16 Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies

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16 Safety Aspects of Biologics: Lessons Learnt

from Monoclonal Antibodies

C.K. Schneider, J. Löwer

16.1 Introduction

Based on his observations from selective histological staining of bacteria, Paul Ehrlich in 1900 published his idea that certain compounds could be used as “magic bullets” to selectively target external pathogens or even tumours (Ehrlich 1900). With the description of the hybridoma technology by Köhler and Milstein in 1975, the production of targeted monoclonal antibodies (mAbs) became possible and Paul Ehrlich’s dream of magic bullets a reality. Currently, more than a dozen mAbs are licensed (Reichert et al. 2005), and more promising products are about to enter clinical development. While the first antibodies were of completely murine origin due to the underlying technology, scien- tists started an “evolution” of mAbs in order to reduce immunogenicity (Borchmann et al. 2001). Murine pro- teins are highly immunogenic, and continuous use in humans in most cases is not possible due to the occurrence of neutralizing antibodies, leading to loss of efficacy and/or safety problems like infusion reactions.

Therefore, chimaeric antibodies such as infliximab (Remicade) were developed, reducing immunogenicity by replacement of the murine Fc part by the human counterpart. Still being considerably immunogenic, monoclonal antibodies were further “humanized” also by replacing large parts of the Fab part of the molecule by human sequences (for example, in trastuzumab, Herceptin). With the latest techniques, the production of fully human mAbs has also become possible, further reducing immunogenicity to a small but still existent extent. The only fully human mAb licensed so far in Europe is adalimumab (Humira), an anti-TNF-[ mAb (Salfeld and Kupper 2007).

Despite these achievements in a more structural

“evolutionary chain” of mAbs, mechanisms of action

are also evolving. While the “classical” mAbs have rather clear-cut hypotheses as regards the way they func- tion, for example targeting tumour epitopes on the surface of tumour cells, newer mAbs appear to be becom- ing more specific, targeting distinct subepitopes of certain structures. A recent example is the anti-CD28 mAb TGN1412, which is directed against a certain substruc- ture of the CD28 molecule, the C’’D loop (Luhder et al.

2003), exhibiting a distinct pharmacodynamic effect, representing a so-called “super-agonist”. This product has gained unfavourable publicity due to serious adverse events during testing in a first-in-man trial (Schneider et al. 2006; Suntharalingam et al. 2006).

Cases like this demonstrate that therapeutic intervention with biologics can be harmful to a considerable extent, and that safety is a central aspect to be considered for these products. In this context it is important to note that possible adverse events might be deducible from the (putative) mechanism of action of a particular compound, for example potential toxicity to skin kera- tinocytes of mAbs directed against epidermal growth factor receptor-1 (EGFR-1), like cetuximab (Erbitux).

However, experience clearly shows that such considerations might not be sufficient, and that other aspects need to be taken into account. mAbs might exhibit a certain fine specificity that discriminates them from others, although directed against the same antigen. As discussed above, TGN1412 might again serve as an example. Another important aspect influencing safety of mAbs is the design of the antibody with respect to the isotype. An IgG1 mAb can be expected to behave differently from, for example, an IgG4 mAb, although directed against the same epitope. This can be explained by the different effector mechanisms mediated by the Fc parts of the IgG isotypes. While IgG1 can fix and activate complement, thereby triggering complement-mediated cytotoxicity when directed against a

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cell-membrane bound antigen, IgG4 might be expected to only bind to and block the epitope, potentially without directly affecting the viability of the target cell, since IgG4 cannot activate complement. Monoclonal antibodies are under development with mutated Fc parts, modulating the interaction with Fc receptors.

For such molecules, the possible effects cannot solely be imputed by considerations about blockage of the epitope. Chemical modification such as PEGylation can change pharmacokinetic behaviour, tissue penetration capacity, and immunogenicity, thereby considerably changing the pharmacodynamic behaviour of a mAb.

Therapeutic intervention with mAbs can be achieved by several principles, for example, neutralization of soluble and/or membrane bound cytokines, blockage of membrane-bound molecules that mediate intercellular communication, and blockage of growth factor receptors with or without triggering of cell death. This chapter highlights recent examples of licensed mAbs representative for each of these mechanisms, discusses specific safety aspects concomitant with these interventions, and briefly describes the regulatory measures aiming at a reduction of these identified risks.

16.2 Intervention with Pleiotropic Cytokine Pathways

One of the major breakthroughs in the development of efficacious medicines against various autoimmune diseases including rheumatoid arthritis and Crohn’s disease (CD) has been the introduction of the anti-TNF-[ principle (Taylor 2007; Salfeld and Kupper 2007). One of the key mediators of autoimmune inflammation is TNF-[ , a multipotent cytokine occurring both in a soluble and a non-soluble, membrane-bound form. Pro- duced mainly by macrophages and T cells, TNF-[ induces a variety of further cytokines, resulting in a pleiotropic biological role in inflammation, host defence including mycobacteria, and cancer immunity (Beutler 1999). With the introduction of TNF-[ inhibitors, the therapeutic arsenal against RA and other autoimmune diseases was amended by a potent new mechanism of action. Three drugs are licensed, two monoclonal antibodies (infliximab, Remicade, and adalimumab, Humira) and one fusion protein consisting of the extracellular portion of the p75 TNF receptor fused to the Fc part of human IgG1 (etanercept, Enbrel). Remi-

cade was the first monoclonal antibody against TNF-[ to be licensed in Europe, and therefore a relatively large safety database exists for this product. Remicade was first licensed in the EU for the treatment of severe, active Crohn’s disease under “exceptional circum- stances” (European Medicines Agency 2006a) due to promising efficacy in this frequently severe autoimmune disease. Remicade was licensed for treatment of patients who have not responded despite a full and adequate course of conventional treatment such as a corticosteroid and/or an immunosuppressant. However, after marketing authorization in this second line setting (i.e. requiring an immunosuppressor OR cortico- steroids), the “efficacy” of Remicade became apparent also in a negative sense, since due to the strong inhibition of TNF-[ serious adverse events were also observed, such as reactivation of tuberculosis (TB), severe infections, and sepsis. Occurrence of serious infections is the key safety problem. Data suggest that TNF-[ plays a physiological role in the formation and maintenance of granulomas, a host defence mechanism against intracellular pathogens that cannot be killed by other mechanisms (e.g. mycobacteria) (Wallis and Ehlers 2005). Treatment with TNF-[ inhibitors apparently leads to delayed formation of these granulomas or the disruption of existing ones, resulting in reduced or even abolished host defence against certain pathogens like mycobacteria. Accordingly, cases of tuberculosis reactivation and other opportunistic infections have been reported in the post-marketing setting. Such reports have also been reported in Germany, as illus- trated in the reports in 2004 of opportunistic infections to the competent national authority responsible for these kinds of products, the Paul-Ehrlich Institut (Table 16.1). These data from the reporting period of 2004 are representative results. A recent report in the literature describes a clinical case of a patient who developed widespread pulmonal TB during long-term treatment with infliximab for ankylosing spondylitis despite a 9-month isoniazide prophylaxis, showing a delayed response even to a five-drug anti-tuberculosis treatment (Vlachaki et al. 2005). Cases of patients like this, which are very difficult to treat, illustrate that the use of biologics like Remicade needs to be thoroughly considered on a case-by-case basis. Based on the observations on serious infections and also other safety issues in the time after marketing authorisation, an expert panel had convened at the European Agency for the Evaluation of Medicines (EMEA), and in early 2002 170 16 Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies

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Table 16.1. Opportunistic infections with infliximab (Remica- de) during February to August 2004 reported to the Paul-Ehr- lich Institut, Germany

Pathogen No. of

reports

Deaths Crohn’s RA Other

Pneumocystosis 13 2 0 11 2

Atypical mycobacteria 8 0 1 5 2

TB 92 11 13 55 24

Histoplasmosis 8 3 1 7 0

Listeriosis 3 0 1 1 1

Aspergillosis 2 0 1 1 0

Coccidioidomycosis 15 2 0 14 1

CMV 14 6 3 7 4

Systemic candidiasis 9 4 6 1 2

Blastomycosis 2 1 1 0 1

Toxoplasmosis 1 0 0 1 0

Nocardia 5 0 2 1 2

Herpes 25 1 6 18 1

Total 200 50 35 122 39

the indication for the treatment of CD was restricted.

This was implemented by the EMEA’s scientific committee, the Committee for Proprietary Medicinal Prod- ucts (CPMP, now CHMP, see below), by an urgent safety restriction (European Medicines Agency 2006a). The resulting indication was a restriction on patients not responding to a full and adequate course of treatment with a corticosteroid AND an immunosuppressant (instead of “and/or”), resulting in a restriction to a third line indication. Narrowing the patient population to a more restricted subset is a strong regulatory mea- sure, and one of the regulatory principles intended to ensure safe use of medicines only for patients where the benefits clearly outweigh the risks. To further enhance patient safety and also awareness both of patients and prescribers, a patient alert card was introduced in 2002, which has proven to represent a powerful tool for this purpose. Meamwhile, the indication for Remicade in the treatment of Crohn’s disease has been changed back to a second line indication, based on a more solid database that has emerged over the years.

16.3 Intervention with Adhesion Molecules

Another promising principle of interference with the immune system is the inhibition of adhesion molecules on T lymphocytes, like integrins. Integrins are hetero- dimeric cell adhesion molecules, playing pleiotropic roles in fetal development, immune reactions, leuko-

cyte migration, haemostasis, and tumour biology (Hynes 2002). T cells in the blood circulation adhere to endothelium by interaction of alpha-beta integrins on their surface (Lobb and Hemler 1994). In 1993 it was established that T-cell migration into brain parenchy- mal tissue is mediated by a distinct subset of these integrins, the [4q1integrin molecule (Baron et al. 1993), interacting with counter-receptors on endothelial cells.

Observations like this lead to the idea of inhibiting T- cell migration in diseases where T cells are key mediators, like multiple sclerosis (MS), which is the most common inflammatory autoimmune disease of the central nervous system (Gold and Hohlfeld 2007). Anti- bodies against [4integrin on T cells were shown to inhibit the MS animal model, experimental autoimmune encephalomyelitis (EAE) (Yednock et al. 1992). A humanized antibody was developed (natalizumab, Tysabri, formerly “Antegren”), which binds to the [4

subunit of human [4q1and [4q7integrin, for the treatment of patients with MS. First clinical data from patients dosed for 6 months revealed a nearly subtotal inhibition of inflammation, as measured by uptake of contrast medium in Magnetic Resonance Imaging (MRI) (Miller et al. 2003). These promising data also translated into clinical benefit, resulting in a significant reduction of relapse rate and delay in the progression of disability as measured by the Expanded Disability Sta- tus Score (EDSS) after 2 years of treatment (Polman et al. 2006). Based on 1-year data, which already showed a clinical benefit in terms of relapse rate reduction, the US Food and Drug Administration (FDA) granted pri- ority review status for the regulatory assessment of the 1-year clinical data in June 2004, and granted an accel- erated approval of Tysabri in November 2004. In mid February 2005, the marketing authorization holder published key 2-year efficacy data, but around 2 weeks later on 28 February 2005 announced a voluntary sus- pension of the marketing authorization due to the occurrence of a serious unexpected adverse event, progressive multifocal leukencephalopathy (PML). This event evolved in two patients in the long-term MS trials, both being concomitantly treated with beta-interferon (Kleinschmidt-DeMasters and Tyler 2005; Lan- ger-Gould et al. 2005). One of the patients died, and one survived with severe disability. All clinical trials were immediately suspended, and no further drugs were administered to patients. PML is a rare non-inflammatory demyelination (Astrom 1958) mediated by reactivation of JC virus, a polyoma virus. PML is mainly seen

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in patients with severe immunosuppression, e.g. indi- viduals with AIDS. The most probable explanation of how PML could have occurred in the two patients is that Tysabri potently suppresses the migration of any T cell, not only pathogenic autoreactive cells, but also T cells circulating for detection and elimination of viruses and other pathogens (T-cell surveillance). As a consequence, reactivation of viruses is thought to become insufficiently controlled by T-cell surveillance.

Following these events, the marketing authorization holder evaluated most patients having been treated with Tysabri and found no other case of PML, despite a retrospective case of a patient who had died of PML in a clinical trial for Crohn’s disease (Van Assche et al.

2005). Based on the observed clinical efficacy and the medical need in the treatment of patients with MS, the Committee for Medicinal Products for Human Use (CHMP, until 2004: CPMP) at the European Medicines Agency EMEA (until 2004: European Agency for the Evaluation of Medicines) issued a positive opinion in April 2006, recommending a marketing authorization for Tysabri for the European Union to the European Commission. This was based on a clear-cut restriction of the patient population, firstly patients with high disease activity despite treatment with a beta-interferon, and secondly patients with rapidly evolving severe relapsing remitting multiple sclerosis. Together with a strict risk management system, educational program- mes including algorithms for the detection of PML and other opportunistic infections, and a patient alert card, the CHMP considered the benefit-risk positive for the aforementioned patient population with more severe MS (European Medicines Agency 2006b). The Europe- an Commission granted a marketing authorization for Tysabri in the aforementioned setting at the end of June 2006. In June 2006, the US FDA also reintroduced Tysa- bri to the US market.

16.4 Intervention with Growth Factor Receptors

Tumour cells frequently harbour growth factor receptors and/or structures that are rather restricted to these cells. The idea of using the immune system by specifi- cally targeting these molecules with mAbs is one of the classical concepts in the therapeutic interventions with mAbs. Several antitumoural antibodies are licensed, e.g. cetuximab (Erbitux), rituximab (MabThera), alem-

tuzumab (MabCampath), and trastuzumab (Hercep- tin). The latter antibody, trastuzumab, is directed against the Her2/neu antigen (human epidermal growth factor receptor 2 protein) expressed on a subset of breast cancers (approximately 30 % of cases), and in Europe until recently was only licensed for the treatment of metastatic breast cancers which express a high level of this antigen, either as a “last line” monotherapy, or in combination with taxanes as first-line combination therapy. Her2/neu is a member of the epidermal growth factor receptor (EGFR) family and is encoded by c-erb B-2 (the human homologue of the rat neu oncogene), a proto-oncogene that codes for a trans- membrane glycoprotein receptor tyrosine kinase (European Medicines Agency 2006c). Its physiological role is thought to be part of tissue homeostasis including lobuloalveolar differentiation and lactation in physiological breast tissue. Further, it participates in normal development and organogenesis of nervous and heart tissue (Olayioye et al. 2000; Yarden and Sliw- kowski 2001). Adding Herceptin to chemotherapy exhibited a potent cytotoxic effect on breast cancers in clinical trials, leading to a market authorization of the mAb in 2000 in the European Union (European Medi- cines Agency 2006c). Soon it became clear that some patients treated with Herceptin showed signs and symptoms of cardiotoxicity, especially in combination or after treatment with anthracyclins. This unwanted effect on the adult organism cannot readily be explained by its physiological role in embryonic development. In preclinical studies during the clinical development programme before marketing authorization application, no tissue cross-reactivity with monkey or human heart tissue was observed, and no cardiotoxicity in a relevant animal surrogate model was noted (European Medicines Agency 2006c). Animal experi- ments performed subsequently show that selective Her2/neu deletion in left ventricular tissue in mice yields an apparently normal phenotype; however, mice with this deletion show signs of dilatative cardiomyop- athy, and cardiomyocytes are more susceptible to anthracyclin-induced cardiotoxicity (Crone et al.

2002). Bioptic evaluations suggest that Herceptin- induced cardiotoxicity could be distinct from that of anthracyclins (Ewer et al. 2005). While most cardiac events are usually asymptomatic and reversible, severe cardiac events have also been reported. Most events occur in combination with anthracyclins, which are themselves cardiotoxic compounds. As a result, the 172 16 Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies

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warning statements in the European Summary of Prod- uct Characteristics (SmPC) were strengthened. Cardiac monitoring before and during therapy with Herceptin has become standard practice. Recently, the license for Herceptin has been extended to the treatment of patients with HER2 positive early breast cancer following surgery, chemotherapy (neoadjuvant or adjuvant) and radiotherapy.

16.5 Conclusion

Biologics like monoclonal antibodies are innovative molecules that have in some cases revolutionized mod- ern medicine due to unprecedented efficacy; however, experience shows that this efficacy also expands to unwanted side effects. Several principles of how mAbs can target human autoimmune diseases are known, ranging from intervention with pleiotropic cytokine pathways to inhibition of T-cell migration. Direct cytotoxicity is a classical principle of targeted therapies against tumours. All of these mechanisms are inherent- ly associated with certain safety problems; however, regulatory measures can be implemented that never- theless allow for a relatively safe administration of such drugs to patients. Such regulatory measures include a restriction of the indication to certain patients, usually those suffering from a more severe stage of the disease, and provision of educational information to prescribers and patients. Further research is needed to elucidate the exact mechanisms of action behind the typical safety problems. While most hypotheses appear convinc- ing, still other unknown mechanisms could be responsible, and detailed knowledge could help further reduce the risks for patients.

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