99 22 Cartron, G. et al. (2007) Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit. Rev. Oncol. Hematol. 62, 43–52 23 Brocchini, S. et al. (2006) PEGylation of native disulfide bonds in proteins. Nat. Protoc. 1, 2241–2252 24 Choy, E.H. et al. (2002) Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II doubleblinded, randomized, dose-escalating trial. Rheumatology (Oxford) 41, 1133–1137 25 Rao, B.M. et al. (2005) Integrating cell-level kinetic modeling into the design of engineered protein therapeutics. Nat. Biotechnol. 23, 191–194 26 Traggiai, E. et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304, 104–107
27 De Groot, A.S. and Moise, L. (2007) Prediction of immunogenicity for therapeutic proteins: state of the art. Curr. Opin. Drug Discov. Dev. 10, 332–340 28 Bourmans, M. et al. (2010) Mechanisms of inhibition of joint destruction by rituximab in rheumatoid arthritis. Arthritis Rheum. 62, 761 29 Silverman, G.J. and Boyle, D.L. (2008) Understanding the mechanistic basis in rheumatoid arthritis for clinical response to anti-CD20 therapy: the B-cell roadblock hypothesis. Immunol. Rev. 223, 175–185 30 Isaacs, J. et al. (2009) Autoantibody-positive rheumatoid arthritis (RA) patients have enhanced clinical response to rituximab when compared to seronegative patients. Ann. Rheum. Dis. 68, 442 31 Taylor, R.P. and Lindorfer, M.A. (2007) Drug insight: the mechanism of action of rituximab in autoimmune disease – the immune complex decoy hypothesis. Nat. Clin. Pract. Rheumatol. 3, 86–95 32 Beers, S.A. et al. (2008) Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood 112, 4170–4177 33 Tak, P. et al. (2010) Efficacy and safety of ocrelizumab in patients with active rheumatoid arthritis who have an inadequate response to at least one TNF inhibitor: results from the Phase III SCRIPT trial. Arthritis Rheum. 62, 909 34 Stone, J.H. et al. (2010) Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N. Engl. J. Med. 363, 221–232 35 Hauser, S.L. et al. (2008) B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N. Engl. J. Med. 358, 676–688 36 Gabay, C. and McInnes, I.B. (2009) The biological and clinical importance of the ‘new generation’ cytokines in rheumatic diseases. Arthritis Res. Ther. 11, 230 37 Schett, G. et al. (2005) Mechanisms of disease: the link between RANKL and arthritic bone disease. Nat. Clin. Pract. Rheumatol. 1, 47–54 38 Tarner, I.H. et al. (2007) Emerging targets of biologic therapies for rheumatoid arthritis. Nat. Clin. Pract. Rheumatol. 3, 336–345 39 Miller, P.D. et al. (2008) Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: a randomized blinded phase 2 clinical trial. Bone 43, 222–229 40 Cohen, S.B. et al. (2008) Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial. Arthritis Rheum. 58, 1299–1309 41 Strand, V. et al. (2007) Biologic therapies in rheumatology: lessons learned, future directions. Nat. Rev. Drug Discov. 6, 75–92 42 Cobo-Ibanez, T. and Martin-Mola, E. (2007) Etanercept: long-term clinical experience in rheumatoid arthritis and other arthritis. Exp. Opin. Pharmacother. 8, 1373–1397 43 Sandborn, W.J. et al. (2007) Certolizumab pegol for the treatment of Crohn’s disease. N. Engl. J. Med. 357, 228–238 44 http://www.arana.com/text/news_media/2008_html/press_release_170308.html (accessed 8 August 2008). 45 Holt, L.J. et al. (2008) Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng. Des. Sel. 21, 283–288 46 Annunziato, F. et al. (2009) Type 17T helper cells-origins, features and possible roles in rheumatic disease. Nat. Rev. Rheumatol. 5, 325–331 47 Ding, C. and Jones, G. (2006) Belimumab human genome sciences/Cambridge antibody technology/GlaxoSmithKline. Curr. Opin. Investig. Drugs 7, 464–472 48 http://www.hgsi.com/latest/human-genome-sciences-reports-positive-long-termdata-for-lymphostat-b-in-patients-with-active-systemic-lupus-erythema-6.html (accessed 8 August 2008). 49 Lin, W.Y. et al. (2007) Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade. Blood 110, 3959–3967 50 Bruce, S.P. and Boyce, E.G. (2007) Update on abatacept: a selective costimulation modulator for rheumatoid arthritis. Ann. Pharmacother. 41, 1153–1162 51 Suntharalingam, G. et al. (2006) Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N. Engl. J. Med. 355, 1018–1028 52 Lainer-Carr, D. and Brahn, E. (2007) Angiogenesis inhibition as a therapeutic approach for inflammatory synovitis. Nat. Clin. Pract. Rheumatol. 3, 434–442
www.elsevier.com/locate/nbt
509
Review
References
REVIEW
53 Strunk, J. et al. (2006) Anti-TNF-alpha antibody Infliximab and glucocorticoids reduce serum vascular endothelial growth factor levels in patients with rheumatoid arthritis: a pilot study. Rheumatol. Int. 26, 252–256 54 Holash, J. et al. (2002) VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. U. S. A. 99, 11393–11398 55 Hurwitz, H. et al. (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, 2335–2342
Review 510
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
56 Parker, M.H. et al. (2005) Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two. Protein Eng. Des. Sel. 18, 435–444 57 Nishimoto, N. et al. (2007) Study of active controlled monotherapy used for rheumatoid arthritis, an IL-6 inhibitor (SAMURAI): evidence of clinical and radiographic benefit from an x ray reader-blinded randomised controlled trial of tocilizumab. Ann. Rheum. Dis. 66, 1162–1167
REVIEW
Review
Recombinant proteins in rheumatology – recent advances Achim Rothe and Andrea Rubbert Department of Internal Medicine I, University Hospital Cologne, Joseph Stelzmann Street 9, 50931 Cologne, Germany
New targeted anti-inflammatory drugs have revolutionized the therapeutic strategies in rheumatology. Recombinant DNA selection technologies have enabled the isolation and humanization of specific antibody fragments of any specificity that can be ‘armed’ to deliver effective anti-inflammatory ‘payloads’. Antibodies and other targeted provide the opportunity to block key soluble mediators of inflammation in their milieu, or alternatively to block intracellular inflammation-triggering pathways by binding to an upstream cell-surface receptor. Designed proteins can be improved with respect to desired pharmacokinetic and pharmacodynamic effects. They facilitate the delivery of the required immunosuppressive effect. However, the individual extent of desired and undesired effects of a particular biologic therapy can be broader than initially predicted and requires careful evaluation during clinical trials. This review highlights advances in the application of recombinant antibody technology for novel biologic therapies in rheumatology. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The engineering of biologic modulators. . . . . . . . . . . . . . . . . . . . . . . . . . . Intact antibodies and antibody fragments . . . . . . . . . . . . . . . . . . . . . . . Fusion proteins with antibody Fc fragments (immunoadhesins). . . . . . . The generation of biologic immune modulators. . . . . . . . . . . . . . . . . . . . . Humanization and transgenic mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . Library and display technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Affinity maturation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preclinical and clinical applications of targeted biologics in rheumatology . Inhibition of cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-cell ablation or modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benefits and risks of biologics in rheumatology . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .
503 503 503 504 504 504 504 504 504 505 505 507 508 508 508 508 509
Corresponding author: Rothe, A. ([email protected])
502
www.elsevier.com/locate/nbt
1871-6784/$ - see front matter. Crown Copyright ß 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.nbt.2011.03.019
REVIEW
Introduction Rheumatic diseases are characterized by aberrant cellular and humoral immune responses. Accordingly, the primary goal for protein-based therapeutics in rheumatology is to target and remediate the disease-causing upregulated cytokines, to block cell surface receptors that are crucially involved in initiating and maintaining the inflammatory cascade and to counteract autoimmune T and B cells. This review summarizes the molecular structures and design of antibody-based immune-modulators and discusses their production and properties as therapeutic agents. In addition, therapeutic formulations, focusing on the clinically most relevant targets and current in-trial status (TNF, IL-6, Table 1) are described.
The engineering of biologic modulators Intact antibodies and antibody fragments Intact antibodies, such as the Y-shaped IgG, are highly specific targeting reagents and provide congenital defense against patho-
genic organisms and toxins. So far, almost all engineered therapeutic antibodies exclusively use the bivalent format of IgG. The antigen-binding domains are located on the two antigen binding (Fab) tips, which enable the antibody to bind to two antigens simultaneously. This feature increases their functional activity and influences their pharmacokinetic profile, as described later. Recruitment of effector functions, such as complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent phagocytosis, is mediated by the fragment crystallizable (Fc) domain through interactions with Fc receptors (Fc receptors for globulins). The Fc domain also provides the antibody with an extended circulation time through recycling within vascular endothelial cells via the neonatal Fc receptor (FcRn) [1]. Because of its two binding surfaces and Fc domain, intact IgG antibodies can result in cross-linking of soluble or cellsurface target antigens. The inclusion of post-translational modification, such as glycosylation, can also be used to profoundly alter the circulation time of recombinant proteins [2].
TABLE 1
Antibody- and receptor-based biologics that have been approved or are preclinical or in-trial for rheumatologic diseases Format
(Generic) name
Target
Maximum serum half-life
Indication in rheumatology
Stage
Company/ developer
Chimeric IgG Humanized IgG ScFv-Fc (SMIP) Human IgG Ligand-Fc Chimeric IgG
Rituximab Epratuzumab TRU-015 Denosumab OPG-Fc Infliximab
CD20 CD22 CD20 RANKL RANKL TNF
16 14 12 32 N/A 10
Approved Phase II/III Phase II Phase III Preclinical Approved
Genentech UCB Trubion/Wyeth Amgen Amgen Centocor
Human IgG
Adalimumab
TNF
13 days
Approved
CAT/Abbott
Human IgG
Golimumab
TNF
13 days
Approved
Centocor
Receptor-Fc
Etanercept
TNF
3 days
Approved
Amgen/Wyeth
Fab-PEG
Certolizumab
TNF
14 days
dAB
TNF
Ligand-Fc
Atacicept (TACI-Fc) Belimumab Tocilizumab ALD518 (BMS-945429) Anakinra
BLyS
Tailore days through protein engineering 63 days
Approved (Phase II/III) Preclinical
UCB Pharma
Human variable domain
RA SLE RA Bone erosion in RA Bone erosion in RA RA, Ankylosing spondylitis, Psoriasis arthritis RA, Ankylosing spondylitis, Psoriasis arthritis RA, psoriatic arthritis, ankylosing spondylitis RA, (ankylosing spondylitis, Psoriasis arthritis) RA (ankylosing spondylitis, Crohn) Autoimmune diseases
RA, SLE
Phase II/III
BLyS IL-6R IL-6
16 days 10 days 30 days
RA, SLE RA RA
Phase III Phase III Phase II/III
Merck Serono/ ZymoGenetics GSK/HGSI Roche/Chugai BMS
IL1-receptor
4–6 h
RA
Approved
Amgen
Canakinumab
IL-1ß
14 days
CAPS, (Gout)
Novartis
Human IgG
Secukinumab
IL-17
14 days
Receptor-Fc
Rilonacept
IL-1
8.6 days
RA, psoriasis, ankylosing spondylitis CAPS (Gout)
Approved (Phase II/III) Phase II
Receptor-Fc Ligand-Fc Receptor-Fc
BR-3-Fc Abatacept Aflibercept (VEGF-Trap) Bevacizumab
BAFF CD80/86 VEGF
15 days 23 days 14 days
VEGF
20 days
Humanized IgG Humanized IgG Humanized IgG Modified receptor antagonist Human IgG
Humanized IgG
days days days days days
Domantis/Peptech
Novartis
RA, SLE RA –
Approved (Phase II) Phase I Approved –
Regeneron Biogen IDEC/Genentech Orencia/Bristol-Myers Squibb Regeneron/Sanofi Aventis
–
–
Genentech/Roche
This table is based on information in the public domain but is not comprehensive, due to the confidential embargo of company commercial information. Abbreviations: BAFF, B-cell activating factor; BLyS, B-lymphocyte stimulating factor; Fc, fragment crystallizable region; IL, interleukin; RA, Rheumatoid arthritis; RANKL, receptor activator of nuclear factor kB ligand; ScFv, single chain variable fragment; SLE: Systemic lupus erythematosus; TACI, transmembrane activator and calcium-modulating and cyclophilin interactor; TNF, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.
www.elsevier.com/locate/nbt
503
Review
New Biotechnology Volume 28, Number 5 September 2011
REVIEW
Review
Antibody fragments (e.g. monovalent Fab fragments) and engineered antibody designs (e.g. single-chain variable fragments [scFv], diabodies, triabodies, minibodies and single-domain antibodies) have proven to be valid alternatives to full-size IgG antibodies [3]. Fab fragments may be coupled to polyethylene glycol (PEG) that extends the half-life of the drug but may avoid potentially Fc-mediated side effects. PEGylation may facilitate accumulation of the compound in inflamed tissues [4]. In contrast to fullsize IgG antibodies, PEGylated Fab fragments will most probably not cross the placenta. Receptor antagonists (nonimmunoglobulin protein scaffolds) protein engineering has enabled the production of a wide range of different nonimmunoglobulin scaffolds with diverse origins, biodistribution and pharmacokinetic properties, such as the recombinant IL-1 receptor antagonist anakinra, which is approved for the treatment of patients with active rheumatoid arthritis (RA) [5]. Anakinra is a small recombinant, nonglycosylated 153-residue (17 kDa) domain of the human IL-1 receptor antagonist. The majority of these nonimmunoglobulin scaffolds, such as ankyrins and anticalins are also small, single protein domains [6] and are less than half the size of the 30 kDa scFv antibody fragment, which comprises two 14 kDa V-domains. Many of these small novel scaffolds have stability and production advantages over antibody domains because they do not require disulfide bonds and posttranslational modifications. Their immunogenicity in therapeutic formulations, however, is still under early evaluation [6].
Fusion proteins with antibody Fc fragments (immunoadhesins) As an alternative to antibodies or scaffolds, the extracellular soluble domain of a cell-surface receptor can be fused with the Fc domain of an antibody, thus combining the unique binding specificity of the receptor domain with the capacity to mediate antibody effector functions or to increase serum half-life. These constructs commonly represent more stable and easier to produce compounds than their original physiological structures. Etanercept is a TNF receptor-Fc fusion protein and the most successful engineered immunoadhesin for RA therapy as yet [7]. Abatacept represents a fusion protein of soluble CTLA-4 (cytotoxic lymphocyte antigen-4) and the Fc fragment of IgG1 and inhibits T cell costimulation. Abatacept is currently approved for patients with active RA who failed to methotrexate and/or TNF inhibitors. Another example is the transmembrane activator and calciummodulating and cyclophilin interactor (TACI)-Fc fusion protein atacicept, which similarly comprises the extracellular binding domain from the TNF receptor family and has been studied for the treatment of arthritis, SLE and multiple sclerosis [8]. Fv-like fragments of T-cell receptors (single-chain T-cell receptor) can also be fused to IgG1 heavy chains to create properties similar to monoclonal antibodies, but that are targeted to antigens derived from intracellular targets [9].
The generation of biologic immune modulators Humanization and transgenic mice Murine monoclonal antibodies provide very effective therapeutics but their deleterious immune response can prevent multiple administrations. A preferred method of reformulation is ‘humanization’, which involves grafting the murine Fab/Fv (targeting regions) onto a human Fc backbone. To generate a fully human 504
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
antibody, transgenic mice have been developed that lack the native mouse immune repertoire and instead harbor most of the human antibody repertoire in their germline. A humoral immune response in these mice, following the injection of an antigen, leads to the development of human antibodies that can be recovered by classic hybridoma technology. The transgenic mice also have a fully operational affinity maturation system of somatic hypermutation and selection, so the resultant human antibodies have high affinity [10].
Library and display technologies As an alternative to using live animals, antibody library selection (entirely in vitro) has become a fast process for the discovery of human antibodies, using either natural or synthetic immune repertoires [11,12]. Specific high-affinity antibodies can be selected by linking phenotype to genotype in vitro, which results in gene isolation after a stepwise enrichment process based on antibody affinity. Adalimumab, a fully human antibody to TNF, which has FDA approval for the treatment of RA was selected from human phage display libraries using a guided selection strategy [11]. In phage display, the recombinant library is expressed as fusion constructs on the surface of phage particles before entering the selection process. Antibodies are generally displayed as monovalent fragments, and in vitro display technology can effectively supersede natural in-cell selection. Several technologies are now available for display and selection of high-affinity antibody fragments, including ribosome, messengerRNA and DNA linkages that lead to the isolation of a vast range of engineered protein-based reagents [12].
Affinity maturation The improvement of the binding characteristics of a low-affinity binding molecule is achieved by an affinity maturation process involving mutation followed by selection of the higher-affinity mutants. Random mutagenesis, as well as engineered interface mutations, have been proven effective for affinity maturation [13– 15]. In vitro display systems provide ideal tools for inherent affinity maturation. The fully human antibody belimumab, which is directed against BLyS, was isolated from a naive human phage display library [12,16]. Affinity optimization was performed by randomization of the last six amino acids of the heavy-chain CDR3 region. Belimumab is currently being tested in clinical trials for the treatment of rheumatic diseases and approval for use in SLE is expected [17].
Production The production of stable, high-affinity antibody fragments in high yield for preclinical and clinical trials can be elaborate and expensive. Expression systems, including bacteria, yeasts, plants, insect and mammalian cell lines and even cloned transgenic animals, have been evaluated and compared [18]. Presently, the preferred media for the bulk production of recombinant proteins are devoid of proteins or components that can elicit deleterious immunogenicity in the final recombinant product. Bacteria are favored for the expression of small, nonglycosylated antibody fragments, but yields can vary for each construct. Mammalian or plant cells are favored hosts for high-yield expression of larger intact antibodies and minibodies due to the efficient protein folding processes
New Biotechnology Volume 28, Number 5 September 2011
provided by the eukaryotic endoplasmic reticulum. Staphylococcal protein A affinity columns facilitate the purification of Fcfusion proteins. Several strategies have been developed to simplify recombinant antibody expression and purification, such as terminal polypeptide tags for affinity purification, although this adds a complication of potential, unwanted immune responses. The production of smaller non-immunoglobulin scaffolds, however, can be cheap and efficient, because they can be efficiently expressed in bacteria or produced by chemical synthesis.
REVIEW
of actions of a new biologic might be well-characterized, acute or long-term adverse events are unpredictable. Initial toxicity and immunogenicity experiments are commonly performed in rodents; however, the rodent immune system is distinct and different to humans. The establishment of settings closer to a human system such as transgenic, knock-in models or human cord-blood transplanted mice [26] would be of great benefit. In addition, computer-based prediction of immunogenicity should be utilized at an early stage in the process of engineering human biopharmaceuticals [27].
Pharmacokinetics
Preclinical and clinical applications of targeted biologics in rheumatology Targeting cell-surface receptors B cells play a crucial role in autoimmune diseases such as rheumatoid arthritis, SLE or ANCA associated vasculitis. They may not only be regarded as the source of autoantibodies, but also function as antigen presenting cells and producers of proinflammatory cytokines [4]. The therapeutic chimeric anti-CD20 antibody rituximab has been approved for the treatment of patients with active RA who have failed to TNF inhibitors. Rituximab does not only improve signs and symptoms of disease, but also inhibits radiographic progression and improves physical function. It has been recently demonstrated that rituximab induces a significant decrease in RANK+ osteoclasts within the synovial tissue [28]. Not surprisingly, the likelihood of achieving a clinically relevant response is significantly greater in patients who are rheumatoid factor and/or CCP positive [29,30]. Rituximab treatment may well be judged as the most efficient blockade against recirculating and potentially pathologic B cells eventually enabling ‘re-booting’ of the immune system. Rituximab can generally be administered repetitively without major side effects; although a noteworthy first-dose reaction has been observed so commonly that the infusion rate is now increased slowly to minimize possible allergic reactions [29]. Antichimeric antibodies to rituximab have been occasionally reported but do not generally seem to cause treatment abrogation. Different mechanisms of action have been proposed, ranging from B-cell depletion to the generation of decoy cellular immune complexes [31]. The most crucial issue for successful CD20 immunotherapy is to dictate the most effective engagement strategy for the surface CD20 epitope as modulated by the choice of antibody and choice of effector function [32]. Therefore, targeting the same cell surface protein will not necessarily result in comparable clinical efficacy and safety. Ocrelizumab as a humanized anti-CD20 directed monoclonal antibody has been evaluated in randomized controlled trials in RA but its development for RA and SLE was recently stopped because of an unfavorable balance between efficacy and safety [33]. However, Ocrelizumab is still being explored for patients with multiple sclerosis. Ofatumumab (HuMax-CD20R) represents a fully human antiCD20 directed monoclonal antibody that is currently in clinical development. SLE represents a prototypic autoimmune disorder that is characterized by potential involvement of multiple organs and the presence of autoantibodies. Retrospective cohort analyses and www.elsevier.com/locate/nbt
505
Review
The evaluation of pharmacokinetics is most easily performed with radiolabeled antibodies, which not only are important clinical therapeutic reagents, but also represent effective tools for in vivo imaging [19]. Intact IgG antibodies have the ability to bind to two antigens, which greatly increases their functional affinity and confers high-retention times (avidity) on many cell-surface receptors and polyvalent antigens. The Fc component of intact antibodies also provides long serum half-lives (>10 days) through interaction with the FcRn, as discussed previously. This FcRn binds and transports IgGs both within and across cells and rescues them from a default degradative pathway [20,21]. For example, serum rituximab levels can still be detected 3 months after the last infusion [22]. By contrast, smaller proteins, such as antibody fragments, exhibit a short serum half-life, which can be extended by the addition of polyethylene glycol (PEG; pegylation). Pegylation of a protein increases the apparent size through hydration and is a simple process that can be completed in 1 day and the pegylated protein can be purified in hours. This approach has been successfully applied to antibody fragments and other proteins and peptides, without destroying their tertiary structure or interfering with their biologic activity [23,24]. Certolizumab pegol consists of a Fab fragment of a humanized anti-TNF coupled to PEG with a serum half life of around 14 days and represents a new TNF inhibitor that is currently approved for rheumatoid arthritis and Crohn’s disease. Pharmacokinetics relies on computational algorithms to assess the appropriate characteristics for cell targeting by inclusion of rates for diffusion, binding, internalization and systemic clearance [25]. In tumor targeted therapy the size of an antibody-based therapeutic is crucial for penetration into the solid tumor mass. This feature is eventually less important in rheumatology, where target antigens are often circulating and may be generally more accessible. However, treatment of arthritis may require that therapeutic antibodies or compounds get access into the synovium where proinflammatory cytokines and activated lymphocytes are abundant. Anti-TNF antibody therapy, for example, is associated with a spectrum of anti-inflammatory effects and the concentration of adalimumab in the synovial fluid after intravenous administration reaches 31–96% of the serum concentration. The chronic nature of rheumatologic diseases necessitates longterm biopharmaceutical intervention; accordingly, any associated adverse events should be carefully considered and addressed. Removing the Fc domain in intact antibodies or immunoadhesins certainly abrogates many of the associated side effects, but might also impair the efficacy of the therapy. This leaves a small therapeutic window between loss of efficacy and the associated toxicity mediated by the Fc-domain. Thus, although the mechanisms
REVIEW
Review
open-label Phase I/II trials suggested efficacy of using rituximab as monotherapy or in combination with cyclophosphamide in patients with active SLE who failed to standard therapy. However, the placebo-controlled Phase II/III trials LUNAR and EXPLORER in patients with SLE failed to meet their primary and secondary endpoints (Genentech/Biogen IDEC press release 2008/2009). Possible reasons may have been the relatively short duration of the trials, the suitability of the outcome parameters selected and the confounding effects of oral steroids that the patients had to take initially. Apart from case reports and case series, a recent randomized controlled trial (RAVE) has demonstrated that rituximab has comparable efficacy to cyclophosphamide in patients with ANCA-associated vasculitis [34]. Based on these results, rituximab will hopefully be approved for use in this indication. The HERMES trial represents a Phase II study in patients with relapsing-remitting multiple sclerosis and has demonstrated significant reductions in gadolinium-enhancing cerebral lesions and prevention of clinical relapses following a single course of rituximab [35]. Of note, a placebo-controlled Phase II/III trial of rituximab in primary progressive multiple sclerosis (OLYMPUS) did not prove efficacious (Press release Genentech/Biogen IDEC 2008).
[()TD$FIG]
New Biotechnology Volume 28, Number 5 September 2011
Numerous case reports and cohort studies have reported on the benefit of rituximab in various other autoimmune disorders such ¨ gren’s syndrome, myositis, mixed cryoglobulinemia, as Sjo myasthenia gravis and pemphigus. Of note, levels of autoantibodies do not necessarily predict response to rituximab or correlate with the clinical response to rituximab. A different approach targeting the CD20 receptor and designed to balance effector functions, such as antibody-dependent cellmediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), uses the fusion protein of an anti-CD20 scFv antibody fragment to the human IgG Fc-domains CH2 and CH3. This so-called small modular immunopharmaceutical TRU-O15 had been evaluated in clinical trials for the treatment of RA. The IL-1 receptor antagonist anakinra represents an example of a modified, nonglycosylated form of a natural ligand (IL-1), in this case for competitive blockade of the IL-1 receptor. Because of a superior efficacy of other biologics in RA, the use of anakinra in RA has decreased. Interestingly, anakinra has been reported to be highly efficacious in Still’s disease and hereditary fever syndromes [36]. Receptor blockade of RANKL activity is a key intervention strategy relevant for both osteoporosis and bone destruction in arthritis [37]. An Fc-fusion protein linked to the RANKL physiological decoy
FIGURE 1
Structural comparison of engineered, targeted monoclonal antibodies, fusion protein and PEGylated Fab fragment directed against TNFa (adapted from Weir et al. Therapy 2006;3:535–545). 506
www.elsevier.com/locate/nbt
receptor osteoprotegerin (OPG-Fc) blockades RANKL activity, and a comparison of this format to the fully human anti-RANKL antibody denosumab is reviewed by Tarner and colleagues [38,39]. Denosumab has recently been approved for the treatment of osteoporosis in men and postmenopausal women. Interestingly, a randomized controlled clinical trial of denosumab in RA revealed that denosumab significantly inhibited erosive joint destruction, but had no benefit regarding disease activity [40].
Inhibition of cytokines TNF is an inflammatory cytokine produced by cell compartments of the innate and adaptive immune system, which contributes to the pathogenesis of various rheumatic diseases. High TNF levels may be detected in the synovial fluid and synovial tissue of patients with RA, where TNF triggers cell proliferation and the production of other proinflammatory cytokines. TNF inhibitors are well-established in the therapy of various rheumatic diseases by neutralizing soluble and membrane-bound TNF and directing cell death either through cytokine deprivation or through Fc-mediated mechanisms [41]. Anti-TNF complexes have been postulated to also have an agonistic effect on cytokine production via binding to cell-surface Fcg receptors, for example on natural killer cells [42]. For clinical use, currently five different TNF inhibitors are available (Fig. 1). The chimeric monoclonal antibody infliximab and the human antibodies adalimumab and golimumab are IgG anti-TNF agents (i.e. intact, full-size antibodies). By contrast, etanercept represents a fusion protein of the soluble TNF-receptor and the Fc domain of human IgG and certolizumab is a pegylated, humanized anti-TNF Fab fragment [42,43]. The latter Fab construct (certolizumab) lacks the Fc-portion and thus does not induce apoptosis through complement activation or ADCC. Differences among the available TNF inhibitors relate to the molecule structure, application (sc versus iv) or half-life. TNF inhibitors have successfully been used in RA, psoriatic arthritis, ankylosing spondylitis, juvenile arthritis, Crohn’s colitis or psoriasis. At present, not all of them are approved for all indications. Of note, if patients show intolerance to one TNF blocker (injection site reaction to the sc compounds, infusion reaction to infliximab) or if they develop antibodies to one compound that results in secondary inefficacy, switching within the class of TNF inhibitors may represent a therapeutic option. An increased risk for serious infections and tuberculosis as well as opportunistic infections, the development of malignant lymphoma and inflammatory demyelinating disease has been reported [17]. Screening for latent tuberculosis before starting a TNF inhibitor is mandatory. In patients with a history of demyelinating disorders or in patients with cardiac insufficiency, TNF inhibitors are contraindicated. Several national registries (Germany, Great Britain and Sweden) have been established and have followed patients on TNF inhibitors for more than 10 years. These registries have not revealed a higher incidence of malignancies associated with the use of TNF blockers. However, potentially other therapeutic options may be preferred in patients with a history of a recent malignancy. The smallest available anti-TNF biologics are single-domain proteins that potentially offer a greater penetration (and hence affinity) to TNF. One human single-domain antibody (14 kDa) originally developed by Domantis, is now undergoing a Phase II trail as ART621 (Arana Therapeutics Ltd.) but very limited data are
REVIEW
currently available [3,44]. Domain antibodies are also engineered for long-term serum half-life either through pegylation, fusion with a carrier protein or through expression as multivalent formats. In one example a domain antibody targeting human serum albumin was recently fused to an IL-1-receptor antagonist and considerably extended the half-life of this receptor blocker resulting in a potentially more efficient arthritis therapy [45]. The blockage of cytokines that increase inflammation represents an attractive strategy for the development of targeted protein-based therapy. IL-1 represents a key player in inflammation; however, the term IL-1 refers to two cytokines, namely IL-1a and IL-1b that are encoded by different genes. IL-1 effects are potentially counteracted by endogenous inhibitors such as the IL-1 receptor antagonist (IL1Ra) and the IL-1 receptor type II (Il-1RII). Inhibition of IL-1 by anakinra or canakinumab, a human antibody directed against IL-1b, has been studied mainly in RA with limited success. Interestingly, the blockade of IL-1 has proven significant efficacy in other disorders, mainly cryopyrinopathies, Still syndrome and IL-1Ra deficiency. Canakinumab and rilonacept (IL-1 Trap) have recently been approved for patients with cryopyrin-associated periodic syndromes (CAPS). More recently, IL-1 appears as an attractive target in patients with refractory gout based on the findings that urate crystals induce inflammation through the NALP3 inflammasome that leads to IL-1 production. Currently, in addition to anakinra, clinical trials have been set up to assess rilonacept and canakinumab on gout flare treatment and prevention. IL-6 has recently been characterized as a pleiotropic driver in acute and chronic inflammation. IL-6 mediates its effects by binding to a soluble or membrane bound IL-6 receptor (IL-6R), that may then assemble with gp130, that is necessary for signal transduction. IL-6 may therefore be targeted by monoclonal antibodies directed to either the receptor, an example of this is the chimeric antibody tocilizumab, or directed against IL6 itself. IL-6 has been shown to be crucial for joint inflammation in arthritis, but is also crucial for systemic features of inflammation such as fever, anemia, fatigue and the raise of acute-phase proteins. Tocilizumab has recently been approved in patients with active rheumatoid arthritis who failed to conventional DMARDs and/or TNF inhibitors and has proven highly efficacious in patients with juvenile arthritis. Tocilizumab represents also a promising therapeutic option for other autoimmune diseases such as adult-onset Still’s disease, Crohn’ disease and Takayasu arteritis. Recent studies have demonstrated the importance of IL-12 and IL-23 in the immunopathogenesis of psoriasis. The p40 subunit common to IL-12 and IL-23 is an attractive target for treatment of psoriasis and psoriatic arthritis. Two compounds, ustekinumab (approved in 2009 for treatment of plaque psoriasis) and ABT-874 have been studied and resulted in significant improvement of cutaneous lesions. Of the cytokines relevant to autoimmunity, IL-17 and its family have recently received attention, as in murine models of autoimmune diseases, IL-17 producing Th17 cells play a pivotal role in pathogenesis. The role for IL-17 in driving human autoimmune diseases is less clear, but IL-17A may be detected in synovial fluid of RA patients and in T cell rich areas of RA synovial tissue. Thus far, monoclonal antibodies have been developed and are currently being assessed in rheumatoid arthritis, psoriasis and ankylosing www.elsevier.com/locate/nbt
507
Review
New Biotechnology Volume 28, Number 5 September 2011
REVIEW
Review
spondylitis. Other IL-17 directed approaches may include subunits of the IL-17 receptor complex (IL-17RA and IL-17RC) [46]. Cytokines such as BLyS and a proliferation-inducing ligand (APRIL) are required for B-cell maturation and may contribute to autoantibody production and disease severity. A fusion protein consisting of the BLyS receptor TACI coupled to a Fc-domain has been shown to decrease serum levels of rheumatoid factors, but significant amelioration of patients’ symptoms was not observed. The humanized anti-BLyS antibody belimumab [47] was isolated from a naive human phage display library [12] and its affinity optimized by randomization and selection of the best six amino acids of the heavy-chain CDR-3 region. Even though initial trials in RA were disappointing, belimumab has achieved excellent Phase III trial for the treatment of systemic lupus erythematosus [48]. A B-cell modulation approach with a construct similar to etanercept uses a fusion protein of the B-cell activating receptor BR3 and a Fc domain of a human IgG1 to target the B-cell activating factor BAFF (BR3-Fc). By trapping soluble BAFF, this construct inhibits B-cell activation and leads to apoptosis [49]. Data from clinical trials have not yet been reported.
T-cell ablation or modulation Monoclonal antibodies targeting T-cell surface antigens, such as CD4, CD5 and CD52 have not shown clinical benefit for rheumatoid diseases [41]. However, abatacept, an Fc-fusion protein targeting CD28 has been approved for the treatment of patients with RA refractory to MTX and/or TNF inhibitors [50]. Abatacept combines the extracellular domains of the cytotoxic T-lymphocyte-associated antigen 4 with the Fc-portion of an IgG1 without the complement activating properties. By binding to the co-stimulatory molecules CD80/CD86 on antigen-presenting cells with greater avidity than CD28, abatacept blocks CD28 binding and increases the threshold for T-cell activation [50]. In 2006, TGN1412, manufactured by TeGenero Immuno Therapeutics, an activating anti-CD28 antibody, came into public prominence when clinical trials led to multi-organ failure and cytokine storm after first administration in human [51]. A massive T-cell activation through FcgR crosslinking by the Fc-domain of the agonistic TGN1412 antibody might have contributed to these drastic effects. By contrast, abatacept offers a different modulation response and only causes serious adverse effects when combined with other disease-modifying biologic agents [50].
Angiogenesis inhibition Angiogenesis is involved in the pathogenesis of inflammatory arthritis. Consequently, the levels of its mediator, VEGF, are elevated in the synovial fluid of RA patients, [52] whereas levels are reduced in patients treated with TNF-antagonists [53]. VEGFantagonists, such as the VEGF-Trap molecule consisting of a bait receptor based on VEGFR1 and VEGFR2 fused to an IgG1 Fcfragment [54] and the anti-VEGF antibody bevacicumab [55] may represent promising biologics for future evaluation in rheumatic diseases [52]. Indeed, single domains are also being exploited to bind and block VEGF activity [56].
Benefits and risks of biologics in rheumatology Engineered proteins, most commonly based on antibodies or antibody-like scaffolds, are now providing a new wave of thera508
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
peutic products. Indeed, the biotechnology industry is embracing this opportunity because designed biologics now outnumber and surpass the number of new small-molecule drugs approved annually by FDA. Antibodies and immunoadhesins that directly target cytokines for their systemic removal (ligand ablation) have become an effective therapeutic strategy (e.g. adalinmumab and infliximab), and in some indications the selective targeting of cytokine receptors (e.g. anakinra) can deliver a highly effective clinical outcome. Powerful display technologies have enabled the rapid isolation of fully human proteins, often with novel and unique targeting specificities. Some display systems, such as ribosome or messenger RNA display, even enable a simultaneous affinity maturation process and have derived new biologics with exceptionally high binding affinities. Antibody fragments can be further engineered as multivalent constructs with increased functional affinity. Fc-fusion proteins mediate effector functions through their constant domain and these properties can now be fine-tuned by molecular design. In addition, Fc-fusions offer an increased size advantage and an extended serum half-life [1]. The high affinity and avidity of engineered protein-based biologics, however, might not always be associated with maximum efficacy and the best safety profile. We postulate that a construct of lower affinity might equally translate its effect and presumably be even more versatile through consecutive binding-release events. When administering biologics, and in respect of the past experiences with intact antibodies and Fc-fusion constructs, one has to consider that in vivo effects are unpredictable. Even when animal trials demonstrate a safe profile, there remains uncertainty when administering a highly active targeted protein into a human, as demonstrated by the extreme reaction to the CD28 activating antibody TGN1412 [57]. Independent of the structure and the origin of a biologic agent, immunogenicity is often unexpected and unpredictable. For example, even the fully human antibody adalimumab invokes an anti-human-antibody response in 12% of patients when administered as monotherapy [41]. Antibodies to therapeutic antibodies may be associated with a secondary loss of efficacy as reflected by lowering of serum concentrations. Antiidiotype-responses against the antigen-binding surface, commonly of neutralizing character, and anti-isotype responses against the constant domains may be detected. Nevertheless, a serious systemic inflammatory response syndrome in humans, such as that found to TGN1412, is rare. Furthermore, engineered protein-based biologics have widened the therapeutic spectrum for rheumatic diseases and facilitate effective immunotherapeutic strategies as a potent addition to the classic disease-modifying antirheumatic drugs. If we learn from previous mistakes made and incorporate the knowledge of protein structure using recent technological advances in protein engineering the impact of engineered biologics in rheumatology in the future will be immense.
Conclusion Biologic reagents have significantly broadened the therapeutic options for patients with rheumatic diseases. New engineered antibody/based drugs aimed at different targets in the inflammation cascade. They are able to block signaling pathways at definite, predicted stages with different efficacies. Safety issues and adverse events such as a possible increased risk of infection or secondary malignancies (resulting from the reduction of natural immuno-
New Biotechnology Volume 28, Number 5 September 2011
surveillance) have occurred and need to be addressed. The limiting effect of downstream alternative signaling, biasing the therapeutic effect, has to be considered when developing biologics blocking pathways at the intercellular or cellular level. Nevertheless, specific
REVIEW
biologic reagents represent novel and powerful mediators of anti/ inflammatorial effects in rheumatological therapy and engineered antibodies are widely used in the clinical routine as essential therapies against a range of rheumatoid diseases.
1 Olafsen, T. et al. (2006) Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment. Nat. Protoc. 1, 2048–2060 2 Walsh, G. and Jefferis, R. (2006) Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol. 24, 1241–1252 3 Holliger, P. and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136 4 Dorner, T. et al. (2009) B-cell-directed therapies for autoimmune disease. Nat. Rev. Rheumatol. 5, 433–441 5 Cohen, S.B. et al. (2004) A multicentre, double blind, randomised, placebo controlled trial of anakinra (Kineret), a recombinant interleukin 1 receptor antagonist, in patients with rheumatoid arthritis treated with background methotrexate. Ann. Rheum. Dis. 63, 1062–1068 6 Hosse, R.J. et al. (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci. 15, 14–27 7 Moreland, L.W. et al. (1997) Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N. Engl. J. Med. 337, 141–147 8 Wang, H. et al. (2001) TACI–ligand interactions are required for T cell activation and collagen-induced arthritis in mice. Nat. Immunol. 2, 632–637 9 Mosquera, L.A. et al. (2005) In vitro and in vivo characterization of a novel antibodylike single-chain TCR human IgG1 fusion protein. J. Immunol. 174, 4381–4388 10 Lonberg, N. (2005) Human antibodies from transgenic animals. Nat. Biotechnol. 23, 1117–1125 11 Hoogenboom, H.R. (2005) Selecting screening recombinant antibody libraries.. Nat. Biotechnol. 23, 1105–1116 12 Rothe, A. et al. (2006) In vitro display technologies reveal novel biopharmaceutics. Faseb J. 20, 1599–1610 13 Lipovsek, D. and Pluckthun, A. (2004) In vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51–67 14 Marks, J.D. (2004) Antibody affinity maturation by chain shuffling. Methods Mol. Biol. 248, 327–343 15 Valjakka, J. et al. (2002) Crystal structure of an in vitro affinity- and specificitymatured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains. J. Biol. Chem. 277, 44021–44027 16 Baker, K.P. et al. (2003) Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum. 48, 3253–3265 17 Bayry, J. et al. (2007) Monoclonal antibody and intravenous immunoglobulin therapy for rheumatic diseases: rationale and mechanisms of action. Nat. Clin. Pract. Rheumatol. 3, 262–272 18 Chambers, R.S. (2005) High-throughput antibody production. Curr. Opin. Chem. Biol. 9, 46–50 19 Kenanova, V. et al. (2007) Radioiodinated versus radiometal-labeled anticarcinoembryonic antigen single-chain Fv-Fc antibody fragments: optimal pharmacokinetics for therapy. Cancer Res. 67, 718–726 20 Ward, E.S. et al. (2005) From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases with the Fc receptor, FcRn, during recycling. Mol. Biol. Cell 16, 2028–2038 21 Woof, J.M. and Burton, D.R. (2004) Human antibody-Fc receptor interactions illuminated by crystal structures. Nat. Rev. Immunol. 4, 89–99 22 Cartron, G. et al. (2007) Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit. Rev. Oncol. Hematol. 62, 43–52 23 Brocchini, S. et al. (2006) PEGylation of native disulfide bonds in proteins. Nat. Protoc. 1, 2241–2252 24 Choy, E.H. et al. (2002) Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II doubleblinded, randomized, dose-escalating trial. Rheumatology (Oxford) 41, 1133–1137 25 Rao, B.M. et al. (2005) Integrating cell-level kinetic modeling into the design of engineered protein therapeutics. Nat. Biotechnol. 23, 191–194 26 Traggiai, E. et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304, 104–107
27 De Groot, A.S. and Moise, L. (2007) Prediction of immunogenicity for therapeutic proteins: state of the art. Curr. Opin. Drug Discov. Dev. 10, 332–340 28 Bourmans, M. et al. (2010) Mechanisms of inhibition of joint destruction by rituximab in rheumatoid arthritis. Arthritis Rheum. 62, 761 29 Silverman, G.J. and Boyle, D.L. (2008) Understanding the mechanistic basis in rheumatoid arthritis for clinical response to anti-CD20 therapy: the B-cell roadblock hypothesis. Immunol. Rev. 223, 175–185 30 Isaacs, J. et al. (2009) Autoantibody-positive rheumatoid arthritis (RA) patients have enhanced clinical response to rituximab when compared to seronegative patients. Ann. Rheum. Dis. 68, 442 31 Taylor, R.P. and Lindorfer, M.A. (2007) Drug insight: the mechanism of action of rituximab in autoimmune disease – the immune complex decoy hypothesis. Nat. Clin. Pract. Rheumatol. 3, 86–95 32 Beers, S.A. et al. (2008) Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood 112, 4170–4177 33 Tak, P. et al. (2010) Efficacy and safety of ocrelizumab in patients with active rheumatoid arthritis who have an inadequate response to at least one TNF inhibitor: results from the Phase III SCRIPT trial. Arthritis Rheum. 62, 909 34 Stone, J.H. et al. (2010) Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N. Engl. J. Med. 363, 221–232 35 Hauser, S.L. et al. (2008) B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N. Engl. J. Med. 358, 676–688 36 Gabay, C. and McInnes, I.B. (2009) The biological and clinical importance of the ‘new generation’ cytokines in rheumatic diseases. Arthritis Res. Ther. 11, 230 37 Schett, G. et al. (2005) Mechanisms of disease: the link between RANKL and arthritic bone disease. Nat. Clin. Pract. Rheumatol. 1, 47–54 38 Tarner, I.H. et al. (2007) Emerging targets of biologic therapies for rheumatoid arthritis. Nat. Clin. Pract. Rheumatol. 3, 336–345 39 Miller, P.D. et al. (2008) Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: a randomized blinded phase 2 clinical trial. Bone 43, 222–229 40 Cohen, S.B. et al. (2008) Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial. Arthritis Rheum. 58, 1299–1309 41 Strand, V. et al. (2007) Biologic therapies in rheumatology: lessons learned, future directions. Nat. Rev. Drug Discov. 6, 75–92 42 Cobo-Ibanez, T. and Martin-Mola, E. (2007) Etanercept: long-term clinical experience in rheumatoid arthritis and other arthritis. Exp. Opin. Pharmacother. 8, 1373–1397 43 Sandborn, W.J. et al. (2007) Certolizumab pegol for the treatment of Crohn’s disease. N. Engl. J. Med. 357, 228–238 44 http://www.arana.com/text/news_media/2008_html/press_release_170308.html (accessed 8 August 2008). 45 Holt, L.J. et al. (2008) Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng. Des. Sel. 21, 283–288 46 Annunziato, F. et al. (2009) Type 17T helper cells-origins, features and possible roles in rheumatic disease. Nat. Rev. Rheumatol. 5, 325–331 47 Ding, C. and Jones, G. (2006) Belimumab human genome sciences/Cambridge antibody technology/GlaxoSmithKline. Curr. Opin. Investig. Drugs 7, 464–472 48 http://www.hgsi.com/latest/human-genome-sciences-reports-positive-long-termdata-for-lymphostat-b-in-patients-with-active-systemic-lupus-erythema-6.html (accessed 8 August 2008). 49 Lin, W.Y. et al. (2007) Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade. Blood 110, 3959–3967 50 Bruce, S.P. and Boyce, E.G. (2007) Update on abatacept: a selective costimulation modulator for rheumatoid arthritis. Ann. Pharmacother. 41, 1153–1162 51 Suntharalingam, G. et al. (2006) Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N. Engl. J. Med. 355, 1018–1028 52 Lainer-Carr, D. and Brahn, E. (2007) Angiogenesis inhibition as a therapeutic approach for inflammatory synovitis. Nat. Clin. Pract. Rheumatol. 3, 434–442
www.elsevier.com/locate/nbt
509
Review
References
REVIEW
53 Strunk, J. et al. (2006) Anti-TNF-alpha antibody Infliximab and glucocorticoids reduce serum vascular endothelial growth factor levels in patients with rheumatoid arthritis: a pilot study. Rheumatol. Int. 26, 252–256 54 Holash, J. et al. (2002) VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. U. S. A. 99, 11393–11398 55 Hurwitz, H. et al. (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, 2335–2342
Review 510
www.elsevier.com/locate/nbt
New Biotechnology Volume 28, Number 5 September 2011
56 Parker, M.H. et al. (2005) Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two. Protein Eng. Des. Sel. 18, 435–444 57 Nishimoto, N. et al. (2007) Study of active controlled monotherapy used for rheumatoid arthritis, an IL-6 inhibitor (SAMURAI): evidence of clinical and radiographic benefit from an x ray reader-blinded randomised controlled trial of tocilizumab. Ann. Rheum. Dis. 66, 1162–1167