Estado del arte:

A striking feature of antibodies is the high specificity, targeting only the microorganism that causes the disease or the tumorigenic cell, which displays distinct properties in contrast to normal cells. The design of an antibody (isotype, coupling of chemical compounds, scFv of IgG) depends on the target, disease and mode of action. Antibodies can function through several mechanisms: selective killing of the target cell by either delivery of toxic payload (radioisotope, catalytic toxins, drugs, cytokines and enzymes) (i), or inducing apoptotic signaling (ii), antibody dependent cellular cytotoxicity (ADCC) (iii), complement dependent cellular cytotoxicity (CDC) (iv), or simply blocking the binding of molecules to a target e.g. a receptor (v).

The isotype offers a variety of possibilities for the design of antibodies to achieve a specific effect. Most therapeutic monoclonal antibodies (mAb) contain the IgG1 isotype, which is able to mediate Fc domain-based functions, such as ADCC and complement fixation. Other isotypes e.g. IgG2 act only through antigen-binding e.g. mAb directed against growth factor receptors on tumor cells. Since antibodies are produced in cell lines or other expression systems, a possible contamination with viruses or other infectious agents cannot be excluded. A further challenge is the appropriate therapeutical antibody concentration, which is crucial for a successful therapy, since the so-called prozone effect occuring after excessive antibody administration, can be detrimental for the host (Taborda et al., 2003). The prozone effect describes the lack of agglutination at high concentrations of antibodies. Since protection against bacterial infection is hampered as a result of high antibody concentration, the appropriate antibody concentration has to be considered for the treatment for infectious diseases.

4.2.3.1 Antibodies for cancer therapy

Several monoclonal antibodies are currently used for therapy of several cancer types (Table 6). The first humanized antibody was alemtuzumab (Campath® anti-CD52) (Albitar et al., 2004; Osterborg et al., 1997), which is now used for the treatment of chronic lymphocytic leukemia (Alinari et al., 2007). In contrast to most anti-cancer antibodies, which are coupled to toxic substances, so-called naked antibodies are in therapeutic use, acting through Fc receptor or complement activation, which induces crosslinking of surface receptors leading to apoptotic signaling. Many solid tumors overexpress growth factor receptors such as EGFR (epidermal growth factor receptor) stimulating cell growth in an autocrine manner. A series of mAbs were shown to inhibit ligand binding and

therefore, receptor activation (Teramoto et al., 1996). Healing from established tumors was achieved when anti-EGFR mAbs were combined with chemotherapeutic agents such as cisplatin (Fan et al., 1993). A novel strategy in mAb tumor therapy describes the antibody-directed enzyme prodrug therapy (ADEPT). Here, an enzyme is conjugated to the antibody. After antibody binding to the tumor cell, the enzyme cleaves a subsequently delivered prodrug, which becomes then active in the tumor cell. This leads to highly specific deposition of an active drug solely in the tumor cell. Clinical study, employing this strategy, have been investigated for colorectal cancer (Francis et al., 2002) (Mayer et al., 2004).

Lymphomas are targeted by mAbs directed against cell surface receptors like e.g. CD20 acting through mechanisms like activation of ADCC, CDC and apoptosis. The affinity of an antibody can also be modified according to the desired effect. For example, in tumor therapy a higher affinity of the antibody to the antigen is not recommended since this correlates with an impeded penetration ability into the tumor mass (van Osdol et al., 1991; Weinstein et al., 1987). This finding was proposed as the binding site barrier, which describes the fact that high affinity interactions between the antibody and its target block the diffusion of the antibody through the tumor mass. The use of a high affinity scFv directed aginst EGF (epidermal growth factor) which exhibited diminished penetration into solid tumor compared to their lower affinity counterparts (Adams et al., 2001) supported this effect. In contrast, if an ADCC response should be achieved, a higher affinity is required. In combination with multivalency, which is also an important feature, the ADCC response can be more efficient. For example bispecific antibodies targeting both EGFR and a distinct Fc receptor reveal more effective mediators of ADCC, in case the antibody affinity is higher (Weiner and Carter, 2005).

4.2.3.2 Antibody based therapy for infectious diseases and other

implications

Passive antibody therapy is currently used to treat and prevent diseases caused by severeal viruses and bacteria. Besides the general properties such as ADCC or CDC, antibodies exhibit also direct toxin neutralization and microbial properties (e.g. generation of oxidants). Human mAb have been generated against e.g. fungal antigens (Matthews et al., 2003). Antiviral active antibodies have been designed against respiratory syncitical virus (RSV) infection, available for prophylaxis (Wang and Tang, 2000) and for treatment (Domachowske and Rosenberg, 1999). Prophylactic antibodies against Hepatitis B virus are currently used (Zuckerman, 2007). A protective effect against the

antibodies (Lang et al., 1993). Recently, immunization of transgenic mice resulted in the generation of shiga-toxin 1 neutralizing human mAbs (Mukherjee et al., 2002). Additionally, in mice, high affinity scFvs have been demonstrated to be protective against anthrax toxin in mice (Maynard et al., 2002). Furthermore, neutralizing Fab fragments against measles virus have been isolated by phage display (de Carvalho Nicacio et al., 2002). Moreover, some antibodies seem to be able to kill bacteria directly by altering their surface structures (Connolly and Benach, 2001). Radioimmunotherapy, succesfully used in cancer treatment, has also been described for treatment of fungal infection (Dadachova et al., 2003).

Antibodies encompassing high affinity can be developed to bind cytokines and their receptors to reduce the inflammatory response. Patients with rheumatoid arthritis for example, can be treated with chimeric anti-TNF α antibodies (e.g. Infliximab; Table 6), which are also available for the treatment of Crohn’s disease (Mikula, 1999).

Fc-IgG interaction has been reported to reduce the inflammatory response and diseasae related damages (Samuelsson et al., 2001).

Antibodies can be applied not only for disease treatment or vaccination, but also for other implications (Table 6). For instance, in organ transplantation, immunsuppression is required and achieved by targeting CD3 (Wilde and Goa, 1996), a T-cell surface receptor required for T-cell activation. Furthermore, antibodies directed against the 37 kDa/67 kDa LRP/LR block both the binding of the prion protein (Leucht et al., 2003; Zuber et al., 2008a; Zuber et al., 2007b) and the binding of laminin-1 to the laminin receptor (Zuber et al., 2008b). Thus, these antibodies can be used for therapeutic application in prion disease (Chapter II-IX) and intervention of metastatic potential of tumor cells (CHAPTER XI).

4.2.3.3 Rediscovery of polyclonal antibodies for therapy

Although designed monoclonal antibodies, Fabs and scFvs are widely used and generated for therapeutic purposes, also the polyclonal format becomes again interesting for therapeutic applications. Polyclonal antibodies (pAb) are recommended for the treatment of diseases associated with complex antigens such as sepsis (Kreymann et al., 2007). A polyclonal ovine anti-TNF fragment antigen binding (Fab) fragment was effective for sepsis (Rice et al., 2006). Additionally, an oligoclonal antibody cocktail has been proven to efficiently neutralize and clear patients from botulinum neurotoxin (Nowakowski et al., 2002). The polyclonal anti-LRP/LR antibody displayed also a more prominent therapeutic effect compared to the single chain format (Zuber et al., 2007b).

Intraperitoneal (ip.) administration in scrapie infected mice prolonged the survival of these mice compared to another study where scFvs were delivered i.p. into prion infected mice. For cancer therapy the polyclonal format is believed to be more efficient in mediating effector functions and escape variants are thought to be minimized by multi-targeting through the polyclonal format (Sharon et al., 2005). Polyclonal antibodies can be produced in transgenic animals or via the combination with mAb production technology. For this, polyclonal antibody libraries (pALs) have been generated (McNichol et al., 2007; Sarantopoulos et al., 1994). Heavy and light chain variable region genes are obtained from immune B cell-containing tissues, by RT-PCR, and cloned in pairs in a Fab phage display vector. The resulting Fab phage display library is then selected for desired specificities and the selected VL–VH region gene pairs are transferred from the phage display vector

to a mammalian expression vector. Full-length antibodies can be produced by transfection of the mammalian expression vector library into mammalian cells (Sharon et al., 2005; Sharon et al., 2002).

Table 6. Available therapeutic antibodies Generic name /

Trade name type target indication

Cancer Alemtuzumab / MabCampath® humanized CD52-antigen on lymphocytes Chronic lymphatic leukemia, T-cell-lymphoma Bevacizumab / Avastin®

humanized VEGF (Vascular

Endothelial Growth Factor)

colorectal cancer, lung cancer

Cetuximab /

Erbitux®

chimeric EGF-receptor (Epidermal

Growth Factor Receptor)

colorectal cancer

2_{Epratuzumab /}

LymphoCide®

humanized CD22-antigen Non-Hodgkin-Lymphoma,

Autoimmune diseases 1_{Gemtuzumab /} Mylotarg® humanized, conjugared to calicheamicin

CD33-antigen acute myelogenous

leukemia Ibritumomab / Zevalin® murine, 90Y- radiolabeled CD20 on B-lymphocytes non-Hodgkin-lymphoma Panitumumab / ABX-EGF

humanized EGF (Epidermal Growth

Factor Receptor)

non-small cell lung cancer (not approved yet) (Phase I/II-Studien)

Rituximab /

MabThera®

Bexxar® labeled

Trastuzumab /

Herceptin®

humanized HER2/neu-Rezeptor breast cancer

Autoimmune diseases

Adalimumab /

Humira®

humanized Tumor Nekrose factor

(TNF- )

rheumatoid arthritis

Basiliximab /

Simulect®

chimeric CD25-antigen (Interleukin-

2-receptor)

Prophylaxis in kidney

transplant rejection

Daclizumab /

Zenapax®

humanized CD25-antigen (Interleukin-

2-receptor)

Prophylaxis in kidney

transplant rejection

Infliximab /

Remicade® chimeric tumor Nekrose factor _{(TNF- )} Crohn’s rheumatoid arthritis disease,

Muromonab / Orthoclon OKT3® murine CD3-antigen (T- lymphocytes) Prophylaxis in kidney/heart/liver transplant rejection 1_{Natalizumab /} Tysabri® (FDA appproved)

humanized CD49d (VLA-4, integrin) multiple sclerosis

Cardiovascular disease Abciximab / ReoPro® chimeric, Fab2- fragment, anti- platelet

GPIIb/IIIa on thrombocytes Coronary intervention and angioplasty

Infectious disease

Palivizumab /

Synagis® humanized anti-F protein, component of respiratory syncytial virus (RSV) Prophylaxis of RSV- pneumonia others Efalizumab / Raptiva®

humanized CD11a-antigen psoriasis

1_{Omalizumab /}

Xolair ®

humanized IgE (Fc-Teil) Allergy, severe asthma

bronchiale (1_{not approved for Germany), indicated types are all full length Igs except Abciximab}