Pasteurized and nanofiltered, plasma-derived C1 esterase inhibitor concentrate for the treatment of hereditary angioedema

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1 For reprint orders, please contact: Pasteurized and nanofiltered, plasma-derived C1 esterase inhibitor concentrate for the treatment of hereditary angioedema Hereditary angioedema (HAE) is a relatively rare autosomal dominant disorder that is typically characterized by recurrent episodes of edema in various body locations. It is most commonly caused by an inherited deficiency of functionally active C1 esterase inhibitor (C1-INH). Replacement therapy with a human plasma-derived C1-INH concentrate is recommended for the treatment and prophylaxis of acute attacks of HAE due to C1-INH deficiency (HAE-C1-INH). This article will discuss the current therapies available for the treatment of HAE-C1-INH, latest treatment guidelines, results of several studies demonstrating the efficacy and safety of the plasma-derived, pasteurized and nanofiltered C1-INH concentrate Berinert (CSL Behring GmBH, Marburg, Germany), and future perspectives for the treatment and management of HAE-C1-INH. Konrad Bork Department of Dermatology, University Medical Center, Langenbeckstr 1, Mainz, Germany Tel.: Fax: konrad.bork@unimedizin-mainz.de Keywords: Berinert C1 esterase inhibitor C1-INH complement system contact system hereditary angioedema pnfc1-inh pdc1-inh concentrate prophylaxis Background Hereditary angioedema Hereditary angioedema (HAE) is an autosomal dominant disorder that is most commonly caused by an inherited deficiency of functional C1 esterase inhibitor (C1-INH), a protein of the complement system [1]. Two types of HAE due to C1-INH (HAE-C1- INH) have been described: the more prevalent HAE type I (approximately 85% of all cases), which is characterized by a deficiency in functional C1-INH; and HAE type II (approximately 15% of all cases), which is characterized by functionally impaired C1-INH [2]. However, a third form of HAE (HAE type III), in which patients have normal antigenic and functional C1-INH levels and which predominantly affects women, has also been described [3]. This type of HAE is either caused by mutations in the gene coding for coagulation factor XII (HAE-FXII) or by a genetic alteration that is still unknown (HAE unknown) [4]. This variant will not be discussed further and the rest of this article will focus exclusively on HAE-C1-INH. More than 250 distinct mutations in the gene that codes for the C1-INH protein, SERPING1, have been identified [5]. De novo mutations of SERPING1 have been shown to account for approximately 25% of HAE-C1- INH cases in patients with no prior family history [6]. It is generally accepted that the prevalence of HAE-C1-INH ranges from 1:10,000 to 1:50,000 persons, with no major differences due to ethnicity or gender being reported [7,8]. C1-INH functions to regulate the activation of the complement and intrinsic coagulation (contact activation pathway) and is a major endogenous inhibitor of the protease plasma kallikrein. In patients with HAE-C1-INH, the absence of functional C1-INH leads to faulty, uninhibited activation of the contact activation pathway, resulting in the unregulated cleavage of high molecular weight kininogen (HMWK) by kallikrein, and the generation of excessive free bradykinin, a key mediator of vascular permeability and subsequent angioedema (Figure 1) [9]. HAE-C1-INH is characterized by recurrent episodes of nonpruritic, nonpitting subcutaneous or submucosal edema that part of /IMT Future Medicine Ltd Immunotherapy (2014) 6(5), ISSN X 533

2 Bork Coagulation pathway Fibrinolysis pathway Fibrinogen FXI FXIa tpa/upa Fibrin Plasmin Plasminogen Fibrin degradation Contact activation pathway HMWK Prekallikrein FXII FXIIa HMWK Cleaved HMWK Bradykinin Kallikrein Kallikrein Bradykinin B2 receptor Complement pathway C2 C4 Increased vascular permeability C1r C1s C1r C1s MASP 1/2 C2a C4a Angioedema Proteolytic activity Indirect proteolytic activity Inhibition by icatibant Inhibition by C1-INH Inhibition by ecallantide Figure 1. C1-INH Inhibition of the complement, contact, coagulation and fibrinolytic pathways. In the complement pathway, C1-INH inhibits the protease component of C1 esterase, C1r and C1s (activation is indicated by horizontal bars over the complement names), as well as the MASPs. In the contact activation pathway, trace amounts of FXIIa activates prekallikrien to plasma kallkrein, which in turn cleaves HMWK to release bradykinin. FXIIa and kallikrein activate each other, generating a positive feedback loop. C1-INH also inhibits FXIa as well as plasmin and tpa/upa, and is therefore involved in the regulation of the coagulation and fibrinolytic pathways. FXI: Factor XI; FXIa: Activated factor XI; FXII: Factor XII; FXIIa: Activated factor XII; HMWK: High-molecular-weight kininogen; MASP: Mannose-binding lectin-associated serine protease; tpa/upa: Tissue/urokinase plasminogen activator. 534 Immunotherapy (2014) 6(5) future science group

3 C1 esterase inhibitor concentrate for hereditary angioedema occurs primarily in the extremities, face, genitals, trunk and abdomen [10]. Laryngeal edema (approximately 1% of all HAE-C1-INH attacks) may occur in up to 50% of patients and is potentially life-threatening owing to obstruction of the upper airways [10 12]. Symptoms of HAE-C1-INH typically appear in the first or second decade of life and persist throughout adulthood. Although symptoms occur early in life, the diagnosis of HAE-C1-INH is often considerably delayed because of a lack of awareness of this disease due to its rarity [13]. Many triggers for HAE-C1-INH attacks have been reported; these include stress, physical trauma, infection, consumption of estrogen-containing or angiotensinconverting enzyme (ACE) inhibitor-containing medications and hormonal fluctuations. Pregnancy can result in an increased frequency and severity of abdominal attacks and skin swellings in patients with HAE-C1-INH [14]. Treatment guidelines HAE-C1-INH is associated with significant morbidity and mortality [12]; treatment and prevention of attacks is therefore of great importance. In recent years, several clinically effective options for treating attacks of HAE- C1-INH have been introduced, including C1-INH replacement therapy with human plasma-derived C1-INH (pdc1-inh) concentrates or recombinant human C1-INH (rhc1-inh), and agents that inhibit kallikrein activity in the contact system (ecallantide; thus decreasing the conversion of HMWK to bradykinin) or act as antagonists of the bradykinin B2 receptor on the surface of endothelial and smooth muscle cells (icatibant) [15]. Several international consensus documents and guidelines regarding the management of HAE-C1-INH have been published or updated in recent years. These include the International Consensus Algorithm for the Diagnosis, Therapy and Management of HAE [16], consensus documents by the International Collaboration in Asthma, Allergy and Immunology [17], evidence-based recommendations for the therapeutic management of HAE-C1-INH by the Hereditary Angioedema International Working Group [18], as well as the first global guideline for the management of HAE-C1-INH by the World Allergy Organization (WAO) HAE International Alliance [19]. A number of documents focusing on more specific aspects of HAE-C1-INH therapy have also been published, including consensus documents/guidelines on home therapy [20] and management of HAE-C1-INH in pediatric patients [21], as well as pregnant or nursing women [22]. Management of HAE-C1-INH can be categorized into on-demand treatment for acute attacks, with a focus on quickly alleviating the progression and severity of attacks, and short- or long-term prophylaxis to reduce incidence of attacks or to prevent attacks altogether. Consensus guidelines generally recommend that acute attacks of HAE-C1-INH should be treated as early as possible with on-demand treatment such as pdc1-inh concentrates, rhc1 INH, ecallantide or icatibant [16 19]. The goal of short-term prophylaxis is to prevent an HAE-C1-INH attack that may be triggered by surgical, dental or other selected medical procedures. Consensus guidelines recommend that patients with HAE-C1-INH should preferably be treated with pdc1-inh concentrate 1 6 h before the procedure [19]. pdc1-inh concentrate treatment is also the first-line short-term prophylactic option in children and pregnant or nursing women [21,22]. Consensus guidelines recommend the use of either pdc1-inh concentrate or androgens for long-term prophylaxis, depending on contraindications, risk factors for adverse events (AEs), tolerance of and response to treatment, and dose required to control attacks [19]. Dosing of pdc1-inh concentrate should be twice weekly. On- demand therapy (pdc1-inh concentrate, icatibant or ecallantide) should be available for all patients on prophylaxis in case of breakthrough attacks. pdc1-inh concentrate therapy is also considered safe and effective for long-term prophylaxis in children and pregnant or nursing women [21,22]. All patients with HAE-C1- INH should be considered for home therapy and selfadministration training with either pdc1-inh concentrate or icatibant [19,20]. As not all treatments are currently approved in all countries, country-specific exceptions to the guidelines apply. Overview of the market Traditionally, attenuated androgens have been used for prophylaxis of HAE-C1-INH attacks. In most patients, androgens are very effective in decreasing the frequency and severity of attacks. Attacks of HAE-C1-INH are completely or almost completely prevented in nearly 50% of patients on androgens [23]. However, a number of side effects, including weight gain, hypertension, menstrual disturbances and virilization, may occur in some patients. Furthermore, androgens can affect serum lipid levels and can be hepatotoxic. Therefore, careful surveillance is generally recommended. Antifibrinolytics are no longer recommended for long-term prophylaxis because data supporting their efficacy are lacking [19]. More recently, new intravenous therapies have been approved for the treatment of HAE-C1-INH (Table 1). They include two human pdc1-inh concentrates pasteurized, nanofiltered C1-INH (pnfc1-inh) concentrate (Berinert, CSL Behring GmBH, Marburg, Germany) and nanofiltered C1-INH (nfc1-inh) concentrate (Cinryze, ViroPharma Inc., PA, USA/Cetor, Sanquin Blood Supply, Amsterdam, The Netherlands), and recombinant C1-INH produced from transgenic future science group 535

4 Bork Table 1. Drugs approved for the treatment of hereditary angioedema due to C1 esterase inhibitor. Drug Berinert (CSL Behring GmBH, Marburg, Germany; pnfc1-inh) Cinryze (ViroPharma Inc., PA, USA; nfc1-inh) Rhucin (Santarus Inc., CA, USA)/ Ruconest, Pharming NV, Leiden, The Netherlands; rhc1- INH, conestat alfa) Kalbitor (Dyax Corp., MA, USA; ecallantide) Firazyr (Shire, Basingstoke, UK; icatibant) Mechanism of action C1-INH replacement C1-INH replacement C1-INH replacement Kallikrein inhibition Antagonism of bradykinin B2 receptor Dosage USA (US FDA) approval status EU (EMA) approval status 20 IU/kg bodyweight iv. Approved for treatment of acute facial, abdominal and laryngeal attacks and self-administration (adults and adolescents) 1000 U iv. Approved for LTP and selfadministration (adults and adolescents) 50 U/kg bodyweight iv. Not approved 30 mg sc. Approved for treatment of acute attacks (administration only by healthcare professional; patients 16 years) 30 mg sc. Approved for treatment of acute attacks and self-administration (adults 18 years) Approved for treatment of acute attacks, STP and selfadministration (adults and children) Approved for treatment of acute attacks, STP, LTP and self-administration (adults and adolescents) Approved for treatment of acute attacks (adults) Not approved Approved for treatments of acute attacks and self-administration (adults) Cetor is approved for acute treatment in The Netherlands and a number of other countries. Age range of patients approved for each product is indicated in brackets. C1-INH: C1 esterase inhibitor; IU: International unit; iv.: Intravenous; LTP: Long-term prophylaxis; nfc1-inh: Nanofiltered C1-INH; pnfc1-inh: Pasteurized, nanofiltered C1-INH; rhc1-inh: Recombinant human C1-INH; sc.: Subcutaneous; STP: Short-term prophylaxis; U: Unit. rabbits (rhc1-inh), conestat alfa (Rhucin, Santarus Inc., CA, USA/Ruconest, Pharming NV, Leiden, The Netherlands). Drugs that can be administered subcutaneously have also been approved for the treatment of HAE-C1-INH attacks; these include icatibant (Firazyr, Shire, Basingstoke, UK), a bradykinin B2-receptor antagonist that blocks the vasodilatation and increased vascular permeability associated with exogenous bradykinin administration [24] and ecallantide (Kalbitor, Dyax Corp., MA, USA), a human plasma kallikrein inhibitor that treats HAE-C1-INH symptoms by decreasing the conversion of HMWK to bradykinin [25]. It should be noted that regulatory approvals for these therapies vary in different countries. Licensing details of these drugs are presented in Table 1. In the EU, Berinert, Cinryze, Ruconest and Firazyr are approved for the treatment of HAE attacks; Berinert is approved for shortterm prophylaxis, while Cinryze is approved for both short- and long-term prophylaxis [26 30]. In the USA, Berinert, Kalbitor and Firazyr are approved for the treatment of HAE attacks; Cinryze is approved for routine (long-term) prophylaxis [31 34]. The continuing search for new therapies in HAE-C1- INH has resulted in a number of drug candidates, which are at various stages of clinical development. The goal is to develop new HAE-C1-INH prophylactic treatments. It is hoped that these new preventive treatments can potentially further reduce the burden of disease with less disruption of daily activities. BCX4161 is an oral small molecule kallikrein inhibitor [35] that is being developed by BioCryst Pharmaceuticals Ltd (NC, USA). A Phase IIa proof-of-concept clinical trial (OPuS-1) of BCX4161 in patients with HAE- C1-INH has recently been initiated [36]. The main goals for the OPuS-1 trial are to estimate efficacy in reducing the frequency of angioedema attacks and to evaluate the safety and tolerability of BCX4161 in patients with HAE-C1-INH. Using a phage display technology, a long-acting inhibitor of plasma kallikrein has been developed by Dyax Corp. (MA, USA). DX-2930 is a fully human monoclonal antibody inhibitor of plasma kallikrein for the treatment of HAE-C1-INH [37]. A low C1-INH functional activity is one of the main criteria for the diagnosis of HAE-C1-INH [19]. Clinical observations have suggested that the protective effect can be achieved with 35 40% C1-INH functional activity [2]. As such, restoration of activity (i.e., to at least 40% of normal activity) is a biochemically plausible target and is expected to translate into clinically relevant efficacy. A recent study has shown that, after a subcutaneous 536 Immunotherapy (2014) 6(5) future science group

5 C1 esterase inhibitor concentrate for hereditary angioedema injection of 1000 international units (IU) Berinert, the mean relative bioavailability of C1-INH was 39.7%, compared with intravenous administration [38]. A Phase I/II clinical trial with a volume-reduced, subcutaneous formulation of Berinert investigating dosing regimens based on a criterion of a trough C1-INH functional activity of at least 40% at steady-state has been recently completed [39]. Part of the COMPACT program, the study evaluated the pharmacokinetics, pharmacodynamics and safety of various doses of this presentation of C1-INH. Results of this study will be presented at the EACCI Congress 2014 in Copenhagen (June 2014). Clinical experience suggests that Berinert is effective in long-term prophylaxis of HAE-C1-INH, and thus the potential exists for self-administration of prophylactic treatment. The subcutaneous administration of pdc1- INH concentrate is therefore being investigated as an alternative approach to intravenous administration for prophylactic treatment. A Phase III randomized crossover study to evaluate the clinical efficacy and safety of subcutaneously administered low-volume Berinert for the prophylactic treatment of HAE-C1-INH has been initiated [40]. ViroPharma Inc. also intends to initiate a Phase III registration study with a recently developed low-volume subcutaneous formulation of Cinryze [41]. CSL Behring GmBH also recently announced that a fully humanized monoclonal antibody against coagulation factor XIIa has been added to the company s research pipeline [42]. Preclinical proof of concept (inhibition of edema) has been demonstrated in a relevant animal model of HAE. Preliminary pharmacokinetic data indicate that less frequent dosing intervals than current therapeutic options may be feasible in humans. Apart from the treatment of HAE, Berinert has been shown to be effective in reversing ACE inhibitor-induced angioedema [43]. A randomized, double-blind, two arm, multicenter, Phase III study of Berinert for the treatment of this disorder has been initiated and is currently recruiting participants [44]. Introduction to pasteurized & nanofiltered C1-INH (pnfc1-inh) concentrate Berinert The use of C1-INH replacement for the treatment of HAE-C1-INH was first described by Brackertz and Kueppers in 1973 [45]; the rest of the article will focus on pnfc1-inh concentrate, Berinert, one of the pdc1- INH concentrates approved for treatment of HAE-C1- INH. Berinert was first licensed in Germany in 1979 as an unpasteurized human C1-INH concentrate. The first human pasteurized C1-INH concentrate (Berinert P) was licensed in Germany in Berinert is a highly purified, virus-inactivated C1-INH concentrate derived from human plasma. The pooled human plasma used for manufacturing Berinert is obtained from rigorously selected healthy donors and is thoroughly screened for the presence of viruses. The plasma is processed using a range of manufacturing steps to purify and concentrate C1-INH and to inactivate and remove contaminants that are potentially present in product intermediates. In particular, three independent virus reduction steps of this process (pasteurization, hydrophobic interaction chromatography [HIC] and 20N/15N virus filtration) have been shown to effectively remove a broad spectrum of viruses. Prion particles are also efficiently removed by the ammonium sulphate precipitation, HIC steps and the virus filtration step [46]. Berinert is produced as a purified, pasteurized, nanofiltered lyophilized concentrate of C1-INH. Each vial contains 500 IU of C1-INH, mg total protein, mg glycine, mg sodium chloride and mg sodium citrate [31]. One IU of C1-INH is equal to the amount of C1-INH in 1 ml of human plasma, which is equivalent to 270 mg/l or 2.5 µm/l. Following reconstitution with 10 ml water, Berinert is administered by slow intravenous injection. The reconstituted solution must be used within 8 h. When stored at temperatures of 2 25 C (36 77 F), Berinert is stable for the period indicated by the expiration date on the carton and vial label (up to 30 months). Currently, no pharmacopeia requirements exist for C1-INH concentrates. In general, it is important that the products should have high purity and that the C1-INH protein should be in its native conformation. In addition, there should be minimal aggregation or degradation of the protein. A comparison of the biochemical properties of four commercially available C1-INH products in vitro showed that they differ in their biochemical composition and purity (Table 2) [47]. Among the plasma- derived products, Berinert had a higher purity profile than Cinryze and Cetor (Table 2). Chemistry & pharmacodynamics C1-INH is a plasma glycoprotein with an apparent molecular weight of 104 kda. C1-INH is primarily synthesized in the liver, but is also produced in monocytes, skin fibroblasts and endothelial cells. It is a member of the serine protease inhibitor (serpin) family a group of plasma proteins that inactivate proteases by forming covalent complexes. C1-INH acts as an inhibitor in four physiological systems made up of serine protease cascades the complement, coagulation, fibrinolytic and contact activation pathways (summarized in Figure 1). C1-INH is the major inhibitor of several proteases in the complement pathway (C1r, C1s and mannose-binding lectin - associated serine protease [MASP] 1 and 2), as well as the contact activation pathway (plasma kallikrein and coagulation factor XIIa) [9,48]. C1-INH also inactivates future science group 537

6 Bork Table 2. Comparison of biochemical properties of the four commercially available C1 esterase inhibitor preparations. Properties Berinert Cinryze Cetor Ruconest Total protein amount (mg/500 U) Specific activity (IU/mg) C1-INH antigen (mg/ml) Native/recombinant C1-INH protein (mg/ml) Ratio antigen/activity Purity (%) Values for Berinert, Cinryze and Ruconest are mean values of two analyzed batches. C1-INH activity converted from IU/ml to mg/ml, 1 IU corresponding to 220 µg. C1-INH: C1 esterase inhibitor; IU: International unit; U: Unit. Data taken from [47]. the coagulation protease factor XIa, and is a relatively minor inhibitor of the fibrinolytic proteases, plasmin and tissue plasminogen activator [9]. During HAE-C1- INH attacks, low levels of C1-INH activity result in the activation of the complement and contact activation pathways. Insufficient inhibition of the contact activation pathway leads to the unregulated production of bradykinin, which has been shown to be the predominant mediator of enhanced vascular permeability and angioedema. C1-INH replacement therapy is able to regulate bradykinin generation [49] and thus prevent or halt the development of angioedema. Pharmacokinetics & metabolism A comparison of Berinert with other pdc1-inh concentrates showed that they all had comparable mean half-lives (Table 3). The mean half-life of Berinert after intravenous administration has been shown to range from 32.7 to 62.0 h, depending on the dose and study. The mean half-life of Cetor/Cinryze ranged from 40.2 to 62.0 h (Table 3). Clinical efficacy Treatment of acute HAE attacks Prior to 2009, only observational studies of Berinert use in HAE-C1-INH had been reported. Numerous observational and descriptive studies have demonstrated that replacement therapy with Berinert was effective in treating HAE-C1-INH attacks at varying body locations such as the face, abdomen, skin and larynx (or upper airway), and significantly shortened time to onset of symptom relief in HAE-C1-INH attacks in adults as well as children [54]. A selection of these studies is presented in Table 4. We reported on 4834 abdominal attacks and 2104 skin swellings treated on-demand with Berinert [55,57]. A total of 500 IU Berinert was sufficient for successful treatment in 68.6% of HAE-C1-INH abdominal attacks and in 80.9% of skin swellings. In another experienced center, 500 IU Berinert was sufficient for successful treatment in 98.1% of the 468 HAE-C1-INH attacks [56]. In 2009, results from a large prospective randomized controlled trial (RCT) comparing Berinert with placebo for the treatment of HAE-C1-INH attacks (IMPACT 1) were published. IMPACT 1 included 125 patients with HAE who were randomized to receive Berinert 20 IU/kg (n = 43), Berinert 10 IU/kg (n = 40) or placebo (n = 42) (Tables 4 & 5) [58]. Berinert provided faster onset of symptom relief compared with placebo (0.5 vs 1.5 h, respectively; p = ) with a single dose of 20 IU/kg, irrespective of the location of the attack (face or abdomen). 1 h after treatment with Berinert 20 IU/kg, >75% of patients had reported onset of symptom relief compared with approximately 40% of patients treated with placebo. The treatment effect with Berinert 10 IU/kg was less than with Berinert 20 IU/kg, but greater than with placebo, for onset of symptom relief. The median time to complete resolution of HAE-C1-INH symptoms was significantly shorter with Berinert 20 IU/kg compared with placebo (4.9 vs 7.8 h, respectively; p = ). No new attacks occurred before the complete resolution of the previous attack in patients treated with Berinert, indicating an absence of rebound angioedema. IMPACT 2 was the open-label extension of IMPACT 1 to evaluate the efficacy and safety of long-term treatment with Berinert for acute HAE-C1-INH attacks at any location (Table 4) [59]. During a median study duration of 24 months, 1085 HAE-C1-INH attacks in 57 patients (10 53 years of age) were treated with 20 IU/kg Berinert. The predominant type of HAE- C1-INH attack was abdominal (68.8%), followed by peripheral (21.7%), facial (4.7%) and laryngeal (4.4%). The median time to onset of symptom relief was 0.46 h (ranging from 0.39 h for abdominal attacks to 0.48 h for facial attacks). The individual average time to onset of symptom relief was <1 h in 89.5% of patients. The median time to complete resolution was 15.5 h (ranging from 5.8 h for laryngeal attacks to 26.6 h for facial 538 Immunotherapy (2014) 6(5) future science group

7 C1 esterase inhibitor concentrate for hereditary angioedema Table 3. Pharmacokinetic parameters for different plasma-derived C1 esterase inhibitor concentrate preparations. Study (year) n Dose of C1- INH T max, h t 1/2, h CL, ml/kg/h Ref. Berinert Martinez-Saguer et al. (2010), on-demand Martinez-Saguer et al. (2010), IRT Martinez-Saguer et al. (2002) IU 3.5 ± 7.3,1.0 ( ) IU 1.3 ± 2.1, 0.5 ( ) 47.8 ± 21.2, 39.1 ( ) 33.3 ± 19.8, 30.9 ( ) IU Median: mild: 46.5; severe: Bernstein et al. (2010) or 20 IU/kg 32.7 Martinez-Saguer et al. (2013) (90% CI: ) 0.9 ± 0.4, 0.9 ( ) 1.1 ± 0.61, 1.0 ( ) [50] [50] [51] 0.92 (90% CI: ) IU 0.5 (0 8) 62.0 ± ± 0.4 [38] [52] Cetor Hofstra et al. (2012) , 1500 or 2000 U Cetor/Cinryze Hofstra et al. (2012) , 1500 or 2000 U Cinryze Cinryze PI (2012) and 2000 U ,# b.w. adj: ,# b.w. adj: U: 3.9 ± 7.3, 2000 U: 2.7± U: 56 ± U: 62 ± U: 51.0 ± U: 70.2 ± 46.8 # b.w. adj: 1000 U: U: 1.00 Values are given as mean ± SD and/or median (range). Older preparation of Cetor prior to implementation of a nanofiltration step. In Europe, Cinryze (ViroPharma) is manufactured by Sanquin Blood Supply (Amsterdam, The Netherlands), who also manufactures and markets Cetor. The manufacturing process of Cetor was recently altered to improve viral safety with the addition of a nanofiltration step, and the resulting product is essentially identical to Cinryze. Therefore, the data presented in this column can be considered applicable to both products. Population pharmacokinetic estimates with 90% CIs (Bernstein et al. 2010), or without 90% CIs (Hofstra et al. 2012). Clearance measured in IU/h/%. # Clearance measured in ml/h. b.w. adj: Reported value adjusted for median weight of subjects in the pharmacokinetic population; CL: Clearance; IRT: Individual replacement therapy; IU: International unit; n: Maximum number of patients with data; t 1/2 : Elimination half-life; T max : Time to reach maximum plasma concentration; U: Unit. [53] [53] [32] attacks). The individual average time to complete resolution was <24 h in 71.9% of patients. This study confirmed the results of IMPACT 1 and demonstrated that a single dose of 20 IU/kg Berinert provides reliable efficacy in the long-term treatment of successive HAE-C1-INH attacks at any location. In addition, a retrospective analysis of 57 patients who were treated for at least 15 attacks in IMPACT 2 showed that repeated administration of Berinert did not affect the frequency of HAE-C1- INH attacks, time to onset of symptom relief or time to complete resolution of HAE-C1-INH symptoms [68]. Recently, a post hoc ana lysis of IMPACT 1 and IMPACT 2 was performed to evaluate the effect of the time from the start of an attack to treatment with Berinert on the resolution of symptoms in more than 1000 HAE-C1-INH attacks [69]. The median time to complete resolution of HAE-C1-INH symptoms was shorter after treatment with 20 IU/kg Berinert within <6 h compared with treatment after 6 h after the start of an attack (IMPACT 1: 2.8 vs 7.9 h; IMPACT 2: 12.6 vs 14.4 h). These results showed that early treatment of HAE-C1-INH attacks with future science group 539

8 Bork Table 4. Clinical studies evaluating the efficacy of Berinert for treatment of acute hereditary angioedema due to C1 esterase inhibitor attacks. Study (year) Bork et al. (2003) Bork et al. (2005) Farkas et al. (2007) Bork et al. (2008) Craig et al. (2009) Craig et al. (2011) Farkas et al. (2012) Czaller et al. (2010) Study description Patients Type of attacks Results Ref. Survey on incidence and treatment of laryngeal edema Berinert dose: IU Retrospective study of abdominal attacks in patients with HAE Berinert dose: IU Retrospective study on use of Berinert in patients with HAE Berinert dose: IU Retrospective and prospective study to evaluate efficacy of Berinert for treatment of skin swellings Berinert dose: IU Randomized, double-blind, placebo-controlled Phase II/III dose-finding study (IMPACT 1) Berinert dose: 20 IU/kg Open-label, multicenter study (IMPACT 2) Berinert dose: 20 IU/kg Prospective study of Berinert in the treatment of HAE attacks in pediatric patients Berinert dose: 500 IU or 20 IU/kg Retrospective study of pregnant patients with HAE Berinert dose: 500 IU laryngeal attacks Time to onset of relief: min in 24/25 patients (symptom resolved within 4 h in one patient) Mean duration of laryngeal edema: 15 vs 103 h (treated vs untreated) abdominal attacks 61 (including 22 children and four pregnant women) 468 laryngeal, abdominal or subcutaneous attacks (94 attacks in children and six in pregnant women) Time to onset of relief: min Mean duration of abdominal attacks: 39.9 vs 92.0 h (treated vs untreated) Time to onset of relief: min No decrease in therapeutic efficacy was observed upon repeated administration of Berinert No adverse reactions were observed in the pregnant women and all gave birth to healthy neonates skin swellings Mean time to onset of relief: 1.1 vs 50.4 h (treated vs untreated) Mean duration of attacks: 1.7 vs 3.2 days (treated vs untreated) abdominal and 25 facial attacks attacks (747 abdominal, 235 peripheral, 51 facial, 48 laryngeal and four other) 42 pediatric patients (6 18 years in age) 12 (nine pregnant and three nursing patients) HAE: Hereditary angioedema; IU: International unit; QoL: Quality of life. 152 attacks (46 upper airway, 49 abdominal and 57 subcutaneous) 36 attacks (mainly laryngeal and abdominal attacks in pregnant patients) 12 abdominal attacks in nursing patients Median time to onset of relief: 30 min Median time to complete resolution of symptoms: 4.9 h Median time to onset of relief: 0.46 h Median time to complete resolution of symptoms: 15.5 h Requirement for second dose in treatment arm: 1.1% Time to onset of relief: min Time to complete resolution: Upper airway attacks: <12 h Abdominal and subcutaneous attacks: up to 48 h Time to onset to relief: min Every woman treated with Berinert delivered a healthy neonate (disregarding inheritance of HAE) [11] [55] [56] [57] [58] [59] [60] [61] 540 Immunotherapy (2014) 6(5) future science group

9 C1 esterase inhibitor concentrate for hereditary angioedema Table 4. Clinical studies evaluating the efficacy of Berinert for treatment of acute hereditary angioedema due to C1 esterase inhibitor attacks (cont.). Study (year) Bygum et al. (2009) Kreuz et al. (2012) Study description Patients Type of attacks Results Ref. Prospective study assessing impact of self-administered home therapy with Berinert on QoL of patients Retrospective, observational study assessing safety and efficacy of home therapy with Berinert in pediatric patients Berinert dosage: IU administered for on-demand treatment 7 Patient receiving home therapy with Berinert showed significant improvements in QoL parameters Mean Dermatology Life Quality Index: 12.6 vs 2.7 (before vs after home therapy self-administration) Cumulative mean score of 36-Item Short Form (before vs after home therapy self-administration): Physical component: vs Mental component: vs pediatric patients ( years in age) HAE: Hereditary angioedema; IU: International unit; QoL: Quality of life. Median time from initial signs of an attack (or edema symptoms) to start of treatment was significantly shorter with home therapy (15 min) than with previous physician-based therapy (67.5 min) Median time to onset of symptom relief: 40 vs 60 min (home therapy vs previous physician-based therapy) Mean annual number of days hospitalized: 0.11 vs 3.8 (home therapy vs previous physician-based therapy) [62] [63] Berinert (within <6 h) provides a better response than late treatment (after 6 h). In 2009, a prospective cohort study evaluated the efficacy of on-demand home therapy with Berinert. A significant improvement in QoL was reported in seven adult patients with frequent and severe HAE-C1-INH attacks who had been initiated on self- administered, on-demand home therapy with Berinert (Table 4) [62]. Following IMPACT 1, a number of RCTs evaluating other HAE-C1-INH treatments such as Cinryze, Firazyr and Ruconest were published (Table 5) [64 67]. No head-to-head trials of the various therapies have been conducted and definitive comparisons of efficacy are challenging in light of the different trial designs and end points. However, a general comparison of the results of these RCTs suggest that the therapeutic efficacy of Berinert is comparable with that of Firazyr, Cinryze and Ruconest, as assessed by time to onset of relief. This observation is reflected in the WAO Guidelines for the Management of HAE [19]. Prophylaxis In addition to treatment of acute attacks, the preventive effects of Berinert in preprocedure and long-term prophylaxis have been well documented [54]. Short-term prophylaxis Numerous studies have demonstrated the success of short-term prophylaxis with Berinert before dental procedures, abdominal surgeries (cesarean section, hysterectomy, gastroscopy, laparotomy, cholecystectomy, appendectomy and endotracheal intubation for general anesthesia before other abdominal surgeries), deliveries or abortions, tonsillectomies/adenoidectomies and orthopedic surgeries. A number of these studies are presented in Table 6. In 2012, results were published from the first large-scale observational study with long-term fol- future science group 541

10 Bork Table 5. Comparison of clinical characteristics of Berinert with other hereditary angioedema due to C1 esterase inhibitor treatments established in randomized controlled clinical trials of on-demand treatment. Treatment Trial Study design Patients (n) Berinert (pnfc1-inh; 20 IU/kg) Cinryze (nfc1- INH; 1000 U) Firazyr (icatibant; 30 mg) Ruconest (conestat alfa; 50 and 100 U/kg) IMPACT 1 Randomized, double-blind, placebocontrolled, multicenter CHANGE Part A FAST-1 FAST-2 FAST-3 C and C Randomized, double-blind, placebocontrolled, multicenter Randomized, doubleblind (vs placebo), multicenter Randomized, double-blind (vs TXA), multicenter Randomized, double-blind, saline-controlled, multicenter Randomized, double-blind, salinecontrolled, multicenter Efficacy end point Median time to end point (h) 85 Median time to onset of relief Median time to complete resolution 68 Median time to onset of relief Median time to unequivocal relief Median time to first symptom improvement Median time to clinically significant relief Median time to almost complete relief Median time to first symptom improvement Median time to clinically significant relief Median time to almost complete relief Median time to first symptom improvement Median time to almost complete relief of all symptoms 70 Median time to the beginning of relief of symptoms Treatment Placebo (50 U/kg) 1.1 (100 U/kg) > p-value Requirement for second dose in treatment arm (%) < < <0.001 < Ref [58] 65.7 [64] [65] [65] [66] SPC pooled: 10 [28] [67] Results should be interpreted with caution owing to the heterogeneity between clinical end points. Patient number = number of patients in 20 IU/kg treatment arm plus number of patients in placebo arm. Second dose was rescue therapy, including C1-INH, antiemetic agents or opiates. IU: international unit; SPC: Summary of product characteristics; TXA: Tranexamic acid; U: Unit. 542 Immunotherapy (2014) 6(5) future science group

11 C1 esterase inhibitor concentrate for hereditary angioedema low-up evaluating the efficacy and safety of the shortterm prophylaxis of HAE-C1-INH before a range of dental and nondental surgical procedures [72]. Berinert was found to provide significantly greater reductions than tranexamic acid and danazol in both the number of patients who experienced postprocedural attacks and the number of procedures followed by an attack (Table 6). Long-term prophylaxis Although Berinert is not currently approved for use in long-term prophylaxis, its efficacy in this setting has been previously demonstrated in a number of nonplacebo-controlled observational, descriptive or cohort studies (Table 6) [54]. In a nonplacebo-controlled, prospective cohort study, 14 patients received long-term prophylaxis with Berinert over an average of 9 years (Table 6) [73]. All patients reported that all or most of their attacks were considerably less severe, indicating that symptoms of HAE-C1-INH can be significantly alleviated by one or more injections of Berinert per week. However, some patients reported an increase in attacks, and in some cases, an increased amount of Berinert was required to control the disease (i.e., the interval between Berinert injections became shorter). Breakthrough attacks developed more rapidly and often at multiple body sites. These observations reflected an increased HAE-C1-INH activity; however, this increased activity was still effectively controlled by Berinert. Special populations: pediatric & pregnant/ nursing patients The efficacy of Berinert in treating acute attacks, as well as its prophylactic efficacy in special populations such as children and pregnant/nursing patients, has also been reported in a number of studies (Tables 4 & 6). Farkas et al. demonstrated the efficacy and safety of Berinert in the treatment of 152 edematous attacks in 42 pediatric patients (Table 4) [60], while Rusicke et al. reported successful short-term prophylaxis with Berinert in 23 pediatric patients (Table 6) [71]. The earliest documented use of Berinert is in a 4-week-old girl; treatment with Berinert resulted in onset of relief within 30 min, with complete recovery after 7 h [74]. In addition to treatment of acute attacks and prophylaxis, Kreuz et al. have also shown that Berinert can be successfully used in self-administration home therapy in pediatric patients (Table 4) [63]. Observational open-label studies demonstrated that Berinert treatment was effective in treating HAE-C1- INH attacks during pregnancy, delivery and postpartum (with no reports of AEs) [14,61] and preventing HAE attacks in pregnant patients when administered for short-term prophylaxis (Table 6) [14]. Although results of observational studies should be interpreted with caution, these results suggest that Berinert is a viable option for the treatment of HAE-C1-INH attacks and for prophylaxis during pregnancy (as well as in nursing patients). Redosing & rebound effects In IMPACT 2, a single dose of Berinert was sufficient for effective treatment in 99% (1073 out of 1085) of all HAE-C1-INH attacks (Table 4) [59]. This suggested that use of Berinert would significantly reduce the need for redosing or the use of rescue medication to achieve symptom relief compared with other HAE-C1-INH treatments [64,65,75]. In addition, Berinert treatment was not associated with rebound effects (recurrence of HAE-C1- INH symptoms at the same or a different location within 48 h after treatment) [58], which have been observed with other treatments for HAE-C1-INH attacks [76]. One study has shown that Berinert might be used as rescue therapy in patients not responding to icatibant [76]: an uncontrolled pilot cohort study of 15 patients with HAE-C1-INH demonstrated that five attacks in four patients (four rebound attacks and one new attack 20 h after treatment with icatibant) were successfully treated with Berinert. Safety & tolerability Adverse events A recent systematic literature review has demonstrated that Berinert is generally well tolerated [54]. The pivotal, multinational, randomized trial IMPACT 1 reported safety outcomes in 126 patients with HAE- C1-INH who received either Berinert (10 or 20 IU/kg) or placebo [58]. Most AEs reported in IMPACT 1 were reflective of the underlying disease and type of attack. The percentage of patients experiencing a possibly treatment-related AE within 4 h after the start of treatment was lower with Berinert 20 IU/kg (five out of 46 [10.9%]) than with placebo (eight out of 41 [19.5%]). The most commonly reported AEs in patients receiving Berinert were nausea, abdominal pain, pain and muscle spasms. No serious AEs due to Berinert were observed during this study. In the open-label extension trial IMPACT 2, Berinert was well tolerated during repeat administrations, with no drug-related serious AEs reported [59]. AEs were reported in 25 out of 57 (43.9%) patients and most were mild or moderate in intensity. AEs possibly related to treatment were reported in eight out of 57 (14.0%) patients and in nine out of 1085 (0.8%) attacks. One patient discontinued treatment owing to an infusion-related reaction. The most commonly reported AE was headache (Table 7). Repeated treatment with Berinert in IMPACT 2 did not lead to the development of inhibitory anti-c1-inh antibodies. future science group 543

12 Bork Table 6. Clinical studies evaluating the efficacy of Berinert for short- and long-term hereditary angioedema due to C1 esterase inhibitor prophylaxis. Study (year) Bork et al. (2011) Farkas et al. (2007) Czaller et al. (2010) Study description Patients (n) Procedures Results Ref. Retrospective study to assess outcome of Berinert STP on patients with HAE undergoing dental surgery Berinert dosage: 500 or 1000 IU Retrospective study of patients who received Berinert for STP Berinert dosage: 500 IU Retrospective study of pregnant patients who received Berinert preprocedure (STP) Berinert dosage: 500 IU tooth extractions Number of patients who experienced edema attacks: 20.8 vs 37.2% (with prophylaxis vs without prophylaxis) Number of edema attacks following tooth extraction: 12.5 vs 21.5% (with prophylaxis vs without prophylaxis) 21 Eight endotracheal intubations, six gastroscopies, six dental interventions and one induced abortion 15 One cesarean section, eight deliveries, six abortions No case of angioedema reported in any patient during surgery or postoperatively No case of angioedema reported in any patient following STP (note: two patients received LTP during the first trimester of pregnancy; no attacks occurred and they both delivered healthy neonates) [70] [56] [61] Martinez- Saguer et al. (2010) Rusicke et al. (2010) Farkas et al. (2012) Prospective/retrospective observational study investigating the characteristics and treatment of HAE attacks before, during and after pregnancy (high frequency on-demand treatment and STP use) Berinert dosage: 500 or 1000 IU Case-series study of patients treated with Berinert for STP Berinert dosage: 500 or 1000 IU Retrospective observational study with long-term follow-up to evaluate efficacy of preprocedure STP Berinert dosage: 500 IU 1 h preprocedure pregnancies All HAE attacks were successfully treated with Berinert None of the patients experienced an attack during delivery 51 (including 23 children) 137 (including 20 children) 71 surgical procedures (including dental interventions, orthopedic surgery, tonsillectomies, adenoidectomies and appendectomies) 113 surgical procedures, 89 diagnostic/surgical interventions No swelling attacks or laryngeal edema during or postsurgery were reported Proportion of patients experiencing an edematous attack despite STP: 36% (danazol) vs 50% (TXA) vs 9% (Berinert) Proportion of surgical/diagnostic interventions followed by an edema despite STP: 13% (danazol) vs 33% (TXA) vs 6% (Berinert) Berinert is significantly (p = , Fisher s exact test) superior to oral agents (danazol and TXA) in reducing the occurrence of postprocedural edema [14] [71] [72] Bork and Hardt (2011) Prospective observational study to evaluate efficacy and safety of LTP with Berinert Berinert dosage: IU weekly 14 (received LTP for 9 years on average) Percentage of severe attacks: 93.3 vs 3.8% (without vs with LTP) Frequency of attacks; eight patients had a decrease, five had an increase and one had no change [73] HAE: Hereditary angioedema; IU: International unit; LTP: Long-term prophylaxis; STP: Short-term prophylaxis; TXA: Tranexamic acid. 544 Immunotherapy (2014) 6(5) future science group

13 C1 esterase inhibitor concentrate for hereditary angioedema An increase of disease activity was noted in some patients with severe HAE-C1I-INH receiving weekly Berinert administration who had been under long-term observation [73] (also see Long-term prophylaxis section). However, as mentioned previously, this increased activity was still effectively controlled by Berinert. Viral transmission & C1-INH autoantibodies Human plasma-derived products such as Berinert carry the risk of transmitting blood-borne viruses. A review of clinical studies has demonstrated that treatment with Berinert was not associated with transmission of viruses [54]. The same review also concluded that administration of Berinert was not associated with the development of C1-INH autoantibodies. C1-INH autoantibodies had been detected upon Berinert treatment in one study, but none of them were found to be inhibitory in nature. However, studies are in progress to investigate this matter further. A prospective, international, multicenter, nonrandomized, single-arm, open-label, postmarketing study is currently in progress to investigate the formation of inhibitory anti-c1-inh antibodies in HAE-C1-INH subjects treated intravenously with Berinert [77]. Thromboembolic events Administration of pdc1-inh concentrates (Berinert and Cinryze) in off-label and supratherapeutic doses have been reported to be associated with the occurrence of thromboembolic events [26,27]. Postmarketing surveillance data on Berinert (see Postmarketing surveillance section) show that 16 thromboembolic events have been reported over a 25-year period and the majority of them occurred with high-dose off-label use [78]. A retrospective data mining ana lysis of the US FDA Adverse Events Reporting System (AERS) database identified potential signals of pdc1-inh-concentrate-associated thromboembolic events among patients with HAE-C1-INH. In this analysis, the ten reported cases of thromboembolic events were associated with the use of Cinryze and no signals were found with Berinert or Kalbitor [79]. It was suggested that these Cinryze- associated thromboembolic events were likely to be related to the use of catheters. Preclinical studies have shown that Berinert treatment up to supratherapeutic doses did not indicate any prothrombotic risk in in vivo models of venous and arterial thrombosis [80]. Postmarketing surveillance Since its launch in 1985, Berinert has had a favorable clinical safety record within the approved indication. Between 1985 and 2011, an estimated 630,000 treatments of Berinert were administered worldwide [78]. In a recent postmarketing surveillance report on Berinert, the adverse drug reaction (ADR) reporting rates were 1 in 8967 treatments between 1 January 1985 and 30 November 2008 (500 IU dose) and 1 in 2052 between 1 December 2008 and 30 June 2011 (1500 IU dose) [78]. The higher ADR reporting rate after 1 December 2008 coincided with the introduction of Berinert in a major new market (USA). A total of 61 of 95 suspected ADRs reported were covered by the known safety profile of Berinert: allergic or anaphylactic-type reactions (nine; in very rare cases involving shock), chills and fever (five), lack of effect (26), suspected virus transmission (five) and thrombosis (16). The remaining 34 cases involved isolated reports of varying symptoms not covered by the known safety profile of Berinert. A causal relationship to the product could not be established for any of these unlisted ADRs. None Table 7. Safety profile of plasma-derived C1 esterase inhibitor concentrates. Adverse reactions that occurred in 2 (Berinert ) or 3 patients (Cinryze ) Patients with adverse reaction, n (%) Berinert (within 72 h after infusion; n = 57) [31] Headache 4 (7) 28 (19) Nasopharyngitis 2 (4) N/A Upper respiratory tract infection 1 (2) N/A Rash 2 (4) 15 (10) Vulvovaginal mycotic infection 2 (4) N/A Nausea 1 (2) 26 (18) Vomiting N/A 15 (10) Pyrexia N/A 7 (5) Dizziness N/A 3 (2) Erythema N/A 3 (2) Pruritus N/A 3 (2) N/A: Not applicable (term not reported). Cinryze (n = 146) [32] future science group 545

14 Bork of the five cases of suspected virus transmission could be attributed to Berinert. Of the 16 reports of thrombosis, 14 reports occurred with high-dose off-label use during cardiac surgery. In one of the remaining two cases, causal relationship could be excluded. In the second case, the patient had a medical history of thrombotic events and prothrombotic risk factors. Regulatory affairs Berinert was first approved for the treatment of HAE- C1-INH attacks in Germany in 1979 and in the USA in It is now approved for the treatment of all types of HAE-C1-INH attacks in adults and children in Europe [26]. More recently, European health authorities approved the extended use of Berinert for preprocedure prevention (short-term prophylaxis) of acute episodes of HAE-C1-INH in adult and pediatric patients undergoing elective dental or surgical procedures [81], as well as for self-administration [82]. In the USA, Berinert is currently approved for on-demand treatment of acute abdominal, facial or laryngeal attacks of HAE- C1-INH in adults and adolescents, and for self-administration for the treatment of acute attacks of HAE [31,83]. Berinert is also currently approved for treatment of acute HAE-C1-INH attacks in Argentina, Australia, Canada, Israel, Japan and Russia. Marketing authorization is being sought in several additional countries in Asia, Central America and South America [81]. Health economics HAE-C1-INH is a chronic debilitating disease that requires lifelong management, resulting in a total disease burden comprising economic (direct and indirect) expenditure as well as costs to the patients quality of life and overall wellbeing. Direct costs of the disease include the cost of emergency department (ED) visits and hospital stays for acute attacks, in addition to the HAE medication. A 2010 study examining the economic burden of HAE-C1-INH [84] found that the average annual direct medical cost was approximately US$26,000 per patient. Indirect costs associated with attacks included missed workdays, reduced productivity and reduced income due to an inability to work fulltime. A significant proportion of patients with HAE- C1-INH report that the disease has impaired their work productivity, career advancement and educational achievement [84]. The total annual cost (combining direct and indirect costs) for an average HAE-C1-INH patient was found to be approximately US$42,000 [84]. Apart from the abovementioned economic costs, patients with HAE-C1-INH also have an impaired quality of life, and frequently suffer from emotional distress and depression [85]. It should be noted that the studies mentioned above were conducted before the FDA approvals of Berinert, Cinryze, Kalbitor and Firazyr for the treatment of HAE-C1-INH attacks. As the introduction of these agents would undoubtedly impact both the patient-centered and economic sides of the condition, the results of these studies should be evaluated with this in mind. Apart from the impact of these newer HAE-C1- INH therapies on patient outcomes and the costs associated with the management of this condition, the approval of some therapies for self-administration have provided excellent opportunities for patients to switch to self-administered home therapy; indeed training programs have been established in a number of countries [86]. This permits rapid administration in the early stages of an attack, improves patients quality of life and reduces the frequency (and the associated costs) of ED visits [86]. Treatment options approved for on-demand use for attacks of HAE-C1-INH provide similar efficacy. They differ, however, by the doses administered per patient and price per administration, weighted in part by the need for redosing. A comparison of costs for on-demand treatment of HAE-C1-INH in the UK demonstrated a lower economic burden for treatment with Berinert 20 IU/kg compared with other treatments in patients 75 kg bodyweight [87]. Conclusion In the past few years, tremendous progress has been made in the treatment of HAE-C1-INH, with pdc1- INH concentrates becoming a mainstay of treatment. An RCT, numerous uncontrolled studies and pharmacovigilance studies have demonstrated that Berinert is a well-tolerated and effective treatment for all types of HAE-C1-INH attacks. It is the only treatment for HAE-C1-INH that is licensed in children and is also an efficacious and well-tolerated option for treating acute HAE-C1-INH attacks in pregnant and nursing women. Clinical experience suggests that Berinert is also effective for both shortand long-term prophylaxis of HAE-C1-INH. Recent publications have also demonstrated the benefits of home therapy and self-administration with Berinert. In addition, accumulated clinical evidence demonstrates that Berinert is reliable and that its efficacy is sustainable. Repeated administration of Berinert did not lead to any decrease in therapeutic efficacy, and rebound effects observed with Berinert treatment were minimal. These clinical observations support Berinert as one of the main therapies for treating as well as preventing HAE-C1-INH attacks, and this is reflected in its recommendation as first-line treatment in international consensus guidelines. 546 Immunotherapy (2014) 6(5) future science group