Therapeutic antibodies mAbs

Mammalian cells are commonly employed for the production of therapeutic and diagnostic proteins, since they are able to correctly synthetize the large and complex structures that the human body requires as medicine [1]. Nowadays, they are employed for the large-scale production of recombinant therapeutic proteins, monoclonal antibodies (MAbs) and viruses used in the preparation of vaccines (e.g. against rabies, hepathytis B, polio, etc) [2]. An overview of some licensed/approved products derived from mammalian cell culture is given in Table 1. 

The cost of some therapeutic antibody products (eg, MABs) is more than 10,000 per year. Pharmaceuticals tend to be the highest out-of-pocket health-related cost because other health care services are covered by health insurance, whereas prescriptions often are not, although this is changing. 

As of 2008, 25 therapeutic monoclonal antibodies (mAbs) mAbs had been approved for clinical use in the United States, and with over 400 antibodies being in preclinical and clinical development further increase of antibody therapies is assured (10, 11). As a general rule, the Fc fragment is a key component of therapeutic mAb design because it extends their pharmacokinetics. Inclusion of the Fc from IgG is also a key component of other bioactive proteins where prolongation of pharmacokinetics is desired, e.g., the tumor necrosis factor receptor (TNFR) fusion protein etan-ercept (Enbrel ) (12). Thus for both therapeutic antibodies and Fc-fusion proteins, the FcRn interaction is a generalized way to exploit FcRn protection to achieve the benefits of extended persistence in vivo. 

The beta phase clearance kinetics of human IgG and therapeutic antibodies is typically monophasic and abides to a log-linear relationship of serum concentration with time after administration. However, in rare cases we have found that certain administered mAbs are immunogenic to the mouse and elicit mouse-anti-human antibodies. In this case, there is a bi-phasic kinetic, typically appearing at days 5-6 after antibody administration and resulting in a precipitous antibody loss. This complication can be overcome by the use of immunodeficient hFcRn transgenic mice, described in Section 3.7. 

Therapeutic antibodies must be distinguished as mAbs (and derived products) and polyclonal antibodies when discussing their different pharmacokinetic properties and therapeutic applications. mAbs are already - and will in the future - be much more important in drug development and applied pharmacotherapy due to their favorable properties and higher clinical success rates compared to polyclonal antibodies. Thus, although both types of antibody will be discussed in the following sections, most emphasis will be placed on mAbs. 

Polyclonal antibodies however, also have some drawbacks. Due to the non-human molecular heterogeneity, unspecific reactions are likely to occur and may cause a large variety of adverse reactions. In addition, the dose to be administered to target a specific antigen is relatively high compared to that for mAbs this is due to the heterogeneity in specificity and affinity. Furthermore, the immune system will produce anti-antibodies to attack the non-human structures on polyclonal therapeutic antibodies, thereby potentially leading to serious hypersensitivity reactions. 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

Compared to other bioanalytical methods such as high-performance liquid chromatography (HPLC), the methods used to quantitate mAbs often display less precision and a higher between-day variability. In choosing a bioanalytical method it must also be considered that some assays measure the unbound fraction, the bound fraction, or both. When using FACS, only the fraction of the therapeutic antibody that is bound to its antigen on the cells is counted. In contrast, ELISA measures only the unbound fraction in serum that can react with the offered antigen. 

Another reason for the different half-lives among mAbs lies in the origin of the therapeutic antibodies. Degradation will be more rapid for more nonhuman anti-... 

The formed anti-idiotype antibodies almost irreversibly bind the therapeutic antibodies and therefore, by neutralization, eliminate them from the body. In total, the formation of anti-idiotype antibodies will alter the pharmacokinetics and consequently the pharmacodynamic effect (loss of effectiveness) of affected mAbs. 

Compared to polyclonal antibodies, mAbs display molecular homogeneity and significantly higher specificity leading to increased in-vivo activity. For example, 100-170 mg serum containing polyclonal antibodies against the tetanus toxin are necessary to achieve the same effect as 0.7 mg of a respective mAb. This section will provide an overview on therapeutic antibodies which have either been approved or are in clinical development, and will classify them according to different pharmacodynamically relevant properties. 

There are several relatively new therapeutic modalities for the treatment of SLE. Trying to eliminate pathogenic anti-dsDNAs, Ferguson etal. developed an antigen-based heteropolymer (AHP) (F3). AHP is a bispecific dsDNA x monoclonal antibody (mAb) complex (dsDNA x anti-CRl mAb) that enables the use of the unique immune complex-binding and clearing capacity of the complement receptor (CR1) on primate erythrocytes. In vitro studies of AHP show a substantial reduction (>90%) of anti-dsDNA titer (F20). In vivo studies in two rhesus monkeys indicate that the erythrocyte-bound antibodies are rapidly cleared from the circulation (F3). 

The first therapeutic antibody approved (Orthoclone OKT-3 or Muromonab CD3, 1986) was indicated not for cancer treatment, but for controlling acute rejection of transplanted organs (kidney, heart, and liver). Nowadays, other clinical indications such as asthma, rheumatoid arthritis, psoriasis, and Crohn s disease are treated with mAbs (see Chapter 17) (Antibody Engineering and Manufacture, 2005 Monoclonal Antibodies and Therapies, 2004 Hot Drugs, 2004 Walsh, 2004). 

Air-lift bioreactors were popular in the 1990s, but currently their large-scale use is limited. Only the company Lonza (originally Celltech) uses this type of bioreactor on a large scale, for the production of monoclonal antibodies (mAbs) and other therapeutic proteins (Lonza, 2006). 

Comparison of sales of therapeutic monoclonal antibodies (mAbs) in 2002 (in white) and forecast for 2008 (in black) (adapted from Reichert and Pavlou, 2004). 

Monoclonal antibodies (MAb) or MAb fragments have been described above as homing devices for soluble and participate carriers however, they can also be used in their own right as soluble carriers. The first marketed (1986) MAb for therapeutic use was the anti-CD3 antibody OKT3, for the prevention of rejection of kidney transplants. More recently, MAb for the treatment of post angioplasty complications (ReoPro) and for the treatment of colorectal cancers (Panorex) have been introduced. 

In the 1980s, monoclonal antibodies were hailed as magic bullet therapeutics for the treatment of cancer, autoimmune disorders, and infectious diseases. Humanized monoclonal antibodies (MAbs) are now succeeding as drugs where mouse MAbs failed. Fully humanized MAbs are in the development pipeline, and therapeutic-biospecific MAbs are extending the versatility of nature s magic bullets. Several MAbs are now FDA-approved drugs (Table 2). Several MAbs are also in development (Table 3).3... 

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