Mouse antibodies

Cetuximab is a human/mouse antibody that binds to the epidermal growth factor receptor to block its stimulation. The pharmacokinetics of cetuximab demonstrate a volume of distribution that approximates the vascular space and a terminal half-life of 70 to 100 hours. Cetuximab has shown clinical activity in the treatment of colorectal cancer. An acnelike rash may appear on the face and upper torso 1 to 3 weeks after the start of therapy. Other side effects include hypersensitivity reactions, interstitial lung disease, fever, malaise, diarrhea, abdominal pain, and nausea and vomiting. 

Denkers, E.Y., Wassom, D.L., Krco, C.J. and Hayes, C.E. (1990b) The mouse antibody response to Trichinella spiralis defines a single, immunodominant epitope shared by multiple antigens. Journal of Immunology 144, 3152-3159. 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

FIGURE 14.7 Schematic illustration of an electrochemical immunoassay based on die poly[G]-func-tionalized silica NPs on carbon electrode. Diamond stands for die mouse IgG Y shape for mouse antibody red strings for poly[G] (reproduced from [62] with permission) (see Plate 16 for color version). 

The antibody preparations could be administered unaltered or (more commonly) after their conjugation to radioisotopes or toxins. Binding of unaltered monoclonal antibodies to a tumour surface alone should facilitate increased destruction of tumour cells (Figure 13.4). This approach, however, has yielded disappointing results, as the monoclonal antibody preparations used to date have been murine in origin. The Fc region of such mouse antibodies is a very poor activator of human immune function. Technical advances, allowing the production of human/humanized monoclonals (see later) may render this therapeutic approach more attractive in the future. 

Antibody immunogenicity remains one of the inherent therapeutic limitations associated with administration of murine monoclonals to human subjects. In most instances, a single injection of the murine monoclonal will elicit an immune response in 50-80 per cent of patients. Human anti-mouse antibodies (HAMA) will generally be detected within 14 days of antibody administration. Repeated administration of the monoclonal (usually required if the monoclonal is used for therapeutic purposes) will increase the HAMA response significantly. It will also induce an HAMA response in the majority of individuals who display no such response after the initial injection. The HAMA response will effectively and immediately destroy subsequent doses of monoclonal administered. In practice, therefore, therapeutic efficacy of murine monoclonals is limited to the first and, at most, the second dose administered. 

Fig. 11.7 Equilibrium assay using immbolized mouse IgG antigen capturing anti mouse antibody from solution. The dynamic range is 300 1, showing 16% biological activity and an affinity of...

Leger, O. J. et al., Humanization of a mouse antibody against human a-4 integrin A potential therapeutic for the treatment of multiple sclerosis, Hum. Antibodies, 8, 3, 1997. 

Incubate with mouse antibodies (in the case of Inada et al. (22) it was mous e monoclonal antibodies raised against human single- and double-stranded DNA) at a dilution of 1 10 in blocking buffer for 1 h at 37°C. 

Ascites Monoclonal 1 10 mg/ml 0.9 9 mg/ml Background from mouse antibodies Max. 90%, cross reactions possible... 

Procedure for generating and using primary mouse antibody Fab fragment complexes on mouse tissues (Adapted from Brown et al. 2004)... 

After preincubation of mouse tissue sections with 10% normal mouse serum for 15 min at room temperature, apply mouse antibody Fab fragment complexes for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

The first monoclonals were murine (mouse) antibodies, because the early experiments involved injecting mice with an antigen, harvesting their B cells, and then fusing a harvested B cell with an immortal tumor cell line. There were immediate ahempts to make therapeutic drugs from murine monoclonal antibodies. Scientists at Harvard Medical School tried to apply murine monoclonal antibodies for heahnenf of tumors as early as 1980. But with a very few exceptions, murine antibodies failed as drugs in humans. 

The problem is the neutralizing or allergic reactions caused by the production of human anti-mouse antibodies because the body treats the MAbs as foreign (refer to Section 4.3.4). The humanization of antibodies, through chimeric to humanized and full human types, helps to address this problem. 

The advent of recombinant DNA technology led to the development of antibodies and fragments that are tailored for optimal behaviour in vivo [7,8]. Humanized and chimeric antibodies can be constructed to circumvent the human anti-mouse antibody response elicited by mouse antibody treatment of patients, which severely hampers the application of these powerful molecules. The treatment of rheumatoid arthritis patients with doses of as high as 10 mg kg cA2 chimeric antibody specific for TNFa [9], emphasizes that at present the production and purification methods for these proteins have been optimized to such extent that clinical studies can be considerably intensified. 

---